MODERN PHARMACOLOGY-TOXICOLOGY

Series oj'Mollograplzs ol7d Texthuoks

Wi!liam F Bousquet
l)ivision ot' Biological Research
S"arlc Searle &
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Roger F Palmer
University 01' Miami
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L!NTERFERON AND
INTERFERONINDUCERS

Edited Dale
Experimcntal Rcscarch
The Upjohn
KCJ1CJmCJzoo. Michigun
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1, INC.
Ma(!ison Avenue, New Yo1'k, New Yo1'k 10016
(:I/I'I'eJ1t p1'inting (last digit):
10 9 8 7 6 5 4 3 2 1
I'l1lNTED lN STATES OF
daughters:
Jennifer, Wendy, and Ashley
During the past two decades interferon research has steadily incrcascu 1
intensified. At first, the discovery of interferon was of interest to
handful of virologists the of viral
more about this unique group molecules, interest Ll!eil'
potential use in treating viral increased, new invcsti1-(al.ol'::
the field. then seemed to wane due to the difficulties utiliy.illJ'.
or its inducers for viral diseases. the past
few years, however, era of research has developed. TI1('
role of cellular control processes has lllOUlIla-
tion of antiviral to include regulation and cancer chcl1\o-
therapy. These developments have rekindled awareness interferon
research and brought about renewed commitment to bring this excitil11-(
group of agents to clinical
When 1 was first asked to plan this volume, 1 realized that prol1l is-
ing approaches that lead to the fulfillment of potential
effective chemotherapeutic agents had neglected. 1 therefore deciUc(J
to emphasize this phase of research.
The authors, leaders in their fields, have prepared chapters 011 areas
il1 which they hold of esteem. Each chapter is designed to
broad enough to of value to the novice yet detailed enough to useful
referel1ce source for the experiel1ced interferon researcher .
Interferon and interferon inducers are presently undergoing clinical
evaluation. This book is intended to serve as for these stuuies,
highlighting our progress and problems currently being encountered.
Dale Stringfellow
CONTENTS
Preface v
Contributors
1 INTERFERONS: AN OVERVIEW 1
Dale Stringfellow
2 PRODUCTION, PURIFICATION,
AND PROPERTIES OF INTERFERONS 21
Kurt Berg, Kurt Osther, and Iver Heron
EXOGENOUSINTERFERON:
AND 57
Stephen Greenberg, Maurice W. Harmon,
and Robert Couch
4 EXOGENOUS INTERFERON: USE IN
FOR OF MALIGNANCIES 89
Michio Ito and Rita F. Buffett
5 CLINICAL USE OF INTERFERONS
IN VIRAL INFECTIONS
Alfons and Piet De Somer
6 INTERFERON INDUCERS:
THEORY AND APPLICATION 145
Dale Stringfellow
7 INDUCTION OF INTERFERON
POLYNUCLEOTIDES 167
Hilton Levy
8 TILORONE HYDROCHLORIDE
AND RELATED MOLECULES 187
Gerald D. Mayer and Russell F.
vi

M01,f';CUJ,f';S
ItoIJCI.'ll<'. Bctls Uoruon Uouglas, J1'.
lN'l'J,;I{,}'J,;H,ON lNDUCERS:
YANIONS AND OTHERS
Mary Breinig and Page S. Morahan
11 AND INDUCERS:
IMMUNE MODULATION
Howard Johnson
Author Index
Subject Index
vi I

KURT BERG Department of Virology, Institute of Medical Microbiology,
University of Aarhus, Aarhus,
ROBERT BETTS Infectious Disease Unit, Department of Medicine,
University of Rochester School of Medicine, Rochester, New York
ALFONS Department of Human Biology, Division of Microbiology,
Rega Institute, University of Leuven, Leuven, Belgium
MAR BREINIG * Department of Microbiology, Medical College of
Virginia, Virginia Commonwealth University, Richmond, Virginia
BUFFETT Department of Viral Oncology, Roswell Park Memorial
Institute, Buffalo, New York
ROBER COUCH Department of Microbiology and Immunology, Baylor
College of Medicine, Houston, Texas
PIET DE SOMER Department of Microbiology, Division of Microbiology,
Rega Institute, University of Leuven, Leuven, Belgium
R. GORDON DOUGLAS, JR. Infectious Disease Unit, Department of Medi-
cine, University of Rochester School of Medicine, Rochester, New York
STEPHEN GREENBERG Departments of Medicine, and Microbiology
and Immunology, Baylor College of Medicine, Houston,
MAURICE W. HARMON Department of Microbiology and Immunology,
Baylor College of Medicine, Houston,
IVER HERON Institute of Medical Microbiology, Department of Immunology,
University of Aarhus, Aarhus, Denmark
*Present Department of Microbiology, Graduate School of Public
Health, University of Pittsburgh, Pittsburgh, Pennsylvania.

)NTltll 11 J '1'( )11,:-;
IVIICIIIO L)epal'(,IIlUII(, 01' Vil'al ItoHwull I'al'k IVIUlllOl'ial
LIlHLiLuLu, New Yot'k

IIOWAltU JOIlNSON Dcpartment of Microbiolo!!,"y, UllivcrHiLy
Mcdical 13ranch, Galvcston, Tcxas
ItUSSELL F. Research Center, Merrell-Natiollal
Division of Richardson-Merrell Inc., inc innati ,
IlILTON LEVY Section of Molecular Virology, National Instii-utc of
Allergy and Infectious Diseases, National lnstitutes of of
Bethesda, Maryland
GERALD D. MAYER Research Center, Merrell-National Laboea-
tories, Division of Richardson-Merrell Inc., Cincinnati,
PAGE S. Department of Microbiology, Medical College of
Virginia, Virginia Commonwealth University, Richmond, Virginia
KURT OSTHER Biological Department, Alfred Benzon Ltd., Hvidovre,
Denmark
DALE Cancer and Virus Research, Experimental
Biology Research, Upjohn Company, Michigan
INTERFERON AND
INTERFERONINDUCERS
1
INTERI"ERONS: AN OVERVIEW
I)alu Stringfellow
Upjohn
!(alamazoo, Michigan
1. INTRODUCTION
DISCOVERY AND PROPERTIES
INDUCTION PROCESS
IV. CONTROL OF INTERFERON PRODUCTION
V. MECHANISM OF ANTIVIRAL
VI. NONANTIVIRAL PROPERTIES OF INTERFERONS
VII. ROLE OF INTERFERON IN HOST RESISTANCE
VIII. CONCLUSIONS
REFERENCES
1. INTRODUCTION
2
"
7

11
12
15
15
Since its discovery in 1957 [1], interest in interferon as mediator of
antiviral resistance has steadily increased. During these years of explora-
tion, the role of interferon as natural regulator of various cellular func-
tions has expanded beyond the initial area of mediation of antiviral resistancc
to include modulation of immune responsiveness and cellular growth and
regulation. Thus its pharmaceutical potential now includes areas of cancer
and viral chemotherapy and immune modulation. Yet the answers to ques-
tions as basic as what is the structure of this intriguing group of molecules
and what is their natural physiological role not clear. This intro-
ductory chapter is background for the chapters that follow and therefore
will deal with interferon' s "past" and how this fits into its future. Particu-
lar attention devoted to the antiviral of interferon, its role
in control of viral disease processes, the mechanism of antiviral action,
1
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DISCOVERY AND PROPERT1ES
Host resistance to and from virus infection is complex phenome-
nOI1 wide ral1ge of known and currently Ul1knOWn factors. One
such determinal1t is the nonspecific resistance initiated and mediated
interferon. 111terferon, or perhaps more accurately, interferons, are
group of substances of cellular origin which render the cells of many verte-
brates il1capable of supporting virus replication. The term interferon was
first applied to soluble factor produced the cells of chicken chorio-
allantoic membranes after exposure to il1activated influenza virus [1].
Exposure of the membral1es to inactivated virus reduced their capacity to
support replicatiotl of il1fluel1Za virus al1d stimulated secretiol1
of the interferol1.
Short1y after its discovery, Lil1denmal1l1 et al. [2] determil1ed that
il1terferol1 was proteil1, or at least was susceptible to the actiol1 of pro-
teolytic el1zymes, and was 11011dialyzable yet could not sedimel1ted
cel1trifugatiol1 at 100,000 g for 4 hr. Antiviral activity was retained when
solutiol1S of il1terferol1 were subjected to extremes of 2 to 10, whel1 heated
at 600 for 1 hr, al1d whel1 il1cubated with antiserum prepared against the
virus that had used to stimulate its productiol1. 1nhibition of both RNA
al1d DNA viruses was also observed [3-5]. The fact that il1cubatiol1 of
virus with il1terferol1 had 110 effect 011 viral il1fectivity led Lindel1malll1 et al.
[2] to assert that il1terferol1 exerted its antiviral effects rendering cells
il1capable of supporting viral replicatiol1 rather than interacting direct1y
with the intact virus particle. Shortly thereafter, Tyrrel1 [6] demol1strated
that il1terferol1 prepared in chickel1s had detectable al1tiviral activity 011
calf cells, thereby il1troducil1g the cOl1cept of species specificity.
the discovery of the origil1al il1terferon, l1umerous reports have
described the il1duction of other viral-inhibitory substances which have
essel1tially the same characteristics. 1n culture, cells from humal1S, pigs,
chickens, cats, dogs, cows, rabbits, mice, rats, guinea pigs, sheep, ham-
"ters, mOl1keys, tortoises, snakes, fi::;h, al1d other al1imal species have
yielded interferon whel1 exposed to an appropriate agent. 111ter-
feron appears il1 the blood, lungs, brail1s, uril1e, spil1al fluid, al1d other
tissues of al1imals following administratiol1 of various il1terferol1 il1ducers.
Although viruses or viral compol1ents were initially used to induce the
peoductiol1 of interferol1, certain nOl1viral Substal1ces also stimulate its
production in vivo and il1 vitro. Amol1g these nOl1viral il1terferol1 il1ducers
are el1dotoxil1s [7,8], polysaccharides [9], phytohemagglutil1il1 [10],
bacteria [11], rickettsiae [12], protozoa [13], sYl1thetic low-molecular-
weight compoul1ds such as bis (diethylamil1oethyl) fluorol1one [14], sYl1thetic
1, i\N (IVI':II,VII':V\-
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sub:otal1cct: terms biolo!!,'ical physico-
clJCmical propcI'tics :OUCll and heat stability, weight, and
specics specificity, it has increasingly apparent that considerable
vaI'iation exists between interferon induced in species or specific
inducer and another induced under different conditions. interferons
have varied in the degree to which they resist heat, inhibit virus production
in ceHs heterologous species, and res ist degradation under either
acid or alkaline conditions [19]. Therefore, interferons are different
from the original interferon isolated from the chorioaHantoic membranes
of embryonated chick eggs. Molecular weight determinations confirm these
differences. Partial purification of interferons has revealed remarkable
heterogeneity in molecular weights and electric charges of different prepara-
tions [20]. IHterferOHS with different molecular sizes have been observed
in the serum of animals or from ceHs in vitro when inoculated
with either virus or nonviral inducers [21,22]. These reports indicated
that there were usually two or more molecular species, one with molecular
weight greater than 100,000 and another smaHer molecule ranging between
20,000 and 40,000. and [23] have reported that the molecular weights
of interferon found in the serum of endotoxin inoculated rabbits were con-
s iderably greater than the 35,000 molecular weight molecule execreted in
the urine. Carter [24] suggested that some interferons multimeric
aggregates of smaHer weight molecule, since shifting the salt
concentration dissociated the high-molecular-weight species of mouse or
human interferon into smaHer common molecular weight components which
were biologically active [25]. It is notable that et al. [26] recently
reported that low-molecular-weight "subunits" of interferons also
display antiviral activity. Furthermore, although it was previously suggested
that viral inducers stimulated the production of relatively low-molecular-
weight interferon (20,000-30,000 daltons) while nonviral inducers, particu-
larly such agents as endotoxins, stimulate the release of larger molecular
species (90,000-120,000 daltons), it is possible that this function
of the conditions under which the interferon was induced. For example, in
an animal given single injection of an inducer, the molecular species of
interferon in the serum differed markedly from those found in different
organs [27], yet the different molecular species were apparently anti-
genicaHy similar as indicated neutralization of biological activity with
anti- interferon antibody.
More recent studies the antigenicity of various interferon prepara-
tions convincingly demonstrate that there are several types of interferon
produced particular animal species. For example, there at least
three antigenically distinct types of human interferon: fibroblast, lympho-
cyte, and immune or type II interferons [28-30; Chapter 2]. It is not clear,
1 :-)'I'ltl Nl: 1,'1': 1,1 ,()W
CaCl1 Liic;lil1cllypc plaYH iJl vivu.
[:31] haH HuggeHlcLi thal fibrublaHtH catl pruLiuce butlJ fibro-
blast lymphocyte illte rfer 011 , cOllversely lymphocytes make
both types as well as type II (immune-induced) interferon. The function
each interferon is not clear. Poss ibly particular (fibroblast) interferon
is preferentially involved in cellular regulation while leukocyte or type II
interferons are involved in immune regulation, although definition each
interferon species has antiviral activity.
Although interferons differ, most meet of the physicochemical
and biological properties which historically have been established to charac-
terize interferons. These include species specificity, stability at
of 2. and nondializability yet when centrifuged at
100,000 g for 4 hr. There are also several properties which substance
must possess it considered to an interferon, Three the
most basic these are (1) inactivation of antiviral activity treatment
with proteolytic enzymes but not nucleases; (2) antiviral activity call1lot
mediated through direct interaction with the illtact virus particle but
must rather function rendering otherwise susceptible cells incapable
supporting virus replication through process requiring cellular protein
and RNA synthesis; (3) the interferon-like substance must active
against wide of viruses . Some of the discrepancies that have
appeared in the literature regarding the properties and characteristics
interferons attributed, at least in part, to the crudeness the inter-
feron preparations used in most studies. variety techniques have more
recently been applied to the concentration and purification interferons.
Characterization of the interferon in these preparations will hopefully re-
solve some conflicting reports, further clarify molecular charac-
teristics, and possibly lead to determination of the amino acid sequence of
various interferons (see Chapter 2).
Lockart [32] [33] have postulated that interferons
family proteins cellular origin which initiating non-
specific intracellular inhibition virus replication. Interferons
differentiated according to their source, their spectrum activity, and
their physicochemical properties. They similar in respects
to enzymes and antibodies which are classified the basis of their biological
activities but include proteins which vary considerably with respect to
their molecular size, physicochemical properties, and biological properties.
INDUCTION PROCESS
Since its discovery, practically every major group viruses has been
found inducing interferon production in suitable in vivo or in vitro
systems. Thus the production interferon is general response of cells
toward viral infections. the other hand, in 1964 [7] interferon was
1. ;.
ill HlJt'(1111 01,' LktL illjlH,Loll illll'avOllotlHly WitJI
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lioll which will expanded to other type8 01 illduccrs.
Ilcllcl" [34] and Wagner [35] furnished the first
mechanisms the of sYl1thesi8 demoll-
that the cellular respol1se to virus 1)0
illllibited pretreating cells with D. The activity
01 this is the of guanine residues of DNA,
thereby the of messenger RNA (mRNA) and sub-
sequel1tly proteil1 This suggested that il1ter-
was UIlder host in response to viral
stimulus. Furthermore, if actinomycin D was introduced after mRNA was
formed, it had effect interferon synthesis [36]. It was therefore
assumed that interferon is produced cells in response to virus infection
mechanism involving derepression of host cistron(s) to form an
mRNA that codes for the production of interferon [37].
precise event or stimulus which initiates cellular mRNA, and
consequently interferon production, is presently unclear and the subject
of considerable controversy. With RNA viruses it appears that the double-
stranded replicative intermediate formed during the replication of single-
stranded RNA virus and the natural double-stranded RNA of other viruses
are potent interferon inducers [38,39]. The induction of interferon
natural and synthetic polynucleotides has also been observed in variety
of mammalian cel1s in vitro or in whole animals [40]. Furthermore, Field
et al. [15] reported that single-stranded polynucleotides were inactive at
concentration 10,000 times greater than that which induced viral inter-
ference with the helical double-stranded form. Likewise double-stranded
DNA or DNA-RNA hybrids also have been reported to
interferon inducers [40].
The importance of the double-stranded form has been challenged, how-
ever, experiments which employed inactivated viruses as the
Dianzani et al. [41] attempted to the replication of Newcastle disease
virus (NDV) RNA with actinomycin D and therefore eliminate the double-
stranded intermediate form. They concluded that, since infectious virus
was produced UIlder the conditions employed, the viral RNA did not replicate
and the induced was due to single-stranded RNA. NDV was later
found to carry its own transcriptase into the with its nucleic
which have led to the formation of double-stranded intermediates even
though infectious virus was formed. and therefore the interferon induced
have been due to small amount of double-stranded RNA. This
also could how ultraviolet-irradiated NDV induce
since the replicase would not affected UV irradiation. More recently
cL :11. t'cpcaLcll LlIUil.' c;tt:ly llhilll-(
:11:: thc il1duccr. L{NA viruH d()c::; c:1rry ()wn
replicase with it, therefore tlle respo11se induced u::;it1g tllis
virus was presumably induced HNA. Although the
troversy has 110t fully settled, HNAs are effective
interferon i11ducers to major degree are probably respo11sible for
most HNA virus-il1duced i11terferot1, although additional processes
required [43,44]
How does our u11dersta11ding of the ability of viral RNA
to induce i11terfero11 fit i11to the mechanism of inductio11 other substances?
The specific answer to this question is not available. It is 110t clear whether
it1ducers 11eed to penetrate the or whether they are active at exter11al
membrane site. Most evide11ce suggests that it1ducers such as polyt1ucleo-
tides need i11teract with the surface to induce it1terfero11 productio11
[45-48]. However, it1 these studies, polynucleotides were attached to
i11s01uble matrices that could not et1ter the but Ui1fortunately
nucleotide was slowly released [46,47]. Therefore, evet1 though the matrix-
attached inducer could 110t e11ter the the slow release of sma11 amounts
of inducer might responsible for the interfero11 respo11se. Recently,
Borde11 and [49,50] have demonstrated that amphoterici11 caused
cellular productio11 of interferon it1 respo11se to polY11ucleotide
inducer. They postulated that amphotericin caused the cellular
to "leaky" thereby permitting greater intracellular accumulation
of inducer. These data, alo11g with the ability of viral RNA to i11duce inter-
fero11, suggest that intracellular 11ucleotides i11duce interferot1.
How double-stranded RNAs, or for that matter other i11ducit1g agents
(Chapter 6), stimulate cells to begin producil1g the mRNA(s) that code for
the interferon protei11 is 110t Ui1derstood. It appears that some inducers do
combine with specific or cytoplasmic receptor sites and that
this attachme11t is agents such as hormones [51]. 1t1terpreta-
ti011 of these studies is complicated the fact that most of the agents used
stimulate membra11e-associated adenylate cyclase an intracellular
i11crease i11 Therefore the decreased responsive11ess of such
cells to il1terferon it1ductio11 might simply due to modulatiot1 of interferon
productiot1 [52-55]. 111 additiot1, treatme11t of cells with
i11terfero11 or interferon inducers stimulates production or cellular release
of molecules [56]. Certain prostaglat1di11s (i. series
stimulate levels in various types of cells,
and the interrelatiot1ship betwee11 nucleotides, and
membra11e receptor sites is still U11clear. Also, do appear
to involved, il1depende11t of 11ucleotides, in modulation of interferon
productio11 at least murit1e cells [57].
The mechanism which various low-molecular-weight compounds,
such as tiloro11e hydrochloride or various pyrimidines (see Chapter 6),
stimulate cellular interfero11 production is even less well defined.
1.
'1
IIHIIICUI'S al'o acLlvu ollly 111 vivo 01' 111 cOlllplex 111l1lLlcelllllae 111 VIL!'o coll
SYSLOII1S I :iH], sovoL'al col,l ]JopulaLlolIs
1.0 IIIILlaLo 1"01." uxamplo, illllllCOL'
stimulatou 11i/.':11 lovols
01' [l1 vivo thu splcuns and thymuscs 01' audc(l to
111l1l'il1o splool1 al1d organ culture systems [11 vitro [58].
11' (glass-adherent were separated from lymphocytcs
the ability of both populatiol1s to produce interfcrol1
111 to vitro additiol1 of was lost. These data suggest that
cull-to-cell interaction or amplification l1ecessary to achieve il1ter-
il1duction with inducil1g agents.
IV. CONTROL OF INTERFERON
PRODUCTlON
As with most biologica11y active molecules, l1atural cOl1trol or regulatory
limit the amoUl1t of interferon 110rma11y make. High
interferon levels appear to trigger the productiol1 of cellular proteil1
that il1hibits further il1terferol1 SYl1thesis, probably through suppressiol1 of
further il1terferol1 mRNA productiol1. The precise l1ature of the cOl1trol
proteil1 is ul1kl1own. Speculation has il1cluded the proteil1(s) that
il1terferol1 il1duces, il1terferol1 itself, or al10ther as yet Ul1idel1tified cOl1trol
proteil1 [59-61]. Evidel1ce for the existel1ce of such cOl1trol system is
based il1 large part 011 il1direct evidel1ce gel1erated through the selective
use of metabolic il1hibitors. Ce11s to produce abl1orma11y
high levels of il1terferol1 (superil1ductiol1) the additiol1 actil1omycil1 D
al1d cycloheximide shortly after (2-3 hr) stimulatiol1 il1ducer [62-66].
the 2- to 3-hr period il1terferol1 mRNA is and
translation is adding actinomycil1 D at that production
the mRNA that codes for the control protein is il1hibited, but
proceed with interferol1 mRNA translation, consequel1tly producil1g much
higher levels of interferon to 10 times more).
The control protein has not been satisfactorily isolated or idel1tified,
although Bordel1 et [67,68] have reported that such protein could
detected [11 vitro il1 murine L929 cultures il1duced to make il1terferol1
with NDV. Duril1g the early stages il1terferon il1ductiOI1 very little cOl1trol
protein was detected, while later 011 whel1 ce11s were less il1terferon,
high levels of the control were detected. 111terferon al1d the
hyporeactivity factor were never completely separated phySicochemica11y,
but biologica11y distinct separation was observed.
upon these observations, Glasgow
in vivo system to determine if such cOl1trol protein could
detected in the serum of mice infected with encephalomyocarditis
virus. They had Her observed that infected mice developed
,.,
S'I'II.IN(:I,'I';I,I,()W
SCVCL'C slalc of lo l' L'OII (aetor ill
SCl'um was idcntiJicd al1d cllaL'actcl'izcd l 70]. It had of the proper-
ties of il1terferol1 (species specificity, stability, molecular weight of
10,000-20,000, etc.) but had 110 demonstrable antiviral activity nor was
its ability to mediate hyporeactivity lost incubation with anti-interferon
antibody. Since that time similar factor has been in the sera
of mice infected with of several viruses or carrying two types of lym-
phomas [71,72]. Tarr et al. [73] have identified similar substance in
the serum of mice infected with cytomegalovirus, although in their studies
the hyporeactive factor was inactivated incubation with anti-interferon
antibody. Whether the substances detected in vivo are the same as the con-
trol protein identified Borden et al., and if these substances are proteins
that naturally control interferon production, is not yet known.
Recently, StringfeHow [57] reported that mice infected with virus
developed hyporeactive interferon response and that the response of these
mice restored to normal if inducers were administered with
prostaglandins. data suggest that the hyporeactive state (at least
under these was reversible and that prostaglandins
involved in regulation of interferon responsiveness (cf. Chapter 6). The
administration of prostaglandins did not appear to influence the levels of
hyporeactive factors in the serum of infected mice but rather appeared to
reverse the effect of the substance. For example, mouse fibroblast
ceHs incubated with hyporeactive factor from EMC-infected mice [57]
develop 90% suppressed response; yet, when PGE2 (2 was added
to such ceHs, only 10-20% suppression was observed. The interrelation-
sMp between prostaglandins, the control protein, and modulation of inter-
feron production is currently under investigation.
V. MECHANISM OF
ACTIVITY
The antiviral activity of interferon is indirect since it prevented
pretreating ceHs with actinomycin D other inhibitors of protein synthesis
[74-76]. Interferon itself causes the host to produce mRNA for yet another
antiviral protein polypeptide which functions as the actual antiviral agent.
Such protein, however, has yet to isolated in ceHs treated with inter-
feron preparations [74-75], although Samuel and Joklik [76] found
ribosomal-associated protein (molecular weight of 48, 000) in interferon-
induced cells. While direct proof for the interferon-induced antiviral
protein is lacking, circumstantial evidence supports this interpreta-
tion [77-80].
Soon after the discovery of interferon, it apparent that it did
not interact with viruses directly nor did it prevent the absorption of viruses
to ceHs, viral uncoating, viral assembly, release of virus particles
[81-84]. Instead, interferon appeared to act preventing the synthesis
\. (IVI-:\tV\I-:W )
()\' vil'\I::J 1:-;:,.1. pt'cci::Jc which the
pI,'oLoil1 il1tluco(1 il1Lorl'cl'Ol1 prcvents synthesis of new
Vil,'LI::J pal'Liclo::J 0[' is !lOt k!lOWn. Marcus and Salb [86-88]
l'cp0l'Lot! derived from interferon-treated were
witll either viral or host mRNA. They observed, however,
tIH.tL tllere w:ts suppression of the translation of viral but not host mRNA.
led them to postulate that interferon-treated produced mRNA
WI1iCll then coded for new protein which they called translation inhibitory
(TIP). TIP supposedly was bound to cellular ribosomes; polysomes
composed of host mRNA and TIP-attached ribosomes translated
while those composed of viral mRNA and TIP-attached ribosomes did not
translate efficiently, resulting in suppression of viral protein synthesis.
Carter and Levy [89], working with different virus system, found
that ribosomes from interferon-treated failed to viral RNA at
but normal ribosomes bound and translated viral RNA to form poly-
Thus, although somewhat in contrast with Marcus and Salb, Carter
and Levy argued that the primary site of interferon action was the
efficiency of viral RNA to ribosomes, not final viral RNA trans-
lation. Both groups did agree, however, that the action of interferon was
directly fue ribosomes. Samuel and Joklik have reported the presence
of ribosomal-associated (48,000 mol. wt.) that was not present
in ribosomal washes from untreated [76] . Whether tl1is is the
TIP described Marcus and Salb is not at present. Additional work
has confirmed and extended these observations [90-92], alfuough infor-
mation is currently fue mechanism wl1ich ribosomes from
interferon-treated selectively distinguish between viral and
mRNAs. In addition, recent studies suggest that interferon' s action
involve modification alteration of tRNA or mRNA
ribosomes, making the mechanism of action more complicated [93-95].
anofuer approach, Marcus et al. [96] have postulated that the
antiviral of interferoll due to an inhibition of transcription
rather than or in addition to translation. Using chick fibroblast
cultures which were infected wifu strain of vesicular stomatitis virus
(VSV), they, did Manders et [97], demonstrated decrease in VSV
polymerase activity in interferon-treated and postulated fuat inter-
feron might interfere with viral transcription rather than inhibition of
translation of viral mRNA previously speculated. Jungwirfu et al. [98]
and et al. [99], while investigating the anti-poxvirus state created
interferon, did not, however, reduction in the amount of early
viral RNA. Therefore, an explanation for fue drastic inhibition of viral
protein syntheais in interferon-treated could not explained in their
systems the basis of reduction transcription of early RNA. Recent
studies [100-106], however, confirmed the original observation of
et al. [96] fuat interferon alter tranacription of viral RNA and that the
reduction was not associated with more rapid degradation of viral mRNA.
Increased degradation of viral mRNA however, in explain
10 S'I'1t1 N(; 1,'1'; I.I.()W
tl1C il11liIJiLiol1 0[' vit'al J!eoLcil1 il1
cntly, nuclcasc i" acLivatcd to tl1C prc"cllcc LtNA
in interferon-trcatcd Hecently, Kerr et al. [111-112]
identified trinucleotide ill interferon-treated cells exposed to double-
stranded HNA tlJat may responsible for activation of nuclease [113]
which degrades mllNA and inhibits protein syntlJesis. AltlJough the mecha-
nism is not understood, evidence from number of virus-cell systems
indicates tlJat tlJe interferon-induced antiviral state is characterized
inhibition of both primary transcription and translation [106].
Hecent studies also suggest that interferon may mediate, in specific
instances, some of its antiviral activity tlJrough modification of membrane
function. et al. [114] and Chang et al. [115] reported tlJat in
mouse cells the release of murine leukemia virus (MLV)
was inhibited and tlJat infectious virus particles accumulated at tlJe
surface. In tlJese studies the ability of ce11s to release virus particles was
inhibited, but viral protein syntlJesis was not affected. This suggests
third mechanism of interferon-mediated antiviral activity, namely alteration
of cellular membrane function.
Interferon produced animal cells apparently does not need to pene-
trate tlJe to initiate establishment of antiviral resistance. HatlJer,
interferon needs to released or excreted the and specifica11y binds
to tlJe membrane before antiviral resistance develops [116-118]. Evi-
dence for this includes blocking tlJe establishment of tlJe antiviral state
cytochalosin [119], cholera toxin, chorionic gonadotropin, and tlJyroid-
stimulating hormone (TSR) [120,121]. Each of tlJese substances was bound
to or altered tlJe membrane in such fashion tlJat attachment of inter-
feron was inhibited. Also, incorporation of anti-interferon antibody into
the culture media in which tlJe cells were induced blocked establishment of
the antiviral state, suggesting tlJat interferon had to externalized before
the antiviral state could initiated [122]. The existence and specificity
of membrane receptor sites may one explanation for the species specifi-
city of interferon.
working hypotlJesis concerning the induction and mechanism of inter-
feron action postulated from tlJe literature reviewed. This hypotlJesis
does not attempt to explain tlJe mode of action of types of interferon
inducers but uses an RNA virus as convenient example. In tlJe simplest
concept single-stranded RNA virus penetrates the is uncoated,
releases its RNA, and, using the original strand of HNA as template,
begins reproducing complementary strands of RNA that are essential for
replication. Double-stranded RNA, formed in tlJe process, is not normal
to tlJe and tlJis is presumed to provide tlJe stimulus for ce11ular forma-
tion of interferon mRNA and interferon production. Newly syntlJesized
interferon is released and either diffuses or is distributed to cells locally
or at diverse sites way of tlJe blood stream, body fluids, or direct
contact. Apparently, interferon then attaches at specific membrane
1, i\N (IVI';HVII';V,'
11
/'puupto/' siws atltl il1itiates (llH'ep/'oHs iOll 01" <.:iStl:OIl tllat <.:o(lu::;
1111tNA, 'l'lli::; 1111tNA ill (lieu<.:tH U1C :tlltiviea\
]!eowill(s) tllaL intcr[urc:; wiL!l productioll or releasc progcllY viru:;.
il1turfcrcnce either at thc level of trallscription,
viral particlc release theoretically does Ilot affect the normal meta-
process of the although some diverse effects ce11ular metabo-
lism and division due to interferon have been reported. These are possibly
to the effect of interferon ce11ular membrane induction of endo-
nucleases.
VI. NONANTIVIRAL PROPERTIES
INTERFERONS
their antiviral activities, interferons appear to have other effects
cells. These regulation of ce11ular functions such as phagocytosis,
antitumor activity of macrophages, production of lymphokines
lymphocytes, expression of ce11ular antigens, cytotoxicity of ce11ular
immunity, in addition to effects cellular macromolecular synthesis and
growth [123-130]. Conflicting reports have described both
ment suppression of cellular functions, probably the impurity
of the preparations employed, different sources of interferon,
and variations in experimental methods and procedures. Nevertheless,
sufficient evidence now supports the notion that interferon plays broader
role in ce11ular regulation simply as modulator of antiviral resistance,
and this has stirred interest the natural physiological role of interferon.
Is its primary function immune regulatory, or critical part of
ce11ular homeostasis? Al1swers to these questions are elusive, but the
insight gained the of interferon shed light
some of its other ce11ular affects. observation that interferons have
multiple effects cells is not too surprising since the action
encompasses at least three of
of mRNA and inhibition of viral particle release from ce11ular
membranes. With such diverse effects interferon hardly considered
to specific in its actions. For example, many of the proper-
ties of interferon associated with effects membranes. Many
of the alterations listed are associated with membrane function;
thus, agent like interferon, which apparently does attach to affect
the ce11ular membrane, could dramatica11y alter functions such as phago-
cytosis activity of macrophages and lymphocytes. ln fact,
it was recent1y reported that activates the tumoricidal activity
of macrophages [131] and that inhibited the activation
process [132]. These results again suggest that acts the
ce11ular that prostaglandins and/or nucleotides
in the activation process. Treatment of ce11s with interferon

iIIU.)l" [u ("011 Lu illllueu J>t"US lJL"OuueLioll
uulls, LillR SLlel1 ttlolueulcs illvol vcu in t'uv;ulatioll
tlle syslcm. Mal1Y prostaglal1dil1s, particularly those thc
series, stimulatc membral1e-associated adel1ylate cyclase activity resu1til1g
il1 il1creased il1tracel1ular levels [133-135]. data suggest
that of the l1ol1antiviral and, for that matter, effects of inter-
ferol1 may modulated cyclic l1ucleotides al1d prostaglandins, al1d future
studies the effects of should cOl1sider this possi-
bility.
VII. ROLE OF INTERFERON
IN HOST RESISTANCE
its very l1ature interferon appears involved in host defense
is present1y known about the role of interferon in the
of immune responsiveness Chapter 11) and the control of
processes, but the role of as barrier to viral
infection is more clear.
For virllS to establish infection successfu11y, it replicate in
the initially infected the resu1ting progeny virus spread to
infect other cells within the same tissue area, subsequently infecting tissues
at diverse sites or target of the first host responses to appear
[n the initia11y infected tissues is interferon [136,137]. Interferon
detected infected cells at about the time that infectious, progeny
viruses are synthesized. Presumably, however, the interferon-sensitive
stage of virus replication already passed the time interferon
is synthesized the and viral resistance mediated interferon is
not established in the initia11y infected As resu1t virus infection is
probably not affected in that since inhibition of interferon production
metabolic inhibitors does not increase the virus yield from
ce11s during single-step gro,vth curves [138].
Theoretically, fo11owing release of interferon from infected cel1s, it
diffuses to surrounding cel1s where it initiates activation of the
state. In this way, reaching as yet uninfected cells, it is thought to
establish barrier to further viral replication that limits viral dissemina-
tion. degree of antiviral activity is probably dependent the extra-
cellular concentration of interferon as we11 as the sensitivity of the virus
to the action of interferon. Therefore, the cells in closest contact with the
infected interferon-prodllcing ce11s are exposed to the highest concentration
of interferon and become more resistant to virus replication [139,140] .
Protection of more cells tissues does occur but apparently to
lesser degree depending the virus or strain of virus being used.
interferon defense mechanism might thought of as in
of two ways. First, interferon could act limiting virus replication
1.
aL HiLu 01' illiLiu.luIlLIY IJI('I'l'I)y 1(,(JlIUillj.\" LIIU 01' Vit"lIH
locally 1l'(llIUillj.\" sysLumiu suuolJd of
al1tiviral aution could to diffuse transported to
Largct organs establish an state these yet
lIllinfcutud thereby localizillg tissue involvement. Obviously the defense
barriers established are in delicate with other host
mechanisms such as the immune response (both ceHular humoral)
as weH as other more nonspecific factors. Furthermore, the degree of
protection is dependent of interferon induced or
present as well as relative susceptibllity of infecting virus to
action of interferon.
characteristic of tissues recovering from virus
is nonspecific resistance to viral superinfection, suggesting that
such as correlated temporari1y with
recovery f:com established infection [141,142].
that interferon is important part of host to virus
is from source s. adequate s
inhiblt of viruses variety of animal tissues
vivo or vitro is tissues of prior
to and during onset recovery from virus infections [143]. These
studies have that interferon produced as early as 1 hr
after virus infection is demonstrable at high levels within
24-28 hr.
comparison, recovery, as measured decreasing virus levels in
infected tissues, usually or more days after is first
detected [144,145]. presence of interferon in target organs
recovery has also observed [146] .
Further evidence relating interferon to recovery is furnished experi-
ments that attempted to decrease the ability of to produce
thereby that corresponding increase in the severity of virus
infections would resu1t. Baron and Isaacs [147] such an experi-
mental approach in attempt to evaluate what ro1e interferon plays in host
resistance to virus infection. demonstrated that chick embryos younger
8 days had "immature" interferon system and were very susceptible
to the lethal effects of influenza virus infection. As the age of embryos
there was corresponding increase in both their ability to pro-
duce interferon and their resistance to lethality of infection. decreased
capacity to produce interferon, caused altered temperature, reduced
tension, psychological stress, chemical inhibitors, different virus
strains, age, and treatment with steroid vasoactive amines,
has also, some cases, led to impaired recovery of animals from virus
infections [148-150]. studies carried out in vitro have
indicated that impairment of ability of ceHs to produce interferon
the virulence of variety of viruses. Two interesting studies were
reported Fauconnier [151,152], who rabbit
1-1
lo l'UULlCU !Jl'olucLivu il1lerfurol1 ilJ in vivo anu
ill vitro Under in vitro eonditions }'auconnie1' was to 1'educe
the of inte1'fe1'ence observed between seve1'al types of vi1'uses (p1'e-
sumably mediated inte1'fe1'on) adding anti-inte1'fe1'on antibody to the
system, thereby enla1'ging the plaque size of the infecting vi1'uses. Simi-
larly, in vivo an augmentation of viral virulence was observed when mice
were treated with 1'abbit anti-mouse interferon antibody and were then
infected with Semliki Forest virus. An increase in both cumulative mortal-
ityas well as virus levels in brain tissues was reported. note of caution
should obse1'ved in the inte1'p1'etation of of these studies, however,
since methods used to inhibit the interferon 1'esponse we1'e not entirely
selective and also have suppressed other host defenses such as the
cellular immune response, which at least in part, have respon-
sible fo1' the enhanced seve1'ity of infection.
Although most attention has directed toward demonstrating
correlation between the amount of interferon produced, the sensitivity of
the virus to interfe1'on, and susceptibility to infection, another aspect of
this relationship might well the susceptibility of the itself to the
action of interfe1'on. For the effect of specific dec1'ease in the
ability of mice to respond to the action of inte1'fe1'on was demonstrated in
sel'ies of studies involving Group arbovirus infection of two strains
of mice. Both strains were of p1'oducing equivalent amounts of
interferon, but was more resistant to the establishment of the antiviral
state interferon and was therefore much more susceptible to the lethal
outcome of infection with the arbovirus. This difference was not
served in vivo, but cells obtained from each mouse strain demonstrated
the susceptibility and lack of interferon protection that had been seen
in the intact animal [153]. Olivie and Boiron [154] have also demonstrated
similar pattern of alte1'ed sensitivity of mouse embryo cells infected in
vitro with murine sarcoma virus. Although both uninfected and mu1'ine
sarcoma virus-infected cells produced equivalent levels of inte1'feron in
response to Newcastle disease virus (NDV), the infected cells were severely
refractile to the antiviral activity of passively transferred interferon.
When both uninfected and murine sarcoma virus-infected cells were treated
with NDV-induced interferon and then challenged with vesicular stomatitis
virus, protection was observed in the mu1'ine sarcoma vi1'us-infected
cells while the uninfected cells were completely protected.
Added c1'edence is given to the 1'elationship of interferon to viral 1'esist-
when studies are considered which utilized passive transfer of exogen-
ous inte1'feron to protect or treat potentially susceptible animals 01' cells.
1n va1'iety of expe1'imental settings exogenous inte1'fe1'on and inte1'fe1'on
induce1's, when administered eithe1' p1'ophylactically or afte1' establishment
of infection with va1'iety of vi1'uses (Chapters 4 and 5), have effective
in vivo and in vitro in both preventing and limiting vi1'us infections (Chapters
6 through 10).
1. i\N ()VI-:ItVII-:\V
VIII. CONCI,USlONS
1\1 bcclI lcarncd in thG last qucsliOlIS
il1lcr[crons are still far from being answcrGd.
tl1e molecular nature or mechanism of antiviral action is rcmaill
Interferon and its accompanying cellular antiviral resistal1co
play significant role in host defense against virus infection, play
1."010 i!l control of metastasis or malignancy, and modulate immune
rcsponsiveness. However, rather than its being considered as the sole
factor involved in host resistance, it is more realistic to visualize inter-
Ieron as member of complex system of cellular and humoral barriers.
The importance of i!lterferon or any other member of the host' s defense
system in any particular disease probably depends upon the !lature of the
invasive organism as well as the conditio!l of the host. depression of
any aspect of the cellular or humoral defense mecha!lisms, including inter-
feron, could (again depending upon the nature of the causative agent) lead
to compromised state of resistance and cO!lcomitant increase in suscepti-
bility to infection. The administratio!l of exogenous interferon or potent
!lontoxic interfero!l i!lducers an ideal chemotherapeutic approach
to the treatment of viral and possible neoplastic diseases. However, as
discussed in the remaining chapters, the practical utilizatio!l of the i!lter-
feron system faces many obstacles, although recent successes indicate
that such an approach holds promise.
REFERENCES
1. Isaacs a!ld J. Li!lde!lma!l!l, Proc. Roy. Soc., Ser. 147: 258-267
(1957).
2. J. Li!lde!lma!l!l, D. Burke, a!ld Isaacs, Brit. J. Pathol.
38: 551-562 (1957).
3. Isaacs, D. Burke, a!ld L. Fadeeva, Brit. J. Pathol. 39:
447-451 (1958).
4. Isaacs, Virology 10: 144-146 (1960).
5. Isaacs and G. itchcock , La!lcet 69-71 (1960).
6. D. J. Tyrrell, Nature 184: 452-453 (1959).
7. J. S. You!lg!ler a!ld W. R. Sti!lebri!lg, Scie!lce 144:1022-1023 (1964).
8. Scie!lce 146: 1472-1474 (1964).
9. W. J. J. a!ld Murphy, Federation
Proc. 23: 507 (1964).
10. F. Wheelock, Scie!lce 149:310-311 (1965).
11. W. R. a!ld J. S. You!lg!ler, Nature 204: 712 (1964).
12. Hopps, S. Koh!lo, Koh!lo, a!ld J. Smadel, Bacteriol.
Proc. 115-116 (1964).
13. Huang, W. W. Schu1tz, a!ld F. Gordon, Scie!lce 162: 123-124
(1968) .
11. 1). May()I.' '{. 1". Scicl1cc (1970).
1[j. 1<'icld, Lampson, and IC Ililleman,
Proc. Nat. Acad. Sci. U.S. 5t;: 1004-1010 (1967).
16. DeClercq and Merigan, Ann. Rev. Med. 21:17-47 (1970).
17. Merigan and J. Finkelstein, Virology 35:363-369 (1968).
18. J. Diederich, Lademaun, and Wacker, Virusforschung 40:82
(1973) .
19. R. Lockart, Jr., in Interferons (N. Finter, ed.), North Holland
Amsterdam, 1966, 1-20.
20. Fantes, in Interferons (N. Finter, ed.), North Holland
Amsterdam, 1966, 119-179.
21. J. Smith and R. R. Wagner, J. Med. 125:559-564 (1967).
22. Cesario, J. Schryer, and J. G. Tilles, Antimicrob. Agents
Chemother. 11: 291-298 (1977).
23. andM. J. Virol.1:883-890 (1967).
24. W. Carter, Proc. Nat. Acad. Sci. U.S. 67:620-626 (1970).
25. W. Carter, Prep. 1:55-61 (1971).
26. W. Stewart, Chudizio, L. S. Lin, and Wiranowska-Stewart,
Proc. Nat. Acad. Sci. U.S. 75:4814-4818 (1978).
27. R. Levy-Koenig, R. R. Golgher, and Paucker, J. Immunol.
104: 791-797 (1970).
28. Berman, Ogburn, Berg, Paucker, and
J. Vilcek, Proc. Nat. Acad. Sci. U.S. 72:2185-2187 (1975).
29. Yip, and J. Vilcek, J. Gen. Virol. 38:51-59
(1977).
30. J. S. Youngner and Salvin, J. Immunol. 111: 1914-1922 (1973).
31. G. Hayes, and J. Vilcek, Abstr. Ann. Meeting
Amer. Soc. Microbiol. 246 (1978).
32. R. Lockart, Jr., Progr. Med. Virol. ,Q:451-475 (1967).
33. R. Hilleman, J. Physiol. 71:43-60 (1968).
34. Heller, Virology 21:652-656 (1963).
35. R. R. Wagner, Trans. Assoc. Amer. Physicians 76:92-101 (1963).
36. R. R. Wagner, Amer. J. Med. 38:726-737 (1965).
37. Buchan and D. Burke, J. 98:530-536 (1966).
38. Field, G. Lampson, Nemes, and
R. Proc. Nat. Acad. Sci. U.S. 58: 2102-2108 (1967).
39. W. F. Long and D. Burke, J. Gen. Virol . ..: 1-12 (1970).
40. Colby, Progr. Nucleic Acid Res. Mol. Biol. 11: 1-21 (1971).
41. F. Dianzani, Gagoni, Buckler, and S. Baron, Proc. Soc.
Exptl. Biol. Med. 133: 324-330 (1970).
42. F. Dianzani, Pugliese, and S. Baron, Proc. Soc. Biol. Med.
145: 428-433 (1974).
43. J. Gauntt, Infec. Immun. 17:711-718 (1973).
44. R. Z. Lockart, Jr., N. L. Bayliss, S. and F. Yin,
J. Virol. 962-970 (1968).
1,
11 . ,1. :oHI ,J . KaLI.os, NaLLIL'c INcw IHol.] 245:
I,I:I-I/I!; (l!)7:1).
,lli. 1'. J. J. Virol. 21: 31-39 (1973).
,1'1. 1';. l)cClc['cq and De Somer, J. Gen. Virol. 22:271-280 (1974).
'IH. 1,. l\acl1llcr, J<;. DeClercq, and N. Thang, Biochem. Biophys.
I{cs. 63:476-481 (1975).
1'7
,1 !). 1';. 130rden and Leonhardt, Antimicrob. Agents Chemother.
Q:551-570 (1976).
Borden, Abstr. Ann. Meeting Amer. Microbiol. 264
(1978).
!il. 1<'. Besancon, Ankel, and S. Basu, Nature 259:576-578 (1976).
!;2. N. Reizin, V. Roikhel, and Chumakov, Arch. Virol. 49:
307-315 (1975).
!):!. Jensen, Proc. Soc. Biol. Med. 130: 1-8 (1969).
!./I. Johnson, Nature 265:154-156 (1977).
[)G. F. Dianzani) Neri, and Zucca, Proc. Soc. Biol. Med.
140: 1375-1380 (1972).
rI(i. Yaron, 1. Yaron, D. Gurari-Rotman, Revel, R. Lindner,
and U. Zor, Nature 267:457-459 (1977).
rl7. D. Stringfellow, Science 201: 376-378 (1978).
!)8. D. Stringfellow, Antimicrob. Agents Chemother. 11:984-992 (1977).
rl9. S. Rousset, J. Gen. Virol. 22:9-20 (1974).
Breinig, J. Armstrong, and J. Gen. Virol. 26:
149-158 (1975).
SehgalandI. Proc. Nat. Acad. Sci. U.S. 73:1621-1625
(1976) .
62. J. Vilcek, G. Rossman, and F. Varacelli, Nature 222: 682-683
(1969) .
63. J. Vilcek, Ann. N. Acad. Sci. 173:390-403 (1970).
64. J. Armstrong, and Ito, Proc. Nat.
Acad. Sci. U.S. 21:464-470 (1970).
65. Sehgal, 1. and J. Vilcek, Virology 70:256-259 (1976).
66. Sehgal, 1. and J. Vilcek, Science 190:282-284 (1975).
67. Borden and F. Murphy, J. Immunol. 106: 134-142 (1971).
68. Borden, V. Prochownik, and W. Carter, J. Immunol.
114: 752-756 (1975).
D. Stringfellowand L. Glasgow, Infec. Immun. 10: 1337-1342
(1974).
D. Stringfellowand L. Glasgow, Infec. Immun . .2.: 743-747
(1972)
70. D. Stringfellow, Infec. Immun. 11: 294-302 (1975).
71. D. Stringfellow, Infec. Immun. 13:392-398 (1976).
72. D. Stringfellow, R. Kern, D. Kelsey, and L. Glasgow,
J. Infec. Dis. 135:540-552 (1977).
73. G. Tarr, J. Armstrong, and Infec. Immun. 19: 903-907
(1978)
IH STII,IN(:I"I';I.I.()W
74. Ll. J. (HHi4).
75. Levine, 5 (1%4).
76. Samuel u-nd W. Joklik, Virology 5tJ:47li-491 (1974).
77. F. Dianzani, Buckler, and S. Baron, Virology
(1968) .
78. W. Stewart and R. Z. Lockart, J. Virol. &.: 795-799 (1970).
79. J. Vilcekand F. Varacelli, J. Gen. Virol. 13:185-188 (1971).
80. W. Joklik, Repts. Biol. Med. 35:364-269 (1977).
81. and J. F. Enders, Proc. Nat. Acad. Sci. U.S. 45:385-389
(1959) .
82. J. Vilcek, Nature 187:73-74 (1960).
83. R. R. Wagner, Virology 13:323-337 (1961).
84. D. Metz, (1975).
85. J. Taylor, Biophys. Res. 14:447-451 (1964).
86. 1. Marcus al1d J. Salb, Virology 30:502-516 (1966).
87. 1. Marcus and J. Salb, Cold Spring Harbor SymP. Quant. Biol.
31: 335-344 (1966).
88. 1. Marcus and J. Salb, in Interferons (G. Rita, ed.),
Academic Press, New York, 1968, 111-127.
89. W. Carter and Levy, Science 155: 1254-1256 (1967).
90. W. Carter and Levy, Biochem. Acta. 155:437-443
(1968) .
91. R. Friedmal1, D. Metz, R. Esteban, D. Torell, L.
andI. Kerr, J. Virol. 10:1180-1198 (1972).
92. S. L. Gllpta, L. Sopori, and Lengyel, Biochem. Biophys.
Res. 54: 777-783 (1973).
93. Zilberstein, Dlldock, Berissi, and Revel, J. Mol. Biol.
108:43-54 (1976).
94. Revel, Texas Repts. Med. 35:212-220 (1977).
95. G. Sen, R. Desrosiers, L. Ratner, S. Shaila, G. Brown,
Leblell, Slattery, Kawakita, Cabrer, Taira, and
Lengyel, Texas Repts. Med. 35:221-229 (1977).
96. 1. Marclls, D. L. Engelhardt, J. Hllnt, and J. SekeHick,
Science 174: 593-598 (1971).
97. Manders, J. G. Tilles, andA. S. Hllang, Virology49:573-580
(1972).
98. 1. Horak, G. Bodo, J. Lindner, and Schllltz,
Virology 48:59-70 (1972).
99. G. Hiller, G. Bodo, and Schllltz, Virology 52:
22-29 (1973).
100. 1. Marclls andM. J. Sekellick, Virology 69:378-393 (1976).
101. W. J. and R. W. Simpson, J. Virol. 2:286-289 (1972).
102. J. Biochem. Res. 47: 1228-1236 (1972).
103. S. L. Gllpta, W. D. Graziadei, Weideli, L. Sopori, al1d
Lengyel, Virology57:49-63 (1974).
1. i\N ()VI';I!VII';W
101\. [). 11. Mel"" ,1. I.evil\, N. ()Xl1lall, J. Virol. 32:
() (1!)7(j).
1()5. N. J. Lcvin, Proc. Nat. Acad. Sci. U.S. 63:
299-:JU2 (1971).
lU(j. N. Texas Biol. Med. 230-238 (1977).
I!)
107. Brown, Lebleu, Kowakita, S. Shaila, G. Sen, and
Lengyel, Biophys. Res. 69: 114-122 (1976).
108. Lebleu, G. Sen, S. Shaila, Carver, and Lengyel,
Proc. Nat. Acad. Sci. U.S. 73:3107-3111 (1976).
109. G. Sen, Lebleu, G. Brown, Kawarita, Slattery, and
Lengyel, Nature 264:370-373 (1976).
110. R. Bacteriol. 41:543-567 (1977).
111. 1. Kerr, R. Brown, and L. Nature 250:57-59 (1974).
112. 1. Kerr andR. Brown, Proc. Nat. Acad. Sci. U.S. 75:256-260
(1978).
113. Baglioni, Minks, and Maroney, Nature 273: 684-687
(1978).
114. illiau , V. G. Edy, Sobis, and DeSomer, Int. J. Cancer
14: 335-340 (1974).
115. Chang, S. J. J. Triche, and R.
J. Gen. Virol. 34:363-367 (1977).
116. and J. Vilcek, Virology 57: 378-386 (1974).
11 7 . Chang, Gregoire, J. Moncuit, Brown, Besan-
con, Surarez, and R. Cassingena, Proc. Nat. Acad. Sci. U. S. 70:
557-561 (1973).
118. Chang, 24: 148-157 (1976).
119. L. Radke, Colby, J. R. Krider, and D.
Prescott, J. Virol. 13: 623-630 (1974).
120. Besancon and Ankel, R. Acad. Sci. Paris 283: 1807-
1810 (1976).
121. L. D. Kohn, R. J. and G. Lee, Proc.
Nat. Acad. Sci. U. S. 73: 3695-3699 (1976).
122. V. Vengris, D. Stollar, and Pitha, Virology 65:410-417
(1975)
123. W. McNeill, and Killen, Immunol. 23:429-437
(1972).
124. W. Braun and Levy, Proc. Soc. Biol. Med. 141: 769-773
(1972) .
125. R. Donahoe and Huang, Scand. J. Infec. Dis. 1: 101-104 (1972).
126. 1. Gresser, Bourali, 1. Chouroulinkov, D. Fontaine-Brouty-Boye,
andM. Ann. N.Y. Acad. Sci. 173:694-707 (1970).
127. J. Cerrottini, Brunner, Lindahl, and 1. Gresser, Nature
[New Biol.) 242: 152-153 (1973).
:->'1'11.1 N(; 1,'1': I.I.()W
12H. [.;. Ul)Mal)YcL', ,J. l)jc;. l:.!2: (1!)7(').
12U. 1'. l,illllabl, 1. alllllVI. '!'()vcy, NaL.
Sci. U.S. 73: (197li).
130. Dahl and Degro, Naturo 257: 799-801 (1975).
131. R. Sohultz, J. D. Papamatheakis, and Chirigos, Science
197: 674-676 (1977).
132. R. Schultz, N. Paulidis, W. Stylos, and Chirigos,
8cienoe 202: 320-321 (1978).
133. R. W. Butcher, Advan. Psychopharmacol. (1970).
134. R. R. Gorman, J. Nuoleotide Res. 1: 1-9 (1975).
135. 8. Bergstrom, Danielson, and 8amuelsson, Biochem. Biophys.
Acta 90: 207-211 (1964).
136. 8. Baron, Advan. Virus Res. 10:39-64 (1963).
137. Isaaos, Advan. Virus Res. 10: 1-38 (1963).
138. R. Friedman, Nature 201: 848-849 (1964).
139. F. DianzaniandS. Baron. Nature 257:682-684 (1975).
140. F. Dianzani, 1. Viano, 8antiano, Zucca, and 8. Baron,
Proc. Med. 155:445-452 (1977).
141. 8. Baron, J. Gen. Physiol. 56: 184-211 (1970).
142. S. Baron, Arch. Intern. Med. 216:84-93 (1970).
143. 8. Baron, duBuy, Buckler, and L. Johnson, Proc.
Med. 117:338-341 (1964).
144. 8. Baron, Buckler, R. V. McCloskey, and R. L. Kirschstein,
J. Immunol. 96:12-16 (1966).
145. R. Murphy and L. Glasgow, J. Med. 127: 1035-1052
(1968).
146. L. DeGuerrero, and V. L. Savy, Arch. Ges. Virus-
forsch. 40: 10-16 (1973).
147. S. Baron and Isaacs, Nature 191:97-98 (1961).
148. 8. Baron and G. Buckler, 8cience 141: 1061-1063 (1963).
149. J. Ruiz-Gomez and J. 80sa-Martinez, Arch. Ges. Virusforsch. 17:
295-299 (1965).
150. J. Mendelson and L. Glasgow, J. Immunol. 96:345-352 (1966).
151. Fauconnier, R. Acad. 8ci. Paris 271: 1464-1466 (1970).
152. Fauconnier, Arch. Ges. Virusforsch. 31: 266-272 (1970).
153. Koprowski, 8. Baron, and Buckler,
Microblos 51-68 (1969).
154. Olivie and Boiron, R. Acad. 8ci. Paris 274: 1106-1108
(1972).
2
PlJRJFICATION,
PROPERTIES OF HUMAN INTERFERONS
!\lII'L Bur[!; Ivcr IIcron
IJllivcrsity of Aarhus
Dcnmark
Kurt Osther
Alfred Benzon Ltd.
Hvidovre, Denmark
1. INTRODUCTION
11,
PRODUCTION AND PURIFICATION OF HUMAN
INTERFERONS FOR CLINICAL PURPOSES;
FURTHER PURIFICATION FOR LABORATORY
EXPERIMENTS
Leukocyte Interferon
Lymphoblastoid (Namal Interferon
Fibroblast Interferon
D. Immune Interferon
Concluding Remarks
OF HUMAN INTERFERONS
(INCLUDING DENATURING CONDITIONS)
IV. ANTIGENIC PROPERTIES
REFERENCES
1. INTRODUCTION
During the last decade the production of human interferons for clinical
purposes has given rise to great deal of effort from several groups.
21
22
22
33
34
38
39
40
46
50
The most successful work until now has unequivocally done Profes-
sor Cantell's group (Helsinki), who, as early as 1967 [1], started the
systematic exploitation of the "art of producing human leukocyte interferon"
using human buffy coats. Since Cantell proved that it was indeed possible
to produce large quantities of human leukocyte interferon [> 1011
interferon units (U)/year], new groups undoubtedly begin to
21
J/ue CX:J.IIlP!c, !cukocylc i"lcl" kl"OII ic; Pl"OUUUCU
in lhu "C:J.lllul! (fol' dctails,
see Section 11. .
Since one limitation to of leukocyte is the number
of buffy coats the use of lymphocytes for the produc-
tion of human interferon has exploeed. Seveeal geoups working
along these lines, for example, Bridgen's group [2] and N. Finter and
Fantes, Wellcome Research Laboratories, United Kingdom (personal
communication). The Wellcome group has now developed techniques that
enable them to produce large of (20,000-40,000 U/ml)
lymphoblastoid interferon in >1000-liter tanks. Finally, the production of
human fibroblast interferon mentioned. Considerable progress has
been made and co-workers (personal communication), who
recent1y found high-producing fibroblast line that is trisomeric
with respect to chromosome No. 5 (associated with interferon production).
Thus, crude, unconcentrated fibroblast interferon now obtained with
titers in the of 200, 000-800, 000 U/ml. It should also pointed out
that interferon may possibly produced bacteria that have had the proper
chromosome(s) incorporated into theie genomes genetic recombination
technique. For example, preliminary reports indicate that insulin produced
such technique for commercial sale in 1980.
PRODUCTION AND PURIFICATION OF
INTERFERONS FOR CLINICAL PURPOSES; FURTHER
PURIFICATION FOR EXPERIMENTS
The aim of this section is to provide brief view of the present state of
"the art of producing interferons in clinical amounts. I! This will described
in some detail for human leukocyte interferon (HuLeIF), human lympho-
blastoid interferon (HuLyIF), and human fibroblast interferon (HuFiIF).
Since HuLeIF is the only species interferon which has used clinically
to great extent, more detailed description of this interferon
presented. After the production has been described, of inter-
ferons in clinical amounts will briefly mentioned. Further experimental
small scale to 107 U) will also considered in some
detail. scheme used for the three types of interferons de-
scribed.
Human Leukocyte Interferon
Production large scale for clinical purposes is essentially done (in
our laboratory) as described Cantell and co-workers [4-6,40,41,71],
with minor modifications. For further information, see the excellent
reviews Strander [39] and Mogel1sen and Cantell [73].
111 I,I()()(I (ill Lakull 1'1'0111 volLlI1Ut('y <\0110(,':-; i:-;
11l1,oly CllillU(1 il1 ice 1II1Lil pu['[o['mu<I,
1[11'0 ill ice to celltra1 1aboratoL'Y
pL'occssing;,) After centrifug'ation (2-40 about 15 1111 of
coal" i:-: cxpressed from the p1astic bag. iderable variations in the
the coat occur (10-30 ml). The buffy coats collectull
tlJe daytime and kept at 2-4 overnight (poo1ed) to used the nexi
1110 . Two-day-01d coats cannot used.
Contaminating erythrocytes must comp1ete1y removed since they
will otherwise react with Sendai virus (hemagglutinate), \vould lyse (proteo-
enzymes would released into the medium), and toxic products will
illlerfere with the interferon yield 500 U/m1). Remova1 of erythrocytes
i:-: performed 1ysis using ammonium ch10ride (9 vo1umes 0.83% NH
4
C1
1.0 one vo1ume bUffy coat for 15 min at

centrifuge at

resuspend
ill PBS, and repeat the procedure). The ceHs resuspended in the incuba-
tiO!l and cou!lted.
The leukocytes finaHy suspended in round-bottom 2-liter flasks
with 107 ceHs/ml. total volume of 900 ml is important, and flask
Sl10Uld loosely covered with alufoil with magnetic stirring at 37.5
(water bath). suspe!lsion is primed with 100 U/ml for 1-2 hr

This step is important. After the priming period, Sendai virus is added
to final concentration of 100-150 hemagglutination units (HAU)/ml of
leukocyte suspension. After 15-20 hr, the suspension is centrifuged
(160 g for 10 min). pellet is discarded. supernatant is kept
at -400 until further processing. titers of crude interferon are
around 25,000-50,000 U/ml. At present, the normal weekly production in
laboratory amounts to 15 bottles, and the total weekly production comes
to 400-700 106 U. Based these figures, it calculated that one
bUffy coat gives about 1-3 106 U.
Interferon is thawed and pooled, and potassium thyocyanate (as 5
solution) is added with stirring until the final concentration reaches 0.5
(4 The is slowly lowered dropwise addition (with stirring) of
1 N HCl and 0.1 N HCl to 4.5, whereby precipitate is formed (more
than 98% of the interferon is fou!ld in the precipitate together with 45% of
the proteins). After centrifugation, the interferon treated in two
ways: (1) The precipitate is dissolved in about one-tenth of the original
crude volume in PBS; the is adjusted to 7.5 means of additional
0.1 N NaOH (important, otherwise the precipitate will not dissolved)
and dialyzed extensively against PBS. type of preparation, which is
called crude leukocyte interferon (CIF) , is normaHy too crude to used
in humans. Specific activity is about 104 U/mg protein. (2) The precipitate
is dissolved in ethanol and, increasing the stepwise to 5.8, the major
part of the impurities precipitate. further increasing the most of
the interferon precipitates out at 8. last precipitate is dissolved
in PBS (about 20-40 ml) and dialyzed extensively against PBS, This partially
purified interferon (PIF) has specific activity of about 0.5-1 106 U/mg
;',1
j)t'olciI1. TI1C t'CCOVCI'Y iH (l>aHC([ ()I1 1I1C
It i" ampul1cd (i11 ;: 10" lL<'U pOI,'liol1H) kcpl LlI1lil '1'1li"
type rnaterial i8 8table for 10
The PIF material has very well tolerated rnore than 15 patients
(administered il1trarnuscularly), only 8ide effect we have noticed l3],
with Torben Fog (per80nal communication), is the well-lmown fever
reaction, which is in accordance with the findings of others, for
Strander' s group (personal communication). fever often reaches
38-390 and lasts for 1-3 hr. After 2-8 injectiol1s, the fever reaction gradu-
disappears.
The PIF preparations from two different laboratories (Cantell' s and
our own) have been compared in sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) (slab gradient) to determine similarities or
differences in the protein composition (Figure 1). protein patterns
are quite similar (the input for both types of PIF preparations had specific
activity close to 1 106 U/mg protein). C1F preparations also show
identity (Figure 1). No antigenic differences were found between the inter-
feron batches (determined interferon neutralization tests) .
1n principle, troubleshooting 100ks relatively simp1e, and that is
probably the reason why many people have started to follow the success
of Cantell. 1n practice, however, relatively few laboratories have succeeded
in getting rea1 steady production going (between 300-1000 million
U/week). reason is clearly harbored in the small and delicate
technicalities which are "hidden" in this type of procedure. For example,
great care must taken to a1most comp1etely remove the erythrocytes.
(and similar divalent ions) must avoided, otherwise clumping will
occur. a1so has to consider the quality of the water, etc. Even the
form of the flask is of great importance: the flask must round-bottomed,
2 liters in size, and have broad neck (5 in diameter). stirring
has to kept at very constant level: not too quickly, which is harmful
to the 1eukocytes, and not too sl0w1y, as c1umping will then occur.
virus is probably the most important variant in the whole "game." Great
care should taken to obtain good Sendai virus [1,4,5, 6] without
bacteria1 contamination. Poo1s should made from 10-12 eggs; keep the
virus at 4 and wait for bacteriological screening (which should nega-
ti . Inc1us ion of egg subs trate should not forgottcn; alternati vely, the
virus batches checked means of special screening plates made
with egg substrates. virus also kept at _700 after addition
of agamma serum (as stabilizer). The diluted virus suspensions should
handled carefully (virus is unstable when diluted).
Although the technique does vary arnong laboratories, it has been
found in three independent laboratories that when virus with an
titer >4000 per milliliter is used will good production of interferon
possible (15,000-100,000 IFU/ml crude interferon). high concentra-
tions egg proteins will probably interfere with the yield. During the
17 t 800
12,400
1 2 3 4 5 6 7
FIGURE 1. SDS-PAGE, tbln slab of HuLeIF (24 cm-long gels). Slots
No. 1 and 2: Molecular markers (mol. wt. 12,400, 17,800, and 25,000).
Slot No. 3: CIF 8 3 (Cantell). Slot No. 4: PIF (Cantell). Slot No. 5:
PIF-1 ( 3), not typical PIF preparation due to variation in the
ethanolic precipitation procedure (the titer was about 1 106 U/ml). Slot
No. 6: CIF ( , ) . Slot No. 7: PIF ( , ) . The titers of CIF in slots
No. 3 and No. 6 were about the same (400,000 U/ml). The corresponding
PIF had titers of 1-4 106 U/ml. Based the protein bands (slots No. 4
and 7) it that the two PIF preparations are very similar with
respect to the impurities.
peullllUliOll pUl'io(1 J.!:1ual cal'u lakull lo Il1illillli:;,u I)aclul'ial
lf luu lliJ.!:ll luvul iH pl'uluulylic will
releaseu, a11U blologiual activity will inevitably uccur. Cruuc i!lter-
fero11 preparatio11s are routi!lely froze!l i!l batches of 10 liters (-400 before
being processed to PIF, but other laboratories (for that of Cantell)
just keep the CIF at

Duri!lg the last eight months, we have had

routine productio11 of crude human leukocyte i!lterferon of 25,000-35,000
U/ml total of about 500 106 U/week).
It should, u!ldoubtedly, possible to improve the above-mentio!led
system considerably further manipulatio11 of the primi11g procedure,
virus, media, etc. For example, consider the possibllity of pre-
treating the leukocytes with polycatio!ls before the virus is added [7]. The
of the leukocyte suspe!lsion is crucial. It should in the vicinity of
7.2 (7. 1-7.3); after the i!lduction of interferon the drop to 6.8.
too high level indicate poor choice of buffer and give rise to
death. There are reports 011 the successful use of the superi11duction
technique in the production of human leukocyte i!lterferon (see Vilcek
the human fibroblast system, Sectio11
Further purificatio11 of huma11 leukocyte i11terfero11 is ofte11 desirable.
When explori11g differe11t aspects of i11terfero11, i11Cludi11g the of
i11terfero11 to membra11es, the actio11 of i!lterfero11, etc., it is most des irable
to have as pure i11terfero11 preparatio11 as possible. laboratories
will very ofte11 asked the 110W classical questio!l the referee: "Is the
phe11ome11011 have observed result of i11terfero11 itself, or is it due to
some u11kllown impurities prese!lt i11 uIlkllown qua11tities?" Exte!lsive efforts
have therefore devoted to purifyi!lg i11terfero11. At prese11t, 110
has reported complete purificatio11 of human leukocyte i11terfero11. The
best specific activity obtai11ed is 108 -109 U/mg proteill. l!l the followi11g
sectio11 several methods will outli11ed that will, hopefully, serve as
guide for those who wish to purify i!lterfero!l to level that the referee
accept.
Gel filtratio!l of c011ce11trated crude leukocyte i!lterfero11 (CIF) has
routi11ely d011e ill our laboratory with very good results, Ultrogel
5/4 (Pharmacia, colum11 2.6/100 at 40 1 urea (i!lclud-
i11g 0.1% etha11olami11e) i11 PBS, = 7.2 (recovery of about 100% i11 each
rU11). typical profile is shown i11 Fig'ure 2. About 90-95% of the
are 10cated i11 the protei11 peak, which is followed the i!lterfero11 activity.
The specific activity is close to 106 U/mg protei11, a11d i11 respects
this type of material resembles the PIF material. Whe11 dealillg with small
amou11ts of crude leukocyte illterfero11 (1-20 106 U/ml), the gel filtratio!l
tech11ique should the first choice. I11cide11tally, this type of material
was tolerated very well i11 patie11t, as was PIF (2 106 U/dose).
Torma a11d Paucker [43] reported similar purificatio11 procedure
for leukocyte i11terfero11 PIF plus sodium dodecyl sulfate (SDS) i!l
gel filtratio11, e11di!lg with specific activity of 5-10 106 U/mg protei11
()I"
5.1
1,.9
3
L..7
2

4.5


-'
4.3
4.1
-------------------------,



LJ')""t'--'"
N <D
f 1'.(
: \ \ \
I }I \ [\ r I
r ,1 I ,\ II
r 11 \ 11 II
; : / \ ; I
I I 1::
,
! I ,1 I l'
\ \ I 1,' 1"
: '-1 j -"\,/ 1, \
/ ; \
3.9
600,000
500,000
z

400,000

lL 300,000
---
::J
200,000
100,000
------,
,
,
MW18,200
,
I
{
I
I
I
1(\ 1\1
11 \ 11'
I J '}I
:: \1
: j }:
, I
I I
, I
80 70 60 50 40 30 20 10
FRACTION NO_
FIGURE 2. Gel filtration of crude leukocyte interferon (CIF). Ten
ml of CIF, 4

U in total (in PBS). was loaded directly an Ultrogel
5/4 column (100 2.5 2.5/100, Pharmacia) at

(buffer: 1
urea including 0.1% ethanolamine, in PBS, 7.2). Fractions size 8 ml;
flow rate 35-40 ml/hr. Recovery 95-100%; specific activity about 106 U/mg
protein. The was equilibrated means of standard markers.
solid curve, U/fraction (in 69/19 units); dashed curve, OD
z80

(100-140% recovery). One major drawback with this method is the presence
of SDS, which has to removed before additional affinity chromatography
performed [8]. How close PIF preparation is related to gel-
filtered CIF preparation remains to seen (experiments in progress).
Hydrophobic chromatography has been extensively exploited Sulkowsky
and co-workers [9-13]. Mostly, they have been working with relatively
small amounts of interferon (10,000-100,000 U) without obtaining great
il1 spccil'ic aeLivily. l11:till PllI'IJOse ol'llleil' w()["k 11aS
been tllC uivul!!:ill!!: tlle IlyueopllOble IlUlllan
leukocyte interferon. Thcy have yet bcen to fillU Wllich
clearly useful in purification when crudc I1uLeIF is the :>tarting
material. For example, phenyl-Sepharose is able to bind interferon (several
hundred thousand units), but unfortunately most other proteins are also
bound and eluted with the interferon-thus the purification factor is only
twofold Berg, unpublished data). Of course, this situation could change
drastically if new matrix which was far more selective would appear.
The whole picture might also change drastically if more purified interferon
preparation were used as starting material (for example, gel-filtered
CIF). Thus, it is too early to rule out this type of technique for large-
scale purification of clinical amounts of material.
Since most interferons are bound to Con it has been widely claimed
that interferons are glycoproteins [56]. Jankowski et al. [10] demonstrated
that leukocyte interferon was not bound to Con column (the same has
been found in our laboratory). One might speculate whether this behavior
of leukocyte interferon in contrast to other interferons could due to the
rather crude material (specific activity = 104 U/mg protein) which has been
used in these experiments. In crude HuLeIF preparation the glycoprotein
content could very high, causing competition between interferon and
extraneous glycoproteins for binding sites Con
using three types of different purity human leukocyte interferon
(specific activity 104, 106, 6 107 U/mg protein), we found that all the
interferon was passed through the Con column as long as the specific
activity was below 106 U/mg protein. At specific activity of 6 107 U/mg
protein, we could indeed bind most of the activity to the Con column, but
subsequent elutions only 5% was recovered. Therefore it is difficult to
make any conclusions since the protein content was rather low (about 3-5
Thus nonspecific binding or inactivation very well have
occurred.
Until now, the most promising technique for the purification of human
leukocyte interferon has been antibody affinity chromatography, which was
introduced Anfinsen et al. [14] and Berg et al. [15]. The principle of
antibody affinity chromatography is that antibodies to interferon are bound
to matrix (for example, Sepharose and packed into column. At
neutral the antibodies will react with the crude interferon
preparation that is loaded the column at 4 lowering the to 2.4,
the antigen-antibody complex is dissociated, and the purified
antigen (interferon) is released. equilibrating the column with "loading
buffer, 11 the column used repeatedly. Antibody columns last
for 1-2 years (and used more than 100 times). When not in use, the
columns are stored in PBS (40 with streptomycin, gentamycin,
and chloromaphenicol (1% of each) . The column should cleaned (loading
buffer plus eluting buffer plus loading buffer) before it is actually loaded
:J. ))'1':11'1'11';:-; IIIIIVI 1 )N:-;
4!)
wll.ll CI'\ttJC illl,CII'("OII. Lllis f:.til'ly la1"!!;c volurnes inte1"fe1"on
Lo ;'00 lO"/U, tl1C size the column and
1.11(' 1"01" ucLails, thc is 1"eferred to Anfinsen et
al. 1\11 J cL [15].
'I'llc c["uci:.tl poinJ in antibody affinity chromatog1"aphy is the antibodies.
111 LIH,'OI'y, antibodies should monospecific, i. the antibodies
HIIOIJIc1 ecact only with the interferon p1"oteins. In practice, antibodies
in sheep or rabbits prolonged injection schedule using only
pltI'Lially purified inte1"feron. In order to remove antibodies that bind
proteins, the antiserum is cleaned absorption. The
in principle, performed two methods: (1) employ-
III!!; Lhose contaminants thought to most important binding them to
matrix and then passing the crude antiserum (or
1-: lobulins) through such column several times [16]; or (2) tinding the
cOlltaminants to Bentonite [17] using batchwise absorption. combination
(1) and (2) used, starting with the batchwise proeedure. The
should include human serum proteins, egg-allantoic fluid,
coat extracts, whole buffy coats, etc., as previously described [14,
I[), 17].
the use of indirect hemagglutination procedures, it is possible to
l'oIIow the removal of the different known contaminants [17], although one
not put too much emphasis this technique [16]. the use of
best absorbed antiserum, column was constructed [15], and crude
leukocyte interferon was purified in step to about 10 106
U/mg protein.
In theory, the very best antigen mixture to use for absorption of anti-
interferon would crude interferon preparation that harbors the
impurities. Such absorption, however, might also remove anti-interferon
antibodies during repeated absorption. Most fortunately, this does not
Berg et al. [18] showed that the major part of the antibodies
remained in solution, after the immunoglobulins were passed through
"CIF column" (CIF bound covalently to Sepharose) for more than 10 times.
The absorbed antiserum was in turn coupled to Sepharose and the column
was loaded with crude leukocyte interferon. Specific activity in the eluate
was about 30-40 106 U/mg protein 1, experiment Nos. 299, 303,
313) .
When gel filtration was combined with affinity chromatography, inter-
feron of two to three times higher specific activity was obtained, as seen
from 1, experiment No. 420 (specific activity about 10 108 U).
It was disclosed that probably 5-10% of the interferon is "masked" as
far as the antigenic properties are concerned. When performiug antibody
affinity chr-omatography of CIF, about 10% of the input was always found
in the wash [8]. If the CIF was first prepurified gel filtration (in 1
urea with 0.1% ethanolamine), dialyzed and loaded the affinity column
(input 100% of the original starting crude material), less than 0.1% was

1. Purification of Human Interferons Antibody-Affinity Chromatography
Specific Specific
activity activity of
Interferon of input Wash Eluate eluate (U/mg Re- Purifi-
of input Volume (U/mg total total protein covery cation
No. interferon (U 10-6) (ml) protein) (U) (IFU 10-6) 10-6)
(%) factor
299 CIF 3 25
104 8 105
4.2 43 140

303 CIF 1.5 2.5
104 6 105
1.8 33 120

313 CIF 2 5 104 6 105
2.4 31 120

420 Gel-filtered
CIF 4.5 50 5 105 2 103 3.0 90 66

285 Crude
6 103 104
=
Namalva 1.6 150 2.6 23 90

296 Crude
.-
.
Namalva 1.05 150 4 103 8 104 0.95 8.6 90

5;
301
...:
fibroblast 4 50 8 105 8 105
3.6 103 90

--
:-
aBased specific activity of peak fraction.
bBased the eluate.
.
--
,:::
z
2. 01" IIlJIVI I
l'OlllHI ill waHII. '1'11 iH ilKlieaLu(l [JlaL (1) tlle a!lLige!lic sites were masked
I)'y HOll1U Wi1iUJl wuru rumoved with 1 urea plus gel filtratio!l,
Ittlt! sitluu thu biological situ was !lot disturbed (the i!lterfero!l was meas-
(II'al>lu), the locatio!l of the antige!lic sites might differe!lt from the
siie(s) (see discussio!l i!l Sectio!l IV). Alter!latively,
La.kus placu duri!lg the biological assay.
Suque!ltial antibody affi!lity chromatography of leukocyte i!lterfero!l
I!aS rece!ltly described [8]. I!litially, !lormal a!ltibody affi!lity
cla'omatography was performed. The eluate was dialyzed agai!lst the 10ad-
buffer, the!l passed through " co!ltrol which was directed mai!lly
the impurities, a!ld the last wash (which i!l pri!lciple cO!ltai!led most
()f the i!lterfero!l) was loaded again directly the anti-interfero!l colum!l.
As far as removal of the impurities was cO!lcerned, the principle worked;
lJUt, unfortunately, most of the interferon activity was 10st the pro-
uedure.
One of the u1timate means to check the purity of given protein prepa-
ratio!l is Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAG Several papers Stewart and co-workers have dea1t with electro-
phoresis of human interferon [19-21,72]. We are at present thin
slab version of SDS-PAGE (20-24 lO!lg gels, 8-20% acrylamide gradient)
10aded with 80-fJl samples. The results of gel-filtered a!ltibody affinity-
leukocyte i!lterfero!l preparatio!l are shown in Figure 3. It
clearly seen that proteins are !lot 10cated at the i!lterfero!l peaks.
Whe!l using shorter gels, the interferon activity and the proteins coincided
(not shown). When the preparation in longer gels (24 the
visible protein bands and fue interferon peaks could separated. This
phasizes that great care should exercised in fue interpretation of these
types of experiments. Interferon recoveries from such experiments are
usually about 50%, based the total input the gel. This is somewhat
10wer than reported others [2]. In order to see stained interferon protein
bands, we would estimate fuat more than 2-5 106 U have to applied
one gel. This amount of interferon happens to fue as was very
recently described De Maeyer-Guignard et al. [22] in the mouse system,
when they obtained visible, stai!led interferon protein bands.
The anticellular effect of leukocyte interferon as described Stewart
and co-workers [20] was also measured at fue same time. As
in Figure 3, the two curves, interferon and thymidine were opposite
as expected, except at fraction No. 34 (mol. wt. 35,000). There was
interferon activity in peak 34, and several occas ions the anticellular
effect could detected. We have, however, not been to isolate the
anticellular activity (at fraction No. 34). One might speculate whether
high concentration of SDS would give rise to the observed phenomenon,
or whether we are dealing wifu protein which is very unstable (present in
very small concentrations).
III':HC. ()S'I'III';H. 1lI111111':ltON

3
4.40

Q
<5
4.20

....J
4.00
20 "10
15,000
"
1',
"
1 ,
'1
11
<f)
, ,
11
1-
10,000
11
11
10,000 3 z
' 1
, 1
Q : 1
11

1-
1"
U
U
"
1
"

"
1
"
1,
,
l

11
1 l
LL
1,
, 1
1
11
::J
5000
' ,
5000
11
:
1
:,

2000
1000 1000
<100
2 10 15 20 25 35 40
FRACTION NO.
FIGURE 3. SDS-PAGE of purified (PIF). About
2 106 U was purified of gel fi1tration, dialysis, and antibody
affinity chromatography. After concentration (visking tube + aquacide II)
and dialysis versus buffel' (diluted threefold),
115 in 175 f.Ll together with 2.5 106 U in total activity about
20 106 U/mg protein); 80 f.Ll were mixed with 10 f.Ll glycerol, including
bromphenol tracking dye. After electrophoresis was performed,
the gel was cut into 2 pieces, eluted in 01% SDS at 4 for 2 days,
and titrated for interferon activity. Low interferon titers (5-40 U/fraction)
were found in fractions 4-15. The anticellular activity was measured
of lymphoblastoid (Daudi), which was cu1tured
in microtiter plates. The interferon samples were added to the at
the beginning of propagation, and proliferation was measured
thymidine uptake. solid curve, interferon units/fraction; dashed
curve, [
14
C]thymidine uptake.
I'H(H'I:H.'I'II':S IN'I'I':IIYI':II()NS
11. 1111111:t11 Ll1Lue[ueOll
SI11CU limiLaLioll Lo amounL 01 that produced is the number
01' l'lll'l:y coaLs availablu, it. wou1d important if substitute source
('0111(1 strander et a1. [42] investigated if it was possible to make
(IHU 01' 1ymphocytes in the production of 1ymphoblastoid
IIILUL"I'uron. soreened about 21 human 1ymphoblastoid cell1ines and
olle which was remarkably good interferon producer, name1y, the
Hne. of about 10,000 U/m1 cou1d obtained. W.
1"iIlLor and Fantes (persona1 communication) have taken the produo-
l,i0l1 of Nama1va interferon in 1arge tanks (> 1000 Hters), and they now pro-
(ILlCC crude interferon with titers of about 20,000-40,000 U/m1. Bridgen
uL a1. [2] have a1so reported production of Nama1va interferon in 800-liter
Lallks using techniques similar to those of Finter and Fantes, a1though their
yiuld per m1 10wer (2000 U/m1).
1n theory, the procedure is very simp1e. Nama1va are grown
suspension culture (from 100 m1 to 1000 Hters) until density of
!) 106 cells/m1 is reached. are resuspended in medium, and
interferon induction is done essentially described in the previous
section, except that twofo1d higher amount of Sendai virus (300 HAU/m1
in the fina1 suspension) is recommended. We have a1so checked the
production "semi-micro" sca1e (50-100 m1) and found it very to
obtain titers of about 12,000-15,000 U/m1. It is advisable before starting
to grow the for interferon production, to c10ne the origina1 suspension
in order to ensure good and healthy starting popu1ation (J. Zeuthen,
persona1 communication).
crude Nama1va interferon is precipitated means of trichloro-
acetic acid follows Fantes, persona1 communication): 1/20
vo1ume of 50% (adjusted to 2.5) is added to the crude interferon
and, after 4 overnight, is centrifuged. precipitate either
disso1ved in PBS (1/10 vo1ume) and dia1yzed extensive1y versus PBS to give
crude concentrated Nama1va interferon, or it disso1ved (homogenized)
in 94% ethano1 using one-fifth of the origina1 interferon vo1ume. raising
the to 5. 1, many contaminating proteins are precipitated together with
amount of interferon. further raising the to 7.5, the inter-
feron is precipitated out of the ethanoHc solution. It is redisso1ved in
vo1ume of PBS and dia1yzed extenSive1y. recovery is normally
about 40-50% when working with crude interferon of reasonable titer
210,000 U/m1). 1f lower titers of crude interferon are obtained, the re-
coveries will drop to be10w 15%.
Such purified Nama1va interferon cou1d suitable for clinica1 purposes.
If the Nama1va are grown in the presence of calf serum, specia1 pre-
cautions must presumably taken against any residua1 amounts of calf
serum proteins that provoke serious allergic reaction even when
present in extreme1y amounts (ng). If media without calf serum pro-
LuilH'; LktL woubl I'isu lo l'cacLiol1s (luvulopu(l,
1'01' Mosi
the danger ulOing i1'anlOJo1'rnud ulua1'ud
shortly, especially if cOnlOidering the use PIF-like rnateriallO for human
trials. As an initial step, "Narnalva PIF" SllOUld used ill cancer patients,
and based these trials ful'ther analyses decisions could rnade.
Further Purification of Narnalva Interferon
Fantes has reported (personal comrnunication) that, in ion
exchange chromatography, crude Narnalva interferon 30-fold purified
loading (100-300 rnl) sulfopropyl (SP-Sephadex colurnn [size
10 ml]) at 4. Recovery is about 50-80%. Sirnilar reports
published Bridgen et al. [2] and G. Bodo (personal cornrnunication).
Gel filtration of Narnalva interferon has also been reported Fantes,
using Ultrogel 5/4 (which proved to the very best gel) with quantita-
recovery (at 3.5). Purification 50-150-fold (specific activity about
106 U/rng protein).
Antibody-affinity chromatography has reported Bridgen et al.
[2], Fantes (personal communication), and Berg et al. [23]. They
found that Namalva interferon purified to 20 106 U/mg protein
with about 80% recovery 1, experirnent Nos. 285 and 296). If gel
fi1tration and antibody-affinity chromatography are combined, 3-6-fold
higher specific activity is obtained Berg, unpublished data).
combining antibody-affinity chromatography with SP-Sephadex
chromatography, together with isoelectric focusing followed SDS-PAGE,
specific activity to 108-109 U/rng protein will most likely obtained.
employing suitable ligand, it rnight possible to rnake some short
cuts in the purification procedure, ending with pure interferon proteins.
Undoubtedly, we shall very soon see the final purification of Namalva inter-
feron.
Fibroblast Interferon
The production of human fibroblast interferon has during the last decade
attracted much attention. Originally, Vi!cek and [24] introduced
"superinduction," which rendered possible the production of large
of fibroblast interferon with titers 10,000 U/ml. et al. [25]
have also the production of fibroblastoid interferon. They screened
18 continuous human lines for the ability to produce interferon, using
Newcast1e disease virus (NDV) or superinduction with poly 1: poly and
found (MG-63) of osteosarcorna origin which was to produce
more than 50,000 U/rnl. It was possible to produce 109 IFU in 5 weeks,
using 364 bottles of cells. If the MG- 63 could adapted to grow
as suspension culture, giving the same high titers, envisage that
COlII(1 jJl'OLlLlucLl ill IlU!,:C similar
iIlLUI"I'eI"Oll). coursc, reservations
I'aisctl wiLIl I'c!':[tl"t[ lo usc il1tcrferoll produced from trallsformed
mClllio!HXl il1 SccLioll II.13.
Mozus ct al. [26] reported that increased interferon production ill
IlUm::ll1 fibroblasts obtailled employi.ng UV-irradiated
itlstcad superillduction (irradiation plus superillductioll gave smaller
yield thall irradiatioll alone). [27] recently published paper
tl1e purification alld production of fibroblast interferon using the
"Vilcek techllique" for the productioll of illterferon.
Very recently, the work of Berthold et al. [28] gave rise to rellewed
speculatioll Oll the productioll of human fibroblast illterferon for clinical
applicatioll. employing that are trisomeric in chromosome No. 5,
they succeeded ill maki.ng crude uncollcentrated illterferoll with titers to
500,000 U/ml. Briefly, monolayer cultures of were induced
with poly 1: poly plus cylohexamide for 4 hr. Three hours later actillomy-
cin D was added, and illterferoll was harvested. The crude illterferoll
preparatioll was fOUlld to very labile. It was stabilized precipitatioll
with 50% ammonium sulfate alld kept such for mOllths at

Prior to
it was redissolved ill PBS alld dialyzed. Agaill it would desir-
if one could accommodate the to suspension culture.
Purification of humall fibroblast interferoll has beell described
several groups. Several techniques have been applied with remarkable
Gellerally speaki.ng, principal difference to exist between
fibroblast and leukocyte illterferOllS. Fibroblast interferon, opposed to
leukocyte interferon, will often billd to wide variety of affinity matrices.
For example, fibroblast illterfel'on billds to COll (stro.ngly), to octyl-
,sepharose, to poly-U Sepharose, to Sepharose, to serum albumin
Sepharose [although this last type ofbindillg is not reproducible Sul-
kowsky, personal commUllicatiOll)], to glass beads, etc. Crude leukocyte
interferoll will, however, ollly bind to one of these matrices Sepharose).
Whether this is the result of ftllldamental molecular differences between
fibroblast interferoll alld humall leukocyte interferoll remaills to seell.
For the time being, one call1lot completely rule out that differellces ill the
impurities present ill the two preparations might somehow illterfere with
the apparently different behavior of the interferons for example,
ullder Sectioll II. the COll column) owing to competition betweell impuri-
ties alld illterferoll proteins with regard to the tinding sites givell
matrix.
[27], for the first time, completely purified humall fibroblast
illterferoll USillg combillation of "classical" physicochemical procedures.
First, the illterferoll was fractionated alld cOllcelltrated meallS of
mOllium sulfate. It was thell fractionated BioGel columll alld
purified Oll carboxymethyl-Sepharose columll followed preparative
SDS-PAGE. After elutioll and cOllcentratioll, it was allalyzed SDS-PAGE
:lli
(lI1il1 sLal) ol11y OIIC' al!.(,, (111OIeCllla,' wuil-(i1L
Spucil'ic acLiviLy was lOH U/1111-( pt'oLciI1, LoLal
was about i)%.
Berthold et al. l have also pUr'ifiuJ
(produced from , the affinity approach comblned
with SDS- PAG First they loaded column with crude, concentrated
interferon and eluted, resulting in 30% recovery; specific activity was
107 U/mg protein. The elution of interferon also gave rise to
protein, which could abolished subsequent phenyl-Sepharose affinity
chromatography. purified preparation was (without 10s ing
interferon activity) , and preparative SDS-PAGE was performed (recovery
about 16% based the input SDS-PAGE; total recovery based original
input was 4%); specific activity of U/mg protein was obtained.
band was found in SDS-PAGE This material
,vas also labelled, and it was shown that the radioactivity was at the same
location as the interferon proteins. Thus it appears pure labelled
human fibroblast interferon now produced in 20 quantities.
columns were introduced Huang et al. [29] with
promising results. They obtained high specific activity (over 1 107
U/mg protein) and quantitative recovery. Presumably, the method involves
hydrophilic Uinding between the matrix and interferon (the interferon is
loaded at 7.2 at 10w ionic strength and eluted with 20% ethylene glycol).
The method was later found to erratic and, for the time has
abandoned Sulkowski, personal communication).
Hydrophobic chromatography, which was originally introduced
Sulkowsky and co-workers [9,10,30,31,36], is very useful method for
purifying small amounts of interferon (5000-100,000 U). The specific
activities obtained are usually about 105 U/mg protein, depending the
amount and source the interferon. Recovery is about 100%. Octyl-
Sepharose, phenyl-Sepharose, w-carboxypentyl-Sepharose, etc.
used as matrices.
The Ciba-cron blue column, introduced Janowsky
et al. [32], is also very useful approach for purifying 50,000-500,000
U/mg to specific activity of about 106 U/mg protein (recovery about 100%).
1n brief, the column is loaded with crude interferon in 0.02 sodium
phosphate at 7.4, including .15 NaCl, washed with the same buffer,
including 1 NaCl, and eluted with 50% ethylene glycol (including
1 NaCl). It is very important that the dialysis is done thoroughly; other-
wise the interferon does not bind to the column. The columns should also
washed extensively with 10-15 bed of buffer before loading the
interferon.
The A-Sepharose column was introduced Besangon and Bourgeade
[33] and Davey et al. [34,35]. The principle makes use of the fact that
glycoproteins very often blnd to due to the rigbl sugar
the interferon molecule (hydrophobic interactioll might also illvolved
I'I()I'I,:I('I'II'::-; ()I' IN'I'I<I{I'I<I(()N:-;
1:1:,1. 111 Illc iH ill sodiurn phosphate
(pll 7.'1) O . .l;) NaCI (loadin/-,: 13y adding 0.1 a-D-
(ulJMl) Lo loading some impurities e1uted
1'1'0111 furLher atlding ethy1ene glyco1, the interferon
oluLcll (quantitaLive with specific activity of 5 107 U/mg
IJloLcitl. SUCll preparation was not homogeneous SDS-PAG
(1';. Sulkow ski, personal communication).
Con co1umn 1eaks off Con proteins during use. minimize
lcakag'e, the co1umn should first washed with the 10ading buffer fo1-
lowctl the elution buffer (if necessary, this "cycle" repeated).
co1umn 1eak Con protein (at 10wer 1evel compared to the
"unwashed" Con column). get rid of the contaminating Con proteins
(il1 purified interferon preparation), the e1uate shou1d dia1yzed back to
PBS, 0.15 NaCl, and then rechromatographed phenyl-Sepharose
Sulkow ski, personal communication). This method process to
about 500 106 U small co1umn (20-m1 size).
Controlled glass beads have recent1y been described Edy et al.
l J 7]. In princip1e, the crude interferon is loaded 10-ml column
with controlled glass (CPG-10-350, Electro-Nucleonics, Inc., Fair-
N. J. ). Most of the impurities escape the column during washing,
and lowering the to about 2, 60% of the interferon recovered
(specific activity 1-5 106 U/mg protein). Since leakage of the column
matri.x occurs, it possible this method to purify fibroblast
interferon for clinical trials.
Z inc chelate affinity chromatography has very recently been introduced
Edy et al. [38] with even greater success. Approximately 60% of the
interferon could recovered (final specific activity close to 108 U/mg
protein). The interferon is loaded small column (6 ml) at 7.2 (in
0.02 sodium, potassium phosphate buffer plus 0.15 NaCl) and eluted
with gradient 6-4). It wiH interesting to see the number of
protein bands that develop an SDS-PAGE of such purified material.
Antibody affinity chromatography of fibroblast interferon has a1so been
employed in laboratory, using an antiserum which was directed against
human leukocyte interferon (cf. Berg et al. [18] and Section IV). The crude
fibroblast interferon was purified to 108 U/mg protein in one step
covery 70%). The eluate was subsequently checked an SDS-PAGE (slab
methods) and several protein bands were revealed, of which one located in
the where blological activity was determined. Thus we observed
specific activity of purified fibroblast interferon in the same range as
[27] found with fibroblast interferon, but contrary to his,
ration was not homogeneous (four bands in Since the recovery in
Knight' s procedure is rather low (5 %) as compared with ours, we would
expect his homogeneous preparation to "contaminated" with but
inactive interferon proteins. Therefore the specific activity of the homo-
geneous preparation could, at least theoretically, the same level as
heterogeneous one.
ul al. I :-;lI/-,:/-,:U:-;lUll IH'U:-;ul1l ill
luukouylc il1lcr[urotl atlli/-,:ulliuatly diHurctll thc
cor-r-csponding itnpurities in Iibrublast illter-fer-on preparation. If so, it
should possible to purify fibroblast interferon antileukocyte column
(the so-called denominator principle). Ber-g et al. [15] could not confirm
this interesting idea, since they obtained almost the same activity
when loading antileukocyte column with fibroblast interferon leukocyte
interferon. We later found, however, that, when using highly absorbed
antiserum (absorbed means of crude interferon immobilized Sepharose
[8]) the "denominator principle" was working as far as the impurities were
concerned. Mock-fibroblast interferon proteins wer-e indeed passing through
antileukocyte column, provided that the absor-ption was pursued to the
extreme [8]. contrast mock-leukocyte interfer-on prepar-ation was
bound to the antileukocyte column.
D. 1nterferon
The induction of interferon(s) was originally described as response
to viral infection [74]. Such interferon is narned "classical" interferon,
antiviral-induced leukocyte interferon or type 1 interferon. "immune"
interferon (also called type interferon) is interferon-like substance
[75-77] produced from immunocompetent cells (in vivo and in vitro). There
are several reports the production of human immune interferon in vitro
[75-79]. brief, lymphocytes are exposed to, for example, antilymphocyte
serum [75], [76], Berg, unpublished
data) , or other mitogens [78,79] with varying degree of success. Nor-
the titers are in the low range (20-500 U/ml).
The criteria used when evaluating whether given interferon prepara-
tion is of immune interferon origin are the same as those used for type 1
interferon, except that type has to destroyed at low (2.5) and
the antigenic properties of type are different from type 1. Very often
the first prerequisite is only partially fulfilled, probably due to partial
contamination of the immune interferon preparation with type 1.
partial (low) neutralization is sometimes obtained when immune
interferon is tried with antitype 1 interferon. Very often the investigator
wants to observe special effect of immune interferon in given system;
therefore it would important to sure that the immune interferon prepa-
ration is not contarninated with type 1. We would expect that passing
mixture of type 1 and through anti-interferon column, the type 1
should bound (provided that type 1, or part thereof, is not "masked";
see Section Thus, after such passage the wash, which in theory
should contain only type II interferon, checked versus low and
in neutralization test. Such procedure is for the time being explored
in our laboratory with promising results (experiments in progress).
I'IUH'I';II.'I'II'}; ()I,' IN'I'I,:IIYI-:II.()NS
lovporLH 11 itlLvl1"vroll binds to
SOpll:Ll0HP l(jll i/l/"alli' s communication) using
()4 HOllilllll 7.4 lt could eluted (and 20-fold
lIHillj2; salt cOllccntration (1 NaCl) and 50% ethylene glycerol o
No rcports 011 purification type II interferon vailable
I,'aloo[[ et alo [75] reported molecular weight of 50,000 obtained gel

1'; COl1cluding Remarks
'1'110 research interferon has expanded during the past 20 years from the
of basic virology to the fields of biochemistry, biology, immunol-
0J2:Y, and clinical medicineo clinical interest in interferon is based
wide spectrum of actions (antitumor and antiviral activity) together with
[cw side effects of this "natural" compoundo
The production of human leukocyte interferon has been thoroughly
described earlier in this sectiono It was mentioned that large amounts of
material, suitable for patients, now producedo The material is very
well tolerated the patients, and the stability of leukocyte interferon
appears to satisfactory Thus HuLeIF soon (1-3 years)
practical clinical tool in medicine
The major limitation for production an industrial scale is the
amount of buffy coats available One way out of this dilemma is most likely
to the Namalva which for the first time make it possible to produce
interferon in "industrial amountso" The main problems remaining to
solved are (1) the "danger" of using proteins from transformed cell-line
and (2) the possible allergic reactions which minute amounts of calf serum
proteins could provoke, a1though this problem might not so great
Fantes, personal communication) For example, it is highly likely that
there are also minute amounts of egg proteins in current PIF material which,
nevertheless, are tolerated very wello There have been reports that fibro-
blast interferon is not stable leukocyte interferon when given to man,
since it was difficu1t to detect any interferon in serum after injection of
5 106 U Whether this is due to an actual breakdown of fibroblast inter-
feron, or if the fibroblast interferon is more readily bound to surfaces
compared to HuLeIF, remains to seeno
Human immune interferon might reveal anticellular and immunoregula-
tory properties which could very useful for the future treatment of
patientso This new aspect of interferon is currently under intense investiga-
tion, and we shall undoubtedly fairly soon see some new principles and
of this interferono
Finally, it should mentioned that the ways and means which inter-
ferons are administered to patients for the being appear too unsophis-
ticatedo large amount of interferon is given to patient, and it is "hoped"
/1
111-:1(,(;, OS'J'III-:It, :tllll III-:I!,()N
ccrtaill paI'L iI1LcI'[CI.'OIl "peeiJ'ie Lal'j2;cL LllaL i" ill-
vo1vcd in illncl:il:i. IL would mUCll aL Ica"L Lo cl1vi"aj2;c
techniques deve10ped that wou1d us to direct io
specific targets. cou1d think ul:iing certain type
(carrying the interferon), which preferentiaHy would absorbed
the liver, for treating hepatitis patients, etc. [80].
HUMAN INTERFERONS
(INCLUDING DENATURING CONDITIONS)
One of the to production, purification, and clinica1
eva1uation of interferon as antivira1 and agent has been its
notorious particu1ar1y in re1ative1y pure (specific activity
above 107 protein, depending the total of interferon present).
The was not to the purified interferon preparations.
We have occasionally experienced that crude leukocyte interferon
batches have sudden1y (over 3-7 days at 4 10st of the activity
25,000 to below 2000 The reason for this 10ss has not
fuHy exp1ained, although there to hints that proteo1ytic
activity cause. of HuLyIF
interferon preparations were checked for proteo1ytic activity
using Azocoll as substrate. The that
practicaHy known proteo1ytic react with the substrate,
which is peptides with dye bound to the peptides.
proteolytic activity split the peptides and release the dye.
ing the optica1 density (OD) at 520 in the supernatant, an "expression"
of the proteolytic activity is obtained. It was hoped that high proteo1ytic
activity (high OD) wou1d correspond to 10w interferon titer. As
seen 2, these expectations were c1early not although
proteo1ytic undoubtedly, play an ro1e
in the of interferon.
Several other interferon batches were a1so checked; these were
PIF and CIF Cantell) and PIF-1 another 1aboratory; see
3). PIF-1 was never used for any clinica1 purposes, since it lost its activity
going 1 106 down to 20,000 during period of three weeks
(at 4 It appeared that proteo1ytic activity have been the deter-
factor for the instability observed. retrospective
of the protoco1 a1so revealed that there had been bacterial
nation during the production (of PIF-1). It is noteworthy 3) that the
level of proteolytic activity in PIF (Cante1l) is lower to CIF
Thus during the procedure (inc1uding the ethanolic precipitation),
proteolytic activity is a10ng with other Crude inter-
feron preparations appear to have the leve1 of proteo1ytic
activity as CIF, despite the fact that interferon activity is increased 10- to
20-fold in the latter.
SLablliLy (01.' Yield)
01' 1IIl,cI'I'uI'ol] all(1 I'r'oLcolyLic
Nalllalva illtcrfurol1 Proteo1ytic "activity"
I\al,ell 110. U/m1 units (OD Mean va1ue
102 5,000 2.279
2.045
2.162
116 5,000 0.460
0.425
0,390
118 1,600 0,494
0.476
0.459
435 11,300 0.370
0.376
0.383
439 20,000 0.729
0.742
0.754
443 15,000 0.542
0.545
0.543
aMeasured using ca1cu1ating absorptiometer (LKB 7400).
number of occasions, proteo1ytic such as soybean
trypsin (STI), Trasy101, etc., were added without the slightest
effect the stability of 1eukocyte interferon. is in contrast to the
findings of Berthold et a1. [28], who reported that they were to stabi-
lize fibroblast interferon means of Trasy101.
proteo1ytic attack of human 1eukocyte interferon has a1so recent1y
been advocated Chadha et a1. [62]. Using molecu1ar sieving of crude
HuLeIF, tlley revea1ed an apparent molecular weight of 26,000. However,
after denaturation (guanine + mercaptoethano1j Mogensen and Cantell [44]),
mo1ecu1ar weight of 21,000 was observed. The reactivated interferon
(mo1. wt. 21,000) gave on1y sing1e peak il1 SDS-PAGE, confirming that
sing1e mo1ecu1ar species was generated during the denaturation and re-
activation procedures. Based 011 these findings, the authors suggested that
the interferon mo1ecule proteolytic cleavage, probably
protease present in the extracellular fluid. Thus peptide fragment dis-
sociates from the parent mo1eculc when HuLeIF is denatured in the presence
of reducing agent, resulting in drop of 5000 in molecular weight (without
any 10ss of antivira1 activity in the parental mo1ecule). No interferon activity
was in the small fragment wt. 5000). It might also argued
that the 10ss in molecu1ar weight (5000) is due to break in an S-S bond.
III;II.{;,
Protoolytic Activity lJotormillod ill Ilu J,oH' 1'1"OlXtealiollH
OD Aotivity Interferon
Samples readinga
10-3
titer
Control 0.100
PIF-1 (unoentrifuged) 1.370 0.035 20.000
PFI-1 (oentrifuged) 1.090 0.028 20.000
CIF 0.800 0.015 400.000
PIF 0.210 < 005
2 106
Crude HuLeIF (HKN) 1.370 0.030 9.000
Crude HuLeIF 1.160 0.025 5.400
Crude HuLeIF 95D 1.388 0.034 5.400
Crude HuLeIF 0.410 < 005 16.000
Crude HuLeIF 0.709 0.005 48.000
Crude HuLeIF 96D 0.706 0.005 5.400
Crude HuFiIF (12099/138) 0.318 < 005 20-40.000
aThe OD reading is expressed as of trypsin per
oomparing the OD value with trypsin standard ourve obtained using
different but known amounts trypsin.
During the reaotivation, the small part is unable to into the right
moleoular oonfiguration, whioh thus resu1ts in the 10ss in molecular weight
5000.
It has been reported that an increase of the serum concentration,
together with decrease of proteolytic activity, might stabilize crude inter-
feron, but the pattern was not Fantes, personal communica-
tion) " We found that CIF and PIF were stable at _200 Cantell et al.
[71J normally keep their interferon preparations at +40 For the last
10 months we have not encountered the problem,
because rigorous rules in the laboratory concerning bacterial
contamination, including more streamlined procedure. Thus we
essentially confirm the findings of Cantell et al. that leukocyte interferon,
when handled properly, appears to stable months (> 10 months).
Several reports have been published in connection with the stability
of human interferon under denaturating conditions. Some the data
summarized in 4. Generally speaking, the interferon activity is
:'.. I'I{(H"-:I!'I'II';S .. 1IIIIVIi\N
'I'AI\ 1,1'; ;j. SLabll iLy
of interferon
Ilcat (1000
.1 min
IIcat (1000 in
Hel
of
SDS
SDS + heat
(1000 1 min
Heat followed
e
2
H
s
SH + SDS
("heat resur-
rection")
Proteolytic
activity in
crude inter-
feron
Inhibition of
proteolytic
activity Tra-
sylal or STlb
Mol wt in
SDS-PAGE
Mol wt gel
filtration
aND = not done.
Iluman
leukocyte
99.5%
destroyed
70%
destroyed
100-150%
recovery
150-250%
recovery
0% recovery
++
No inhibition
17,800 and
21,000
26,000 (21,000)
(17,800)
bSoybean trypsin inhititor.
Namalva
>98%
destroyed
ND
a
100%
recovery
ND
ND
++
No
18,000 and
21,000
23,000
Human
fibroblast
>98%
destroyed
100%
destroyed
20-60%
recovery
25-100%
recovery
25-125%
recovery
+

20,000
20,000-
25,000
lost when interferon is heated to 1000 (1 min). Partially purified leuko-
cyte interferon (PIF) sho\ved remarkable at 560 [44]. Mter
three days at 560 10ss was seen (eIF dropped one log in activity in
12 hr at 370 These results [44] are in concord with t11e data shown in
3 concerning the proteo1ytic activity of different interferon prepara-
tions. A1though it is tempting to specu1ate whether the higher
,1,1
111';11(;, ():-;'I'III';II" :tI1<1111';I{()N
il1 uUHuevu<1l)y 1'111 j was (llIU to jJr'otuo-
lytic activity cOlllpaeu<1 Lu it iH uaely to l1laku COf]ClllHiOll.
For exalllple, it Illight expected paetial il1acLivaLiul1 peutculytic
would occur at 560
It is clear ly doculllented [44] that interferon contains S-S bonds which
are illlportant for the expression of the biological activity. breaking
these bonds means of mercaptoethanol alone) the Wological activity dis-
appears [44]. breaking the S-S bonds in the presence of 8 urea (or
5 guanidine chloeide), the biological activity recovered after
dialysis against PBS. The urea (or guanidine) functions as protein-folding
agent which permits the eeduced interferon molecule to return to the proper
configuration before reoxidation (done dialysis against PBS) has occurred.
Based anti-interferon neutralization tests, the antigenic composition of
leukocyte interferon seems to remain unchanged after such treatment
Berg, unpubl ished data) .
In 1974 Stewart et al. [45] introduced an interesting principle
ing the "resurrection" of interfeeon. Interferon was boiled (and inactivated);
Illercaptoethanol, urea, and SDS were added, and the mixture was boiled
again. After subsequent dialysis the investigators [45] found that most of
the activity was recoveeed in stable Resurrection worked only for
fibroblast interferon and mouse), but not for HuLelF (Table 4).
Although recovery does vary (also in hands), it is interesting observa-
tion that it is possible to resurrect "dead" (boiled) interferon.
of occasions, we have tried to see if such resureected interferon prepara-
tions stable in subsequent purification steps (affinity chromatogra-
phy, etc.). We have not, however, found any Illarked illlprovements, and
since recoveries from the resurrection procedure generally were low
(25-30%) we stopped pursuing this technique. The antigenic properties of
interferon assessed antibody affinity chromatography remain unchanged
after such treatment.
The fact that the biological activity of interferons either
completely or, at least, partiaHy recovered after SDS treatment (see Table
4) has aided considerably in the molecular weight determination of types
of interferon means of SDS-PAGE. Treatlllent of protein mixtures with
SDS, followed boiling modest heating, for example, to 560 breaks
noncovalent bonds whereby proteins, cOlllplexing with masking interferon,
are dissociated from the interferon molecule. lt seems likely that such
random binding of foreign proteins to interferon was responsible for the
variation in the reported molecular weights obtained means of g'el filtra-
[46-48].
Stewart et al. [49,50] were the first to report calculation of interferon
molecular weight means of SDS-PAGE. They found biological activity
in two peaks of interferon, corresponding to lllolecular
weights of 21, 000 and 15,000, respectively. addition to these findings
they also disclosed that the two peaks differed, as far as stability was
I'I{()I'I,:I!.'I'II':S 11l11Vli\N IN'I'I':I!.I"I':I!.ONS
ill SI)S il1 their uegt'ee of hetet'o-
()Il eafJf)iL
Wu also examil1ed SDS-PAGE ge1, 24
polyacry1amide 9-20%) found slight1y higher va1ues
wL. 17,1100 Thus we
() [SLuwart. l27) reported that mo1ecu1ar weight of
pure SDS-PAGE is about 20,000 peak).
is with others, Pitha [51) Vilcek
uLal. l52].
Edy et [53] reported that of
blast resembled leukocyte renaturable
after with hydroch10ride. However,
its 10w activity heterologous it resembled fibroblast

Cartwright et [54] reported that fibroblast could
stabilized forces
processes, etc.) of simp1e su1fhydry1 such
The of such removable re1ative1y
toxic help of fibroblast
for
No reports available stability of Nama1va
(Nama1va PIF); but most 1ike1y stability properties similar to those of
1eukocyte (PIF) would expected, although there have
a1coholic procedure to Nama1va
Fantes,
proteins to
et [55] observed decreased charge of rabbit
isoe1ectric with
They proposed that most like1y was
varying of sialic acids). This was further
et [56], who, after of rabbit with
found (broad) peak at 6.3. This type of
its character after of
sialic acid residues the of sia1y1 The studies of
et [55] et [56] showed that sialic
residues removed without of activit.y. et
[56] showed that the carbohydrate residue the po1ysaccharide
ga1actose, cou1d successive1y oxidized with ga1actose oxidase
reduced with sodium borohydride without 10ss of activity.
blast interferon has a1so suggested to glycoprotein [27,30,33,34,
55-59). rabbit, and human fibroblast interferon to
with sugar It is therefore to specu1ate
whether 1eukocyte interferon should The
for this is 1ess Neither crude 1eukocyte inter-
feron [59, 73) PIF wOll1d to [73] .
Isoe1ectric profilcs 1cu].;ocyLc al1l1 al'Lct.'
treatment with neuraminidasc
obtained after similar treatment rabbit interferon [56,60, G1, 73].
et [58] were able to fragment (mol. wt. 4000)
interferon molecu1e treating g1ycosidases, thus indicating
the presence of sugar moiety the interferon the other
hand, it cannot comp1etely ru1ed out that possible proteolytic activity
present in the crude interferon preparation cou1d responsible for the
in molecu1ar weight. wou1d, in way, in harmony with the
recent resu1ts of Chadha et [62].
Recent1y, et al. [63] demonstrated that it was possible to
convert the heterogenous 1eukocyte interferon to one peak (in isoe1ectric
focusing) of chemica1 oxidation (using periodate). They cou1d
show that the 21,000-mo1ecu1ar-weight species was converted to the
15,000 species. 1atter was suggested to the most stable species of
HuLeIF. The (in molecular weight) was reported et
[58] and Chadha et [62], using different approaches. At present,
it that the t,vo species 1eukocyte interferon wt. 15,000
and 20,000) converted to one species wt. 15,000) varied
treatments (1) chemica1 c1eavage "oxidation" (using periodate), (2)
protein denaturation "reduction" (using guanidine and mercaptoethanol),
and (3) enzymatic c1eavage (using g1ycosidases and neuraminidase). It
wou1d interesting to combine treatments 1-3 to whether the
phenomena described unre1ated whether we dealing with different
aspects the mo1ecule.
IV. ANTIGENIC PROPERTIES
For it has been widely accepted that interferons were species-
specific far the antiviral activity was concerned. That is to
interferon will only protect not human etc.
theorem has gradually modified, especially after Gresser et al.
[64] showed that HuLeIF had higher antiviral activity bovine
compared to human With regard to antigenicity of interferons, the
"species specificity theorem" still to valid, since antiserum
against interferon from another species has been shown to neutralize, for
HuLeIF vice versa). In 1964, Paucker [65] showed that
interferon failed to neutralize chick interferon.
Ogburn et [17] reported that interferon (from L
duced of two different inducers 1: NDV) was bound
equally we11 to anti-mouse interferon column, suggesting that there
were antigenic differences among interferons produced in response to
different inducers acting the was also found
Havell et al. [66] in the human system.
I'H()PI':H:I'II':S ()I,'
17
l,aLel' i L W:LH [Ollllll LlraL exist
pl'olltlcell ill cell LypeH 01' species. This was first
Ll'aLe(1 [J.Y Sal l ()7], who
PI'OHUIIL Hurum from mice after with the
ILIILiI4UII. which presumably is produced from
cou1d with raised virus-
IIHJuccd mouse fibroblast (which is surprising, as we
shortly). The authors the substance made
Ulis immunostimulation type II ("immune interferon") as
to the conventiona1 type 1 interferon ("c1assica1 or viral interferon").
I3erg et [15] showed that 1eukocyte and fibroblast interferons
were dissimilar with respect to antigenicity. This was probably most
ulear1y demonstrated leukocyte and fibroblast interferons to
:tl1tibody affinity chromatography, using two different columns made
of interferon and antifibroblast interferon. When
loading 1eukocyte interferon antifibroblast c01umn,
1-2%) was When 10ading fibroblast interferon
alltileukocyte c01umn, more than 95% of the interferon was to the
e01umn. Based these findings, it was suggested that the antigenic
position of fibroblast interferon form) was different from 1eukocyte
interferon (L form). This was a1so confirmed interferon neutralization
si-udies [15,66]. That the antileukocyte serum neutralize both types
of interferon probably ascribed the "buffy coat preparation," which
is composed of wide variety of whereas the fibroblast be10ngs
to much more homogeneous popu1ation. Thus during the
induction, both forms of (L and F forms) are produced in the
buffy coat system, and only form (the F form) is produced the fibro-
blast system. Of the tota1 amount of interferon activity in 1eukocyte inter-
the L form amounts to about 99% and the F form to 1%.
this small amount of the F form is sometimes sufficient to evoke antibody
response in rabblts. This was documented, for et
[66] Berg et [15] of the interferon test:
The serum (for as twofold is mixed
with 10 U of the If the the
the mixture + protect against
virus (or vice versa).
It might seem peculiar that call1lot
1eukocyte interferon, the very same serum is used to
the F form [15]. These results are,
nevertheless, good using the
as in chromatography, the total
amount of the F form put the c01umn. Thus
this behave as fibroblast with respect to
When crude 1eukocyte interferon is diluted to concentration
of 10 U/m1, the F form is not recognized the antifibroblast interferon,
111':11(;,
sincc it is U/ll1l) COll1jJar:Cl! wiLll
L form (about 9.9 U/m1).
HaveH et [66] were to is01ate the antifibroblast intcrflJron
activity that was present in se1ected antileukocyte interferon "reverse"
affinity chromatography. First, they fibroblast interferon to
Sepharose (they could demonstrate that the beads had titer of about 400
U/O.1 m1 reacted beads). After loading tbe antileukocyte (which had
titer of about 100-200 against both forms of interferon), they were
to elute antifibroblast interferon (which could not neutralize leukocyte inter-
feron) .
In conclusion, it has thus proven that human leukocyte interferon
is made up of two species: the L form and the F form %) . The
latter is most likely identical with, or very closely related to, the fibroblast
interferon produced fibroblast ceHs, as summarized in 5.
Recently, Paucker et [68] showed that the F and L forms a1so
distinguished using rabbit ceHs instead of human ceHs in the neutral-
ization of 1eukocyte interferon (when employing sheep anti-interferon).
They proposed mode1 in which sing1e interferon m01ecule contains
multip1e binding sites, each of which is of interacting with ceHs
of different species.
Namalva interferon, which is produced transformed
(Nama1va), is very similar to 1eukocyte interferon with respect to m01ecu1ar
weights and isoe1ectric points, as shown in 5. far as antigenic
properties are concerned, however, there is distinct difference between
leukocyte and Namalva interferons. About 15% of the total interferon activity
in Nama1va interferon is represented the F form, whereas, in 1eukocyte
interferon, about 1% of the activity is represented the F form.
This was elegantly shown HaveH et [69], employing antibody affinity
chromatography with "monospecific" antisera. passing crude Namalva
interferon antifibroblast c01umn, about 13% of the interferon could
bound subsequently eluted lowering the This corresponds very
weH with the neutralization results. when using mixture of
interferon sera (antifibroblast plus antileukocyte sera) was it possible to
neutralize Nama1va interferon (using "monospecific" sera; J. Vilcek, per-
sona1 communication).
It might asked if the F form (in Namalva interferon) is identical
with fibroblast interferon regards m01ecu1ar weight and isoe1ectric
points. This identity cou1d easily demonstrated first passing crude
Nama1va interferon through antifibroblast column. This purified F form
could then further ana1yzed to see if full (or partia1) identity exists.
Very few reports have published the possible correlation
tween tbe antiviral activity the antigenicity of interferon. An important
question might asked: Will the antigenic the inter-
feron molecule 10cated the same site as the biologica1 (antiviral)
activity? Mogensen and Cantell [70] reported that carboxymethy1ation
>i>-

5. Antigenic Properties of Interferons
Neutralized with
Anti-
Anti- Anti- fibroblast
of fibroblast leukocyte + anti- SDS-PAGE Isoelectric F form L
interferon IF (%) IF (%) leukocyte vvt) point(s), pI 1: I
Fibroblast 100 0-100 100 20,000 6.8-7.3 100

Leukocyte 100 100 21,000 (23,000) 5.4-6.4 (7) 1 99
15,000 (17,000) Three components
Namalva Partial Partial 100 21,000 (23,000) 5.4-6.3 (7) 15 55
15,000 (17,500) Three components
Immune NDb
ND ND ND
aln preliminary report, et al. (Amer. Soc. Microbiol. Abstr., 1978) find that fibroblast interferon contauls
small portion of the L form.
bND = not done.
llu:-;Lt'oyc(l iL:-; acLiviLy
locaiioll Lwo 111 tJJis
laboratory, we have several obscrvull
could compete tests with active similar
purity Berg, data). order to explore possible relation-
ship activity, the
was performed. Crude HuLeIF was bOUl1d to two different matrices: acti-
vated Sepharose (AS) epoxy-Sepharose (ES), both purchased from
Pharmacia. About 50% of the were bOUl1d both CIF-
ES had absolutely activity the beads were tried
cells. CIF-AS had activity (in higher 1: 100).
were made the two gels. (titer about
100,000 Ul1its/ml) was loaded each
after thorough wash, the were eluted activity
could the very last of the wash). From both
could eluted specifically. Thus of the gels
(CIF-ES) had biological activity but was to neutralize
(No activity was fOUl1d
eluate after the with rabbit
could that the biological site(s) might have
the molecule. If this proves to
it makes the very difficult-if
with pure

work was supported from The Society.
REFERENCES
1. Cantell, G. Hadhazy, and R. How
much prepared leukocyte
The of the Symposium held
at Siena, 1967 (G. Rita, ed.), Academic Press, New York, 1968,
223-232.
2. J. Bridgen, L. Corby, S. Zoom,
V. Ruegg, Buckler, Lymphoblastoid
Large scale production partial purification. J. Biol. Chem. 252:
6585-87 (1977).
3. I. S. Christophersen, R. Jordal, Osther, Hammer, J. Linden-
berg, Berg, of
diseases: report. Med. 204: 471-476 (1978).
<>1" IN'I'I':HYI':I!()NS
:>1
,1. "'. CatlLcll, 1'ecp:H'aLioll 01 lcukocyte inteeferon. Standardiza-
Lioll 01 111Lce1ceoll alld llltee[eron Inducers: Proceedings of the Inter-
Symposium held in London, 1969 (F. Perkins alld R.
ltc!!;amcy, eds.), Karger, 1970.
:>. 11. Strander and allte 11 , Further studies the productioll of
[ceoll humall leukocytes ill vitro. Anll. Med. Biol. Fellll. 45:
(1967).
ll. Strander and Callte11, Productioll of illterferon human leuko-
cytes invitro. Anll. Med. Felln. 44:265-273 (1966).
7. 13. Larsell, personal communicatioll (1978).
Sequential antibody affinity chromatography of human leuko-
cyte interferon. Scand. J. Immullol. .: 77-86 (1977).
9. J. Chell, W. J. Jankowski, J. Sulkowski, alld
W. Carter, Nature of the molecular heterogelleity of human leuko-
cyte interferoll. J. Virol. 19: 425-434 (1976).
10. W. J. Jankowski, W. Davey, J. Sulkowski, and
W. Carter, Moleculat> of llumall and leukocyte
illterferons: lectin and J. Virol.
16: 1124-30 (1975).
11. J. W. Huallg, I. Hejna, Sulkowski, W. Carter, G.
and
The influence of albumine Virology 65: 268-271 (1975).
12. J. Chen, W. J. Jankowski, J. Sulkowski, and
W. of the moleculat> of human leuko-
cyte J. Virol. 19:425-434 (1976).
13. W. Davey, J. W. Huang, Sulkowski, and W. Hydro-
phobic tinding sites human J. Chem. 250:348-349
(1975) .
14. S. L. Corley, and D. Gurari-Rotman,
purification of human affinity
Nat. Acad. Sci. U. S. 71: 3139-42 (1974).
15. Berg, Ogburll, Mogensen, and Cantell,
Affinity of human leukocyte and diploid
antibodies. J. Immunol. 114: 640-644 (1975).
16. Paucker, Berg, and Purification of mouse
antibody affinity In Effects of Interferon
and the Immune System Geraldes, ed.), Academic
New York, 1975, 639-657.
17. and of mouse interferon
affinity globulin-sepharose.
J. Immunol. 111: 1206-1218 (1973).
18. R. HamiIton, and Heron, Purification of human
antibody affinity using highly absorbed anti-
Scand. J. Immunol . ..:429-436 (1978).
19. W. 1.;. Stuwari, J. MOluclll.at' 01'
human leukocyto intcrIerOtl: Two populatiOlls ditTurillf!: ill tnolucular
weights, requirements for renaturation and cross-specics antiviral
activity. Virology 67: 68-73 (1975).
20. W. Stewart, 1. Gresser, G. Tovey, Bandu, and
S. LeGoff, Identification of tlle multiplication inhibitory factors
in interferon preparations as interferons. Nature 262: 300-302 (1976).
21. W. Stewart, L. S. Liu, Wiranowska-Stewart, and Cantell,
Elimination of size and charge heterogeneities of human leukocyte
interferons chemical cleavage. Proc. Nat. Acad. Sci. U.S. 74:
4200-4204 (1978).
22. J. G. Tovey, 1. Gresser, and DeMayer,
Purification of mouse interferon sequential affinity chromatography
poly(U) and antibody-agarose columns. Nature 271: 622-625 (1978).
23. Berg, R. R. D. and 1. Heron, Repts.
Med. 187-193 (1977).
24. J. Vilcek and HaveH, Stabllization of interferon messenger
RNA activity treatment of witl1 metabolic inhibitors and lower-
ing of tl1e incubation temperature. Proc. Nat. Acad. Sci. U.S. 70:
3909-3913 (1973).
25. Billiau, V. G. Edy, Heremans, J. van Damme, J. Desmyter,
J. Georgiades, and De Somer, Human interferon: Mass produc-
tion in newly established cellline, MG-63. Antimicrob. Agents
Chemother. 12: 11-15 (1977).
26. L. W. Mozes, HaveH, L. Gradoville, andJ. Vilcek,
Increased interferon production in human cells inadiated witl1 u1tra-
violet light. Infec. Immun. 10: 1189-91 (1974).
27. Interferon: Purification and initial characterization from
human diploid cells. Proc. Nat. Acad. Sci. U.S. 73:520-523 (1976).
28. W. BertllOld, and Purification and in vitro labeHing
of human fibroblastoid interferon. J. Biol. Chem. in press (1978).
29. J. W. Huang, W. Davey, J. Hejura, W. van Muenchhausen,
Sulkowsky, and W. Carter, Selective binding of human interferon
to albumin immobilized agarose. J. Chem. 249:4665-4667
(1974) .
30. W. Davey, W. Sulkowski, and W. Carter, Binding ofhuman
fibroblast interferon to concanavalin A-agarose: Involvement of carbo-
hydrate and hydrophobic interaction. Biochemistry 15:
704-713 (1976).
31. Sulkowski, W. Davey, and W. Carter, Interaction of human
interferons with immobilized hydrophobic amino acids and dipeptides.
J. Chem. 251: 5381-5385 (1976).
32. W. J. Janowsky, W. von Muenchhausen, Sulkowski. and W.
Carter. Binding of human interferons to immobilized Cibacron Blue
F36A. Biochemistry 15:5182 (1976).
J>H()J>I';II.'I'II';S ()I,' IIIJJVIAN
:1:1. ,,'. 1Vl-"'. I\OLIL"!,:C:tL!C, of and inter-
'"Ct'OII '"01" J. Immunol. 113: 1061-1063 (1974).
W. lJavcy, 1<;. Sulkowski, and W. Carter, Hydrophobic inter-
of interferon with concanavalin A-agarose. J. Biol.
249: 6354-6355 (1974).
:1;). W. Davey, Sulkowski, and W. Carter, Binding of
fibroblast interferon to concanavalin A-agarose: Involvement of carbo-
hydrate recognition and hydrophobic interaction. (Manuscript in
preparation, 1976.)
:I(j. W. Davey, Sulkowski, and W. Carter, Hydrophobic inter-
action of human, and rabbit interferons with immobilized
hydrocarbons. J. Biol. 251: 7620-7625 (1976).
:17. G. Edy, 1. Brande, De Clercq, and De Somer, Purifica-
tion of interferon adsorption chromatography controlled pore
glass. J. Gen. Virol. 33:517-521 (1976).
:ltJ. G. Edy, illiau , and De Somer, Purification ofhuman fibro-
blast interferon zinc chelate affinity chromatography. J. Biol.
252: 5934-5935 (1977).
Strander, Production of interferon suspended leukocytes.
Thesis, Balder Stockholm, 1971.
40. Strander and Cantell, antiviral and effects
of leukocyte interferon in vitro and in vivo. 1n Production
and Use of Interferon for the Treatment and Prevention of Virus
1nfections: Proceedings of Tissue Culture Association Workshop
held at Lake Placid, 1973 Weymouth, ed.), Tissue Culture
Rockville, Md., 1974, 49-56.
41. Cantell and S. Hirvonen, Large-scale production of human leukocyte
interferon containing 108 units per ml. J. Gen. Virol. 39: 541-543
(1978).
42. Strander, Mogensen, and Cantell, Production of
lymphoblastoid interferon. J. Clin. Microbiol.1:116-117 (1975).
43. Torma and Paucker, Purification and characterization ofhuman
leukocyte interferon components. J. Biol. 251:4810-4816
(1976).
44. Mogensen and Cantell, Human leukocyte interferon: role
for disulphide bonds. J. Gen. Virol. 22: 95-103 (1974).
45. W. Stewart, De Clercq, and De Somer, Stabilisation of
interferons defensive reversible Nature 249: 460-461
(1974) .
46. J. S. Youngner and W. R. Stinebring, Comparison of interferon pro-
duction in mice bacterial endotoxin and statolon. Virology 1: 310
(1966) .
47. Merigan and W. J. Kleinschmidt, Different molecular s pecies
of interferon induced statolon. Nature 208: 667 (1965).
;,1 III':I!.(;, ()S'I'III':lt,
413. Mcrigan alld W. ,J. I\JciI1Hl:JlIIl itll, HpceicH
of interferon in illjcl:ted Nalurc 212: l:JH:J
(1966) .
49. W. Stewart, Distinct species of interferons. Virology
61: 80 (1974).
50. W. Stewart, De V. G. Edy, Paucker, Berg,
and Ogburn, Distinct species of illterferons:
for stabilization and reactivation of leukocyte
and fibroblast interferons. J. Gen. Viral. 26:327-331 (1975).
51. F. Reynalds and Pitha, Molecular weight study of
fibrablast interferon. Biophys. Res. 65:107 (1975).
52. J. Vilcek, Havell, alld S. Antigenic,
and biological characterization of interferons. Ann. N.
Acad. Sci. 284: 703-710 (1976).
53. V. G. Edy, J. Billiau, and De Stable alld
unstable af fibroblast interferon. lnfec. lmmun. 16:
445-448 (1977).
54. Cartwright, Sellussi, and D. Grady, Reagellts which inhibit
disulphide balld formation stabilize fibroblast interferoll.
J. Gen. Virol. 36: 323-327 (1977).
55. Schonne, illiau , and De Somer, The properties of interferon
IV. Isoelectric focusing of rabbit interferoll (NDV-RK13). In Standard-
ization af Interferon and Interferon Inducers: Proceedings of the Inter-
national held in London, 1969 (F. Perkins and R.
eds.), Karger, Basel, 1970, 61-68.
56. F. Dorner, Scriba, and R. Weil, Interferon: Evidence for its
glycopratein nature. Proc. Nat. Acad. Sci. U.B. 70: 1981-1985 (1973).
57. Heterogeneity of purified interferons. J. Biol.
Chem. 250:4139-4143 (1975).
58. S. D. U. Ruegg, L. Corley, and
Anfinsen, Apparent dispensability of the carbohydrate of
interferon far antiviral activity. J. Biol. 251: 1659-1662 (1976).
59. W. J. Janowski, W. Davey, J. Sulkowski, and
W. Carter, Molecular structure af fibroblast and leukocyte
interferons: Prabe lectin and hydrophobic chromatography. J. Virol.
16: 1124-1130 (1975).
60. Mogensen, L. and Cantell, No evidence
for carbohydrate affecting the clearance of circulating
leukocyte interferon in rabbits. Acta Pathol. Microbiol. Scand.
305-310 (1974).
61. J. Morser, J. and D. W. Hutchinson, Differences in sialic
content of interferons. J. Gen. Virol. in press (1978).
62. Chadha, Sclair, Sulkowski, and W. Carter, Molecular
size heterogeneity of leukocyte interferon. 17:
196.::.200 (1978).
INTI':Hlo'l':H()NS
li;). W. 1';. SkW:tI'L, 11, 1,. S. I,iu, Wieanowska-Stewart, and Cantell,
,1';1 i 111 s [:.-;u hutel'ogeneities of leukocyte
il1Lce[cL'ol1S cleavage. Proc. Nat. Acad. Sci. U.S. 74:
(1977).
li'l. 1. Ul'essel', D. and Tovey, Pronounced
activity of interferon bovine and cells.
543-545 (1974).
(j!). Pauckel', The serological specificity of interferon. J.
94: 371-378 (1964).
(j(j. Havell, Ogburn, Paucker, and
J. Vilcek, Two antigenically species of interferon.
Proc. Nat. Acad. Sci. U. S. 2185-2187 (1975).
(i7. J. S. Youngner and S. Salvin, Production and properties ofmigra-
tion factor interferon in the circulation of with
delayed J. Immunol. 111: 1914-1922 (1973).
(j8. Paucker, J. Dalton, Ogburn, and Torma, Multiple
active sites interferons. Proc. Nat. Acad. Sci. U.S. 72:
4587-4591 (1975).
69. Havell, Yip, J. Vilcek, Characteristics of
lymphoblastoid (Namalva) interferon. J. Gen. Virol. 38:51-59 (1977).
70. Mogensen and Cantell, Loss of antigenicity of car-
boxymethylation of interferon human
leukocyte origin. 52-55 (1974).
71. Cantell, S. Hirvonen, Mogensen, and L. Pyhala, Human
leukocyte interferon: Production, purification, stability and
experiments. In The Production and Use of Interferon for the Treat-
and Prevention of Virus Infections: Proceedings of
Tissue Culture Workshop held at Lake Placid, 1973
Weymouth, ed.), Tissue Culture Assoc., Rockville, Md., 1974,
35-38.
72. W. Stewart, and J. Desmyter, Molecular modification of inter-
feron: Attainment of interferon in conformation active cat
cells, but inactive human cells. Virology 70:451-458 (1976).
73. Mogensen and Cantell, Production and preparation of human
leukocyte interferon. Pharmacol. Therap. 1: 369-381 (1977).
74. Isaac and J. Lindenmann, Virus interference. 1. The interferon.
Proc. Roy. Soc. 147: 258 (1957).
75. Falcoff, R. Falcoff, L. De Vomecourt, and J. Sancean,
Synthesis of interferon in lymphocytes stimulated in vitro
antilymphocytic Eur. J. Clin. Res. 1: 20-26 (1972).
76. J. Valle, G. W. Jurdan, S. Haahr, and Merigan, Charac-
teristics of immune interferon produced lymphocyte cultures
compared to other interferon. J. Immunol. 115: 230-233 (1975).
77. F. Wheelock, Interferon-like virus induced in
leukocytes phytohemagglutinin. Science 169: 310 (1965).
78. L. 13. l<:PStCill, uf 011 illll1lLlIlC irl
vitro and in vivo. In IntcrIerolls alld Thcir' (W. 1<:. SLcwat'L,
ed.), Press, 1977, 92-126.
79. Hirth, Schwenteck, Becker, and Kirchner, Interfcron
production and lymphocyte cultures stimulated Corynebacterium
parvum. Clin. Immunol. in press (1978).
80. La Bounardiere, Donnees preliminaires sur l'effet protecteur
de l'interferon couple: Des liposomes dans le modele souris-virus de
l'hepatite murine. Ann. Microbiol. (Inst. Pasteur) 397-402
(1978) .
3
EXUGENUUS INTERFERON:
STABILITY AND PHARMACOKINETICS
13. Greenberg, Maurice W. Harmon, and Robert Couch
lIu.ylor College of Medicine
Iluuston,
1. INTRODUCTION

Thermal
Mechanical Stability
Stabilization to Thermal and Mechanical Stress
D. in Body Fluids
Conclusions
III.
In Animals
In Humans
Mechanism of Serum Clearance
D. Side Effects
Conclusions
REFERENCES
1. INTRODUCTION
57
58
59
62
62
65
66
67
67
74
79
81
82
83
The prospect for of exogenous interferon in human disease necessitates
an evaluation of the relative stability of different types of preparations and
of the pharmacokinetics of this antiviral agent. Not only is this information
needed for clinical trials of preparations, but any eventual preparation,
distribution, and of commercial material will require knowledge of
of the preparation. In addition, design of schedules for optimal
clinical uti1ization must based understanding of the absorption,
distributiol1, metabolism, al1d excretion of this biological substance within
the host animal.
Although originally described stable in acidic solution, studies
have demol1strated the susceptibility of interferon to inactivatiol1 thermal
and mechal1ical stress. The instability of interferon preparations in
57
various fluids UUUll UUlllOI1Hlt.'aLuu. illCOlllpkLu,
information tlle stabHity of iIl1UL'[U['OIl peupat'aLiollH 10
variety of is
reviews have emphasized our lack of fue
of indicated need for additional researcll
in fuis area [1.-3]. Those factors affecting fue absorption, distribution,
and metabolism of as well as pharmacological a11d clillical
ditions which define its utility, need to These two subjects,
the of human interferon and its pharmacokinetics in animals and
are fue subjects of this review.

It has known for some that disti11ct structural differences exist
among interfero11s of differe11t species. Antibody directed against
from animal species generally fails to neutralize illterferon
produced other anima1 species [4]. Moreover, and Sa1vin [5]
recognized fuat than structurally distinct interferon could made
fue same animal species when they demonstrated fuat interferon induced
in sensitized mice with tuberculill was not neutralized antiserum against
virus-induced mouse L-cell
rece11tly, it was found that human prodl1ced 1el1ko-
cytes differed in several respects from human interferon produced
blast cells. Antiserum against fibroblast interferon neutralized homo10gous
interferon but not leukocyte interferon [6,7]. Antiserum to leukocyte inter-
feron showed fue greatest neutralizillg activity homologous interferon,
but some neutralizillg activity toward fibroblast was also evident
[6,8]. was ShOWll to due to minor component in the leukocyte
interferon which had fue antigenic specificity of fibroblast interferon and
thus acted as separate immunogen [8-10] .
to antigenically distinct human interferons (leukocyte and
fibroblast), differences in molecular weight and/or charge within
each type. Leukocyte interferon is heterogeneous wifu respect to bofu
molecular weight [10-13] and charge [14-16]. Fibroblast illterferon thus
far has appeared to homogenous in molecular weight [17] but hetero-
geneous in charge [18, 19] .
Because of this known heterogelle ity of interferon preparations, any
consideratioll of fue stabllity of should keep ill that each
the molecular species that comprise interferoll preparatioll
exhiblt unique stabllity. what would to partial
to certaill treatment fact complete stabllity of component(s)
and total 1ack of stabllity allofuer compollent(s) fuat preparation.
'I'11U'"lllal SlalJilily
I';ael.y eUpOrlH c()Ilccrning stability of human 1eukocyte and fibroblast
i Illu 1" [C.I"OIlS that both were somewhat [20]. recent
(laLa, 11Owever, indicate that interferon produced 1eukocytes is generally
IllOru stablc than interferon produced fibroblast cells 1 [17,20-26]).
Ceude interferon preparations heated at resulted in 50% 10ss of 1euko-
uytc interferon activity after 10 min, while fibroblast interferon 10st 50%
its activity after on1y 2-3 min [21]. De Somer et [23] found that
crude fibroblast interferon exposed to 56 10st 90% its activity after
:30 min, although it was stable at Havell and Vilcek [24]
found 50% 10ss of crude fibroblast interferon activity after 30 min at
ln contrast, Mogensen and Cantell [25] found crude 1eukocyte interferon
required exposure to 56 for 12 hr before 90% reduction in activity was
noted.
Mogensen and Cantell a1so presented evidence that partially purified
1eukocyte interferon was stable to heating than the crude preparation
[25]. For the purpose of this review, interferon preparations with
specific activity equa1 to 01' greate1' than 106 U/mg protein considered
pU1'ified. In partially purified state, 1eukocyte inte1'fe1'on was
comp1ete1y stable to heating at 70 for 100 min, whe1'eas c1'ude 1eukocyte
inte1'fe1'on lost than 99% of its activity under the same conditions.
Evidence suggested that agg1'egation of interfe1'on with other proteins was
responsible fo1' the appa1'ent 10ss of activity, since centrifugation of heated,
crude inte1'feron at 80,000 g fo1' 1 hr 1'esu1ted in pellet (inte1'fe1'On does
not normally sediment upon high-speed centrifugation) which, when t1'eated
with 4 guanidine hyd1'och101'ide, 1'esu1ted in full recove1'Y of interfe1'on
activity. This agg1'egation phenomenon of c1'ude 1eukocyte interfe1'on
responsible fo1' the limited heat stability of the 1eukocyte interfe1'on
p1'eparations 1'epo1'ted in othe1' studies [21].
It was 1'ecently shown that the concentration of inte1'fe1'on p1'eparations
a1so influence thei1' stability towa1'd heat. [17] indicated that
c1'ude fibroblast inte1'fe1'on was stable fo1' 1 to 2 months at 4 while par-
pU1'ified inte1'fe1'on in dilute s01utions 10 10st 50-75% of its
activity in 24 h1' at This observation was confirmed and extended
Sedmak and G1'ossberg [26]. purified fibroblast inte1'fe1'on, in
concent1'ations of 500, 50, and 5 protein/m1, was incubated at 37 for
24 h1' and the amount of activity 10st was 57%, 90%, and >97%, respective1y.
The effect of concentration heat stability was a1so shown to to
tially purified 1eukocyte inte1'fe1'on [26]. After heating at 68 fo1' 30 min,
interfel'on in concentrations of 500, 50, and 5 f.!g p1'otein/m110st 24%, 42%,
and 83% of thei1' activ ity , 1'espective1y.
1. Therma1 Stability of Human Interferon8
Condition8 and re8u1t8
Interferoll concentration
a
1-9 105 U/mg protein
2-8 102 U/mg protein
1-2 103 U/m1 (crude)
1-2 103 U/m1 (crude)
Not 8tated (crude)
Not 8tated (crude)
1.8 104 U/mg protein
1 106 U/mg protein
Leukocyte interferon

200 min: 50% 1088

5-7 min: 50% 1088

10min: 50%1088

6 week8: 90% 1088

3 week8: 90% 1088

560 12 hr: 90% 1088

3 daY8: 1088
Fibrobla8t interferon

200 50% 1088

5-7 50% 1088

560 2-3 50% 1088

30 min: 50% 1088

month8: 1088

month8:

8 daY8: 30% 1088

200 8 daY8: 30% 1088

1-1.5 daY8: 50% 1088

30min: 90%1088
Ref.
20
21
Z
24
--
23
::::
:::
::;
z
25

-
;:::.
-
Not 8tated (partially purified)
Not 8tated (crude)
4-20 103 U/m1 (crude)
1 107 U/mg protein
2 106 U/mg protein
Not 8tated (partially purified)
450 1 hr: 1088
560 1 hr: 1088
680 30 min: 24% 1088
(500
680 30 min: 42% 1088
(50
680 30 min: 83% 1088
(5
450 1 hr: 1088

1hr: 70%1088
40 1-2 month8: 1088
40 24 hr: 50-75% 1088
10

24hr: 57%1088
(500

24 hr: 90% 1088

(50

24 hr: >97% 1088

(5
22
17
26
aThe concentration8 of interferon preparation8 were expre88ed unit8 (U)/mg protein (8pecific activity)
U/m1. Interferon preparation8 were cOn8idered partially purified if the 8pecific activity wa8 equal to or greater
than 106 U/mg protein.
bThi8 preparation wa8 te8ted for thermal 8tability in the freeze-dl'ied 8tate. othel' preparation8 were te8ted
in the liquid 8tate.

'"

;::.
:r:
z
-:

z
...::
>
.-
$
;:::
7.
z
-:
:r:
(
")

13. t>l.alJilil.y
Human interferon has also S!lOWIJ to subjccl. 1.0 illactivatiol1 lJY
mechanical stress, partieular problem durillg proeeSsill!4' and purifieatioll
of fibroblast interferoll. alld Vilcek [24] that vigorous meehall-
ieal stirring of erude fibroblast interferon resulted in an 80% 10ss of aetivity
after 5 min. DeSomer et [23] found the aetivity of crude fibroblast
interferon was redueed 99% after rotation in tubes at 25 revolutions per
minute (rpm) for 24 hr at

Edy et [27] eompared the suseeptibility

of fibroblast and leukoeyte interferon to mechanieal stress. Interferon
preparations were rotated end-over-end at 50 rpm in glass-stoppered glass
tubes at temperature of 40 greater t!lan 90% 10ss of aetivity was noted
with fibroblast interferon, while leukoeyte interferon was eompletely stable
to the same treatment.
Cartwright et [28] observed meehanieal inaetivation of partially
purified fibroblast interferon in rotational viseometer. Depending upon
the exposed and the shearing rate, fibroblast interferon lost from
70 to > 99% of the original aetivity. Loss of aetivity was not due to absorption
to surfaees, sinee inaetivation oeeurred wheIJ silieonized glass and various
plastie or stainless steel tubes were used. Denaturation at gas/liquid
interfaee and oxidation were also eliminated as probable eauses for inaetiva-
tion, sinee aetivity was lost in eompletely filled and sealed vessels as well
as when inert gases were used in the eontainer. Inaetivation appeared to
solely eaused shearing forees. Changes in either of the two variables
governing the of shear foree in the viseometer (rotational speed
and the width of the gap) had the predieted effeet inaetivation of
fibroblast interferon. Crude human leukoeyte interferon was eompletely
stable the same eonditions.
Stabllization to Thermal and Meehanieal Stress
The demonstration of the of human interferons to heat and
meehanieal stress stimulated t!le seareh for agents or eonditions whieh
would proteet interferon from inaetivation. Marshall et [20] were the
first to demonstrate that heating of interferon at low (3.5) tended to
preserve biologieal aetivity when eompared to heating at neutral Based
their studies of the stabilizing effeet of low mouse interferon [29,
30], Sedmak et [31] evaluated the stabilizing effeet of 10w (2.
partially purified human fibroblast interferon. interferon preparation
added eytochrome to prevent absorption to surfaees [18]. At
- 200 differenee was noted between the preparations at
2. and 7. However, at 40 the 2. preparation lost only 5 % of
its aetivity after 46 weeks of storage, whereas the 7. sample lost 93%
of its poteney. At 200 the 2. sample was again the most stable.
01' Llre arrLivieal a<.:Livily was 10st at as opposed
LI) aeliviLy a[Lcr 1 week the sample held at 7.0. This
of 10w was evident at va1ues of 2 and 3 but was 10st
plJ was increased to 4 [31]. noted earlier, fibroblast interferon
ill rliluLc solution was less stable to heat than interferon in more concentrated
Holutiol1s; this was a1so ShOWl1 to the case at 2. [26,31].
detergent sodium dodecy1 su1fate (SDS) has also ShOWl1 to
human 1eukocyte interferon agail1st heat inactivation [32]. Stewart
cL a1. [33] reported that crude human 1eukocyte and fibroblast il1terferol1s
rliffer il1 their stabllity to heat (1000 for 2.5 in the presel1ce of SDS
ul1der reducil1g or nOl1reducing conditiOI1S. Leukocyte il1terferol1 was stabi-
lized SDS a1one, whereas fibroblast interferon was on1y partial1y pro-
tccted from il1activatiol1. Leukocyte il1terferon was protected 1ess efficiel1tly
in SDS under reducing conditions, while fibroblast interferon was more stable
il1 SDS with mercaptoethano1 and urea added. These results were confirmed
Vilcek et a1. [10].
Two mo1ecu1ar weight subspecies of 1eukocyte interferon
described. Stewart and Desmyter [11] reported that these two subspecies
were a1so distinguishable the basis of their to heat in SDS a10ne
or SDS under reducing conditions. Leukocyte interferon boiled in SDS and
electrophoresed in SDS-po1yacrylamide gels revealed 2 peaks of activity:
major peak at 15, 000 daltons, and minor peak at 21, 000 daltons. When
1eukocyte interferon was boiled in SDS under reducing conditions and sub-
jected to electrophoresis, only the minor peak (21,000 daltons) of activity
was obtained.
As noted, fibroblast interferon was not stabllized against
SDS alone. However, at

SDS a10ne did purified

human fibroblast [17] .
Experience with mouse suggested that chaotropic salts, i. ,
salts whose anions increase the solubllity of the nonpolar regions of proteins,
would protect interferon against therma1 inactivation [30]. This was found
to the case with partially purified human fibroblast in high
temperature

test [31]. However, at the temperatures most like1y

to used for storage of interferon for clinica1 (40 200 chaotropic
sa1ts did not fibroblast interferon.
Freeze-dried preparations of crude fibroblast interferon retained 50%
of their origina1 activity after 24 of heating at 900 [31]. Thus they
more stable to heating than liquid preparations. No difference
was noted between 2. and 7. freeze-dried preparations.
therma1 stabllity of crude freeze-dried fibroblast interferon was
not enhanced addition of extraneous proteins. However, in the case of
immunoaffinity purified interferon (which contained 25 cytochrome
therma1 was increased added protein. 1n nonisotherma1
test [34] in which the temperature was increased from 50 to 900 freeze-
dried purified interferon (with 25 cytochrome was near1y comp1ete1y
1;\
inacLivaLcd Limc LCl11pCl'aLLII'() l'caC\ICI\ ,'j()"
protein in the form 01 bovillC albumin, cyLocl1l'0I1lc I4claLill, Ol' ovalfJUmin
to freeze-dried purified intcrfcroll csscntially complctc stability
to

[31J.
Another that distinguishes between human leukocyte
alld fibroblast interferoll is hydrochloride ill cOllcelltratiolls of
4-8 Humall leukocyte interferoll is stable to this reagellt, vvhile the
majority of fibroblast illterferon is illactivated [27]. The same millor
portion of fibroblast illterferoll that was stable to guallidille hydrochloride
was also stable to mechallical stress [27]. Edy et al. [35] recelltly pro-
vided evidence for stable alld ullstable forms of fibroblast The
stable fraetioll of fibroblast illterferon differs from the bulk of fibroblast
illterferoll ill the extellt Ilature of glycosylatioll.
Proteetion of fibroblast illterferoll from mechallical-stress-indueed
illaetivatioll was aceomplished usillg reagellts that inhibit disulfide bond
formation. Cartwright et al. [36] demollstrated that DL-thioetie ill
eOllcelltratiolls of 0.1-1. eompletely crude alld partially
purified fibroblast illterferon to meehallical inaetivatioll. Thioetie
however, divergellt effeets t11ermal stability. At 56 alld

it
aceelerated inaetivatioll, but at 40 it biological aetivity [36] .
It was shown that thioetic aeid easily removed from the preparation
dialysis, whereas other stabilizers, such as SDS, call1lot removed.
The Ilollionic detergellt Tween 80, in eOlleentratiolls of 0.1-1.0%, also
protected fibroblast illterferoll from meehanical illaetivatioll [23]. Sedmak
et al. [31] confirmed that observatioll with Tween 80 eOlleelltratiolls as 10w
as 0.001%. They also demollstrated with 10 thioetic
alld 0.01-0.1% SDS. However, they were UIlable to demollstrate the protec-
tive aetion of low 011 meehanieal illaetivatioll [31] .
It is apparent that rapid inactivatioll of humall fibroblast illterferoll
results from thermal and meehallieal stresses. Leukocyte interferoll is
much stable to these forces. Reagellts that proteet illterferoll from
thermal inaetivation do not Ileeessarily provide proteetioll agaillst mechall-
ical shear forees, which suggests unique mechanisms responsible
the two types of inactivatioll. Thermal illactivatioll due to changes
in the eovalellt structure of the proteills [37]. Instead of illter-
feroll ill its native configuration, thermal eOllditiolls generate
ullfolded moleeules whieh, upon eooling and Ileutralizatioll removal of
the reagent, refold illto the biologieally active form [30]. For
ple, Stewart et al. [33] demollstrated that leukoeyte illterferon whose
logical activity had beell destroyed heat could completely reactivated
brief reheatillg in the presellee of SDS. Inactivated fibroblast illterferon
required reheatillg ill the presenee of SDS under reducillg eonditiolls [33].
Illaetivatioll meehallical stress appears to due to shearillg forees
that promote formatioll of molecular aggregates through the gelleratioll of
intermoleeular disulphide bOllds [28,36]. Thioctic effeetively prevented

.,.
01' c;IICII I)(HIlIc;. '1'11 ic; II'yjJoUlUsis ic; compatiblc with the protective
'l'WCCII allll acill 1>11. TWCCIl i>U, its dctergent action, and
Itc i( 1 pll, !J.Y pL'oLlJllaLiOIl, prcvolli formatioll of illtermolecular disulphide
!1O!\CIS 1<'.;;, <'.7].
1) s ill I30dy Fluids
'I'IIU illhibitioll illterferoll actioll 1l0lltoxic biological fluid was first
Vilcek alld Lowy [38], who fOUlld that 40% fetal calf serum
s illhitited the activity of chick embryo interferon.
illllibitioll was greatest if the fetal calf serum was added to along with
illtcrferon rather thall immediately before or after interferoll treatmellt.
Itossmall alld Vilcek [39] fOUlld that chick illterferOll was also
il11libited normal sera from variety of other allimal species, including
Ilumall serum. The amoullt of suppressioll of interferoll action increased
with illcreasillg COllcelltrations of serum and decreased with increasing
eentrations of interferon. Prelimillary characterization of the inhibitioll
Hug'gested it associated with lipoprotein.
Cesario alld Tilles [40] found that humall fibroblast interferon iIlcubated
with human urine expressed less antiviral activity than iIlterferon exposed
io control medium. The 10ss of interferon activity began immediately
cxposure to urine and was completed wit1lin 30 Dialyzed urine did not
illactivate interferoll, alld, of the dialyzable components of urille tested,
ollly phenol reduced interferon titers. Cesario et [41] subsequently
demonstrated that fibroblast interferon was inactivated several human
body fluids, includiIlg serum, cerebrospiIlal fluid, stool extract, and
saliva. Pleural fluid did not appear to inactivate fibroblast interferon.
Work in our laboratory indicated that nasal secretions contained
illhibitor of fibroblast iIlterferon [42J. Increasing quantities of nasal
secretions resu1ted in increased inhibition of fibroblast interferon but showed
illhibitory activity toward leukocyte interferon (Figure 1). The illhibitory
activity toward fibroblast iIlterferon could overcome with increasing
concentrations of fibroblast interferon. of the major proteins intrinsic
to nasal secretions (i. albumin, lysozyme, and IgA) did not appear to
responsible for the inhibition. Preliminary characterization of the iIlhibitor
suggested it could lipoprotein.
Cesario [43] has recently tested several human body fluids against
both fibroblast and leukocyte interferons. Leukocyte interferon was quite
resistant to cerebrospinal fluid serum (10-17% inactivation), while
fibroblast interferon 10st 74% 97% of its activity, respectively. In
response to and urine, leukocyte interferon 10st 56% and 57% of its
activity, while fibroblast interferon 10st nearly 97% 93% of its activity,
respectively. Saliva reduced the leukocyte interferon titer 45 %, while
fibroblast interferon lost 67% of its initial titer.
(i(i
NS +
109 .-.. _ .. _ .. _ .. ..- .. _ .. _ .. _ .. .-.-.. _ .. _ .. _ .. --

108
FI BROBLAST IF ... NS
::J
u..


--'
LU
>-
LEUKOCYTE I F + NS
>
107
v>
>
LEUKOCYTE IF ...


0---

106
_---6---_
&-_ -----t:r-- FIBROBLAST IF + --6
0.5: 1 1: 1 2: 1 4: 1
VOLUME NASAL SECRETION OR VOLUME INTERFERON 150 UNITS)
FIGURE 1. Effect of increasing vo1umes of nasa1 secretions (NS) or mini-
essentia1 the antivira1 activity of fibroblast
and leukocyte interferon (IF). (Reprinted from Ref. 42, courtesy of the
Society for Experimenta1 Bio1ogy and Medicine.)
Conc1usions
From the data reviewed, it is apparent that the two major conditions respon-
sible for 10ss of interferon activity are therma1 and mechanica1 stress. It
is a1so evident that of factors influence the stability of interferon
to these stresses; these inc1ude the type of interferon (leukocyte or fibro-
blast), the concentration, and the re1ative purity of the preparation. Fibro-
blast interferon is quite susceptible to inactivation heat and mechanica1
(;'/
Wllilt) lt)llkocyLt) ilIU'I,'lcl'OII is sLaJJlc Lo HtreHSeS, even when
p:tl'(.i:tll.v jJlll'i
that stabilize interferon to therma1
Hircss. Maintenance of 10w and incorporation of
LII iocLic acid SllOUld particu1ar1y usefu1 in the purification and concentra-
(,iOlI interferon. Storage of interferon in dilute solution, even
1'01' HllOrt periods of time, shou1d avoided.
The human interferon preparations tested for stability were not purified
Lo 110 mogene ity . It is evident that when comp1ete1y purified interferon
pl'cparations available, studies of stability to therma1 and mechan-
ical stress, as well as responses to stabilizing conditions, will have to
I'cpeated.
Loss of interferon activity during storage 1argely avoided
lIHing freeze-dried preparations. Purified interferon preparations
L'cquire the addition of stabilizing protein. Accelerated storage tests
IH!.ve established that freeze-dried preparations have suitably shelf
I,ives for the long-term storage of interferon for clinica1 use [31].
With respect to stability in body fluids, there appear to major
drawbacks to the use of leukocyte interferon. However, clinica1 use of
fibroblast interferon limited if activity must maintained in cere-
brospina1 fluid, serum, urine, or nasa1 passages.
III.
Data the pharmacokinetics of interferon induced in anima1s both vira1
and nonvira1 inducers have been reported previous1y [44]. These ear1y
observations were made assaying for interferon in various body fluid
compartments. Over the past 10-15 years, exogenous interferon prepara-
tions have been given to anima1s, and antivira1 activity has been measured
under variety of conditions. review will summarize those studies
in anima1s and humans that emp10yed on1y exogenous interferon and provided
information the pharmacokinetics of this antivira1 agent (see Tables 2
and 3, respective1y).
In Anima1s
Initia1 experiments the pharmacokinetics of interferon in anima1s
p10yed interferon prepared in mouse brain tissue 2). The rapid 10ss
of intravenous1y administered interferon in the mouse was demonstrated in
four different reports [45-48]. These studies suggested that interferon
was distributed to some but not tissues of the body and that repeated
did not 1ead to accumu1ation of interferon. However, Nuwer et
[49], using mouse L-cell interferon injected intravenous1y every hour
:
TABLE 2. Pharmacokinetics of Exogenous Interferon (IF) Preparations in Anima1s
Interferon Anima1 Dose and Serum 1 1
preparation mode1 route of administration (half-life) Comments Ref.
Mouse brain Mouse NS
a
1. . <30 U/3 1 at 1 hr
c
30-f01d 1 per hour; 45
c1earance equiva1ent to
96.6% per hour
Mouse brain Mouse 18,000 U

1 . . U at 15 min Repeat i. . dose 2 hr 1ater 46
78 U at 1 hr showed simi1ar pattern of
11,400 U i. . >100 U at 1 hr 1
U at 2 hr
Mouse brain Mouse 13,000 U i. . 3,840 U at 1 min After i. . injection, IF 47
80 U at 1 hr measured in peritonea1 z
< 10 U at 3 hr fluid; after intraperitonea1

..
l
< 13 U at 1 min injection, serum 1 1 of ;::::
20 U at 3 hr IF detected, well in
extracts of 1 , kidney,
>
and1ung
-
~
Mouse brain Mouse 340 U i. . 28 U at 5 min Approximate1y 40% re- 45
z
150 U i. . 20 U at 5 min covered in kidney, liver,
1ung; IF recovered in
brain, 1 , or musc1e
-
Mouse L l l Mouse 750-4,500 U i. . (1 min) C1earances s10\ver after 49
-
(q.1hrX5) second to seventh injections
Mouse l l Mouse 3,850 U . . 4U Similar results \\-ith
5(!
(q. 1hr 7) 5- and 8-day-old mice, but
1,400 U . . 2U not with adu1t mice; rabbit

IF, given orally, also ga\-e

low serum levels
--
~
Chick embryo Chicken 5,000 U i. v. 217 U/4 ml at 40 sec No evidence of accumula- S7
;:;
J:
(q. 1 hr 5) 70 U/4 ml at 10 min tion with repeated doses
U at 1 hr
z
--
Rabbit Rabbit 100,000 U i. v. (11 min) 7-13% recoverable at 1 min 51
(NDV induced)b after inoculation
Rabbit Rabbit 119,500 U i. v. 356 U at 1 min 6.9-12.5% inplasma, 87-93%
;:::
52
z
(NDV induced) 163,840 U i. v. 256 U at 1 min in tissues, distribution
119,500 U i. v. 256 U at 1 min throughout entire extracellular
...::
space
>
....
Rabbit Rabbit 1.5 106 U i. v. 7, 945 U at 1 min 56
~
::::
1.5 105 U i. v. 1,000 U at 1 min
-
1.5 104 U i. v. 82 U at 1 min
7-
Rabbit Rabbit 4.3 103 U
.
1. . ND
d
z
150-250 U at 30 min after 61
1.8 103 U IVent
b
ND injection in CSFe, half-life
-:
NS
a
i. v. (11 min) in CSF is approximately
J:
1 hr; tailing effect with re-
s pect to concentration and
time
Human Mouse 17,500 U i.m. 600 U at 1 hr Mean level of IFU/ml of 57
leukocyte serum 348
-
-,
Table 2 (continued)
Interferon Animal Dose and Serum level
preparation model route of administration (half-life) Comments Ref.
Human Guinea 175,000 U i.m. 200 U at 1 hr Mean level of IFU/ml of 57
leukocyte pig serum 221
Human Sheep 30 106 U i.m. 600 U at 1 hr Mean level of IFU/ml of 57
leukocyte serum 168
Human Rabbit 1.75 106 U i.m. 150 U at 1 hr Mean level of IFU/ml of 57
leukocyte serum 148
Human leuko- Rabblt 30 106 U i.m. 800 U at 1 hr "Purified" IF preparation;
- ,
..
cyte (crude) 3 106 U i.m. 200 U at 1 hr faster than
z
leuko- 30 106 U i.m. 2,000 U at 1 hr "crude" IF
cyte (purified) 3 106 U i.m. 600 U at 1 hr
;::::
leuko- Rabbit 2 106 U
i. v. 8, 900 U at 1 min Early affected 56
cyte 2 105 U
i. v. 1,100 U at 1 dose; in
>
2 104 U
i. v. 86 U at 1 results using "crude" or
$:
"purified" IF; tailing :z
.
effect more pronounced \vith
increasing dose; after re-
peated (1 q. 24 5)
of 3 106 U, change in

peak or trough
Human Rabbit 3 106 U i. . (13 min) IF leyels not directly pro- "J"-,
leukocyte 0.3-30 106 U i.m. 200 U at 1 hr portional to dose giyen; i. .
3 106 U s . .

200 U at 3 hr IF measured in serum longer
;,-:
2. 5 and 6 106 U . . Not detectable than i. . IF; repeat injec-

tions demonstrated
--
~
clearance rate
5 105 U
3:
Human Rat i. . 150 U at 6 hr Significant levels of IF in .56
leukocyte 5 105 U 600 U at 1 & 6 hr lymph after i. . or i. .
z
i.m.
injection
~
Human Rabbit
106 U
i. . 200 U at 1 hr 30 min after i.v. dose, <30 ; ) ~
fibroblast i.m. 64 U at 1 hr U/g of IF detected in liver -
Human 106 U
i. . 1,000 U at 1 hr when serum was 800 U/ml
z
leukocyte i.m. 64 U at 1 yr
-=
-
Human Monkey 30 106 U i. . 60 U at 24 hr 30-fold difference between 59
>
leukocyte (7.1 hr)f IF concentration in serum
3:;
30 106 U i.m. 600 U at 24 hr and CSF; 20 U/ml in CSF at
>
~
10 106 U i. . 600 U at 2-12 hr 24 hr after i.v. or i.m.
~
7
dose; 20,000 U/ml in CSF
z
at 24 hr after i. . dose
Human Gibbon 3 106 U i. . 4 U at 24 hr Almost complete recovery 60
:;
leukocyte (17 min) of drug at 3 min; tailing effect
3 106 U i.m. 40 U at 24 hr noted after 3 hr; i.m. re-
3 106 U . . 20 U at 48 hr sponse similar to . .
-.:)
2 (continued)
Interferon Animal Dose and Serum level
preparation model route of administration (half-life) Comments Ref.
Human Rhesus 5 105 U i.m. 200 U at 2 hr 88
leukocyte monkey
Human Chimpan- 4,000 U
.
1. n. ND 4 and 40 U recovered after 63
leukocyte zee 5 min; 5- to 50-fold reduc-
tion in recovered IF over
1 hr
aNS = not stated.
bi.v. = intravenous; i.m. = intramuscular; s.c. = subcutaneous; . . = orally; i.p. = intraperitoneal; i.c. = intra-
cisternally; IVent = intraventricularly; i. n. = intranasally; NDV = Nevvcastle disease virus.
Cu = units of IF in reference standard units for species of interferon used, unless othervvise stated.
dND = not done.
eCSF = cerebrospinal fluid.
fMethod of interferon assay and use of 69/19 reference standard are not stated.
-.::
tc
z
;::
>
~
:::;
z
~
:1, I-:X()(:I-:N(IIJS IN'I'I':H,I'I-:II,(IN:
1'01' J'iv(, illjecLiollC:, t,l,e paLLel'll Lo slower after repeated
IlIj('ct,iOllC: ,
Tlle fil:c:L orally administeeed interferon suggested that
l'ouLc adrninistration would not feasible. Adult mice were given
1l1Ouc:e L mouse serum interferon, or rabblt interferon orally [50].
Lhe recipient animals had detectable serum interferon at 1 hr after
last Ieeding. However, when 5- or 8-day-old mice were inoculated
with interferon every hour for seven doses, low levels of both mouse
atld rabblt interferon were detected in serum. This finding is consistent
Witll the apparent of young animals to absorb proteins from the intes-
Linal tract.
The distribution of rabblt interferon injected into rabblts extended the
il1itial observations made with mice. 1n the earliest reports, the ha1f-life
intravenously administered rabblt interferon was calculated to 11 min.
These single injection studies showed that there was rapid disappearance
detectable interferon and that the interferon was distributed in plasma
and tissues, as well as throughout the entire extracellular space [51,52].
Human leukocyte interferon prepared from the buffy coats of blood
donors has been injected into various animals and the
serum levels assayed with time [53-58]. Cantell and [55] demon-
strated similar distribution pattern of human leukocyte interferon in mice,
guinea pigs, rabblts, and sheep. Peak serum levels in animals were
found some 1-3 hours after injection of the interferon preparations, and
low levels were found between 12 and 24 hours later.
It was also shown that the rapid clearance of the human leukocyte inter-
feron was not affected the dose injected and followed pattern similar
to that of rabblt interferon [56]. serum ha1f-life of 13 min was found,
and repeated injections (1 every 24 hr 5) did not alter the peak or trough
levels of interferon. When the interferon preparations were administered
intravenously, Cantell and [56] observed delayed final disappear-
ance of interferon, or tailing effect, which was more pronounced with
increas ing interferon doses.
1n dose response experiment, Cantell and [55] injected
0.3 to 30. 106 units of human leukocyte interferon intramuscularly into
rabblts and tested their serum for interferon at various times after inocula-
tion. Although the serum levels were not directly proportional to the dose
employed, increasing peak levels of interferon were found with the higher
administered dose. 1n the same series of studies, comparison was made
between the intramuscular and subcutaneous routes of administration. Both
intramuscularly and subcutaneously injected interferon gave similar curves,
although the peak level of interferon later with the subcutaneous route.
1n addition, interferon could detected in serum for longer time when
given either intramuscularly or subcutaneously than when given intraven-
ously.
'1-1
J;;dy et al. l t-;avc illLcl'[Ct'OII Lo
rabbits both intravenously alld il11 ilar'
was for each ihey
detect the liver, which was removed the serum level
was U/ml.
Studies primates leukocyte have
results. Habif et al. [59] half-life of 7.1 hr
while Skreko et al. [60] half-life of 17 The
for these results is completely clear, although 10
times more interferon was into the the gibbons.
Nevertheless, after of leukocyte
the peak levels 10ss were similar both and

Two studies have attempted to look at the of interferon
across the barrier. the first study et al. [61], inter-
feron was or into the
system of rabbits. In both cases low levels of were the
fluid for several hours after The half-life in the
fluid appeared to approximately 1 hr, but there was
similar delayed as described earlier for serum levels with
respect to both time. s imilar study
was the serum from 2 to
12 hr after injection, although 30-fold was found between the
the serum the fluid [59] .
From these results, Habif et al. [59] suggest that barrier
exists for
There have several studies in which has applied
topically to mucous [62, 63]. In these studies,
of the rate of or the detection of in serum have
limited. et al. [63] measured the recovery of leukocyte
after topical application to the nasal mucosa of
10 there appeared to 5- to 50-fold reduction in the re-
covered over 1 hr. These results suggested that
applied locally to the nasal mucosa is rapidly removed that the
of locally applied might alter the dose of
for antiviral activity in the cavity.
Humans
Most of the human studies with administered interferon have
measured serum levels after single dose and have employed patients with
some disease, such as Hodgkin' s disease, lymphoma, or leuke-
mia 3). most studies, information in animals was used
as guide in dosage and routes of administration.
:1.
7
"
.)
Val"yillj!; SUI"LII1I levcls 01' luukouyiu have been found
Itl1ol" :HllI1illisll"aliol1. el [64] found peak serum
U OIlly 1 min aftcr administration of4.5 105 U,
HlLJ!;j!;uslillJ!; 8horl half-lifc. !<;modi et [65] a1so found evidence of
after intravenous administration. These investigators a1so
(1t\1l101l8trated peak of 800 U about 8 hr after 30 106 U were given
illlravunou81y over an 8-hr period. When patients with herpes zoster and
Ilodj!;kill' s disease were given human 1eukocyte interferon intravenous1y
ovcr 12-hr period, an initia1 ha1f-life of 2.8 hr was observed [66]. Jordan
el [66] suggested that their 10nger half-life was the result of tissue bind-
re1ease of interferon and wou1d not have been apparent in the sing1e-
(lo8e experiments.
1ntramuscu1ar administration of interferon in humans resu1ted in
pattern of distribution and c1earance similar to that found in the anima1
8tudies. peak 5-8 hr after injection with detectable 1eve1s present
at 24 hr has been demonstrated. Cantell et [57] predicted from their
studies that 150,000-200,000 U/kg would needed to maintain
at 100 U/m1. These predictions have been confirmed recent studies
in herpes zoster patients given varying doses of human 1eukocyte interferon
intramuscu1ar1y [67]. peak serum interferoll of 238 U/m1
was found in patients who had received 1.7 105 U/kg intramuscu1ar1y.
No accumu1ation of interferon was found with repeated doses except when
the highest dosage schedu1e (5.1 105 U/kg/day) was used. With this
dosage, the serum interferon feH only 5% during the third to sixth
day of treatment when 50% lower dose of interferon was being adminis-
tered.
Three patients with advanced breast cancer who received daily injections
of human 1eukocyte interferon (4.0-6. 104 U/kg per day) intramuscu1ar1y
were studied for serum interferon 1eve1s W. Harmon, S. Greenberg,
and J. Gutterman, unpublished observations). Serum interferon 1eve1s were
measured before and 3, 6, 12, and 24 hr after injection (Figure 2). After
the first injection, mean peak 1evel of 55 U/m1 was found 3 hr after inocu-
1ation. mean peak increased to 330 U/m1 after the seventh injec-
tion, and there was evidence of serum accumu1ation over the next 21
daily intramuscu1ar injections. These data are consistent with those obtained
other investigators and indicate that significant accumu1ation of inter-
feron occurs in patients with intact hepatic and rena1 function.
Other preparations of interferon have only recently been shrdied in
human subjects, and resu1ts suggest that there are differences in
the pharmacokinetics of human 1eukocyte and human fibroblast interferon.
1n recent study Edy et [68], interferon was found in the serum
of individua1s who were given 3 106 U of human fibroblast interferon intra-
muscu1ar1y. 1n addition, only one of five patients who were given 1.8 107
U of human fibroblast interferon had low but detectable 1eve1s of interferon
in the serum 1 and 3 hours after injection. The reasons for the apparent
TABLE 3. Pharmacokinetics of Exogenous Interferon Preparations in Humans
-:
Interferon Underlying Dose and Serum level
preparation disease route of administration (half-life) Comments Ref.
Human Malignancy 4.5 105 U
.
1. v. 200 Ub at 1 min Results from one patient 64
leukocyte 65 U at 15 min
Human Virus 30 106 U i. v. (15 min) Tailing effect between 1 and 4 hr; 65
leukocyte infections (over 5 min) i. v. dose disappeared 36 hr;
and 30 106 U i. v. 800 U at 8 hr s.c. and i.m. gave similar curves;
tumor (over 5 hr) IF detected in CSFd when serum
1 106 U
.
1.m. 100 U at 2 hr level 150 U/ml
1 106 U s.c.
a
100 U at 4 hr
Human Hodgkin's 5 106 U i.m. 50 U at 5 hr Calculated that between 150,000- ;);)
leukocyte disease; 5 106 U i.m. 42 U at 5 hr 200,000 U/kg would needed to
osteogenic 2.5 106 U i.m. 60 U at 5 hr maintain serum level at 100 U/ml
sarcoma
z
Human Malignancy 80 106 U i. v. 300 U at 12 hr Initial half-time was 2.8 hr after 66 ;:::
leukocyte with (over 12 hr) i. v. and 4.8 hr after i.m.; urine
herpes 80 106 U i.m. 200 U at 8-12 hr level of 24-50 U/ml in 1 pt; CSF
>
zoster 6.4 106 U i.m. 32 U at 10 hr level in the patient with 278 U/ml
6.4 106 U i.m. 17 U at 14 hr in serum
::;:
4.2 104
:z
Human Malignancy i.m. 52 . 61 U at 4 hr No accumulation except at highest 6-:- -
leukocyte with U/kg/day dosage schedules
herpes 1.7 105 i.m. 238 . 21 U at 4 hr
zoster U/kg/day
;::;
5.1 105 i.m. 455 . 48 U at 4 hr
U/kg/day
Human Congenital 1.7-3.5 105 i.m. 145 U and 460 U 40 and 60 U in urine of t,,o patients
leukocyte cyto- U/kg/day
megalovirus
Human NS
f
3 106 U i.m. 10 U at 1, 3, Only one of patients given highest
fibroblast or 6 hr dosage of fibroblast IF had detectable
1.8X10
7
U i.m. 8 U at 1 hr serum levels
20 U at 3 hr
Human Normal 35,000 U/dose
.
l.n. NDc 300 U in nasal wash 2 to 15 hr
leukocyte volunteers after IF administration
Human Normal 6 105 to i.n. ND N nasal secretion IF detected
fibroblast volunteers 48 106 U 2-14 hr after intranasal application
Human Normal 4000 U i.n. ND 200 U at 30 and 60 min after
leukocyte volunteers inoculation
Human Disseminated 6 105 U i. t. 100 U at 1 hr Patient also received IUDR
e
treat-
leukocyte neonatal (q. 12 hr 6 day) after 7th dose ment at autopsy, herpes virus re-
herpes covered from brain, although CSF
titers varied from 800-8,000 U/ml;
1000 U/ml at 12 hr in CSF after
first dose; 10,000 U/ml at 12 hr in
CSF after second dose
a i . . = intravenous; i.m. = intramuscular; s.c. = subcutaneous; i. t. = intrathecally; i.n. = intranasally.
bU = units of IF in reference standard units for the species of interferon used, unless otherwise stated.
cND = not done.
dCSF = cerebrospinal fluid.
eIUDR = idoxuridine.
fNS = not stated.
S('
>-:

6S
..
*
J:
:z:
62
69 ,...
:z
..
63
-"
>
71 $
:=:
-
7-
z
-:
'.i.
-1
-1
)N, :LIHI (:( )IJCII
1000

;:
100
Q.>
(/")


'"

=>
z:

""
UJ
u..
"" UJ
1-
z:
10
12
24
HOURS
FIGURE 2. Serum interferon levels (U/ml) 3, 6, 12, and 24 hr after
intramuscular injection of 3 106 U daily. Levels were measured before
and after the first dose ( .... ), the seventh dose ( 0-0 ), and the fourteenth
dose ( ...... ), the twenty-first dose ), and the twenty-eighth dose
(f:::r-1:,. ). W. Harmon, S. Greenberg, and J. Gutterman, unpublished
observations. )
differences in preparations are not fully known, but inhibitor of human
fibroblast interferon has demonstrated to active in serum [41,43].
Little information is presently available the pharmacokinetics of
topically applied human interferon in humans. Merigan et al. [62] gave
human leukocyte interferon to normal volunteers nasal spray prior to
rhinovirus challenge. In those studies, 300 U of interferon were recovered
in nasal washes from 2 to 15 hr after dose of interferon. recent study
with human fibroblast interferon given nasal drops to human volunteers
indicated that interferon could detected from 2 to 14 hr after intra-
nasal application [69]. This is in contrast to the findings with human leuko-
:1. 1;X()(;I';N()IJS
7!!
iI1Lvl"I"CI"OII fibroblast interferon
pl"('pal'aLioll llil"fveH ill ways human leukocyte interferon.
111 rvvvl1L ill 110rmal voluntecrs in which human leukocyte inter-
waH applivu locally to the nasal mucosa nasal drops, we found
;,- Lo reuuction in recovered interferon from 5 to 60 min after
caLiOll l G3]" These results are similar to those found in the chimpanzee
HLuuy and suggest that clearance factors are important in the
l'ate of disappearance of locally applied preparations . Although serum
illLcrfcron was detected from 10 to 60 min after local application of 80,000
lJ of human leukocyte interferon in three volunteers [70], additional studies
arc needed to see if locally applied interferon detected in the blood.
Although there are few instances in which cerebrospinal fluid levels
of interferon were measured after parenteral administration, only one
published report has appeared in which human leukocyte interferon has
administered intrathecally [ 71]. In the one reported patient, human
leukocyte interferon was administered intrathecally in dose of 6 105 U
cvery 12 hr for 2 days and then daily for 4 days. Because the patient was
suffering from disseminated herpes simplex infection, he was also receiving
idoxuridine treatment. Repeated measurements of the serum revealed
approximately 100 U of interferon after injection. At the same time, the
cerebrospinal fluid levels of interferon varied from 800 to 8000 U/ml. The
investigators believe these results indicate that blood-brain barrier does
exist for interferon but that the barrier overcome increasing the
dose.
Mechanism of Serum Clearance
Studies in both animals and man have demonstrated that interferon is rapidly
cleared from the blood stream after intravenous injection. This rapid
clearance has been thought to due to the glycoprotein nature of interferon
l 72-74]. As glycoprotein, interferon contains sialic the terminal
end of the carbohydrate chain [75,76]. It was recently shown that asialo-
interferon disappears from the blood more rapidly than native interferon
[ 72]. In contrast, another study reported effect the clearance of
human leukocyte interferon in rabbits after neuraminidase treatment of
the interferon preparation [16]. Because of conflicting resu1ts, Bose and
Hickman [77] determined whether removal of the major portion of the
carbohydrate moiety would alter the survival of interferon in the circulation.
The rate of disappearance of three different interferon preparations, native
lymphoblastoid interferon, neuraminidase-treated, and glycosidase-treated
interferon, from the serum of rats was measured (Figure 3). half-life
of 9 min was found for both the native as well as the deglycosidated inter-
feron. The half-life of the asialo-interferon was approximately 3 min, and
it appeared to disappear more rapidly than the native interferon. Treat-

100
10
*'
:

Q)
't
Q)
.

:1
~
(/)
1.0

20 40 80 100 120
Minutes After Injection
FIGURE 3. Clearance rates of native ( 0-0 ), neuraminidase-treated
( : - ), and glycosidase-treated ( 0-0 ) human lymphoblastoid interferon
preparations after their intravenous administration at 5 105 U doses in
rats. Each point is the average value obtained from six animals. (Reprinted
from Ref. 77, courtesy of the American Society of Biological Chemists.)
:1. I':X()(; 1': N( HIS 1 N'I' 1':I!.I,'I':lt( )N: 1'11 N I':TICS
11)('111, wiLll L'CI1IOVCH Hialic acid [rorn the native interferon
1111(( rCHiduc thc carbohydrate moiety. These termi-
rCH appoar to specific determinants for the hepatic
illtorforon and other normally circu1ating serum g1ycoproteins
I 7;,J. these investigators agree with the work of Bocci indicating the
irnportance of the liver as the organ for c1earance of interferon
I'rorn the circu1ation [74]. In their results indicate that the carbo-
11ydrate moiety of the present1y used interferon preparations removed
without impairing the of interferon to survive in the circu1ation.
1). Side Effects
Various side effects in patients receiving human 1eukocyte interferon
been reported. The most common1y reported clinica1 side effect has been
febrile response occurring 2-4 hr after the initia1 parentera1 dose of inter-
feron [54,64-66]. This febrile reaction appears to somewhat dose-re1ated
and decreases with repeated injections [3]. Fever has been observed in
tnost patients who received 4.2 104 U/kg per day or greater and with
high doses of interferon (1.7 105 U/kg per day). The first injection
a1so associated with nausea, vomiting, mya1gias, and chills. One patient
who received rapid intravenous injection of human 1eukocyte interferon
experienced hypotensive episode [64], but this has not been observed after
s10w intravenous infusions in other patients. Transient hypotension was
a1so seen in two patients after they received their first dose of 3 106 U
intramuscu1arly; however, subsequent doses had effect their blood
pressure (J. Gutterman, persona1 communication). ln patients who
received repeated injections over weeks to months, feeling of 1assitude
and ma1aise has been reported to occur, and the magnitude of these symp-
toms appeared to 1essened when more purified preparations of interferon
were emp10yed [3].
Merigan [3] has observed tender erythematous skin reactions at the
injection site when interferon was given subcutaneous1y. These skin reac-
tions were limited in size (1-2 appeared within the first day, and
disappeared over 48-72 hr. Similar skin reactions have a1so been observed
at the injection site of human fibroblast interferon [3]. Administering the
daily dose of interferon in two divided doses appears to have decreased the
incidence and severity of these skin reactions [3] .
After receiving human 1eukocyte interferon for severa1 days to weeks,
patients exhibit an increasing effect the hemopoietic system [66,78,79] .
After some 3-5 days of therapy, po1ymorphonuc1ear leukocyte counts begin
to fall, and this followed decrease in p1atelet and reticulocyte
counts. These apparent bone marrow suppressive effects are reversible
discontinuing the interferon, since p1atelet, reticulocyte, and polymorpho-
nuc1ear leukocyte levels return to pretreatment values.
U')

NeOl1aLcH wiLll cyLOI11cl2,"aluvit'UH WllO rcccivu(! Illlll1:l11 IcukocyLu il1Lurl'cL'0I1
developed pyrol2,"ctlic cffccL, dccrcaHc ill wcil2,"i1L 12,"ait1, at](! clcvaLcd
serum levels [80, 81J. WhCtl the waH
the infants increased their food intake and gained wCil2,"ht.
date, evidence of transmission hepatitis virus
leukocyte interferon has been reported. 1n addition, apparent effect
renal function has been discovered. Whether the side effects observed in
the clinical trials reported thus far limit the usefulness and efficacy
of exogenous interferon therapy must await further studies. Nevertheless,
the observed hematopoietic suppression related to interferon's
depressive effect proliferation, since in vitro effects bone marrow
cultures of animals and humans have been demonstrated. This pose
special problems for long-term therapy [82,83], Only studies
with interferon preparations of increasing purity determine if these
side effects eliminated.
Conclusions
The studies of exogenously administered interferon in both animals and
man have helped to define the variables influencing of this anti-
viral agent. Studies in animals demonstrated the basic pharmacokinetic
principles found later to operative in humans. 1ntravenouslyadministered
interferon was rapidly cleared from the serum regardless of the animal
model employed. half-life of few minutes was found when one dose was
given. Although the observed rapid clearance of intravenously administered
interferon has been thought to due to tissue binding, the reasons for the
delayed disappearance or tailing effect are not so apparent.
effect due to different molecular forms with different
clearance rates, to interferon reentering from tissue to serum, or to tem-
porary of organs responsible for removing interferon. Recent
studies have suggested that the Hver is responsible for clearing parenteral
interferon and functions to remove the glycoprotein from the circulation.
1ntramuscular or subcutaneous administration of interferon leads to
more sustained serum level that returns to low levels between 24 and 36 hr
after single dose. No significant accumulation of human leukocyte
feron has been observed after repeated intramuscular injections in animals
and in humans. 1n both animal and human studies, appears to
distribute poorly into the respiratory tract, cerebrospinal fluid, [84],
and across the placenta [85]. It penetrates poorly into fluids
and is rarely recovered from the urine.
The presently available, partially human interferon prepara-
tions have not been without certain side effects when administered to
patients. Constitutional symptoms and suppression have
been very common with prolonged courses of therapy. Long-term evalua-
:1. 1':X()(;I-:N()lJS
LiOl! U'uaLuu Ila:,5 only be!!:un; thus, presently unidentified
si!!:lli1'iuant in the future. It remains to seen
purifieu or synthesized human interferon would have similar

Although major pharmacokinetic differences were found when crude
partially purified interferon preparations were compared in humans,
will have to repeated when more purified or synthesized inter-
/'uron preparation is available. Since the pharmacokinetic resuIts of human
luukocyte interferon in rabbits have been similar to those in humans, future
pharmacokinetic information might more easily obtained with rabbit
model.
The determinants of drug activity include study of the distribution,
metabolism, and excretion of the biological substance in the host animal.
Past studies have defined the distribution and excretion of interferon, but
little information is available its metabolism in vivo. Although the
antiviral effects of interferon are apparent for some time after it has dis-
appeared, factors that could alter the pharmacokinetics of interferon
result in diminished or inconsistent antiviral activity [86]. date, there
is little or information the importance of such factors as nutritional
state, age, race, sex, environmental factors, concurrent disease, and
interferon-drug interactions in altering the pharmacokinetics of interferon.
As additional clinical studies in humans are performed, the importance of
each of these factors apparent.
ACKNOWLEDGMENTS
Our work interferon has supported National Institutes of Health
Contract No. AI-42530.
We gratefully acknowledge the excellent help of Linda Simmons in pre-
paring this manuscript.
REFERENCES
1. in Interferons and Interferon Inducers (N. Finter, ed.),
American Elsevier, New York, 1973, 241.
2. HoandJ. Armstrong, Ann. Rev. Microbiol. 29:131 (1975).
3. Merigan, Texas Repts. Biol. Med. 35:541 (1977).
4. Paucker, J. Immunol. 94:371 (1965).
5. J. S. Youngner and S. Salvin, J. Immunol. 111: 1914 (1973).
6. Berg, Ogburn, Paucker, Mogensen, and Cantel1,
J. Immunol. 114: 640 (1975).
7. J. Vilcek, Havel1, L. W. Mozes, and Berman, Proc. First
Intersect. Congr. IAMS, Science Council of Japan, Tokyo 1975, Vol.
4, 65.
(:I{.I':I:NIlI-:I!t.:, ;111,1 C(llJCII
8. 1<::. llavcll, 13. l'auckcl',
J. Vilcek, Nat. Acad. Sci. U.S. 72:2185 (1975).
9. J. Dalton, and 1..:. , Nat.
Acad. Sci. U.S. 72:4587 (1975).
10. J. Vilcek, Havell, and S. Yamazaki, Ann. N. Acad. Sci. 284:
703 (1977).
11. W. Stewart, and J. Desmyter, 67:68 (1975).
12. J. Desmyter and W. Stewart, Virology 70: 451 (1976).
13. Paucker, J. Da1ton, Torma, and Ogburn, J. Gen.
Virol. 35:341 (1977).
14. S. Bose, D. Guarari-Rotman, U. Ruegg, L. Corley, and
Anfinsen, J. Biol. Chem. 251: 1659 (1976).
15. J. Chen, W. J. Jankowski, J. Sulkowski, and
W. Carter, J. Virol. 19:425 (1976).
16. Mogensen, L. Pyhala, Torma, and Cantell, Acta Pathol.
Microbiol. Scand., Sect. 82: 305 (1974).
17. Jr., Proc. Nat. Acad. Sci. U.S. 73:520 (1976).
18. Anfinsen, S. Bose, L. Corley, and D. Gurari-Rotman, Proc.
Nat. Acad. Sci. U.S. 71:3139 (1974).
19. D. Stancek, Gressmerova, and Paucker, Virology 41: 740 (1970).
20. L. W. Marshall, Pitha, and W. Carter, Virology 48: 607
(1972) .
21. Cesario, J. Schryer, and J. G. Tilles, Antimicrob. Agents
Chemother. 11: 291 (1977).
22. J. Valle, G. W. Jordan, S. Haahr, and Merigan, J. Immunol.
115: 230 (1975).
23. DeSomer, Joniau, V. G. Edy, and Billiau, in Production
and U of Interferon for the Treatment and Prevention of Human Virus
Infections Waymouth, ed.), Tissue Culture Rockville, Md.,
1974, 39.
24. vell and J. Vilcek, in The Production and U se of Interferon
for the Treatment and Prevention of Human Virus lnfections Way-
mouth, ed.), Tissue Culture Assoc., Rockville, Md., 1974, 47
25. Mogensen and Cantell, Pathol. Microbiol. Scand. ,
Sect. B.l: 382 (1973).
26. J. J. Sedmak and S. Grossberg, Texas Repts. Biol. Med. 35: 198
(1978).
27. V. G. Edy, Billiau, Joniau, and DeSomer, Proc. Soc.
Med. 148: 249 (1974).
28. Cartwright, Senussi, and D. Grady, J. Gen. Virol. 36:317
(1977) .
29. R. Jariwal1a, S. Grossberg, and J. J. Sedmak, Arch. Virol. 49:
261 (1975).
30. R. J. Jariwalla, S. Grossberg, and J. J. Sedmak, J. Gen. Virol.
35: 45 (1977).
:.14.

v;).
:.16.
:.17.
:.18.
:.19.
40.
41.
42.
43.
44.
45.
,J. J. i:'>cum:.t!,, JamCHotl, anu i:'>. 1<;. GrOHsberg, in Human Interferon:
PL"ouucLiotl Clitlical Use (W. Stillebring and J. Chapple, eds.),
Plcllum, Ncw York, ] 978, 133.
1<;. MogensenandK. Cantell, J. Gen. Virol. 22:95 (1974).
W. 1<;. 8tewart, De80mer, V. G. Edy, Paucker, Berg,
and Ogburn, J. Gen. Virol. 26:327 (1975).
D. Greiff and Greiff, 34 (1972).
V. G. Edy, J. Desmyter, illiau , and De80mer, Infec. Immun.
16: 445 (1977).
Cartwright, 8enussi, and D. Grady, J. Gen. Virol. 36:323
(1977) .
Tanford, Advall. Proteill Chem. 23: 122 (1968).
J. Vilcek alld D. R. Lowy, Arch. Ges. Virusforsch. 21: 253 (1967).
G. Rossman and J. Vilcek, Arch. Ges. Virusforsch. 31: 18 (1970).
Cesario and J. G. Tilles, J. Infec. Dis. 127:311 (1973).
Cesario, Mandell, and J. G. Tilles, Proc. Biol.
Med. 144: 1030 (1973).
W. Harmon, 8. Greenberg, and R. Couch, Proc.
Med. 152: 598 (1976).
Cesario, Proc. Biol. Med. 155:583 (1977).
Merigan, DeClercq, and 8. Finkelstein, Ann. N. Acad.
8ci. 173: 746 (1970).
8. Baron, Buckler, R. V. McCloskey, and R. Kirschstein,
J. Immunol. 96: 12 (1966).
46. N. Finter, Brit. J. Pathol. 47:361 (1966).
47. 1. Gresser, D. Fontaine, J. R. Falcoff, and Falcoff,
Proc. Biol. Med. 124: 91 (1967).
48. 8ubrahmanyanandC. Mims, Brit. J. Pathol. 47:168
(1966)
49. R. Nuwer, DeClercq, and Merigan, J. Gen. Virol. 12:
191 (1971).
50. W. 8chafer, Lieberman, Cohen, and 8cience
176: 1326 (1972).
51. and PosHc, Nature 214: 1230 (1967).
52. and Postic, in First International Conference Vaccines
Against Viral and Rickettsial Disease of American Health
Organization/WHO Publication 147 , 1967, 632.
53. L. Pyh1il1i and Cantell, Proc. Med. 146:394 (1974).
54. Cantell, 8. Hirvonen, Mogensen, and L. Pyh1il1i, in
Production and Use of Interferon for the Treatment and Prevention of
Virus Infections Waymouth, ed.), Tissue Culture Assoc.,
Rockville, Md., 1974, 35.
55. Cantell and L. Pyh1il1i, J. Gen. Virol. 20: 97 (1973).
56. Cantell and L. Pyhal1i, J. Infec. Dis. (1976).
57. Cantell, L. and 8trander, J. Gen. Virol. 22:453 (1974).
58. V. I<.:dy, lHlli:.l.u, :.1.11<11'. l)USOI11UI', ,1. llll'l:e. IJiH. (1!J7(i).
59. V. Lipton, al1d tull , Soc. BioJ. Mud.
149:287 (1975).
60. F. Skreko, 1. Zajac, Bahnsen, !i'. and Cantell,
Proc. Soc. Biol. Med. 142: 946 (1973).
61. Nash, W. Morgan, J. Armstrong, R. Carroll,
and Postic, Infec. Immun. 286 (1973).
62. Merigan, S. Reed, S. andD. J. Tyrrell, Lancet1:
563 (1973).
63. Johnson, S. Greenberg, W. Harmon, Alford, and
R. Couch, J. Microbiol . .1: 106 (1976).
64. StraJ1der, CaJ1tell, G. Carlstrom, aJ1d JakobsSOJ1,
J. Nat. Cal1cer Inst. 51: 733 (1973).
65. G. Emodi, Just, R. Herl1andez, and J. R. Hirt, J. Nat. CaJ1cer
Il1st. 54: 1045 (1975).
66. G. W. Jordal1, R. Fried, al1d Merigan, J. Il1fec. Dis. 130:
56 (1974).
67. MerigaJ1, Rand, R. Pollard, S. Abdallah, G. W.
JordaJ1, andR. Fried, New Engl. J. Med. 298:981 (1978).
68. V. G. Edy, and DeSomer, Lancet 1:451 (1978).
69. G. Scott, S. Reed, Cartwright, al1d D. J. Tyrrell,
paper submitted for publication iJ1 IJ1terferol1 Scientific Memoral1dum.
70. S. Greel1berg, W. Harmol1, Johl1s0J1, and R. Couch,
Antimicrob. Agents Chemother. 14: 596 (1978).
71. DeClercq, V. G. Edy, DeVlieger, and DeSomer, J. Pediat.
86:736 (1975).
72. V. Bocci, PaciJ1i, G. Pessina, V. Bargigli, aJ1d Russi,
J. Gel1. Virol. 35: 525 (1977).
73. V. Bocci, Pacini, G. Pessina, V. Bargigli, al1d Russi,
Experimentia 15: 164 (1977).
74. V. Bocci, Texas Repts. Med. 34:436 (1977).
75. G. Ashwell and G. Morell, Advan. Enzymol. 41: 99 (1974).
76. F. Dorner, Scriba, and R. Weil, Proc. Nat. Acad. Sci. U.S. 70:
1981 (1973).
77. S. Bose and J. J. Chem. 252:8336 (1977).
78. Greel1berg, R. Pollard, L. 1. Lutwick, W. Robinson, aJ1d
Merigan, New EJ1gl. J. Med. 295:517 (1976).
79. S. J. Urbal1iak, 1. Halliday, G. W. Beveridge, and
LaJ1cet 1: 553 (1978).
80. Arvil1, S. Yeager, and MerigaJ1, J. Il1fec. Dis.
(1976).
81. G. Emodi, R. O'Reilly, Miller, L. Eveson, U. ,
al1d Just, J. Infec. Dis. (1976).
82. W. Fleming, McNeill, al1d Immunology 23:429
(1972) .
:1. I':X()(;I':N()IIS
K:I. \,. <lIlLl S. ltcs. :37: 1794 (1977).
,J. 011 J. J. Bacteriol. 91:251 (1966).
J. Ovcrall and L. Glasgow, Science 167: 1139 (1970).

J. Wagner, Drug Metabolism and Drug Interactions (F. G. McMahon,
cd.), Futura Mount Kisco, N. 1974, 1.
J. Portnoyand Merigan, J. Infec. Dis. 124:545 (1971).
HI:i. D. Neumann-Haefelin, Shrestha, and F. Manthey, J. Infec. Dis.
(1976).
4
EXOGENOUS INTERFERON: USE IN HUMANS
H)R TREATMENT OF MALIGNANCIES
Michio and Rita F Buffett
l{oswell Park Memorial Institute
Buffalo, New York
10 INTRODUCTION
RATIONALE FOR INTERFERON TREATMENT
Evidence from In Vitro Studies
Evidence from In Vivo Studies (Nude Mice)
Clinical Trials
Do Why Exogenous Interferon Treatment?
PROBLEMS SOLVED
Large-Scale Production of Interferon
Purification of Interferon
Quality Control of Interferon for Clinical Use
Do Development of Simple, Reliable Test for
Determining the Susceptibility of Tumor Cells
to Interferon
Fibroblast Interferon vs Leukocyte Interferon
IV DISCUSSION
Vo
REFERENCES
10
89
91
91
94
96
99
100
100
101
101
101
103
104
106
107
The inhibltion of growth in L cells treated in vitro with interferon was
first described Paucker and his associates [1] interesting finding
did not attract much attention at that time, because interferon was con-
sidered not to have any effect normal physiological functionso However,
number of years later, the antiproliferative function of interferon was
studied extensively Gresser and his co-workers [2,3] Continuous
89
t1lUrillU ill SLISPCIISioll CULtLII"US, witll at I"ela-
tive1y high concuntratiol1s, WCI"U to 1J1L11tiply at slowur" I"atu to
reach lower fina1 dcnsity ulltl"catcd [1,1-(j]. Slowur !!;rowLII
of was a1so observed in culillres both 1l0rma1
formed l 7-9]. The saturation densities of trans[ormell cells were
lower in the presence of interferon tl1an in untreated cu1tures, whereas fue
densities of norma1 were not affected [8]. Co1ony formation
L1210 in soft agar was significant1y reduced [10,11]. variety
of mefuods have been used for fue quantitation of the antiproliferative and
cell-regu1atory activities of interferon; these inc1ude direct counting
[1,12-15], co1ony formation wifu [10] or without agar [16], macro-
mo1ecu1ar synfuesis measured incorporation of radioactive precursors
[ 9, 17-19], etc. Whefuer the growfu inhibitory effect is associated wifu
interferon itse1f or with other contaminants present in the preparations has
long been argued. Recent1y, severa1 reports [8,16,20-22] appear to in
good agreement that tl1e antiproliferative effects caused tl1e inter-
feron mo1ecu1es themse1ves. The mode of action of interferon in the inhibi-
tion of growth has been investigated. Evidence has been accumu1ated
that interferon exerts its effect fue

and/or ear1y S phase of the
multiplication cyc1e, resu1ting in e1ongation of fue growfu cyc1e and thus
subsequently in decrease in the growfu rate ([ 9, 17,23] ; Ito and R. F.
Buffett, unpublished data).
The antitumor effects of interferon in vivo have a1so been docu-
mented [2,3]. Many of fue ear1y studies were carried out Gresser and
his co-workers. They investigated fue effect of interferon vira1 carcino-
genesis in the Friend and Rauscher murine 1eukemia virus systems. The
effect of interferon was observed on1y when interferon treatment
was continued in Swiss and DBA/2 mice for long period of time [24-26],
and it was suggested fuat the continuous suppression of virus growfu
interferon was prerequisite for the prevention of 1eukemogenesis in mice.
It was pointed out, however, fuat direct action of interferon the
liferation of virus-transformed cells cou1d not exc1uded. From fuese
series of experiments, Gresser and his first introduced long-
range, 1arge-dose administration scheme of interferon treatment for malig-
nancies. The effect of interferon treatment fue deve10pment spontaneous
1eukemia, virus-associated disease, in mice was studied [27,28] .
Daily exogenous interferon fuerapy from fue time of birth was continued
for one The surviva1 time mice was considerably pro1onged and
the incidence of 1eukemia was decreased from 95% in untreated contro1s to
63% in interferon-treated mice [22]. The deve10pment of
in mice (anofuer instance of spontaneous virus-associated tumor) was
de1ayed fue repeated inocu1ation of interferon [29]. Of interest was fuat
there was decrease in the amount of mammary tumor antigens in the
milk of interferon-treated mice, despite the c1ear-cut inhibitory effect of
interferon tumor growfu. In order to confirm fue direct action of inter-
,1. 1';X()CI';N(JlIS IN'I'I';H I'I';H()N: IIIIIVIAN MA),\(;NANCJ/<;S !JJ
1'01'011011 I1copla:-:Li(, ill vivo, :tIHJ also studied
clTicacy 01' tllCl'apy thc growth various types
01' though virus implicated as
oLiologieal il1 tlle development some mouse tumors, the presence
viru:-: totaHy excluded, since practicaHy mouse are
as carriers of both ecotropic
IIOUS viruses.) repeated daily intraperitoneal administration of inter-
prevented the growth of several ascitic tumors, including
ItC19, EL-4, and L1210, in different strains of mice [30,31]. most
dl'amatic effect ,vas observed in the Ehrlich ascites tumor-BALB/c mouse
:-:ystem [31]. survival of BALB/ mice with 104 tumor
ceHs was 18 days; and of the untreated mice survived 22 days,
whereas 90% of interferon-treated mice survived more than six
without evidence of tumor. From these experiments, several
ples, important to the practical application of interferon therapy, have
emerged: (1) the greatest antitumor effects were obtained when contact
interferon and tumor ceHs was maximal, i. when both interferon
tumor ceHs were intraperitoneaHy; (2) interferon treatment
was ineffective when it was limited to the period preceding inoculation of
tumor ceHs; and (3) therapy was less effective in mice
solid tumor nodules in mice which were with ascites tumor
cells. However, it has reported that daily intravenous inoculation of
interferon resulted in suppression of the growth of solid subcutaneous
malignant tumor, Lewis lung mice [32]. Also, the develop-
ment of metastasis was blocked. Even when the initiation of
interferon therapy was delayed until six days after tumor inoculation and
palpable nodules had already developed, the growth of both primary and
metastatic was inhiblted.
Thus, the rationale for the application of interferon therapy
to human cancers is the demonstration of effective
activity against variety of murine neoplasms.
RATIONALE FOR INTERFERON TREATMENT
Evidence from In Vitro
In paraHel with the results of investigations with murine interferon, evi-
dence has accumulated that interferons also possess antipro-
liferative and cell-regulatory activities for both normal and neoplastic
ceHs of humans. Growth inhibltory effects variety of human ceHs
have reported to date [18-22,33-46]. Adams et al. [37] tested
several human lymphoblastoid lines, which were established from
Burkitt's lymphomas, for susceptibllity to the antiproliferative effect of
interferon. Some ceHlines were found to quite sensitive to the effect
IT():tIHIIIIII,'I,'I':TT
leukocyLc oLI1CI.' eell lillCS :tlHI
his coHeaguos [18] usod sCllsiLivc lYl1lpllOlJlasLoill 1.iIlC,
studying the inhibition 01 growth of illtcr[crot1,
simp1e alld quite sensitive assay for the al1LiprolifcraLivc activiLy
of interferon using incorporation of [14 thymidil1C. this mothod,
approximate1y 15 U/m1 of 1eukocyte interferon were found to sufficient
to reduce the uptake of radioactive precursor 50%. It has shown
that human chromosome 21 is responsible for determining the susceptibility
of ceHs to the growth-inhibitory effect as weH as to the antivira1
activity of interferon [20]. number of studies have reported c10se
corre1ation between antivira1 and antiproliferative activity of interferon
[16,18,20-22]. Dah1 and Degre [38] c1aimed to have successfuHy separated
these activities from 1eukocyte interferon. However, evidence from
1aboratories suggests that the growth-inhibitory effect is direct1y
associated with the interferon and both antivira1 and antiprolifera-
tive activities are inseparable [16,18,20-22].
paraHe1 with the first systematic tria1 of treatment of patients suffer-
ing from osteogenic sarcoma (see be10W), Strander and Einhorn [41] investi-
gated the growth inhibitory effect of human leukocyte interferon in vitro
nine ceHlines derived from osteosarcomas. the tumor ceHlines tested
proved to susceptible to the antiproliferative effect of 1eukocyte interferon.
cultivation methods, i. treatment of cultures with interferon for
4-8 weeks, it was found that the growth rates of osteosarcoma ceHs were
inhibited strong1y in the presence of 10-100 U/m1 of 1eukocyte interferon,
whi1e norma1 fibroblasts were not affected s ignificant1y.
the authors' 1aboratory, cu1tured ceHlines derived from various
human neop1asms [47] have tested for their susceptibility to the anti-
proliferative activity of human fibroblast interferon (specific
activity 2 107 U/mg protein) using method similar to that reported
Hilfenhaus et [18]. Dip10id fibroblast strains initiated in our
department [48] were used as norma1 contr01s. 1ncorporation of
[ 3 thymidine (TDR) was measured in the acid- ins01uble fraction following
1abeling of both interferon-treated cells and untreated controls for 20-24
hr. The patterns of growth inhibition of tumor ceHs were distinct from
those of nonmalignant fibroblasts. The inhibition interferon was most
prominent in norma1 fibroblasts when TDR uptake was measured
during the initia1 24-26 hr after subcu1turing and addition of interferon;
this is comparable to results obtained from mouse interferon-BALB/c
fibroblast system Ito and R. F. Buffett, unpublished data). However,
stronger reduction in incorporation of TDR was observed in tumor
ceHs between 72 and 96 hr after seeding and the addition of interferon
than in the initia1 24 hr. 1 summarizes the data obtained to date.
Dependiug upon the 1eve1s of to the antiproliferative effect of
interferon, the malignant tumor lines cou1d divided readily into two
groups; some celllines were highly sensitive while others responded poor1y.
1. Antiproliferative Activity of Fibrobla"t lnterferon
Variety of Cultured Neoplastic and Normal Cells in Vitro
a
Cellline
or
strain
Incorporation of [3 TDR
Origin
Neoplastic ceHs
R Carcinoma of urinary bladder
SAOS-2 Osteosarcoma
5959
Daudi

MeWo

Osteosarcoma
Lymphoma
Colon carcinoma
Melanoma
Rhabdomyosarcoma
N onmalignant cells
HF604 Diploid foreskin fibroblast
Diploid foreskin fibroblast
Diploid foreskin fibroblast
Diploid foreskin fibroblast
type

Ff
F
Lyg

F
F
F
F
F
F
50% inhibition Inhibition at
endpoint (U/ml)c (:)d
28 96
17 94
82 90
130 91
>5000 40
>5000 43
>5000 25
53 82
110 73
130 61
48 82
aUnpublished data from our laboratory. the tumor lines employed, except were kindly given
Dr. J. Fogh [47], Sloan-Kettering Memorial Institute; cells were obtained from Dr. J. Whitman,
Electro- Nucleotics Laboratories, Inc.
bMeasured the four-day assay: uptake of TDR during 16-20 hr after 72-78 hr of cu1tivation with inter-
feron was compared. The method described in our previous report [16] was employed with change of time
labelling.
cExpressed as concentration of interferon, international reference units per milliliter; mean value from two or
more independent experiments.
dpercent inhibition at the in the presence of to 5000 U/ml of interferon.
Epithelial.
f Fibroblastic.
gLymphoblastoid.
''I'() ILII,I 1\111,'1,'1-:'1''1'
Whcl1 illLcr[Ul"OIl luvulH 1'01' il1lli1JitiOlI 01" TI)lt Llpl.aku WUI'U
compared, 17-130 U/tnl uf illLUL'l'ueOll WUI'U I'UUUCU upLaku
in the highly sensitivu group, 6000 U/tnl failuu Lo illI1i!>iL 60%
the poorly responding group (l<'igure 1), luvuls of il1hibltion
to 5000 U/ml of interferol1 varied from one tumor cellline to al1other,
even though clear-cut difference was observed between the two groups.
When tested same method, the susceptibility normal diploid fibro-
blasts to human fibroblast interferon was intermediate between the two
groups of neoplastic (Table 1). Figure 1 indicates the dose-
response curves of seven tumor to the al1tiproliferative effect
purified fibroblast interferon. The two osteosarcoma studied in
our laboratory were definitely sensitive to human fibroblast interferon,
confirming and expanding earlier observations [41]. The high
of Daudi to the antiproliferative activity of interferon was also veri-
fied [18,37].
present studies imply wide range of susceptibility to the anti-
proliferative activity of among different types of human neo-
plasms. For this reason, the in vitro screening of neoplasms for degree
of sensitivity of tumor to interferon provide important information,
prior to clinical application, for the selection of the types of human malig-
nancies which respond favorably to exogenous interferon treatment.
Evidence from 1n Vivo Studies (Nude Mice)
nude mouse, because it is "athymic" and therefore
severely immunodeficient, has increasingly popular during the
last decade as an animal model for the heterotransplantation of human
tumors [49,50].
Tygaard and Povlsen [51] were the first to report the successful trans-
plantation of solid human tumor, highly differentiated adenocarcinoma,
in nude mice, This tumor grew rapidly and was carried in serial passage
for more than 30 passages in nude mice [50], Since then, variety of
solid human tumors have been grafted in nude mice [52-59].
The percentage of successful "takes" from implants of individual human
tumors obtained at surgery or autopsy vary conSiderably, depending
presumably upon grade of malignancy, size of inoculum, and variations in
the tissue make-up of the graft. However, the implantation of cells from
human tumors which have been successfully propagated in vitro appears to
resu1t quite consistently in successful "takes", which then carried
in serial in vivo passage in nude mice [58,60-63]. Fogh et al, [47] have
listed 127 individual cultured human tumor 90% of which produce
tumors within the first month after grafting and 10% within 2 to 5 months.
The tumors grown in nude mice, whether from specimens obtained direct1y
from the patient cu1tured in vitro, maintain their human morphological
and functional characteristics [53- 64] .
w
z


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I
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Z

i=
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z
z
Q
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z

100
50
..... ___ RT-4
.-_-_ SAOS-2

MeWo


10 100 1,000 10,000
INTERFERON (units/ml)
FIGURE 1. Dose-dependent of uptake of thymidine in various
human tumor celllines human fibroblast interferon. The method
ployed is described in footnote of 1. The inhibition curves were
obtained from the experimental data of two or more independent assays.
nude mouse system should useful for the study of in vivo effects
of chemotherapeutic agents human tumors. The responses of variety
of tumors to drug therapy are being studied in number of laboratories
[65-68]. Although these studies have not been extensive to date, the results
obtained thus far suggest that this animal model system is suitable for
screening the sensitivity of individual human tumors to drug therapy. In
general, the results obtained from the nude mouse resemble those from
clinical reports, suggesting that the of human tumors is re-
tained after transplantation into the nude mouse. However, comparison
of the responses of the same tumor in humans and nude mice has not as
yet been made, undoubtedly because of the time required to establish the
reproducible growth of the primary human tumor in the nude mouse.
111 ucpal'll1lcl1l, v:1I'icly 01,' c"lalJl i"IICll 1,1111101' I
well as i!\Oculalcl! i!1lo \\1 icc, allu :colul!ic:co
the in vivo activity fibL'oUlast havc
initiated. results illdicate that the !!,'rowtll humall neo-
p1astic RT-4, which were established carcinoma the urinary
bladder in vitro [47] and xenografted in nude mice, was inhiblted
daily of fibroblast interferon [69]. Although these
resu1ts preliminary, there is doubt about the efficacy of purified
interferon as an in vivo antineop1astic drug.
nude nlOuse system is an exce11ent one for studying the effects
chemotherapeutic agents but it does not duplicate condi-
tions that influence the response of the cancer patient to the tumor 01'
to If one considers, rather simplistica11y, that the
activities of exogenous interferon expressed in two ways- (1)
direct (anticellu1ar) effect tumor cells, and (2) an indirect effect host
defense mechanisms (see Section 1V)-then in the nude mouse system the
direct effect tumor ce11s but the indirect effect
call1lOt.
Clinica1 Tria1s
than 70 in Sweden have received intramuscu1al' of
human 1eukocyte interferon to date [70-74]. The concentrations of
feron that have been administered have varied fL'om 2 106 to 2 107 U
daily, 01' 2 to 3 times weekly. Some patients have received interferon for
as as 1i years. the stages, the emp10yed had
10w specific activity, 104 U/mg so that certain side effects such
as transient fever, pain, mild a11ergic reactions were frequently
observed. However, since the specific activity has been increased to 106
U/mg improved methods of purification, a1most adverse
reactions fo11owing the of interferon have been essentially
eliminated. This is in sharp contrast to other drugs,
which a1ways associated with strong undesirable side effects,
cause interferon is physi010gica1 substance, produced cells.
first systematic study of the effect of exogenous treat-
ment for malignancies in humans has been done Strander and his
1eagues [73-75]. Their clinica1 tria1 was begun at the Hospita1
(Stockho1m) in 1971, in which osteosarcoma patients received exogenous
interferon therapy. date, than 28 patients have treated with
1eukocyte interferon [73]. Strander's group se1ected osteosarcoma for
the first tria1 for the following reasons: (1) its prognosis; (2) the
capability of easy remova1 of tumors surgery to decrease the tumor
burden; and (3) the predictability of rapid deve10pment of metastasis within
which allowed them to set fixed endpoint for the study. The
) '1
(,I'ea(.I!1UI!(. 0(' illjecLiolls 0(';\ l()" U lcukocyte inLcrferon intra-
1IILIsclllal'Iy llaily OIIC IIIOIILlI, whit:h the patients stayed in
LlIC either exarticulation, amputation,
r'csccLio!l was performeu. After the first month, administration of inter-
was continued three times weekly for an additional period of 17 months
(Lotal 18 months, approximately 25 injections, and total of 7.5 108 U
patient). According to recent review Strander [73], life-table
of the study reveals the following: (1) 64% of the interferon-treated
patients were free from metastasis at 2i years, whereas 30% of the con-
current control group consisting of 23 were free from metastasis;
(2) 73% of the interferon-treated group were surviving after 2i years, as
compared to 35% in the concurrent control group. The resu1ts obtained at
that time appeared so promising that the experimental groups were being
expanded. This first systematic trial of exogenous interferon treatment
was initiated without any of the susceptibility or resistance of
osteosarcoma to the growth-inhibitory activity of interferon. Soon,
however, it was found Strander and Einhorn [41] that they had been lucky
enough to choose neoplasm which was quite sensitive to interferon in in
vitro studies. the first clinical application of interferon therapy in
osteosarcoma has been successful with adverse reactions. In addition,
the average incidence of symptoms considered to typical of acute viral
infections (sore throat, congh, nausea, diarrhea, headache, fever, etc.)
in eight children and adolescents was compared with the
average incidence of these symptoms in 21 adu1t members of their immed-
iate [76]. individuals were ambulatory and performing daily
duties. The interferon-treated patients remained symptom-free (at least
one symptom was scored) for five out of six months, whereas family
bers remained symptom-free for only one out of six months. Also, the
duration and severity of most persistent symptoms were less in the treated
patients. Therefore, treatment with exogenous interferon also appeared
to provide other benefits.
Other studies have shoWll temporary benefit from exogenous inter-
feron therapy. Blomgren et [77] have reported case of Hodgkin' s
disease (lymphocyte predominance, stage IV purified leuko-
cyte interferon was injected intramuscularly, 5-7 106 U daily for 7 months.
During interferon treatment, the peripherallymph nodes decreased in size,
the general conditions of the patient improved, and symptoms disappeared.
However, the interferon therapy resulted in remission of the disease that
lasted for six months only. similar delay in the progression of disease
was observed in case of mu1tiple (in the review written
Strander [73]). The patient, who suffered from far-advanced
received 6 106 U of leukocyte interferon daily, and improve-
ment in clinical symptoms as well as in laboratory parameters occurred
before the remission ended. At Roswell Park Memorial Institute, pre-
liminary studies in which human fibroblast interferon was injected directly
2. Clinical Trials of Exogenous Interferon Treatment for Human Neoplastic Diseases
a
No.
patients Interferon theraE.l
Tumors involved Source Dose
b
Duration Results References
Osteosarcoma 28 Leukocyte 3 106 months Increased survi val Strander [73]
Hodgkin' s disease 1 Leukocyte 5-7 106 7 months Remission for 6 Blomgren et al.
months
Multiple myeloma 1 Leukocyte 6 106 ? Clinical improve- Strander [/3]
ment
Malignant melanoma 2 Fibroblast 5 105 1 montl1 of 01'08 ze\\'ic z
(skin metastatic lesions) (local) 01' reduction in etal. [69]
size of skin
number of other clinical trials have done in various of the world (Belgium, Japan, the l:nitec.
Yugoslavia, etc.). Unfortunately, detailed was not at the time of writing. these
omitted from the table.
brnternational units (U) per day per patient.
'1. I':N( )1/:-: IIIJIVI IVIAI.I(:NANCII'::-: !)!)
1111.0 IlIetasL:ttic 01.' lesiol1s two patients
antitumol'
Ili!)I. I{eslllis olJiail1eu to the time of
ill TaIJ]e
1). WllY l:;xogenous Interferon
ucscribed large amounts of interferon must administered for
period of time in the treatment of malignancies with exogenous inter-
I'eron. The task of preparing large of interferon is

An alternative approach, of course, is to apply inducers of endogenous
production. However, treatment with inducers has always been
the appearance of refractory phase in the response of host
cclls, during which interferon production becomes minimal even in the
presence of adequate amounts of inducer. Furthermore, Stringfellow found
serum hyporesponsive factor to interferon production in leukemic mice
transfer the hyporeactivity to interferon inducers to normal
mouse cells in vitro [78]. direct relationship was reported to exist
iween the concentration of the serum factor and the development of hypo-
responsiveness to interferon induction in vivo. The discovery of this factor
in leukemic animals appears not only to discourage attempting induction of
interferon for treatment of but also to reduce
the potential therapeutic value of interferon inducers for neoplastic diseases.
Another complication that must interferon are
used to stimulate the of is the toxicity
that is always associated with of [79,80].
after injection with poly 1: poly variety of side effects such as
fever, mild elevation of liver laboratory
ties, etc., were observed [81].
Clinical trials for of malignant diseases with poly 1: poly
have reported number of [82-85]. et al.
s tudied the therapeutic effect of poly 1: poly [85].
They the multiple doses of 0.3-75 mg/m 2 of body
surface area to 37 with variety of tumors, 26 solid
tumors, 9 acute leukemias, 2 leukemias blast
crisis. Low levels of serum greater 10 U/ml, were de-
tectable following the first in 24 of 38 trials. It was reported that
one of the patients favorable tumor to the
tration of poly 1: poly of disease in most of them
while they were the drug.
From these results, the administration of exogenous is
considered definitely more promising than interferon inducers for the treat-
ment of though the preparation of adequate
100 I'I'() :tllll 11111,'1,'1';'1''1'
illLcl'[CI'OII all Ilowcvcl', l110sL
recenLly, Strill!!:[cllow LllaL 01' 1J1'osLa!!:lall
dins with four dif1'crcnL il1ducers prcvel1Lcd 0['
the refractory phase micc I If aIlLa!!:OIli:-:-
tic effect prostaglandins tlle Lo inierferon induccl';:
in tumor-bearing mice is confirmed, interfcron thcrapy
various interferon inducers will merit serious reconsideration for treat-
ment of human
III. PROBLEMS SOLVED
Large-Scale of
future of with interferon will depend
simply upon the successful mass production of date, with
fewexceptions [87], almost trials have been performed using
human leukocyte because pooled leukocytes from blood donors
are the most readily available source for the large-scale production of
human for the production of sufficient supply
of leukocyte illterferon for the first systematic trial in humans was developecl
Cantell his co-workers [88,89].
the other hand, large-scale production of human fibroblast inter-
feron has been accomplished in our [48], and outline of our
system (Figure 2 in Section HI. will discussed briefly. As
potential sources for interferon production, 15 new foreskin fibroblast
were isolated, cryopreserved, characterized. The following
criteria were used for the selection of suitable efficiency of
interferon production, saturation denSity, karyology, freedom from adventi-
tious agents, and others that are listed in Figure 2. The cultures are
scaled in large glass roller bottles with 1585

growth surface area.
The roller bottle cu1tures are superinduced poly I: poly (75
with cycloheximide (60 and actinomycin D 75 After wash-
ing three times, medium containing 5% serum is
added, and cells are incubated for 22 hr at 34 Approximately 2 106
U of interferon are per bott1e. crude from
of bottles is pooled, clarified and stored at -900 for
later.
Most recently, attempts have been made to mass produce
with lymphoblastoid Namalva; this cellline has been selected
from more than 100 similar celllines and does not produce Epstein-Barr
virus [90- 93]. Since the cells can propagated in tank cultures, this
method appears extremely promising for the industrialization of
interferon production. produced Namalva cells resembles
leukocyte interferon [94] .
101
11. l'LII'il'ical,ioll 01' LIII,UI'I'uI'oll
1IlII\l:tll I'iIJl.'oblasL iIlLUL'[Ul'UII purified in department several
Ill4ltlHls: albumin, and micro-
(Ilydrocarbons, F3GA, aromatic amino acids, etc.)
I Currel1tly, purification of large quantities of human fibroblast
IIILur[oron involves utilization of concanavalil1 A-Sepharose [95] and phenyl-
CL-4B column chromatography J. Mikulski, J. S. Horosze-
wicz, 1<::. Sulkowski, and W. Carter, personal communication, Interferon
Memoral1da, October, 1976). direct combination of the two
highly purified preparation is eluted from the second column in
narrow and highly concentrated fractiol1 (specific activity approxi-
llIately 2 107 U/mg protein).
1n contrast, purification of leukocyte interferon is more difficu1t to
and awaits the development of new s imple technology.
Quality Control of 1nterferon for Use
'L'he safety potency of interferon, biological product, should certi-
for administration to humans extensive tests under an established
quality control scheme. The scheme might differ slightly depending upon
the purpose for which the drug is to used, i. for treatment of viral
diseases of children and for admil1istratiol1 to patients with far-advanced
neoplasms. Unfortunately, federal regulations inter-
feron products are not available to date. We prepared quality control
scheme for production of human fibroblast interferon, adapted from U. S.
Food and Drug Administratiol1 (FDA) and Medical Council
guidelines for vaccine production as well as of human diploid fibroblasts
(Figure 2) [48]. It identified specific steps production, purification,
lyophilization of interferon at which the product is evaluated for potency
and other features and is desigl1ed for the general use of fibroblast
feron. Since the quality control procedure accommodates both safety as
as the new technology for production and purification of interferon,
it valuable prototype for future establishment of formal regula-
tions different types of interferon products.
D. Development of Simple, Reliable Test for
the of Tumor to 1nterferon
Our recent results, described earlier (Section indicate that the sus-
ceptibility of neoplastic cells to the effect of fibroblast
interferon differs from one tumor type to another. As repeatedly
emphasized, the exogenous interferol1 treatment for requires
enormous amounts of purified interferon, and not only is its supply limited

(
Ol VL lUPML N' ANO St L I t: IION 01
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UP GROWTH' ROLLER BOHLES

CONIACIINIIIIIIIION

IUN N 111\1
(){iY
ONC()(Jf NICI rv MICI )
LlfE SPAN
l' 1 '( ) 1l11.1 1111 1,' 1" 1'; '1''1'
AGENTS VIHtJSES
MYCOPLASMAS

FlJNGI
AND OF HUMAN 5EHUM DONDHS
IF
IF
,
PURIFICATION


r-..... REAGENTS
STORAGE

PURlflED IF POOllN STQRAGE
4 MONTHS (LOr)
TOTAL
!
flNAL PRODUCT IN AMPUlES I
TOTAL

ANIMAL GENERAL SAFETY (MICE. GUINEA P!GS)
PYROGENICITY (RABBITS)
GENERAL TQXICITY (RATS)
FIGURE 2. Scheme of quality control of human fibroblast interferon based
FDA and regulations. (Reprinted from Ref. 48, 724, courtesy
of American Society for Microbiology.)
but its production is expensive as well. From practical point of view,
therefore, each malignant neoplasm (or type of neoplasm) should screened
carefully for its sensitivity to interferon prior to initiation of treatment of
tumor-bearing patients. The development of simple in vitro screening
test using clinical specimens (or established tumor celllines) would of
tremendous value in the selection of patients for clinical trials. While the
model system of use of xenografts in nude mice has its value, it is not an
ideal one because of the long and large amounts of interferon that are
required for definitive screening tests. Also, as noted previously, it has
been reported that the rate of tumor "takes" in nude mice is not very high
following heterotransplantation of clinical materials, and establishment of
the transplantability of tumor, therefore, becomes major prerequisite
for studies in this in vivo model system. In vitro assays, similar to the
methods for cultured cells described earlier (Section appear to
-1. 1':X()CI':N()IIS IN'I'I':I!YI':I!,()N:

l'UUHiblu up)Jr"OUell ("Ol" HCl"UUrlil1!!: HUHeepiibllity 01" tumors to the anti-
IH"o!i[ur"ulivu aelivity, parLieularly since there is some indication that in
v i,vo al1d vilro rusults the effect of interferon are comparable ([41,
7;)] ; data from our department). Bech- Hansen et al. [100]
described similar method, using incorporation of radioactive pre-
uursors, for measuring drug sensitivity of tumor used
plastic directly from pleural and abdominal perfusions of
cancer patients. simple, reliable in vitro test for determil1ing the
to il1terferol1 of tumor from clil1ical specimel1s is
il1 the developmel1tal stage.
Fibroblast 1l1terferol1 vs. Leukocyte 1l1terferol1
Einhorl1 al1d [44] were the first to 110tice that fibroblast il1terferol1
inhibited the growth of fibroblastic osteosarcoma to much greater
degree thal1 leukocyte il1terferol1. reverse was true in the case of
lymphoblastoid lil1es. Accordil1g1y, they cOl1cluded that il1terferol1 might
ha tissue-type specificity. resu1ts il1dicate l1ew biological properties
of il1terferol1 al1d are of importal1t sigl1ifical1ce for selectiol1 of the type of
il1terferol1 that may useful il1 clil1ical applicatiOl1S. However, more
tel1sive studies are l1eeded before drawil1g defil1ite cOl1clusiol1s 011 the sig-
l1ifical1ce of this phel1omel1ol1, because crude fibroblast interferol1 that was
prepared the superil1ductiol1 method usil1g actil1omycil1 D was employed.
Recel1tly, our group discovered that crude superil1duced interferol1 cOl1tail1ed
actil1omycil1 D-like 11011specific for growth; but after purifi-
catiol1, this activity could 110t detected (8. 8. LeOl1g, J. 8. Horoszewicz,
al1d W. Carter, ul1published data).
Future experimel1ts should dOl1e using purified preparatiol1s. Our
prelimil1ary resu1ts il1dicate that both purified fibroblast leukocyte il1ter-
ferol1s are of reducil1g the growth of 110rmal fibroblasts
at presel1t, additiol1al data are 110t available 011 the tissue-
type specificity of purified il1terferOl1S.
111 additiol1 to fibroblast al1d leukocyte il1terferol1s, there is al10ther
type of interferol1, i. type II or immul1e interferol1, which is produced
lymphocytes stimulatiol1 with either al1tigel1s or mitogel1s
[101-110]. the best of our there have reports of
experimel1tal studies 011 the al1tiproliferative activity of immul1e il1terferol1.
Also, the combil1ed effect of two or more differel1t types of il1terferol1 011
growth inhibitiol1 has 110t described as yet.
From the practical poil1t of view, fibroblast il1terferol1 is prepared
using m0l101ayer cultures of selected strail1s of humal1 fibroblasts, whereas
leukocyte il1terferol1 is il1duced pooled peripheral leukocytes from blood
d0110rs. Fibroblast strail1s therefore, characterized prior to
their use for il1terferol1 productiol1 al1d for this reaSOl1 are cOl1sidered to
safer thal1 per ipheral leukocytes. The purificatiol1 of fibroblast inter-
1 ()/I
I'I'() 1\111" 1,'1';'1''1'
feron is easily accomplislHx! !JY eoLUlI1I1 witll vaL"ious
affinity ligands, described earlieL" (Sectioll COllSidc/"illj4
source of for induction of leukocyte interferon is limited, and
sidering the magl1itude of the effort required for inductiol1 of fibroblast
interferon, production systems for either type of interferon 110t
adequate to produce sufficient quantities of the drug for large-scale clil1ical
applications.
1V. DISCUSSION
early studies interferon therapy in animal systems, the effects of
treatment established virus-induced tumors as well as the progressioll
of viral carcinogenesis in animal systems, i.e., antiviral activity, were
emphasized and provided strong background for the rationale of interferol1
therapy. Now, it has clearly demonstrated that interferon itself is
growth-regulatory substance produced cells and that it affects cells
directly, without apparel1t il1volvement of viruses. It should stressed
that interferon is normal physiological substance with little if toxic
effect normal ful1ctions. It is, therefore, quite unique among
anticancer drugs, which are always associated with strong toxicity causing
severe s ide effects. pursue exogenous il1terferon treatment, prepara-
tion of large quantities of purified il1terferon is prerequisite. This is
the most critical step to overcome for the clil1ical application of inter-
feron to practical mode of therapy. At present, leukocyte inter-
feron is produced using pooled materials of peripheral leukocytes from
blood donors, and fibroblast interferon is prepared the superinductiol1
procedure with poly 1: poly in diploid human fibroblast cultures.
methodology developed thus far is inadequate for the production of the
massive of required for large-scale application.
For this the development of technology for the production
of interferon is critical for the future of interferon the treat-
of
It has that the efficacy of interferon treatment depends
the tumor load in each patient [2,78]. Accordingly, the tumor
burden must minimized other treatments such surgical
From the practical point of view, speculate that interferon therapy
alone might powerful enough to cure certail1 neoplasms in
humans. The development of modalities of combining the of
interferon with other methods of treatment, such chemotherapy, immuno-
therapy, therapy, radiation therapy, etc., should pursued
urgently in model systems. our there are very few
reports in the literature with this approach. Chirigos and Pear-
[111] studied the effect LSTRA leukemia mice of
treatment with and the chemotherapeutic agent bischloroethyl-
10;;
1111.I'osOlII'ea (I\CNU). LL Ilas LktL BCNU W;t:,; c;tp::tble of a1'1'esting
1.lliH sysl.olllic ICllkclllia ill CI)I<'-lll1ico Lcmpora1'ily, with 70-80% ofthe
1.I't,al.c<1 111 i<,c eolap:,;c Iol1owing 2-3 weeks 1'emission in the p1'og1'es-
iJ 1011 Ui:,;c;t:,;c. inte1'fe1'on t1'eatment was combined with BCNU
t.I<,aLlI1cI1L, :,;u1'vival of the leukemic mice imp1'oved conside1'ably
1'1'0111 25% to mo1'e than 90%. We have to emphasize that, in this pionee1'ing
IILlI<ly, inte1'fe1'on t1'eatment alone had effect at this systemic leu-
ia in mice. Most 1'ecently, the efficacy combined of inte1'-
l'et'oJl with cyclophosphamide has 1'epo1'ted [112]. Female mice
(Icvcloped palpable lymphomas p1'io1' to initiating t1'eatment. The
time t1'eated mice, which 1'eceived single injection of
(,yclophosphamide int1'ape1'itonea11y, was 25-29 days, whe1'eas it was 17
(I;tY8 unt1'eated animals. The su1'vival time was fu1'the1' elongated to
;;;! days in the case of combining chemothe1'apy with inte1'fe1'on t1'eatment
(uaily administ1'ation of 3.2 106 U of mouse inte1'fe1'On). The antiprolife1'a-
Live effect of human leukocyte Illterfero11 111 vit1'o has 1'epo1'ted to
with the treatment of ce11s ,vith chemicals such as ouabain, cyclo-
Ileximide, and pu1'omycil1 [45,46].
Su1'p1'isingly el1ough, the1'e a1'e references available in the lite1'atu1'e
eegarding combination of the modalities of i11terferon with immunothe1'apy.
Seve1'al inte1'esting obse1'vations, however, have described that imply
significant 1'ole of the immune systems of the tumo1'-bea1'il1g host 1'eceiving
il1terfe1'on therapy [113]. Inte1'fe1'on inc1'eased phagocytosis of ca1'bon
pa1'ticles macrophages [114,115]. It has 1'epo1'ted that phagocytosis
of tumo1' cells peritoneal macrophages was obse1'ved toward the sixth
day following i11oculatio11 of Eh1'lich ascites 01' RC 19 tumor ce11s in il1terfe1'on-
t1'eated mice, but not in unt1'eated mice [31], Fu1'the1'm01'e, surviving
interfe1'ol1-treated mice showed enhal1ced specific resistal1ce to 1'e-
injectiol1 of tumo1' ce11s. Othe1' important fil1dil1gs suggest that the antitumor
action of interfe1'ol1 might mediated in part host facto1's [116,117].
clone of inte1'fe1'on-1'esistant ce11s was isolated f1'om mu1'il1e leukemia
L1210 cells prolonged cultivation in interfe1'ol1-col1taini11g medium.
Inte1'fe1'on was to inhibit the multiplication of these 1'esistant ceHs
il1 vitro. However, mice il10culated with the 1'esistant ce11s we1'e protected
against neoplastic disease daily administratiol1 of i11te1'fe1'o11. Since these
resistant ce11s did not 1'eve1't to inte1'ferol1 sensitivity in vivo, the results
were i11te1'p1'eted as evide11ce that the a11titumor effect of interferon in this
system was mediated the host rather tha11 di1'ect actio11 011 tumo1'
growth.
Evidence has accumulated that i11terfe1'on is of modulating host
immU11e mechanisms, involving activatio11 of macrophages and va1'iety of
lymphocytes (see Chapte1' 11). Inte1'fe1'on activate syngel1eic 01'
geneic, se11sitized or 110nsel1sitized lymphocytes, subsequentlyenhal1ce
the lysis of tumor ce11s, and the1'eby supp1'ess tumor g1'owth [118,119].
Recel1t studies 1'evealed that mac1'ophages played important 1'ole
[0(;
mcdiated wcll I"ot' (Ii:-;-
eases [120]. It is well knowt1 eCL'lait)
such as BCG and products derived from them, tLlO
tem is stimulated, resulting in intcnse hypertrophy 01 Hplcen and liver.
Animals treated in this way are subsequently more resiHtant to challengc
with microorganisms as well as tumor cells. This nonspecific immuno-
therapy for malignant neoplasms has been extensively studied in recent
years [121]. the other hand, the view that specific immunological
reactions play an important role in modifying, or even controlling, neo-
plastic diseases, has now gained wide acceptance. Many experimental
tumors, including those induced viruses and chemical carcinogens, and
those of unknown etiology elicit immune-rejection responses against tumor
cells [122]. Animals previously immunized against tumor cells are found
to reject subsequent challenge with the same tumor. investi-
gation of the combination of specific and nonspecific immunotherapy with
interferon therapy for human malignancies merits serious consideration.
Results obtained from such studies should provide more effective and more
powerful ways to apply exogenous interferon in the treatment of human neo-
plastic diseases.
V.
Interferon is physiological substance, produced cells, which does not
cause strong side effects. Interferon is also promising new antitumor
drug. This is supported the results of numerous studies the anti-
proliferative activity of interferon in vitro, as well as its antitumor activity
in vivo. Evidence has been obtained in vitro studies that the susceptibility
of human neoplastic cells to interferon differ from one tumor to another.
Results obtained fL'om clinical test with osteosarcoma argue for the in-
crease of therapeutic trials variety of neoplasms in humans. Exogenous
interferon treatment consists of frequent long-term, large-dose administra-
tions of the drug. Lack of practical method for efficient mass production
of interferon has thus far prevented sufficiently large-scale clinical trials.
Thus, new technology for pursuing the mass production of interferon is
essential and of first priority. Simple and practical procedures for large-
scale of human fibroblast interferon have been developed in
our department, whereas simple purification methods for leukocyte inter-
feron are not described yet. The development of simple screening test
using clinical specimens for determining the susceptibility to interferon
would of tremendous value in the selection of patients for clinical trials.
Studies combining modalities of interferon treatment with other anti-
neoplastic therapies are warranted in order to investigate the capabilities
of increasing the efficacy of exogenous interferon treatment of human malig-
nancies.
10'/

'1'110 LII:LII], of interferon production unit in our depart-
Ur. J. S. l1oroszewicz) for supplying purified human
l'IIJI'ol)lasl They also express their appreciation to Dr. J. S.
Ilot'OHzcwicz for providing normal and neoplastic cultures.

1. Paucker, Cantell, andW. Henle, Virology17:324 (1962).
I. Gresser, Advan. Cancer Res. 16:97 (1972).
3. I. Gresser, in Chemotherapy Cancer: Comprehensive Treatise
(F. F. Becker, ed.), Vol. 5, Plenum Press, New York 521, 1977.
4. I. Gresser, D. Brouty-Boye, Thomas, and Macieira-Coelho,
Proc. Nat. Acad. Sci. U.S. 66: 1052 (1970).
5. I. Gresser, D. Broute-Boye:-M. Thomas, and Macieira-Coelho,
J. Nat. CancerInst. 45:1145 (1970).
6. I. Gresser, Thomas, and D. Brouty-Boye, Nature 231: 20 (1971).
7. Lindahl-Magnusson, Leary, and I. Gresser, Proc. SOC.
Med. 138: 1044 (1971).
8. Jr., J. 56:846 (1973).
9. O'Shaughnessy, Easterbrook, S. S. Lee, L. J. Katz,
and R. Rozee, J. Nat. Cancer Inst. 53: 1687 (1974).
10. I. Gresser, Thomas, D. Brouty-Boye, and Macieira-Coelho,
Proc. SOC. Med. 137: 1258 (1971).
11. I. Gresser, Bandu, and D. Brouty-Boye, J. Nat. Cancer Inst. 52:
553 (1974).
12. Ohwaki and Kawade, Acta Virol. 16:477 (1972).
13. Matsuzawa and Kawade, Virol. 18:383 (1974).
14. L. Borecky, N. Fuchsberger, and Hajnicka:, Intervirology 3:369
(1974).
15. N. Fuchsberger, Hajnicka, and L. Borecky, Acta Virol. 19:59
(1975).
16. R. F. Buffett, Ito, Cairo, and W. Carter, J. Nat.
Cancer Inst. 60:243 (1978).
17. and I. Gresser, Proc. Nat. Acad. Sci.
U.S. 72: 2265 (1975).
18. J. Hilfenhaus, Damm, Karges, and F. Manthey, Arch.
Virol. 51: 87 (1976).
19. Fuse and Kuwata, J. Gen. Virol. 33: 17 (1976).
20. Nature 260:141 (1976).
21. W. Stewart, I. Gresser, G. Bandu, and S. L.
Goff, Nature 262: 300 (1976).
22. Jr., Nature 262:302 (1976).
JOH I'I'(J 1\\11,'1,'1':'1''1'
23. 1';. 1'. Nlalaisu, alltl 1. (;I'USSt,,',
105: 73 (1977).
24. I. Gresser, J. }'alcoff, and }'olltail1c, Pr'ou. Sou.
Biol. Med. 124: 84 (1967).
25. I. Gresser, J. D. }'ontaine-Brouty-Boye, and l,'alcoff,
Nature 215: 174 (1967).
26. I. Gresser, R. Falcoff, D. Fontaine-Brouty-Boye, F. Zajdela,
J. and Falcoff, Proc. Soc. Biol. Med. 126: 791
(1967) .
27.1. Gresser, J. andC. Bourali, J. Nat. Cancerlnst. 43:108;\
(1969).
28. S. Graff, R. Kassel, and Trans. N. Acad. Sci. 32:
545 (1970).
29. Came and D. Moore, J. Nat. Cancer lnst. 48: 1151 (1972).
30. 1. Gresser, Bourali, J. Levy, D. Fontaine-Brouty-Boye, and
Thomas, Proc. Nat. Acad. Sci. U.S. 63:51 (1969).
31. I. Gresser and Bourali, J. Nat. Cancer lnst. 45:365 (1970).
32. 1. Gresser and Bourali-Maury, Nature [New Biol.] 236: 78 (1972).
33. Cantell, N. Acad. Sci. 173: 160 (1970).
34. S. S. Lee, V. O'Shaughnessy, and R. Rozee, Proc. Soc.
Bi01. Med. 139: 1438 (1972).
35. V. Gaffney, Picciano, andC. Grallt, J. Nat. Cancerlnst.
50: 871 (1973).
36. J. Hilfenhaus and Z. Naturforsch. 618 (1974).
37. Adams, Strander, and Cantell, J. Gen. Viro1. 28:207 (1975).
38. DahlandM. Degre, Nature 257:799 (1975).
39. Kuwata, Fuse, and N. Morinaga, J. Gen. Vir01. 33: 7 (1976).
40. Dahl and Degre, Acta Path01. Microbi01. Scand., Sect. 84:
285 (1976).
41. Strander and S. inhorn , Int. J. Cancer 19: 468 (1977).
42. J. Hilfenhaus, Damm, and R. Johannsen, Arch. Vir01. 54: 271
(1977) .
43. Dah1, Acta Pathol. Microbi01. Scand., Sect. 85: 54 (1977).
44. S. Einhorn and Strander, J. Gen. Vir01. 35:573 (1977).
45. Kuwata, Fuse, and N. Morinaga, J. Gen. Virol. 34:537 (1977).
46. Kuwata, Fuse, and N. Morinaga, J. Gen. Vir01. 37: 195 (1977).
47. J. Fogh, J. Fogh, and Orfeo, J. Nat. Cancer lnst. 59:221
(1977).
48. J. S. Horoszewicz, S. S. Leong, Ito, L. and
W. Carter, Infec. Immun. 19: 720 (1978).
49. S. Flanagan, Genet. Res. (Cambridge)..: 295 (1966).
50. J. Rygaard, Thymus and Self: Immunobiology of the Mouse Mutant
Nude, Wiley, New York, 1973.
51. J. Rygaard and Povlsen, Acta Patho1. Microbio1. Scand. 77:
146 (1969).
I
Povl:-;vll aJlll ,J. At;la Mkrouiol. 8cand., 8ect.
(1!)7.l).
1.:1. 11. J. Mach, S. Carrel, L. Ozzello, and
J. Cvrotlini, Pl'ov. l'il'st Internat. Workshop Nude Mice,
l<'ischer, Stullgal't, Germany, 1974, 269.
1.1. Shimosato, Nagai, S. Hirohashi,
Ilayashi, and Nomura, J. Nat. Cancer Inst. 56:1251 (1976).
:)!). 11. Maguire, Jr., Outzen, Custer, and R .. Prehn,
J. Nat. Cancer Inst. 57: 439 (1976).
;,(;. J. Visfeldt, Povlsen, and J. Rygaard, Acta Pathol. Micl'obiol.
Scand., Sect. 80: 169 (1972).
!;7. Povlsen and J. Rygaard, Acta Pathol. Microbiol. Scand.,
Sect. 80: 713 (1972).
I'H. Giovanella, J. S. Stehlin, and L. J. Williams, Jr., J. Nat.
Cancer Inst. 52: 921 (1974).
:>9. Povlsen, J. Fialkow, G. Klein, J. Rygaard, and
F. Wiener, Int. J. Cancer 11:30 (1973).
Giovanella, S. Yim, J. S. Stehlin, and L. J. Williams, Jr.,
J. Nat. Cancer Inst. 48: 1531 (1972).
Giovanella, S. D. Yim, Morgan, J. S. Stehlin, and L. J.
Williams, Jr., J. Nat. Cancer Inst. 50: 1051 (1973).
Shimosato, Tumuraya, N. Ohsawa, and Nomura,
J. Nat. Cancer Inst. 56: 325 (1976).
R. Schlesinger, J. Gerson, S. Moorhead, Maguire, and
Hummeler, Cancer Res. l.: 3094 (1976).
S. Carrel, Sordat, and Merenda, Cancer Res. 36:3978 (1976).
PovlsenandG. Jacobsen, CancerRes. 35:2790 (1975).
66. D. Houchens, Ovejera, R. Johnson, Bogden, and G. Neil,
Proc. Amer. Assoc. Cancer Res. 19:40 (1978).
67. W. R. Proc. Amer. Assoc. Cancer Res. 19:41 (1978).
68. W. Shapiro, G. Basler, Horten, N. Chernik, and J. Posner,
Proc. Amer. Assoc. Cancer Res. 19:153 (1978).
69. J. S. Horoszewicz, S. S. Leong, Ito, R. F. Buffett, Karakousis,
Holyoke, L. Job, J. G. Dolen, and W. Carter, Cancer Treat-
ment Repts. 62: 1899 (1978).
70. Strander and Cantell, In Vitro Monograph 3:49 (1974).
71. Strander, Cantell, G. Carlstrom, and Jakobsson,
J. Nat. Cancer Inst. 51: 733 (1973).
72. L. Ahstrom, Dohlwitz, Strander, G. Carlstrom, and Cantell,
Lancet.!: 166 (1974).
73. Strander, 35: 277 (1977).
74. Strander, Cantell, Jakobsson, Nilsonne, and G. Soder-
berg, Acta Orthop. Scand. 45: 958 (1974).
75. Strander, Cantell, S. Ingimarsson, Jakobsson, U. Nil-
sonne, and G. Soderberg, Fogarty Int. Center Proc. (Washington,
D.C.) 28:377 (1977).
"
76. (;aIlLclJ, S. l'l!!:i'lla"HH0I1, 1'.
Jakobsson, and U. Nilsonac, J. 111fc\;. OiH. 1:\:\ (Sllppl. (1\)7(;).
77. Blomgren, Caatell, 13. Johanssoa, U. .
and Straader, Acta Med. Scand. 199:527 (1976).
78. D. Stringfellow, 1afec. 1mmlla. 13:392 (1976).
79. W. Carter aad De Clercq, Scieace 186: 1172 (1974).
80. F. Torrence and De Clercq, Pharmacol. Therap. 1 (1977).
81. 1. Freeman, N. Al-Bussam, J. L. Stutzman,
S. Bjornsson, and W. Carter, J. Med. Virol. 1.: 79 (1977).
82. W. Young, Med. Clinics N. 55: 721 (1971).
83. Field, in Selective 1nhibltors of Viral Function (W. Carter,
ed.), CRC Press, Cleveland, 1973.
84. Merigan, Cancer Chemother. Repts. 58:571 (1974).
85. R. Roblason, V. DeVita, Levy, S. Baron, S.
and Levine, J. Natl. Cancer 1nst. 57:599 (1976).
86. D. Stringfellow, Science 201: 376 (1978).
87. J. Desmyter, Ray, J. DeGroote, F. Bradburne, V. J.
Desmet, V. G. Edy, ill iau , DeSomer, and J. Mortelmans,
Lancet 11:: 645 (1976).
88. Cantell, S. Hirvonen, Mogensen, and L. 1n Vitro
Monograph 35 (1973).
89. Mogensea and Cantell, Pharmacol. Therapeutics 369
(1977) .
90. G. L. Dombos, and Gothoskar, 1nt. J. Cancer 10:44 (1972).
91. Mogensen, and Cantell, J. Clin. Microblol.
1.: 116 (1975).
92. J. Bridgen, Anfinsen, L. Corley, S. Base, Zoon,
U. Ruegg, and Bucker, J. Chem. 252:6585 (1977).
93. Zoon, Buckler, J. Bridgen, and D. Gurari-Rotman,
J. Clin. Microblol. 1: 44 (1978).
94. Havell, Yip, and J. Vi1cek, J. Gen. Virol. 38:51 (1978).
95. W. Davey, Sulkowski, and W. Carter, Biochemistry 15:
704 (1976).
96. Sulkowski, W. Davey, and W. Carter, J. Biol. Chem.
251:5381 (1976).
97. W. J. Jankowski, W. von Muenchhausen, Sulkowski, and W.
Carter, 15: 5182 (1976).
98. W. Davey, Sulkowski, and W. Carter, J. Biol. Chem.
251: 7620 (1976).
99. J. Chen, W. J. Jankowski, J. Sulkowski, and
W. Carter, J. Virol. 19:425 (1976).
100. N. Bech-Hansen, Sarangi, D. J. Sutherland, and V. Ling,
J. Nat. Cancer 1nst. 59: 21 (1977).
101. F. Wheelock, Science 149:310 (1965).
1. 1':NI)()(a:No(IS IN'f'I':H I'I':I!()N:

11. 1VI. atKll'. Proc. Biol.
Ml)ll. (1%7).
10:\. J. CrCl)I1, 8. Coopl)rband, and S. Kibrick, Science 164:1415
(1%!J).
101. L. Epstein, J. and Merigan, J. Invest. 50:
744 (1!J71).
105. G. Gifford, Tibor, and D. L. Infec. Immun. 164
(1971) .
J06. R. Falcoff, J. Gen. Virol.l.:251 (1972).
107. J. S. Youngner and S. Salvin, J. Immunol. 111: 1914 (1973).
108. W. WaHen, J. Dean, and D. Lucas, Immunol . ..: 110
(1973).
109. J. Stobo, 1. Green, L. Jackson, and S. Barron, J. Immunol. 112:
1589 (1974).
110. G. R. Klimpel, D. Day, and D. Lucas, Immunol. 20: 187
(1975).
111. Chirigos and J. W. Pearson, J. Nat. Cancer Inst. 51: 1367
(1973) .
112. 1. Gresser, Maury, and Eur. J. Cancer 14: 97 (1978).
113. 1. Gresser, Immunol. 34:406 (1977).
114. Huang, R. Donahoe, F. Goldon, and R. Dressler,
Infec. Immun. 1,: 581 (1971).
115. J. Imanishi, Yokota, Kishida, Mukainaka, and Matsuo,
Acta Virol. 19: 52 (1975).
116. 1. Gresser, Bourali, 1. Chouroulinkov, D. Fontaine-Brouty-Boye,
andM. Thomas, N.Y. Acad. Sci. 173:694 (1970).
117. Lindahl, Leary, and 1. Gresser, Proc. Nat. Acad. Sci. U.S.
69:721 (1972).
118. G. J. Svet-Moldavsky and 1. Chernyakhovskaya, Nature 215: 1299
(1967) .
119. 1. Chernyakhovskaya, G. Slavina, and G. J. Svet-Moldavsky,
Nature 228: 71 (1970).
120. Alexander, Rev. Med. 26: 207 (1976).
121. J. F. Lancius, J. Bodurtha, J. Mastrangelo, and R. Creech,
J. Reticuloendothelial Soc. 16: 347 (1974).
122. R. W. Baldwin, F. R. Path, and R. Price, Ann. Rev. Med.
26: 151 (1976).
5
CLINICAL USE OF INTERFERONS
IN VIRAL INFECTIONS
Alfons and Piet De Somer
[{cga Institute
University of Leuven
Leuven, Belgium
1. INTRODUCTlON
RATIONALES FOR INTERFERON APPLICATION
IN VIRAL DISEASES
RELEVANT ANIMAL MODEL SYSTEMS
IV. TOPICAL APPLICA OF INTERFERON
IN HUMANS
Dermatological Affections
Infections
Stomatological Affections
D. Respiratory Affections
V. SYSTEMIC APPLICATION
Therapeutic Studies
Prophylactic Studies
VI. OF EFFECTS
Fever and Malaise
Hematological Side Effects
Skin Reactivity
VII. DIFFERENCES IN OF
LEUKOCYTE AND INTERFERONS
RECENT STUDIES
IX. DISCUSSlON AND CONCLUSlONS
REFERENCES
113
114
115
116
120
120
121
121
122
122
122
131
133
133
133
135
136
136
137
140
11-1 1111,1,11\11 all<l 1>1': S()IVII':I!.
1.
It has often been said and written that thc clinical illtCI'fcrOll
has so far not materialized because sufficicnt amounts of purificd and
centrated have not been available to pcrform adcquatc clinical
trials. 1n the last five years sizable quantities of both leukocyte and fibro-
blast interferons have available. This has confronted interferon-
ologists with the choice of one or more viral diseases for their clinical
This choice has not an easy especially since initial
pilot experiments have not yielded the spectacular results one had hoped
for. The older workers in the interferon community have nostalgically
recalled the era of the first antibiotic, the clinical potential of
which was demonstrated the spectacular improvement in
just few patients suffering from severe bacterial infections. should
recognize that, in spite of its broad antiviral spectrum in vitro, the clinical
spectrum of interferon is narrow Ol1e, situatiol1 embodied thc epi-
gram "1nterferon is drug looking for disease. "
Most virus infections il1 humal1s, such as rhinovirus and enterovirus,
are self-limited and cause only mil10r symptomatology. The few that
arc life-threatening or invalidating, e.g., poliomyelitis and variola, have
been mastered effective vaccinations that and easy to
implemel1t. Until recently it was thought that, logical basis, the
clil1ical application of interferon (except for its in oncology)
would limited to two kil1ds of diseases:
1. Respiratory and infections, both extremely common diseases
which have eluded the vaccil1ation approach
2. Rare life-threatening conditions such as rabies, Ebola, Marburg,
or Lassa fever, herpes virus infection, extel1sive varicella
or zoster
The rather poor results of the 1975 controlled trial with respiratory infec-
tions [11 reduced hopes for broad application of interferon and temporarily
reoriel1ted the attention to the application in maligl1ancy and in viral
plications of malignal1CY, such as extensive varicella or zoster. suddel1
revival occurred recently when it was found that systemic interferOl1
administration, rather unexpectedly, affected viral parameters il1 chrol1ic
virus infections [2,3]. Agail1, with this disease, the interferon
munity is confronted with rather peculiar dilemma. As will shown,
large amoul1ts of much larger thal1 those currently
available-will needed to establish whether or not interferon therapy
eradicate the virus il1fectiol1 from chrol1ic carriers with or without
signs of disease. For this, large efforts will have to made,
while it is becomil1g increasingly clear that efficiel1t vaccines for virus
!., CI,INI('AI, \1:->1-: IN VlltAI, INI,'I-:C'I'I()N:->
\vill 1)l:COlllC availaJ)le ill fuLure. might there-
CUlTellL e[[oetH il1 carry tl1e risk of serving
of interferon, rather tl1an to broad
11l<tekcL.
111 reviewil1g the available evidel1ce we will try to demol1strate that the
eurrent1y put il1to clinica1 tria1s are worthwhi1e indeed
of bel1efit to 1arge group of patients.
HATIONALES FOR INTEHFERON APPLICATION
lN VlRAL DISEASES
lnterferol1s are biomo1ecu1es evo1ved l1atura1 se1ectiol1 to serve certain
purpose. It is 10gica1 to derive their clinica1 potentialities from the function
they exert in natura1 situation. Therefore, it is important to know which
interferon plays in spontaneous recovery from virus infections.
reviews exist this matter [4,5], and we limit ourse1 ves to
restate the genera1 consensus in this respect. The organism possesses
two important defense mechanisms against vira1 infection: the interferon
mechanism al1d the immune respol1se, cellu1ar as well as humora1, These
mechanisms are comp1emel1tary to each other. lnterferon is produced in
immediate1y after virus infection; in contrast, the immune
response is specialized at site distant from the vira1
infection and requires severa1 days to active. Interferon acts
and de1ays virus replication especially in adjacent to those
ready infected; the immune response, the other hand, kills extracellu1ar
virus partic1es at site in the organism. Certain immune active
are attracted to the site of virus replication. It thus seems that the
essentia1 task of interferon consists in de1aying the progress of viraI inva-
sion, the response to mounted and to effective.
It is that in some virus infections the immune response do more
harm than good [6]; therefore possible ro1e of interferon to limit
the production of vira1 components which constitute targets far immune
attack, and thus to alleviate the immune-mediated inflammatory responses.
designing the interferon nature has apparent1y
fronted with the problem as biochemists current1y invo1ved in designing
antivira1 agents: It is extreme1y difficu1t to interfere with virus replication
without damaging the hast. The fact that the interferon mechanism is
switched off in between virus infections suggests that interferons have
undesirable effects or organ physio10gy. In fact, it seems not
improbable that some of the unexp1ained symptoms of acute virus diseases
are caused interferans. In this context it is worthwhile to note that
interferon to produced during chronic vira1 infections such
the virus carrier state.
.11 (;
itl\iIIH:
In applying exogonous il1ior[erot! ill olillkal silualiollH, HI1Oll1d
expect it to achieve than what is acllieved illlor[orot!
ously produced during virus infections, i.e., delay in virus roplication
and virus spread and alleviation of symptoms. Also, after systemic
administration, one might expect undesired side effects, most likely re-
sembling some of the symptoms occurring during virus infectiolls. Further-
more, one only expect beneficial effect from exogenous interferon if
one can reach local concentrations exceeding those resulting from spontane-
ous interferon production. Local concentrations in infected tissues are
extremely lligh, due to the fact that the interferon is concentrated in the
narrow intercellular spaces [8]. Concentrations reached at distant s ites
are mainly determined the blood levels. In those cases where interferon
applied topically to the site to protected, it quite possible
to reach tissue levels superior to those resulting from the virus infection
itself. For systemic administration to effective, the doses of interferon
must necessarily at least an order of magnitude larger than the total
amount of endogenous interferon. Currently available preparations are
not always potent enough to acllieve this goal.
RELEVANT ANIMAL MODEL SYSTEMS
Various animal studies have been undertaken to demonstrate prophylactic
or therapeutic effects of exogenous interferon. In these studies has
attempted to answer questions of principle. interferon act in vivo?
Does it act when given before the virus or also when the virus has
already replicated? One has also asked questions of more practical
nature. What is the dose necessary? What is the best sequence if repeated
dosages given? Finally, some workers have attempted to use animal
systems which faithfully reflect defined human diseases .
It is of obvious clinical importance to whether exogenous inter-
feron therapy exert beneficial effect when started after the virus has
already multiplied in the target organ. Tllis question has dealt with
in studies involving topical as well as systemic application of interferon.
In earlier studies, as reviewed Finter [9], it appeared that systemic
administration of interferon afford protection against various acute
experimental viral infections. In mice infected with Semliki Forest virus
(SFV) or virus, judiciously chosen regimens
of interferon treatment could increase survival rates when started shortly
after virus inoculation. Tllis is not strictly to say that interferon treat-
ment was effective when virus replication had already occurred. More
recently the question has reevaluated using lligher doses of interferon
and different animal systems. Olsen et [10] found that daily doses of
40,000 to 200,000 U could protect mice against intranasal or
challenge with virus when started 1 hr after inoculation. When started

111' a['Lct' wat; cffective against intranasa1 but
il1trapcritoncal Conceivably, seeding of the target
occurred after than after intranasa1 challenge.
Worthington et [11] found that interferon did not alter mortality in mice
when treatment was initiated two days after infection with SFV. De
and De Somer [12] and Gresser et [13] used intranasa1 of
mice with vesicu1ar stomatitis virus (VSV). Repeated intraperitonea1
injections of surprising1y small doses of interferon (300 arbitrary units)
were found to increase the surviva1 rates even when the treatment was
started five days after virus [12]. Similar results, a1though
with higher doses of interferon, were described Gresser et [13],
when the treatment was started at day 4 after virus challenge. These
investigators a1so determined the time of onset virus replication in the
brain, and they conc1uded that interferon still had effect when given at
time when virus replication had a1ready occurred, However, about 30%
of the mice had evidence of virus replication in the brain at day 4, and
it is 10gica1 to suppose that those mice wou1d preferentially protected
1ate interferon therapy. It seems safe to conc1ude that interferon therapy
does have effect when given in time to inhibit replication at the porta1
of entry; the important question whether it a1so protect when the virus
is a1ready replicating in the remains essentially unsett1ed. However,
it must remarked that, of organs, the brain is probably the most
difficult to reach with exogenous interferon.
same basic question, i. whether interferon has therapeutic
potentiality, was approached using topica1 application. et [14]
were unsuccessfu1 in attempting to protect rabbits against rabies virus
infection intraventricu1ar administration of interferon. 1n contrast
Hilfenhaus et [15] reported partia1 protection against 1etha1 rabies in
monkeys receiving intramuscu1ar intra1umbar injections of human inter-
feron starting 2:24 hr after inocu1ation. Rabies virus replicates and spreads
slow1y. the effect seen in this study shou1d considered as
phy1actic rather than therapeutic. Significant1y the surviving monkeys,
which had received intramuscu1ar injections of interferon, failed to develop
antibody, indicating that periphera1 virus replication had inhibited;
intra1umbar interferon therapy did not prevent the antibody response,
indicating that the target organ was protected.
Experimenta1 infections are particu1ar1y re1evant to the clinica1
applicability of interferon in humans. Recurrent herpes is problem of
major importance in ophtha1mo10gy. Both rabbit and monkey mode1 sys-
tems have used to study the effect of application of interferon.
Regu1ar treatment started short1y before, but not after, herpes simp1ex
virus (HSV) inocu1ation is to protect the eyes monkeys against
u1ceration [16]. fibroblast and 1eukocyte interferons were found
to essentially equiva1ent in the monkey model [17]. Of especial interest
is rabbit recurrence used Sugar et [18]: 70 survivors out of 200
1.l11 1111.1.11\11 1l11(11)1'; S()IVI1;I{.
H8V-inoculated rabbitc; wcrc trcatcu ()!" !"aIJIJil illlC!"I"Cl"OI1
observed for recurrence tl1C ocular lec;ionc;. No c[[cel waH
seen, despite the use of rather potent interfcrol1 prcparatiol1. '1'110
vestigators pointed out, however, that interferol1 also had 110 effect
primary infection in rabbits, while good results were obtained il1 monkeyc;.
et al. studied the effect of human leukocyte interferon
primary herpetic keratitis in rabbits and found reduction in the infectivity
of the inoculum which was dependent the concentrations of interferon in
the drops but not the total dose administered.
80 far we have discussed experimental models in which the disease
(or death) of the animal is due to rather sudden replication of virus in
target organ and to the resulting acute inflammatory response. It is sur-
prising to note how little attention has paid in these studies to the
question of whether endogenous interferon was produced as result of the
virus challenge. It is that some of the viruses used as challel1ge
il1 protectiol1 experimel1ts, g., virus, il1duce circulatil1g il1ter-
ferol1. Perhaps late il1terferon therapy is ineffective because it has litt1e
to add to the il1terferol1 which is already present.
In recent years it has increasingly clear that some pathological
conditions il1 humans are due to persistel1t, oftel1 low-grade, replicatiol1 of
certain viruses. The tissue damage occurring il1 these persistel1t viral
il1fections is often ascribed to the everlastil1g immul1e attack 011 the viruses
al1d the virus- il1fected cells, rather thal1 to the cytopathic effect of the
viruses themselves [6, Various al1imal model systems exist il1 which
chronic viral il1fectiol1s lead to tissue damage al1d early death. effect
of il1terferol1 il1 chrol1ic il1fection of mice with muril1e leukemia viruses
(MLV) was studied several groups, as reviewed No
el1dogenous il1terferol1 is produced in these al1imals. adminis-
tration of large of exogel1ous interferol1 delayed the development
of disease but did not cure the animals, of which evel1tua11y died of their
leukemia. It is 110t clear whether the delay il1 disease was to
attributed to suppressive effect of virus replication or
of tumor growth.
chronic mice is that lactic dehydro-
genase virus was detected for 48 hr after
of the virus [20 When interferon was gi ven from
days 5 to 8 after the blood titers of LDV were lowered. Arrest
of resulted rise to titers. This seems to
that inhibit the of virus
cells vivo. This relevant to the clil1ical
of Reduced productiol1 of viral
alleviate the immune-mediated inflammatory respol1ses to the persistel1t
However, this pos sibility , one should extremely
cautious that reduction infectious titers is necessarily due to
reduction the synthesis of viral It has amply been
: . CI,INICi\I, 11:->1': IN VIH.i\I, INI"I';('TI()N:-> 1 J
ill viLto, LllaL lloUH 110L <t[[ut.:L ratc the major viral
1)I'oLeillH ill ellrol1kally witll L V, a1though it reduces the
11UI1lIJC1' of parLkle::; rc1eased these [21-24].
elaH::;it.:a1 l1lode1 of vira1 persistence is the persistent
illfection of mice with lymphocytic choriomeningitis (LCM)
r01e of interferon in this mode1 has not studied to
extent. Wagner and 8nyder [25] failed to detect interferon in mice
acute1y or persistently infected with LCM virus. contrast, Riviere et
l26] did fil1d interferon during acute infection. effect of interferon
the deve10pment of rena1 involvement occurril1g late during has, to our
knowledge, not studied. 8crapie is the animal model system for the so-
called "subacute spongiform virus encephalopathies," of which Creutzfeldt-
Jakob disease and kuru are the representatives. No interferon was
found in serum, spleen, or brains of mice affected with scrapie, and long-
term admil1istration of interferon did 110t affect the progression of scrapie
in mice [27,28].
the therapy, we
that the possesses to switch off
synthesis in virus thereby implying that
ul1desired effects or physiology. harmful effects
of associated m01ecules) exemplified in two al1imal
model systems. Gresser et al. [29] that high doses of
caused liver death mice. When
was limited to the first week of life, most mice re-
covered. However, several of these survivors developed
progressive complex
[30J. disease symptoms mice resembled those occurring
after with LCM virus. Therefore the investigators hypothesized
that is responsible for part or of the symptoms
of acute LCM virus mice [28]. Their hypothesis
was supported the observation that high levels of were being
produced during acute LCM virus and that specific
antibody aHeviate the symptoms reduce the mortality in LCM
virus suckling mice. other experimental g.,
mice, of newborn adult mice with serum
the attesting to the of in the
of the host [31,32]. Thus the effect of
associated molecules seems to most
further test this hypothesis, mouse eggs were treated with
order to detect possible effect the blastula
effect was BiHiau, De80mer,
data) .
80 far effects of il1terfe1'ol1
ported in adult except for study, Heremans' g1'oup
in which NZB NZB/NZW-F
j
mice we1'e t1'eated fo1' seve1'a1 with

higll doses of illier[erol1. NZB alld NZU/NZW-I"l lIIice
develop an auto-immulle disease some aspeets wlliell l'eHCmble
lupus erythematosus (SLE) in humallS. One hypothesis is that
oncornavirus involved in the pathogellesis of the disease. Treatmeni
with illterferon might inhibit the replicatioll of this virus alld thereby delay
the development of the disease. However, in contrast to this expectation,
both NZB and NZB/NZW-F 1 mice developed disease symptoms faster when
they were treated with interferon thall when mock preparation or plain
salille was givell. In this regard it is perhaps worthwhile to mention
hypothesis holding that the pathogellesis of SLE in mice and humans is pri-
marHy due to imbalallce in prostaglandin (PG) metabolism [33]. It has
recently found that interferon stimulates the synthesis of prostaglandin
(PG)E [34]. It interesting to find out whether the toxic effects of
interferon in young and adult mice are mediated the prostaglandin system.
IV. TOPICAL APPLICATION OF INTERFERON
IN HUMANS
Dermatological Affections
Although topical application to the skin constituted the first attempt to
demonstrate protective effect of interferon in man, this approach has not
been followed many investigators. Early studies, as reviewed
Finter [35] and Merigan et al. [36], showed that intracutaneous injection
of monkey interferon prevented the development of vaccinia lesions in
injected area. An attempt to treat single case of vaccinia gangrenosa
failed. early studies were done with interferon preparations of low
potency and were not repeated using higher dosages. In any case,
injection of interferon in the skin is inpracticable for controlling extended
virallesions of the skin. Topical application in form of an ointment
was attempted Ikic and his co-workers for number of viral skin lesions.
In uncontrolled study treated 11 patients with persistent ulcers after
revaccination with vaccinia virus [37]. Treatment was started around
42 after vaccination; improvement was seen around day 4 of treatment,
and healing was complete around day 12. Another uncontrolled study in-
volved 69 patients with recurrent labial herpes and 69 with recurrent genital
herpes [38]. The general impression from this study was that the develop-
ment of blisters could aborted early application and that the healing
of existing lesions was accelerated and intervals between recurrences
wel'e prolonged. The same authors also attempted to cure condylomata
accuminata (genital warts) [39]. It is generally assumed that these benign,
but potentially malignant, tumors are caused
persist for months or years but eventually regress spontaneously. The
usual surgical and chemical treatments do not always yield satisfactory
:'. IJSI-: IN INI,'I,:C'I'IONS
1:6.1
I'CHL11 LH ;tllll ;tt'C il1 [;tt:L iI11POHHi[J1c for' localizatiOnH. Papillomatosis
iH frcqucntly Iound in children to mothers who had
I4cl1iLal aL tlle delivery. double-blind, p1acebo-controlled
Htudy :60 patients, lkic et al. found regression of fue tumor (in 4 to 12
in 10 patients treated with interferon-containing ointment. Re-
I4ression occurred in 3 out of 10 p1acebo-treated patients [39].
These results should encourage further investigation. view of the
unpredictable c1inical course of the skin 1esions, the need for double-blind,
placebo-controlled studies is evident.
lnfections
of in the has used severa1 groups to treat
viral infections of the cornea or the conjunctiva. Earlier studies, as
viewed Merigan et al. [36], yie1ded favorable impressions for herpetic
or vaccinal keratitis. double-blind, p1acebo-controlled study involving
95 patients, Kaufman et al. [40] did not find prophylactic effect of leuko-
cyte interferon (potency 64,000 U/ml) fue of herpetic kera-
titis. Jones et [41] treated 38 patients with mechanica1 debridement
and eifuer 1eukocyte interferon to see whether the addition of
interferon would reduce the recurrence rate. Although the potency of the
interferon preparation in this study was high, i.e., 10 million units
the recurrence rate was not significant1y reduced. Sundmacher et al. [42-
45] did similar stLtdies comparing the effect of debridement with
debridement plus interferon, and wifu interferon only. first study [42],
interferon (potency 62,000 U/ml) was found to have effect. 1ater
studies [43-45] these authors found that 1eukocyte fibroblast interferons
of higher potency (2:,1 Significantly accelerated healing and inhibited
shedding when given topically in to debridement.
Interferon instillation has a1so used in trial wifu keratoconjunc-
tivitis adenovirus [46]. During epidemic in Zagreb (Yugoslavia), 70
patients were given 1eukocyte interferon while 72 received conventiona1
treatment. When fue treatment was started in the phase of disease,
healing was observed. During the same epidemic, three cases
occurred in clinic; prophylactic instiHation of interferon was given
to 28 contacts, four of which seroconverted but did not develop clinical
disease. These resu1ts need confirmation in controlled double-blind
randomized trial, especiaHy since rafuer weak preparation (4000 U
gram of suspension) was used.
Stomato10gica1 Affections
Topica1 application fue mucosa has used in controlled study,
involving 34 children, to demonstrate beneficial effect the course of
herpetic gingivotomatitis [47]. The authors c1aimed statistically
1111,1,11\\/ alllll)I'; S()IVII';lt
ill 01' Ic:;ioll:; ill Cllil(II'()11
12 daily doses of 2000 U of lyoplliliz()u [Ol' /1-(; uay:;,
D. Res piratory
Attempts to control respiratory tract of aerosolizeu
were after decisive trial l1] was
pleted at the Unit of the British Medical Research
This and trials have reviewed elsewhere [35,36]. 111
summary, sil1g1e day of pre-exposure treatment (16 doses of 50, 000 U
of leukocyte did reduce the severity of al1 influenza virus
infectiol1. greater daily dosage al1d combil1ing day of
pre-exposure with three days of post-exposure treatmel1t (total of 14 MU
patient), statistically s reductiol1 il1 clil1ical symptoms and
virus sheddil1g was achieved il1 volul1teers challel1ged with rhil10virus 4.
This of bel1eficial effect of il1terferon frequel1t,
distressil1g, al1d ecol1omically important disease has, to knowledge,
not beel1 put into practice; 110r have attempts to further improve the
treatment schedule made. The reasons for this obvious. First,
the treatmel1t schedule is tedious, requir il1g 10 applicatiol1s day.
tlle treatment would have to started before exposure to the virus.
in l1ature, exposure at al1Y particular time call11ot foreseen, the
treatmel1t would have to givel1 the whole autumn winter period,
whel1 respiratory most Third, at 10 MU
sure, would cost about $500; six-mol1th continuous treat-
mel1t would cost ul1realistic $30,000. The most optimistic perspective
would that the costs of il1terferon drastically (at least 100-fold)
reduced and that suitable vehicle cal1 developed which allow
ferol1 to kept in permal1el1t with the l1asal mucosa usil1g 0111y Ol1e
two per day. Only 011 these terms cal1 Ol1e expect il1terferol1
to broadly used prophylactic agail1st commOl1 cold or other respira-
tory infectiOl1S.
V. SYSTEMIC APPLICATION
Therapeutic Studies
The idea that systemic application of il1terferol1 might useful il1 the
therapy of gel1eralized viral disease stems from the cOl1cept that, duril1g
l1atural virus il1terferol1 is released early at Ol1e site of virus
replicatiol1 with the "purpose" of protectil1g secol1dary sites of viral inva-
al1d Exogel1ous il1terferol1 givel1 at al1 early phase of
virus disease, i.e., before il1vasiol1 of critical target organ, there-
; . ('I,INi('!\I, IN VIH.!\I,
l'OI'c <Jel:ty il1vu.siol1 Lllu.L ocvcL"al days, allowing the organism
Lo <levclop and to eradicate the virus before extensive
Lisouc occurred. This reasoning is limited several considera-
Liollo:
1. The amount of exogenous interferon should larger an order
of magnitude than the amount of endogenous interferon generated
as result of the infection.
2. The exogenous interferon should reach the target organ in due time.
The shorter the time interval between the first signs of disease
(symptoms resulting from infection of the portal of entry) and inter-
feron administration, the better the effect
3. exogenous administration of interferon should resu1t in suffi-
ciently high tissue levels in the target organ. Some organs, such
as the central nervous system, not reached at inter-
feron injected intravenously intramuscularly.
Studies the systemic use of interferon in the therapy of viral dis-
eases have been confined to varicella zoster and hepatitis virus infections.
Patients with other virus infection been treated sporadically.
Varicella Zoster Studies
At Stanford University large trial was conducted to evaluate the use-
fulness of systemic interferon administration in varicella zoster infections
in immunosuppressed patients. 1n preliminary study done mainly to
evaluate tolerance and pharmakokinetics, the experimenters were encour-
aged the finding that rather high blood interferon levels could realized
[48]. However, the interferon did not penetrate in the vesicle fluids. Sub-
sequent placebo-controlled, double-blind trials [49,50] involved three
groups of patients:
1. Early disseminated zoster in malignancy (first two days). These
patients were treated intramuscularly for three days with either
0.04, 0.17, 0.51 MU/kg/day. No benefit was noted with the
lowest dose. With the middle and highest dosage schedules there
was significant reduction in disease progression as measured
of patients and of days.
2. Early localized zoster in malignancy (45 interferon-treated and
45 placebo-treated patients, mostly lymphoma, first 7 days [50]).
These patients received the same treatment schedules as the first
group. With the highest dose distal cutaneous dissemillatioll
occurred; with the middle dose level the incidence of dissemination
was reduced. Patients receiving the highest dose also had shorter

:(1)(1 1>10: S()lvll';II,
course new iclc llc L'maLOI1\C. 'l'11c
incidence of viscera1 complications toLalleu ouL 1"
interferon-recipient groups against six out of 45 il1
groups. Postherpetic neura1gia appeared reduccd in
receiving the middle and higher dosage schedules.
3. Early (first three days) in children with malignant diseasc
most1y 1eukemia) received either of the two lower dosage leve1s
for three days [49]. Visceral complications were reduced from
six out of nine to two out of nine. The numbers were too smaH to
distinguish differences between the low and high dose interferon
groups.
An open placebo-controlled study Emodi et [51] inv01ved 37
patients with herpes zoster, six of which had confirmed tumor. l11terferon
was given to 28 patients i11tramuscu1ar1y in one daily dose of 1 MU for 5-8
days. Interfero11 was detectable i11 the skin 1esions (vesic1e fluid) before
the injections; 1 hr after the injectio11s the titers were increased approxi-
mate1y 10-f01d. 111 the interferon-treated group the duration of pain was
shortened and the lesio11 seemed to hea1 more quickly.
Studies Viral Hepatitis
In number of infected patie11ts, hepatitis virus virus)
persists i11 the liver cells in as-yet-u11defined form. Its activity is
manifested the prese11ce of 1arge amounts of vira1 surface antigen (HEsAg)
in the blood a11d sometimes the presence of virio11-associated DNA poly-
merase. In some patie11ts the virus persiste11ce leads to chro11ic active
liver disease (CALD). This condition ultimately leads to porta1 cirrhosis
of the liver. Childre11 born from mothers WllO carry infectious virus
duri11g pregnancy are i11variably i11fected a11d deve10p chronic carrier
state. The prognosis of this condition is 110t fully known as yet, except that
small percentage of such childre11 deve10p hepatoma.
The idea to treat these patients with interferon stems from three con-
s iderations:
1. Although active virus replication takes in DNA polymerase
positive patie11ts, e11dogenous interfero11 seems to produced.
U11der these c011ditio11S, exogenous interferon is more like1y to
be11eficia1 than in acute infections where sizable amounts of en-
dogenous interferon are generated the infection itself.
2. In some chronicaHy infected interferon inhibits virus
despite the fact that the viral genome is i11tegrated i11 that of
the which are infected with
Retroviridae show reduced release of virus particles after treat-
!'. 11:-;1': IN INI,'I';C'I'loN:-;
1IlUI1L wiLlI slIIaH l
that the synthesis of the
proteins and vira1 RNA is not inhibited [22,23,24].
inhibition of partic1e re1ease is thought to result dis-
turbances in processing of vira1
3. presence of HBsAg and of DNA in the enables
one to quick1y detect an inhibitory effect of interferon therapy
vira1 functions.
Studies invo1ving the of interferon to patients with positive
HB-vira1 are in progress in severa1 centers. We here
rize the evidence available through publications and or informa1
as well. When this book appears, of this information and
possibly been published. conc1usions of this survey
necessarily tentative.
first, and so far convincing, evidence of an effect of inter-
feron vira1 activity in chronic virus infection has workers
at Stanford University. Greenberg et a1. [3] treated four patients
with 1eukocyte interferon. Three of had constant and high 1eve1s of
partic1e-associated DNA in the Doses of 0.3 to 3 MU/day
were associated with rapid and reproducible in DNA
activity, HB-core antigen and Dane-partic1e-associated DNA in
the effect was transient when the interferon was given for 10
days or 1ess but appeared to permanent when the interferon
tration was pro1onged for or In further studies, the
group of inves tigators Merigan, persona1 found that
DNA-partic1e-positive patients treated for with doses
in the order of of 3 MU/day (e.g., 800 MU over period of 8
responded in three possible ways. Out of 11 patients six showed
reversible, partia1 reduction in the of Dane partic1es in the blood
with change in 1eve1s. Three showed disappearance
of detectable Dane partic1es and partia1 reduction in 1eve1s. Finally,
two out of the 11 patients showed permanent disappearance of detectable
Dane partic1es and HBsAg. Scullard and his co-investigators [5la] re-
ported five patients with active virus infection (three with CALD
and two with e-antigen positive persistent infection) they treated
for five weeks with 3.5 MU of 1eukocyte interferon. Transient
decreases occurred in the of circu1ating Dane partic1es, in the
1eve1s of DNA and of e-antigen.
So far two research groups used fibroblast interferon to treat
CALD patients. As of this writing nine patients received fibro-
blast interferon prepared at our Institute (Table 1). Four of these patients
(Nos. 017, 018, 019, and 020) were followed Weimar et a1. [52-54] at
the Dijkzigt Hospita1 (Erasmus University, Rotterdam, Nether1ands)
1. Viral and Disease Parameters in Nine Patients \vith Treated with Interferon
Patient Concurrent favorable evolution of
code Consecutive Polymerase Transaminase in
No. treatments
a
levels levels hepatocytes Follow-up
031 F
a

No Yes Normalized transaminases; perma-
nent decrease in hepatocyte
003
Fb
Yes Yes Yes Polymerase remains undetectable;
transaminases normalized; arthriti"
019 F
c
and Ld Yes (L) (F/L) ? Polymerase lo\vered during interim
period (F Ld)' remaining undetect-
in follow-up
018 F
e
, F
c
' and Ld Yes (F/L) Yes (F) ? Improvement transient
020 F
c
and Ld Yes (F/L) Yes (F) ? Improvement transient
033 F
f
Yes No ? Improvement transient
034
Ff
No Yes ? Improvement transient
002 F
g
, Fh' and L
f
No No ? Polymerase lowered 10-fold
interim period (F
g
, Fh); change
during follow-up
017 F
e
No No ? No
aF = L = leukocyte F
a
= 7 10 in 2 weeks; Fb = daily injections 4 \\'eeks,
7 1 7 3 7 1 and 7 3 F = 14 2. 2 (2 weeks); Ld = 14 3 (2 \veeks); F = 3 :2
3 4 and 3 8 in 3 consecutive weeks; Ff. Lf = 7 3 in 1 week; F
g
= daily injections for 3 \yeeks,
7 0.3 7 1 and 7 3 Fh = daily injections for 2 weeks, 7 3 and 7 10
be-Antigen undetectable before treatment.
: . IN

00:;, Uei:5myter et al. [2; also
il1 lJl'cparatiorl] aL LJlC Avadcmic llospital of the University of Leuven.
801nc paLients rcceived more than treatment course, including
Icukocyte interferon for comparison. two out of the nine patients,
occurred in the disease parameters; in seven out of nine, favor-
evolution of viral or disease parameters occurred during or shortly
after of the treatment courses; in three of these seven patients, the
improvement was permanent for the of follow-up. evolution of
patient No. 031 has described in detail elsewhere [2]. During and
shortly after the treatment decrease in staining of hepatocytes
was observed. Over three years following treatment this patient has evolved
into chronic HBsAg carrier; at the time of this was
detectable in the hepatocytes, and serum transaminase levels were normal-
ized.
Patient No. 003, 50-year-old had suffered from chronic active
liver disease for 2 years before interferon treatment. Serum transaminases
were elevated [glutamic oxaloacetic transaminase (GOT) , 80 U/liter;
glutamic pyruvic transaminase (G - 70 with two episodes of
aggressive disease and 15 months before treatment with interferon)
characterized peaks in serum transaminase levels to 1500 (GPT) and
2000 (GOT) For this the patient was being treated with prednisone
(15-30 mg/day). decision to administer interferon was taken the
basis of high particle-associated DNA polymerase level in the serum.
1ncreasing doses (Figure 1) were given over period of 4 weeks, and serum
samples were taken daily. samples were assayed s imultaneously. 1t
appeared that DNA polymerase levels had already decreasing before
the treatment was started. During the first three weeks of treatment (0.1
to 1 MU/day) further decrease was seen. However, in the last week of
treatment (3 MU/day) DNA polymerase undetectable. Simu1taneously,
increase in level was observed. HBsAg levels were increasing
steadily as were the levels of serum transaminases. the follow-up period
(seven months, at the time of this DNA polymerase remained un-
detectable, decreased, and transaminases normalized. It
important to note that nondestructive developed in the course of
this period.
third patient (code No. 019), in whom favorable evolution occurred
during interferon treatment, was 35-year-old woman with confirmed
for years. She received 14 daily injections of 2.2 MU of fibroblast
interferon and 14 daily injections of 3 MU of leukocyte interferon, with
interval of two months. During the treatment with fibroblast interferon the
DNA polymerase level did not change, but the transaminase levels were
transiently decreased. the interval between the two treatments the DNA
polymerase levels decreased and e-antigen undetectable. During
the second tl"eatment course (leukocyte interferon) DNA polymerase levels
remained low and transaminases decreased. Shortly after the treatment

1111,1,11111 :llllll)/'; S()!VII';!1.
3
2 -
100
75
50
25


-5
-1

-30 -10
5 10 15 20 25 40 80
I (days after first interferon dose)
FIGURE 1. Evolution of viral and clinical parameters in patient with
(code No. 003) during and after greatment with fibroblast interferon.
!>oIYll1()I,'aH() cOlltillued to llor-
maJiz(), Lim() UJiH wl'iLi!lf.\' (thl'ee mOllths after the last illterferoll
(JOHC) levelH were llormal,
111 patic!lts (Nos. 018, 020, 033, alld 034) the changes ill viral and
lul1ction parameters were ambiguous. trallsiellt decrease in trans-
arnillase levels occurred in thl'ee out of the four patiellts treatment
with fibl'oblast but 110t with leukocyte il1terferol1. Also ill three out of the
four patients the DNA polymel'ase levels showed and unconvincing
decrease.
Finally, in two patients (Nos. 002 al1d 017) fibrob1ast il1terferol1 had 110
effect 011 polymerase or transamil1ase levels. of these refractory
patients (002) also received leukocyte interferol1 without effect.
Fibroblast has also used Kil1gham et al. [55] to
treat two patients ;vith documellted The patiel1ts received 10 MU
daily for two weeks. Both patiel1ts were e-alltigel1 llegative before treat-
mellt. The levels showed decrease duril1g treatrnel1t.
The most strikil1g fillding was 64-fold faH ill agaillst
occurring durillg treatmel1t and beillg the period.
Furthermore, the transamil1ase levell10rmalized il1 patiellt.
It is impossible to say whether the favorable evolution seell ill some of
the patiellts was effect of illterferon treatmel1t. little is about
the spolltalleous evolutioll of DNA polymerase levels ill patiellts to
exclude the possibility that the llegativatiol1 occurred spolltalleously rather
as result of the illterferoll admillistratioll. However, the
resu1ts suffice to justify large-scale, double-blilld, placebo-colltrolled
trial. It also appears that fibroblast illterferoll is probably as effective
(or as illeffective) as leukocyte illterferoll; future trials should therefore
illclude both il1terferOllS. could save time alld effort cOllcertil1g the
trials USillg sillgle placebo group.
Pilot 8tudies ill Various Acute Viral Illfectiolls
Drug trials illvolvillg relatively lal'ge of patiel1ts are usually
preceded pilot tests with single patiellts. Ulldoubtedly, number of
patients with various affectiol1s have received il1tel'fel'ol1, but they were
l1ever reported ill the literature because 110 effect was seell. Leukocyte
illterferoll was used to treat c0l1ge11ital or acquired cytomegalovirus illfec-
tiol1s [56,57]. These u11co11trolled trials yielded el1couraging results i11
that viral and disease paral11eters evolved favorably. As fibrob1ast i11ter-
feroll available ill large qualltities, we have treated few patiellts
with various diseases of viral or presumed viral origil1. The purpose of
these studies was to obtaill prelimi11ary i11dicatiolls which could orient us
to cOllce11trate l110re efforts 011 or al10ther disease. The resu1ts of
these illvestigatiolls are sUl11marized i11 2. The diseases treated
illcluded acute alld fu1l11inal1t hepatitis virus cuta11eous,
larYl1geal, alld vellereal alld patiellt with l11ultiple sclerosis.
2. Pilot Trials with Fibroblast Interferon in Patients with Various Diseases of Proven or Viral Etiology
Patient Interferon treatment
code No. of Avg. dose Duration
No. (yr) Sex Diagnosis injections (days) Outcome
025 43 F Acute hepatitis 14 2.0 14 Recoveryas expected, HBsAg
positive for. 3 months
026 40 F Acute hepatitis 14 2.0 14 Recoveryas expected, HBsAg
positive for 3 months
027 48 F Acute hepatitis 14 2.0 14 Recovery as expected,

for 2 months
024 20 Fulminant hepatitis 10 3.5 15 Recovery, HBsAg positi\-e for ;)
months
028 40 F Fulminant hepatitis 6 6.0 6 Died 011 day 7
029 25 F Fulminant hepatitis 6 3.5 6 Recovery
030 31 F Fulminant hepatitis 5 4.4 5 Died day 5
032 45 F larynx 13 3.5 18 Partial regression, follo\\-ed :'.-
rejectioll
005 35 F Multiple warts in renal 14 5.9 24 Regression of small
transplant carrier planae, not of large \\-arts
037 25 Multiple \varts 8 3.5 14 No effect
038 18 F Multiple plantar warts 8 3.5 14 No effect
035 23 F Condyloma 8 3.5 14 No effect
036 80 F Condyloma 8 3.5 14 No effect
001 40 Osteosarcoma 15 3.5 15 No effect
006 13 Osteosarcoma 42 3.5 90 Transient delay in progressio!l I -?,
007 3 F N euroblastoma 18 1.7 28 No effect
:., ll:->I': IN INI,'I';(''I'I()N:;
No c;peCLaClllal' wo 1'0 c;OOll ill 01' Wc were
IJY LllO t:o}';t'CooiOll 01 planae in
l'ollal (paLicl1L Nu. 005) with mu1tiple cutaneous warts
:tI1U lJY parLial anu subsequent of
(paticJlt No. 0:32), which had been after surgical
t'cocotions.
Prophylactic Studies
is designed nature to protect cells against exogenous
For tilis reason, it is more logical to use interferon as prophylactic
than as therapeutic agent virus infection. However, very few
clinical situations such tilat tile exact of viral infection
One such situation in patients receiving immunosuppres-
sive tilerapy for organ transplantations. About 84% of renal transplant
patients show seroconversion ag'ainst or more viruses witilin tilree
montils More ilian half of these seroconversions are
against HSV or (CMV) , Both of these are tilought to resu1t
[rom activation of latent infection, existing since early lifetime. patho-
genesis of this activation is not lmown. Cellular immunity ratiler than cir-
culating antibody seems to tile most important factor keeping latent
infection in check. It is not lmown which cells tile viruses are
during latency; in animal models HSV reactivated from the sensory
ganglia. Finally it is lmown whether prevent activation
of HSV in cells. In any case it seems safe to assume tilat
the situation of herpetic infections in renal patients differs from
tilat occurring in the animal model systems tile effect
of interferon virus infection has been demonstrated.
double-blind clinical employing fibroblast in
transplant patients has recently been completed et al. [58] at
the Erasmus University of Rotterdam Netherlands). similar trial
using leukocyte interferon is being conducted Dr. S. Hirsch (Harvard
Medical School, Boston, Mass.). renal transplant trial
was in February 1976 and included 16 non-diabetic, HBsAg negative
patients between 18 and 45 years of age kidney at tile
Dijkzigt Academic Hospital. immunosuppressive regimen consisted
of prednisone (35 mg daily) and (1-2 mg/kg daily). study
was set in double-blind, placebo-controlled fashion in consecutive
pairs to allow early detection of possible side effects of the interferon
tilerapy. In pair tile patients received either placebo or fibroblast
(3 MU, twice weekly) tilree months, starting 1-2 hr before
transplantation. patients were observed for six montils, Hematological
and liver function parameters of interferon-treated patients did 110t differ
from those of placebos. virological observations are summarized in
3. No differences were seen betweel1 interferol1- and placebo-treated
:tllll 1)10; S()IVII';I{.
3. il1 [{cHu.l l'aLicI1LCi 'l'1.'caLcu
P1'ophylactically with I<'ib1'oblast Inic1'fcronU.
Number of g1'OUP
viral


with vi1'al


vi1'al
with herpes vi1'uS
Clinical

Treatment
Mock- in terferon
8
4
9
4
6

3
(seve1'e)
4
lnte1'
8
4
10
4
6
2


1

1
trial pe1'formed Weimar et al. [58];
serology for HSV, CMV, rubella vi1'us, virus, respiratory
vi1'US, va1'icella zoster virus. 3 MU
of fibroblast twice weekly,
for 6 weeks, starting the day of Follow-up period:
6
the total of viral
the of Two
occurred the group, the group
placebo. The for effect of was
the occurrence of HSV the group sero-
occurred four out of eight three
of these had severe orofacial Of the eight who received
placebo, showed this was
fever bliste1'. these results should take
the relatively small dose of It is possible that better
results might have had the dose 3- to 10-fold.
Furthermore viral occurred period of 3 weeks, start-
the thi1'd week after Hence, future trials might
the possibility of the to this period.
:,. CI.lNICi\I, [ISI': IN VII(i\I, INI"I';("I'I()NS 1;;;;
COl1vil1cillf.( WUL"C ol)taillcu [59] ill propl1Ylaxis IlSV activa-
(,i()I] surf.(ical lL"Catmunt condition called douloureux, "
rcsulting" compression the g"anglion nervi trigemini.
paticnts wcre given intramuscular injections of either placebo or 5 MU
leukocyte interferon days -1, 1, 2, and 3 after operation.
out 11 placebo-treated patients showed reactivation (cold sores or virus
sllCdding or both); only five out of 12 interferon-treated patients showed
signs of reactivation.
VI. NONTRIVIAL1TY OF SIDE EFFECTS
Fever and Malaise
Fever and malaise occurring short1y after each injection were reported in
studies [3,48,60-62] involving systemic administration of leukocyte inter-
feron in humans. These reactions were thought to rather aspecific and
were assigned to trivial impurities in the interferon preparations; as purer
preparations available, less fever and malaise seemed to occur.
Recent1y it has clear that fever does occur with relatively pure
preparations of leukocyte interferon [36] and with fibroblast interferon as
well [63].
From group of 27 patients who received intramuscular injections of
fibroblast interferon (2: 2 MU per injection), 11 developed fever peaks
ceeding 38.00 (see 4). interferon was prepared at our 1nstitute
and purified absorption and elution from controlled pore glass (CPG
method) [64]. 1nterferon preparations which underwent additional puri-
fication using affinity chromatography zinc iminodiacetate Sepharose
method) [65] were less pyrogenic. This indicates that the pyrogenic
effect is not due to interferon itself but to molecule(s) of similar chemical
nature. If this is so, it would unjustified to consider fever as aspecific
and trivial side reaction of interferon therapy; interferon of
family of molecules which responsible for some of the unexplained
systemic symptoms of acute virus infections, g ., fever, muscle pains,
headache, malaise, and others.
Hematological Side Effects
Interferon has shown to inhibit the proliferation of marrow cells
in vitro [66-69]. However, in the majority of patients who have treated
with leukocyte or fibroblast interferon, gross signs of marrow sup-
pression have observed. two patients receiving interferon for
infection complicating marrow transplantation, suppression
of marrow engraftment was observed [58]. animal systems inter-
J:H 1111.1.1 :tll' I 1 S( )IVII':H.
TA13LI<: 4. l'cvce LleaeLioll ill l'aLicl1L:-; (; ivell
Injeetions ]<'ibroblast Intcrfcroll
Inter
Number
Patient Avg.
eode eation injee- dose
No. Sex Diagnosis
a
method
b
tions
Fevcr'('

42 CALD AS 7 10.0
009 35 Renal transplant AS 26 3.0
010 31 Renal transplant AS 26 3.0
011 24 F Renal transplant AS 26 3.0
012 37 Renal transplant AS 26 3.0
013 37 F Renal transplant AS 26 3.0 +
014 26 F Renal transplant AS 26 3.0
015 34 F Renal transplant AS 26 3.0 +
016 19 Renal transplant AS 26 3.0
017 57 F CALD CPG 9 4.6
018 21 F CALD CPG 16 4.5
002 36 CALD CPG 37 3.6 +
003 50 CALD CPG 28 1.2
019 35 F CALD CPG 14 2.2
020 39 CALD CPG 14 2.2 +
025 43 F Aeute hepatitis CPG 14 2.0
026 48 F Acute CPG 14 2.0
027 40 F Acute hepatitis CPG 14 2.0
024 20 Fulminant CPG 10 3.5
023 36 Renal transplant CPG 26 3.0
001 40 Osteosarcoma CPG 15 3.5 +
006 13 Osteosarcoma CPG 42 3.5 +
005 35 F Multiple warts CPG 14 5.9 +
004 30 F Multiple sclerosis CPG 7 4.7
008 6 Avian tuberculosis CPG 37 1.5 +
022 9 SSPE CPG 12 4.6 +
007 3 F Neuroblastoma CPG 18 1.7 +
aCALD = ehronic active liver disease; SSPE = subacute sclerosing panen-
cephalitis.
AS = zinc iminodiacetate Sepharose method; CPG = controlled pore glass
method.
cPeaks exceeding

;." IISI'; IN INlo'l,;(''I'I()NS
ICI"OI1 Lo ill1l11U11C pllcllOmena Cllapter 11)" 1n
paLiOllLH LI"eaLe<l WiLIl of cellular responsive-
exiellsively studied. et al" reported suppression
response to in two patients (out of an
studied) who received leukocyte interferon for CMV
illfection l57]" It would to krlOW whether this is gelleralized
finding in illterferon-treated patiellts. Failure of illterferoll to act pro-
phylactically against CMV and HSV infections in renal transplant patients
due to the cumulative lmmunosuppressive effects of interferon and
azathioprine-prednisone therapy.
1n patients receiving intramuscular injection of fibroblast interferon
we observed an increase in the percentage of granylocytes and decrease
in the percentage of lymphocytes, most pronounced 4-8 hr after each injec-
tion. blood formula restored to normal within 24 hr. The relative
numbers of and cells, as determined rosette assay, remained un-
altered. One CALD patient 1, No. 018) developed overallleukopenia
after each injection of either fibroblast (2.2 MU) or leukocyte interferon
(3 MU). With the latter, the total white blood count decreased to .$ 3000
cells/mm 3. necessitating arrest of treatment. Since the reaction occurred
with both fibroblast and leukocyte interferon, it seems highly probable that
it was due to interferon.
Skin Reactivity
1ntracutaneous injection of fibroblast interferon preparations (;::20,000 U
in 0.1 to 2. ml) was found to provoke erythema and induration at the site
of injection [63,70,71]. The reaction is painless.
it resembles delayed type hypersensitivity response, being characterized
the presence of monolluclear infiltrate especially around the blood
vessels, few polynuclear cells, very little edema, and absence of vascular
lesions. The reaction peaks at 12-16 hr after illjection and subsides in 26
to 48 hr. The size of the reaction is remarkably constant within single test
persons but varies from one individual to another. Patients with depressed
cellular immullity react as normal persons. It is not with certainty
whether the reaction is caused interferon itself or associated mole-
cules. Fibroblast interferon purified the Zn-chelate method was less
reactive than that purified the CPG method. Leukocyte interferon was
also less active in provoking the skin reaction. the basis of these
findings we proposed [71] that the skin reaction is caused molecules
resembling interferon, rather than illterferoll itself.
illICIIH:
VH. lN PllAltMACOKlNE'l'lCS 01"
LEUKOCYTE ANV
Recently it has apparent that there are difference::; in the pharmac()-
kinetic behavior of leukocyte and fibroblast interferons. J>atients injecteu
with leukocyte interferon at dosages of > 10, 000 U/kg show measurable levcl::
interferon activity in the blood (>50 U/ml) [3]. In contrast, patients
injected with as much as 40,000 U/kg of fibroblast interferon failed to demOlI
strate detectable serum levels 10 U/ml) [72]. Although earlier informa-
tion [ 73,74,75] obtained in rabbits suggested the pharmacokinetic::; of
fibroblast and leukocyte interferon would s imilar, more recent work [ 7 JI
confirmed that intramuscular injection of fibroblast interferon in rabblts
resulted in 10wer serum titers than those obtained with similar doses of
leukocyte interferon. In monkeys the difference was also present but 1ess
pronounced [77]. Experiments with intravenously injected rabbits [71]
failed to support the concept that fibroblast interferon was removed more
rapidly from the circulation. Rapid destruction at the site of injection
remains
VIII. RECENT STUDIES
The following recent information has Scott et [78]
showed protection of human against vaccinia virus scarification
prophylactic intradermal administration of human fibroblast interferon.
Similar experiments were done in rhesus monkeys [77]: intramuscu1ar
doses of leukocyte as well fibroblast interferons provided protection
against vaccinia virus scarification. The interferon was given daily for 7
days starting the day of scarification. dose of 0.4 MU/kg of fibroblast
interferon was about as effective as 0.1 MU/kg of leukocyte interferon. The
protection obtained in this model cannot explained the direct antiviral
effect of interferon since vaccinia virus replication was found not to
inhitited either interferon in cells in vitro [791. lt was suggested
that the protection is mediated activation of aspecific resistance the
of the immune system.
Additional uncontrolled studies chronic HB-virus infection were done
Dolen et [80,81]. Fibroblast interferon was administered at daily
doses of 1 MU intramuscularly for periods up to 82 days. In 2 out of 3
patients, rapid and apparently permanent decline in viral markers was
observed. In another uncontrolled study [82] leukocyte interferon was used
in conjunction with adenine arablnoside over periods varying from 4 to 6
months. Complete 10ss of viral markers was seen in 1 out of 6 patients
while in the 5 others decrease occurred. Clinical parameters a1so tended
to improve. double-blind placebo-controlled study was done Weimar
et [83]. Leukocyte interferon treatment was given for 6 weeks, 12 MU
per day in the first week, 6 MU in the second week, and so rapid
IN INI,'I';C'I'I()NS 1:; '1
(IOl:l'uase tTull itl (J:tt"L iu [e-assoe iaLell UN levels in the
viral clinical parameters were not signifi-
tllOSO in the group. Sinoe polymerase levels
itll;reasod again in spite of high daily doses of interferon, the authors
peessed doubts as to whether the effeot polymerase was to ascribed
to interferon rathee than to impurities causing aspeoific eeactions such as
the basis of theie eesults they also pleaded against implementation
of long-term interferon therapeutic trials in patients with chronic hepatitis.
Tl1is, however, remains matter of judgment rather than of solid logic.
Romane, Revel, D. Gurari-Hotman, Blummenthal, Soko-
10vsky, and Stein (personal communication) performed controlled trial
011 group of 50 patients with adenovirus conjunctivitis and reported signifi-
cant shortening of disease and reduction in the incidence of corneal involve-

Cheeseman et al. [84] completed their double-blind placebo-controlled
trial renal transplant patients and reported reduction in cytomegalo-
virus shedding. As there was apparent clinical benefit from this effect,
the practical conclusion from this trial agreed with that from the trial with
fibroblast interferon [58] mentioned earlier.
DISCUSSlON AND CONCLUSlONS
Clinical trials with any drug are necessarily preceded animal studies to
test efficacy and safety. 1n the case of interferon the number of animal
studies preceding clinical application is remarkably small when compared
to drugs used in other areas of medicine. We see several explanations
for tl1is. First, the1'e are only few model systems involving smalllabora-
tory animals that faithfully reflect those human viral infections that one
would like to t1'eat with interferon. Thus, there good model systems
for viral hepatitis, for acute respiratory diseases, for 1'ecurrent herpes,
or fo1' CMV recurrency. Second, for each animal species one needs the
homologous interferon, which is expensive to produce. Therefore is
limited to the use of only few animal species, preferentially small ones.
Safety assessments in animals have not been done to any great extent
cause it has been postulated that interferon would definition have toxic
side effects. Recent animal and clinical studies have provided ample evi-
dence to the contrary.
The efficacy studies in animals irrefutably prove that interferon given
before virus inoculation favorably influence the evolution of several
acute viral infections. 1t would seem that in wel1-defined conditions inter-
feron still act when given at time when viral replication in the target
organ has already begun. Probably this important question will have to
reevaluated for each type of viral infection. 1t is clear, however, that for
one particular model, higher doses of interferon are necessary and results
are less favorable when the treatment is started later during the course
:tllll 1>1': S()IVII':11
infcction. The c[[ccL sysLcmic ;lllll1il1isLtal,ioll 011 vital ill
establislled virus il1Eectiol1 il1 al1ill1als :oucll ;1::; (Jc1":oistcl1t Lolce:tl1l
LCM vi1"uS infection, LDH, 01" MIJV virus Ilas tlOt beell CXlCll:oiv,'I\'
studied. Yet studies of this type would ext1"emely w01"thwhile ill vicw
of current inte1"est in the effect of interferon ill chrollic active hepatitis
other chronic diseases of proven or suspected viral origin. J<'inally, topical
application of interferon ill of herpetic keratitis
yielded rather discouraging results. Poss ibly the doses used in
were too In clinical trials, topical application of interferon follow-
ing of corneal herpes lesions accelerated healing
and inhibited virus shedding. In contrast, high doses of interferon
did not prevent recurrence. Possibly this failure was due to the
of interferon to penetrate into the deeper layers of the cornea.
controlled trial in upper respiratory disease established prophylac-
tic but not therpeutic effect of topical interferon. However, the doses
needed were high that practical of interferon for the
of colds held in until better techniques for
the production of interferon
Encouraging results were obtained in controlled study involving
cation of interferon-containing in
controlled study using interferon in
was done in renal transplant recipients in to pro-
phylactic effect the occurrence of viral infections due to
sive therapy. In view of the of patients included in this study,
the results could hardly expected to significant. Its was
to prove the of double-blind, studies in renal
transplant patients and to provide orienting indications for further trials of
this kind. the results it would that little effect is to
pected CMV infections; HSV infection, the contrary, reduced
interferon
Treatment with leukocyte interferon was shown to reduce the incidence
of herpes virus reactivation in patients operated for condition called
"tic douloureux."
Another controlled study demonstrated that cancer patients treated
with interferon had less dissemination and complications with herpes zoster,
as well as less postherpetic neuralgia, than controls. Leukemic children
with early varicella also could protected against visceral complications.
other trials done so far essentially uncontrolled.
most significant ones perhaps those chronic virus infection,
cause it is possible to study the effect of interferon administration the
evolution of viral activity in time. levels of viral activity first
established, and changes occurring after interferon
then studied. Such studies are currently in progress in severallabora-
tories. those reviewed in this paper, it would appear that about two-
thirds of the patients react favorably to or another regimen of treatment.
:'. IISI': IN INI"I':CTI()NS
Cle;tr'[Y, patiul1ts ;tr'u t'USistallt to lcss cluar1y, are
TIJC tllese studies suffers heavily from the
l'act study vira1 parameters in chronic virus infection
ollly recent1y possible and, hence, that very little background in-
formation this subject is available yet. Perhaps the merit of the
clinica1 tt'ia1s done far will have been to provide of that background
and to orient investigators toward the establishment of suitable treatment
protoco1 with which double-blind, p1acebo-contro11ed studies under-
taken.
Systemic administration of interferon has been attempted to treat
sporadic cases of severa1 other vira1 infections, inc1uding acquired or
congenital eMV infection and acute and fu1minant hepatitis. perform-
ance of such sporadic tests criticized the basis that they are
waste of time, effort, and money which wou1d better spent if concentrated
one proper1y controlled tria1 in one particu1ar disease. However, both
the choice of particu1ar disease to studied and the choice of particu1ar
treatment protocol to used must decided the basis of pre1iminary
pilot experiments. If one were to make these choices mere1y arbitrary
basis, sing1e contro11ed tria1 wou1d certain1y use1ess and wou1d at the
time consume more effort than number of individua1 pilot tria1s.
1nterferon is exception in this regard; contro11ed tria1s with any drug
are preceded pilot tria1s individua1 cases. However, with rather
inexpensive drugs these preliminary tests are never brought to the attention
of the scientific community, for the simp1e reason that the ensuing contro11ed
tria1s performed without de1ay. 1n the case of interferon the decision
to start contro11ed tria1 is painstaking it does invo1ve an enor-
mous financia1 effort. 1n the meantime it is usefu1 to co11ect and carefu11y
study the fragmentary evidence from uncontro11ed tria1s done in severa1
centers, in order to make as educated decision possible.
While the clinica1 tria1s were being performed, it c1ear that
current interferon preparations exert some unexp1ained side effects: fever
and ma1aise, transient 1ymphopenia, and skin reactivity. question
whether these effects are due to the interferon mo1ecu1e or to some impuri-
Hes remains unreso1ved. We favor the view that they are due to mo1ecu1es
that resemble and hence tend to copurify with interferon. These mo1ecu1es
might as re1evant interferon to exp1ain some of the beneficia1 efforts
seen in vivo, g., the antitumor effect. An important task wi11 to find
anima1 mode1 systems to study these side effects and then to iso1ate and
identify the substances responsible for these effects.
Fina11y, whi1e in the past 1eukocyte and fibroblast interferons were
mere1y seen as a1ternative techno1ogies for preparing the drug, it
has now c1ear that these interferons have quite different physico-
chemica1, sero1ogical, bio1ogica1, and pharmacokinetic properties. Per-
haps of them will a1so turn out to have its own p1ace in the prophy1axis
or therapy of human disease.
itllll IH: S()IVH:lt
ACKNOWLEDGMENTS
The studies from the authors' laboratory, commented upon in this papcr,
were made possible grants from the Belgian A.S. L.K. (General Savin!!,';;
and Retirement Fund) and from the Belgian Ministries of Economic Affair;;
and of Science Administration
authors are indebted to colleagues from St. Academic
Hospital (Leuven, Belginm), Academic Hospital (Ghent, Belgium), and
Dijkzigt Hospital (Rotterdam, The Netherlands), who cooperated in the
clinical trials.
Editorial help of Christiane Callebaut is gratefully acknowledged.
REFERENCES
1. Merigan, S. Reed, and Tyrrell, Lancet.!..=
563 (1973).
2. J. Desmyter, Ray, J. De Groote, F. Bradburne, V. J.
Desmet, V. G. Edy, Billiau, and De Somer, Lancet.!i: 645
(1976).
3. Greenberg, R. Pollard, L. I. Lutwick, Gregory,
W. S. Robinson, and Merigan, New Engl. J. Med. 295: 517
(1976).
4. S. Baron, in Interferons and Interferon Inducers (N. Finter, ed.),
American Elsevier, New York, 1973, 267.
5. L. Glasgow, in Interferons, Proc. Symp. New York Heart Assoeia-
tion, 1970, 212.
6. D. Gilden, in Viruses and Immunity Koprowski and Koprowski,
eds.), Aeademie Press, New York, 1975, 83.
7. D. Steinberg, in Viruses and Immunity Koprowski and
Koprowski, eds.), Aeademie Press, New York, 1975, 95.
8. F. Dianzani and S. Baron, Nature 257:682 (1975).
9. N. Finter, in Interferons and Interferon Indueers (N. Finter,
ed.), Ameriean Elsevier, New York, 1973, 295.
10. G. Olsen, R. Kern, L. Glasgow, and J. Overall, Jr.,
Antimierob. Agents Chemother. 10: 669 (1976).
11. Worthington, Levy, and J. Riee, Proe. Soe. Biol. Med.
143: 638 (1973).
12. De Clereq and De Somer, Proc. Soe. Biol. Med. 138:301
(1971).
13. I. Gresser, G. Tovey, and Bourali-Maury, J. Gen. Virol. 27:
395 (1975).
14. Nash, W. Morgan, J. Armstrong, R. G. Carrol,
and lnfee. lmmun. J!: 286 (1974).
15. J. Hilfenhaus, Weinman, Majer, R. Barth, and Jaeger,
J. lnfec. 135: 846 (1977).
: . IN INI,'I':CTIONS ).11
J(j. It. D. anu Shrestha, Albrecht
Arch. I<;xp. Ophthalmol. 195: 263 (1975).
17. D. Sundmacher, R. Skoda, and Cantell,
Infec. Immun. 11.:468 (1977).
18. J. Sugar, and Varnell, Invest. Ophthalmol. 12:
378 (1973).
J. 1. Collins, Cantell, R. Jones, and N. Finter,
J. Infec. Dis. (1976).
19. N. in Interferons and Interferon Inducers (N. Finter,
ed.), American Elsevier, New York, 1973, 391.
20. Dubuy, S. Baron, Uhlendorf, and L. Johnson, Infec. Immun .
..: 977 (1973).
21. V. G. Edy, Sobis, and De Somer, Int. J.
14: 335 (1974).
22. R. Friedman, Chang, J. Ramseur, and W. Meyers,
J. Virol. 16:569 (1975).
23. Heremans, Allen, J. De Maeyer-Guignard, and
De Somer, Virology 73:537 (1976).
24. S. Z. Shapiro, Strand, and Infec. Immun. 16: 742
(1977) .
25. R. R. Wagner and R. Snyder, Nature 196:393 (1962).
26. Riviere, 1. Gresser, J. Cuillon, and G. Proc.
Nat. Acad. Sci. U. S. 74: 2135 (1977).
27. J. Field, G. Joyce, and Keith, J. Gen. Virol. 2.: 149 (1969).
28. Worthington, Infec. Immun. &.: 643 (1972).
29. 1. Gresser, G. Maury, and I. Chouroulinkov, Nature
258: 76 (1975).
30. I. Gresser, Maury, L. Morel-Maroger, and F. Pontillon,
Nature 263:420 (1976).
31. 1. Gresser, Bandu, Maury, and D. Brouty-Boye,
J. Med. 144:1305 (1976).
32. I. Gresser, and Bandu, J. Med.
144:1316 (1976).
Heremans, Billiau, Colombatti, J. Hilgers, and De Somer,
Infec. Immun. 21: 925 (1978).
33. D. F. Horrobin, R. S. 1. Ally,
Kamazyn, R. Morgan, Swift, and Zinner, IRCS J. Med.
Sci. 2.:547 (1978).
34. 1. Yaron, D. Gurari-Rotman, Revel, R. Lindner,
and V. Zor, Nature 267:457 (1977).
35. N. Finter, in Interferons and Interferon Inducers (N. Finter,
ed.), American Elsevier, New York, 1973, 363.
36. Merigan, G. W. Jordan, and R. Fried, in Perspectives in
Virology Pollard, ed.), Vol. 9, Academic Press, New York,
1975,
;(111110; :-!()IVII';lt
37. D. Ikic, 1. Pctriccvic, Cupak, U. Teajct', [. Sol(!o, 1';. SOOH,
D. Jusic, and S. Smerdel, in Proc. Symp. Clitlical U8C Il1Lc [' [Cl'OIJ
(D. Ikic, ed.), Yugoslav Acad. Sci. al1d Arts, Zagreb, 1975,
38. D. Ikic, S. Smerdel, Hajninger-Miholic, S008, and D. JU8ic,
in Proc. Symp. Clinical Use oflnterferon (D. Ikic, ed.), Yugoslav
Acad. Sci. and Zagreb, 1975, 195.
39. D. Ikic, N. Bosnic, S. Smerdel, D. Jusic, Soos, and N. Delimar,
in Proc. Symp. Clinical Use oflnterferon (D. Ikic, ed.), Yugoslav
Acad. Sci. and Zagreb, 1975, 229.
40. R. F. Meyer, R. Laibson, S. R. Waltman,
Nesburn, and J. J. Shuster, J. Infec. Dis. 133: (1976).
41. Jones, D. J. Coster, G. Falcon, and Cantell, J. Infec.
Dis. 133: (1976).
42. R. Sundmacher, D. Neumann-Haefelin, F. Manthey, and MU11er,
J. Infec. Dis. (1976).
43. R. Sundmacher, D. Neumann-Haefelin, and Cantell, Albrecht v.
Graefes Ophthalmol. 201: 39 (1976).
44. R. Sundmacher, D. Neumann-Haefelin, and Cantell, Lancet.i: 1406
(1976).
45. Sundmacher, Cantell, Skoda, Hallermann, and
D. Neumann-Haefelin, Albrecht v. Graefes Opthalmol.
208, 229 (1978).
46. D. Ikic, Cupak, D. Trajer, Soos, D. Jusic, and S. Smerdel,
in Proc. Symp. Clinical Use oflnterferon (D. Ikic, ed.), Yugoslav
Acad. Sci. and Zagreb. 1975, 189.
47. D. Ikic, Prazic, S. Smerdel, D. Jusic, N. Delimar, and Soos,
in Proc. Symp. Clinical U se of Interferon (D. Ikic, ed.), Yugosla v
Acad. Sci. and Zagreb, 1975, 203.
48. G. W. Jordan, R. Fried, and Merigan, J. Infec. Dis. 130:
56 (1974).
49. Merigan, Rand. Pollard. S. Abdallah.
G. W. Jordan. and R. Fried, New Engl. J. Med. 18: 981 (1978).
50. N. Arvin, S. Feldman, and Merigan, Antimicrob. Agents
Chemother. 13: 605 (1978).
G. Scullard, Alberti, Wansborough-Jones, R. Howard,
L. W. F. Eddleston, J. Zuckerman. Cantell, and William8.
Clin. Lab. Immunol. in press.
52. W. Weimar, Heijtink. S. W. Schalm, and Schellekens,
Gastroenterology 74: 1150 (1978).
53. W. Weimar, Heijtink, S. W. Schalm, van Blankenstein,
Schellekens, N. Masurel, V. G. Edy, ill iau , and De Somer,
Lancet 1!: 1282 (1977).
54. W. Weimar, Heijtink, S. W. Schalm, and Schellekens, Eur.
J. Clin. Invest. 151 (1979).
!'. 11:-;1': IN INI"I':CTI()N:-;
1,1;;
;,;,. ,J. N. Z. D. t;hari, ll. Mendelson, Cart-
WL'if2;11L, t;cott, 13. al1dR. Wright, Gut
(1978).
56. ll. J. L. Everson, G. Emodi, J. Hansen, Smith-
wick, Grimes, S. Pahwa, R. Pahwa, S. Schwartz, D. Armstrong,
F. Siegal, S. Gupta, Dupont, and R. Good, Immunol.
Immunopathol. Q: 51 (1976).
57. G. Emodi, R. O'Reilly, L. Everson, U. Binswanger,
and Just, J. Infec. Dis. 133: (1976).
58. W. Weimar, Schellekens, L. D. F. Lameijer, N. Masurel,
V. G. Edy, illiau , and De Somer, Eur. J. Invest. 8: 255
(1978).
59. G. J. Pazin, J. Armstrong, Lam, G. Tarr, J. Janetta,
andM. New Engl. J. Med. 301:225 (1979).
60. L. Ahstrom, Dohlwitz, Strander, G. Carlstrom, and Cantell,
Lal1cet i: 166 (1974).
61. G. Emodi, Just, R. Hernandez, and R. Hirt, J. Nat. Cancer
Inst. 54: 1045 (1975).
62. Strander, Cantell, G. Carlstrom, and Jakobsson,
J. Nat. Cancer Inst. 51: 733 (1973).
63. De Somer, V. G. Edy, and Billiau, Lancet Q:47 (1977).
64. V. G. Edy, 1. Braude, De Clercq, illiau , and De Somer,
J. Gen. Virol. 33:517 (1976).
65. V. G. Edy, Billiau, and De Somer, J. Chem. 252:5934
(1977).
66. McNeillandI. Gresser, Nature 244:173 (1973);
67. L. GreenbergandS. Mosny, CancerRes. 37:1794 (1977).
68. Nissen, Speck, G. Emodi, and N. N. Iscove, Lancet Q: 203
(1977).
69. Van 't Schellekens, Lowenberg, and J. de Vries,
Cancer Res. 38, 911 (1978).
70. G. Scott, J. Butler, Cartwright, Richards, J. G.
R. Wright, and D. J. Tyrrell, LallcetQ:402 (1977).
71. Billiau, De Somer, V. G. Edy, De Clercq, and Heremans,
Antimicrob. Agellts Chemother. 16: 56 (1979).
72. V. G. Edy, and De Somer, Lallcet (1978).
73. V. G. Edy, Billiau, and De Somer, J. Infec. Dis. 133:
(1976) .
74. Calltell alld L. Pyhala, J. Gell. Virol. 20:97 (1973).
75. Cal1tell and L. pyliiri, J. Infec. Dis. (1976).
76. Cesario, Proc. SOC. Biol. Med. 155:583 (1977).
77. W. Weimar, Billiau, Cantell, L. Stitz, and Schellekel1s,
J. Gen. Virol. in press (1980).
78. G. Scott, Cartwright, G. Le Du, alld D. Dicker, J. Biol.
Standardization Q: 73 (1978).
:LIIIII>I';
79. Schellekens, W. Wcimar, CallLcll, :1.11(11,. SLiL;.-;, NaLul"c
742 (1979).
80. J. G. Dolen, W. Carter, J. S. Horoszewicz, Vladutiu,
1. Leibowitz, and J. Nolan, Amer. J. Med. 67: 127 (1979).
81. J. G. Dolen, S. S. Leong, Vladutiu, 1. Leibowitz, and
J. S. Horoszewicz, J. Clin. Haematol. Oncol. 63 (Abstr.) (1979).
82. G. Scullard, Makal, S. Sacks, Gregory, W. Robinson, and
Merigan, J. Clin. Haematol. Oncol. (Abstr.) (1979).
83. W. Weimar, R. Heijtink, Cantell, F. J. ten Kate,
S. W. Schalm, N. Masurel, and Schellekens, Lancet in press
(1979) .
84. S. Cheeseman, R. Rubin, J. Stewart, N. Tolkoff-Rubin,
Cosimi, Cantell, J. Gilbert, S. Winkle, J. Herrin,
Black, S. Russell, and S. Hirsch, New Engl. J. Med.
300:1345 (1979).
6
INTERFERON INDUCERS:
THEORY AND EXPERIMENTAL APPLICATION
Dale 8tringfellow
The Upjohn Company
Kalamazoo, Michigan
1.
CHEM1CAL REQUIREMENT8
V
1V. HOW MUCH 1NTERFERON 18 ENOUGH?
V. HYPOREAC
VI.
VI1. EFFECT OF INDUCER8 ON
OUTLOOK
REFERENCE8
1.
145
147
149
151
152
160
161
162
162
Following the discovery of interferon great deal of speculation has cen-
tered around its use as an effective antiviral and antineoplastic agent and,
more recently, as modulator of the immune response. Essentially two
approaches had been taken toward the development of this agent. The first,
as described in the preceding portion of this book, involved mass produc-
tion of enough interferon for tl1e direct treatment of virus-infected individ-
uals or those bearing various types of malignancies. At present, this
approach has as major limitation the amount of interferon which
produced. Currently available methods do not appear sufficient for the
successful production of enough interferon for routine clinical use. This
situation obviously reversed if interferon or an active component
of the interferon molecule synthesized synthetically or if inter-
145
feron gene to procaeyotic cells tJlat pl'OC[LlC() it.
The theory behind second appeoacl1 to tlle illtcr""ct'Oll SYStcll1
has, therefore, centered around the identification anu ucvelopmenL of
of stimulating the host's own cells to produce interferon, thcreby
circumventing the necessity of producing large quantities exogenous
feron.
Over the past several years variety of agents have been found
of stimulating interferon production both in vivo and in Besides
types of viruses, many bacteria, and bacterial wall extracts, synthetic
and naturally occurring nucleic acids (i.e., double-stranded RNA) , protozoa,
mytogens, antigens, and variety of high- and low-molecular-weight
pounds with diverse chemical structures have been found to effective
interferon inducers in the appropriate (in vitro) animal (in vivo)
tems 1 [1-19]). The number ofagents which have found to
effective interferon inducers increases each year, yet at present havc
been successfully employed clinically useful antiviral agents; however,
only very few (three or four) have evaluated in humans. area i::;
in its infancy, and the purpose of this and the next few chapters is to identify
1. Major Classes of Agents That Induce Interferon
Class Example Reference
Naturally occurring
Viruses, RNA Influenza 1
Viruses, DNA Vaccinia 2
Bacteria Brucella abortus 3
Bacterial extracts abortns 4
Rickettsia R icketts ia 5
Protozoa Taxoj21asma gondii 6,7
Endotoxin (LPS) Escherichia 8,9
Mitogens Phytohemagglutinin 10
Poke weed 11
Antigens Viral 12
Bacterial (BCG) 13
Synthetic
Polynucleotides Poly 1: poly 14
Polycarboxy lates Pyran copolymer 15
Low molecular weight Tilorone 16
Propanediamine 17
Acradines 18
Pyrimidines 19
'l'AB
Sll<Jllld lVlccL Use
1. Safc;
cleared from the does
Prophylactically, and therapeutically, active
4. to induce interferon in the animal species to treated
5. Effective practical route of administration
6. of adventitious agents
7. Relatively inexpensive to prepare
the progress so far achieved and the problems being encountered in the
development of effective antiviral agents.
An inducer must meet certain minimal criteria to considered for
human use 2). It must relatively nontoxic for the treatment of
life-threatening diseases and entirely safe if it is to used in the treat-
ment of what are considered to self-limiting infections. It must
effective prophylactically and preferably therapeutically, must
to induce interferon in or the animal species in which it is to
used, and should relatively inexpensive as well as effective practical
routes of administration. It should relatively nonirritating, should
cleared from the animal fairly rapidly, and not contain adventitious
agents. Certain of the agents to considered in this and later chapters
possess but not of these characteristics, while others have not
tested clinically; thus, of the are yet
CHEMICAL REQUIREMENTS
The chemical structures of molecules that are diverse.
Various RNAs [14], lipopolysaccharides [8,9],
polycarboxylates [15], [20], [16],
amines [17], [19], etc., are to high serum
the appropriate species. however, it
that of the structure of various low-molecular-
weight disrupts the antiviral activity [21,22]. For example,
series of isocytosine clearly 3)
that minor structure drastically affect biological activity.
in 3, occupation of the (R
j
) with
the (R
z
) with hydroxy were for activity. If
3. Effcct of Structural 011 AbiliLy l'yt'il1lillil1c
Molecules to Protect Micc Against l,cthal
Virus Infection

R
2
R
1
R
z

R
4
MPDa






NH
z


250
NH
z




NH
z
NH
z


NH
2
1

200
NH
z
Cl

NH
z




NH
z


NH
z


500
NH
z



NH
z


NH
z
1

400


1


1000
NH
z
1

NH
2
1
CH
z
C
6
H
s 800
aMinimum dose (mg/kg, i.p.) needed to protect mice against virus
infection. Drugs were administered 18 prior to infection.
(i. TIII';()I{Y .l1!)
WaH altut"uu HubHtitutcu, alltiviral activity
WaH [OHt. blkcwiHC thc hau to occupied halogen
(prefcrably chlori11e, bromine, or iodine) while as the 6-position (R4) alkyl
side chain was lellgthened, alltiviral activity progressively decreased. This
type of selective activity of specific structures appears to the rule rather
the exceptioll for i11ducers in general. At prese11t precise definitio11
of structural characteristics i11ducer requires is not available. slightly
better is available for polYllucleotides with regard to how
structural a11d physical alteratio11s affect biological activity [23-29].
SPECIES-TO-SPECIES V
Not only are illducers difficult to predict in terms of activity of specific
structures, but it is very difficult to predict if of in-
ducing interferon in, for mice also active in other a11imal
species. For example, tilorone hydrochloride (Chapter 8) i11duces very
high levels of serum i11terferon in mice but is l10t active in chickens, cats,
dogs, rabblts, a11d apparently l10t inhumans ([30,31]; see also Chapter 8).
Simi1ar results i11 terms of i11ability to i11duce i11terferon in huma11s have
bee11 reported with few other i11ducers, although l10t ma11Y have yet bee11
tested clinically. Some inducers however, do have broad species
For example, I:poly induces high levels of interfero11 in variety
cells (in vitro) a11d a11imals, i11Cludi11g huma11s a11d primates
([32-34]; see Chapter 7). new polynucleotide complex, poly ICLC,
developed at the Natio11al I11Stitutes Health (Chapter 7), appears to a11
effective i11terfero11 inducer in humans a11d protected monkeys against
otherwise lethal virus infection [35]. Likewise, pyrimidi11e interfero11
i11ducer 2-ami11o-5-bromo-6-methyl-4-pyrimidinol ca11 i11duce higll
levels of i11tel'feron in mice a11d is very good i11terferon inducer in cats
[36]. We have therefore bee11 interested i11 system ill which
we ca11 predict whether such agents wi1l to i11duce i11terfero11 i11
huma11s. 111 prelimi11ary studies we evaluated the rhesus monkey as such
system. Duri11g these studies variety viruses [Newcastle
disease virus (NDV)], polynucleotides (poly 1: poly and low-molecular-
weight compounds were evaluated without success. This raised the questio11
of whether the monkey was good species to predictor of huma11
activity. try to a11swer this question, well possibly develop
better system, we turned to the ability of human tissues cultured
i11 vitro to respo11d to various compounds and age11ts previously fOU11d to
active in rode11ts. Since several differe11t types appear to i11volved
i11 the in vivo induction process [37-40], mixture of several types
cultured together in vitro seemed to better simulation of in vivo
and predictive system than types of cells cultured
1;'0
,
Human Tonsillar h .. " "" .. """,, .. ;:;! Human Amnion
Tissue ~ . . . . .. ",,: "0--=. .:.....: __ Monolayer
1
24 hr Incubation 3 7
0

VSV /. ~ u
'====""!':::::':::===-=:::':::::':=:JI v- '"'.""0"
. Human amnion cells
challenged with vesicular
stomatitis virus to determine
direct antiviral effect.
. Growth medium
collected for
interferon assay.
FIGURE 1. In vitro system to determine the ability of compounds to induce
interferon or antiviral resistance in human cells as predictive tool for
future evaluation in humans.
alone. Since the lympho-reticuloendothelial system has been implicated
as major contributor in the induction process with low-molecular-weight
compounds [37-38], system consisting of human tonsillar or splenic
tissue cultured alone or with monolayer of human cells was investigated.
After several unsuccessful attempts to induce interferon, it was found that
human tonsillar tissue minsed in small pieces and incubated over confluent
monolayer of human amnion cells (Figure 1) was consistently good re-
sponder to poly 1: poly and NDV, yet it appeared to selective to induc-
tion various low-molecular-weight molecules. Also, using this system,
both direct antiviral activity ( amnion cel1s) and ability to induce inter-
feron could measured. In this system induced antiviral resistance
at 50 jJg/ml and stimulated moderate (50-100 U/ml) levels of interferon.
NDV and poly 1: poly were also very active and tilorone was inactive.
Present1y it is not known if this system is predictive of activity in humans.
We obviously w not know until more compounds have been evaluated clin-
ical1y. It does not seem unreasonable, however, to assume that an agent
that induces interferon and antiviral resistance in human cells in vitro will
(i. INIHJ('I';II,S: TIII';()lty
HLallll IJuil1!!: acLivu ill cOlllpoul1d that had
acLiviLy ulIly ill micc ur uxpurimelltal anilllals. The predictability of
compOUllds, therefore, relllains key ullallswered question in the search
for a!!:ellts that will effective, clillically useful interferon inducers.
1V. HOW MUCH INTERFERON 18 ENOUGH?
Problems raised in the previous section are even more complicated than
they lllight first appear. Even if inducer stimulate interferon produc-
tion in humans, the questions relllain as to how much is needed for in vivo
antiviral activity and whether serum interferon levels are reflection of
antiviral activity. Dianzani and Baron [41,42] and Dianzani et al. [43]
have addressed this problem using cu1ture systems that simulated in
vivo conditions. Their data suggest that extremely high interferon levels
are achieved in extracellular spaces in various tissues after induction and
that in cases serum interferon levels are misleading in terms of the
actual concentration that cells are exposed to. some cases the serum
interferon titers might higher than the concentration in certain tissues
such as the central nervous system, and the reverse might also true
where cells in the immediate vicinity of the site of interferon production
might exposed to much higher levels of interferon than detected
systemically in serum. Also, cells only needed to exposed to interferon
for very short period of time (30 min) for development of full antiviral
resistance. Using mice infected with either 8emliki Forest virus (8FV) or
encephalomyocarditis virus, we have addressed this question; and
our data would suggest that there is rough correlation between circulating
interferon levels and protection against these neurotropic diseases 4).
Concentrations of inducers that stimulated peak interferon levels of greater
than 50-100 U of interferon/ml of serum protected animals against 8FV
and virus infection. Less than 50 U/ml did not seem to protective.
Whether the correlation will observed in humans is not yet answered
(Chapter 5 deals more with this question). Also, local administration of
inducer at the site of infection stimulate high enough local illterferon
titers to protective without need for systemic (serum) response.
The important point to made is that inducers apparent1y do not have to
stimulate serum interferon titers of 5000-10,000 U/ml for extended periods
of time to protective but that lower titers of 50-100 U/ml of short dura-
sufficient. 1n the future, instead of searching for agents that
stimulate more total interferon, more practical approach might to
search for compounds that are more active at lower concentrations and
treatment regimens that provide constant antiviral state for an extended

S' 1 '1 t 1 N( : 1" 1'; 1 ,1,( )W
4. Comparisol1 of lll(]U(;(;/'
Stimu1ates Interferon Produ(;tion and Antiviral ltcsistall(;C ill Mi(;(;
Dosage
Inducer
a
(mg/kg)
Tilorone 200
100
50
25
12
1000
500
250
120
60
1: 5
1
0.5
0.1
0.05
Maximum
interferon
response
(U/ml)
6500
1300
500
120
25
7300
1500
70
< 10
< 10
5500
4000
510
80
< 10
Antivira1 protection
SFV
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
.
aTilorone and I:poly (i.p.) were administered
18 hr prior to infection with SFV. Serum was collected for inter-
feron assay 4 hr after I:poly 6 hr after 18 hr after
tilorone administration.
V. HYPOREAC TIVITY
One of the main problems confronting the development of inducers has been
that anima1s progressively 10se their ability to respond to various compounds
following repeated administration [3,44,45]. For example, the serum
interferon response of mice to inducers given day for five consecutive
days is illustrated in Figure 2 [55]. In each case, the of mice to
respond producing serum interferon 1evels progressive1y decreases.
The rate of onset of the hyporeactive state was, however, inducer dependent.
That is, mice were hyporeactive to second dose of tilorone the second
day but were still responsive to or 1: through the third
dose of compound. Others have a1so reported that the rate and severity
of the hyporeactive state depends upon the inducer, dose, and route of
administration [46-49]. small initia1 dose of inducer caused moder-
(i. INIH!CI':II,S: 'l'111':OI!,Y
4,0


!::
z
:::>



z
3,0


w
u..

w
1-
Z
:;;
:::>

w

2.0
2 3
DOSE
IJJ POLY 1: POLY


4 5
FIGURE 2. Development hyporeactivity in mice following daily doses
poly I:poly (100 jJg/mouse, i.p.), (500 mg/kg, tilorone
(250 mg/kg, (Reprinted from Ref. 55 with permission of the Ameri-
Society for icrobiology.)
ate suppression in responsiveness, whereas larger doses increased the
severity the hyporeactive state. These data suggest that judicious
selection of inducers and treatment regimens extremely important
in controlling the development hyporeactivity. was in fact found to
the in children given various dosage regimens of poly 1: poly [39].
Another approach around the problem concerns whether alteration
between different inducers avoid development of hyporeactivity. In
early studies it ,vas found that in general the hyporeactive state had
established, the response to other inducers was also inhitited [45,50].
More recently, however, it demonstrated that alteration inducers
in completely avoided the development of hyporeactivity [51-53].
in 5, mice given an initial dose of inducer, for
example, progressively less responsive (decreased with
each injection) to induction the inducer, but they remained respon-
sive to other unrelated compounds. Likewise, mice rendered hyporeactive
with anthraquinone interferon inducer were hyporeactive to the or
related compound (ti1orone) but remained responsive to These
data suggest that proper selection of treatment regimens critical
in developing the most effective methods of using agents.
I I S'I'IIIN(; I,'I-:I,I,()W
TAl3Lj,; 5. l)uvulopl1lul1L 01' IIYPol'uauLiviLy Lo lIIlILlC[.iol1
in Mice Given Daily l)osus 01" Sal1lu 0[' ll1ULlCUI,a
Inter- Inter-
Dose feron Dose feron Dose Dosc
1 (U/m1) 2 (U/m1) 3 (U/m1) 4 (U/rnl)
1200
500
Tilorone 4600
4500
PBS <50
300
Tilorone 4100
3100
PBS <50
<50
Ti1orone 4000
2500
PBS <50
3500
400
5000
Tilorone 50
550
730
Tilorone <50
<50
900
Tilorone <50
= 2-amino-5-bromo-6-methyl-4-pyrimidino1; =
morpholinopropy1)amino] anthraquinone.
Part of this same question revo1ves around how fast anima1s recover
their ability to respond to inducers after onset of hyporeactive state. In
other words, how often inducers given without of responsiveness.
With most inducers (Figure 3) four to six days between injection of the
inducer were required before anima1s (or were fully responsive to
the next dose of compound [34,49,54,55]. Based these data we have
eva1uated the ability of cats to respond to compounds which were given
li. IN'I'I':ltl"I':II.()N INIIIICI':I\S: 'l'11I':Olt\'
4.0


.....
2:
::::1

t.:J

.....
2:
3.0


.....
.....

.....
.....
2:

::::1

.....

2.0
2
EJ POLY .: PDLY


3
DAYS
4 5 6
FIGURE 3. Recovery of interferon responsiveness in mice that received
single dose of (500 mg/kg, poly I:poly (100
i.p.), or ti1orone (250 mg/kg, day and second dose ofthe
same inducer either 1, 2, 3, 4, 5, or 6 days later. (Reprinted from Ref.
55 with permission ofthe American Society for Microbiology.)
weekly or biweekly basis [36]. As summarized in 6, was
given biweekly basis to cats over an 18-week period. Animals remained
fuHy responsive to subsequent injection of inducer. Since and
other inducers create an antiviral state that persists for to several
days [55], the possibility exists that giving such compounds weekly
basis could maintain high nonspecific, antiviral state for an extended
period of .
Animals also develop hyporeactivity to interferon induction as conse-
quence of viral infections. Mice infected with one of several viruses
been found to have suppressed ability to respond to interferon inducers
[56-60]. In 7 the abi1ity of virus-infected mice to respond to
battery of inducers is summarized [55]. In general, as the infection pro-
gressed the ability of mice to respond to interferon inducers decreased.
Again the development of hyporeactivity created virus infection was
inducer-dependent. For example, animals remained quite responsive to
poly 1: poly for longer time-periods than to inducers such as tilorone
hydrochloride. Development of such hyporeactive state could severely
1;)(;
:->'1'1!.1 N(: 1,'1'; 1 ,1,()W
TAl3LI<; 6. lIlLcr[crot1 CaLH (J1.'ally Civctl
(200 mg/kg) Other Weck
Cat Weight Experimental Hours after inducer was given
No. (kg) week 6 12 24 48
1 3.0 <

2560 80 10 < 10
3.2 2 <10 1280 40 < 10 < 10
3.3 4 10 5120 640 20 < 10
3.2 6 10 2560 160 40 <10
3.3 10 10 1280 160 20 < 10
3.2 12 10 2560 320 20 <10
3.2 14 20 2560 160 10 < 10
3.2 16 10 1280 80 40 <10
3.2 18 <10 2560 160 < 10 <10
2 3.0 <10 320 20 < 10 < 10
3.5 2 < 10 40 20 <10 < 10
4.1 4 < 10 320 20 < 10 < 10
4.3 5 <10 160 40 < 10 <10
4.9 10 <10 320 80 10 < 10
4.9 12 <10 640 40 10 < 10
4.9 14 10 80 40 40 20
5.0 16 <10 1280 40 10 <10
5.0 18 < 10 640 160 10 <10
aSerum interferon titers ml).
restrict the use of interferon inducers as therapeutically active antiviral
agents, since in cases virus-infected mice developed hyporeactivity
before symptoms of illness were observed. series of studies designed
to determine why animals developed hyporeactivity, it was found that cells
from virus-infected mice (peritoneal cells) were functionally normal except
in their to respond to interferon inducers 8). This suggested
that specific control process was opcrating and that the hyporeactive state
might reversible. 1n series of studies it was found that the only
sistently observed difference between normal and hyporeactive cel1s (cells
from virus-infected mice) was that hyporeactive cells consistently had
lower membrane-associated adenylate cyclase levels and intracel1ular
levels than did normal cells 8). If suppres-
sion was modulating the hyporeactive then stimulating
might to restore the interferon response of hyporeactive cel1s.
Catecholamines, theophylline, and other agents that had the ability to in-
crease intracellular levels, however, in general did not affect
the response of normal or hyporeactive cells. ProstaglandinS, the
(i. TIII';()ltY
TA13Ll<: 7. IntGr fcron Lcvcls 1l1duced Poly 1: poly
(100 l.tg, i.p.), (1000 mg/kg, Tilorone HCl
(250 mg/kg, in SFV-Infected Uninfected
Control Mice
a
Days Serum interferon resQonse (U/ml)
Post- Uninfected SFV
Inducer infection control infected (%) infected (%)
Poly 1: poly 1 3,500 9,500 (270) 5,000 (140)
2 4,300 4,300 (100) 2,100 (49)
3 2,100 850 (40) 720 (34)
4 2,900 270 (9) 300 (10)
1 5,500 9,500 (170) 4,200 (76)
2 4,300 4,500 (104) 1,500 (35)
3 3,700 1,100 (30) 900 (24)
4 4,800 120 (2) 250 (5)
Tilorone HCl 1 10,000 1,500 (15) 1,200 (12)
2 6,500 350 (5) 190 (3)
3 7,000 150 (2) 100 1)
4 6,500 100 (1.5) 100 1)
1 <50 150 350
2 <50 100 100
3 <50 <50 <50
4 <50 <50 <50
aAnimals received single injection of inducer 24, 48, 72, 96 hr after
infection. Blood and subsequent serum samples were collected 4 hr after
poly I:poly 6 hr after phosphate-buffered saline (PES), and
18 after tilorone HCl.
bNumber in parentheses is percentage of uninfected control response.
Source: Reprinted from Ref. 55 with permission of the Americal1 Society
for Microbiology.
other hal1d, could increase the il1terferon response of hyporeactive cells
animals but had little effect the responsivel1ess of normal cells
animals (Figures 4 al1d 5) [61]. The prostaglandin restoration phel10mena
appeared to ubiquitous il1 that animals il1fected with of several viruses
including SFV, A
z
influenza virus, and Friend leukemia virus were
restored in their ability to respond to variety of interferol1 il1ducers.
These results particularly important, they suggest that prostaglan-
dins to significal1tly e11hance therapeutic efficacy of interferon
inducers as antiviral agel1ts. At present, correlation does not exist
between the state of hyporeactivity that develops as consequence of mu1tiple
8. Comparison of lJyporcaciivc
Interferon-Producing Cells
a
Macromolecular synthesis
DNA
RNA
Protein
Morphology (SEM)
Inducer uptake
NDV
CV
Doubling time
Adenylate cyclase

same
same
same
same
same
same
same
Hyporeactive cells
(lower 70% of normal)
Hyporeactive cells
(20-25% of normal)
S'I'ltl 1,'1'; 1 ,1 ,( >W
aHyporeactive cells were peritoneal cells from EMC-virus-infected mice
(96 hr) mouse embryo cells incubated with serum from EMC-virus-
infected mice. Normal cells were from uninfected mice or cells incubated
with normal mouse serum.
doses of inducer and prostaglandins. Preliminary evidence indicates that
prostaglandins in specific instances, restore the ability of mice
rendered hyporeactive multiple doses of poly 1: poly to respond to
second dose of the same or another inducer, for example, tilorone hydro-
chloride. Prostaglandins have not, however, consistently been to
increase responsiveness. This indicate that other mechanisms
involved in the development of hyporeactivity following multiple doses
of inducer. Development of hyporeactivity reflection of several
mechanisms, including the of animals to clear compounds from the
circulation and the reticuloendothelial system. For example, many
low-molecular-weight inducers, such as anthraquinones and tilorone
hydrochloride, not rapidly cleared the animal and persist in the
reticuloendothelial system for extended periods of time (Chapter 7). Ani-
mals dosed with such agents not amenable to prostaglandin restoration
of interferon responsiveness, whereas poly 1: poly is rapidly cleared and
animals injected with it appear to the most sensitive in their to
restored prostaglandin administration. At present, evidence indicates
that prostaglandins restore the response of hyporeactive animals and
to significantly improve the therapeutic efficacy of interferon
inducers in experimental viral infections neoplastic diseases.
(i. INIJlJCI':II.S: 'l'111<;()I(Y /lNI)
4.0

3.0
;;;
!::
2:
=>
'"


-'
...
CI')
2:

"- 2.0
CI')
...
cr:
4.0
2:

cr:
...
...
cr:
...
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2:

=>
cr:
...
3.0
CI')
2.0
PBS
PGEt PGAt
PROSTAGLANDIN
D
POLY I:POLY

PGFta
I!,!)
FIGURE 4. Effect of prostaglandins the of peritoneal cel1s from
normal or hyporeactive (EMC-virus-infected) mice to respond to il1terferon
il1ducers in vitro. Prostaglandins were added to cel1s 30 min after inducer
(Newcastle disease virus, NDV). Growth medium was col1ected 24 hr after
the addition of inducer and was assayed for interferon.
.1
4.0 NORMAL PCs


!::
z
::::1
>
CI
Z
:= 2.0 L--L....L_-'-
....
'" 4.0
z


'"
....

z


....
...

....
3.
1.0
D



PGF1a
PBS
5.0
PROSTAGLANDIN (/Lg!ml)
STltl N(; 1,'1': 1 ,1 ,()W
FIGURE 5. Prostaglandin enhancement of the interferon
of hyporeactive (EMC-virus-infected) but not uninfected mice.
Inducers were injected 96 hr after infection, and prostaglandins (1 mg/kg,
i.p.) were administered min later.
VI. TOXICITY
Another facing the development of most of the presently known
interferon inducers is toxic side effects associated with drug administration.
Only few inducers have given to humans. The chief side effects
observed in ha ve included, depending the inducer,
of the animal to clear the compound from its system and toxic manifesta-
tions such leukopenia, fever, headache, nausea, lethargy, insomnia,
and changes in hematopoietic and liver function ([ 31-34,62); also
(j. INTI';H.I,'I';HON INIHICI';H.S: TIII';OHY I(jl
7, JO). IJaLa [1"0111 CXjJ()l"iHIC!lLal suggest
LlI::tL kl1uWI1 too
side 1<'01" example, tilorone caused 01 the toxic mani-
listcd above in dogs and cats, and the drug was apparently
depositcd in various tissues for extended periods of time ([ 63]; see also
Chapter 8). Poly 1: poly likewise, has caused of the side effects
listed above in experimental animals. It had maximum tolerated dose
less than 4 mg/kg (i. v.) in cats [64]; yet it appeared to we11 tolerated
at low doses in children, with little toxicity being observed [34], although
low levels of interferon were induced at the doses given. series
of pyrimidine molecules that we have developing toxic mani-
festation that appears to due to insolUbility. As reported Levine
[65] and confirmed in our own laboratories (J. Gray, unpublished results),
fol1owing systemic or oral administration of fairly large doses of 2-amino-
5-bromo-6-methyl-4-pyrimidinol 300 mg/kg) , the drug crystallized
in the kidneys of rats, causing tubular obstruction. rat appears to
very sensitive to development of this side effect, since rabbits, cats, and
dogs maintained similar treatment regimens did not develop tubular
obstruction even though in some cases slight crysta11ization was observed
in kidneys and urine. Not members of this group of inducers caused
this side effect, and specific analogs have that have the
same or greater antiviral activity yet do not crysta11ize in kidneys [66-67].
New inducing molecules-or analogs of currently inducers with
less toxicity and fewer side effects-need to developed if these agents
are to used in the treatment of mild, self-limiting infections such as
the cold. Side effects that have reported with most inducers
probably would acceptable if they were being used in treatment of gravely
il1 persons with various life-threatening viral infections or neoplasias.
VlI. EFFECT OF INDUCERS ON IMMUNE SYSTEMS
great deal of attention has recently focused interferon as key
regulator of the immune response. As might expected, inducers like-
wise to affect the immune response and appear to have actions
separable from those mediated interferon (see Chapters
10 and 11). Inducers appear to affect the humoral and ceHular immune
response and also macrophage function, either enhancing or depressing
activity depending dosage or route of administration with regard to
immunogen and the parameter being investigated. The fact that inducers
mediate macrophage function is particularly interesting with the current
interest in modulators. The development of agent that could
have at least three modes of action that could account for the antiviral and
antitumor activity not unrealistic. The agent could induce inter-
feron, have direct anLiviral activity, l"CSPOIISU.
The potential of inducers in treating tral1splant paticllis parLiculaely
important to investigate, if compound could doprossos
function while inducing interferon having other antiviral activity.
VIII. OUTLOOK
The future for the development of interferon inducers appears to bright,
particularly if molecules identified that devoid of the problems
currently being encountered. Toxicity must decreased without markedly
reducing, and hopefully increasing, the antiviral activity of such agents.
This particularly difficult to do, since minor changes of the struc-
ture of most interferon-inducing molecules markedly affects (generally
depresses) antiviral activity. This is further complicated the fact
that simply because interferon inducers effective in mice is guaran-
tee that the same compound will have equivalent activity in humans.
system for predicting the ability of molecules to induce in
humans therefore needs to established, and then enough
ducers have been evaluated in humans will it known whether the system
is accurate. of hyporeactivity due to multiple
doses of would to avoidable reversible event.
of regimens, inducers given
with other agents in time sequence that will
allow avoidance of the hyporeactive state. The that
restore the interferon of hyporeactive
animals suggests that the therapeutic efficacy of inducers
of prostaglandins with
Reports that and its modulate
have broadened the horizon of develop-
They suggest that the future should developed not
only for their ability to stimulate production conversely mediate
activity, but that their ability to macrophage activity
suppress the cellular humoral immune response just
as critical to Answers to many of the remaining should
the next few years as compounds at various stages of
preclinical development toward
REFERENCE8
1. Isaacs and J. Roy. 147:258 (1957).
2. Kujima, R. Biol. 154:2172 (1958).
3. J. 8. and W. R. 8tinebring, 8cience 144: 1022 (1964).
1;. IN'I'I,:II.I"I':II.()N 'l'1I1,:()IIY
IJ. 1'. lJc SOl)1CI, 1':. l)cClCLcq, Cociio, and lHlliau, Ann. N.
Acad. Sci. 173: 274
5. 11. lJopps, S. Kohno, Kohno, and S. Smadel, Bacteriol.
l'roc. 115: 92 (1964).
6. W. llytel and Jones, Proc. Soc. Biol. Med. 123:859
(1966).
7. Freshman, Merigan, J. S. Remington, and I. Brown-
lee, Proc. Soc. Med. 123: 862 (1966).
8. W. R. Stinebring and J. S. Youngner, Nature 204: 712 (1964).
9. Science 146: 1472 (1964).
10. F. Wheelock, Science 149:310 (1965).
11. R. Friedman and L. Cooper, Proc. Soc. Biol. Med. 125:
901 (1967).
12. L. Glasgow, J. Bacteriol. 91:2185 (1966).
13. J. Green, S. R. Cooperband, and S. Kibrick, Science 164: 1415
(1969).
14. Field, Tytell, G. Lampson, and R.
Proc. Nat. Acad. Sci. U.S. Q..:1004 (1967).
15. Merigan, Nature 214:416 (1967).
16. G. D. Mayer and R. F. Science 169: 1214 (1970).
17. W. W. Hoffrnan, J. J. Korst, J. F. Niblack, andT. Cronin,
Antimicrob. Agents. Chemother. (1973).
18. Glaz, Szolgay, I. Stoger, and Talas, Antimicrob. Agents
Chemother. l:537 (1973).
19. F. R. Nichol, S. D. Weed, andG. Underwood, Antimicrob. Agents
Chemother. 3!.: 433 (1976).
20. J. Grisar, R. Hickey, R. W. Fleming, and G. D. Mayer,
Biophys. Res. Commun. 73: 149 (1976).
21. Carr, J. F. Gunwell, D. D. R. Meyer, F. W. Sweet,
J. Schweve, J. Grisnar, R. W. Fleming, and G. D. Mayer,
J. Med. Chem. 19: 1142 (1976).
22. Chandra and Woltersdorf, FEBS Lett. 41: 169 (1974).
23. Colby and J. Chamberlin, Proc. Nat. Acad. Sci. U.S. 63: 160
(1969) .
24. DeClercq, F. Eckstein, and Merigan, Ann. N. Acad. Sci.
173:444 (1970).
25. DeClercq and Merigan, Nature 222: 1148 (1969).
26. F. Torrence and Witkop, Proteins, Nucleic Acids and Enzymes
21: 536 (1976).
27. F. Torrence and DeClercq, 16: 1039 (1977).
28. F. Torrence and DeClercq, Pharmacol. 1 (1977).
29. W. Carter, Pitha, L. W. Marshall, 1. Tazawa, S. Tazawa,
and Ts'o, J. Mol. 70:567 (1972).
30. J. Portnoy and Merigan, J. Infec. Dis. 124:545 (1971).
1<.:. 1). III'OWI1,
Proc. Soc. 1:37: :357 (1971).
32. Field, W. Young, 1. Krakoff, Tytcll, 1'.
son, Nemes, and Hilleman, Proc. Soc. Biol, MCll,
136: 1180 (1971).
33. Field, Piperno, G. Lampson, NemcH,
and R. Hilleman, Medicine 51: 169 (1972).
34. Guggenheim and S. Baron, J. Infec. Dis. 136: 50 (1977).
35. Levy, W. London, D. Fucillo, S. Baron, and J. Rice,
J. Infec. Dis. (1975).
36. D. StrmgfellowandS. D. Weed, J. Vet. Res. 38:1963 (1977),
37. D. Stringfellowand L. Glasgow, Antimicrob. Agents Chemother.
73 (1972).
38. Siminoff, J. Infec. Dis. (1976).
39. Ito, 1. Nagata and Kunji, Virology 52:439 (1973).
40. Breinig and N. Maehara, J. Infec. Dis. (1976).
41. F. Dianzani and S. Baron, Nature 257: 682 (1975).
42. F. DianzaniandS. Baron, Proc. Soc. Biol. Med. 155:562 (1977).
43. F. Dianzani, 1. Viano, Santiano, Zucca, and Baron, Proc.
Soc. Biol. Med. 155:445 (1977).
44. and J. Clin. Invest. 44: 1059 (1965).
45. Kono, and Brening, Proc. Biol. Med.
119: 1227 (1965).
46. Buckler, G. DuBuy, L. Johnson, and S. Baron, Proc.
Soc. Biol. Med. 136: 394 (1971).
47. G. DuBuy, L. Johnson, Buckler, and S. Baron, Proc.
Biol. Med. 135:340 (1970).
48. DeClercq, Proc. Soc. Biol. Med. 141:340 (1971).
49. Breinig, J. Armstrong, and J. Gen. Virol. 26:
149 (1975).
50. G. Bansek and Merigan, Proc. Soc. Biol. Med. 134:
672 (1970).
51. Glaz and Talas, Arch. Virol. 48:375 (1975).
52. F. 1. Yershov, Tazulakhova, and S. Novokhatsky,
Virol. 20: 15 (1976).
53. D. Stringfellow, S. D. Weed, and G. Underwood, Antimicrob.
Agents Chemother. 15: 111 (1979).
54. D. J. Giron, J. Schmidt, F. F. Pindak, and J. Acta
Virol. 17: 209 (1973).
55. D. Stringfellow, Antimicrob. Agents Chemother. 11: 984 (1977).
56. J. Science 177: 797 (1972).
57. D. Holtermann and Havell, J. Gen. Virol. 101 (1970).
58. D. Stringfellowand L. Glasgow, Infec. Immun . ..: 743 (1972).
59. J. Osborn and D. N. Medearis, Proc. Soc. Biol. Med. 124:
347 (1967).
1). 1';. It. !\.UI.II, 1). J\.. KuIHC'y, alld 1,. Ulasgow,
J. lllfuu. Vit>. (1!177).
D. Suit)llue 201: 376 (1978).
62. J. L. Alderfer, J. Levy, L. W. Marshall, J.
J. S. and W. Cal'ter, Mol. Pharmacol. 12:299 (1976).
63. W. Rohovsky, J. W. Newberne, and J. Gibson, Toxicol. Appl.
Pharmacol. 17: 556 (1970).
64. McCullough, J. lnfec. Dis. 125: 174 (1972).
65. S. Levine andR. Sowinski, Toxicol. Appl. Pharmacol. 42:603 (1977).
66. D. Stringfellow and S. D. Weed, J. Clin. Hematol. Oncol. in press
(1980).
67. D. Stringfellow, Vanderberg, and S. D. Weed, Curr.
Chemother. Infec. in press (1980).
7
INDUCTION OF INTERFERON
POL YNUCLEOTIDES
Hilton Levy
National Institute of Allergy and Infectious Diseases
Bethesda, Maryland
1.
EARLY STUDIES
DOUBLE-STRANDED RNA: EARLY WORK
IV. DOUBLE-STRANDED RNA AND
V. POLY 1: POLY IN PRIMATES
VI. POLY 1: POLY (POLY ICLC)
VII. STUDIES IN WITH POLY 1: POLY
TOXICITY OF POL 1: POL
IX. OTHER MODIFICATIONS OF POLY 1: POLY
GENERAL
REFERENCES
1.
167
168
169
172
172
172
179
180
181
182
182
Interferon was first named in 1957 Isaacs and Lindenman [1]. At about
the same time Nagano and Kojina [2] and Chany [3] were reporting analo-
gous findings. While many were skeptical about the existence of interferon,
it was realized some that interferon potentially represented broad-
spectrum antiviral agent that could have widespread clinical application.
The realization of this potential has been painfully slow. The difficulty of
preparing large quantities of interferon has kept the supply at very low
level and the price very high. lt has been estimated that mouse might
make about 1-2 106 units (U) of interferon in response to virus infection.
If one wanted to treat the infected mouse with interferon, the slope of the
167

dose-response curve would pcrhaps 10" a<blitiomtl
of interferon should given to augmcnt significal1tly tllC antiviral
Until recently this represel1ted severalliters of il1terferon.
humans, the situation would more dramatic. Hecently, as rcsu[t
of the work of laboratories, the situation has good deal
better, and very limited, very costly experiments in humans are
now, for the foreseeable future, the use of interferon itself in human
disease severely limited cost and availability.
EARLY STUDIES
Attention was turned, therefore, to finding nonviral inducers that would
cause the host to synthesize large quantities its own interferon, and
number of such materials were found. The types tabulated in 1.
Of the several inducer types, some are both in tissue culture
and in vivo, and others are effective primarily in vivo. latter, in
some instances, shown to induce interferon in cultures of leuko-
cytes, macrophages, or spleen
far the most effective nonviral interferon inducers have the
double-stranded ribonucleic acids (RNA). Two series of experiments led
to their discovery. Isaacs [4] postulated that interferon production is the
response to the presence of foreign nucleic presented
data that indeed indicated that treatment of tissue culture with nucleic
acids extracted from heterologous induced the formation of interferon.
chemical modification of homologous nucleic acids with nitrous
was sufficient to make the nucleic acid an interferon inducer. However,
the amount of interferon induced was very the experiments were
hard to reproduce, and the question of nucleic acid induction of interferon
was held in More direct evidence leading to the development of
1. Nonviral Inducers of Interferon
1. Endotoxins 7. Fungal viruses
2. Bacteria 8. Natural and synthetic nucleic acids
3. Trachoma-inclusion 9. Synthetic polymers and other
conjunctivitis agents chemicals
4. Mycoplasmas 10. Mitogens
5. Protozoa 11. Polysaccharides
6. Rickettsiae 12. Antiblotics
'1. INI>lJ(''I'ION 1\)' l'ol.YN'JCI,I';O'l'II>I';S ,(,!)
ItNAH V;lIllC wUl'k [5], crude
fountl cu!Lurcs PCl1icillium fUl1iculosum, which shows
viral aciivity 16]. [7] that stimulates
tissuc culture cells mice to make [8J. When helenine was
extracted phenol and the resulting product purified, double-
stranded llNA was obtained which was to induce interferon [9J. It was
later ShOWl1 that the double-stranded RNA was derived from virus that
infected the funiculosum [10].
III. DOUBLE-STRANDED RNA: EARLY WORK
series of papers from workers at Merck, Sharp and Dohme [11-15]
showed that variety of both natural and synthetic double-stranded RNAs
were effective interferon inducers in tissue cultures in rodents. The
homopolymer pair, was t11e most
effective of the synthetics, with polyriboadenylic-polyribouridylic acid being
significantly less so. As little as 0.5 of 1: poly intravenously
to rabbit induced detectable interferon. some tissue culture
of the is effective. RNAs
are 1ess as inducers; a1though under some conditions, they cause
the production of amounts.
observations regarding poly 1: poly r s capacity to induce
in triggered search for that would more effec-
tive. From these studies there has emerged the that number
of structura1 requirements must combined in compound in order for it
to good interferon inducer. For more detailed review of the struc-
tural that have examined, see the review DeClercq
[16] .
(1) There needs to structure that is stable at the tempera-
ture of the test. The two of each double-stranded RNA disassociate
from each other at specific temperature, the me1ting tempera-
ture

which is measure of the of that double-stranded RNA.

If attempts to correlate the

va1ues of group of double-stranded
RNAs with their ability to interferon, discern trend toward
correlation. However, tl1ere are so that it is obvious that
other factors in addition to the degree of thermal are important.
RNAs that have secondary structure induce
bofu vitro and tissue cultures although, genera1, so well
as double-stranded RNA [17,18,19,20]. It wou1d appear that secondary
structure, not necessarily double-strandedness, is requirement for activ-
ity.
(2) Another factor that is important, though not dominant, in determin-
ing the degree of activity of RNA is its resistance to the
action of ribonucleases. The RNA, AU, is poor
17()
inducer intcr fCrOH. WllCll 8lll)8 li lu lCll [ot"
groups, the resultallt poly (AsUs) is re" i"Lal1l to l1uclcasc actioll
alld is better inducel' thal1 poly AU [21]. Ifowever, are cxccptio!ls
to this gellerality. The effect of differences ill the amoullt nuclease actiOll
in the sera of diffel'ellt animal species wi1l melltiolled later.
111 the where it has possible to study series of chemi-
idel1tical double-strallded RNAs with differing molecular weights, it
appears that minimum molecular weight is Ilecessary for actiol1
[22]. Poly 1: poly with molecular weight of 1.5 105 is illactive, but
the compound with molecular weight of 2.7 105 higher is active.
However, the differellces accordil1g to others are small [16]. In
particular the size of the poly 1 stralld is more important thall that of the
poly strand; complexes made with poly 1 of s values equal to 12.5 S il1
tissue culture yield slightly more illterferol1 than do complexes made with
poly 1 of s = 2.5 S. Here s is the sedimel1tatioll coefficiel1t alld S is the
svedberg (10-13 sec).
(3) ribose backbone is needed. Sillgle- or double-strallded DNAs
induce 1ittle or illterferol1 [23] il1 spite of gel1erally high

values.
If the 2'-hydroxy groups the ribose are esterified with methyl group.
the RNA loses its activity.
list of al1tiviral drugs of type that are effective il1 vivo is
illdeed. Poly 1: poly is the most successful. effect has largely
limited to rodellts and rabbits. This fact will referred to later.
Two examples of the action are as follows. Whel1 rabbit eyes
abraded illfected with herpes virus, they develop keratoconjunc-
tivitis the human disease. Figure 1 shows the data obtailled
Park and Baron ill treating this disease with poly 1: poly in the form
of drops [24]. The abscissa is the time ill days after the
is Ilumber obtained combining the evaluation of several clinical
parametel'S. highel' the Ilumber, the more the disease. It
seell that if tl'eatment is begun the day infectioll, 110 disease
develops. wait as long three days after illfection to begin treat-
ment still have curative effect. However, if treatment
begins day 4, the drug is without therapeutic value.
1n other experiments Worthington Baron [25], mice were in-
fected with Semliki Forest virus (SFV) , virus that fatal
litis. Figure 2 plots the percentage of the animals that die function
of days after infection, with and without treatment poly 1: poly It
seen that untreated animals were day 5. Virus was replicat-
ing in the brain day 4. However, initiation of treatment after virus
was in the resu1ted in significant decrease in the mOl'ta1ity l'ate.
'/. INI>lIC'I'I()N IIY I'()I,YNIICI,I':()'I'IIH:S 17.\
011.'( 4
4 +-

+
3+
f/
.,.
I


w


'-'

2+
u
g;
v>
:z
Q::
Q

v>
w
---'
1+

2 3 4 5 7 8 9 10 11 12 13
DAYS INJECTION
FIGURE 1. Response of herpetic keratoconjunctivitis to topical treatment
with poly 1: poly Rx indicates treatment.
100
90

75

>-
Controls
f--
...J

60
f--
ct:

:::;
f--
45
z
w
u
ct:
w
30 (L
15
00
2 4
DAYS INFECTiON
FIGURE 2. Treatment of Semliki Forest virus (SFV) infection il1 mice with
poly 1: poly
172
DOUBLE-STH,ANDEU
Levy et studied the effect of 1: [26]. Tablc 2
gives partiallist of the 100ked at. The of different
tumors to the drug is quite variable, \vith being quite sensitivc,
the fast growing being affected just barely
[27,28,29]. With the more sensitive tumors, there were to one-third
survivors. The mechanism of this actioll is complex will
not discussed in detail, but there appears to at least three factors
involved. The first is that poly 1: induces the formation of interferoll
in mice, and interferon has antitumor action [30]. Second, poly I:poly
is potent enhancer of immulle reactivity, particularly cell-mediated
immunity, the type that is thought to important role in natural host
defense mechanisms against tumors [31,32]. Third, there is more or
specific inhibition macromolecule in some the
animal tumor systems [33]. These three elements illterplay in differ-
ent degrees in the different
V. POLY 1: POLY IN PRIMATES
human response to 1: poly is very weak, with
high doses of the drug [34-36]. Levels 50 U/ml of serum were found,
but not much more, contrasted with perhaps 50,000 U/ml in
mice. Rhesus and chimpanzees were totally nOllresponsive to
1:
possible explanation for poor primate response to 1: poly is
that there is present ill primate relatively high concentration of
llucleolytic enzymes that hydrolyze and inactivate poly 1: much more
is present in rodent serum [37]. and large those species animals
whose sera showed large capacity to hydrolyze were poor responders,
and good res ponders had 10w hydrolytic capacity. Efforts were made, there-
fore, to develop derivative that would more resistant to hydrolysis.
VI. POLY 1: POLY (POLY ICLC)
1: poly forms with poly-lysine, but the complex is not
and, therefore, is 1l0t useful clinically. However, if hydrophilic
between poly-L-lysine and carboxymethyl cellulose is first formed,
then poly 1: with this hydrophi1ic poly-L-lysine to give
derivative (poly ICLC) that is in alld partially resistant to
hydrolysis (Figure 3) [38].
That poly 1CLC is more stt'ucture to thermal
is 1: poly is shown in Figure 4. Poly ICLC in 15 NaCl
'1. INIJlI(''!'I!)N 1'()i,YNII(:I,I';()'I'II>I';:-;
l'ol.y 1: l'oly (; 011 Allil1la!
Tumor
J96132 reticulum sarcoma (subcutaneous)
J96132 reticulum sarcoma (ascites)
Carcinosarcoma Walker 256
Reticulum sarcoma RC8L
Ehrlich ascites tumor
891 Melanoma
Fibrosarcoma
lymphoma (ascites)
L1210 leukemia
Plasma
lymphoma (subcutaneous)
tumor (subcutaneous)
Reticulum sarcoma ovarian
Leukemia
Leukemia
Percentage
increase in median
survival over control




100
89
70
55
52
45
42
39
28
26
20
16
12
aTreatment, in most cases, was 150-200 three times weekly,
intraperitoneal route. With the exception of the J96132 reticulum
sarcoma, some Ehrlich ascites tumors, and few Walker carcinosarcoma,
animals ultimately died.
bMean day of death the animals that died. About 30% of the animals
treated have survived, although treatment had been stopped at about day 50.
does not denature below

whereas poly I:poly has

of

It is necessary to dilute the salt to 0.015 to obtain

for the
complex, while plain poly 1: poly melts at about 490
The compound was slightly more effective in mice as an interferon
inducer than poly 1: poly as shown in Figure 5. 8erum interferon was
detectable slightly earlier, rose to somewhat higher titer, and was
present longer. Not surprisingly, poly ICLC is somewhat better antiviral
agent in mice than is poly l:poly (L. Glasgow, unpublished data).

'"
N


1 100
1050 -
0900
30
I,I':VY

Poly 1: Poly
/
/
/
/
/
--//:/
__________ ___
Complex 2
60 90 120 150 180 360
TIME AFTER ADDITION OF ENZYME(in minufes!
FIGURJi; Hydrolysis of poly 1: poly and two different lots of the poly-
L-lysine complex of poly I:poly pancreatic RNase. The complexes,
at concentration of 50 poly 1: poly in 0.15 NaCl and 0.001
phosphate buffer 7.2), were exposed to 5 pancreatic RNase/ml at
room temperature (about 240 Optical density (OD) readings at 260 nm
were taken at 10 min intervals.
Of greater interest was the fact that poly ICLC was effective inducer
in monkeys (Figure 6) and chimpanzees. Interferon levels high 15,000
U/ml of serum have found in cynomolgus monkeys under conditions
where interferon was induced poly 1: poly However, levels between
200 and 2000 U are more regularly
new compound is effective antiviral agent in monkeys. Monkeys
infected with street rabies virus could effectively protected poly ICLC
together with antirabies shown in 3 [39]. Vaccine alone
had little or protective effect in several experiments.
Comparable results were obtained with yellow fever virus, Tacharibe
virus, Japanese encephalitis virus, Pichinde virus, and with simian hemor-
rhagic fever virus, ofwhich ordinarily lethal for [40,41],
well with Russian spring-summer encephalitis and vaccinial keratitis
[42,43] .
There is animal model of chronic hepatitis in chimpanzees.
Figure 7 shows the effect of treatment of such infected with poly
'1. INIHI(''I'I()N I'(H,YNIJCI,I':()'I'II)/';S
0.300
0.200

---
//
/ (
0.100
/ I
I
/ J
I I
I
, 1------ =

! I m
I I
PIC-L
/ I
// I
0.000
." I
"",,/ //
---"
-------

30 40 50 60 70 80 90
Temperature
FIGURE 4. Thermal denaturation of poly 1: poly and the poly- L-lysine
complex of poly 1: poly (PIC-L). The compounds, at concentration of
50 poly 1: poly C/ml in 0.1 standard saline-citrate, were heated to the
indicated temperatures in recording spectrophotometer set at 243 nm.
m = melting temperature.)
5
4

LLJ
1- 3
1-
LL.
(!)

...J
I
I
I
I
I

.",.----,,-
/' - - -
--

--
-#---
....
......
......
*,
"-
"-
"-
,


HOURS POSTINJECTION
FIGURE 5. Kinetics of induction of serum interferon (IF) in mice after
intravenous administration of 5 mg/kg poly 1: poly or poly ICLC.
] '7(i
6000
4000
2000

2' 1000
"
800
::J
'-"
:
600
UJ
1-
400
i=
z

:
UJ
200
lL.
:
UJ
1-
Z
100
80
60
40
20
I
I
I
I
I
I
I
I
,
I
I
I
I
I
10
"
\
\
\
\
\3mg/kg
5 mg/kg
\
20 30 40 50
TIME AFTER INJECTION
\
\
\
,
\
\
\
\
\
60
\
\
\
\
\
\
\
'.
70
FIGURE 6. Kinetics of induction of m interferon in rhesus monkeys
administration of 3 5 mg/kg poly ICLC.
T A L E 3. Effects of Poly ICLC Treatment in Postexposure
Prophylaxis of Rabies in Monkeys
Treatment No. dead/No. treated
Poly ICLC + vaccine
24 and 72 hr postinfection
Vaccine
24,48, and 72 hr postinfection
Controls, untreated
1/8
7/8
8/8
'1. INIHJCTI()N I'OI,YNIJCI,I';()TII)I<S
1'1'1
2.0
z



LLa:




;?
1.0
(:J

<0.7
-"
300
Q..
1-
200
1-
1
'I'

::2: 100
Q..
U
1975
1976
FIGURE 7. The treatment of chronic in young chimpanzees with
1: The interferon titer is shown and the polymerase
activity is shown below. The beginning and end of the treatment period are
indicated arrows the abscissa. dot and bar the graph of DNA
polymerase response indicate the (+1 SD) of polymerase activity
detected in six samples obtained during the weeks immediately
preceding the experiment.
ICLC [44]. It seen that when the animals are injected with poly
ICLC, they produce interferon. marker of the progress of the infection
is the level of DNA-dependent polymerase in the blood. During the course
of the treatment the polymerase activity fell to background level. When
treatment stopped, evidence of the disease returned. Whether really pro-
longed treatment would have more permanent effect has yet to deter-
mined.
Vaccinia virus infection in the skin in rabbits severe, some-
times fatal infection. Rabbits were injected intradermally with vaccinia
virus. After the lesions visible, some rabbits were treated daily
with ointment containing the drug. comparison of the treated and the
nontreated rabbits is seen in Figure 8. The infection essentially did not
progress after initiation of treatment [45].
last effect of poly ICLC should mentioned. It has proven to
potent booster of antibody response to at least two virus vaccines,
FIGURE 8. Effect of poly ICLC ointment cutaneous vaccinia infection in t s . (--\1 Pol',- ICLC !:'ea::::.
( ) Placebo tl'ea,:ed,
-J

<
-<
'1. \N\HII ''I'\()N \\ \' \'()\ ,\'NIII' \,1';0'1'11
\'I!I
1000

IJJ
1-

Z
<t
IJJ
::1:
u 100

I;j
::1:



10
--.
-,
10 20 30 40 50 72
DAYS POSTIMMUNIZATION
FIGURE 9. Adjuvant effects of poly ICLC inactivated Venezuelan equine
encephalitis virus vaccine in rhesus monkeys. Standard errors are shown
where significant differences occur as compared to controls.
KVEE + 3 mg/kg;+ ' KVEE + 1 mg/kg; 8, KVEE controls; *, = <0.01.
zuelan equine encephalitis virus vaccine [46] and swine flu vaccine [47],
and to bacterial vaccine, Hemophilus influenzae Levy, Stephen,
and Anderson, unpublished data). Figure 9 shows some of the resu1ts
obtained with Venezuelan equine encephalitis virus vaccine. It seen
that one dose of the drug, together with the vaccine, boosts antibody re-
sponse about 80-fold and increases the during which antibody
detected.
VII. STUDIES IN HUMANS WITH POLY 1: POLY
Several studies in humans are ongoing. Levine et al. have completed
phase 1 study of human response and toxicity in terminal cancer patients
[48] to determine (1) dose response curve and (2) the highest tolerated
level of drug. In this group of 35 patients, serum interferon levels of about
100 U/ml were seen at dose of about 100 body weight. Peak levels
of to 15,000 U/ml of serum were seen at 510 and 770 body weight.
These latter levels of drug were associated with unacceptable levels of
toxicity; 350 was judged to the highest acceptable dose, with mean
peak titers of interferon of about 2000 U/ml of serum being achieved.
JHO
relative absence of W;L[-; Icvcl[-; ill
l'hesus and cynomolgus monl,cys L .
We Lerner, J. Cranc, and Lcvy)
the drug as applied ointment in double-blind study herpcs
genitalis. Champney, Lerner, and Levy [50] gave the drug
to few patients with St. Louis encephalitis (as descl'ibed later). Levels
of serum interferon to those noted Levine et al. [48] were
found. Engel et al. [51] have given poly ICLC to two patients with
tropic lateral sclerosis (ALS) and to one patient with chl'onic l'elapsing
neuropathy, possibly associated with an immunological distrophy. Thcsc
patients l'esponded with slightly higher levels of serum interferon than did
the other groups. There was clinical improvement in the ALS patients.
However, the patient with chronic relapsing neuropathy showed dramatic
clinical improvement. In addition, his cerebrospinal fluid protein from
105 to 40 At this writing he is continuing to improve.
TOXICITY OF POLY 1: POLY
Some problems with poly ICLC should mentioned. double-stranded
RNAs are pyrogenic, and this one is exception. In humans, tempera-
tures to

have been seen, although

is usual. The fever

usually is gone in 5-6 hr. Some differences have been found in other re-
activities the three human studies mentioned above. Levine and
Levy found, particularly at the higher levels of drug, that leukopenia (down
to 2000 WBC/mm 3) and hypotension (to below 90 systolic) occurred in
about 30% of the Lerner and Levy found leukopenia and hypotension
in most of their cases (patients with high fevers from the disease at the
onset of treatment), and Engel et al. [51] found leukopenia but hypoten-
sion. frequent finding is myalgia and group of flu-like symptoms.
There are suggestions that the of lower-molecular-weight poly 1: poly
in the complex result in drug with fewer side effects Lerner,
Gatmaitan, and Levy, unpublished data). It might noted that
treatment of humans with interferon has itself elicited of the same
undesirable effects as those mentioned with poly ICLC. In of
the studies has it been necessary to abandon treatment except with the
highest levels of drug.
It was mentioned earlier that primates hydrolyze poly 1: poly faster
than do rodents, and the suggestion was made that this increased hydrolysis
might explain the weak response of primates to poly 1: poly Cons istent
with this suggestion are the observations made with poly ICLC preparations
which have been made with different molecular weights of poly-L-lysine.
With poly-L-lysine of molecular weight 2000, the complex formed is per-
'1. INIJII("I'I()N l'O),YNIICU:UI'IIJI':S
11'1
Lwiee l'csisLallL Ily(ll'olysis ;LS plaill !!oly [: poly (;,
Lo 1'-10 Limcs ['csisLatlL 1'01' complux maLlu witll poly-L-lysine of
muluuulaL" weiglJt induces perhaps as
inturfuron in does the complex made with high-molecular
weight puly-lysine.
IX. OTHER MODIF1CATIONS OF POL 1: POLY
1n the past the induction of interferon any means has generally been
associated with certain level of undesirable side effects. 1n humans and
in many animal species, at least some of these symptoms were found when
interferon itself was administered. Attempts to dissociate the reactogenicity
from the interferon or interferon induction not been particularly re-
warding. Recently group of workers have reported some success in such
dissociation [52-54]. These workers postulate that the several blological
effects of the polynucleotide inducers-interferon induction, pyrogenicity,
possible effects blood elements, etc. -require different lengths of
time of contact with the responsible specific population, and that inter-
feron induction requires the shortest length of of contact. They
prepared complexes of poly 1 with other copolymers containing either 13
cytosine to uracil molecule or 29 cytosine to guanine, to yield
r1
n


U)n' or r1
n
r(C
29
, G)n. These compounds are thermodymically
less stable than poly 1: poly and more readily hydrolyzable RNase.
According to the hypothesis of these workers, when these compounds are
administered to animal, they hydrolyzed and eliminated rapidly,
with existence only sufficiently long to stimulate the production of inter-
feron and resistance to viral infection. The data presented are indeed
consistent with their hypothes is. These authors point out the difficulty in
defining toxicity and therefore take as their definition that toxicity is
biological alteration other than the induction of interferon. of the mis-
matched analogs of 1: poly induced significant interferon and resist-
to virus infection, both in tissue culture and in rode11ts, although
perhaps 110t quite so i11terfero11 as poly 1: poly [53]. The mismatched
compou11ds appeared less pyroge11ic i11 rabbits, caused less stimulatio11 of
s plee11 cells, as measured thym idi11e uptake, a11d required larger amou11ts
for acute toxicity i11 mice whe11 compared with poly 1: poly 111 additio11,
i11 rode11ts there were somewhat less chronic effects, such as lymphope11ia
a11d a11emia, occasio11ed the mismatched a11alogs tha11 poly 1: poly
No studies were made i11 primates. It i11deed that the ease of hydrol-
ysis of these compounds does reduce their half-life i11 rode11ts, a11d C011se-
que11tly their reactoge11icity. However, it also prove to that the ease
of hydrolysis re11der them i11effective i11 primates.


There are severa1 general SllOUld lJC il1
cussion of po1ynuc1eotide inducers of 111 limiteu
use of the inducer thought of as just a1terBative way of aumil1ic;l!""-
ing interferon. As such, tlle drug" some it is very mucl]
cheaper, it is sca1e tilat makes it practica1 item in matceia
medica, and it is certain conditions, to resu1t tile
of higher serum 1eve1s of than presently obtained
tile use of exogenous
the other hand, there are some major the ad-
ministration of and tile of the
While does cause the same type of side
effects that po1y 1CLC does, it that the inducers show stronger
reactions. It is possible that these differences are associated
witil the higher 1eve1s of induced, or it that tile drug per
se is more reactogenic. It does seem that the drug more fever
interferon. 1n addition, interferon, at 1east in tissue cu1ture systems,
appears to inhibit most immune tested. Whether this hold
true in the host is Po1y 1CLC, the other hand, acts
as where it has tested. this turn
out to clinically usefu1 feature, because such has
found at 1eve1s of drug well be10w those tilat adverse re-
actions.
drawback of tile inducers is that they require bio1ogica1
response the part of tile host to produce interferon. There some
patients who make tile response. If the undesirable
of inducers are separable from the induced, furtiler work
might produce more effective, 1ess reactogenic
REFERENCE8
1. 1saacs and J. Virus 1. interferon.
Proc. Roy. 8er. 147:258 (1957).
2. Nagano and Kojima, de par
facteur liquide dans 1e tissu infecte par 1e virus homo1ogue. R.
152: 1627 (1958).
3. An inhibiting factor of intracellu1ar multiplication of viruses
called interferon from cancer cells. R. Acad. 8ci. 250:
3903 (1960).
4. 1saacs, 1nterferon. 8ci. Amer. 204: 51 (1961).
5. R. 8hope, An from funicu1osum.
Effect of he1enine in mice with 8emliki- Forest virus.
J. Med. 97: 627 (1953).
'1. INIHI(''I'I(IN II\'

(j. W. COClll':lII :01(1 '1'. 1,'I':lIICiH, ,11'., AIlLivit'al acLioll of IlGlenine
pO\iOlll'yc1iLiH. 1'1,'OC. Soc. 13iol. Med. 92:230 (1956).
'7. U. ,1. J,CWiH, L. L. N. G. Purifi-
of thc agent, helenine. J. Amer.
CJlCl1l. Soc. 81:4115 (1959).
W. l{ytel, Shope, and D. Kilbourne, An antiviral
stance from V. 1nduction of interferon
J. Med. 123:577 (1966).
9. G. Tytell, Field, Nemes, and
R. Hilleman, 1nducers of interferon and host resistance. 1. Double-
stranded RNA from extracts of Penicillium funiculosum. Proc. Nat.
Acad. Sci. U.S. 58: 782 (1967).
10. G. Banks, W. Buck, Chain, F. Himmelweit, J. Marks,
J. Tyer, Hollmgs, and F. Lost, Viruses in fungi and inter-
feron stimulation. Nature 218:542 (1968).
11. Field, Tytell, G. Lampson, and illeman ,
1nducers of interferon and host resistance. Multistranded
polynucleotide complexes. Proc. Nat. Acad. Sci. U.S. 58: 1004 (1967).
12. Field, G. Lampson, Tytell, Nemes, and R.
1nducers of interferon and host resistance. 1V. Double-
stranded replicative RNA (lVIS 2-FF-RNA) from infected with
MS2 coliphage. Proc. Nat. Acad. Sci. U.S. 58:2102 (1967).
13. Field, Tytell, G. R.
1nducers of host resistance, V. vitro studies.
Proc. Nat. Acad. Sci. U.S. 61:340 (1968).
14. Tytell, G. Field, and R.
of host resistance. RNA
from reovirus type 3 Proc. Nat. Acad. Sci. U.S. 58: 1719
(1967) .
15. Field, Tytell, G. Nemes, R.
Hilleman, Double-stranded polynucleotides inducers.
J. Physiol. 56: 905 (1970).
16. DeClercq, Structural of inducers. 1n
Symposium and Clinical Use of 1nter-
feron, Zagreb, 1977, Yugoslav Acad. Sci. Arts, Zagreb.
17. S. N. N. Bogomolova, Billiau, Levy, Buckler,
R. Naylor, of preparations of
synthetic single-stranded RNA. Proc. Nat. Acad. Sci. U. S. 64: 67
(1969).
18. illiau , Buckler, F. Dianzani, and S. Baron,
1nduction of interferon mechanism single-stranded RNA: Potentia-
polybasic substances. Proc. Soc. Biol. Med. 132: 790
(1969).
19. , Buckler, F. Dianzani, Uhlendorf, and S. Baron,
1nfluence of basic substances the induction of the interferon mecha-
nisms. Proc. Soc. Biol. Med. 132: 790 (1969).
20. I3illiau al1d 10;. lrHJucLiurl irrl,cr'l'cr:OIl rllcclr:lIlislll IJY
l1aturalllNA. Life Sci. 2,: 69 (1970).
21. DeClercq, ]<'. Ecksteil1, al1d J. Merigal1,
il1creased through chemical modificatiol1 of sYl1thetic polYl1uclcoLidc.
8cience 165: 1137 (1969).
22. Jameson and 8. Grossberg, 1I1terferol1 il1duction in mice
complexes of polYl1ucleotides of varyil1g sizes. Bacteriol. Proc.,
L55 (1970).
23. J. Vilcek, Ng, Friedmal1-Kein, and Induc-
tion of interferol1 synthesis sYl1thetic double-stral1dod polYl1ucleo-
tides. J. Virol. 648 (1968).
24. J. Park and 8. Barol1, Herpetic keratoconjul1ctivitis therapy with
synthetic double-stranded RNA. 8cience 162: 811 (1968).
25. Worthington and 8. Baron, Late Therapy with interferol1 stimula-
tor il1 an arbovirus encephalitis in mice. Proc. Biol. Med.
136:323 (1971).
26. Levy, L. W. Law, and 8. Rabson, Inhibltion of tumor growth
polyinosil1ic-polycytidylic Proc. Nat. Acad. 8ci. U.8. 62:
357 (1969).
27. V. al1d Levy, acid
chemically il1duced tumorigel1esis in skin. 8ciel1ce 167: 205
(1970) .
28. 8. 8arma, G. 8hiv, 8. Barol1, Il1hibltory effect of interferol1 011
murine sarcoma al1d leukaemia virus il1fectiol1 il1 vitro. Nature 223:
-----
845-846 (1969).
29. L. D. Zelezl1ick al1d Bhuyan, Treatment of leukemia (L-1210)
mice with double-stranded polyribonucleotides. Proc. Biol.
Med. 130: 126 (1969).
30. 1. Gresser, L. Berman, G. De The, D. Brouty-Boye, J. and
Falcoff, Interferon and muril1e leukemia. V. Effect of interferol1
preparatiol1s 011 the evolutiol1 of Rauscher disease in mice. J. Nat.
Cal1cer Inst. 41: 505 (1968).
31. Cantor, R. Asofsky, and Levy, effect of polyinosinic-
polycytidylic graft-vs-host aotivity il1 BALB/c mice.
J. Immunol. 104: 1035-1038 (1970).
32. W. Turner, 8. Chal1, and Chirigos, 8timulatiol1 of humoral
al1d cellular al1tibody formatiol1 in mice Poly Ir: Cr. Proc.
Biol. Med. 133: 334-338 (1970).
33. Levy and F. Riley, The effect of polyil1osil1ic: polycytidylic
tumor metabolism. Proc. Biol. Med. 135: 141-145
(1970).
34. D. S. Baron, Levy, J. Bellal1ti, Buckler,
G. Canl1ellos, Carbone, R. Chanock, V. DeVita, Guggen-
heim, Z. R. L. J. Mills, J.
Vankirk, al1d Worthingtol1, Clil1ical studies of il1duction of interferon
'1. INIHI(:TI()N
IJY aci!l. 111 irl
Vol. 7: Moluculus to ed.), Acadernic
Nuw 1971, 198-222.
35. [{. l{obinson, V. Devita, Levy, S. S. Hubbard,
and S. Levine, phase trial of multiple-dose polyriboinosinic-
acid in patients with leukemia or solid tumors.
J. Nat. Cancer Inst. 57: 599 (1976).
36. W. Young, Interferon induction in cancer, with some observations
the clinical effects of poly 1: Med. Clinics N. Amer. 55: 721-728
(1971).
37. J. J. Nordlund, S. Wolff, and Levy, Inhibition ofbiologic
activity of poly 1: poly human plasma. Proc. Soc. Med.
133:439-444 (1970).
38. Levy, G. Baer, S. Baron, Buckler, J. Gibbs, J.
Iadarola, W. London, and J. Rice, Amodified polyriboinosinic-
polycytidylic acid complex that induces interferon in primates. J. Infec.
Dis. 132:434-439 (1975).
39. G. Baer, J. Shaddock, S. Moore, Levy, and S. Baron,
Successful Prophylaxis against rabies in mice and rhesus monkeys:
interferon system and vaccine. J. Infec. Dis. 136: 286-271 (1977).
40. Levy, W. London, D. Fuccillo, S. Baron, and J. Rice,
Prophylactic control of Simian hemorrhagic fever in monkeys an
interferon inducer, polyriboinosinic-polyribocytidylic acid poly-L-
lysine. J. Infec. Dis. 133: (1975).
41. L. Stephen, L. Sammons, W. L. Pannier, S. Baron, R.
Spertul, and Levy, Effect of nuclease-resistant derivative of
poly I:poly yellow fever in Rhesus monkeys. J. Infec. Dis. 136:
122-126 (1977).
42. Burgasova, D. G. Andzaparidze, Bektemirov, N. N.
Bogomolova, and S. Boriskin, Influence of Poly
complex with poly-L-Lysine the experimental tick-Eorne encepha-
litis. Virusologie 4: 438-441 (1977).
43. D. G. Andzhaparidze, Bektemirov, and Burgasova,
The effect of poly complex with poly-L-Lysine the course
of vaccinia infection in monkeys. Virusologie 3: 339-343 (1977).
44. R. Purcell, W. London, J. McAliffe, Palmer,
Kaplan, Levy, J. L. Gerin, J. Wagner, Popper,
Lvovsky, and D. Wong, Modification of chronic hepatitis-B
virus infection in chimpanzees administration of interferon
inducer. Lancet, 757 (Oct. 9, 1976).
45. Levyand Lvovsky, Topical treatment of vaccinia virus
infection with an interferon inducer in rabbits. J. Infec. Dis. 137:
78-81 (1978).
46. W. Houston, L. Crabbs, L. Stephen, and Levy,
Modified polyinosinic-polycytidylic an immunological adjuvant.
Infec.lmmun. 14:318-319 (1976).
JH(i
47. L. Stephcn, D. J. 11. 11. I,ovy,
Swine influenza viruc; vaccinc: Potontiation of ill
rhesus monkeys. Scienoe 197: 1289-1290 (1977).
48. S. Levine, Wiernick, and Levy, Phaso
trial of stabilized polyriboinosinic-polyribocytidylic acid in leuketnia
and solid tumors. AACR in press (1978).
49. L. Sammons, L. Stephen, Levy, S. Baron, and D.
Hilmas, Interferon induction in cynomolgus and rhesus monkeys after
repeated doses of modified polyriboinosinic-polyribocytidylic
complex. Antimicrob. Agents Chemother. 11: 80-83 (1977).
50. Champney, Levy, and Lerner, Sustained interferon
in human serum following Poly ICLC. Proc. Soc. Clin. Invest.
Clin. Res. 24:451 (1976).
51. W. Engel, R. Cuneo, and Levy, Treatment of neuropathy
with stabi1ized polyinosinic-polycytidy1io Lancet t 503 (1978).
52. W. Carter, Pitha, L. W. shall, 1. Tazania, and
Tazaia, Synthes is of inte!'feron induction in mismatohed
analogues of polycytidy1ic J. Mol. Biol. 70: 567-587 (1972).
53. Ts'o, J. L. Alderfer, J. Levny, L. W. Marshall, J.
J. S. Ho!'oszewicz, and W. Ca!'ter, Antiviral and other biological
properties of PolyI. PolyC and its mismatched analogues. Mol.
Pharmacol. 122: 299-312 (1976).
54. W. Carte!', J. Beesan, Cunnington, Kelvin,
Ver-Hodge, J. L. Alderfer, and Ts'o, Mismatched analogues
of PolyI. PolyC. Mol. Pharmacol. 123:440-453 (1976).
8
TILORONJ<: HYDROCHLURIDJ<:
AND RELATED MOLECULES
Gerald D. Mayer and Russell F. Krueger
Merrell-National Laboratories
Division of Richardson-Merrell Inc.
Cincinnati, Ohio
1. INTRODUCTlON
INTERFERON INDUCTION
ANTIVIRAL ACTIVITY
IV. 1MMUNOLOGICAL
V.
VI. CLINICAL STUDIES
VII. CONCLUSION
REFERENCES
1.
187
191
201
205
209
210
213
215
Among the large number and variety of agents that induce interferon, tilorone
hydrochloride represented significant advance. The first synthetic
pound of low molecular weight recognized to induce interferon in vivo
the oral route [1,2], it was also demonstrated to an orally and paren-
terally active broad-spectrum antiviral agent in the laboratory [3,4].
Subsequently, it was found to stimulate the reticuloendothial system [5]
and influence the primary immune response to sheep red blood cells in mice
[6]. These initial observations were vastly extended Megel and associates
through their studies the selective effects humoral and cell-mediated
immunity 1 7] .
A1though tilorone has been the most prominent member of t!le
molecular-weight compounds synthesized at the Merrell Research Center
and otller laboratories, it was not the first important compound. initial
187
IHH
TAl3LE 1. J,J)'iO ValuuH
Compound
a
Tilorone
hydrochloride
Structure
=0 2

N- CH
2
eH
z
o
RMI 10,024 I
0= =
RMI 10,874
O-CH
2
CH
2
-N

'/ '\
=0
RMI 11,002
2
Mol.
wt.
b
439
2
370
LlJ'iO (11l/-';/k/-';)c
()ral
1520 111
1560 110
1780 353
392 >4000 353
'l'11,(III,()NI'; 11"1>lI,()('III,()II.lIII';
'l'able .1
Compoul1d
Q
ltMI 11,513
RMl11,567
RMI 11,645
Strueture
.2
2 CI
fi-
CH
2-
N
(CH
3
)2

2


N

Mol. Mouse LD
so
(mg/kg)C
wt. Oral Subcutaneous
352 1410 304
338 2700 1000
373 2590 930
j !)()
1\1111)'/-;1/ IIIIII,\I/IJI-;(;,-:,t
1 (collLiIlUO<l)
Mol. LlJ.,o (1I1!.ojk!!:(
Compoulld
a
Structure wL.!J ( ) l Su!JcuLat100lIH
RMI 11,877 354 .2930 8.20
2 CI
RMI 12,358 380 >1000 420
compoUllds are dihydrochloride sa1ts, as the free bases are illsoluble ill
aqueous solutiollS at physiologically compatible .
bMo1 . wt. = molecular weight of free bases.
After 7-day observatioll period.
cOlnpound, desigllated Richardsoll-Merrelllllc. RMI 2557 DA (2 HCl),
was bis-basic ester of fluorellolle that had modest antiviral activity but
only the parenteral route [8]. Furthermore, its interferon- inducing
capacity was minimal. Following the discovery of RMI 2557, more exten-
sive chemical synthesis program was undertaken to elucidate the structure
activity relationships of bls-basic tricyclic compoUllds and to develop
pOUllds that were orally active, more potent, and which possessed other
pharmacodynamic differences. Over 800 compounds were synthesized,
including representatives of different chemical series of bls-basic
substituted polycyclic aromatic compounds. The structure activity relation-
ships of of the chemical series were first reviewed in presentations
at the 1970 meeting of the American Chemical Society [9] and in 1971 at
Leuven, Belgium [10].
,'j. '1'11 ,( l/i,oN 1< ")'1 i1{(I( '/1 1,( 11(/1 1 1
1,1),,(\ 01' l)il1c ol'ally acLivc incluu-
WUL'U uvaluatiollH HllOWll in 1.
WUl"U 01) HLruuLural diffcrellce8, relative toxicity, alld
potcnuy a{.!;ainst ellcep!Jalomyocarditis virus; compoullds were
also acLi differellt tricyclic nuclei are
HUllted, llamcly, fluorellolle (tilorOlle), allt!Jraquillone (RMI 10,024), xant!Jolle
10,874), fluorelle (RMI 11,002), xanthelle 11,513), dibenzofurall
11,567), fluorallthelle (RMI 11,645), dibellzothiop!Jene (RMI 11,877),
and acellaphthelle (RMI 12,358). Side chain lillkages are ethers, represented
tilorolle, RMI 10,024, alld RMI 10,874, and ketolles, represellted the
rest of the analogs shown. Tilorone, RMI 11,002, RMI 11,567 alld RMI
11,877 appeared to good repl'eselltatives with potelltial for clillical activity
alld so were evaluated more extellsively thall the other five. of the
compounds were tested as dihydrochloride salts, which improved their
solubility for testing ullder physiological cOllditions. Physicochemical
properties were reported Albrecht [11]. The structure activity relation-
ships of each chemical series these particular
antiviral agellts are reported elsewhere [9, 10, 12-16] .
The size of the molecule appeared to importallt for activity.
Molecular weights of these oral1y active illterferoll illducers ranged from
338 to 439. Many allalogs with 10nger side chains or substituents that added
to their molecular weight were active subcutaneously but not The
limit for subcutal1eously active compoulld to display activity
appeared to about 450. No 10wer molecular size limit was established
except that which was required to maintail1 the functional integrity of the
basic compound structure l1ecessary for interferol1 inductioll.
lNTERFERON INDUCTION
The interferon-inducil1g of tilorol1e-related compoul1ds were
compared in mice 2) al1d rats 3). Sera from treated rodel1ts
displayed characteristics associated with il1terferon, species
specificity, broad al1tiviral spectrum, 1l011dialyzability, trypsin sensitivity,
and resistance to ribol1uclease. of the compoUl1ds il1duced high levels
of interferol1 il1 mice oraHy yet
RMI 11,002, and RMI 11,513 were good il1ducers in rats either route.
Although showl1 1, 6-hr titers after oral subcutal1eous treat-
ment of mice were <25 with of the compoullds. Titers ill mice 12 24
hr after treatment were gel1erally comparable with each other
11,645was the significant exception). In cases of subcutalleous adminis-
tratiol1, 12-hr titers were higher thall those at 24 hr. Our experiellces
J
1I1LCl'(Cl'OIl 1,cvclH 01' Tl'caLcll IVI
InLcrfcron tiLcrH
Oral (250 mg/kg) (100
Compound 12 hr hr 48 hr hr hr 48 hr
Ethers
Tilorone 6,400 6,400 50 12,800 800 < 25
RMI 10,024 3,200 12,800 100 12,800 1,600 <25
RMI 10,874 6,400 3,200 25 1,600 800 <25
Ketones
RMI11,002 3,200 1,600 <25 3,200 200 <25
RMl11,513 6,400 3,200 <25 3,200 800 <25
RMI 11,567 12,800 25,600 50 6,400 3,200 25
RMI 11,645 400 1,600 200 3,200 1,600 <25
RMI 11,877 6,400 6,400 100 12,800 800 < 25
RMI12,358 3,200 3,200 < 25 800 400 < 25
with tilorone have shown that intcrferon titers reached maximum some
16-18 hr after oral treatment and 12 hr after
Stringfellow and Glasgow, however, reported similar patterns of interferon
induction tilorone orally or subcutaneously [17]. Polymeric
inducers such as poly 1: poly and endotoxin are "early" (within 6 hr) in-
ducers. Tilorone then must considered "late" Declercq
Me1'igan suggested that slow abso1'ption of tilo1'one f1'om the gast1'ointestinal
t1'act accounted fo1' the dclay in the onset of tilo1'one [18].
We have not in stimulating p1'oviding
antiviral p1'otection with intravenous administration of tilo1'one to mice.
Significant p1'otection against Semliki Fo1'est vi1'us (SFV) was obtained
int1'avenous administ1'ation of tilo1'one to 1'ats, but inte1'fe1'on measu1'e-
ments we1'e made.
Diffe1'ences in potency of the compounds we1'e evaluated single o1'al
t1'eatment of mice with th1'ee diffe1'ent dose levels and selecting se1'um
samples at the p1'edete1'mined maximal time fo1' inte1'fe1'on to assayed
(Table 4). this study nine compounds induced good inte1'fe1'on levels
at 250 mg/kg, with RMl 11,567 eliciting the highest RM111,645
was the most potent induce1' at 50 and 10 mg/kg. No inte1'fe1'on was detect-
able with 50 01' 10 mg/kg of RMl 11,877.

"-
""
3. Serum Interferon Kinetics in Rats
z
--
Interferon titers (hour after treatment)
------
Oral (250 mg/kg) Subcutaneous (100 mg/kg)
-:
Compound 12 hr 18 hr 24 hr 30 hr 6 hr 12 hr 18 hr 24 hr 30 hr
;:::
...:
Ethers
"-
Tilorone 400 200 200 25 100 400 200 25 25
""
RMI10,024 < 25 50 50 50 <25 50 50 25 < 25
RMI10,874 <25 50 50 50 <25 50 < 25 <25 < 25
RMI11,043 < 25 <25 25 < 25 <25 < 25 50 <25 < 25
Ketones
50 2:200 100 25 <25 400 < 25 < 25 < 25
RMI11,513 50 100 200 50 25 400 100 < 25 <25
RMI 11,567 <25 50 25 <25 < 25 25 25 <25 <25
RMI 11,645 < 25 <25 <25 <25 <25 <25 <25 <25 < 25
11,877 <25 <25 25 <25 < 25 <25 < 25 <25 < 25

TAl3bl': 4. iec lo 'l'leaLI11uI1l
Ilour after Uo:-;u
(mg/kg)
Compound treatment 250 50 10
Ethers
Tilorone 24 6,400 400 25
RMI10,024 24 12,800 200 25
RMI10,874 12 6,400 400 25
Ketones
RMI 11,002 12 3,200 100 25
RMI11,513 12 6,400 100 25
RMI 11,567 24 25,600 50 25
RMI 11,645 24 3,200 1600 25
RMI 11,877 12 6,400 25 25
RMI12,358 24 3,200 25 25
DeClercq and Merigan found that thymus and lymph nodes con-
tained the highest levels of interferon organs after oral administra-
tion of tiloronc [18]. Analys is of the kinetics of interferon induction in
and rat organs offered interesting observations. Figure 1
shows that spleen, kidney, and lung interferon titers in mice were maximum
18 hr after oral tilorone treatment; peak thymus levels occurred 6-12 hr
later. appearance and duration of interferon in organs of oraHy dosed
rats is seen in Figure 2. It is significant that rats, which been shown
to produce much less serum interferon than mice in response to tilorone
and analogs, showed detectable thymic interferon in direct contrast to
the high levels found in mice. Again, spleen, kidney, and lung interferons
peaked at 18 hr after oral treatment of both mice and rats with tilorone.
We thought perhaps that serum interferon might due to organ "spillover"
(this yet the in mice). Although splenectomy reduced serum
interferon levels in mice significantly, splenectomized rats treated with
tilorone had much serum interferon tilorone-treated, sham-operated
controls. No evidence for interferon stimulation in the gastrointestinal
tract of mice was found after orally administered tilorone [17, 18] .
interferogenic responses to tilorone of different strains and ages
of mice, well mice subjected to various types of stress,
investigated. C
57
BL mice less interferon than Swiss outbred albino
mice [19]. Nude athymic mice produced respectable levels of interferon

3.0 -
..
2.8
-- SPLEEN
- - KIDNEY
......
g 2 .
- - LUNG ...J
'"
L.U
.-- THYMUS
t:; 2.4
1-
...J
'"
;: 2.2
.------.
. . ~
~
z
--::
;::
,...
:=.
1-
z
'" 2 u...

...J
;5 1.8

'"
~
> . .
~ 1 .
'"
1.4
1 . 2

~ -
12 18 24 30 3 42 42
HOURS AFTER 250 mg/kg DOSE
FIGURE 1. Interferon titers of mouse organs after tilorone treatment.
2.6
2.4 J
~ ~ 2 2 j
w 2.0
>--
>--
~ 1.8
~
::-
~
!z: 1.6
ct
u..

-' 1.4
ct
u

~ 1.2
u
w
cr:
1.0
0.8
0.7
.-
SPLEEN

-
KIDNEY
.-
LUNG
8-
THYMUS
I
6 12 18 24
HOURS A F E R 250 mg /kg DOSE
FIGURE 2. Interferon titers of rat organs after ti1orone treatment.

~
>
..".
7.
~
TII,()J{,()NI': 11\'I>I{()('III,()j{IIH<
I!)'I
wllicll we/"e cOlIsisl,elll.ly lllOse IOlllIlJ ill
l.itCel'llIaLes 21 j. 'I\'"-;-;I'alll IlIice I.ess il1LerJ'erol1
25-g l1liue ;-;ivel1 sallle :tlllOlIIlL LiJo/"ot1c; agc a1so
in ti1orolle's uapaciLy lo protect mice [rom S}'V infection [22].
We that adrcna1ectomized mice l1lade significant1y 1ess interferon in
rcsponse to orally tilorone than did their sham-operated
counterparts.
The induction of interferon tilorone in mice depends protein syn-
thesis [18,23,24]. Non1etha1 doses of given during the
initia1 and peak phases of interferon induction, reduced the amount of inter-
feron induced tilorone. If cyc10heximide was de1ayed
until the declining phase was in progress, however, the protein synthesis
inhibitor enhanced the interferon response. Although interferon 1eve1s were
cycloheximide given 1 hr prior to tilorone did not de1ay the
onset of interferon induction. The degree of inhibition
was age-dependent; young mice were 11l0re adverse1y affected than older
mice. In spite of the apparent requirement for protein synthesis for in
vivo interferon induction tilorone, it is still uncertain whether this re-
flects direct consequence of new protein synthesis or if the effect
is an event preceding actua1 interferon
5 the weights of interferons elicited
tilorone and of its congeners in and rats. In mice, tilorone,
ether, induced interferon of sing1e species. The
weight of interferon induced the ether RMI 10,024 was not deter-
Another ether ana1og, not among tlle nine compounds,
induced interferons characteristic of the ti1orone type. contrast, the
ketone compounds induced two distinct species of interferons in
mice. Rat interferons of two species were found regard-
1ess of which inducer was used.
5. Mo1ecu1ar Weights of Mouse and Rat Interferons
Inducer
Ethers
Tilorone
RMI10,024
Ketones
RMI11,002
RMI 11,877
RMI11,645
RMI11,513
RMI11,567
Mo1ecu1ar weights
Mouse
34,800
32,000 and 69,000
31,000 and 68,000
35,500 and 67,000
35,500 and 62,000
Rat
27,000 and 80,000
27,000 and 81,000
33,000 and 85,000
32,000 and 77,000
l!m
6. 1n Vitro Intcl' [crot1 Ltcs lo i Lor'olltP
1nduction
embryo lung fibroblasts
[28]
Normal leukocytes [28]
Normal lymp]lOcytes [29]
Leukemic lymphocytes [29]
Lymphoblastoid celllines [28]
and

Mouse embryo fibroblasts [30 J
No
Mouse L 929 cells [18,27]
Mouse embryo fibroblasts [18]
Mouse spleen lymphocytes and
[18 J
Mouse thymus lymphocytes and
macrophages [18]
Mouse peritoneallymphocytes and
macrophages [17,18]
Rabbit kidney (RK
13
) [31]
Human skin fibroblasts [27]
Mouse peripheral leukocytes
b
Rat peripheral leukocytes
b
Monkey peripheral leukocytes
b
alleference numbers are shown in brackets.
bpersonal observation of author (GDM).
No interferon stimulation orally or parenterally administered tiloronc
could demonstrated in rabbits, hamsters, ferrets, cats, or dogs. Only
one report describes interferon induction tilorone in monkeys [25]; we
ha been unable to confirm or expand upon such data with orally or sub-
RMI 11,002, RMI 11,567, RMI
11,877. Furthermore, could rabbits
treated orally with RM1 11,002 or RM1 11,567. Topical of
tilorone to eyes did stimulate secretion tears [26].
The that low-molecular-weight do not
stimulate production vitro is valid 6 [17,18,27-31]).
1n most cases, however, cytotoxic of were
required to elicit positive
as simply matter of different used. Furthermore,
the cytotoxic of tilorone has varied among different types.
6 those types from which interferon was directly
sought and does not include observations relating to protection of cells
from virus infection compound itself.
Tilorone and poly 1: poly in the of diethylaminoethyl (DEAE)-
dextran were for interferon induction in mouse L929 cells and
'l'11,()I(()NI'; IIYI)I!()('III,()HII)I';
I!)!)
ellllH"YO cells I II()wevt\l', Lllis t"esulLetl wiLll
poly 1: poly 1';AE-clexlxal1 111 ixLul"cs il1uuccu low lovols
[01'011, tilO poLcllLiatioll was to the tilorono
concentration to cytotoxic No synergism was in
!lUman foreskin fibroblasts, nor in primary rabbit kidney Although
mixtures the individual 1'ibonucleotides, polyinosinic and polycytidylic
acids, were effective, mixtures of polyadenylic and polynridylic acids were
not. Individual ribonucleotide homopolymers, well extracted
brain RNA and RNA, were ineffective. DEAE-dextran
has been shown to enhance infectivity of vi1'al nucleic acids and increase
the activity of synthetic polynucleotides interferon inducers [30].
Hyp01'eactivity, condition whereby an initial dose of compound inter-
fe1'es with the response of the host to subsequent dose, is to
interferon inducers. The overall subject is dea1t with in detail [32],
but information tilorone is lacking.
extent and length of the hypo1'eactive period depends at least
the dose level, the host itself, and the induce1'. Fo1' example, dose of
50 mg/kg tilorone given although inducing substantiallevel of
interferon, did not establish hyporeactivity in mice to subsequent 250
mg/kg dose. Hyporeactive states established in mice inducers
were not in rabbits [32]. We found that prio1' administration
tilorone failed to establish hyporeactivity to poly 1: poly in rats but did
for another dose of tilorone. It is obvious from the information presented
in 7 [18,33,37,76-78], which shows hyporeactivity of tilorone with
nonviral and viral inducers, that the extent of hypo1'eactivity varied with
the inducer. Stringfellow [33] proposed that the hyporeactive phenomenon
was responsible for the lack of therapeutic activity of interferon inducers.
infections caused virus, SFV, influenza A
z
, and murine
cytomegalovirus (CMV) made the host refracto1'Y to subsequent dose
of tilorone, poly 1: poly Newcastle Disease virus (NDV), or Rochester
murine virus (RMV). Mice inoculated with herpesvirus hominis (HVH)
type 2, either intraperitoneally or intravaginally, were hyporeactive to
subsequent dose of NDV or RMV but not to the nonviral inducers poly 1: poly
or tilorone. NDV used the first inducer did not establish hyporeactivity
to tilorone. In mice we found that tilorone used the first inducer estab-
lished severe hyporeactive state to statolon given 72 hr later. Statolon
the first inducer, the other hand, failed to estab1ish hyporeactive
state to tilorone given 72 h1' later.
The time of administration between the first and the second dose of
inducer was important. development of the hyporeactive state
in mice occurred with tilorone when the two doses were separated 72
hr. normal interferon response of the host returned 144 hr after the
first dose. Although of the established hyporeactivity
to themselves and tilorone in mice, the length of of the hyporeactive
periodwas variable. Thus, 10,874, RMI 11,002, RMI 11,567, and

7. Tilorono lIyporoaolivo Mieo
First inducer Second inducer ltesponse Ite[erencoH
Nonviral inducers
Tilorone Poly 1: poly Moderate
18
Tilorone Endotoxin None 18
Tilorone Tilorone Severe 18,37
Poly 1: poly Tilorone Severe 18
Endotoxin Tilorone Moderate 18
Maleic acid divinyl ether Tilorone Moderate 18
M;ycoElasma arthritidis Tilorone Moderate 76
M;ycoElasma arthritidis Tilorone None 76
Viral inducers
Tilorone Severe 33,77
SFV Tilorone Severe 33
Influenza A
z
Tilorone Severe 33
CMV Tilorone Severe 33
HVH, type 2 Tilorone None 33
NDV Tilorone None 33,78
RMI 11,877 induced their maximum hyporeactive state earlier in mice
than did tilorone and the other analogs.
et al. [34] suggested that hyporeactivity resulted from reduced
capacity of the reticuloendothelial system to clear inducer from the blood.
Metabolic studies have suggested that the length of the hyporeactive period
coincide with the disappearance of the compound from the host.
hyporeactive state could not established in mice with single doses of
non-interferon-inducing compounds given 72 hr earlier than their active
analogs. Invariably, the inactive congeners were cleared more rapidly
than the analogs. inactive Merrell analog that, like tilorone,
was cleared relatively slowly did establish hyporeactivity to tilorone.
Hyporesponsiveness, then, does not appear to depend the quantity of
interferon induced because it established with structurally related
compounds that are ineffic ient interferon inducers. Thus, it appears that
the faster the clearance, the shorter the hyporeactive period. However,
more extensive work is indicated to support this hypothesis. Hyporeactivity
is not an absolute condition. Tilorone incorporated into the drinking water
of mice provided continual low level of serum interferon while the mice
were supplied with the treated water [35]. We found that incorporation of
low levels of tilorone into their drinking water week before using mice
experimentally for other studies protected mice from the lethal effects of
infection of unknown etiology that appeared to transmissable. Hypore-
'l'11.()I!.()NI: IIYI)II()('III.()IIIIII':

:tcl,iviLy, 11Ie:tc;IIl'e(1 I,,Y 1IIItll'I'(1I'OII vot'L"vLaku clitucLLy wiUJ
a<:LiviLy Vil'llH, Wu WUl'lJ alJlu Lo c;how invorporation
Li.!orol1u /'uuu lIL"oLccl,lJ<] 111 i v ira1 challenge 1 week 1ater.
Sevu1"a1 other thuo1"iuc; advanced to account f01" hyporeactivity.
SLringfellow et [;);)] founu of serum hyporeactive factor
(SIIF) in virus, SIi'V, CMV, and influenza A
z
vi1"us-infected mice,
which caused severe state to induction poly 1: poly
NDV, aMV, tilorone. the othe1" hand, type 2, which did not
cause hyporeactivity to poly 1: poly tilo1"one interferon induction, but
did to the virus inducers, had the 10west SHF 1evels. We looked for
similar SHF that might stimulated tilorone but found Further-
more, we found tilorone not to antigenic. With serum collected from
tilorone-treated during the period of maximum hyporeactivity, we
could not (1) prevent tilorone from inducing interferon in when such
serum was mixed with tilorone and administered or subcutaneously
to (2) suppress the in vitro activity of serum interferon derived from
tilorone-treated (3) suppress stimulation of interferon mouse L929
cells poly 1: poly in the presence of D EAE-dextran. oungner and
[36] could not transfer the hyporeactive response passively in
serum taken from animals treated with endotoxin 48 hr earlier. Cyclo-
heximide given 1 hr before initial dose of tilorone failed to prevent the
induction of the hyporeactive state in It inferred, then, that
new protein synthesis is not required for the development of hyporeactivity.
ANTIVIRAL
Antiviral activity with tilorone and congeners has been demonstrated
rats, rabbits, and monkeys as judged byan increased number of
survivors, increased length of the survival time, prevention of viremia,
and prevention of antibody response to virus or attenuation of and
skin lesions. were primarily seen with prophylactic regimens
and could obtained with oral, topical, or parenteral treatment, depending
the type of infection. Viruses against which some of these compounds
were found to effective 1aboratory animal experimentation include:
SFV [4,22,31,38]; viruses, including
and Mengo [4,8,9,10,11,12,13,14,15,16,31,37,38,39,40,41,42]; Vene-
zuelan equine (VEE) virus [43,44,45]; influenza viruses
[4,37,46,47,48]; herpes viruses [37,49,50,51,52]; vi1"uS [4,37];
vesicular stomatitis (VS) virus [18,37,52]; tick-borne encephalitis
virus [53,54]; foot and mouth disease (FMD) virus [55]; Friend leukemia
virus [56,57,58,59,60,61,62,63]; rabies virus [64]; scrapie virus [65];
spring-summer virus [66]; and flaviviruses [67].
8 summarizes the of compounds against key virus
types.



8. In Vivo Antiviral Spectrum of Analogs
Influenza Influenza
Compound SFV A
z
PR
8
Vaccinia

Tilorone
+ + + + + + +
RMI 10,024 + + NT NT +
RMI 10,874 + + + NT NT +
RMl11,002 + + + + +
RMl11,513 + + NT + NT NT
RMl11,567 + + + + + +
RMI11,645 + + NT NT NT +
RMI 11,877 + + + + + +
RMI 12,358 + + NT + NT NT +
$
>
..-:
.-
1, topical treatment.
= active; NT = not tested; 0= il1active.
::.
7
"'"
.-
'I'II,()I!(INI'; II\'IJH()('III,(II!IIII';
wa:-; ill wiLll :-;ill!-(lc ol'alllu:-;c
caely 111" pr"ior" 1.0 illOeLllaLioll wiLll viruH. activity
Wa.H HCCIl WllCll alll\1 illiHlcrcd approximately 24 hr before sub-
illoculatioll. tl1C virus was the
best activity was obtailled with compound administered 48 hr before
In general, the minimal orally effective dose in fatal virus illfectiolls
from 10 to 25 mg/kg, alld maximal effectivelless occurred witil 100-
250 mg/kg. Similar data were obtailled SFV, except tilat activity
could demonstrated when tilorolle was given as early as 120 hr before
virus and as late as 24 hr after inoculation. Hyporeactivity was not nearly
so with tilorolle whell measured against SFV infectioll as it was
against virus Tilorolle and its congellers were
foulld to less active agaillst tile influellza viruses than against tile
virus SFV" greater of mice were protected
orally tilorone if tiley were vaccinated with
(Jap/305) or B/Mass later witil homologous
virus tilan mice that were (1) (2) treated witil
(3) with heterologous virus. Altilough
usually less active was more active tilan tilis
if tile virus was instead
of
effects were additive both and
were given to tile same mice. However, tilorone had to
multiple-dose to activity.
of witil RMI 11,002, RMI 11,567, RMI 11,877
revealed that multiple-dose were required to optimal
activity against (Jap/305) virus. order of
activity of tilese compounds did follow the order of potency for
was active 1\0 PR
8

mice were
Activity with tilorone to mice was also seen against
VEE, viruses but HVH type 1. Topical
of to hairless mice infected with HVH type 1 was
effective but somewhat to tile No activity mice could
demonstrated with fatal established
of type or coxsackie viruses.
tested for the ability of tile compound to protect cells from viral
was active vesicular stomatitis virus (VSV) at 1
2 L929 or 2 cells. level of 5 was cytotoxic. No
was detectable in cells that were treated with for 24 hr.
No activity was demonstrated in rabblt kidney (RK13) cells.
Tilorone had antiviral activity nonconfluent L929 mouse cultures in
direct to duration of exposure of cells to
pound. Tilorone was also active vitro HVH type 1 [68] tile
monkey herpes virus saimiri [69] .
RMI 11,002 was activc :.1.1\(1 TI\/<; vi,uc;oc; ill IlIice
and against SFV in micc and rats. Although oral activity wac; ucmollc;t,'atc(1
against VEE virus in both mice and monkeys, WJ.S uctcct()(1
only in mice L 43]. Evell with evidence of interferon in the serum of
activity against influenza viruses or vaccinia virus was Topical
application of 11,002 suppressed the development of u1cerated zosteroill
lesions in mice caused types 1 and 2; however, systemic adminis-
tration was ineffective.
When given systemically, RM1 11,002 did not prevent the development
of blister-like lesions caused type 1 mouse tails. No activity
against HVH type 1 was seen in rabbits when RM1 11,002 was given sub-
cutaneously or applied topically [51] .
RM1 11,567 is as potellt in mice as but the spectrum of activity
differs. Like tilorone, it was against VEE, SFV, HVH
type 1, and vaccinia viruses but was not against influenza A
z
(Jap/305)
and

PR
8
viruses. RM1 11,567 had oral ED
50
of 15 mg/kg against
SFV in mice. Activity against VEE virus was also demonstrated ill both
mice alld rhesus monkeys; however, as with RMI 11,002, illterferon was
detected only in mice [43].
Oral doses of RMI 11,567 were ineffective in reproducibly influencing
HVH types 1 and 2 cutaneous lesions in mice and rats. An investigation of
mice treated with the compound showed high levels of serum interferon.
However, in tissue culture, type 1 was not sensitive to mouse infer-
feron induced RMI 11,567.
RM1 11,567 was found to inactivate HVH type 1 direct exposure.
This demonstration of activity prompted topical evaluation of the
against this virus in mice. The compound was formulated in polawax
and propylene glycol at 1, 3, and 5% concentrations. Three applications
of 3 or 5% given at 2, 4, and 6 hr after inoculation of the mouse tail with
type 1 were as effective as when treatment was continued q.i.d. for 1, 2,
3, or 4 days. Topical of 3 and 5% concentrations of RM1 11,567
at 2, 4, and 6 hr after inoculation plus q. i. d. for the next 4 days also pre-
vented full skin lesion development and paralysis in hairless mice. 1n either
the tail or skin lesion models of infection, 1% concentration was not
tive.
Antiviral efficacy was obtained with RMI 11,877 in rodents and monkeys.
As with the previously described compounds, interferon was detected only
in mice and rats. RM1 11,877 was orally, topically, and parenterally
active.
Mice were protected oral and subcutaneous administration of RMI
11,877 against infections caused virus. It also was active in mice
and rats against SFV. Protection was dose- and time-dependent with maxi-
mal activity achieved with administration 24 hr before virus inoculation.
The minimum effective dose orally in mice was 21 mg/kg, which was
parable to that seen with ti1orone. However, when the LD
50
was considered,
'1'11.()I(lNI< IIYI)I(,()CIII,(I!IIIH':
ItMl JJ, 1'77 II;H] Ili.,;IIl'I ill(lox tllc mil1imum
oral lloHa,,;cH, detected
il1 t!Je of micc }'atH. '1'110 was a1so shown serum
antibodyal1d virus til.crs 1.0 orally active in mOl1keys against VEE virus.
Alfuough interferon was detected in fue monkey, it was detected in mice
at dose effective against fata1 infection of VEE virus. Mice were a1so
protected against virus. RMI 11,877 was active orally in mice infected
wifu influenza A
z
(Jap/305) but not against influenza 1\0 PR
s
.
severity of vaccinia-virus-induced tail1esions of mice was reduced
over 50% with prophy1actic and fuerapeutic regimens using mu1tip1e ora1
doses of RMI 11,877. sing1e prophy1actic dose was ineffective. RMI
11,877 was eva1uated against herpes viruses rafuer extensive1y. No activity
cou1d demonstrated against fue deve10pment of 1esions the tails of
white mice infected subcutaneous1y with HVH types 1 or 2. Despite high
serum interferon 1eve1s induced ora1 administration of RMI 11,877, fue
deve10pment of 1esions in hair1ess mice was not pre-
vented. Topical application, however, was as effective as RMI 11,567.
RMI 11,877 a1so direct1y inactivated HVH type 1. In rabblts, 5% aqueous
solution of RMI 11,877 applied direct1y to the when HVH-type-1-induced
1esions were first detectable effective1y prevented progressive 1esion
deve10pment after eight doses were given.
The remaining five compounds are somewhat different chemically and
were evaluated against spectrum of RNA and DNA viruses in vivo and in
vitro. All of the compounds showed dose-dependent antivira1 response
wifu maximal activity obtained with prophy1actic treatment.
RMI 11,645 was active in mice against virus and SFV. No activity
was seen against influenza A
z
(Jap/305) or vaccinia viruses. RMI 11,645
inactivated viruses exposure to light [70]. 10,024 and RMI 10,874
were similar in their antivil'a1 spectrum, with activity seen against
SFV, and vaccinia viruses but not agail1st influenza A
z
(Jap/305). RMI
10,874 was active in mice against VEE virusj RMI 10,024 was not evaluated
in this test. RMI 11,513 and RMI 12,358 were bofu active against
SFV, and influenza A
z
(Jap/305) viruses, but on1y RMI 12,358 was active
against vaccinia virus.
IV. IMMUNOLOGICAL EFFECTS
Interferon inducers usually impose other effects the host fuat inv01ve
the immune system and tilorone and ana10gs are exception. Mege1 et
have recently reviewed many of fue immun010gica1 responses elicited
tilorone [71,72] .
The initia1 report immunomodulation tilorone was Hoffman
and Ritter [6], who showed that single dose of tilorone to mice enhanced
the primary immune response to sheep red blood cells and increased serum
9. Summary of willl 'l'iIOl'OIl(:
and Related Compounds
med iated
Humoral immunity immunity
stimulation sU22ression
Compound IgG IgM
Tilorone Yes Yes Yes
RMI 10,024 Yes No No
RMI 10,874 Yes Yes Yes
RMI 11,002 Yes No Yes
RMl11,513 Yes No No
RMl11,567 Yes Yes Yes
RMI 11,645 Yes Yes Yes
RMI 11,877 Yes No Yes
RMI 12,358 Yes Yes Yes
hemolysin titers as well. Subsequent reports confirmed these findings
[59,67,73-75] .
9 summarizes the consistent immunomodulatory effects of
tilorone and some of its analogs in rodents. of the compounds were
selective in their effects, stimulating humoral immunity, indicated
elevated IgG and IgM antibody to sheep red blood cells, and suppressing
cell-mediated immunity, indicated reduced incidence of paralysis in
the Lewis rat experimental allergic encephalomyelitis model [7,71,74,79] .
RMI 10,874, RMI 11,567, RMI 11,645, andRMI 12,358, like tilorone,
enhanced IgG arrd IgM antibody response arrd suppressed cell-mediated
immunity. Tilorone also has shown to stimulate IgE antibody levels
[71], RMI 10,024 and RMI 11,513 stimulated IgG antibody levels but had
effect IgM levels or cell-mediated immunity. RMI 11,002 arrd
RMI 11,877 differed from tilorone only in that they failed to stimulate
duction of IgM arrtibody. Interferon inducers of the synthetic polymeric
type enhanced antibody production [80,81] and cell-mediated immunity [82] .
Interferon itself has shown to stimulate antibody productiol1 [83] .
Administration of tilorone to mice produced anergy to tuberculin-
mediated delayed hypersensitivity [84]. Tilorol1e and some of its analogs
have shown to cause transient lymphopel1ia in mice and rats with
depletiol1 of lymphocytes in areas of the spleel1, thymus, and lymph
,'j. '1'\\ ,( )\()N \< \\ \'\ 1II,()( :\\\ ,( )\1,\\ )\<

0\' J}]ice :0)(1 J'aL,c; 1 H;J-!)()J. ill aJld
WUJ'U )'api<lly J'upoj>lJlaLu<J \JY [20,86,
Alllymic l1udc mice Lo <Juvulop lyrnphopel1ia t1'eatrnent
121J. AlUlOugh Lhe of the data
laI" irnmunity, others have ::;hown effects
with tilorol1e [92-94].
extel1ded the surviva1 of tral1splants. Mouse tail skin
were prolonged tilorol1e given cOl1tinuously in the
wateI" [95]. NDV and statolon also pr010nged surviva1 of such allografts
but not as long as tilorOl1e. Megel et al. [72,96] showed that given
to donor for tllree cOl1secutive days graft-versus-host
(G reaction in rec ipient rats, but or two-day treatments were
ineffective. Prolongation of skin al1d heart allografts in rats [97-99] and
rena1 allographs in dogs [99,100] was demonstrated in tilorone-treated
anirnals. Tilorone administered orally to 6-week-01d chickens suppressed
de1ayed but not the G VH reaction. Hemagg1utination anti-
body was not stirnulated in chickens [101].
Stimu1ation of hurnora1 immunity tilorone-1ike compounds in mice
is cause to consider such cornpounds as potential adjuvants.
with, distal to, A
z
influenza vaccine significantly elevated
hemagglutination-inhibltion titers in guinea pigs those achieved with
aqueous influenza vaccine of content in guinea pigs
not given tilorone [71]. Concomitant use of with water
in emu1sion showed adjuvant effect. The p01ymeric interferon inducer
poly 1: was used as an adjuvant with VEE virus in mice [102].
Tilorone has used successfully as an against murine
turnors [103,104].
Sp1een weights of tilorone-treated mice were increased [5,20,86,91] .
Tilorone chromated sheep red blood vascu1ar clearance and
hepatic in mice [5]. Interferon has been shown to phago-
cytosis peritoneal macrophages in vitro [105].
Several interferon inducers, including tilorone, enhanced the spreading
of peritoneal macrophages freshly plated glass surface, but
cant corre1ation between interferon titers and percent of spread macrophages
cou1d not determined [106]. Sp1enectomy did not influence the spreading
response of macrophages to tilorone. 1n the presence of tilorone peritonea1
exudates, containing macrophages lymphocytes from
mice, comp1etely protected embryo fibroblast from de-
struction HVH type 2 or vaccinia virus [107]. Macrophages with or
without lymphocytes were protective while lymphocytes in the absence of
macrophages were not. Tilorone inhiblted endotoxin-induced production
of 1eukocyte-inhititing factors in human 1ymphocytes [108].
Tilorone has shown to stimu1ate comp1ement, presumably
comp1ement consurnption, in vivo and in vitro [72]. RMI 11,645
inhitited comp1ement to some extent in vitro. RMI 11,002, RM1 12,358,
\':\t "\tlll':(; \':11.
llM1 11,5li7, HMI LtMI10,1)7iJ, \tMll1,;'J;I,
effect complemellt ill vitro.
McGuire et al. [109] found that tilorone and
modulators suppressed the yield of prostacyclin [rom activated mousc
peritoneal cells. Prostacyclin formation was not blocked immunomodu-
lators applied directly to homogenates, therefore they were llot prostacy-
clin synthetase inhititors.
Although antibody levels did not seem to altered in NZB/NZW mice
treated with tilorone, Walker [110] suggested that tilorone- induced suppres-
sion of cell-mediated immunity have caused young NZB/NZW mice to
die prematurely with severe glomerulonephritis and vasculitis. Tilorone
enhancement of tumor growth in mice and rats, which could accounted
for stimulation of blocking antibody and/or suppression of cell-mediated
immunity, seen Gazdar and co-workers [111-113].
The anti-inflammatory properties of tilorone have been extensively
reviewed Megel [72]. 1n summary, tilorone (1) inhitited the primary
inflammatory the secondary
tory the arthritis test in rats; (2) inhibited carrageenan-
induced paw edema and abscess ill rats; (3) inhibited the direct passive
Arthus reaction rats. HM1 11,002 inhibited paw
edema but not abscesses nor the Arthus reaction.
HMI 12,358 inhibited paw edema but not the direct
Arthus HMI 11,567 modestly inhibited paw
edema but the abscess. HMI 11,645 did not inhibit
paw edema the direct Arthus reactioll. HMI 10,024
had effect carrageenan-induced abscess the direct Arthus re-
action. RMI 10,874 inhibited paw edema but not
abscess. RM1 11,513 inhibited neither carrageellan abscess nor
the direct Arthus reaction. HM1 11,877 inhibited neither
paw edema l10r abscess formatiol1.
of did l10t il1crease the suscepti-
bility of mice to with Staphylococcus aureus [114]. cOl1trast,
of mice to il1fectiol1 with Listeria was sigl1ifi-
il1creased [114]. [115] found l1umbers of
il1 livers spleel1s of tilorol1e-treated mice il1fected with L. monocyto-
genes, Mycobacterium tuberculosis, enteritidis. Tilorone
increased antibiotic dosage require-
ments for [116]. We found that tilorone administration to mice
24 hr prior to challenge with aureus ablated the protective response of
chloramphenicol. The effect was not when sulfonamide
were substituted for Neither tilorone itself nor serum
collected from mice 24 hr after treatment affected the in vitro
action of chloramphicol against aureus. Munson et al. [59] found that
although tilorone did not protect against aureus challenge 24 hr later,
RMI 11,002 did.
'I'\\n\!()N \'; \\ \'\ )\!()C\I\ ,III!\\ )\-:
TAB1,\'; 10, \to(\eIIL \tUSPOIISUS Lo 'l'ilo]"ol1c
\tcsPOI1SC References
Activc Mammary carcinoma 13762 117
sarcoma A-RCS 118
Walker carcinosarcoma 256 118, 120-123
Mammary adenocarcinoma R35 117,123
Morris hepatoma 120,121
Morris hepatoma 7777 118,121
Morris hepatoma 7794 121
Lewis lung carcinoma (oral treatment) 124
16 melanoma 125
Friend leukemia 56-63
Leukemia 1964 126
Leukemia YLL 126
Leukemia YALL 127
Spontaneous lymphoma; development
in immunosuppressed mice 128
Equivocal 16 melanoma 117,123,129
Murine sarcoma 111
Inactive Ehr lich adenocarc inoma 59
Prostatic adenocarcinoma 130
Ependymoblastoma 123
Mast 118
Plasma tumor 118
Lewis lung carcinoma (parenteral treatment) 123,129
L1210 leukemia
118,123,124
leukemia
118,123,124
V.
Antitumor activity was demonstrated with tilorone in mice and rats.
10 provides the references for individual studies with tilorone [56-63,111,
117,118,120-130]. Onlyan occasional discrepancy was observed, as noted
the 16 melanoma studies and subcutaneous vs. oral treatment of I,ewis
lung carcinoma. Oral administration provided better activity than did
parenteral treatment. Generally, single daily dose of tilorone was active
in mice with responsive tumors, and in some cases, even single weekly
dose was Activity was reported against four of six leukemic and
against eight of 14 solid tumors evaluated, excluding

melanoma and

IVI 1':1(. :LIH I 1':11.
sarcoma, WIJiCll l11ust lJC COllsiuCl'CU WllCll llSCll ill
quence with t'Clllissioll
of murine lymphoid long-term survival rcsulLcu. 01"
analogs were effective than tilorone.
The anticancer properties of RMl 11,002 were st:lldied in sevet'a1
search laboratories in various animal Activity was
in mice against Ehrlich and Friend leukemia [59], Lewis
1ung carcinoma and B
l6
[124], and 1ymphocytic 1eukemia
[117). No activity was in mice against L1210 1eukemia [59,117,124]
or in rats against R35 mammary adenocarcinoma [59].
RMI 11,567 was evaluated in vitro and il1 vivo for anticancer activity.
Activity in at 2.6-3.6 jJg/ml was demonstrated against human
epidermoid carcinoma of the nasopharynx [119]. mice, ora1 administra-
tion inhibited sarcoma 180 [131]. An increase in survival time was obtained
when RMl 11,567 was given intraperitoneally to lymphocytic leukemic
mice [117, 119]. No activity was when intraperitoneal therapy was
used for L1210 leukemic mice [117]. compound was not in
mice when evaluated against fibrosarcoma which was
duced metllylcholal1tl1rene [131] .
RMI 11,877 also was evaluated in vitro al1d il1 vivo for al1ti-cancer
activity. lt was active il1 cultures agail1st epidermoid car-
cinoma of the nasopharynx [119J. mouse models of L1210 lymphoid
leukemias, activity was found with intraperitoneal
therapy.
The other four have tested for
tumor activity in mice. RMI 10,024 was active Ehrlich adenocar-
cinoma and lymphocytic leukemias and the solid sarcoma 180,
B
l6
melanoma, fibrosarcoma. RMI
10,874 was active in mice against Friend leukemia, Lewis lung carcinoma,
and Ehrlich adenocarcinoma but was inactive against L1210 and leuke-
mias well sarcoma 180 and the fibrosar-
RMI 11,513 al1d RMl 11,645 were active in mice agail1st El1rlich
adenocarcinoma and were inactive against L1210 al1d leukemias and
sarcoma 180, B
l6
melanoma, and methylcholanthrene-induced fibrosarcoma.
RMI 12,358 was tested only against L1210 leukemia was il1active.
VI. CLINlCAL STUDIES
The laboratory have suggested that tilorone hydrochloride
and its analogs might have clinical utility against viral diseases, cancer,
and in various immunologic disorders. Tilorone was the initial well
most extensively studied representative investigated humans.
Phase 1 clinical trials of tilorone were conducted in 110rmal subjects
[132] al1d il1 stage IV cancer patients who were refractory to modes of
,,\. 'I'II,()II,()NI': IIYIHI(H'III,()IIII)I';
11
'l'AI\I,J.; 11. SlII\1mal'Y 01' il1
P1accbo Ti1orone
Total symptomatology 19.5 days 16.3 days
Average antibody response 299 68
Average 1ength of time virus shed 8.0 days 5.0 days
Number of virus iso1ations
(first passage) 13 3
therapy previously attempted with them [133]. The tolerated ora1 daily
dose was about 500 mg, and the weekly tolerated single dose was 1500 mg.
Side effects inc1uded nausea, vomiting, diarrhea, and stimu1ation of the
central nervous system (CNS). After long-term therapy, trans ient cornea1
dysplasia deve10ped in three of 120 patients [134]. The only clinicallabora-
tory abnormality observed was the development of 1eukocyte inc1usion bodies.
Efforts to detect circu1ating after ora1, 500-, 1000-,
1500-mg dose of were
was eva1uated against virus in
study hea1thy male [135]. ora1 750-mg dose of
was given to four subjects 24 hr before with
1000 TCID
so
of virus. Four p1acebo subjects were treated similar1y.
1aboratory measurements that did
vent but appeared to shorten 3 days, suppress
virus shedding, and full antibody 11). The
of virus iso1ations first passage indicated higher virus titers in contro1s.
No remarkable side effects associated with therapy were observed. No
was in the serum of subjects given tilorone 24 hr
Lymphocyte transformation, derma1 hypersensitivity, peripheral blood
were assessed in these individua1s [136].
with had effect of the parameters
studied.
Since tilorone ,vas very active group arboviruses causing
neurotropic SFV and VEE virus, attempt to show clinica1
activity against similar type virus was conducted [137]. Sandfly fever
is self-limiting febrile of vira1 etio1ogy transmitted the
botomus fly. The virus is ungrouped arbovirus. Temperatures of about
1020 F after infection, with fever from 1 to 3 days.
gained the U. S. has made it possible to
and characterize vira1 so that it described with
dence as acute vira1 infection, se1f-limiting, with morta1-
ity seque1ae and very predictable clinica1 course. The appears
to we11 suiteu for aSHCHCiil1[.'; acLiviLy 01.' COIl1!JOLlIlIIH likc
tilorone in humans. Tilorone was [.';iven ora11y low
500 mg to 10 ma1e subjeets, six of whom werc inoculaLcu
with p1asma containing sat1dfly fever virus day 1ater. subjectH
were infected but not treated. Conc1usive evidence of interferot1 in tilorotlc-
treated t10ninfected subjects was not found. The proportiot1 subjects
with viremia was approximate1y the same among the tilorone and non-
tilorone-treated individua1s. Both groups showed essentia11y the same
tempora1 onset and persistence of viremia. It was conc1uded that tilorone
was inactive against sandfly fever virus itlfection in humans.
Five chi1dren with subacute sc1erosing panencephalitis were treated
with tilorone [138]. Two who sing1e week1y dose
of 20 mg/kg than year showed definite improvement. others
the regimen have been treated for 1ess than six months
and cannot yet conc1usive1y evaluated at this stage. The fifth child with-
drew because of nausea and vomiting.
renal transplant patient, who had been immunosuppressive therapy
and developed progressive mu1tifocalleukoencephalopathy (PML), was
placed twice-a-week regimen of 750-mg tilorone dose after cessa-
tion of azathioprine but continuance of prednisone [139]. After 16 months
of tilorone treatment, T-ce11 rosettes increased and phytohemagglutinin
T-ce11-stimulated mitogenesis improved. De1ayed hypersensitivity to
Candida appeared. B-ce11 stimulation to S. aureus antigen was greater
than in norma1s. Serum immunog1obulin normalized and cerebrospina1
fluid (CSF) IgG was increased. Neuro1ogica1 status and rena1 function re-
mained unchanged in condition that genera11y gets progressive1y worse.
It was felt that continua1 immunosuppressive therapy prompted the expres-
sion of PML. Tilorone was chosen to treat this particu1ar patient because
it suppresses function, stimu1ates humora1 immunity, and is anti-
vira1. Although the findings were positive and tilorone cou1d have accounted
for the enhanced humora1 response and arrest of virus activity, cessation
of azathioprine a10ne cou1d possibly have accounted for the improvements
measured.
therapeutic, double-blind, p1acebo-contro11ed tria1 with 16
trophic 1atera1 sc1erosis patients showed evidence of any benefit from
tilorone treatment [l40]. Po1y 1: po1y po1y lysine was a1so not effective
in patients with this disease [141] .
In stage IV cancer, positive responses were seen in patients with
malignant melanoma [133]. No bone depression was seen, and
there to transient stimu1ation of plate1et production. Because
of the encouraging responses seen in this initial and limited investigation,
phase II trials were conducted in 66 patients who were beyond hope of cure.
No significant benefits were observed in 13 me1anoma patients. Partial
responses were seen in patients with and renal cancer [142]. As
part of the Eastern Oncology Multi-Clinic Cooperative Study Group, 25
,,\. TII,()I)NI':
CallCUr' paLiullLs sLuuiuu. No si{.!;l1ificanL
at1U was 110L of value when used sing1e
3.{.!;onL l113) .
was a1so eva1uated against in humans in France (144).
It was given a10ne or in combination with other therapy to 48 patients
with disseminated 01' inope1'able cancer. on1y detectable response was
the temporary arrest of tumor growth in six of 11 patients with measurable
metastatic 1ung lesions where tumor doubling time was
In ongoing investigation of tilorone in children with acute 1eukemia
in re1apse, changes in 1eukocytes other than bodies have
reported. transient decrease in percent of was found in five of
seven patients, in three there were increases in active and in four
transient increase in (145).
The on1y other tilorone-re1ated compolll1d where data have
lished invo1ves RMI 11,002 (146). drug was given to advanced
cancer patients who were beyond surgica1, or chemo-
therapeutic cure. In 32 patients, given 1.6-25 mg/kg of the daily
for to 91 days, tumor regression nor stabllization was observed.
Side effects were similar to those seen with tilorone. In addition, three
cases of acute dermatitis were noted that responded prompt1y to
drug withdrawa1. Maximum tolerated dose was 22 mg/kg per day, which
was greater than that with ti1orone.
VII. CONCLUSION
The antiviral chemotherapist prefers to deal with chemica1
entities, and the advent of the 10w-molecular-weight interferon inducer
offers promising possibilities to explore new classes of compolll1ds and
new concepts of antiviral chemotherapy. The opportlll1ities for mo1ecu1ar
modification to achieve optima1 structure activity relationships and mini-
mize toxicity are manifold. Tilorone hydrochloride, the first, and
of the most potent 10w-mo1ecu1ar-weight interferon inducers, repre-
sents the standard which other compounds of its kind are measured.
Dosage requirements for expression of activity these compounds
are high. Toxicity in higher anima1s is increased considerably
goes the phy10genetic ladder, the therapeutic ratio against
infection is like1y to diminished. Just as variation viruses
in interferon sensitivity exists [14 7], is there order of virus sensi-
tivity to inducers in vivo [148]. Add to this the fact that the dose
of virus used to assure infection sure1y is greater than that encountered
under natura1 conditions and the chances for c1inica1 success are reduced
substantially. The requirement for prophy1actic treatment,
other than short1y after viral exposure, is another handicap to clinica1
interest and usage.
illturfcrolJ il1l1ul:tioll ic: :.t majol." I"actot" ill LI1C allLivil"al acLiviLy 01"
tilorolle rc1atcu l:OIllPOUI1US Icvelc:
and the degree of activity against vesicu1ar stomatitic: virus, SClllliki l'ot"cc:L,
alld encepha1omyocarditis viruses showed c10se corrc1ation [11),37], a1-
though Giron et a1. could not establish that point with 10w doses
against virus [40]. We have shown the
ship in severa1 ways with Semliki Forest viruses,
through (1) dose response curves, (2) time response curves,
(3) hyporeactive responses. Furthermore, we have to
hamsters with this protect
hamsters against para1ysis caused Semliki Forest virus,
the most sensitive virus to interferon vivo. Herpes viruses
were to stimu1ated in mice and re1ated
compounds. The same active when applied topica11y to hair1ess
mice infected with herpes viruses, failed to if
they were given orally subcutaneous1y.
A1though these most assured1y p1ays
protective role some it obviously does not the
who1e story. Direct inactivation of viruses and topical activity against
herpes viruses with some of the the or absence of
wmte light do not an Tilorone, RMI 11,002,
11,567, llMI 11,877 of
mice, yet tilorone was influenza PR
s
. No interferon
was detected in treated with these four yet were
active against equine encepha10myelitis virus.
There is ample reason to suspect that the immunoregulatory and even
the anti-inflammatory properties of tilorone re1ated compounds cou1d
influence certain other disease states. Also, temp1ate binding tilorone
and some of its related affected polymerase of oncogenic
viruses, wmch might account, in part, for some of the antitumor
observed [149-155]. The ultimate success of these 10w-mo1ecu1ar-weight
interferon then, not marked their
potential but their other actions. Their broad biological profile must
considered. Besides interferon and direct virus inactivations,
some decrease ce11-mediated immunity while others stimu1ate it (this
dosage regimen stimu1ate humoral immunity,
phagocytosis, inhibit reverse and complement
prostacyclin formation and/or stimu1ates complement.
The specific clinica1 where compounds with such actions might
useful include, of course, acute viral, bacterial, and fuugal
slow virus tumors, transplants, arthritis, and possible use as
vaccine adjuvants.
laboratory curiosity, these low-molecular-weight inter-
inducers seek their niche among the drugs of the future.
'l'11.()I()NI: IIYIH!()(:III.()I!II)I';
1. Maycr l<'il1k, l'ed. Proc. 635 (1970).
Maycr alld L{. 1'. Krueger, Sciellce 169: (1970).
3. L{. }'. Krueger alld Yoshimura, Fed. Proc. 29: 635 (1970).
4. 1<'. alld D. Mayer, Sciellce 169: 1213 (1970).
5. w. llegelsoll, J. MUllSOll, R. F. MUllSOll, alld
D. Mayer, Preselltatioll at the lllterllatiollal Meetillg the Reticulo-
elldothelial Society, Freiburg, GermallY, 1970.
6. F. Hoffmall alld W. Ritter, J. Ret. Soc. 638 (1971).
7. Megel, Raychaudhuri, S. Goldsteill, R. Killsolvillg,
1. Shemallo, alld J. Michael, Proc. Soc. Biol. Med. 145:
513 (1974).
8. D. W. L. Albrecht, R. Alldrews, R. W. Flemillg, S. W.
Horgall, Roberts, W. Sweet, J. Med. Chem. 16:240 (1973).
9. W. L. Albrecht, R. Alldrews, R. w. Flemillg, J. Grisar,
S. W. Horgall, D. F. W. Sweet, alld D. L. Wellstrup,
Preselltatioll at the the Americall Chemical Society, Chicago,
1970.
10. R. W. Flemillg, W. L. Albrecht, R. Alldrews, J. Grisar,
S. W. Horgall, D. F. W. Sweet, alld D. L. Wellstrup, Illt.
Colloq. Oll lllterferoll alld Illterferoll Illducers, Leuvell, Belgium (1971).
11. W. L. Albrecht, ill Modulatioll of Host Immulle Resistallce ill the Pre-
velltioll or Treatmellt Illduced Neoplasias Chirigos, ed.),
u. S. Goverllffiellt Prilltillg Office, Washillgtoll, D. 1974, 83.
12. W. L. Albrecht, R. W. S. W. Horgall, J. alld
D. Mayer, J. Med. Chem. 17:886 (1974).
13. W. L. Albrecht, R. W. Flemillg, S. W. Horgall, alld D. Mayer,
J. Med. Chem. 20:364 (1977).
14. R. Alldrews, R. W. J. Grisar, J. D. L.
Wellstrup, andG. D. Mayer, J. Med. Chem. 17:882 (1974).
15. Carr, J. F. Grunwell, D. D. R. Meyer, F. W. Sweet,
J. Scheve, J. Grisar, R. W. Fleming, J. Med. Chem. 19:
1142 (1976).
16. D. R. Alldrews, F. W. Sweet, J. W. L.
Tiernan, J. Grisar, R. W. Fleming, and D. Mayer, J. Med.
Chem. 17: 965 (1974).
17. D. L. Glasgow, Alltimicrob. Agents Chemother.
73 (1972).
18. DeClercq and Merigan, J. Illfec. Dis. 123: 190 (1971).
19. J. Kanadyand W. R. Smith, Proc. Soc. Biol. Med. 141:794
(1972) .
20. Megel, Raychaudhuri, and J. GibsOll, in Control Neoplasia
Modulation the Immune System Chirigos, ed.), Raven
Press, New York, 1977, 409.
21. J. 11. Mc!-\,cl, 1\:. alHI ,1.
Med. 151: (197(;).
22. R. F. G. D. Mayer, Yoshimura, l"udwi!-\,, il\
Antimicrob. Agents Chemother. 1970 (G. L. cd.), Amcricall
Society of Microblology, Bethesda, Md., 1971, 41:3(;.
23. G. D. R. F. Krueger, Camyre, F. Bray, and LC
Bacteriol. Proc. 195 (1971).
24. DeClercq and Merigan, Virology 42: 799 (1970).
25. Z. V. Ermolieva, L. Korneeva, G. 1. Balezina, D. V. Nikolaeva,
I. Gvazava, and L. L. Fadeeva, Antiblotiki 18: 517 (1973).
26. Centifanto, D. Ellison, and D. Brown,
Proc. Med. 137: 357 (1971).
27. J. W. Groelke, andG. D.
Med. 148: 1044 (1975).
28. Degre and G1az, Pathol. Microblo1. Scand. 189
(1977) .
29. J. Dennis, Wi!son, D. Barker, and Rheins,
Proc. Med. 141: 782 (1972).
30. F. Dianzani, Cantagalli, Gagnoni, and G. Rita,
Bio1. Med. 128: 708 (1968).
31. R. F. Krueger, G. D. Camyre, and Yoshimura,
Presentation at 11th Intersci. Conf. Antimicrob. Agents Chemother. ,
Atlantic City, N.J., 1971.
32. in Interferons and Interferon Inducers (N. Finter, ed.), Ameri-
Elsevier, New York, 1973, 100.
33. D. Stringfellow, R. Kern, D. Kelsey, and L. Glasgow,
J. Infec. Dis. 135:540 (1977).
34. Postic, and in Interferon: March 1967 Ciba
Foundation Symposium (G. W. Wolstenholme and
eds.), London, 1968, 19.
35. GlazandM. Talas, Virol. 17:168 (1973).
36. J. S. Youngner and W. R. Stinebring, Nature 208:456 (1965).
37. G. D. and R. F. Proc. Int. Congr. Infec. Dis. ,
Vienna 245 (1970).
38. W. L. Albrecht, Presentation at Univ. ofIllinois Pharm.,
Urbana, 1972.
39. W. L. Albrecht, R. Andrews, R. W. Fleming, J.
Grisar, S. W. Horgan, D. Sill, F. W. Sweet, and D. L. Wenstrup,
Med. Chem. Symposium, Iowa City (1972).
40. D. J. Giron, J. Schmidt, al1d F. F. Pindak, Antimicrob. Agents
Chemother . .!: 78 (1972).
41. D. Stril1gfellow, J. Overall, Jr., and L. Glasgow, J. Infec.
Dis. 130:470 (1974).
42. Vickenstedt and ChemotherapY (Basel) 20:235 (1974).
43. G. D. Hagan, al1d F. Bray, Fed. 32:704 (1973).
'1
1/1. l{. W. W. L. l'alllliur, 1<:. L. Antimicrob. Agents
11: (;1);) (1977).
45. 1.;. 1 . Stephen, tt. Spertzel, W. L. Pannier, and R. L. Mundy,
l<'ed. Proc. 33:555 (1974).
46. R. F. and S. Yoshimura, Bacteriol. Pl'OC.
188 (1972).
47. J. Portnoy and Merigan, Res. 19: 139 (1971).
48. J. Portnoy and Merigan, J. Infec. Dis. 124:545 (1971).
49. J. F. FitzwilliamandJ. F. Griffith, J. Infec. Dis. 133 (Suppl.):A221
(1976) .
50. Katz, Margalith, and Winer, Antimicrob. Agents Chemother.
1!.: 189 (1976).
51. G. D. Mayer, F. Bray, and Camyre, Presentation at 14tll
Intersci. Conf. Antimicrob. Agents and Chemother., San Francisco,
1974.
52. Tokumaru, Res. Chem. Pathol. Pharmacol. 11:.:289 (1975).
53. Hofmann and Kunz, Arch. Ges. Virusforsch. 37:262 (1972).
54. L. Vi1ner, V. Finogenova, N. S. Tikhomirova-Sidorova,
1. Rodin, V. Kropachev, Kogan, and L. Trukhmandva,
Vop. Virusologie 1: 70 (1976).
55. J. Richmond and Arch. Ges. Virusforsch. 42:
102 (1973).
56. D. Barker, S. Rheins, and Wilson, Bacteriol. Proc.
188 (1971).
57. D. Barker, S. Rheins, and Wilson, Proc. SOC.
Biol. Med. 137: 981 (1971).
58. D. Barker, Diss. Abstr. Int. 2881 (1971).
59. Munson, J. Munson, W. Regelson, and G. L. Wampler,
Cancer Res. 32: 1397 (1972).
60. S. Rheins, Barker, and Wilson, J. Microblol.
11: 1257 (1971).
61. W. Rana and Pinkerton, Anat. Rec. 178:444 (1974).
62. W. Rana, Pinkerton, and Rankin, Proc. Soc. Biol.
Med. 150: 32 (1975).
63. S. Rheins, D. Barker, and Wilson, Bacteriol. Proc.
188 (1971).
64. F. Fornosi, Talas, andG. Weiszfeiler, ActaMicrobiol. Acad.
Sci. Hung. 18:327 (1971).
65. W. Cochran, Abstr. Amer. Soc. Microblol., 72nd Meeting (1972).
66. HofmannandC. Kunz, Zbl. Bakteriol. (Orig. 225:305 (1973).
67. V. V. Vargin, W. Zschiesche, and F. Semenov, Acta Virol. 21:
114 (1977).
68. Katz, Winer, and Margalith, J. Gen. Virol. 31:125 (1976).
69. R. Adamson, D. V. Ablashi, G. R. Armstrong, and V. W.
Antimicrob. Agents Chemother. 1.: 82 (1972).
IVI :11111 I\H.11 1';(:
J. W. al1ll W. 1,.
Soc. Microbiol., 7:3ru McC)Lil1g
71. Megel, I. Shemano, al1uJ. Uilmoll, il1
Modulation Host B,esistance in the Prevel1tiol1 TrcaLIIH'I11
Induced Neoplasias Chirigos, C)d.), U .8. PL'iIIL-
ing Washington, D. 1974,
72. Megel, Presentation at the International Symposium New
inflammatory Drugs, Pisa, Italy, 1976.
73. Immunology 24: 771 (1973).
74. Megel, Raychaudhuri, S. Goldstein, Kinsolving,
I. Shemano, and J. G. Michael, Fed. Proc. 32: 1021 (1973).
75. Munson, J. Munson, and W. Regelson, Collog.
Interferon and Interferon Inducers, Leuven, Belgium (1971).
76. Cole, J. Overall, Jr., S. and L. Glasgow,
Infec. 12: 1349 (1975).
77. D. Stringfellowand L. Glasgow, Immun. &:743 (1972).
78. Buckler, G. Du Rafajko, and S. Baron, Int.
Collog. Interferon and Interferon Inducers, Leuven, (1971).
79. S. Levine and Sowinski, Amer. J. Pathol. 82:381 (1976).
80. W. BrownandM. Makano, Science 157:819 (1967).
81. J. Chester, DeClercq, and Merigan, Infec.
516 (1971).
82. Cantor, Asofsky, andH. Levy, J. Immunol. 104:1035 (1970).
83. J. Chester, Paucker, and Merigan, Nature 246:92 (1973).
84. Massanari, Clin. Hes. (1977).
85. S. Levine, Sowinski, and W. L. Albrecht, Toxicol. Appl. Pharma-
col. 40: 137 (1977).
86. Megel and Raychaudhuri, Presentation at meeting of the
Reticuloendothelial Society, Miami Beach, Fla., 1975.
87. J. Gibson and J. W. Newberne, Lab. Invest. 34:316 (1976).
88. G. Zbinden and Emch, Acta Haematol. 47:49 (1972).
89. S. Levine, J. Gibson, and Megel, Proc. SOC. Biol. Med.
146: 245 (1974).
90. S. Levine, in Modulation of Host Resistance in the Prevention
or Treatment of Induced Neoplas ias Chirigos, ed.), U. S.
Printing Office, Washington, D. 1974, 89.
91. HaychaudhuriandH. Megel, J. Ret. Soc. 20:127 (1976).
92. S. Johansen, S. Johansen, and D. W. Talmadge, J. Allergy
54:86 (1974).
93. G. Freidlaender, Mosher, and S. Mitchell, Cancer
Res. 34:304 (1974).
94. Kavetsky, Vop. Onkol. 23: 88 (1977).
95. L. Mobraaten, DeMaeyer, and J. Trans-
plantation 16:415 (1973).
%. 11. ('1,>;('1, 1 taycl!;llH IlllIl' i , <1.IICI (,. (,. TIIOIII<1.H,
21:
(] .
W ilLiH Lc ill, .
(.'
llashirn, Clin. lles. 23:
(1
WildsteilJ, L. 1<;. 8tevens, andG. Hashim, TransElantation 21:
129 (1976).
99. Wildstein, L. Stevens, Applebaum, R. Hashim,
and Fitzpatrick, Proc. Clin. Dial. Transplant Forum (1975).
100. Wildstein, L. Stevens, Applebaum, R.
Hashim, and Fitzpatrick, TransElantation 22: 205 (1976).
101. J. Donahue, J. Giambrone, J. Fletcher, and S. Kleven,
Amer. J. Vet. Res.l.:2013 (1977).
102. W. Houston, L. Crabbs, L. Stephen, and Levy,
Infec. Immun. 14: 318 (1976).
103. S. J. Mohr and Chirigos, in Modulation of Host Immune
Resistance in the Prevention or Treatment of Induced NeoElasias
Chirigos, ed.), U.S. Governrnent Printing Washington,
D. 1974, 131.
104. J. W. Pearson, S. D. Chaparas, Chirigos, and N. Sher,
J. Nat. Cancer Inst. 52: 463 (1974).
105. Huang, R. Donahue, F. Gordon, and R. Dressler,
Infec. Immun. !: 581 (1971).
106. Rabinovitch, R. Manejias, Russo, and
Immunol. 29: 86 (1977).
107. D. Stringfellow, Abstr. Amer. SOC. Microbiol., 77th Meeting
(1977).
108. D. Fumarola, R. Monno, and 1. Munno, SOC. Ital.
SEer. 53:20 (1977).
109. D. Stringfellow, F. Fitzpatrick, F. F. Sun, and J. McGuire,
Prostaglandins 16: 901-910 (1978).
110. S. Walker, Clin. Immunol. lmmunopathol . ..: 204 (1977).
111. F. Gazdar, D. Steinberg, and S. Baron, Int. Collog. Inter-
feron and lnterferon Inducers, Leuven, Belgium (1971).
112. F. Gazdar, D. Steinberg, F. Spahn, and S. Baron, Proc.
SOC. Med. 139: 1132 (1972).
113. F. Gazdar, J. Nat. Cancer Inst. 49: 1435 (1972).
114. R. Gruenewald and S. Levine, Infec. Immun. 13: 1613 (1976).
115. F. Collins, Antimicrob. Agents Chernother. 1:447 (1975).
116. F. Durr and N. Kuck, Abstr. Amer. Soc. Microbiol., 73rd
Meeting 251 (1973).
117. NCI: CCNSC Screening Report to Merrell-National Laboratories,
Merrell-National Laboratories Project Rept. No. 0-72-32 (1973).
118. R. Adamson, J. Nat. Cancer Inst. 46:431 (1971).
119. NCI: CCNSC Screening Report to Merrell-National Laboratories,
Merrell- NatLonal Laboratories Project Rept. No. 0-73-08.
IV1i\YI,:II. 1lI\ll 1\ltIIl'Xa:lt
120. U. Ituyu, W. allull. 1'. I'I'OC. 1(;211(1
Meeting Arner. Soc. (1971).
121. R. Rhoads, W. L. anu
Chem. Path. Pharmacol. Q: 741 (1973).
122. R. Adamson, Arch. Int. Pharmacodyn. 192: 161 (1971).
123. NCI: CCNSC Screening Report to Merrell-National Laboratories,
Merrell-National Laboratories Project Rept. No. 0-71-46 (1971).
124. L. vVampler and W. Regelson, in Modulation of Host Immune
Resistance in the Prevention or Treatment of Induced Neoplasias
Chirigos, ed.), U.S. Government Printing
D. 1974, 123.
125. V. Turner and J. Wallace, Abstr. Arner. Soc. Microbiol. ,
77th Meeting (1977).
126. D. Johns, S. Sieber, and R. Adamson, in Modulation of
Host Immune Resistance in the Prevention or Treatment of Induced
Neoplasias Chirigos, ed.), U.S. Government Printing Office,
Washington, D.C., 1974, 117.
127. S. YanceyandW. CancerRes. 34:1866(1974).
128. S. Walker and R. Anver, Clin. Res. 22: (1974).
129. S. Morahan, J. Munson, L. Baird, Kaplan, and
W. llegelson, Cancer Res. 34: 506 (1974).
130. ll. Burleson and Pollard, Fed. Proc. 35:624 (1976).
131. Sloan-Kettering Institute for Cancer Research, Merrell-National
Laboratories Project Rept. No. 0-73-75 (1973).
132. Schiff, Linnemann, Rotte, Mayer, and S. Trimble,
Presentation at Symposium Antivirals with CHnical Potential,
Stanford Univ., Stanford, CaHf., 1975.
133. L. Wampler, Kuperminc, and W. Regelson, Cancer Chemo-
ther. Rep. 57:209 (1973).
134. Cawein, Lampkin, 1. Tsukimoto, Mayer, W. Regelson,
and Schiff, Presentation at Symposium Antivirals with CHnical
Potential, Stanford Univ., Stanford, CaHf., 1975.
135. Schiff, Linnemann, Rotte, Mayer, and S. Trimble,
CHn. Res. 21: 882 (1973).
136. Kauffrnan, J. Phair, Linnemann, Jr., and
Schiff, Inf. Immun. 10: 212 (1974).
137. J. Ann. Progr. Rept. FY 1973 189 (1973).
138. Fenichel, Saavedra, and N. Cooper, Presentation at
Child Neurology Society Conference, MOllterey, CaHf., 1976.
139. J. Selhorst, F. Ducy, J. Thomas, alld W. Regelson,
Preselltatioll at Arnerican Academy of Neurology, 1978.
140. W. Olson, J. Simons, and W. Halaas, Neurology 28: 1293
(1978) .
141. W. Ellgel, R. Cnneo, and Levy, Lancet l= 503 (1978).
142. Richter, L. Wampler, Kupermillc, and W. Regelson, ill
Modulatioll of Host Immulle Resistance in the Preventioll or Treat-
'I'II,()II,()NI';
I11Ul1L 1I1uuuuu Nuuplasias Cllirigos, uu.), U.S. Govcrnment
Officc, Washington, D. 1974, 141.
143. Kupcrminc and Gelber, Proc. Assoc. Cancer Res. 17:
260 (1976).
144. L. Israel, Miller, and Edelstein, in Modulation of Host
Resistance in the Prevention or Treatment of Induced Neoplasias
Chirigos, ed.), Government Printing Office, Washington,
D.C., 1974, 145.
145. Lampkin, D. Hake, and Granger, Abstr. 16th Int. Congr.
Hematol. 224 (1976).
146. Richter, G. L. Wampler, Kuperminc, and W. Regelson, in
Modulation of Host Immune Resistance in the Prevention or Treat-
ment oflnduced Neoplasias Chirigos, ed.), Government
Printing Office, Washington, D. 1974, 139.
147. W. Stewart, W. D. Scott, and Sulkin, J. Viro1. 1:147 (1969).
148. G. D. Mayer and R. F. Krueger, in Antimicrob. Agents Chemother.
1969 (G. L. ed.), American Society of Microtio10gy, Bethesda,
Md., 1970, 182.
149. Chandra, F. and Gotz, FEBS Lett. 22: 161 (1972).
150. Chandra, F. Zaccara, and Wacker, FEBS Lett.
23: 145 (1972).
151. Chandra, F. V. Gaur, Zaccara, and G. Luoni,
FEBS Lett. 28: 5 (1972).
152. Chandra and Woltersdorf, FEBS Lett. 41: 169 (1974).
153. Chandra, G. Will, D. Gericke, and Gotz, Biochem. Pharma-
23: 3259 (1974).
154. Green, G. F. Gerard, D. Grandgenett, Gurgo,
Rankin, R. Green, and D. Cassell, Cancer 34: 1427 (1974).
155. Green and G. F. Gerard, in Progress in Nuc1eic Acid Research
and Mo1ecu1ar Bio10gy, Vo1. 14 (W. Cohn, ed.), Academic Press,
New York, 1974, 255 and 322.
9
INTJ.t:RIJ.t:RON INDUCERS:
PROPANEDIAMINES AND RELATED MOLECULES
Hobert F. Betts and Gordon Douglas, Jr.
University of Rochester School of Medicine
Hochester, New York
1. INTHODUCTION
CHEMISTRY
III. ACTIVITY IN EXPERIMENT AL ANIMALS
961

IV. FEATURES OF EXPERIMENTAL
VS. NATURAL VIRUS INFECTIONS IN
STUDIES OF 961 IN EXPERIMENTAL
RESPIRATORY VIRAL INFECTIONS IN VOLUNTEERS
The Preparation in Rhinovirus Infection
Microdispersed Preparation in Rhinovirus
and Influenza Virus Infection
VI. STUDIES OF 888 IN EXPERIMENTAL
INFECTIONS IN VOLUNTEERS
SYNOPSIS OF STUDIES DATE
DIFFERENCES IN INTERFERON INDUCTION
IN MICE AND HUMANS
IX. ALTERED RESPONSIVE STATE PRODUCED
INTERFERONINDUCERS
POTENTIAL USEFULNESS OF INTERFERON INDUCERS
XI.
REFERENCES
223
224
224
225
225
225
226
228
228
229
232
232
232
233
234
235
236
1\ 1':'l''l'S all< I I )()!)(;
1.
Because of the diversity of viral families and serotypes producc
colds and related syndromes, control specific vaccine seelllS unlikcly.
Therefore, the use of exogenous interferon or interferon inducers to pre-
vent or treat respiratory viral infections is particularly attractive. Some
success already has been achieved with the use of interferon and interferon
inducers in this setting. For example, in studies in volunteers, the intra-
nasal application of large quantities of human leukocyte interferon has been
shown to reduce the symptoms of rhinovirus [1], and topical
cation of poly 1: poly was shown to have some effect in reducing incidence
of rhinovirus infections [2].
This success has stimulated further efforts, since respiratory viral
infections constitute our most common afflictions and are the most important
cause of days lost from school and industry in this country. Further trials
with human leukocyte interferon, as discussed elsewhere in this book, have
been limited to few life-threatening because of the cost and
limited supply, and it is generally agreed that poly 1: poly is too toxic
for use in acute self-limited infections. Thus, search for other, cheaper
sources of human interferon, or less toxic inducers continues.
series of low-molecular-weight interferon inducers was developed
Pfizer Research, Groton, Connecticut. Of these, two that appeared
most promising-a propanediamine called 961 [3] and xylenedia-
mine, 888 (J. F. Niblack, personal communication)-have been
tested clinically.

961 * is white microcrystalline solid with melting point between
370 and 390 (see Figure 1). It is lipoidal diamine with an aqueous solu-
so low that using it requires formulation in an aqueous emulsion using
surfactive dispersants. For clinical trials, it has been fused with equal
weights of polysorbate 80 and glycerol [3].
*N, N-Dioctadecyl-N' ,N'-bis(2-hydroxyethyl)propanediamine.
FIGURE 1. Chemical structure of (N,N-dioctadecyl-N',N'-
bis (2-hydroxyethyl)propanediamine).
!).
}<'IGURE 2. Chemical structure of 888 (N, N-dihexadecyl-m-
xylylenediamine) .
is molecule related to 961, but it is substituted
xylenediamine rather than propanediamine, as shown in Figure 2 (J. F.
Niblack, personal communication). Like 961, it possesses low
aqueous solubility and also requires for clinical trials.
III. ACTIVITY IN EXPERIMENTAL ANIMALS

Although oral administration has not been effective, administration of
961 to mice intraperitoneally markedly reduces mortality of these
animals from challenge with encephalomyocarditis virus [3]. Semliki
Forest virus infection in mice is even more sensitive to the effects of
961, and resu1ting from injection of vaccinia virus into the tail
of weaning mice are suppressed intraperitoneal administration of this
drug. However, protective effect was observed in mice when 961
was tested against two RNA viruses, influenza A/Hong Kong/68 H3N2 virus
and pneumonia virus of mice [3]. In the encephalomyocarditis virus model
infection, 961 is more effective with lower mu1tiplicity of infection,
and its dose response curve is linear. It is most effective when administered
between 24 and 6 hr prior to infection. Administration of drug 3 hr post-
infection reduces its effect 10-fold, and effect is discernible if drug is
administered 24 hr postinfection. Toxicity in the mice is not detectable
except for low-grade peritonitis which develops in those animals who
receive doses greater than 200 mg/kg intraperitoneally [3].

appears to more potent interferon inducer in mice than
961 (J. F. Niblack, personal communication). Also, interferon
stimulation in plasma of rats was demonstrated with 888 but not
with 961 (J. F. Niblack, personal communication), when given in
*N, N-Dihexadecyl-m-xylylenediamine.
cquivalcnt of I1lI4/kl4 lo 111 icc. L't'olcclioll al4ail1:-:L
challenge with vaccinia allu was Willl
1n hamsters, reduction of lo vieu:-:
was demonstrated with 888 but not with 961.
Several features suggest that the drug' s effect is illterferol1.
First, interferon-like activity demonstrated in the of mice
who injected with these d1'ugs. This activity appears 12 hr after ad-
ministration of drug and persists for 20 hr. Second, interferon
levels about 10-fold higher following 888 than
with 961, and the higher interferon levels correlate with greater
This interferon-like activity is inactivated if held at
2 for 24 hr at 5 and treatment of the preparation with 0.25 % trypsin
for 1 hr at 370 eliminated its activity. These are properties
consistent with type II immune interferon. Third, the drug itself has
direct antiviral effect at concentration of 40 f1g'/ml, and fillally, it does
not stimulate cell-mediated antiviral activity.
1n spite of this in vivo effect, neither 961 nor 888
lates interferon in cultures, nor do they protect cultures against
chaHenge with vesicular virus ([3]; J. F. Niblack, personal
munication). However, both compounds enhance the protective activity of
1: in such cultures, and they equally potent in this regard
(J. F. Niblack, personal communication).
lack of toxicity of these compounds and their demonstrated efficacy
as inducers of interferon in animal systems prompted initial toxicity studies
in humans; and when these drugs to safe, subsequent studies in
experimental infection in humans were carried out.
COMPARAT1VE FEATURES OF EXPERIMENTAL
VS. NATUllAL VIRUS INFECTIONS IN HUMANS
Clinical evaluation of antiviral substances, including interferon and inter-
feron inducers. in respiratory viral illfectiolls out ill llatural1y
occurrillg or experimentaHy induced infections. 1n addition, one has to
make choices about which of the serologicaHy distinct viruses should
studied. Most oftell, rhinovirus and influenza virus
infection chosen, rhinovirus infectioll because of its major role in the
etiology of the most frequellt respiratory tract syndrome, the common cold,
and influenza virus infection because of its unpredictable epidemic nature
and resultant mortality. few studies have been performed in respira-
tory syncytial, parainfluenza, coronavirus, othe1' types of
respiratory viral infections, despite the importance of these agents.

1':xpel'il!lcl!LaL uesieaJ)Lc 101' sLuuy suitable
sLlJJjccLs sLLluiclL at single POillt ill time so that well-
placebo evaluatioll carried out. III additioll,
01 urug admillistratioll il1 relatioll to virus illoculatioll
oasily mal1ipll1ated. Study of llatural cases of c01ds, or other
respiratory sYlldromes, wou1d take mOllths to complete becallse of
the diverse etiology of such illfectiOllS. the other halld, llaturally occur-
ring illflllellza, because of its epidemic llature high attack rate, 1ellds
itse1f to study.
Sillce most studies are performed experimelltal ill
teers, must ullderstalld the closelless with which experimelltal illfectioll
ill volullteers approximates llatllral illfectioll to illterpret the results. Illtra-
llasa1 illocll1atioll of with rhillovirus results ill illfectiol1
which closely mimics llatural illfectioll ill humalls [4]. The illfectious dose
for humalls is very small alld has estimated for several serotypes to
0.01-1.0 fifty-percellt tisslle cultllre illfectious dose (TCID
so
)' Illl1ess
very similar to llatura1 illlless cOllsisting of rhillitis, pharYllgitis, alld, ill
low freqllellCY, cOllstitutiollal symptoms fever, develops 24-48 hr
postchallel1ge alld lasts two to days. Virlls sheddillg il1 llasal secretiol1s
is detected simllltalleously with the developmellt of symptoms, alld l1asal
secretory alltibody alld illterferoll-like activity recovered from llasal
wash material. Artificially illdllced illfectioll cOllfers agaillst
subseqllellt exposllre to llatural illfectioll. MlIch of the illformatioll
llatural rhillovirlls illfectioll sllggests that virlls is illocll1a-
illtO the allterior llares alld, thllS, the experimelltal illfectioll approxi-
mates llatural illlless both ill its mode of acquisitioll alld clillical expressioll.
Experimelltal il1flllel1Za virus illfectiol1 ill humallS, the other halld,
has dissimilarities compared to llatural illfluellza [5]. First, high illOCU-
1um of virus illtO the llose is reqllired to prodllce illfectioll. The resultillg
illlless is milder thall llatural illfectioll ill that fever alld cOllgh are 1ess
frequellt al1d 1ess severe, alld pu1mol1ary fullCtioll abllormalities that occllr
regularly ill llatllra1 illfluellza are llOt detected experimelltal illflllellza [5] .
the other halld, the titer of virlls shed ill the llasal wash is
higher alld persists for lOllger period ill experimelltal illfectioll compared
to llatllral illfectioll. III fact, much of the data cOllcerlling trallsmissioll of
natural influellza suggests that llatllral influenza is transmitted small
particle aeros01s, which deposit and initiate illfection in the lower respira-
tory tract [6]. Aerosol illoclllatioll of v01unteers, which results ill more
severe illlless and requires low illoculum to illitiate infectioll, more close1y
mimics natural infectioll [7]. However, most investigators feel that the
resllltant illness is too severe for widespread use of this method of illocula-
in volllnteer stlldies.
01<' 9(;1 lN
INl<'ECTlONS IN
The Initial Preparation in llhinovirus Infection
of these studies used placebo-controlled, double-blind design, and the
placebo consisted of the vehicle used to emulsify the drug: equal weights
polysorbate 80 and glycerine homogenized in hot buffered saline solution.
The first two studies, which differ only in minor respects, were done using
rhinovirus chaHenge of volunteers who were free of antibody to the adminis-
tered virus strain [8,9]. In both, drug was administered nasal spray
in dose of 50 mg per treatment. ln one, the drug was administered four
time day for days beginning one day prior to virus challe11ge [8], and
in the other it was administered three times the day prior to and
two times the day of [9]. 111 both studies, plaque reductio11 of
vesicular stomatitis virus (VSV) assays were used to detect i11terferon, a11d
in the study performed Gaitmaitan et al. [9] the nasal secretions were
treated at 1.5-2. overnight before testi11g for interferon content. In
the former study [8], rhinovirus type 13 was used as and in the
latter [9], type 21 was used.
In both studies, nasal interferon was stimulated the drug. In the
study Douglas and Betts [8], interferon production was significantly

25
R.-P<002S
1\
I \
!
I \
I \
I \
I \
p<o.os-'r )\
I \
I \
,)... \
/ /r ./.,...,!.\
5
.-- / /. \/ \
\.'/' 0'0
1
";:::::::' v "-
.ti
2 -1 6 i 2 . 3 4 5 6 ;,
DAYS
FIGURE 3. Comparison of mean total sign scores of 961-treated
14 patients) and placebo-treated (0--0, 10 patients) subjects. value
calculated from paired sample t tests. (Reprinted from Ref. 8, with per-
mission of the American Society for Microbiology.)

il1 l"uuipiul1t::> ueug (.;olllpared to plaucbo recipients, and there
W:l::> 110 tllat thc stimu1ated intel"feron. 1n the study
Gaitmaitan et [9], among individua1s who were chal1enged with virus,
there was significant difference in the frequency of interferon production
those who received vehic1e versus those who received vehic1e with drug.
IIowever, buffer-chal1enged, drug-treated group deve10ped interferon
responses, whereas buffer-chal1enged, untreated group did not. Both
studies showed that interferon was detected earlier after infection in the
drug group than in the group, and that interferon titers in nasa1
washes were higher in the subjects who received drug. There appeared to
mild therapeutic effect of 961 in both studies, manifested
decreased symptoms in the first study [8] (see Figure 3) and more rapid
recovery from in the 1atter study [9]. However, in the 1atter study,
c1earing of the symptoms occurred equal1y rapid1y in drug-treated individua1s
who were interferon-negative as those who were interferon-positive, suggest-
ing that the drug might produce its effect mode of activity other than
interferon production.
Microdispersed Preparation in Rhinovirus
and 1nfluenza Virus lnfection
As result of these encouraging findings, and because of concerns about
bioavailability of the highly ins01uble drug, new preparation was prepared
a1tering the formation of the drug so that it was presented in micro-
dispersed form 2 diameter/droplet). Three studies were performed
with the microdispersed preparation in volunteers with experimenta1 rhino-
virus infections and one in vo1unteers with experimenta1 influenza.
1n the rhinovirus studies carried out Panusarn and colleagues [10]
and Wa1dman and Gangu1y [11], there were some similarities in mode
of administration of drug and in resu1ts, but there were important differ-
ences as well. 1n both studies, the drug was administered the day before
and the day of virus challenge. 1n the study carried out Panusarn and
co-workers [10], the method for measuring interferon was altered slight1y
from their previous work. 1nstead of the p1aque reduction of 10w inocu1um
of VSV, prevention of cytopathic effect caused 500 fifty-percent tissue
cu1ture infectious doses (TC1D
so
) of VSV was used to assay interferon
activity. 1n their studies, drug-treated individua1s produced significant1y
higher interferon 1eve1s and significant1y more pr010nged interferon in
secretions compared to Virus titers in nasa1 washes were not
significant1y 10wer in the treated group, but the l1umber of virus-positive
days was significant1y 1ess. 1n both studies the most striking effect was
an amelioration of symptoms (see Figure 4). 1nfected individuals who
received drug had symptoms identica1 to the uninfected group who were
challenged with buffer. Analysis of virus shedding, clil1ical symptoms,
-_._----
V/RUS + DRUG
[] Symptomatic
Response
.Virus Shedding (%)
- Interferon Level
80 - (U/ml)
/
/
1--
40: 1. \
20 /1
I
-
111':'1''1':-> :tllll I)()[J<;
V/RUS -f PLACEBO
-1 1

23456
DAYS
7 8 -1 1 2 345678
DAYS

TREATMENT TREATMENT
t t
CHALLENGE CHALLENGE
FIGURE 4. Time relations of symptoms, viral shedding, and nasal inter-
feron titer in groups of volunteers without specific prechallenge
antibody. Shown is the effect of drug or placebo treatment for one day
before and after nasal instilation (day of approximately 1 TCD
so
(WI-38
cells) of rhinovirus type 21. (Reprinted from Ref. 10 with permission of
the New England Journal of Medicine 291: 57 (1974).)
and interferon production suggested that amelioration of symptoms resulted
from presence of interferon. In addition to prophylactic administration of
drug, both groups carried out additional studies in which drug was adminis-
tered to subjects following challenge [11,12]. In such studies, the time of
administration was important. If drug was administered 8 hr after challenge,
then the rate of infection, the rate of illness, and the severity of symptoms
were diminished without any effect the frequency of positive speci-
mens, but of these differences were significant [12]. as shown
Waldman and Ganguly [11], drug was begun 24 and 48 hr after challenge,
there was effect the development of symptoms or virus shedding. In
fact, these individuals had somewhat greater symptoms than placebo recipi-
ents.
Other minor variations of drug administration and virus challenge have
been carried out in humans [12].
I .
N N 1'::-; 1
5120
2560



z

640

z
320


160
w
!:::


t-

u..
40



20

..
10




-1 2 :3 r.f3
DAYS AFTER INOCULATION
FIGURE 5. Interferon titers, international units per of nasal
wash (NW), detected in relation to days after virus inoculation. Symbols:
= 961, = placebo. (Reprinted from Ref. 13 with permission of
the American Society for Microbiology.)
The microdispersed form of the 961 was also tested in experi-
mental influenza A/England/42/72 H3N2 virus infection in 20 volunteers.
In that study Douglas and co-workers [13], the drug was administered
for days beginning one day before virus challenge. No appreciable
effect symptoms or virus shedding was detected. In fact, virus titers
and mean total symptom scores were somewhat higher in the 961
group compared to the placebo group, but these differences were not
significant. Interferon levels were high for the first day after inducer was
begun and then the levels diminished, suggesting that with more than two
days of treatment, hyporesponsiveness to the inducer developed Fig-
ure 5). The apparent hyporesponsive state did not explain the failure of
the drug, s ince virus shedding and clinical symptoms were prominent
the first and second day after initiation of inducer, days which interferon
titers were at their peak. The failure of 961 in influenza infection
in volunteers is in keeping with results obtained in experimental influenza
infection in mice. However, the difficulty of demonstrating efficacy in
acute, mild, self-limited and the numbers of volunteers should
kept in mind in interpreting these results.
VI. Olo' 888 IN
INli'ECTIONS IN
111':'1''1':-1 :t1)(1 I)()l}(: I,A:-I
of the apparent superiority in interferon induction 01' 888
compared to 961 in anima1s, two studies of thc clinica1 effectivcness
of 888 in experimenta1 rhinovirus infection in vo1unteers were under-
taken. Since the design of the two studies was identica1 and the resuIts
indistinguishable, the studies were combined for purposes of publication
[14]. Drug or were administered intranasally spray to 60
teers, three times day the day prior to challenge and two times the
day of virus chal1enge. The tota1 dose was 50 mg.
Eva1uation of tota1 signs and symptoms, frequency of virus shedding,
and serum antibody revea1ed differences between the two groups. Inter-
feron content of washes was determined for 30 vo1unteers: five of 15
in the 888 and two of 15 in the group had interferon detected
in their nasal washes. The distribution of nasa1 wash
specimens accordil1g to day after illocu1ation is shown in 1. Titers
were 10w and ranged between 5 and 10 U. Most vo1unteers had sing1e speci-
mens containing interferon except for two patients in the 888 group,
each of whom had two positive s pecimens. the frequency and quantity
of interferon in nasa1 secretions was much 10wer than after 961
administration, findings ill contrast to studies in the mouse and the rat.
It is c1ear that the failure to ameliorate was associated with failure
to induce interferon. These studies emphasize the significance of species
variation in response to interferon inducers and the 1ack of predictability
of response in humans from anima1 data. Furthermore, it is apparent that
such compounds must tested in humans to determine potentia1 usefu1ness.
VH. SYNOPSIS OF STUDIES DATE
961, when applied intranasal1y, appeared to nontoxic and induced
interferon in 50-70% of uninfected recipients and in 80-100% of infected
recipients. It is most effective when administered drop1ets 1ess than
2 in diameter. microdispersed form of the drug dramatically
reduces symptoms of rhinovirus but not influenza infection when adminis-
tered prior to challenge. Its effect is 1ess when administered 8 hr after
challenge, and it has effect when administered 24 or 48 hr postchallenge.
VIII. DIFFERENCES IN INTERFERON
IN MICE AND HUMANS
As noted, mice responded to sing1e dose of drug with production of inter-
feron that deve10ped 9 hr after drug and persisted for about 24 hr. In
humans, sing1e intranasa1 dose did not result in interferon production [12] ,
!J.
1,1'; 1. iu
Itll illuvit"us
Distriblltion of Interferon-
No. with positive nasal wash specimens
interferon accordil1g to day after inoclllation
No. of
subjects
tested in nasal wash 1 2 3 4 5 6 7

Placebo
15
15
5
2
SOllrce: Adapted from Ref. 14.
1
1 1 3
1
2
bllt doses over two days reslllted sllstained interferon prodllction
after 24 hr persisting for 96 hr. This lag period and prolonged production
of interferon from brief exposllre to indllcer would not have been antici-
pated from animal data. It is also uncertain whether the physical charac-
teristics of the interferon prodllced in mice and in humans are the same.
ln mice, interferon produced is atypical compared to that stimulated
other inducers in that it is partially inactivated at 2. In hllmans,
detailed analysis of properties of the interferon induced 961 have
been carried out. However, in the stlldy Gaitmaitan et al. [9], nasal
secretions were treated at 2 prior to assay for interferon, and levels
measured were nearly identical to those studies where treatment was
not llsed [8]. Results in experimental infections appear to similar in
mice and in hllmans. 961 had its greatest effect when given prior
to infection, and it had diminished effect when given 3 hr (in the mice)
and 8 hr (in humans) infection. It was ineffective when given 24
hr (both mice and humans) or later after infection. The most striking differ-
ence the of mice humans was the discrepancy of
reslllts experimental with 888.
IX. ALTERED RESPONSIVE STATE
PRODUCED INTERFERON INDUCERS
From the available stlldies there has some that administration
of render the host more susceptible to viral
or result in more severe symptoms. the studies of Douglas
and Betts [8], three of 15 of drllg who did shed virus
after day 4, whereas of 14 placebo subjects did.
the swdy of et al. [9], eight subjects were treated with drug
with bllffer. FOllr of these eight developed virlls
(two of these acqllired challenge virus
1 \ 1':'1''1':-; :tlll 1 I)()\ J<:
Ll':lJ1smissioll). l'ivc alHl
challenge, vi1'US.
single dose of dl'ug was i1r
pa1'adoxica1 1'esu1t occu1'1'ed. Subjects who 1'eccived
dose 01 d1'ug 01' 01 vehicle did not sec1'ete intel' aftel' vil'US
lenge, and who 1'eceived the single dose of d1'ug we1'e mo1'e
symptomatic following vi1'us challenge than individua1s who had
saline 01' vehic1e on1y [12]. In Wa1dman' s study, individuals who
induce1' beginning 24 and 48 h1' afte1' challenge seemed to have slight1y
symptoms than p1acebo [11].
the othe1' hand, subsequent expe1'iments showed that with
fou1' days of d1'ug 01' vehic1e, both of which induced
state of when these subjects we1'e challenged with
[12]. In spite of this, these subjects we1'e 1ess symptomatic following
challenge than we1'e unt1'eated subjects.
When 961 was tested in expe1'imenta1 influenza
in nOl'ma1 high-titel'ed was induced and it
appea1'ed vi1'al infection. Howeve1', day 3 of tl'eatment, inte1'fel'on
1eve1s had at the time when subjects l'emained symptomatic. In
fact, a1though the diffe1'ences wel'e not significant, day 3 of t1'eatment,
d1'ug had both 10we1' inte1'fe1'on 1evels and highel' symptom sco1'es
than p1acebo 1'ecipients. Taken togethe1', these data suggest that the1'e is
complex inte1'action between the of inte1'fe1'on and susceptibllity
to infection which 1'equi1'es Thus, a1though it is
att1'active to p1'esume that eithe1' bette1' fo1'mu1ated 01' mo1'e active
fe1'on induce1' would have efficacy, it that these mo1ecu1es
have ti1e undesi1'able featu1'e just alluded to. Pe1'haps, at 1east, the
hypo1'esponsive state shou1d have been anticipated since oti1e1's have obse1'ved
this in anima1s [15] .
USEFULNESS OF
INTERFERONINDUCERS
In the case of vi1'a1 illnesses, p1'ophy1axis is imp1'actica1 except
diseases witi1 epidemic behavio1' because d1'ug would have to adminis-
yea1' 1'ound. Thus, ti1e1'apeutic effectiveness is essentia1 fo1' most of
these infections. Howeve1', in the of influenza, 1'espi1'ato1'Y syncytia1
vi1'us, pa1'ainfluenza types 101' 2, p1'ophy1axis is possible because of the
epidemio1ogica1 behavio1' of the vi1'uses. Thus, p1'ophy1axis
wou1d not administe1'ed yea1' 1'ound, but on1y fo1' few weeks. Howeve1',
ti1e of hypo1'esponsiveness and potentia1 heightened
must ove1'come. It shou1d noted ti1at an agent of 1'ende1'ing
the1'apeutic effect wou1d a1so highly desi1'able in these infections. In
eithe1' case, topica1 administ1'ation wou1d desi1'able to minimize toxicity
!}.
ize Altl10ugh siudies io dalc shown that these
itldueers ineffective whcn given therapeutically, that is, after onset of
sumptoms, it shou1d noted that therapeutic effects difficult to demon-
strate in acute self-limited illness, and that vo1unteer studies done to date
1imited in scope and numbers of subjects. prove efficacy, it might
necessary to study 1arger numbers of subjects with natura1 rhinovirus
infections before it conc1uded that these agents are ineffective thera-
peutically. 1n small, double-blind, p1acebo-controlled study, 43 of 171
p1acebo-treated subjects deve10ped "co1ds" as opposed to similar number
and severity in 171 who prophy1actically received 961 once daily for
30 days [16]. Viro1ogica1 studies were not done, and measures of inter-
feron production and/or hyporesponsive states were carried out.
vira1 diseases in which interferon has been shown to therapeu-
tically effective not 1end themse1ves to the use of interferon inducers.
For these compounds to effective in chronic vira1 infections such as
hepatitis, the hyporesponsive state that has been noted with pro1onged
administration wou1d need to overcome. In addition, because the infec-
tion is disseminated to severa1 organ systems, such inducers shou1d
of being administered parenterally.
studies to date, a1though not dramatic, do demonstrate some
encouraging findings. Symptoms in subjects receiving drug for intermediate
periods of time were 1ess than p1acebo-treated subjects. Usually, a1though
not a1ways, amelioration of symptoms was associated with presence of
interferon in nasa1 secretions. 1n the instances where there is 1ack of
corre1ation, the exp1anation might the method of treatment of specimens
before testing, the presence of substance that does not inhibit VSV but
does inhibit rhinovirus, or the fact that another mechanism other than inter-
feron production account, in part, for the drug effect.
XI.
Two low-mo1ecu1ar-weight interferon inducers, and
have tested in experimenta1 respiratory vira1 infections in humans.
Both are inso1uble, required emu1sification for delivery as nasa1 sprays,
and microdispersed form appears to have optima1 effect.
was shown to induce nasa1 wash interferon and to decrease symptoms of
rhinovirus, but not influenza virus, infection in vo1unteers when given
prior to or to 8 hr after virus challenge. When administered for more
than 24 hr prior to challenge, the interferon response postchallenge was
decreased, but drug efficacy persisted. although more
potent inducer in mice and rats, failed to induce significant amounts of
interferon in humans, and prophy1actic efficacy in experimenta1 rhinovirus
infection in vo1unteers was 1acking.
III';'I''I'S 1)()lJ(;
B.J<:E'EH.J<:NCJ<:S
1. Merigan, S. Heed, 8. and D. Tyrcll,
of respiratory virus infection applied interferon. Lancct.!..=
563-567 (1973).
2. D. 8. Baron, J. Perkins, Worthington, J. VanKirk,
J. z. and R. Chanock, an inter-
feron inducer in vira1 respiratory disease. J. Amer. Med. Assoc.
219: 1179-1184 (1972).
3. W. W. Hoffman, J. J. Korst, J. F. Niblack, and Cronin,
N, N-Dioctadecy1 N, 'N'-bls (2-hydroxyethy 1) propanediamine: Antivira1
activityand interferon stimu1ation in mice. Antimicrob. Agents
Chemother. 498-502 (1973).
4. R. G. Doug1as, Jr., Pathogenesis of rhinovirus co1ds in
humanvo1unteers. Ann. Oto1. Rheno1. Laryngo1. 79:563-571 (1970).
5. J. W. Litt1e, R. G. Doug1as, Jr., W. J. and F. Roth,
Attenuated influenza produced experimental intranasa1 inoculation.
J. Med. Viro1. 167-176 (1979).
6. R. G. Doug1as, Jr., Influenza in in Influenza Viruses and
Influenza D. Kilbourne, ed.), Academic Press, New York, 1975,
395-447.
7. R. Alford, J. J. Gerone, and V.
influenza resu1ting from aeroso1 inhalation. Proc. Bio1.
Med. 122: 800-804 (1966).
8. G. Douglas and F. Betts, Effect of induced interferon in experi-
menta1 rhinovirus infection in vo1unteers. Infec. Immun.
(1974).
9. G. Gaitmaitan, D. 8tan1ey, and G. G. Jackson, The limited
effect of nasal interferon induced rhinovirus and topical chemical
inducer the course of infection. J. Infec. Dis. 127:401-407 (1973).
10. Panusarn, D. 8tan1ey, V. Dirda, Rubenis, and G. G. Jack-
son, Prevention of iHness from rhinovirus infection topica1 inter-
feron inducer. New Engl. J. Med. 291:57-61 (1974).
11. R. Wa1dman and R. Gangu1y, Effect of 961, interferon
inducer, upper respiratory tract infection due to rhinovirus type 21
in vo1unteers. J. Infect. Dis. 138:531-535 (1978).
12. D. 8tanley, G. G. Jackson, V. Dirda, and Rubenis, Effect
of topica1 interferon inducer rhinovirus infections in vo1unteers.
J. Infec. Dis. 1338: (1976).
13. R. G. Doug1as, Jr., F. Betts, L. 8imons, W. Hogan, and
F. Roth, Evaluation of topical interferon inducer in experimental
influenza virus infection in volunteers. Antimicrob. Agents Chemother .
..: 684-687 (1975).
14. R. G. Douglas, Jr., R. Wa1dman, R. F. Betts, andR. Ganguly,
Effect of interferon inducer, substituted xylenediamine, rhino-
v i "(11-> i,\ l\tII 11 . ieeu!J. L\j!;cnts 15: 269-
27!)
15. J. S. Youngncr W, Stine!Jurg, Interferon appearance stimulated
bacteria or vil'uses in mice pretreated with Escherichia
endotuxin or infected with Mycobacteeium tuberculosis. Nature
208:456-458 (1965).
16. J. F. Niblack, Studies with low molecular weight of inter-
Texas Repts. Med. 35:528-534 (1977).
10
INTERFERON INDUCERS:
POL YANIONS AND OTHERS
Mary Breinig* and Page S. Morahan
Medical College of Virginia
Virginia Commonwealtb University
Richmond, Virginia
1. INTRODUCTION
AND CHARACTERIZATION OF
POLYANIONS
INTERFERON WITH POLYANIONS
Interferon Induction in Vivo
Interferon Induction in Vitro
IV. OTHER PROPERTIES OF CARBOXYLIC POLYANIONS
Effect of Molecular Weight Biological Activity
and Toxicity
Immunomodulator Activity
Antimicrobial Activity
D. Antitumor Activity
V. OF CARBOXYLIC POLYANIONS
VI. FUTURE PERSPECTIVES
REFERENCES
1. INTRODUCTION
239
240
241
241
245
245
245
246
249
249
251
255
257
Since the discovery 16 years ago that substances otber tban viruses could
stimulate tbe production of interferon, the number of nonviral inducers
has increased great1y. list now includes microorganisms such
*Present Graduate School of Public Health, University of Pitts-
burgh, Pittsburgh, Pennsylvania.
239
11111-:1 NH:
bacteria, chlamydia, mycopla:oma:o, pl'oLozoa
and fungal extracts, endotoxin:o, mitogcn:o, al1LibloLic:o, pOlYHaCC/lal'idCH,
l1atural al1d synthetic acids, and synthetic polymers. thesc 110l1Vil'al
inducers are, or cOl1tain, polyal1ions, and these inducers are the subjeci
this chapter. Polyaniol1s of both natural and sYl1thetic origil1 have
reported. first part of the chapter il1cludes brief description
of their to induce il1ter-
feron. 1n the latter part of the chapter, carboxylic polyanions
are discussed with respect to antiviral activity properties other than
pyran copolymer as model, the role of the
macrophage is possible for the
immunomodulator, activities of these polyanions.
AND
OF POLYANIONS
have wide of biological activity. Besides the
of various polyanions exhiblt [1-21], anti-
tumor [22-40], antibacterial [35,41-43), [35), and antiprotozoal
[35,41,44] activities. Polyanions also are to stimulate the reticulo-
endothelial system (RES) al1d modulate humoral- and/or cell-mediated
immune [45,46]. Many of these
properties, as well the toxicities of the polymers, resemble
bacterial endotoxin, which was the first
to described [47,48].
TABLE 1. Examples Polyanions with
Activity
Synthetic
Polycarboxylates
(maleic ether)
Polyacrylic acid
(chlorite-oxidized oxyamylose)
Polymethacrylic acid
Polysulfates
Polyvinyl sulfate
Polyphosphates
Dextran phosphate

Natural
(LPS)
Lipid
Bacterial products
Heparin
polysaccharide
10.
varicty polyanionic natural and synthetic,
rcported to induce interferon and/or antiviral effects 1). major
emphasis in this chapter will the synthetic polyanions; discussion of the
natural polyanions will limited to bacterial endotoxin/lipopolysaccharide
(LPS), for which considerable tiological data are available. Of the synthetic
polyanions, the carboxylic acid polyanions have studied in most detail.
more important features of the carboxylic polymers for interferon
induction and antiviral activity are high density of carboxylate groups
pendent from long-chain polymeric backbone. Polyanionic polymers, in
general, have found to fairly stable and are not readily biodegradable,
which account for the ir prolonged activity [49]. such polyanion
which has studied in considerable detail is pyran, random copolymer
of maleic anhydride and divinyl ether. Figure 1 relates the struc-
tural features between pyran copolymer and several other polyanions. Poly-
acid-maleic anhydride copolymer has biological activities similar
to but is not as biologically effective [50]. Maleic anhydride homo-
polymer is much less toxic than copolymer but also much less effective
against tumor growth and viruses [51]. Polyacrylic acid, polymethacrylic
acid, acrylic acid-maleic anhydride copolymer, and maleic anhydride-furan
copolymer among the other purely synthetic polyanionic polymers which
have evaluated [50]. Several polysaccharide derivatives have
studied, and the most active biologically was chlorite-oxidized oxyamylose
[4]. is polymer chain with fairly high group
density and possibly more biodegradable polyacetyl backbone, which
account for its rather limited antiviral activity. appears to rela-
tively nontoxic and also inhibits growth [28,29].
Several in structure apparent in the polymers just
described. these fairly flexible polymer chain and high
density of carboxyl groups situated along the backbone the polymer. In
general, the toxicity of polyanions increases with molecular weight, particu-
larly greater than 50,000 [49]. Fortunately, substantial al1titumor activity
is retained in lower-molecular-weight fractiol1s, less than 10,000 [49].
These fractions are relatively less toxic and are associated with macrophage
activation; however, they usually also exhibit less antiviral activity than
higher-molecular-weight fractions.
INTERFERON INDUCTION WIlli POLYANIONS
Interferon Induction in Vivo
The of synthetic polyal1ions to induce circulating interferon has
shown most frequently in the mouse, although pyran copolymer has also
reported to induce interferon in humans [52]. Pyran copolymer [53,
54], polyacrylic [55], polyvinyl sulfate [12], and [7] have






/








Pyran
(maleic anhydride -divinyl ether)
"


"

/"

/

'"

"-

/ "-



/

"



/ "-
Polyacrylic acid

"- / "

/
/0

""

"- / "-

/
/0

"

(chlorite-oxidized oxyamylose)

t-;
z
3:::
.-
>
>
z

I
0= - 0-
I

I
0= - 0-
I
HEprOSE
ETHANOLAMINE
I

RZ
0= - 0-
1

I
0= - 0-
I

I
GLUCOSAMINE -RI
I
GLUCOSAMINE - RI
I
0= - 0-
I

1
0= - 0-
I

I
GLUCOSAMINE - R I
I
R 3- R
Z
- GLUCOSAMINE -R
1
pol ysaccharide core lipid
LPS
( I i popolysaccharide )
I I

I I

I I
RIBOSE - BASE ---- BASE - RIBOSE
I I

I I
- = 0= - 0-
I I

I I
RIBOSE - BASE ---- BASE - RIBOSE
I I

I I
- = = - 0-
I I

I I
RIBOSE - BASE----BASE -RIBOSE
I I

I
- =
I

I
RIBOSE - BASE ----BASE
I
?
- =
I
I
0= - 0-
I

I
-RIBOSE
I

I
0= - 0-
1
Pol ynucleotide
FIGURE 1. Structures of three synthetic polycarboxylates are compared with schematic representations of
polynucleotide and of part of the lipid and polysaccharide core of LPS. Figures are drawn to illustrate the high
density of anionic groups pendent from the various long-chain polymeric backbones. In LPS, R
1
= long-chain
fattyacids, R
2
= the (2-keto-3-deoxyoctulonic containing part of the polysaccharide core, and

= rest
of core and terminal sugar residues.
"'"
>
z
z
:r..
t:=
I\I!.I';INI(; :LIIII
SllOWn to illducc ill illjeeLiOll,
and the amoullLs of [oUlKI IH'.
inocu1ation of micc with a1so itl
of [54], with peak at 24-48 The po1yallioll,
endotoxin, a1so inducos in the [48], [5 and
[47] when injected 1n contrast to synthetic-po1yanion-inducct\
interferon, hovvever, maximum serum interferon is attained
much earlier, within 2
Po1yanions are frequent1y antivira1 out of proportion to the amount of
interferon induced and, in fact, induce antivira1 activity in the absence
of detectable interferon. Po1yacrylio acid protected rabbits against vaccinia
virus infection [21] and protected swine against hog cho1era virus
infection [8], although neither of these po1yanions induced detectable inter-
feron in the respective host.
There have been few attempts to characterize the interferons induced
polyanions. due in part to the low 1eve1s 100 units)
found in vivo. Endotoxin is interferon inducer. Para-
doxically, a1though LPS induces 10w 1eve1s of interferon itself, it has the
capacity of inducing hyporeactivity state of to1erance, which the
is marked1y reduced) to variety of strong interferon
inducers. 1n the injection of particu1ar interferon inducer renders
the anima1 to interferon production to second injection with
the inducer. Endotoxin, however, confers broad hyporeactivity.
Following the injection of endotoxin, there is period of week or more
of marked hyporeactivity not on1y to endotoxin, but to other
well, inc1uding viruses and po1ynuc1eotides [57-59]. Whether
01' not synthetic po1yanions are to confer similar hyporeactivity
has not been examined. however, do follow the
of in which injections of the
results in [5,55]. With the
1asted weeks [5].
It wou1d to determine the of synthetic-
po1yanion-induced to endotoxin-induced both of which
produced in 10w 1eve1s. It is po1yanion-induced inter-
fe1'on is typical of the "class ical-vi1'us- induced inte1'fe1'on" (type 1)
01' "immune (type stable 01' unstable,
01' unstable, etc. For example, induced pY1'an
was found to inactivated 2 [54],
se1'um induced acid 01' po1yviny1 sulfate
was resistant to 2 [12,60]. The the circu-
1ating interferon a1so not yet been dete1'mined. however,
sp1enectomy on1y the amount of interferon in
mice after injection of pyran copo1ymer [5], sp1enectomy g1'eat1y
reduced the amount of injection endotoxin [61]. 1n vivo,
the sites of endotoxin-induced synthesis inc1ude the
sp1een, 1ymph nodes, liver, thymus, and 1ung [62-64].

lIlLu!"[uroll IllduuLiotJ in
While polycarboxylates and endotoxin induce in animals, they
are, in general, not very effective inducers in cultures of many
lines, primary fibroblasts, or epithelial [4, 7, 12]. Endotoxin is
to induce interferon in cultures of peritoneal [65-67] or spleen
[64,67,68], and the adherent produced more interferon than the non-
adherent predominant producing interferon incubation
with endotoxin in vitro appear to the macrophage and lymphocyte.
Interestingly, the interferons produced these two have different
stability and blological properties [67]. Peritoneal macrophages produced
amounts of interferon in response to pyran copolymer, but only if
the pyran was complexed with arginine [5]. interferon-producing
of lymphocytes cultured with pyran or other polyanions not been
well studied.
Synthetic polyanions and endotoxin are therefore, in general, poor
inducers of interferon and are effective only in the animal, resulting
in low yields of interferon. antiviral protection seen after prophylactic
administration of synthetic polyanions been observed to persist
longer than the interferon response [4,5,20], which suggests that the anti-
viral protection is probably mediated factors other than the production
of interferon.
IV. OTHER PROPERTIES OF CARBOXYLIC
POLYANIONS
Effect of Molecular Weight Biological
Activity and Toxicity
Activities of anionic polymers vary according to the average molecular
weight of the molecular weight distribution of particular samples [49].
effect of molecular weight blological activity most exten-
sively studied with pyran, where variety of fractionation, degradation,
and synthesizing techniques been used to produce different molecular
weight fractions. procedures were used in attempts to remove the
toxicity inherent in the high-molecular-weight, especially >50,000,
pounds. Toxicities associated with high-molecular-weight polyanions have
included elevation of liver transaminases, hepatosplenomegaly, thymic
involution, anemia, leukocytosis, and decrease in marrow [49,
69-71,103] .
Low-molecular-weight 10, 000) samples of pyran copolymer with
narrow-molecular-weight distribution exhiblt less toxicity while retaining
the antitumor activity exhibited higher-molecular-weight samples [49,
70]. Low-molecular-weight fractions, well high-molecular-weight
IIltl<INI<:
fractiollS, activatc pcritol1cal I :JHj.
destroy tumor wbllc ccLIc:
[ 72]. Phagocytosis is stimulated low-molecular-weigllt polymers
depressed high-molecular-weight polymers [49,70]. Tbls
the biphasic followed elevatioll)
effects the system of the p01ydisperse
[73]. These data also emphasize the difficulty determillillg
the precise of of the they are well
purified characterized. regard to toxicity, with low molecu-
lar weight polydispersity isolated from the polydisperse
compound showed greater 50% lethal doses the causcll
did mice to bacterial
Whi1e these retained antitumor activity, they had very limited
activity [49}. Similar have made with
pounds with m01ecu1ar weights [70}. These data suggesL
that structura1 features required for the
tumor activities of the activity of
has yet to dissociated
the toxicity of the compound [49,70, 74}.
Activity
copolymer, as well as polyacrylic acid, have shown to have
activity severa1 mode1 systems. Both of these
. the primary of mice
to sheep erythrocytes vivo. P01yacrylic acid a1so the back-
p1aques similar to observations reported
for [75]. In vivo of the other hand, did
not stimu1ate the of background plaques
mice [l,46}.
mechal1isms of adjuvant activity of polycarboxylic for
humora1 responses have partial1y defined thymec-
tomized, irradiated, al1d mice that
are deficient cells. The respol1se
of p01yacrylic-acid-treated mice that were with sheep
erythrocytes was 1ess thal1 that observed il1 norma1 mice that
were immul1ized [76]. However, the respol1se il1 the p01yacrylic-
acid-treated mice was as measured p1aque-forming
cells hem01ysi11 titers, as compared with mice which had received
the antigen without resu1ts suggested that this p01ycarboxylic
similar to p01ynuc1eotides [76,77], was to partial1y
replace or residual ful1Ctiol1 il1 T-dep1eted
mice.
111 cOl1trast to the resu1ts obtail1ed with p01yacrylic acid, was
to specific al1tibody-forming activity to sheep erythrocytes
10. i\NI< )NS
'1
(plaquuH PUl' HJ" HpluUI1 itl Tx13M llliuu [4uJ. HuuHtalltial
of[eoL ill tlormal mioc appeared to depclldellt
'1'-0011 fullctioll. did slight illcrease in total plaques spleen
in fue immunized mice. This minimal increase in total antibody
plaques in the mouse treated wifu was consistent wifu other
data showing that the polyanion caused modest, nonspecific blastogenesis
of fue entire spleen population in and normal mice [46].
Data in other systems provide additional evidence that fue humoral
immunoenhancing effect of is primarily mediated through lympho-
cytes. was of increasing the secondary response of mice
to sheep eryfurocytes in normal mice, suggesting that the immunomodulator
expanded the of antibody-producing ce11s ce11s in
this T-dependent humoral immune response [46]. adjuvant effect of
in T-dependent humoral immune responses to two different a110geneic
cells was variable. Treatment of mice wifu after immunization with
the allogeneic MEL-2 tumor cells caused enhanced production of comple-
ment dependent cytotoxic antibody [46]. However, when was adminis-
tered prior to immunization wifu a110geneic mastocytoma there
was difference in the cytotoxic antibody response between untreated and
pyran-treated animals [46]. Immunomodulator treatment at different
time during immunization necessary for immunoenhancement.
effect of cells is supported other data showing fuat
did not the antibody response to LPS, T-independent antigen
[46] .
The effects of polycarboxylate polyanions immune
responses have investigated primarily wifu pyran. Alfuough the effects
complex, the general pattern is immunomodulator-induced inhibition of
various aspects of cell-mediated immunity. lntravenous treatment of mice
wifu pyran prior to immunization with the a110geneic mastocytoma
cells caused decrease in spleen cytolytic activity, in addition to delay-
ing the time of the peak response [46]. Unfortunately, experiments were
not performed to determine whefuer the inhibition in this in vitro test
of activity was correlated with enhanced tumor growth
in vivo. ln fue MEL-2 a110geneic tumor system, intraperitoneal treatment
of mice with pyran prior to immunization did not alter the growth
and subsequent ce11-mediated immune regression of fue tumor [78]. If,
however, mice were treated with fue immunomodulator 6 days after immu-
nization, fuere was inhibition of fue ce11-mediated immunological regression
of the tumor and the tumor grew progres s i vely . ParadOXica11y,
this inhibition of fue in vivo ce11-mediated immune response was not due to
inhibition of splenic lymphocyte-mediated cytolytic activity, which was
similar in untreated or pyran-treated animals. Moreover,
caused increase in specific macrophage-mediated tumoricidal activity.
The only index fuat correlated with fue decreased in vivo ce11-mediated
immune response was decrease in the of macrophages that nor-
Illtl':INI(: :tII(1
il1filLratcd 010 tlllllOl:" spuculatt:
that the reduced 010 tlllllOL" r"ulatull Lo
the increased macrophage activity in tlle WI1OL"U
the immunomodulator was i.e., that is chal1go l!lU
110rmal macrophage migration
Pyran administration has also reported to cause delay in skin
graft or tumor allograft rejection [79], although the of the
inhibition of this cell-mediated immune response has not established.
The administration of pyran has, in few instances, oaused enhanced growtll
ofchemical-induced [80] or virus-induced [24,25,81] tumors inmice.
Whether the mechanisms underlying these adverse immunomodulatory
events involve effects ceHs or macrophages or other
physiological processes is not yet clear.
In effort to define some of the immunological bases responsible for
the observed effects, the effect of pyran mitogenic responses has
measured. As mentioned earlier, in vivo administration of pyran does
cause modest mitogenic effect both and cells [46]. similar
slight mitogenic activity (stimulation ratio of 8) is observed in vitro when
normal spleen cells are inoubated with pyran. However, single intravenous
injection of pyran depressed significantly the in vitro blastogenic response
of spleen cells to phytohemagglutinin and LPS from 2-14 days after
in vivo administration of pyran. This is similar to data presented for the
biological immunomodulator Corynebacterium parvum. However 5 days
after pyran administration, thymidine incorporation in mitogen-treated
cultures of cells from pyran-treated mice was significantly higher than
that of normal cells incubated with mitogen. As this corresponded to the
time of peak increase in pyran-induced background incorporation of
thymidine, it was possible that pyran-primed spleen cells were more
susceptible to the action of or LPS and/or were more resistant to the
inhibitory effect of activated maorophages.
Pyran-induced inhibition of blastogenesis is macrophage-dependent.
Peritoneal exudate cells from pyran-treated animals were more effective
than were normal macrophages in abrogating the or LPS response of
normal spleen cells. Removal of glass adherent oells from the spleens of
pyran-treated mice restored the response to and LPS [82].
In summary, these results are consistent with dual action of pyran
lymphocytes and maorophages in the modulation of the immune response.
The enhancement of the humoral immune responses is with
direct action lymphocytes [46]. The inhibitory effect of pyran
mediated immunity could due to direct action cells or, most likely,
indirect effect mediated macrophages [78,82]. Further experiments
are needed to clarify these hypotheses. It is likely that some of the carboxy-
polyanionic immunomodulators act more than one type. More-
over, the target oell(s) for specific polyanion is (are) probably to certain
extent dependent the model system used to determine immune reactivity.
10.
[JL'CHCIIL L'cvicw ilHlicaLcH, pulyalliolls
widc uf effects itnrnune and the effect
the to given antigen even
than anticipated. Po1yanions of compounds
with potent activity. These agents have potentia1 ad-
juvants with bacteria1, vira1, and vaccines and stimu1ators
of the immune in clinica1 situations where inhibition
is present.
Antimicrobia1 Activity
Po1yanions to resistance to bacteria1, funga1, and protozoa1
infections. FoHowing with copo1ymer acid,
mice exhibited enhanced to both and gram-negative
bacteria, inc1uding Listeria monocytogenes [41], Dip1ococcus pneumonia
[35,43], pneumonia [42], and PasteureHa tu1arens is [43]. The
antifunga1 activity of pyran was in mice using
neoformans [35]. Enhanced surviva1 of mice chal1enged with Toxop1asma
gondii [41], Trypanosoma duttoni [35], the stage of P1as-
[44] was seen foHowing with
acid.
Certain features of the antimicrobia1 activity of po1yanions
to those observed concernillg the antivira1 activity. examp1e, greatest
protection is observed when the po1yanion is administered
and po1yanion treatment provide pro1onged protection
to L. monocytogenes for 10ng two months) [41].
D. Activity
Synthetic po1yanions such
and acid polymers with weight of 1000 to
100,000 01' greater have been tested against of subcutaneous rodent
[31-34]. In each systemic drug administration was associated
with significant tumor without excessive weight 10ss. The higher
the mo1ecu1ar weight, the toxic was the ethy1ene-ma1eic anhydride
po1ymer for both the and the dog. Optimum activity was obtained
with compounds where carboxamide and ionizable
a10ng the po1ymer backbone. Monomers were inactive, and
when carboxy1 groups were converted to carboxamides, significant
inhibitory activity was 10st. This is similar to the
action of these compounds.
Of the po1yanions, has the greatest atten-
tion with respect to its activity. Treatment with prior to
inocu1ation of mice with the solid inhibited subse-
IIIII';INIC iLlHI
qUCI1LLllIIlO1" I :J:>J. ill11ilJilClI LllI1101.'S iIHllICO(1 IJY l"I'iolll1 10ll-
kemia virus sa1"COI11:l virus Icukcl11ia vir'lIs
polyoma virus, and Uross leukcmia virus il1 l1Orl11al 01"
suppressed mice l Systemic administratiol1 of this polyanion also
inhiblted the growth of several syngeneic solid metastasizillg
including the first generation transplant of the carcil10ma
Lewis lung carcilloma [36], melanoma [36], and Madisolllung
cinoma [37], as as methylcholanthrene-induced fibrosarcomas
[38]. It should noted that several of these tral1splantable tumors, such
as the Lewis lung and Madisol1 lung carcinomas, are quite resistant to
conventional chemotherapy, emphasizing the potential in the anti-
tumor effects of pyran. Pyran was effective against some of these tumors
even when its administratiol1 was delayed until 8 days after tumor implanta-
tion, time when the tumors have already metastasized in many animals
[36] .
While administration of pyran alone has produced significant tumor
growth inhibltion and prolonged survival, the combination of pyran treat-
mel1t with other treatment modalities has provided long-term
survivors. Mohr et al. [39] were the first to demol1strate that pyran could
produce complete cures of mice bearing the LSTRA leukemia, when the
tumor burdel1 was reduced with chemotherapy. We have demon-
strated pyran increase survival in mice bearing the Lewis lung
carcinoma, when the primary tumor is removed surgery after it has
metastasized l70]. The comblnation of pyran with other treatments, such
as radiation, to reduce the tumor burden has also proved effective [83].
Moreover, pyran potentiate the specific immune protective effects
produced vaccination with killed tumor vaccines [84].
The mechanism of antitumor activity of pyran has received considerable
attention from various laboratories. Possible a1ternative or cooperating
concepts of antitumor action include immunoenhancing coupling of the poly-
anion to tumor antigen, direct effect tumor action of polyanions
wide variety of enzymes, alteration of the isoelectric point of proteins,
displacement of nucleohistones, antiviral activities, interferon induction,
enhancement of specific immunity, and macrophage activation. The
antitumor activity does not appear to due to direct tumor cytotoxicity;
studies of cytotoxicity in vitro l1ave shown that levels greater than 1 mg/ml
of drug are required to destroy 50% of either the tumor or normal
targets [36]. These observations of lack of cytotoxicity, together with
the known reticuloendothelial-stimulating activity of pyran, have suggested
that the antitumor activity mediated macrophages. This concept
received additional support the finding that only those polyanionic
rations which activated macrophages for tumoricidal activity exhibited
antitumor activity in vivo [38].
experimental lines of evidence that implicate pyran-activated
macrophages in resistance to the Lewis lung carcinoma and other solid
10. I'()I,YANION:->
l,lllllOl,:-; ille[lIlle: (1) uf witlll1iStiOCytcs foHow-
iHI!: :-;yslclllie [:37,85]; (2) of activated
pcrit(mcal with tumoricidal activity tumo1' bea1'i!lg a!ld
pyr:.tl1-treated [37, 85]; (3) i!ll1ibitio!l tumo1' g1'owth whe!l activated
pe1'itoneal we1'e witll tumor ceHs i!l vit1'o a!ld tra!ls-
pla!lted i!lto sY!lge!leic 1'ecipie!lts [38,40]; (4) isolatio!l of
activity of [86],
p1'ovidi!lg sig!lifica!lce to the mo1'pllological of
[37,85]; (5) cytotoxicity a!ld tumo1' g1'owtll i!l
vit1'o activated [38,40]; a!ld (6) lack 1'ole fo1' specific
t1'a!lspla!ltatio!l i!l pY1'a!l- i!lduced 1'esista!lce to tumo1's [87].
Ma!lY of these a!ld otlle1' app1'oaclles that
othe1' sucll as pa1'vum, also exe1't thei1' a!ltitumo1'
eMects via activated mac1'opllages [88]. I!l fact, the activated mac1'opllage,
1'athe1' tha!l lympllocytes, p1'ovide the i!l tlle a!lti-
tumo1' of polya!lio!lic
I!l few i!lsta!lces, pY1'a!l has bee!l associated with
ment of tumo1' g1'owth. This has bee!l obse1'ved
skiIl tumo1's [80], the Molo!ley vi1'us iIl ce1'tai!l st1'ai!ls
[28], a!ld with i!lt1'ave!lous pY1'a!l agai!lst F1'ie!ld vi1'us
[24]. The these adve1'se effects is la1'gely u!lk!low!l. I!l the
latte1' situatio!l, we have that iIlt1'ape1'ito!leal
of pY1'a!l, which is protective agai!lst F1'ie!ld vi1'us [89], is asso-
ciated with the i!lc1'eased mac1'ophage activity i!l the pe1'ito!leal cavity a!ld
the splee!l. I!l cO!lt1'ast, the adve1'se i!lt1'ave!lous pyra!l 1'egime!l is associated
with i!lc1'eased e1'yth1'ocytic stem p1'olife1'atio!l i!l the spleen, which p1'o-
vides inc1'eased numbe1' of ta1'get ceHs fo1' g1'owth of the vi1'us [25].
These seemingly paradoxical effects exhibited pY1'a!l the
po1'tance of deli!leating the and of actio!l befo1'e
cli!lical 1'atio!lale fo1' ca!l developed.
v. OF CARBOXYLIC
POLYANIONS
Ma!lY dive1'se, !latu1'ally occu1'1'i!lg, a!ld synthetic polya!lio!ls exe1't ma1'ked
activity agai!lst vi1'us 1'eplicatio!l iIl vit1'o [90], agai!lst plant vi1'uses
[91,92], a!ld a1'e !lOW bei!lg developed fo1' pu1'ificatio!l of wate1' f1'om CO!l-
tami!lati!lg vi1'uses. of a!ltivi1'al activity i!l these systems
has !lot bee!l defi!led, but it i!lvolve di1'ect i!lactivatio!l of
vi1'uses i!l situatiO!lS.
I!l rega1'd to va1'ious vi1'us i!lfectio!ls i!l pY1'a!l a!ld
othe1' polyca1'boxylic polya!lio!ls exhibit b1'oad spect1'um of a!ltivi1'al activity
2). Polya!lio!l p1'otects mice from mo1'tality foHowi!lg
lethal i!lfectio!l both RNA a!ld DNA cytopathic vi1'uses a!ld tumo1'
I\I!.I';INI<: Itllll
TAI3L1<; 2. Antiviral AcliviLy il1 Vivu of PYl'al1 OLIICI.
Similar Polyanions
a
Vil'uS
Cytopathic vi1'uses
Encephalomyoca1'ditis

Mengo
Foot and mouth
disease
Vesicula1' stomatitis
Influenza
Semliki Fo1'est
Hog chole1'a
He1'pes simplex
Vaccinia
Tumo1' vi1'uses
F1'iend leukemia
Rausche1' leukemia
G1'oss leukemia
Moloney sa1'coma
Mamma1'Y tumo1'
Polyoma
Adenovi1'us 12
Host
Mouse
Mouse
Mouse
Mouse
Guinea pig
Mouse
Mouse
Mouse
Pig
Mouse
Rabbit
Mouse
Rabbit
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Hamste1'
lloute vil'uS
administl'ation
.
1. v.
i.p.
i.p.
i.p.
Footpad
i.n.
i.n., ael'osol
i.
i.m.
i. v., i.p., i. vag.
Co1'neal
i.p., i. v.
i.d.
i.p.
i.p.
Spontaneous
i.m.
Spontaneous
i.
s.
Selected
l'efel'ences
1
2,3
4,5,6,7
8,9
8,10
11
4,12,13
4
8
14,15,16,17
4
4,7,12,18,19,20
21
22,23,24,25
23,26
27
28
29
26
30
aln most cases the polyanion was administe1'ed the systemic 1'oute (i. 01'
i. v.).
bAbb1'eviations: i.d., int1'ade1'mally; i.m., int1'amuscula1'ly; i.n., int1'a-
nasally; i.p., int1'ape1'itoneally; i.v., int1'avenously; i. vag., int1'avaginally;
s. subcutaneously.
fo1'mation and delays mo1'tality afte1' infection with DNA 01' RNA tumo1'
vi1'uses. This is in cont1'ast to the na1'1'ow antivi1'al spect1'um of most con-
ventional chemothe1'apeutic d1'ugs.
Ou1' investigations [70,74] have substantiated the impo1'tance of molecu-
la1' weight and specific polyanionic cOl1figu1'atiol1 in the al1tivi1'al activity
[5,49]. Howeve1', complete st1'uctu1'e activity 1'elationship in 1'ega1'd to
al1tivi1'al activity still needs to pe1'fo1'med. G1'eatest p1'otection is
se1'ved with p1'ophylactic t1'eatment. The1'apeutic t1'eatmel1t often does not
a1te1' the cou1'se of disease, pa1'ticula1'ly with 1'apidly fatal il1fections [15].
10.
that polyalliollH act vcry early tlle viral
illfcctioll, the drugs need to activated the animal, the drugs
act modulation of host responses. Greatest protection is
observed when drug is administered at site where virus is
subsequently injected [5,11,20]. There is often, however, significant pro-
tection, especially with pyran, when the polyanion and virus are injected
at different sites. For example, intravenous or intraperitoneal administra-
tion of pyran protects mice against lethal vaginal infection with herpes
simplex virus [16,17].
contrast to standard antiviral chemotherapeutic agents, polyanions,
especially when the polyanion and virus are both inoculated intraperitoneally,
provide prolonged protection [5,20]. Mice treated with pyran were
protected against subsequent picornavirus infection for two months [2] and
were resistant to Friend leukemia virus infection for 3 weeks [24]. 1n
contrast to intraperitoneal route, animals inoculated intravenously with
pyran lost protection against picornavirus infection within week [1] .
Considerable emphasis has been directed toward defining
nism of antiviral action of polyanions, particularly for pyran
major modes of antiviral action that been considered are listed in
3. Polyanions directly inactivate viruses [5, 15]. However,
levels required are greater than those required for antiviral activity in
vivo. 1nhibition of virus replication mechanisms similar to conventional
chemotilerapeutic drugs also does not appear to play an important part in
resistance induced polyanions in vivo. Pyran does inhibit the RNA-
dependent DNA polymerase of avian myeloblastosis virus in vitro [93], but
the enzyme is present only in oncornaviruses. Moreover, the drug does
not morphologically toxic effect normal or transformed mammalian
cells at doses far the effective antiviral dose [36,37].
Considerable effort has been directed toward determining whether
induction of antiviral protein, interferon, accounts for antiviral
action of polyanions [1,2,5,6,11,20,23]. There is clear evidence tilat
3. Possible Modes of Antiviral Action of Polyanions
1. Direct inactivation of virus
2. 1nhibition of virus adsorption and/or replication
3. 1nduction of interferon
4. Stimulation of phagocytos is and inflammation
5. Enhancement of virus-specific, humoral- and/or cell-mediated
immune responses
6. Enhancement of macrophage antiviral functions
IIIII':INI(; iLlHI
systemic induciiol1 of plays 1'olc il1 al1livil.'al aclivily uf
[1,2,5,G,15,20,24]. slimulalcs
cytosis the 1'eticuloendofuelial system, the1'e is e01'1'elation with anti-
vi1'al activity. The 10we1'-molecula1'-weight compounds, which exe1't potenl
RES-stimu1ating activity, exhibit mucll 1ess 01' antivi1'a1 activity [49,70].
M01'eove1', fue kinetics of antivi1'a1 activity do not c01'1'e1ate wifu inc1'eased
global phagocytic activity [1,5, 73] .
Because po1yanions immunomodulato1's fo1' va1'ious humo1'al and
cell-mediated immune 1'esponses, attention has di1'ected towa1'd specific
as the mode of antivi1'a1 action. The protective effect
of F1'iend leukemia vi1'us has associated with in-
c1'eased antibody p1'oduction di1'ected to the vi1'ion antigen [94]
Specific immunostimulation, howeve1', at 1east in fue case of does
not to play p1'ominent in fue antivi1'al activity. is
effective antivi1'al agent in animals depleted of lymphocytes [14,26],
indicating fuat fue d1'ug acts independently of fuese immune cells.
is also effective against pico1'navi1'us infection in splenectomized
animals (autho1's' unpublished observations). Animals protected t1'eat-
ment with possess 01' low levels of p1'otective antibody against
he1'pes simplex vi1'us, not 1'esistant to subsequent wifu fue
vi1'us, and have dec1'eased vi1'us-specific 1'esponse
[17]. The po1yanion to inhibit vi1'uS 1'eplication ve1'Y and
efficient1y [15, 17], so fuat the1'e is p1'obably little vi1'us antigen to stimu1ate
vi1'us-specific immune 1'esponse.
The majo1' featu1'es of pyran-induced p1'otection Hgainst he1'pes Simplex
vi1'us type 2 and/o1' encepha1omyoca1'ditis vi1'us a1'e shown in 4. Ou1'
1'ecent data substantiates the hypothesis fuat activated mac1'ophages
involved in fue antivi1'a1 action of polyanions [17]. pe1'i-
toneal t1'ansfe1' 1'esistance to susceptib!e 1'ecipient mice to infection
witl1he1'pes simplexvi1'us 01' F1'iend1eukemiavi1'us [17,89]. Mo1'eove1',
pe1'itoneal mac1'ophages exhibit potent antivi1'al activity in
vit1'o [95]. Thus, we have demonst1'ated fuat activated mac1'ophages have
the capacity fo1' antivi1'a1 activity; howeve1', p1'oof is stil1 needed fuat these
fue majo1' mode of action in vivo in anima1s t1'eated with po1yanions.
This concept is suppo1'ted mo1'phologica1 identification of inc1'eased
of mac1'ophages in fue spleen, ta1'get o1'gan fo1' F1'iend leukemia vi1'us,
in tI1at we1'e protected treatment witl1 pY1'an [24,25]. Mo1'eove1',
pe1'itoneal mac1'ophages f1'om these mice exhibited antivi1'al
activity F1'iend leukemia vi1'us, fuat fue vi1'us injected
into the pe1'itoneal cavity might 1'apidly inhibited eifue1' pe1'itonea1
01' splenic-activated mac1'ophages [89]. P1'elimina1'Y obse1'vations indicate
the p1'esence of mac1'ophages in tI1e vagina1 in mice
p1'otected against vagina1 he1'pes simp1ex vi1'us (aufuo1's' unpublished
se1'vations) .
P1'ophy1actic t1'eatment wifu po1yanions pa1'ticu1a1'1y
in special situations witl1 immunosupp1'essed patients at 1'isk to va1'ious vi1'a1
10.
'l'AJJLt<; 4. 1,'caLul'cs t!lC Atlti vil'al lksistatlce Itlduccd Pyratl
Agaitlst llerpos Simplex Virus 2 alld/or Ellcephalomyocarditis
1. Illductioll of or millimal levels of illterfel'OI1
2. No direct antiviral
3. Early illhiWtion of virus pathogenesis
4. Lack of resistallce in
5. Less neutralizing antibody respollse than in Ul1treated mice
6. Antiviral effect in allimals depleted of lymphocytes
7. Antiviral effect in splenectomized animals
8. Antiviral effect in animals treated with
9. Antiviral effect in neollatal animals
10. Systemic as well as local protection
11. of pyran-activated peritoneal cells to transfer resistance ill vivo
12. antiviral activity of adherellt peritoneal
cells itl vitro
infections (e.g., cancer patients, renal transplant patients, other immullo-
deficiellt patients). Pyran treatment has provell effective ill llaturally
immullodeficient neonatal animals [16,96], in animals rendered deficient
in cell-mediated respollses [14,26,27], ill allimals suppressed
treatmellt with [16], alld in animals treated with steroids [3]. It is
illteresting that the polyallion was active against herpes simplex [16]
or Friend virus [89] ill animals treated with agent toxic
for macrophages. The data suggest that the caused
illfltlX of macrophages into the peritoneal cavity and an hepatosplenomegaly
associated with increased reticuloendothelial capacity, which decrease
the effectiveness of silica. Moreover, pyran as has been reported
for as amplify the residual reserves
of macrophage stem cells, so that it impossible to deplete mice
of macrophages in the presence of the [97] .
VI. FUTURE
As previously discussed, the polycarboxylic
effective in patients. Prophylactic treatment has clearly
been demollstrated in T-depleted in macrophage-deficient allimals [14,
16,26,27,89]. Moreover, several illCludillg pyran
III!.I:INI(; IlIIII
increase rcsistance of to
viral infections. Examples include pyran and Mycobacterium bovis
against herpes simplex virus [16,98] and picornaviruses ([9(j]; also
authors' unpublished observations) in neonatal mice and levamisole against
herpes simplex virus in neonatal rats [99]. Not immunomodulators
have such activity. parvum, typhoid vaccine, vaccine, staphagc
lysate, and levamisole, for example, were without effect in neonatal mice
[98]. mechanisms of the protection in neonatal animals are not known,
but they involve maturation of immature macrophages, as has been
demonstrated for several of these immunomodulators in regard to various
immune responses [100].
The clinical future of polyanions lies with the separation of the toxicity
from the antiviral, antitumor, and immunological effects [49]. Previous
clinical studies with pyran used the drug in direct cancer chemotherapeutic
regimen in advanced cancer patients and were limited drug side effects
[49,69,103]. In contrast, proposed trials with less toxic fractions are
aimed at adjuvant therapy with the polyanion combined with standard cyto-
reductive therapy, i. surgery, chemotherapy, or radiation. Such
protocols, using less drug over longer periods, should have much less
associated toxicity. Moreover, although polyanions exert beneficial
effects, treatment with polyanions has also exacerbated tumor formation
in few instances [24,80,81]. As mentioned earlier, protection against,
or exacerbation of, Friend-leukemia-virus-induced leukemogenesis depends
upon the route of administration of pyran. In the protective (intraperitoneal)
regimen, the drug appears to activate the macrophage in the peritoneal
cavity and the spleen, while in the adverse (intravenous) regimen, the drug
appears to stimulate in which the virus replicates. results
also related to the presence of both low- and high-molecular-weight
species in the preparation, which are known to have different
pharmacological effects [49,70]. illustrates the "yin-yang" aspect
of treatment with polyanions, and the absolute necessity of developing
defined molecular-weight species and of elucidating the parameters affecting
the pharmacology and mechanism of action of polyanions before rationale
for clinical treatment established.
Perhaps the greatest potential use for polyanions is in combination
with virus vaccines. In this situation, the polyanions could act both in an
antiviral capacity and as an immunoadjuvant. and Richmond [101] ,
in series of studies, have documented that polyanions increase the effec-
tiveness of various foot and mouth disease virus vaccines. Moreover,
coadministration of pyran with tumor vaccines enhanced the specific
vaccine-induced protection of the weakly immunogenic L1210 tumor [84].
The tumor vaccine and immunomodulator had to administered
the same route and at the same time, preferably being mixed together.
was not necessary with the virus vaccine; while simultaneous adminis-
tration was most effective, administration of the immunomodulator prior
10.

Lo I'outc wa:::; [101]. The immuno-
logical ulldeI'lyillg the:::;e positive immunomodulatory activities,
alld thc possible illvolvement macrophages, have Ilot established.
The new virus vaccilles that are currently being advocated are those
tnade of virlls pI'otein sllbullits, without the possible cance1' causillg or
othe1'wise delete1'iolls nucleic acid [102]. Unfortunately, these vaccines
are not as effective as live attenuated vi1'us 01' whole inactivated virus
vaccines. However, in conjunction with polyanions, such vaccines might
p1'epared that possess adeqllate effectiveness.
ACKNOWLEDGMENTS
This wo1'k was sllppo1'ted Health Se1'vice Resea1'ch G1'ant 16193
f1'om the National Cancer Institllte, T1'aining Grant AI 00382 f1'om the
National Institute of Allergy and Infectiolls Diseases, G1'ant IN-105A from
the Arnerican Cancer Society, and the Grants-in-Aid Prog1'am for Faclllty
of ViI'ginia Commonwealth UniveI'sity.
was recipient of National Research Se1'vice Award AI 05431 and
PSM I'ecipient of Health Service Research Career Development
Award AI 70863 from the National Institute of Allergy and Infectious Dis-
eases.
REFERENCES
1. S. Mo1'ahan, W. Regelson, and Mllnson, AIltimicrob. Agents
Chemothe1'. 16 (1972).
2. F. F. Pindak, J. Schmidt, D. J. Giron, Allen, P1'oc.
Soc. Med. 138:317 (1971).
3. D. J. Giron, Allen, F. F. Pindak, and J. Schmidt, Infec.
Immun. 318 (1971).
4. J. Desmyter, and DeSomer, J. Virol. 2,:321 (1970).
5. Merigan and S. Finkelstein, Virology 35:363 (1968).
6. J. J. and DeSome1', Natllre 232: 183 (1971).
7. Claes, Billiau, DeCle1'cq, J. Desmyter, Schonne,
Vallde1'haeghe, alld DeSomer, J. Vi1'ol. 2,:313 (1970).
8. J. Leunen, J. Desmyter, and DeSomer, Appl. Microbiol. 21:203
(1971).
9. J. Richmond, Infec. Immlln. (1971).
10. R. F. Sellers, J. Hernitnan, and W. Hawkins, Res. Vet.
Sci, 13: 339 (1972).
11. DeCle1'cq and Merigan, J. Gen. Virol. 2,:359 (1969).
12. Liebe1'man, Pascale, and G. Shimonaski, P1'oc.
Soc. Med. 131:443 (1969).
:1II(1
1:3. J3illiau, J. J. Appl. Mict'oJJiol. 21:
580 (1971).
14. S. Morahan and ll. 8. McCord, J. Immunol. 115: 311 (1975).
15. R. 8. McCord, Breinig, and 8. Morahan, Antimicrob.
Agents Chemother. 10: 28 (1976).
16. S. Morahan, R. Kern, and L. Glasgow, Proc.
Biol. Med. 154: 615 (1977).
17. Breinig, L. L. Wright, McGeorge, and 8. Morahan,
Arch. Virol. 57: 25 (1978).
18. DeClercq and DeSomer, Appl. Microbiol. 16: 1314 (1968).
19. illiau , J. J. and De80mer, Infec. Immun.
(1972).
20. DeClercq and De8omer, Biol. Med. 132: 699
(1969)
21. DeClercq and De80mer, Infec. Immun . ..: 669 (1973).
22. W. Regelson, Advan. Med. Biol. 1:315 (1967).
23. Chirigos, W. J. Pearson, and W. Griffin, Int. J.
Cancer 4: 267 (1969).
24. G. 8chuller, 8. Morahan, and 8nodgrass, Cancer Res. 35:
1915 (1975).
25. 8. Morahan, G. 8chuller, J. 8nodgrass, and
J. Infec. (1976).
26. 8. Hirsch, Black, L. Wood, and Monaco,
J. Immunol. 108: 1312 (1972).
27. 8. Hirsch, Black, L. Wood, and Monaco,
J. Immunol. 111: 91 (1973).
28. DeClercq and DeSomer, J. Cancer ..:535 (1972).
29. Billiau, R. Leyten, Vandeputte, and De80mer, Life 8ci. 10:
643 (1971).
30. V. Larson, W. R. Clark, and R. Hilleman,
Biol. Med. 131: 1002 (1969).
31. G. Franchi, S. Garattini, L. J. and L. VanPutten,
J. Cancer (1973).
32. J. 8andberg and Goldin, Cancer Chemother. Rep. 55:233 (1971).
33. Ferruti and F. Danusso, J. Medicinal Chem. 16:496 (1973).
34. Smith, and Rubin, J. Reticuloendothelial
447 (1971).
35. Munson, W. Regelson, and W. R. Wooles, J. Reticuloendothelial
. ..: 623 (1969).
36. 8. Morahan, J. Munson, L. G. Baird, and
W. Regelson, Cancer Res. 34:506 (1974).
37. R. 8chu1tz, J. D. Papamatheakis, J. Luetzeler, Ruiz, and
Chirigos, Cancer Res. 37:358 (1977).
38. 8. Morahan and Int. J. Cancer 17:82 (1976).
39. 8. J. Mohr, Chirigos, F. 8. Fuhrman, and J. W.
Cancer Res. 35: 3750 (1975).
\0. \'( INS
40. [{. 1>. llarrncl, Jr. Zbar, J. Nat. Cancer Inst. 54:989 (1975).
41. J. S. l{ernington Merigan, Nature 226:361 (1970).
42. F. F. Pindak, Infec. Immun. 1.: 271 (1970).
43. D. J. Giron, J. Schmidt, R. J. and F. F. Pindak, Anti-
microb. Agents Chemother. 1.: 80 (1972).
44. J. Van Dijck, Claesen, and DeSomer, Trop. Med.
Parasitol. 64:5 (1970).
45. W. Braun, W. Regelson, Yajima, and Ishizuka, Proc. Soc.
Biol. Med. 133: 171 (1970).
46. L. G. Baird and Immunol. 20: 167 (1975).
47. Science 146:1472 (1964).
48. W. R. Stinebring and J. S. Youngner, Nature 204: 712 (1964).
49. D. S. Breslow, 1. Edwards, and N. R. Newberg, Nature 246: 160
(1973) .
50. R. Ottenbrite, Goodel1, and Munson, Polymer 18:461
(1977) .
51. Goodel1, R. Ottenbrite, and Munson, J. Reticulo-
endothelial Soc. 23: 183 (1978).
52. Merigan and W. Regelson, New Engl. J. Med. 277: 1283 (1967).
53. W. Regelson, Proc. Int. Symp. Atheroscler. Reticuloendothelial
Syst., Lake Italy (1966).
54. Merigan, Nature 214:416 (1967).
55. DeSorner, DeClercq, Billiau, Schonne, and Claesen,
J. Virol. 886 (1968).
56. DeSorner and Arch. Ges. Virusforsch. 19: 143 (1966).
57. and Breinig, Proc. Soc. Biol. Med.
119: 1227 (1965).
58. J. S. Youngner and W. R. Stinebring, Nature 208:456 (1965).
59. Breinig, Postic, and J. Armstrong, Ann. N.
Acad. Sci. 173: 680 (1970).
60. DeSorner, DeClercq, il1iau , Schonne, and Claesen,
J. Virol. 878 (1968).
61. Ito, N. Mori, and 1. Nagata, Virology 44: 638 (1971).
62. Kojima, Arch. Med. 43:35 (1970).
63. andJ. Armstrong, J. Infec. Dis. 128:212 (1973).
64. Breinig, and N. Maehara, J. Il1fec. Dis. 133:
(1976).
65. J. Smith and R. R. Wagl1er, J. Med. 125: 559 (1967).
66. S. Finkelstein, G. Bausek, and Merigan, Science 161:
465 (1968).
67. N. MaeharaandM. Infec. Immun. 15:78 (1977).
68. S. Kobayashi, Yasui, and Masuzumi, Proc. Soc.
Med. 131:487 (1969).
69. J. Leavitt, Merigan, and J. Freeman, Amer. J. Dis.
Child. 121: 43 (1971).
2GO IIII.I';INI<: ItII(1
70. S. Morahan, D. W. 13arnes, and
Repts. 62: 1797 (1978).
71. Munson, W. Regelson, and J. Munson, J. Toxicol. Appl.
Pharmacol. 22: 299 (1972).
72. Kaplan, S. Morahan, and W. Regelson, J. Nat. Cancer
Inst. 52: 1919 (1974).
73. Munson, W. Regelson, W. Lawrence, Jr., and W. R. Wooles,
J. Reticuloendothelial Soc. 1: 375 (1970).
74. S. Morahan, Munson, W. Regelson, S. L. Commerford,
and L. D. Hamilton, Proc. Nat. Acad. Sci. U.S. 69: 842 (1972).
75. Diamantstein, Wagner, 1. Beyse, V. Odenwald, and
Schulz, Eur. J. Immunol.1.:335 (1971).
76. Diamantstein, Wagner, V. Odenwald, and Schulz,
Eur. J. Immunol. 1.:426 (1971).
77. R. Cone and Johnson, Immunol. (1972).
78. R. Schultz, W. Woods, S. J. Mohr, and Chirigos,
Cancer Res. 36: 1641 (1976).
79. S. J. Mohr, Chirigos, F. Fuhrman, and Smith, Cancer
Res. 36: 1315 (1976).
80. L. Kripke and Borsos, J. Nat. Cancer Inst. 53: 1409 (1974).
81. F. Gazdar, D. Steinberg, F. Spahn, and S. Baron, Proc.
Soc. Med. 139: 1132 (1972).
82. L. BairdandA. Immunol. 28:36 (1977).
83. L. and W. Song, Radiat. Res. 70: 688 (1977).
84. S. J. Mohr, Chirigos, Smith, and F. S. Fuhrman,
Cancer Res. 36: 2035 (1976).
85. J. Snodgrass, S. Morahan, and Kaplan, J. Nat. Cancer
Inst. 55: 455 (1975).
86. S. Morahan and J. Reticuloendothelial Soc.
(1976) .
87. S. Morahan and in Progress in Cancer Research
and Therapy Chirigos, ed.), Vol. 2, Raven Press, New York,
1977, 499.
88. Scott, J. Nat. Cancer Inst. 53: 861 (1974).
89. Schuller and S. Morahan, Cancer Res. 37:4064 (1977).
90. W. Regelson, Advan. Chemother. 303 (1968).
91. S. Gianinazzi and J. Gen. Virol. 23: 1 (1974).
92. D. S. Breslowand Chadwick, U.S. Patent 3,996,347 (1976).
93. S. Papas, W. Pry, and Chirigos, Proc. Nat. Acad. Sci.
U.S. 71: 367 (1974).
94. Marx, Jr. and F. Wheelock, Abstr. Ann. Meeting, Amer.
Soc. Microbiol. 240 (1976).
95. S. Morahan, L. Glasgow, J. L. Crane, Jr., and R. Kern,
Immunol. 28:404 (1977).
10. 2(;1
%. J. 11. Arch. Virusforsch. 36:
:::32 (197:::).
97. N. Wobnark and Fisher, Cancer Res. 34:2869 (1974).
98. 8. 8tarr, Visintine, Tomeh, and J. Nahmias,
Med. 152:57 (1976).
99. W. Fischer, J. Podgore, J. W. J. L. and
Kobayashi, J. lnfec. 132:578 (1975).
100. R. in The Phagocytic and Host Resistance (J.
and D. Dayton, eds.), Raven Press, New York, 1975,
309.
101. Campbell and J. Richmond, Infec. Immun. 1: 199 (1973).
102. R. illeman , Cancer Res. 36: 857 (1976).
103. W. Regelson, 1. 8hnider, J. Colsky, Olson, J. F. Holland,
L. Johnston, and L. Dennis, in Progress in Cancer Research
and Therapy Chirigos, ed.), Vol. 7, Raven Press, New York,
1978, 469.
11
INTERFERON AND INTERFERON INDUCERS:
IMMUNE MODULATION
Howard Johnson
University of Texas Medical Branch
Galveston, Texas
1. 1NTRODUCTlON
The Interferon System
The Immune System
STUDIES WITH INTERFERON
In Vivo Systems
In Vitro Systems
STUDIES INTERFERON INDUCERS
In Vivo Systems
Vitro Systems
IV. INTERFERON AND CELL-MEDIATED IMMUNITY
V. INTERFERON AND GRAFT-VS.-HOST (GVR)
REACTlON
V1. OF CYCL1C IMMUNE
INTERFERON, AND IMMUNE RESPONSE
VII. OF INTERFERONS OTHER
MEDIATORS OF IMMUNE FUNCTlON
MODEL
REFERENCES
1.
The Interferon System
263
263
268
269
269
270
275
275
276
281
281
282
288
288
290
Interferon was first recognized as antiviral system some two decades
ago when Alick Isaacs and Jean Lindenmann observed that virus-infected
produced protein substance of rendering surrounding
263
2
-!
I
I
I
I IF
I
,

D
IF
,
-----'

,/
----
,
,
FIGURE 1. Cellu1ar events of the induction and action of interferon (IF).
Virus in contact with the (1) and penetrates the membrane.
The virus then re1eases its genetic materia1, and replication of the virus
occurs (2). The new virus 1eaves the (3), enters the fluid around the
first and of the replicated virus infects second (4), where
the re1ease of the genetic material again takes p1ace (5). During the ear1y
stages of infection of the first event (vira1 nuc1eic acid?) stimu-
1ates gene in the DNA which contains the stored genetic information for
interferon leads to the production of RNA (mRNA)
for interferon, which 1eaves the nucleus and is trans1ated the
ribosomes into the interferon protein. Several events now occur more
1ess simu1taneously. Interferon is secreted the first infected
(D), enters the surrounding fluid, where it into contact with and
stimulates the second interacting with membrane receptor for
interferon. The second is thereby induced to produce new RNA (F),
which is trans1ated to new protein(s) (G), the antivira1 protein (AVP).
This in turn modifies the s protein-synthesizing machinery such that
mRNA is translated into protein, but viral RNA is poor1y bound or
trans1ated, or both. the first processes F, and G in
instances, a1so operate to form AVP and thereby reduce the virus yield in
the first Shortlyafter interferon is synthesized into the first
another mRNA is believed to synthesized from the s DNA which
is trans1ated (1) into regulatory protein (RP) (hypotheSized). This regu1a-
tory protein with the mRNA for interferon, thereby preventing
the further synthesis of more interferon (J). It has not determined
whether regu1ation of the AVP invo1ves mechanisms in addition to the extra-
concentration of interferon. From Ref. [Reprinted with
permission from Johnson and S. Baron, Pharmacol. Ther . .1 (1977),
Pergamon Press, Ltd.]
11. IIVIIVIIINI';
to viral 1957, governing this
antiviral interferon systern have unveiled in laboratories around the
world [1]. Evidence indicates that interferon produced during virus infec-
tion is of the rnore important body defenses against these infections.
Figure 1 depicts the function of interferon Fundamentally, the inter-
feron system consists of two parts: (1) production of the interferon the
virus-infected and (2) reaction of the secreted interferon with surround-
ing to induce intracellular antiviral protein(s) that inhibit virus multi-
plication within the This mechanism of controlling viral infections
suggests that interferon have other natural cell-regulatory functions.
We present data that indicate that interferon play important role
in regulating the response in doses that occur naturally.
Based recent findings, the human and interferon systems
could provisionally classified into two groups. These are virus-type
interferons [2-4] and immune interferons [5-10] 1). Virus-type
interferons are classicaHy induced viruses or synthetic polyribonucleo-
tides, while immune interferons are induced in primed lymphocytes
antigen or in unprimed lymphocytes mitogens (usually).
Virus-type interferons are heterogenous, and at least two antigenically
distinct types exist [11,12]. They are caHed fibroblast interferon and
leukocyte interferon, indicating their cellular source. Immune interferon
is antigenicaHy distinct from the virus-type interferons [13,14]. Antibodies
to mitogen-induced immune interferon neutralized both mitogen-induced and
antigen-induced interferons to the extent , so immune interferons
induced under various conditions appear to antigenically the same or
similar.
1. Provisional Classification of Interferon
Virus-type (type 1)
1. Antigenic types
Fibroblast origin
Leukocyte origin
2. Inducers
Virus
Polyribonucleotide
Chemicals (tilorone, etc.)
Immune-type (type II)
1. Antigenic types-only type known to date
2. Inducers
Antigen
Mitogens (primarily mitogens)
STEM CELL (From Morrow)
1/i)\2
I
I THYMUS I

LYMPHOCYTE
/ \
4
INTERACTION BETWEEN CELLS AND
ANTIGEN IN PERIPHERAL LYMPHOID
SSUE (HELPER OR SUPPRESSOR
FUNCTION)
KILLER, HELPER, AND 8'F:J 8E=t
SUPPRESSOR L YMPHOCYTES ..
(8,
Y-L
'{ /.;..
( 8)
t



LYMPHOCYTE
(.) PLASMA
CELL
t
'(
SECRETE)

..:..\-
t<:
;:;
z

Z
FIGURE 2. Simplified scheme for the origin and function of the cells involved in the i u n response. The bone
marrow contains stem cells that are destined to and lymphocytes. When the stem migrates (1) to
an organ under the breast bone called the thymus, it undergoes differentiation to lymphocyte and becomes the
effector for the various aspects of cellular immunity. The lymphocyte leaves the thymus and migrates to
the lymph nodes and spleen, where it is of responding to antigen. Another stem from the bone mar-
row migrates (2), to an organ in the lower gut of the chicken called the bursa and in humans probably to the
narrow, liver, and spleen, where it undergoes differentiation and becomes the effector l l for the production of
antibody. The differentiated lymphocyte migrates from the bursa or its equivalent to the lymph nodes and
spleen, where, like the lymphocyte, it is of responding to antigen recognition. Both and lympho-
cytes have specific receptors their surfaces for antigen recognition and subsequent response. Upon exposure
to antigen, the lymphocyte(s) is , with the aid of phagocytic called the macrophage, of being
activated (3) to differentiate and to expand in number. The lymphocyte interact with the antigen directly and
inactivate or k the antigen. It is also , along with the macrophage, of interacting (4) with the lympho-
cyte through its helper and suppressor function, either enhancing the differentiation of the lymphocyte to
plasma (5), which is responsible for antibody production, or suppressing the lymphocyte from differen-
tiating into plasma . Soluble mediators have been obtained from lymphocytes, some of which are
of mediating the helper and suppressor activities. There is evidence that interferon is of mediating the
suppressor activity of lymphocytes.
:5:
:5:
z
:5:
=
>:
-:
z
t,;
,!< )11 NS()N
Immul1e System
FUl1ctiol1ally, two major types of lymphocytes are il1volved in immul1c
respol1se, the humoral and the cellular mode of respol1se to
antigenic stimulation (Figure 2). Both types of lymphocytes originate
stem in the marrow, but they take differel1t paths to maturity.
From both the historical al1d anatomical poil1ts of view, the paths of differ-
entiatiol1 are most clearly il1 the chickel1.
One group of migrates to organ knoWl1 the bursa of Fabricius
il1 chickel1S. of this orgal1 in mammals is 110t known, but
bursal ful1ctiol1s to reside il1 the fetal liver, spleel1, al1d marrow.
After arriving in the bursa of chickel1s, or its equivalent il1 mammals, stem
differentiate into bursal lymphocytes. differel1tiated lympho-
cytes leave the bursa, or its equivalent il1 mammals, al1d seed regiol1al
lymph 110des al1d the spleel1. result of genetic codil1g, these
certail1 immul1og10bulil1s their surfaces. surface al1tibody serves
the receptor for al1tigel1, al1d after reactiol1 with its specific al1tigel1
the ul1dergoes clol1al expal1siol1 al1d differel1tiation il1to al1tibody-
secretil1g known plasma
The lymphocyte that is responsible for cellular immul1ity (delayed-type
hypersel1sitivity) is called the thymic lymphocyte, which develops al1d
differel1tiates in the thymus from marrow precursor
differentiated lymphocytes leave the thymus al1d, like lymphocytes, seed
regiol1allymph 110des al1d the spleel1. lymphocytes are the most l1umerous
lymphocytes il1 the circulatil1g lymphocyte pool. The T-lymphocyte receptor
for al1tigel1 does 110t appear to like that of the
receptor, but its nature is ul1known. Whel1 the is stimulated inter-
actiol1 of its receptors with al1tigel1, it reacts il1 several ways. It performs
"helper" il1 that it-or substance or substal1ces that it
releases-interacts with to tral1sform il1to al1tibody-
secreting plasma Also, stimulatiol1 of al1tigel1s results
in the developmel1t of cytotoxic or "killer" ceHs, which carry out the
cellular immul1e respol1ses. well-known example is the delayed hyper-
sel1sitivity to mycobacteria). The also appears to have the additiol1al
property of "suppressor" activity, which is the opposite of the helper ful1c-
and is due primarily to al1tigel1 stimulatiol1. It has the effect of inhibitil1g
both al1tibody respcnse activity) and forms of ceHular immul1ity
ful1ctions). Together, the helper al1d suppressor
of are thought to play major role il1 the re-
These regulatory activities are thought, il1 cases,
to mediated soluble factors called lymphokil1es [15]. Evidel1ce
presel1ted il1dicating that il1terferol1s are cal1didates for mediatiol1 of
some forms of suppressor activities.
11. IIVIIVIIJNI,;
WITH
In Vivo Systems
Moderate and high doses of virus-type interferon have reported to
the plaque-forming (PFC) to sheep red-blood
(SRBC) in mice [16-18]. Plaque-forming that produce anti-
body against red against antigen coated to red
action of antibody with red in the of complement results in
plaque hole in the lawn of red When mice were injected with
1.5 105 U of interferon 2 days prior to antigen injection, the
PFC response was suppressed >80% when assayed 6 days after SRBC
injection [17]. immunosuppressive effect of virus-type interferon
was effective against low doses of antigen, and both IgM and IgG
antibody synthesis were affected [18] .
antibody of mice to SalmoneHa typhimurium lipopoly-
saccharide (LPS), thymus-independent antigen, was also significantly
inhibited virus-type interferon preparations [19]. removing the
adherent ceHs from spleen ceHs of mice treated with interferon and stimu-
lation of the nonadherent LPS in vitro, data were obtained that
suggested that interferon act directly lymphocytes. The immuno-
suppression effects of the interferon preparations used for the studies
were shown several criteria to due to interferon.
Recent1y, at 200 U/ml was showl1 to significantly
inhibit heterologous adoptive cutaneous anaphylaxis [20]. Briefly,
mice were injected with antigen in that elicited antibody of the
IgE class. Seven to ten days prior to removal of the responding lymphoid
the mice were boosted with the priming antigen. After removal
from the mice, the antigen-sensitized lymphoid were treated with
interferon and injected intradermaHy into Wistar rats. Twenty-four hours
later the site of il1jection was c]lallenged with the specific al1tigen for cutane-
ous anaphylaxis. The findings would suggest that virus-il1duced interferon
was of suppressing IgE al1tibody productiol1 plasma ceHs. The
stage of differentiation of the treated ceHs, however, is unknown.
could ceHs from the mice that have subsequently undergone
"activation" the xenogeneic conditions after injection in the rat.
Related to the above studies IgE al1tibody production is the recent
finding that interferon and viral al1d 110nviral inducers of interferon
of enhancing antigen al1tibody release of histamine
from human leukocytes [21]. Thus, new biological role for interferon
that is related to immune functiol1 has discovered. This finding has
clinical potential in virus-induced upper-respiratory aHergic reactions.
Very little information is available the effect of interferon
the in vivo antibody response. study employed mice sensitized with
)11
the attenuated vaccillc wiLl1 olLl
culin 1'esults in hig"ll lcvcls
When these we1'e with SHBC at thc of i1ig11 Icvcl:-;
of immune inte1'fe1'on, significant suppression of the splenic
was obse1'ved. FU1'the1', the supp1'ession of the anti-SHBC 1'esponse was
1'elated to the se1'um concent1'ation of immune inte1'fe1'on. Although sugges-
tive of supp1'essive 1'ole of immune inte1'feron in the in vivo antibody
1'esponse, studies of this type need to monito1' the p1'esence and
function of othe1' mediato1's 1'eleased into the the with

In Vitro Systems
Most of the info1'mation the of interfe1'on in antibody formation
been obtained in vit1'o systems. The in vivo studies 1'equi1'e la1'ge
amounts of inte1'fe1'on, and the and kinetics of administe1'ed
inte1'fe1'on in the mic1'oenvironment of the a1'e un-
known. In one se1'ies of in vit1'o studies app1'oximately 3000 U of vi1'us-
type inte1'fe1'on/ml we1'e 1'equi1'ed to Significantly supp1'ess the in
vit1'o PFC 1'esponse of BALB/c spleen to SRBC [23]. The
effect was when the cultu1'es we1'e p1'et1'eated with inte1'fe1'on fo1' 6 h1',
01' when inte1'fe1'on was added up to 40 hr afte1' addition of SRBC. The
factor 1'esponsible fo1' the could not quantitatively dissociated
the antivi1'al activity of inte1'fe1'on either in te1'ms of the specific
activity of the inte1'fe1'on 01' standa1'd physiocochemical such
t1'eatment of inte1'fe1'on with t1'ypsin, l' iodate, RNase, and DNase. T1'ypsin
and pe1'iodate t1'eatment, which dest1'oyed the inte1'fe1'on, also dest1'oyed
the PFC inhibito1'Y effect of the inte1'fe1'on p1'epa1'ations. the use of
sepa1'ated populations, it was claimed that inte1'fe1'on acted di1'ectly
and had effect macrophages 01' helpe1'
effect. latte1' obse1'vation 1'ega1'ding helpe1' effect is significant,
since it is the only function 1'epo1'ted not to affected inte1'fe1'on
and the1'efo1'e needs to investigated fu1'the1'. The in vit1'o
study 1'equi1'ed lesse1' of inte1'fe1'on fo1' PFC inhibition than did the
in vivo studies, but the inhibito1'Y concent1'ations we1'e considerably highe1'
than the physiological no1'mally obtained stimulation of
cultu1'es with inte1'fe1'on induce1's [1].
Othe1's [24,25] have demonst1'ated with mouse spleen
that 20-60 U of vi1'us-type inte1'fe1'on f1'om va1'ious sou1'ces and specific
activities inhibited the in vit1'o PFC 1'esponse to SRBC 90% 01' m01'e.
An example of such inhibition is shown in the inhibition data with
pu1'ified mouse vi1'us-type ascites tumo1' inte1'fe1'on p1'esented in Figu1'e 3.
Inte1'fe1'on, 20 U/cultu1'e, inhibited the PFC 1'esponse 92%. in-
hibitory concent1'ations of pu1'ified inte1'fe1'on did not affect viable
11. IIVII\IIINI';


0.0002 0.002 0.02 0.2 2.0
INTERFERON (UNITS/CULTURE)
20.0
w
107

:::1
U
......

w

w
>

106

(/)
...1
...1
W
U
w
...1
m
ct
:>

FIGURE 3. The effect of highly purified mouse ascites tumor virus-type
interferon (3.2 108 reference units/mg protein; Lengyel, Yale)
the primary in vitro PFC Direct anti-SRBC PFC/culture
and viable recovered culture (.-.) determined
day 5. From Ref. 25. [Reprinted with permission from Johnson,
G. Smith, and S. J. Immunol. 114:403 (1975), &
Wilkins Baltimore.]
recovery. Although the above studies invol ved interferon preparations of
different potencies and specific activities, the interferons inhibited the
PFC in proportion to their antiviral activities. In addition, both
the antivil'al activity and the PFC inhibitory activity of the interferons were
neutralized antibody specific for mouse interferon. Both activities were
partially completely inactivated heating at 600 for 1 hr. Human
interferons at the concentrations used had J1either antiviral activity
,J()IINS()N
PE'C-inhibitory activity in Limitcu (1 0[' ccll"
to interferons significantly inhibits both viral inl'cctio11 a.llu 1110 l' l<'C
sponse. Both the antiviral activity and the inhibitory acti 01" thc
interferon preparations are acid stable. It was c011cluded, there1"orc, that
the inhitition of the primary in vitro PFC response was due to interfer011
in the preparations [25].
Kinetic data showed that the greater the concentration of interferon
added to the cultures, the earlier the effect the PFC response [25].
Also, the presence of interferon in the culture for the first 4 hr was suffi-
cient to inhibit the PFC response. I11terfero11, the11, appeared to affect
some early events which lead to inhibition of the PFC response.
events do not appear necessarily to i11volve a11tige11 "processing" macro-
phages or induction of lymphocytes "processed" antigen, si11ce kinetic
data showed that the could induced SRBC to produce PFC 011
days 3 and 4 in studies involving low dosages of interferon (50 U/ culture).
effect of virus-type interferon the in vitro PFC response to
thymus- and macrophage-independe11t antigen has studied [26]. It
required about twice as much interferon (100-200 U) to inhibit the PFC
response to Escherichia 0127 LPS (thymus-i11depende11t) as it did to
inhibit the anti-SRBC response. the of splee11 from nude
(athymic) mice and spleen cells depleted of macrophages, it was shoW!l
that lymphocytes and macrophages were 110t required for i11terfero11 to
exert its inhibitory effect lymphocytes. Si11ce it is impossible to
remove macrophages completely from the cu1tures, possible role for
residual macrophages must considered.
nature of the cel1ular events in virus-type i11terfero11 inhibition
of the in vitro PFC response has recently further clarified [27].
findings suggest that interferon appears to affect only "nonactivated" (un-
committed) precursors preventing them from becoming activated.
Early responding precursor cells, which are already committed to the
cycle, are refractory to interferon-i11duced suppression of the in vitro
response. Thus, clonal expansion of these refractory precursors
is not affected late in the immune response and not at the peak of the
response. These findings are in agreement with preliminary observations
the effect of virus-type interferon immunoglobulin production
mouse plasmacytomas, where the early production (first 4 or
5 days in culture) of immunoglobulin was not suppressed interferon
Johnson, Blalock, and S. Baron, unpublished data).
From the foregoing studies the general picture emerges of population
of precursor lymphocytes that are of responding to given antigen
(SRBC) . The cel1s are at various stages of differentiation, which in turn
determines their relative abilities to differentiate into antibody-producing
plasma in the presence of various concentrations of virus-type (and
perhaps immune) interferon. The lesser differentiated precursor cells
are more susceptible than are the highly differentiated precursor cells to
11. IIVII\II/NI';
illlJi/Jiiiotl /JY illtcrferOll. Precursor 13 arc differell-
tiatcd llormal clollal expansion for Hmited time in the presence
interferon.
Virus-type interferon inhibltion of the primary in vitro PFC response
in mice involves dynamic relationship between the nature the
antigen, the concentration of interferon added to antigen-stimulated cultures,
and the time of addition of interferon relative to antigen addition [14]. The
PFC response to thymus-dependent antigen, SRBC, was more
suppressed interferon than was that to thymus-independent antigen,
0127 LPS, both in terms of inhibltory concentrations of interferon
and the time at which the interferon could added to cultures after antigen
and inhiblt the PFC response. The anti-SRBC response was effectively
inhibited 150 U of interferon when the interferon was added to mouse
spleen cu1tures to 16 hr (87% inhibition) after SRBC. At 500 U of inter-
feron, effective inhibition (76%) was obtained when the interferon was added
to 24 hr after In the anti-E. 0127 response, 150 U of inter-
feron effectively inhibited the PFC response ollly when added at the time of
antigen, and not 8 hr later. With 500 U of interferon, effective inhibition
(70%) was obtained when interferon was added to cultures to 16 hr after
0127. The anti-SRBC, PFC response was inhibited more extensively
both 150 and 500 U of interferon than was the anti-E. 0127 response
when interferon was added to cu1tures at either 8, 16, or 24 hr after antigen.
Evidence has been presented that suggests that splenic memory lympho-
cytes represent population that differs quaHtatively from that of
unstimulated spleen cells [28]. Virus-type interferon has been shown to
effective in inhibiting (91% with 120 U of interferon) the generation of
PFC from this memory pool. data do suggest that the memory
cells are slightly more resistant to interferon inhibition than are virgin
lymphocytes. Both virgin and memory lymphocyte populations, then, are
inhibited virus-type interferon in the in vitro PFC response. This is
in agreement with the inhibitory effects of interferon the secondary anti-
body response in in vivo studies [18]. It is of interest that the in vitro
PFC inhibition of memory lymphocytes was obtained with physiological
concentrations of interferon.
Both the in vivo and in vitro studies cited present data that
suggest that certain conditions interferon also exert
enhancing effect the antibody response in addition to its suppressive
effects. Injections of interferon into mice or additions to mouse spleen
cultures 2 to 3 days after antigen have been shown to sHghtly enhance tlle
antibody response [19,25]. Similarly, suboptimal immune responses in
vitro have elevated to optimallevels interferon [23]. Some en-
hancement has also been noted with low concentrations of interferon [16] .
enhancement data are not as impressive as the data showing the
immunosuppressive effects of interferon. Some of the in vitro studies
[25] reported have recently been confirmed [29].
,1< >11 NS< >N
In related in viteo study l'cpoet
vieus-type and immune crude inter.fol'On pecparatioJls
migration inhibitory properties. Although interesting, this study must
repeated with more interferon preparations in order to more
meaningful. Highly virus-type interferon is currently
for such studies. The study does point the need for pueification of
immune interferon.
Very little information is the immunosuppressive property
of immune interferon. This is primarily due to the lack of immune
interferon for study. One study used sera that were obtained from BCG-
sensitized mice that were with as the source of immune
interferon [22]. The immune-interferon-containing sera suppressed the
in vitro PFC response to SRBC when compared to corresponding concen-
trations of mouse sera that did not contain interferon. The immunosuppres-
sive factor(s) was found primarily in the lower-molecular-weight fraction
(40,000) from Sephadex G-100 column, with minor suppressive activity
in the 90,000 fraction. This corresponded to the distribution of the antiviral
activity from the column. Other biological activities, such as macrophage
migration inhititory factor and lymphotoxin, have been found at these same
or similar molecular weights [31,32]. Since data were provided
these other biological activities, it is not possible to attribute
the immunosuppressive property of the sera to immune interferon. It has
been suggested that immune interferon possess several biological
activities in to its antiviral property, such as macrophage migration
inhibitoryand soluble immune response suppressor properties [10].
recent separation of mouse immune interferon and macrophage migration
inhititory activities, however, would suggest that some lymphocyte mediator
activities are due to distinct molecules [33 J .
It is possible to differentiate the immunosuppressive and antiviral
effects of virus-type interferon. Suppression of the in vitro antibody re-
sponse of mouse spleen to SRBC was blocked 5 10-5
2-mercaptoethanol [34]. The blockade was not due to direct effect
interferon, since was of blocking the suppression when
added to cu1tures after interferon had established the immunosuppressive
state (48 hr after interferon). Similar protective effects of were
observed during immunosuppression virus-type interferon inducers,
but not mitogen inducers of interferon (immune interferon). Virus-
type interferon inhitited DNA synthesis in unstimulated spleen cultures
and in stimulated cultures, and the degree of inhibition of DNA synthe-
sis appeared to t'elated to the immunosuppressive property of interferon
in the absence or presence of did not affect the antiviral
properties of either virus-type or immune interferon in nonlymphoid
Further, the induction of virus-type interferon in spleen was neither
inhibited nor enhanced while the induction of immune interferon
was enhanced. enhancement was consistent with enhancement
of the immunosuppressive effects of immune interferon inducers.
11. IIVIIVIIINI':
aru two possibllitius [or [)loukade
sive virus-type while the property.
Icirst, the properties of virus-type
at the subcellular level.
the selectivity of the blockade cOHld due to the fact that
cells are the target cells in while L cells are the
target cells of virus Thus, virus-type
suppress the at the level of the macrophage,
reverse this effect blocked macrophage
Related to this is the that virus-type
are of macrophages as their
effects [35,36]. Activated macrophages, as result of
appropriate have very motile cytoplasmic processes show
well-developed They are of more active
phagocytosis the activated state the macrophage have
suppressive effect the thus
suppressor [37]. Thus virus-type affect several
types the
STUDIES WITB INTERFERON
INDUCERS
Vivo Systems
of as illustrated 1, classified the
basis of ,vhether they cells to produce virus-type or
of both types of have studied vivo
systems. Virus-type have reported to have both
suppressive effects the vivo.
thetic, virus-type
are well as or of the vivo
[38-40]. This has ascribed to the effect of these poly-
the lymphocytes [41]. Less is the fact
that also the vivo
[40,41]. There is temporal
ship and suppression of the
mice are with polyribouridylate
(poly r poly rU) at various times to of
gamma [40]. Poly rA:poly rU, day to 12 hr
before suppressed the response.
2 hr before, or at the same time as of
the was observed.
The T-lymphocyte
which were ShOWl1 earlier to of lymphoid

,1( 1 NS()N
cu1tures, of il1 111 icc I ,1:\ I
and rabbits [44] if the mitogen::> injcctcd to Lwo day::> ltllLil4cl1.
It was found in the rabbit that injection of Con PlIA at tirnc of
antigen had an enhancing effect the antibody response [44].
1n Vitro 8ystems
1n vitro antibody induction studies have shown that rA:po1y rU havc
both modest enhancing or inhibiting effect the immune response, depend-
ing the po1yribonuc1eotide concentration and the 1ength of the cu1ture
period [45-47]. We have obtained consistent suppression of the in vitro
antibody PFC response with po1yribonuc1eotides. r rU and
po1yriboinosinate: po1yribocytidy1ate r1: rC), at 0.1-1.
inhibited the anti-8RBC PFC response (Figure 4) in sp1een cu1tures
>90% when the po1yribonuc1eotides were added to cu1tures a10ng with antigen
[48]. Functiona1 1ymphocytes were required in the cu1tures for the
ribonuc1eotides to effective as inhibitors, thus demonstrating the thymus
105
105


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104
104


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103 10
7
1iJ 103 107 1iJ

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(f)


u
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1iJ
LI.I
....J
....J

LI.I

LI.I

u
u

102 106
102

0.01 0.1 1.0 10.0 100.0
POLY rA:POLY rU (/lg/CULTURE)
ID
<t
:>
0.0001 0.001 0.01 0.1 1.0 10.0
POLY rl: POLY rC (pg/CULTURE)
FlGURE 4. The effect of r rU and r1: rC
the primary in vitro PFC response to 8RBC in sp1een
cu1tures. Po1yribonuc1eotides were added at the time of 8RBC

:>
and direct anti-8RBC PFC/cu1ture and viable ceHs recovered/cu1ture
were determined day 5. From Ref. 48. [Reprinted with permis-
sion from Johnson, J. Bukovic, and G. 8mith, Proc.
Bio1. Med. 149: 599 (1975).]
10
4
f
w 104
W



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::::>
w u w
u
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"'-
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103 107
103 107
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w

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u
u

w

w
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1- (/) 1- (/)
102
106 ...j U 102
106 j
...j w

w w
iS
u

u


w

w
...j

...j
CI) CI)

:>
11
:>
0"0.0001 0.0010.01 0.1 1.0 10.0 00.00010.0010.01 0.1 1.0 10_0
POLY rA: POLY rU (,JJg/CULTUREJ POLY rA:POLY rU (,JJg/CULTUREJ
FIGURE 5. effect of poly rA:poly rU the primary in vitro PFC response to 0127 LPS
in and nude (athymic) spleen cultures. The polyribonucleotide was added at
the time antigen, and direct anti-E. 0127 LPS PFC/culture and viable
recovered/culture were determined day 5. From Ref. 48. [Reprinted with permission
from Johnson, J. Bukovic, G. Smith, Proc. Biol. Med. 149:599 (1975).]
$
$
=
z
--
$
-


-
>
-
z

-:
-:
,1( )11 NS<)N
2. Neutralizatioll Il1Lcl'[cl'()))
Inhibitory Effect of Poly r Poly rU ill vitro 1'1<'C
Spleen CeHs to
Percentage 01"
PFC/culture
Poly poly rUa SD relative to
Antiserum, dilution
a
(2 for duplicates) control
None 26,750.:!. 1,061
None + 1,075.::!: 742 96
Anti- interferon, 1: 100 12,025.::!: 177
Anti- 1: 100 + 16,650.::!: 530 -29
Anti- interferon, 1:1000 21,550.::!: 636
Anti - interferon, 1:1000 + 18,325 .::!: 2,298 15
NRS, 1: 100 12,925 .::!: 1,237
NRS, 1: 100 + 2,400.::!: 283 81
aAntisera and poly rA: poly rU were added to cu1tures at the same time.
Antisera dilutions represent final cOllcentrations after addition to cultures.
bNormal rabbit serum (NRS) is pool of four normal rabbit sera.
Source: Reprinted with permission from Ref. 14.
dependellce of the This is illustrated ill Figure 5, where
poly poly rU was used to inhibit the PFC response to the T-independent
antigen 0127 LPS in mouse spleen cu1tures and in nude
(athymic) spleen cultures. At 10 IJg per cu1ture, poly rA:poly rU
inhibited the response in cultures 84%, while having effect
in the nude spleen cultures. The cu1tures contained functional
ceHs but lacked functional ceHs. to 100 times more po1yribonuc1eo-
tide was required in order to inhibit the in vitro PFC response to the
independent antigen 0127 LPS than was required for the T-dependent
SRBC antigell. This latter observation is further evidellce of differentia1
effect of interferon and interferon inducers the in vitro PFC response
to T-dependent and T-independent antigen. The in vitro PFC response
to SRBC is macrophage-dependent, while the response to 0127 LPS
is 1ess dependent macrophages [49]. findings, thus, are
consistent with the earlier data, which suggested that interferon
suppressed the PFC response blockade of macrophage function.
11. 11\l11\l1I1NI';
lJala ill show tllC inl1ibitory offoct poly r poly rU
the in vitro rcsponsc to was neutralizcd antibody to virus-type
interferon [14]. The effect was observed with coli LPS
antigen. The antiviral property of interferon stimulated in spleen cultures
polyribonucleotidc is also neutralized thc antibody to interferon.
The findings suggest that the in vitro immunosuppressive effect of
double-stranded polyribonucleotides is due to their early stimulation of
virus-type interferon production lymphocytes.
mitogens are potent inhibitors of the in vitro PFC response
[50-53]. They also induco lymphocytes to produce immune interferon
[5,8-10]. These lymphocytes have been shown to possess histamine
receptors [54]. In an attempt to obtain some insight into the possible role
immune interferon in the mediation of suppressor effects, the
mitogens and SEA were compared for their
to inhibit the in vitro antibody response and to stimulate the production of
interferon in mouse spleen cultures (Figures 6 and 7) [10].
w

::>

::>
u
"-
u
1.1-

r-
u
w



10'
00.001 0.01 100.0
MITOGEN (!'g/ml)
FIGURE 6. The suppressive effect of various T-lymphocyte mitogens
the primary in vitro PFC response to Mitogens were added at the
time of SRBC and direct anti-8RBC PFC/culture were determined
day 5. PFC responses are representative of three experiments. From
Ref. 10. SEA, [Reprinted with
permission from Johnson, G. J. Stanton, and Baron, Proc.
Biol. Med. 154: 138 (1977).]

.....
z





u)
t:



MITOGEN
,1< 111 NS< IN
FIGURE 7. Stimulation of the production of mitogen-type interferon in
mouse spleen cultures various T-lymphocyte mitogens.
Spleen cells and mitogens were incubated for 48 hr under conditions as
described for the PFC response. Interferon concentrations are expressed
as the mean of duplicate determinations . SD. The SD (not plotted) for
0.001 SEA is 146. The responses are representative of three experi-
ments. From Ref. 10. SEA, Con
[Reprinted with permission from Johnson, G. J. Stanton, and
S. Baron, Proc. Soc. Med. 154:138 (1977).]
It was found that the to inhibit the PFC response to SRBC was pro-
portional to the of these mitogens to induce interferon in the cu1tures.
Staphylococcal enterotoxin (SEA), simple protein toxin of molecular
weight approximately 28,500 that is produced Staphylococcus aureus,
was the most effective inhibitor of the PFC response (Figure 6) and the
best inducer of immune interferon (Figure 7), followed Con with
being the least effective. The data are supportive of previous
studies suggesting role for immune interferon in regulation of the immune
response via suppressor cells [14]. data also suggest that SEA
would the most suitable inducer of immune interferon in quantity as
prerequisite to purification and characterization of the molecule.
Studies in humans, using peripheral lymphocytes, also suggest that
SEA is more suitable than Con and for induction of immune
interferon .
11. IIVIIVIlJNI'; IVIOIHJ
IV. AND
IMMUNITY
Along with its inhibitory effects antibody production, virus-type inter-
feron has also been shown to inhibit cellular immune responses or delayed-
type hypersensitivities. Interferon (2500 U/ml) was shown to inhibit DNA
synthesis of PHA-stimulated and mixed lymphocyte-stimulated mouse spleen
suspensions [55]. It is well known that these effects involve the or
thymus-derived lymphocytes. s imilar inhibitory effect has been noted
Con A-stimulated lymphocytes [56]. Interferon also inhibited the cellular
immune response of mice to allografts [57-59]. Mice with contact sensitivity
to picryl chloride and delayed-type hypersensitivity to SRBC were suppressed
3.6 105 and 2.1 106 U/ml of interferon, respectively, when the inter-
feron was given just prior to challenge with the specific antigens [60].
Sensitization with SRBC was also blocked virus-type interferon and in-
ducers of virus-type interferon [61]. function, both afferent and
efferent, then, is also affected interferon.
Recent1y, partially purified human interferon was found to inhibit the
response of human lymphocytes in the mixed lymphocyte reaction, while at
the same enhancing the activity of these cells [62]. The mecha-
nisms involved are not known, but the findings are consistent with data that
indicate that interferon increases the surface density of some membrane
antigens [63].
Both human leukocyte and fibroblast interferons were found to suppress
mitogen stimulation of DNA synthesis in peripherallymphocytes [64]. Inter-
feron had to continuously present in order to suppress DNA synthesis.
The induction of the antiviral state and the immunosuppressive state in the
in vitro PFC response do not require the continuous presence of virus-type
interferon once these states have been induced [25]. This due to
the fact that interferon is much more effective in inducing the antiviral and
immunosuppressive states than in blocking DNA synthesis.
V. INTERFERON AND GRAFT-VS.-HOST
(G REACTION
Poly rI: poly rC was employed in studies with mice to determine its effect
the graft-vs. - host (G reaction [65]. This is the reaction of injected
immunocompetent cells against the cells and tissues of genetically non-
identical recipient. The recipient is unable to reject the graft because of
its immunoincompetence as result of immaturity or immunosuppression.
When effector spleen cells were removed from mice three days after injec-
tion with poly rI: poly rC, the G VH reaction was enhanced in allogenic new-
born mouse recipients of these cells. the other hand, if the effector
,/( HINS( IN
wcrc 7 1;) al"Lcl' illjecl,ioll 01' jJoly ,'1: jJoly ,'('.
the G VH was supprcsscu. poly l'l:
suppressed the G VIl timc rClIloval
effector cells after of fue mice with poly r1: poly
111 related study, treatmel1t of mouse spleel1 cells to L!l()il'
admil1istratiol1 to lefually irradiated resulted 80% 01.'
greater of fue of overt dis-
ease VH over 100-day period [66]. based its
suppressor properties, is good as possible mediator of the
of fue G VH another study [67],
the of the G was reduced when the
mice were injected wifu large of interferon prior to removal of
cells for graftil1g.
Virus-type from several sources and of different specific
activities inhibit the in vitro PFC response il1 the mouse 8ystem
tion to the antiviral activity [25]. system where donor spleen
cells were treated wifu various iI1terferons prior to into F 1 hybrid
mice, it was observed fuat some virus-type preparations
blocked fue GVH reaction while others were wifuout effect
J. Georgiades, unpublished data). Blockade was related to the source
of fue specific activity. Thus virus-type
affect the il1 vitro PFC res and the G VH reaction
VI. RELATIONSH1P OF CYCLIC
INTERFERON. AND 1MMUNE RESPONSE
It has been proposed fuat adenos 3': 5' - phate
has an inhibitory effect fue immunological and iI1flammatory of
lymphocytes [68]. Evidence has been obtained fuat suggests fuat
play role in regulation of interferon production lymphocytes
and of the in vitro PFC response [69]. Figure 8 presents data
showing dibutyryl inhibition of the production of immune inter-
feron in mouse spleen cultures fuat were stimulated the
two mitogens Con and SEA. Dibutyryl 2 10-5
inhibited stimulation of interferon production 96%, while 1 10-4
inhibited SEA stimulation of interferon production 85%. When fue same
concentrations of dibutyryl were added to fue supernatant fluids
of mouse spleen cultures after complete interferon production, inhibi-
tion of antiviral activity of fue interferon was observed, 80 it is concluded
that dibutyryl blocked fue production of immune interferon,
the established activity of produced interferon.
3 presents data dibutyryl blockade of mitogen-
induced suppre8sion of fue in vitro PFC response to SRBC. SEA inhibited
the PFC response >90% when compared to controls. Protection of fue
11. IIVllvIlINI-:
100

......
z


w
u.
10
w
1-


!:::
z
::>
2xlO-
5
2xlO-
4
2xlO- 3
cyclic
FIGURE 8. Dibutyryl inhibition of the production of interferon
in concanavalin (Con and staphylococcal enterotoxin (SEA,
stimulated female (8-weeks-old) mouse spleen cu1tures.
Cu1tures consisted of 1.5 107 spleen cells/ml and 2 and 1 Con
and SEA, respectively. Dibutyryl and mitogens were added to
cultures at the same time, and the cells were incuba ted for 48 hr. Super-
natant fluids were obtained centrifugation of the harvested cultures at
1000 rpm in RC-3 Sorval centrifuge at 70 Data are plotted as units of
interferon per milliliter . SD for duplicated samples. From Ref. 69.
[Reprinted with permission from Johnson, Nature 265: 154 (1977).]
PFC responses was essentially complete when dibutyryl
(1.4 10-4 was added to cultures along with SEA. Dibutyryl
protection of the PFC responses from Con suppression was also
observed. Con at 1.0 and 2.0 inhibited the PFC response
>90% when compared to the controls. Dose response studies with various
concentrations of dibutyryl showed that the concentrations
(1 10-4 to 2 10-4 of the ribonucleotide that blocked the develop-
ment of suppressor activity correlated with those concentrations that blocked
the production of interferon in spleen cultures stimulated the
I N;-)()N
3. af 011
Sllppressian af the in vitra Pl<'C to
Mitogen
SEA
SEA
SEA
SEA
Con
Con
Con

Mitogen
concentration

0.25
0.50
0.25
0.50
1.0
2.0
1.0
2.0
Diblltyryl

(1.4 10-4
+
+
+
+
+
+
SOllrce: Reprinted with permission from Ref. 69.
Pl<'C/10
6
viable
2. SD
642.8
1012. 11
5080. 820
5508. 1406
41482.296
52062.3626
72.9
162. 11
15772.505
14562.307
3146.537
2571. 691
mitogens (Figure 9) [70]. Dibutyryl guanosine 3': monophosphate
GMP), at the concentrations, had effect either mitogen
stimulatian of interferon production or mitogen-induced suppression of the
in vitro PFC response.
The effect of dibutyryl SEA-induced immunosuppression
and interferon production was further explored adding dibutyryl
(1.4 10-4 to cultures at variolls relative ta SEA (0.5
and determining the PFC response (Figure 10) [70]. ln parallel
stlldy, SEA induction of interferon under the conditions was determined
(Figure 10). When dibutyryl was added to the cultures at either
-1 or hr, complete blockade af the SEA-induced immunosuppression was
observed. An enhancement of the PFC response was obtained when dibutyryl
was added to the cu1tures at hr; however, subseque!lt experi-
ments did not always show this enhancement. With increasing time between
mitogen and dibutyryl to cu1tures, there was
an increasing amount af interferon produced and carresponding increase
11. IIVII\IIINI':


.------
.---------8.. __


CYCLIC RI80NUCLEOTIDE
FIGURE 9. Dibutyryl blockade of SEA (0.5 suppression
of the in vitro anti-SRBC, PFC response in female (8-weeks-old)
mouse spleen cultures. SEA, SRBC, and ribonucleotides were
added to cultures at the same time, and direct anti-SRBC, PFC/culture
were determined day 5. The data are expressed as the of duplicate
determinations. coefficient of variation for determinations was
27%. 6, SRBC; SEA + SRBC; dibutyryl +
SEA + SRBC; .---., dibutyryl GMP + SEA + SRBC. From Ref. 70.
[Reprinted with permission from Johnson, J. Blalock, and
Baron, Immunol. 33:170 (1977).
in the suppressiol1 of the anti-SRBC, PFC response. The data suggest
direct relationship, then, between the effect of dibutyryl
SEA-induced immunosuppression and production of interferon SEA-
stimulated cultures. This is further evidence that mitogel1-induced suppres-
sion of the in vitro response is related to mitogen inductiol1 of interferon
in the cultures.
Cholera toxin (an enterotoxin produced Vibrio cholerae) raises the
endogenous level of stimulating adenylate cyclase activity.
Adenylate cyclase converts to The methyl xanthine
3-isobutyl-1-methyl xanthine raises the endogenous level
I N:-I()N
5
10

>3.5

1
4
3.0

10
w
<5

3
::::>
1-

--'
::::>
u
2.5
"-
"- z
u
LL

Q
w
1-
LL

u
w
w
3
2.0
1-

10



1-
Z
::::>
1.5
-4 4 8 12 16 20 24
TIME OF cyclic (hr)
FIGURE 10. Determination of the PFC response and interferon production
after addition of dibutyryl to spleen cultures at various
times relative to SEA addition. SEA (0.5 jJg/ml) and SRBC were added at
hr to the spleen cells for the PFC response, al1d dibutyryl
(1.4 10-4 was added at the indicated times relative to time
Direct anti-SRBC responses were determined day 5. PFC
responses/culture. SD for the SRBC control and the SEA-suppressed
cOl1trol were 4610. 834 and 350. 353, respectively. Parallel studies
were carried out interferon production under the same cu1ture conditions,
except that SRBC were absent from the cultures and the cu1tures were
il1cubated 48 hr after SEA addition at time From Ref. 70. [Reprinted
with permission from Johnson, J. Blalock, and S. Baron,
Immunol. 33: 170 (1977).]
11. IIVIIVIIINI';
TABl,i': 4. 01 Agcl1Ls i':l1dogcl1ous Cyclic
Levels 011 SI..:A-1I1duced Suppressiol1 of the il1 vitro PFC
to SRBC
Agel1t

Cholera toxil1 1)
Cholera toxil1 1)

(12.5)
SEA
5
+
+
+
+
Source: Heprinted with permission from Ref. 69.
PFC/I0
6
viable
SD
431. 135
2984 . 596
4791. 165
4326
91 . 42
3973 . 477
1366.571
2294. 1238
inhiblting phosphodiesterase activity. Both agents blocked SEA suppres-
sion of the in vitro anti-SRBC, PFC response in manner similar to that
observed for dibutyryl 4) [69]. Further, they blocked
SEA stimulation of interferon production in mouse spleen cultures at
concentrations that blocked SEA suppressor activity.
Dibutyryl GMP (10- 3 to 10-7 did not affect the of
dibutyryl (1.4 10-4 to block the suppression of the PFC
response SEA; nor was the effect of dibutyryl mitogen
stimulation of interferon affected dibutyryl GMP [70]. This is
further evidence that the phenomena reported here are due to
and are not influenced GMP in this system.
data suggest that immune-induced interferon is associated with
mitogen-induced suppressor activity, and that the interferon may
possibly mediator of such activity. 1n related studies, it has been
shown that high concentrations (10-3 of dibutyryl inhiblt
the yield of interferon from phytohemagglutinin-stimulated human peripheral
leukocytes [71] and from virus- or polyribonucleotide-stimulated mouse
cultures [72]. The data presented here show logical relationship
between immune interferon activity, suppressor activity,
and regulation of the immune response. Preliminary studies the effect
of induction of virus-type interferon in mouse spleen cultures
suggest that this interferon system may slightly more resistant to blockade
of induction.
VII. 01"
IMMUNE FUNCTION
,J()IIN:->()N
Immune interferon, induced in leukocyte cultures is Ll1C
earliest described lymphokines [5]. plethora of lymphocyte-reg"ulatil1f2,",
soluble factors have subsequently been described [15]. Several of these
factors ultimately shown to represent additional biological properLi(,::
of immune interferon. Mitogen-stimulated lymphocytes, for example,
produce soluble immune response suppressor(s) (SIRS), which inhibits
the in vitro PFC responses [73]. Sensitized lymphocytes that are stimu-
lated antigen also produce an inl1ibitor of the in vitro antibody
response, which the as SIRS [74]. Contained within the
supernatant fluid are immune interferon and macrophage migration inhibitol"Y
factor [10,31]. date the three have not been separated, and
thus they represent different biological activities of the same moleculc.
Recently, mouse fibroblast interferon has been purified to homogeneity
[75]. The purified interferon was of inhibiting multiplication.
This is probably the most convincing evidence that virus-type interferon
possesses tiological activities in addition to being antiviral. It would
interesting to determine the relationship of virus-type interferon to various
lymphokines that are of il1hibitil1g DNA sYl1thesis [15].
VIII. MODEL
In Sectiol1 1 we discussed the cooperatiol1 that occurs between lymphocytes
al1d betweel1 lymphocytes al1d macrophages to help or suppress the immul1e
respol1se. questiOll is how do interferol1s, with their immunosuppres-
sive properties, fit into the suppressor picture of immU110regulation?
al1swer is that we do not but if we focus the cells and al1ti-
body productiol1 al1d helper and suppressor effects, it might help
glue together the interferol1 data. cOl1tact induces the helper or
suppressor It is assumed that the presel1ce of receptor for the
al1tigen both the al1d bril1g the two cells into close proximity
when they bind to the same al1tigel1 so that mediator, after release from
the is in sufficiel1t cOl1cel1tratiol1 in the microel1vironment of the
to exert its effect.
This model app1ied directly to explain how al1tigel1-induced
mediators (such as immul1e il1terferol1) exert their suppressor effects.
Assumil1g similar interactiol1s of al1d cells al1d al1tigen, the suppressor
substance (possibly il1terferol1) could released from the appropriate
antigel1-stimulated in the microenvironment of the al1d inhibit
the antibody response to the same al1tigel1 (Figure 11). Thus, the
kil1etics of the al1tibody respol1se could the reflectiol1 of inter-
action of "helper" al1d "suppressor" activities with the respol1ding
11'11',111:1',1
1'11'1111',111
I
I I y"'pl")(:yl"

. ----- etc.).::
....
xU' U ( / /

xxxAntlgenxx
1:,

// / 6
..)),! /..j)..3 / '
(
() ................i. > ...... : .... .: ...... i.
'6 2 4 5
Precursor
Lymphocyte

.
Sensitized

Lymphocyte
Plasma
Cell
FIGURE 11. Cellular events in the induction and immunosuppressive action
of interferons. Antigen comes into contact with precursor lymphocyte
which undergoes differentiation to sensitized lymphocyte
driven antigen becOlne memory or it release
mediators known as lymphokil1es (D). AmOl1g the mediators produced
the lymphocyte is immul1e il1terferon*. Antigen also reacts with pre-
cursor lymphocyte (1), which Ul1dergoes differel1tiatiol1 to sel1sitized
lymphocyte (and memory (2). The sensitized is further
drivel1 antigel1 (3) to plasma (4), which is responsible for
most of the al1tibody (5) that is produced. This al1tibody reacts with the
specific al1tigen (6). Both antigel1-induced al1d virus-induced
il1terferol1s are of reactil1g with precursor (E
j
) al1d (E
z
) lympho-
cytes well as sel1sitized

and

lymphocytes. As differentia-
tiOl1 progl'esses, in part as resu1t of cOl1til1ued al1tigen presel1ce, it
progressively more difficu1t to inhibit lymphocyte ful1ctiol1
interferOl1S. productiol1 of antibody is res istant to
interferol1. The macrophage is not mcluded in the figure, but interferons
exert their immunosuppressive effects via required macrophage
ful1ctiol1 il1 the immul1e response. The diagrammatic scheme does 110t
l1ecessarily imply, therefore, direct effect of the il1terferons 011 the
lymphocytes. From Ref. [Repril1ted with permissiol1 from
Johnson al1d S. Baron, Pharmacol, Ther . .1 (1977), Pergamol1 Press,
Ltd.]
,)( )11 NS()N
The nlOde1 101' postulaiil1l4 cclls il1Lcl'acL ill
suppressor effects illvo1villg virus-type illterferoll. Lcl'l'c
is produced vil'tuaHy evel'Y type ill the body [1].
fo1' examp1e) that is of the p1'oduction of vil'us-typc
theoreticaHy shut doWll the 1'espollse, p1'ovided thaL
the illterfe1'oll-producing produces an effective concent1'ation of inter-
fe1'on in the of the Viruses with
that are intimate1y associated with the 1ymphoid tissues
have, in gene1'a1, greate1' suppressive effect the immune 1'esponse
vi1'uses not associated with tissue [76]. Because the
intimate association of macrophages ceHs with the ceHs,
these types would expected to p1ay p1'ominent ro1e in supp1'essing
the immune through vi1'us-type interfe1'on. As mentioned above,
it has been that cells are requi1'ed fo1' the virus-type interfe1'on
po1y rA: po1y rU and po1y r1: po1y 1'C to inhibit the in vit1'o PFC
1'esponse to 0127 LP8 [48]. We have shoWll that this is
blocked antise1'um to vi1'us-type inte1'feron [14] .
Given this broad1y outlined mode1, more work needs to done
in o1'de1' to p1'ecise1y dete1'mine the ro1e of various types of interferons in
the regu1ation of the immune 1'esponse. It l'emains to dete1'mined whethel"
other reported suppressions a1'e re1ated to interferon. This wo1'king mode1
provides g'uide fo1' determining the ro1e interfe1'on in the
of of the immune 1'esponse. The data inco1'-
po1'ated into this hypothesis without sigllificant modificatioll.
REFERENCE8
1. N. (ed.), in alld Ame1'ican
E1sevie1', New Yo1'k, 1973.
Johnson and S. Ba1'on, Pha1'maco1. The1'.,1:349 (1977).
2. lsaacs, Virus Res. 10: 1 (1963).
3. Fie1d, TyteH, G. Lampsoll, and R.
P1'oc. Nat. Acad. 8ci. U.8 . ...: 1004 (1967).
4. Paucker, Texas Repts. Bio1. Med. in press (1978).
5. 1". Whee10ck, 8cience 149:310 (1965).
6. J. G1'een, S. R. Coope1'band, 8. Kibrick, 8cience 164: 1415
(1969).
7. 8. J. 8. Youngne1', and W. Lederer, 1nfec. Immun.
1: 68 (1973).
8. J. 8tobo, 1. Green, L. Jackson, and 8. Baron, J. Immuno1. 112: 1589
(1974).
9. L. Epstein, Texas Repts. Med. in press (1978).
10. Johnson, G. J. and 8. Baron, P1'oc. Bio1.
Med. 154: 138 (1977).
11. Ilvll\iIlINI': IVIOIHJ
11. 13. Ogburn, Bcrg, Pauckcr, alld
J. Vi!cek, Proc. Nat. Acad. Sci. U.S. 72: 21t;5 (1975).
12. N. Maehara, and J. Armstrong, Infec. Immun. 17:572
(1977).
13. J. Youngner and S. Salvin, J. Immunol. 111: 1914 (1973).
14. Johnson and S. Baron, Immunol. 25: 106 (1976).
L. Osborne, J. Georgiades, alld Johnson,
Immunol. in press (1980).
15. Waksmanalld Namba, Immunol. 21:161 (1976).
16. W. Braun and Levy, Proc. Soc. Med. 141: 769 (1972).
17. J. Chester, Paucker, and Merigan, Nature 246:92 (1973).
18. R. Brodeur and Merigall, J. Immunol. 113: 1319 (1975).
19. R. Brodeur and Merigan, J. Immunol. 114: 1323 (1975).
20. J. Ngan, S. S. Lee, and L. S. J. Immunol. 117: 1063 (1976).
21. S. Ida, J. J. Hooks, R. Siraganian, and L. Notkins, J.
Med. 145: 892 (1977).
22. G. Sonnenfeld, D. Mandel, and Merigan, Immunol. 34:
193 (1977).
23. R. Gisler, Lindahl, and 1. Gresser, J. Immunol. 113:438 (1974).
24. Johnson, G. Smith, and S. Baron, IRCS Med. Sci. 1616
(1974).
25. Johnson, G. Smith, and S. Baron, J. Immunol. 114:403
(1975).
26. Johnson, J. Bukovic, and S. Baron, Immunol. 20:
104 (1975).
27. R. J. Booth, J. Booth, and J. Marbrook, Eur. J. Immunol . ..:
769 (1976).
28. W. Pierce, J. Med. 130:345 (1969).
29. R. J. Booth, J. Rostrick, R. and J. Marbrook,
Aust. J. Med. Sci. 54: 11 (1976).
30. Clinton, J. Mogoc, R. L. Aspinoll, and N. Rapoza,
Immunol. 27: 60 (1976).
31. L. Kuhner, R. R. Rich, J. R. David, and W.
Pierce, J. Immunol. 117:323 (1976).
32. G. Trivers, D. Braungart, and J. Leonard, J. Immunol. 117:
130 (1976).
33. J. Georgiades, L. Osborlle, R. Moulton, and Johnson,
Proc. Soc. Biol. Med. 161: 167 (1979).
34. Johnson, Immunol. 36: 220 (1978).
35. R. Schultz, J. D. Papamatheakis, and Chirigos, Sciellce
197: 674 (1977).
36. R. Schultz, Chirigos, and U. 1. Heine, Immunol. 35:
84 (1978).
37. D. S. Nelson, in Immunobiology of the Macrophage (D. S. Nelson, ed.),
Academic Press, New York, 1976, 235.
,/< >I I NS< JN
38. W. Braun, Nakano, L. YajiI1l:t, arlll1,.
in Nucleic Acids in Immunology J. ia W.
Springer-Verlag, New York, 1968, 347.
39. G. Johnson, J. Schmidtke, Merritt, and in Nucleic
Acids in Immu.nology J. Plescia and W. Braun, eds.), Springer-
Verlag, New York, 1968, 379.
40. J. R. Schmidtke and G. Johnso.n, J. lmmunol. 106: 1191 (1971).
41. G. Johnson, in Immune RNA Cohen, ed.), CRC Press,
Cleveland, 1976.
42. Markowitz, D. Person, F. L. Gitnick, and R. Ritts, Sciencc
163:476 (1969).
43. S. Ega.n, W. J. Reeder, andR. D. Ekstedt, J. lmmunol. 112:63
(1974).
44. G. Romball and W. Weigle, J. lmmunol. 11:556 (1975).
45. W. BraunandM. lshizuka, Proc. Nat. Acad. Sci. U.S. 68:1114 (1971).
46. lshizuka, W. Braun, and Matsumoto, J. Immunol. 107: 1027
(1971) .
47. R. a.nd J. J. Marchalo.nis, Aust. J. Biol. Med. 50:69
(1972) .
48. Johnson, J. Bukovic, a.nd G. Smith, Proc. Soc.
Biol. Med. 149: 599 (1975).
49. Chused, S. S. and D. Mosier, J. Immunol. 116:
1579 (1976).
50. R. W. Dutto.n, J. Med. 136: 1445 (1972).
51. 11. R. Rich, and W. Pierce, J. Med. 137: 205 (1973).
52. J. R. Epstei.n, 1. Nakoinz, a.nd Ralph, J. Immunol. 110:
43 (1973).
53. G. Smith and Joh.nso.n, J. Immu.nol. 115:575 (1975).
54. R. Brodeur, Weinstei.n, L. Melmon, a.nd Meriga.n,
Immu.nol. 29: 363 (1977).
La.ngford, G. J. Sta.nton, a.nd Joh.nson, I.nfec. Immu.n.
22: 62 (1978).
55. Li.ndahl-Mag.nusso.n, Leary, a.nd 1. Gresser, Nature [New
237: 120 (1972).
56. R. Rozee, S. S. Lee, a.nd J. Ngan, Nature [New Biol.] 245: 16
(1973).
57. L. obraate.n , De Maeyer, a.nd J. De Maeyer-Guig.nard,
Tra.nspla.nt . ..:1&: 415 (1973).
58. J. Cerotti.ni, Bru.n.ner, Li.ndahl, a.nd 1. Gresser, Nature
[New 242: 152 (1973).
59. S. Hirsch, D. EHis, Black, a.nd L.
Wood, Tra.nspla.ntatio.n 17: 234 (1974).
60. De Mayer, J. De Maeyer-Guig.nard, a.nd Va.ndeputte, Proc. Nat.
Acad. Sci. U.S. 72: 1753 (1975).
1 1. 11'111'1111 N 1'; I ON
G1. J. l)c Maeyer-Guigllard, atld J<;. l)e Macycr, SCicllCC
574 (1975).
G2. I. Heroll, Berg, alld CallteH, J. Immullol. 117: 1370 (1976).
63. D. Ki11allder, Lindahl, L. LUlldin, Leary, and I. Gresser,
J. Immullol. 2,:56 (1976).
64. MiOrer, L. Lalldstrom, Larner, 1. Larsson,
alld Strallllegard, Immunol. 35: 15 (1978).
65. Calltor, R. Asofsky, and Levy, J. Immunol. 104: 1035 (1970).
66. L. Tyan, Proc. Soc. Biol. Med. 150: 628 (1975).
67. S. Hirsch, D. EHis, R. Proffitt, Black, and
Chirigos, Nature [New 244: 102 (1973).
68. R. Bourlle, L. Lichtellsteill, L. Melmoll,
Weinsteill, alld G. Shearer, Science 184: 19 (1974).
69. Johnsoll, Nature 265: 154 (1977).
70. Johnson, J. Blalock, and S. Baroll, Immunol. 33: 170
(1977).
71. L. Epstein and R. Bourne, in Mitogells in Immunobiology
(J. J. Oppenheim alld D. L. Rosellstreich, eds.), Academic Press,
New York, 1976, 453.
72. F. Dianzani, Neri, alldM. Zucca, Proc. SOC. Biol. Med.
140: 1375 (1972).
73. R. R. Rich and W. Pierce, J. Immullol. 112: 1360 (1974).
74. D. W. Thomas, W. Roberts, and D. W. Talmage, J. Immullol.
114: 1616 (1975).
75. J. De Maeyer-Guignard, G. Tovey, I. Gresser, and De Maeyer,
Nature 271: 622 (1978).
76. J.-L. Virelizier, Virelizier, alld Allison, J. Immunol.
117: 748 (1976).
AUTHORINDEX
Numbers in brackets are reference numbers and indicate that author' s
work is referred to although his is not cited in the text. Underlined
numbers give the page which the complete reference is listed.

, . . , 207 [106],219
Abdallah, . s., 75 {67], 76 [67],
86, 123 [49], 142
Ablashi, D. V., 203 [69], 217
Adams, . , 91 [37], 94 [37], 108
Adamson, R. . , 203 [69],209
[118,122,126], 217, 219, 220
Ahstrom, L., 96 [72], 109, 133
[60], 142
Alacam, R., 34 [23], 52
Albrecht, W. L., 191 [8,9,10,11,
12,13],201 [9,10,11,12,13,38,
39], 205 [70], 215, 216, 218
AI-Bussam, N., 99 [81], 110
Alford, . , 74 [63], 75 [63], 77
[63],79 [63], 86
Alford, R. . , 227 [7], 236
Alderfer, J. L., 160 [62], 165, 181
[53,54], 186
Alexander, . , 106 [120], 111
Allen, . . , 119 [23], 125 [23],
140,240 [2,3],252 [2,3],253
[2], 254 [2], 255 [3], 257
s , . . , 290 [76], 293
l l , . 1., 120 [33], 141
Andrews, . R., 191 [8,9,10,11,
14,16], 201 [8,9,10,11,14,16,
39], 215, 216
295
Andzaparidze, D. G., 174 [42,43],
185
Anfinsen, . . , 21 [2],28 [14],
29 [14],31 [2],33 [2], 34 [2],
45 [58], 46 [58], 50, 51, 54, 58
[14,18],62 [18], 84, 100 [92],
110
Ankel, . , 6 [51], 10 [120], 17"
19
Anver, . R., 209 [128], 220
Applebaum, . , 207 [99, 100], 219
Armstrong, D., 129 [56], 142
Armstrong, G. R., 203 [69], 217
Armstrong, J. . , 7 [60,64],8
[73], 17,58 [2],59 [61],74
[61], 83, 86, 117 [14],133 [59],
140, 142, 152 [49], 164, 244 [63],
259, 265 [12], 291
Arvin, . N., 77 [80], 82 [80],
86, 123 [50], 142
Ashwell, G., 79 [75], 81 [75], 86
Asofsky, R., 172 [31], 184, 218,
281 [65], 293
Aspinoll, R. L., 274 [30], 291

Bachner, L, 6 [48], 17
Baer, G., 172 [38,39],185
Baglioni, 10 [113], 19
Bahnsen, 71 [(0), 74 [(0),
86
Baird, L. G., 209 [129), 220, 240
[36,40), 246 [46), 247 [46], 248
[46,82],250 [36], 253 [36], 258
259, 260
Baldwin, R. W., 106 [122], 111
Balezina, G. I., 198 [25], 216
L. 9 [91), 10 [111), 18,
19
R. J., 240 [43], 249 [43],
259
Bandu, 31 [20],46 [64],
52, .2., 90 [11,21], 91 [21], 92
[21], 107, 119 [31, 32), 141
Banks, G. 169 [10], 183
Bansek, G. 153 [50], 164
Bargigli, V., 79 [72, 73], 86
Barker, D., 198 [29], 201 [56,
57,58,60,63],209 [56,57,58,60,
63], 216, 217
Barnes. D. W., 245 [70], 246 [70],
250 [70], 252 [70], 254 [70], 256
[70], 260
Baron, S., 5 [41],6 [42],8 [77],
12 [136,139,140], 13 [141,142,
143,144,147,148],14 [153), 16,
1.., 20,67 [45],68 [45], 85, 99
[85], 103 [109], 110, 111, 115
[4],116 [8],118 [20], 140, 149
[34,35],151 [41,42],152 [46,47],
154 [34], 160 [34], 161 [34], 164,
169 [17,18,19],170 [24,25],172
[28,34,35,38], 174 [39,40,41],
180 [49], 183, 184, 185, 186, 199
[78], 200 [78], 208 [111,112],
209 [111], 218, 219, 224 [2], 236,
248 [81],256 [81], 260, 265
8,10,14],270 [24,25],271 [25,
26],272 [25],273 [14,25], 274
[10],278 [14],279 [8,10,14],
280 [10,14],281 [25],282 [25],
284 [70],285 [70], 286 [70],287
[70],288 [10],290 [14], 290, 291
}N})}:X
I3artl1, lt., 117 11;)], 110
Bean, W. J., 9 [101],
Bech-Hansen, N. 103[100],
110
Becker, 38 [79], 56
Beesan, 181 [54], 186
Bektemirov, 174 [42,43],
185
R., 273 [29], 291
Bellanti, J., 172 [34], 184
Berg, 3 [28], 16, 24 [3], 27
[8],28 [15],29 [8,15,16,17,18],
34 [23], 37 [18], 38 [8,15], 44
[50],46 [17,66], 47 [15,66],
48 [66], 50, 51, 52, 54, 55, 58
[6,8], 63 [33], 64 [33], 83, 84,
85,265 [11],281 [62], 291, 293
Bergstrom, S., 12 [135], 20
Berissi, 9 [93], 18
Berman, 10 [116], 19, 58 [7,
8], 83, 84, 265 [11], 291
Berman, 3 [28], 16
Berman, L., 172 [30], 184
Berthold, W., 35 [28], 38 [28],
41 [28], 52
Besanfton, F., 6 [51], 10 [11 7 ,
120], 17, 19,36 [33], 45 [33],
53
Betts, R. F., 228 [8],229 [8],
231 [13],232 [14], 233 [8,14],
236
Beveridge, G. W., 81 [79], 86
Beyse, I., 246 [75], 260
Bhuyan, 172 [29], 184
Billiau, 10 [114], 19, 34 [25],
37 [38],45 [53,55], 52, 53, 54,
59 [23],60 [23],62 [23,27],
63 [27],64 [23,35],65 [23,27],
71 [58], 73 [58], 74 [58], 75
[68],77 [68],77 [68], 84, 85,
86, 100 [87], 110, 114 [2], 119
[21,23,24],125 [21,23,24,53],
127 [2], 131 [58], 133 [58,63,
64,65], 135 [63,71], 136 [72,
73,77], 139, 140, 142, 143, 146
INIH:X
J
[4], 163, 169[17,18,19,20], 183,
184,240 [4,6,7,13,19,29],241
[4,7,29,55],244 [56,60], 245 [4,
7],250 [29],252 [4,6,7,13,19,
29],253 [6],254 [6], 257, 258,
259
U., 82 [81], 86, 129
[57], 135 [57], 142
Bjornsson, S., 99 [81], 110
Black, 137 [84], 143, 240
[26,27],250 [26,27],252 [26,
27],254 [26],255 [26,27], 258
281 [59], 282 [67], 292, 293
Blaese, R. 256 [100], 261
Blalock, J. 284 [70],285 [70],
286 [70], 287 [70], 293
Bleyer, w. 209 [127], 220
Blomgren, 97 [77], 98 [77],
110
Bocci, V., 79 [72,73,74], 81 [74],
86
Bodo, G., 9 [98,99],
Bodurtha, J., 106 [121], 111
Bogden, 95 [66], 109
Bogomolova, N. N., 169 [17], 177
[44], 183, 185
Boiron, 14 [154], 20
Booth, J. 271 [27], 291
Booth, R. J., 271 [27], 273 [29],
291
Borden, 6 [49,50],7 [67,
68],17
Borecky, L., 90 [14,15], 107
Boriskin, S., 174 [42], 185
Borsos, 248 [80], 251 [80],
256 [80], 260
S., 21 [2], 28 [14], 29 [14],
31 [2],33 [2],34 [2], 38 [l4],
45 [58], 46 [58], 50, 51, 54, 58
[14], 79 [77],80 [77], 84, 86
Bosnic, N., 120 [39], 121 [39],
141
Bourali, 11 [126], 19, 90 [27],
91 [30,31,32],105 [116], 108, 111

I3ourali-Maury, 91 [32J, 108,
117 [13], l40
Bourgeade, F., 36 [33], 45
[33], 53
Bourne, R., 282 [68], 287 [71],
293
13 [146], 20
Bradburne, F., 100 [87],110
114 [2], 127 [2], 139
Brande, I. 37 [37], 53, 133
[64], 142
Braun, W., 11 [124], 19,240 [45],
259, 269 [16], 273 [16], 275
[38], 276 [45,46], 291, 292
Braungart, D., 274 [32], 291
Bray, F., 197 [23],201 [43,51],
204 [43,51], 216, 217
Breinig, 7 [60], 11, 152
[45,49], 153 [45], 164, 240 [15,
17],244 [57,59,64],245 [64],
252 [15,17],253 [15,17],254
[15,17], 258, 259
Breslow, D. S., 241 [49],245
[49],246 [49],251 [92],252
[49], 254 [49], 256 [49], 259,
260
Bridgen, J., 21 [2], 31 [2],
33 [2],34 [2], 50,100 [92,93],
110
Brink, N. G., 169 [7], 183
Brodeur, R., 279 [54], 292
Brouty-Boye, D., 46 [64], 55,
90 [4,5,6,10,11,17,23],91 [30],
105 [116], 107, 108, 111, 119
[31], 141, 172 [30], 184
Brown, D. 149 [31], 160 [31],
164, 198 [26], 216
Brown, G. 9 [95],10 [107,
109], 18, 19
Brown, R. 10 [111,112], 19
Brown, W., 206 [80], 218
Brownlee, I. 146 [7], 163
Brunner, 11 [127], 19,
281 [58], 292
Buchan, 5 [37], 16
Buck, W., 169 [10], 183
Buckler, 5 [41],8 [77],
13 [143,144,148], 14 [153], 16,
18, 20, 21 [2],31 [2],33 [2],
34 [2], 50, 67 [45], 68 [45], 85,
100 [92,92], 110, 152 [46,47],
164, 169 [17,18,19], 172 [34,35,
38], 183, 184, 185, 199 [78], 200
[78], 218
Buffett, R. F., 90 [16], 92 [16],
93 [16],95 [69],96 [69],98 [69],
107, 109
Bukovic, J. 271 [26], 276 [48],
277 [48], 290 [48], 291, 292
Burgasova, 174 [42,43], 185
Burke, D. 2 [2,3], 5 [37,39],
15, 16
Burlesol1, G. R., 209 [130], 220
Butcher, R. W., 12 [133], 20
Butler, J. 135 [70], 143

Cabrer, 9 [95], 18
Cairo, 90 [16],92 [16],
93 [16], 107
69 [50], 73 [50], 85, 90
[29], 108,240 [12],241 [12],244
[12], 245 [12], 252 [12], 257
201[55], 217,
255 [96],256 [96,101],257 [101],
261
Camyre, 197 [21,23],198
[27,31], 199 [27], 201 [31,51],
204 [51], 205 [70], 207 [21], 216,
217, 218
Call11ellos, G., 172 [34], 184
198 [30], 199 [30],
216
Cal1tell, 21 [1],22 [4,5,6,40,
51,71],24 [1,4,5,6],28 [15],29
[15],31 [21],33 [42],38 [15],
41 [44], 42 [71], 43 [44], 44 [44],
46 [60,63,73], 47 [15],48 [70],

[Cal1tell,
50, 51, 52, 53, 54, 55, 58 [6,16],
59 [25],60 [25], 63 [32],69
[56,57],70 [54,56,57],71 [55,
56,59,60], 73 [53,54,55,56],
74 [59,60],75 [57,64],76 [55,
64], 79 [16], 81 [54,64], 84,
85, 86, 89 [1],90 [1],91 [33,
37],94 [37],96 [70,71,72,74,
75],97 [76,77],98 [77],100
[88,89,91], 107, 108, 109, 110,
117 [17], 118 121 [41,43
44,45],133 [60,62],136 [74,75,
77,79,83], 137 [84], 140, 141,
142, 143, 281 [62], 293
Cal1tor, 172 [31], 184, 218,
281 [65], 293
Carbol1e, 172 [34], 184
Carlstrom, G., 75 [64], 76 [64],
81 [64], 86, 96 [71,72], 97 [76],
109,110,133 [60,62], 142
Carr, 147 [21], 163, 191
[15],201 [15,39], 215, 216
Carrel, S., 94 [53,64], 109
Carroll, R. G., 59 [61], 74 [61],
86, 117 [14], 140
Carter, W. 3 [24,25],7 [68],
9 [89,90], 16, 17, 18,27 [9,10,
11,12,13],31 [8],36 [9,10,29,
31,32,34,35,36], 41 [62],45
[ 62], 48 [59], 58 [15], 59 [20],
60 [20], 62 [20], 84, 90 [16],
92 [16,48],93 [16],95 [69],
96 [69], 98 [69], 99 [78,81],
100 [48,94], 101 [48,95,96,97,
98,99],102 [48],103 [48], 107,
108, 109, 110, 136 [80], 143,
149 [29],160 [62], 163, 165,
181 [52,53,54], 186
Cartwright, 45 [54], 54, 62
[28],63 [28],64 [36],77 [69],
78 [69], 84, 85, 86,125 [55],
135 [70], 136 [78], 142, 143
Carver, 10 [108], 19
Cassell, D. 214 [154,155], 221

10 [117], 19
Catinot, L., 38 [75], 39 [75], 54
Cawein, 211 [134J, 220
Centifanto, 149 [31J, 160
[31), 164, 198 [26J, 216
Cerrottini, J. 11 [127J, 19,
94 [53), 109,281 [58J, 292
Cesario, 3 (22), 16, 59 [21],
60 [21],65 [40,41,43],78 [43),
84, 85, 136 [76J, 143
Chadha, 41 [62], 46 [62],
54
Chadwick, 251 (92), 260
Chain, 169 (10), 183
Chamberlin, J., 149 [23), 163
Champney, 180 [50), 186
Chan, S. 172 [32), 184
Chandra, 147 (22), 163, 214
[149,150,151,152,1(3), 221
Chang, 10 [117,118), 19
Chang, 10 (115), 19, 119
(22), 125 (22), 140
R. 172 [34], 184,
224 [2), 236
Chany, 167 [3), 182
Cheeseman, S. 137 [84], 143
Chen, J. 27 [9,12], 36 [9), 51,
58 [15], 84,101 [99), 110
Chernik, N., 95 (68), 109
Chernyakovskaya, 1. 105 [118,
119], 111
Chester, J., 218, 269 (17), 291
Chirigos, 11 [131,132),
104 [111), 111, 120, 172 [32],
184,207 [103,104], 219, 240 [23,
37,39),247 [78J, 248 [78,79],
250 [23,37,39,84],251 [37],252
[23],253 [33,37,93J, 256 [84),
258, 260, 274 [35,36],282 [67],
291, 293
Chouroulinkov, 1., 11 [126), 19,
105 [116), 111,119 [29], 141
Cllristophersen, 1. S., 24 [3J, 50
Chudizio, 3 [26J, 16
6 [52J, 17
Chused, 277[49], 292
Claes, 240 [7], 241 [7], 245
[ 7], 252 [7], 257
Claesen, 240 [44], 241 [55 J,
244 [60], 249 [44], 259
Clark, W. R., 240 [30], 252 [30J,
258
Cline, J. 2 [9], 15
Cline, J., 103 [104],111
linton , 274 [30], 291
W. R., 95 [67], 109
Cochard, 281 (61), 293
Cochran, W., 169 [6), 183,
201 [65J, 217
Cocito, 146 [4], 163
Cohen, 69 [50], 73 [50], 85
Colby, 5 [40J, 16, 149 [23],
163
199 [76],200 (76),
218
Collins, L., 250 [82], 260
Collins, 208 [115J, 219
Collins, 118 140
d'Hooghe, 90 [23], 108
Colsky, J., 245 [103J, 256 [103J,
261
Commerford, S. L., 246 [74J, 252
[74], 260
Cone, 246 [77], 260, 276
[47], 292
J. 154 [54], 164
Cooper, L., 146 [11], 163
N., 212 [138), 220
Cooperband, S. R., 103 [103], 111,
146 [13J, 163, 265 [6), 290
J., 67 [47],68 [47], 85,
90 [24,25,26,27), 108, 172 [30],
184
L., 21 [2], 28 [14), 29
[14],31 [2),33 [2J, 34 [2],38
[14],50,51
Corley, L., 28 [14),29 [14], :JH
[14J, 45 [56], 46 [56], 51, 5/1,
58 [14], 84, 100 [92], 110
Cosimi, 13711'/11, JJ..:.I.

Coster, D. J., 121 l41], 141
Couch, R. 65 [42],66 [421,
85, 74 [63], 75 [63],77 [63], 79
[63,70], 86
Crabbs, L., 179 [46], 185, 207
[102], 219
Crane, J. L., Jr., 254 [951, 260
Creech, R. 106 [121], 111
Cronin, 146 [17],147 [17],
163, 224 [3], 225 [3], 226 [3],
236
Cuillon, J. 119 [261, 141
Cuneo, R., 180 [51], 186, 212
[141], 220
Cunnington, 181 [54], 186
Cupak, 120 [37], 121 [46], 141
Custer, R. 94 [55), 109
D
Dahl, 11 [130], 20, 91 [38,40,
43], 108
Da1ton, J., 48 [68], 55,58 [9,
13],
Damm, 90 [18], 91 [18,42],
92 [18],94 [18], 107, 108
Danielson, 12 [135], 20
Danusso, F., 240 [33], 249 [33],
258
Davey, W., 27 [13],31 [8],36
[29,31,34,35,36],45 [30], 51,
52, 53
David, J. R., 274 [31], 288 [31],
291
Day, D., 103 [110], 111
Dean, J. 103 [108], 111
Declercq, 3 [16],6 [47,48],
16, 17, 37 [37], 44 [45], 53, 67
[44,49],68 [49], 85, 99 [79,80),
110, 117 [12], 133 [64], 135 [71],
140, 142, 143, 146 [4], 149 [24,
25,27,28], 152 [48], 163, 164,
169 [16],170 [16,21], 183, 184,
192 [18], 194 [18], 197 [18,24],
INIJI';X
1';. J
llb1, 199 I
201 214 215, 21(;,
240 [7,11,18,20,21,28],241 l7,
28,55],244 [21,60],245
251 [28], 252l7,11,18,20,21,28],
253 [11,20],254 [20], 257, 258,
259
Degre, 11 [130], 20, 91 [38,
40], 108, 198 [28], 216
DeGroote, J., 100 [87], 110, 114
[2], 127 [2], 139
Deguerrero, L. 13 [146], 20
Delimar, N., 120 [39], 121 [39,
47], 141, 142
De Maeyer, 11 [128], 20, 31
(22), 52, 207 (95), 218, 281
[57,60,61],288 [75], 292, 293
De Maeyer-Guignard, J., 31 [22],
52, 119 [23], 125 [23], 140, 155
[56], 164, 207 [95], 218, 281
[57,60,61],288 [75], 292, 293
Dennis, J., 198 [29], 216
Dennis, L. 245 [103], 256
[103], 261
Desmet, V. J., 100 [87], 110,
114 [2J, 127 [2], 139
Desmyter, J., 31 [19,72], 34 [25],
45 [53], 52, 54, 55,58 [11,12],
63 [11], 64 [35], 84, 85, 100
[87], 110, 114 [2], 127 [2], 240
[4,7,8],241 [4,7],244 [8], 245
[4,7],252 [7,8],254 [4], 257
De Somer, 6 [47],10 [114],
17, 19, 34 [25),37 [37,38], 44
[45,50],45 [53,55], 52, 53, 54,
59 [23], 60 [23], 62 [23,27], 63
[27],64 [23,35],65 [23,27],71
[58], 73 [58], 74 [58], 75 [68],
77 [68,71],79 [71], 84, 85, 86,
100 [87], 110, 114 [2],117 [12],
119 [21,23],125 [21,23,53],
127 [21,131 [581,133 [63,64,
651, 135 [63,71],136 [72,73],
139, 140, 142, 143, 146 [41, 163,
INI>I';X

240 [4,6,7,8,13,18,19,20,21,28,
29,44],241 [4,7,28,29,55,56],
244 [8,21,60],245 [4,7,20], 249
[44], 250 [29], 251 [28], 252 [4,
6,7,8,13,18,19,20,21,29],253
[6,20], 254 [6,20], 257, 258, 259
Desrosiers, R., 9 [95], 18
The, G., 172 [30], 184
Devita, V. 99 [85], 110, 172
[34,35], 184, 185
Devlieger, 77 [71], 79 [71], 86
Vomecourt, 38 [75], 39 [75],
55
de Vries, J., 133 [69], 143
Diamantstein, 206 [73], 218,
246 [75,76], 260
Dianzani, F., 5 [41],6 [42,55],
8 [77],12 [139,140], 16, 17, 18,
20, 116 [8], 140, 151 [41,42,43],
164,169 [18,19], 183, 198 [30],
199 [30], 216, 287 [72], 293
L. 92 [48], 100
[48],101 [48],102 [48],103 [48],
108
Dicker, 136 [78], 143
Diederich, J., 3 [18], 16
Dirda, V., 229 [10], 230 [10,12],
232 [12], 234 [12], 236
Dohlwitz, 96 [72], 109, 133
[60], 142
Dolen, J. G., 95 [69],96 [69],98
[69], 109, 136 [80,81], 143
Dombos, L., 100 [90], 110
Donahoe, R. 11 [125], 19, 105
[114], 111, 207 [105], 219
Donahue, J. 207 [101], 219
Dorner, F., 28 [56], 45 [56], 46
[56], 54, 79 [76], 86
Douglas, R. G., Jr., 227 [4,5,6],
228 [8],229 [8],231 [13],232
[14],233 [8,14], 236
Dressler, R., 105 [114], 111,
207 [105], 219
:101
13 [113], 20, 118
[20], 140, 152 [46,47], 164, 199
[78], 200 [78], 218
F., 212 [139], 220
Dudock, 9 [93], 18
Dupont, 129 [56], 142
Dutton, R. W., 279 [50], 292
Durr, F. 208 [116], 219

Easterbrook, 90 [9], 107
Eckstein, F., 149 [24], 163, 170
[21], 184
Edelstein, R., 213 [144), 221
Edwards, 1.,241 [49],245
[49],246 [49],252 [49],254
[49], 256 [49], 259
Edy, V. G., 10 [114], 19, 34 [25],
37 [37,38], 44 [50],45 [53], 52,
53, 54, 59 [23], 60 [23], 62 [23,
27],63 [27],64 [23,35],65 [23,
27], 71 [58], 73 [58], 74 [58],
75 [68],77 [68,71],79 [71], 84,
85, 86, 100 [87], 110, 114 [2],
119 [21], 125 [21], 127 [2], 131
[58],133 [58,63,64,65],135
[63,71],136 [72,73], 139, 140,
143
Egan, S., 276 [43], 292
Einhorn, S., 91 [41,44], 92 [41],
94[41],97 [41],103 [44], 108
Ekstedt, R. 276 [43], 292
281 [59], 282 [67],
292, 293
149 [31], 160
[31], 164, 198 [26], 216
Ellmore, V. W., 203 [69], 217
Emch, 207 [88], 218
Emodi, G., 75 [65],76 [65],81
[65],82 [81], 86, 124 [51],129
[56,57],133 [61,68],135 [57],
142
Enders, J. 8 [81], 18
Engel, W. 180 [51], 186,212
[141], 220
Engelhardt, D. L., 9 [96], 18
Epstein, L. 38 [78], 56,103
[104], 111, 265 [9], 279 [9], 287
[71], 290, 293
Epstein, R., 279 [52], 292
Ermolieva, Z. 198 [25], 216
Esteban, R. 9 [91], 18
Everson, L. 129 [56,57], 135
[57], 142
Eveson, L. 82 [81], 86
F
Fadeeva, L., 2 [3], 15, 198 [25],
216
Falcoff, 38 [75], 39 [75], 55,
67 [47], 68 [47], 85, 90 [24], 108,
172 [30], 184
Falcoff, R., 38 [75],39 [75], 54,
67 [47],68 [47], 85, 90 [25,26],
103 [106], 108, 111
Falcon, G., 121 [41], 141
Fantes, 3 [20], 16
Fauconnier, 13 [151,152], 20
Feldman, S., 123 [50], 142
Fenichel, G. 212 [138], 220
Ferruti, 240 [33], 249 [33],
258
Fialkow, J., 94 [59],95 [59],
109
Field, 3 [15], 5 [15,38],
16,99 [83], 110, 146 [14],147
[14],149 [32,33],160 [32,33],
163, 164, 169 [9,11,12,13,14,15],
183, 265 [3], 290
Field, J., 119 [27J, 141
Fink, 187 [1], 215
Finkelstein, J., 3 [17J, 16
Finkelstein, S., 67 [44J, 85,
240 [5], 244 [5], 245 [5], 252
[5J, 253 [5],254 [5], 257, 259
INI>I';X
l<'inogCllova, V., 201 154], 217
Finter, N. 13.,67[46], 14(i] ,
85,116[9], 118 120 [35],
122 [35], 140, 141,265 [1],270
[ 1], 290 [1], 290
Fischer, G. W., 256 [99], 261
Fisher, 255 [97], 261
Fitzpatrick, 207 [99,100], 219
J. F., 201 [49], 217
Flallagan, S. 94 [49], 108
Fleming, R. W., 147 [20], 163
Fleming, W. 11 [123], 19, 82
[82J, 86,191 [8,9,10,12,13,14,
15,16],201 [8,9,10,12,13,14,
15,16,39], 215, 216
Fletcher, J., 207 [101], 219
Fogh, J. 92 [47],93 [47],
94 [47], 96 [47], 108
Fontaine, D., 67 [47], 68 [47],
85, 90 [24], 108
Fontaine-Brouty-Boye, D., 11
[126], 19, 90 [25,26],91 [30],
105 [116], 108, 111
Fornosi, F., 201 [64], 217
Franchi, G., 240 [31],249 [31],
258
Francis, Jr., 169 [6], 183
Freeman, I., 99 [81], 110
Freeman, J. 245 [69], 256
[69], 259
Freidlaender, G. 207 [93],
218
Freshman, 146 [7], 163
Fried, R. 75 [66,67],76 [66,
67], 81 [66], 86, 120 [36], 121
[36], 122 [36], 123 [48,49], 133
[36,48], 141, 142
Friedman, 103 [102], 111
Friedman, R. 8 [74],9 [91],
10 [110,121], 12 [138], 18, 19,
20, 119 [22], 125 [22], 140, 146
[11], 163
Friedman-Kein, 170 [23],
184
Fritsche, R., 94 [53], 109
1\11'1'1 J( II( INI )I';X
N., [14,15],
D. 149 [35], 164, 174
[40], 185
F. 240 [39J, 248
[79], 250 [39], 258, 260
Fumarola, D., 207 [108], 219
Fuse, 90 [19],91 [19,39,45,
46], 105 [45,46], 107, 108
G
Gaffney, V., 91 [35J, 108
Gagoni, 5 [41], 198 [30J,
199 [30], 216
Gaitmaitan, G., 228 [9J, 229
[9], 233 [9], 236
Ganguly, N. 129 [55], 142
Ganguly, R., 229 [11], 230 [11],
232 [14], 233 [14], 234 [11], 236
Garattini, 240 [31], 249 [31],
258
Gauntt, J., 6 [43], 9 [102], 16,
18
Gaur, V. 214[151], 221
Gazdar, F., 208 [111,112,113],
209 [111], 219, 248 [81], 256
[81], 260
Gelber, R., 213 [143], 221
Gelboin, V., 172 [27], 184
Georgiades, J. 34 [25J, 52,
265 274 [33J, 291
Gerard, G. F., 214 [154,155], 221
Gericke, D., 214 [153J, 221
Gerin, J. L., 177 [44], 185
Gerone, J., 227 [7], 236
Gerson, J. 94 [63], 109
Giambrone, J., 207 [101], 219
Gianinazzi, 251 [91], 260
Gibbs, J., 172 [38J, 185
Gibson, J. 161 [63], 165, 197
[20,21J, 205 [71],206 [71],207
[20,21,71,87,89], 215, 216, 218
Gifford, G. 103 [105], 111
Gilbert, J., 137 [84], 143
1)., 115 [5], 118 [G], 140
J:<;. J., 82[84],
Giovanella, 94 [58, GO,
95 [60], 109
Giron, D. J., 154 [54], 164, 201
[40J, 214 [40J, 216, 240 [2,3,
43],249 [43J, 252 254 [2],
255 [3], 257, 259
Gisler, R. 270 [23], 273 [23],
291
Gitnick, F. L., 276 [42J, 292
Glasgow, L. 7 , 8
[72],13 [145,151], 17, 20, 82
[85J, 87,115 [5],116 [10J, 140,
146 [12J, 149 [37], 150 [37],
155 [58,60], 163, 164, 165, 192
[17],194 [17],198 [17], 199
[33,76,77],200 [33,76,77],201
[33,41], 215, 216, 218, 240 [16],
252 [16],253 [16],254 [16,95],
255 [16], 256 [16], 258, 260
Glaz, 146 [18],153 [51],
163, 164, 198 [28], 200 [35], 216
Goldin, 240 [32], 249 [32],
250 [32], 258
Goldon, F., 105 [114], 111
Goldstein, 187 [7], 206 [74],
215, 218
Golgher, R. R., 3 [27], 16
Good, R. 129 [56], 142
Goodell, 241 [50,51], 259
Gordon, F. 2 [13], 15, 207
[105],219
Gorman, R. R., 12 [134], 20
Gothoskar, 100 [90], 110
Gotz, 214 [149,153], 221
L., 35 [26], 52
Grady, D., 45 [54], 54, 62
[28], 63 [28], 64 [36], 84, 85
Graff, S., 90 [28], 108
Grangenett, D. 214 [154],
221
Granger, 213 [145], 221
Grant, 91 [35], 108
Graziadei, W. D., 9 [103], 19

Green, I., 111,265 [8],
279 [8], 290
Green, J. 103 [103], 111, 146
[13], 163,265 [6], 290
Green, 214 [154,155], 221
Green, R., 214 [154,155], 221
Greenberg, 114 [3], 125 [3],
133 [3],136 [3], 139
Greenberg, L., 82 [83], 87, 133
[67], 142
Greenberg, S. 65 [42], 66 [42],
74 [63], 75 [63], 77 [63], 79 [63,
70],81 [78],85,86
Gregoire, lo[ 117], 19
Gregory, 136 [82], 143
Gregory, 114 [3], 125 [3],
133 [3], 136 [3], 139
Greiff, 63 [34], 85
Greiff, D., 63 [34], 85
Gresser, 1., 11 [126,127,129], 19,
20,31 [20,22],46 [64], 52, 55,
67 [47],68 [47], 85, 89 [2,3],
90 [2,3,4,5,6,7,10,11,17,21,23,
24,25,26,27], 91 [21,30,31,32],
92 [21], 105 [112,113,116,117],
107, 108, 111, 117 [13], 119 [26,
29,30,31,32],133 [66], 142, 172
[30], 184, 270 [23], 273 [23],
281 [75], 291,
292, 293
Gressmerova, 58 [19], 84
Griffin, W., 240 [23),250 [23),
252 [23],253 [23], 258
Griffith, J. F., 201 [49], 217
Grimes, 129 [56], 142
Grisar, J. 147 [20], 163,191
[9,10,14,15,16],201 [9,10,14,
15,16,39], 215, 216
Groeike, J. W., 205 [70], 218
Grossberg, S. 59 [26], 61
[26],62 [29,30,31],63 [26,30,
31],64 [30,31],67 [31], 84,
170 [22], 184
Gruenewald, R., 208 [114], 219
Grunwell, J. F., 191 [15],201
[15], 215
I NI
11!) 1;31 J ,
154[31], 1:31],161[31],
164, 172 [34], 181
Gunwell, J. F., 147 [21], 163
Gurari-Rothman, D., 6 [56], 17,
28 [14],29 [14],38 [14], 45
[56], 46 [56], 51, 54, 58 [14],
84, 100 [93], 110, 120 [34], 141
Gupta, S. L., 9 [92,103], 18, 19,
129 [56], 142
Gurgo, 214 [154], 221
Gvazava, 1. S., 198 [25], 216

Haahr, S., 38 [76], 55
Habif, D. V., 71 [59], 74 [59], 86
Hadhazy, G., 21 [1], 24 [1], 50
Haff, R. F., 71 [60],74 [60], 86
Hagan, 201 [43], 204 [43],
216
Hajnicka, V., 90 [14,15], 107
Hake, D. 213 [145], 221
Halaas, G. W., 212 [140], 220
S., 74 [62],77 [62],78
[62], 86, 122 [1], 139
W. J., 227 [5], 236
Hallermann, 121 [45], 141
Halliday, 1. 81 [79],
Hamilton, L. D., 246 [74], 252
[74], 260
Hamilton, R., 29 [18],34 [23],
37 [18], 51, 52
Hammer, 24 [3], 50
Han, 275 [39], 292
Hal1sen, J., 129 [56], 142
Hal1sol1, 14 [153], 20
Harmel, R. 240 [40], 251
[40], 259
Harmon, W., 65 [42], 66 [42],
74 [63], 75 [63],77 [63], 79
[63,70], 85, 86
Hashim, G., 207 [97,98,99,100],
219
INIH:X
1.<;. 3 l.28,.29], 4 l31],
16, 34 [.24], 35 [.26], 45 l5.2], 48
[69], 52, 54, 55, 58 [7,8,10], 59
[24], 60 [24], 62 [24], 63 [10],
83, 84, 100 [94], 110, 155 [57],
164, 265 [11], 291
Hawkins, W., 240 [10],252 [10],
257
Hayashi, 94 [54], 109
Hayes, G., 4 [31], 16
Heijtink, R. 125 [52,53,54],
136 [83], 142, 143
Heine, U. 1., 275 [36], 291
Hejna, 1., 27 [11], 51
Hejura, J., 36 [29], 52
Heller, 5 [34], 16
Henle, W., 89 [lJ, 90 [1], 107
282 [68], 293
Heremans, 34 [25], 52, 119
[23],125 [23],135 [71], 140, 143
Hernandez, R., 75 [65],76 [65],
81 [65], 86,124 [51],133 [61],
142
Herniman, J., 240 [10], 252
[10], 257
Heron, 1., 29 [18], 34 [23], 37 [18]:
51, 52, 281 [62], 293
Herrin, J. 137 [84J, 143
R., 147 [20], 163
Hickman, J., 79 [77],80 [77],86
Hilfenhaus, J., 90 [18],91 [18,36,
42], 92 [18], 94 [18], 107, 108,
117 [15], 140
D. 172 [34], 184, 224 l.2],
236
J. 211 [137], 220
R., 3 [15], 4 [33],
5 [15,38], 146 [14],147 [14],
149 [32,33],160 [32,33], 163,
164,169 [9,11,12,13,14,15], 183,
.240 [30], 252 [30], 257 [102],
258, 261, 265 [3], 290
G., 9 [99], 18
Hilmas, D. 179 [47],180 [49],
186
Ilimmelweii, }'., 169 l10], 183
S., 94 l54], 109
Hirsch, S., 137 l84], 143, .240
[.26,27],250 [26,27],25.2 [26,
27],254 [26], 255 [26,27], 258
281 [59], 282 [67], 292, 293
R., 133 [61], 142
J. R., 76 [65],81
[65], 86
38 [79], 56
S., 22 [41,71]'" 42 [71],
53, 55, 100 [88], 110
Hitchcock, G., 2 [5], 15
2 [8],3 [23],7 [60],
8 [73,81], 12., 17, 18,58 [1],
58 [2],59 [61],69 [51],73 [51],
74 [61], 83, 84, 86, 117 [14],
133 [59], 1.1.2., 142, 146 [9], 147
[9],149 [40],152 [44,45,49],
153 [45], 164, 199 [32], 200 [34],
216,240 [46],244 [57,59,63,64],
245 [64,67],246 [46],247 [46],
248 [46], 259, 265 [12], 291
Hoffman, J. W., 191 [16],201
[16], 215
Hoffman, F., 187 [6],205 [6],
215
Hoffman, W. W., 146 [17], 147
[17], 163, 224 [3], 225 [3], .226
l3], 236
Hofmann, 201 [53,66], 217
Hogan, W., .231l13], 236
Holland, J. F., 245 [103], 256
[103], 261
169 [10], 183
Holmes, J. 10 [121], 19
Holtermann, D. 155 [57],
164
Holyoke, 95 [69], 96 [69], 98
[69], 109
172 [34], 184
Hooks, J. J., 269 [20], 291
Hopps, 2 [12], 15, 146 [5],
163
Horak, 1.,9 [98], 18
llorgan, S. W.,
[8,9,10,12,13,39], 215,
201 [42], 216
Horoszewicz, J. S., 92 [48], 95
[69],96 [69],98 [69],100 [48],
101 [48], 102 [48], 103 [48], 108,
109,136 [80,81], 143, 160 [62],
165, 181 [53], 186
Horrobln, D. F., 120 [33], 141
Horten, 95 [68], 109
Houchens, D., 95 [66], 109
Houston, W. 179 [46], 185, 207
[102], 219
Huang, S., 9 [97], 18
Huang, J. W., 27 [11,13],36 [29],
51, 52
Huang, 2 [13], 11 [125], 15,
19,105 [114], 111,207 [105], 219
Hubbard, S. 99 [85], 110, 172
[35], 185
R., 197 [23], 216
Hummeler, 94 [63], 109
Hunt, J. 9 [96],
Hutchinson, D. W., 46 [61], 54
1
S., 269 [20], 291
Ikic, D., 120 [37,38,39],121 [39,
46,47], 141, 142
Imanishi, J., 105 [115], 111
Ingimarsson, S., 96 [75,76,97],
109, 110
Isaacs, 1 [1], 2 [1,2,3,4,5],
12 [137], 13 [147], 15, 20, 38
[74], 55,146 [1], 162, 167 [1],
168 [4], 182, 265 [2], 290
Iscove, N. N., 133 [68], 142
Ishizuka, 240 [45], 259, 276
[ 45,46], 292
Israel, L., 213 [144], 221
Ito, 7 [64],11,90 [16],92
[16,48],93 [16], 95 [69], 96
[69],98 [69],100 [48],101 [48],
INIH:X

[413], [413], 107, 1013,
109, 149 [39], 153 [39], 154 [39],
164
Ito, 244 [61], 259
J
Jackson, G. G., 228 [9,10],229
[9,10],230 [10,12], 232 [12],
233 [9],234 [12], 236
Jackson, L., 103 [109], 111, 265
[8], 279 [8], 290
Jaeger, 0., 117 [15], 140
Jakobsson, 75 [64], 76
[64], 81 [64], 86
Jakobsson, 96 [71,74,75],
97 [76], 109, 110, 133 [62], 142
Jameson, 62 [31], 63 [31],
64 [31],67 [31], 84,170 [22],
184
Janetta, J., 133 [59], 142
Jankowski, W. J., 27 [9,10,12],
36 [9,10,32], 51, 52,58 [15),
84, 101 [97,99], 110
Jaraskova, L., 275 [38], 291
JariwaHa, R. J., 62 [29,30],63
[30], 64 [30], 84
Jensen, 6 [53], 17
Jimenez, L., 275 [38], 292
Jirillo, 207 [108], 219
Job, L., 95 [69], 96 [69],98 [69],
109
Johannsen, R., 91 [42], 108
Johansen, S., 207 [92], 218
Johansen, S., 207 [92), 218
Johns, D. G., 209 [126], 220
Johnson, G., 246 [77], 260,
275 [39,40,41], 280 292
Johnson, 6 [54], 17, 265
10, 14, , 103 [102], 111,
270 [24], 271 [25,26], 272 [25],
273 [14,25], 274 [10,33,34], 276
[48], 277 [48], 278 [14), 279
1111'1'1 11{ INI>I';X.
11.
[10,14,53],280 [10,14], 281[25],
282 [25,69],283 [69], 284 [70],
285 [70], 286 [70],287 [69,70],
288 [10], 290 [48,14], 290, 291,
292, 293
Johnson, L., 13 [143], 20, 118
[20], 140, 152 [46,47], 164
Johnson, 74 [63],75 [63],
77 [63],79 [63,70], 86
Johnson, 95 [66], 109
Johnston, L., 245 [103],256
[103], 261
Joklik, w. 8 [76,80], 18
Jones, 118 121 [41],
140, 141
Jones, 146[6], 163
Joniau, 59 [23],60 [23],62
[23,27],63 [27],64 [23], 65 [23,
23], 84
Jordal, 24 [3], 50
Jordan, G. W., 38 [76], 55, 59
[22],61 [22], 75 [66,67], 76 [66,
67[, 81 [66], 84, 86, 120 [36],
121 [36], 122 [36], 123 [48,49],
133 [36,48], 141, 142
Joyce, G., 119 [27], 141
Jungwirth, 9 [98,99], 18
Jusic, D., 120 [37,38,39], 121 [39,
46,47], 141, 142
Just, 75 [65],76 [65],81[65],
82 [81], 86, 124 [51], 129 [57],
133 [61],135 [57], 142

J. 41 [62],46 [62],54
J., 6 [45], 17
120 [33], 141
94 [54,62], 109
Kanaday, J., 194 [19], 215
Kapikian, Z., 172 [34], 184
Kapila, 240 [34],249 [34], 258
Kaplan, 177 [44], 185, 209
[129], 220, 240 [25,36,38,46],
:I()'i
1 Kaplal1,
246 [3t:1,46,72], 116],
[25,46,t:l2], 250 [36,38],251
[25,38,85,86,87],252 [25],253
[36], 258, 259, 260
Karakousis, 95 [69], 96 [69],
98 [69], 109
Karges, 90 [18],91 [18,
36],92 [18],94 [18], 107, 108
Karmali, 120 [33], 141
Kasel, J. 227 [7], 236
Kassan, S. S., 277 [49], 292
Kassanis, 251 [91], 260
Kassel, 90 [28], 108
0.,90 [28], 108
Katz, 201 [50], 203 [68], 217
Katz, L. J., 90 [9], 107
Kauffman, 211 [136], 220
Kaufman, 117 [18], 121
[40], 140, 141, 149 [31], 160
[31], 164, 198 [26], 216
Kavetsky, 207 [94], 218
Kawade, 90 [12,13], 107
Kawakita, 9 [95], 18
10 [109], 19
81 [79], 86
3 [23], 7 [64], 16,11,
200 [34], 216, 244 [63], 259
Keith, 119 [27], 141
J. L., 256 [99], 261
Kelsey, D. 8 [72], 17, 155
[60], 165, 199 [33], 200 [33],
201 [33], 216
Kelvin, 181 [54], 186
Kern, 8 [72], 17, 116 [10],
140,155 [60], 165, 199 [33],
200 [33], 201 [33], 216, 240
[16], 252 [16], 253 [16],254
[16,95],255 [16], 256 [16],
258, 260
Kerr, 1. 9 [91],10 [111,112],
18, 19
S., 103 [103], 111, 146
[13], 163, 265 [6], 290
Kihm, J. 191 [12,14], 201
[12,14], 215
Kilbourne, 1<:. D., 169 l8], 183
Killander, D., 281[63], 293
Killen, 11 [123], 19
Killon, 82 [82], ...
L. S., 269 [19], 273 [19],
288 [19], 291
J. G., 129 [55], 135
[70], 142, 143
R., 187 [7], 206
[74], 215, 218
Kirchner, 38 [79], 56
R., 67 [45], 68 [45],
85
Kirschstein, R. L., 13 [144], 20,
172 [34], 184
105 [115], 111
94 [59], 95 [59], 109
G., 94 [59],95 [59],100
[90], 109, 110
W. J., 2 [9], 15,44
[47,48], 53, 54
S. 207 [101], 219
G. 103 [110], 111
Jr., 35 [27], 37 [27],
45 [27,57], 52, 54, 58 [17], 59
[17],61 63[17], 84, 90 [8,
22], 91 [22], 92 [22], 107
227 [7], 236
Kobayashi, G. 256 [99], 261
Kobayashi, S., 245 [68], 259
Kogan, 201 [54], 217
Kohn, L. D., 10 [121], 19
Kohno, 2 [12], 15, 146 [5], 163
Kohno, S., 2 [12], 15,146 [5], 163
Koide, 94 [54], 109
Kojima, 167 [2], 182, 244 [62],
259
Kono, 152 [44,45], 153 [45],
164, 244 [57], 259
Koprowski, 14 [153], 20
Korneeva, L. 198 [25], 216
Korst, J. J., 146 [17], 147 [17],
163, 224 [3], 225 [3], 226 [3],
236
Kowakita, 10 [107], 19
INIH;X
L. 11., I :!2], I :!2],
164
L. J., 240 l31], 2;19
[31], 258
170 [23], 184
Kripke, L., 248 [80J, 251 [80],
256 [80], 260
Kropachev, 201 [54], 217
Krueger, R. F., 2 [14], 16, 146
[16],147 [16], 163, 187 [2,3,4,
5],197 [22,23,37],198 [31],
200 [37], 201 [4,22,31,37,46],
213 [148], 214 [37], 215, 216,
217, 221
Kuck, N. 208 [116], 219
Kuehne, R. W., 201 [44], 217
Kuhner, L., 274 [31], 288 [31],
291
Kujima, 146 [2], 162
Kunji, 149 [39], 153 [39],
154 [39], 164, 244 [61], 259
Kunz, 201 [53,66], 217
Kuperminc, 211 [133], 212
[133,142],213 [143,146], 220,
221
Kuwata, 90 [19],91 [19,39,
45,46], 105 [45,46], 107, 108
L
La Bounardiere, 40 [80], 56
Ladarola, J., 172 [38], 185
Lademann, 3 [18], 16
Lagergren, 97 [77], 98 [77],
110
Laibson, R., 121 [40], 141
Lam, 133 [59], 142
Lamaitre-Moncuit, J., 10 [117],
19
Lameijer, L. D. F., 131 [58],
133 [58], 142
Lampkin, 211 [134], 213
[145], 220, 221
1\11'1'11( 111. INIH;X
G. 3 [15], 5 [15,38],
16, 146 [14], 147 [14], 149 [32,
33], 160 [32,33], 163, 164, 169
[9,11,12,13,14,15], 183, 265 [3],
290
Lancius, J. F., 106 [121], 111
Landstrom, L. 281 [64], 293
Langford, 280 292
Larner, 281 [64], 293
Larsen, 26 [7], 51
Larson, V. 240 [30], 252 [30],
258
Larsson, I., 281 [63], 293
Law, L. W., 172 [26], 184
Lawrence, W., Jr., 246 [73],254
[73], 260
Leary, 11 [129], 20, 90 [7],
107,281 [55,63], 292, 293
Leavitt, J., 245 [69], 256 [69],
259
Lebleu, 9 [95], 18, 10 [107,
108,109], 19
Lederer, W. 265 [7], 290
Le Du, G., 136 [78], 143
Lee, G., 10 [121], 19
Lee, S. S., 90 [9],91 [34],
107, 108, 269 [19], 273 [19], 281
[56], 291, 292
Leibowitz, I., 136 [80,81], 143
Lengyel, 9 [92,95,103], 10
[107,108,109], 18,
Leonard, J., 274 [32], 291
Leong, S. S., 92 [48],95 [69],96
[69], 98 [69], 100 [48], 101 [48],
102 [48], 103 [48], 108, 109, 136
[81], 143
Leonhardt, 6 [49], 17
Lerner, 180 [50], 186
Leunen, J., 240 [8], 244 [8], 252
[8], 257
Levin, J., 9 [104,105], 19
Levine, 99 [85], 110
Levine, S., 172 [35],179 [48],
180 [48], 185
Levillc, 8 [75], 18, HilIGG],
165,206[79],207[85,89,90],
208 [114], 219
Levny, J., 181 [53], 186
Levy, 9 [89,90], 11 [124],
18, 19, 99 [85], 110, 117 [11],
140,149 [35], 164, 169 [17],
172 [26,27,31,33,34,35,37,38],
174 [39,40,41], 177 [44,45],
179 [46,47,48], 180 [48,50,51],
183, 184, 185, 186,207 [102],
212 [ 141], 218, 219, 220, 269
[16],273 [16],281 [65], 291,
293
Levy, J., 160 [62], 165
Levy-Koenig, R. 3 [27], 16
Lewis, V. J., 169 [7], 183
Leyten, R., 240 [29], 241 [29],
250 [29], 252 [29], 258
Lichtenstein, L. 282 [68], 293
Lieberman, 69 [50],73 [50],
85, 240 [12], 241 [12], 244 [12],
245 [12],252 [12], 257
Lin, L. S., 3 [26], 16
Lindahl, 11 [127,129], 19, 20,
270 [23],273 [23], 281 [58,63],
291, 292, 293
Lindahl-Mag'nusson, 90 [7],
107, 281 [55], 292
Lindenberg, J., 24 [3], 50
Lindenmanll, J., 1 [1], 2 [1,2],
15,38 [74], 55,146 [1], 162,
167 [1], 182
R., 6 [56], 17, 120
[34], 141
J., 9 [98], 18
Ling, V., 103 [100], 110
Linneman, 210 [132], 211
[135,136], 220
Lipton, R., 71 [59], 74 [59], 86
Little, J. W., 227 [5], 236
Liu, L. S., 31 [21], 46 [73], 52,
55
Lockart, R. Z., Jr., 3 [19],6
[44],8 [78], 16, 18
:!]
Loekhart, lC Jr., 4 L 32], 1(j
Lombardi, S., 199 [76], 200
[76], 218
London, W., 149 [35], 164, 172
[38], 174 [40,43], 185
Long, W. F., 5 [39], 16
Lost, F. 169 [10], 183
Lowenberg, 133 [69), 143
Lowy, D. R., 65 [38], 85
Lueas, D. 103 [108,110], 111
Ludwig, 197 [22], 201 [22],
216
Luetzeler, J., 240 [37], 250 [37],
251 [37], 253 [37], 258
Lundgren, 281 [63], 293
Lundin, L., 281 [63], 293
Luoni, G., 214 [151), 221
Luovsky, 177 [44], 185
Lutwiek, L. 1., 81 [78], 86, 114
[3], 125 [3], 133 [3], 136 [3],
139

Maell, J. 94 [53], 109
Maeieira-Coelho, 90 [4,5,10],
107
Maehara, N., 244 [64], 245 [64,67],
259, 265 [12], 291
Maguire, Jr., 94 [55,63], 109
Majer, 11 7 [15], 140
Makal, 136 [82], 143
Makano, 206 [80], 218
Malaise, 90 [23], 108
Mandel, D., 269 [21], 291
Mandell, 65 [41], 85
Manders, 9 [97], 18
Manejias, 207 [106], 219
Mangeafieo, J. 179 [47], 186
Manku, S., 120 [33), 141
Manthey, F., 72 [88], 87, 90
[18], 91 [18, 92 [18), 94 [18),
107, 121 [42], 141
Marbrook, J., 271 [27],273 [29],
291

J. ,J., 27(; 147], 2D2
Mareus, 1., 9 [ti6, ti7, 96,
100], 18
Margalitll, 201 [50], 203 [68],
217
Markowitz, 276 [42], 292
Marks, J. 169 [10], 183
Maroney, 10 [113], 19
Marshall, L. W., 59 [20], 60
[20], 62 [20], 84, 149 [29], 160
[62], 163, 165, 181 [52,53], 186
Marx, Jr., 254 [94], 260
Massanari, R. 206 [84], 218
Mastrangelo, J., 106 [121],
111
Masurel, N., 131 [58], 133 [58],
136 [83], 142, 143
Masuzumi, 245 [68], 259
Matsuo, 105 [115], 111
Matsuzawa, 90 [13], 107
Maury, 105 [112], 111, 119
[29,30,31,32], 141
Mayer, G. D., 2 [14], 16, 146
[16], 147 [16,20], 163, 187 [1,
2,4,5,12,13,14,16],191 [12],
197 [22,23],198 [27,31],199
[27,31,37],200 [37],201 [46,
51],204 [43,51],210 [132],211
[134,135],213 [148], 214 [37],
215, 216, 217, 220, 221
MeAliffe, V. J., 177 [44], 185
R., 207 [99,100], 219
MeCleeland, L., 169 [7], 183
MeCloskey, R. V., 13 [144], 20,
67 [45[,68 [45], 85
MeCord, S., 240 [14,15],252
[14,15],253 [15],254 [14,15],
255 [14], 258
MeCullough, 161 [64], 165
MeGeorge, 240 [17],252
[17],253 [17],254 [17], 258
MeGill, J. 1., 118 140
MeNeill, 11 [123], 19, 82
[82], 86, 133 [66], 142
Medearis, D. N., 155 [59], 164

Mcgcl, 11., 187 [7], 197 [20,21],
205 [71,72], 206 [71,74], 207
[20,21,71,72,86,91,96],208 [72],
215, 216, 218, 219
Melmon, L., 279 [54], 282 [68],
292, 293
Mendelson, J., 13 [151], 20
Mendelson, R., 129 [55], 142
Merenda, 94 [64], 109
Merigan, 3 [16,17], 16,38
[76],44 [47,48], 53, 54, 55, 58
[3],67 [44,49],68 [49],69 [47],
74 [62],75 [66,67], 76 [66,67],
77 [62,80], 78 [62],81 [66,78],
82 [80], 83, 85, 86, 87, 99 [84],
103 [104], 110, 111, 114 [3],120
[36],121 [36],122 [1,36],123
[48,49,50], 124 [49], 125 [3],
133 [3,36,48], 136 [3,82], 139,
141, 142, 143, 146 [7,15], 147
[15],149 [24,25,30],153 [50],
163, 164, 170 [21], 183, 192 [18],
194 [18], 197 [18,24], 198 [18],
199 [18],200 [18],201 [18,47,
48],214 [18], 215, 216, 217, 218
224 [1], 236, 240 [5,11,41],244
[5,54],245 [5,69], 249 [41],251
[52,54],252 [5,11],253 [5,11],
254 [5], 256 [69], 257, 259, 269
[17,18,19,22],273 [18,19],279
[54], 288 [19], 291, 292
Merrell-National Laboratories, 209
[117,123],210 [117,119,131],
219, 220
Merritt, 275 [39], 292
Metz, D. 8 [84], 9 [91,104],
18, 19
Meyer, D. R., 147 [21], 163, 191
[15],201 [15], 215
Meyer, R. F., 121 [40], 141
Meye.rs, W., 119 [22], 125 [22],
140
Michael, J. G., 187 [7],197 [21],
207 [21], 215, 216
Miller, 82 [81],...
:111
Miller, 213[144], 221
Mills, J., 172 [34], 184, 224 [2],
236
Mims, 67 [48], 68 [48], 85
Mims, S. J., 10 [115], 19
Minks, 10 [113], 19
MiOrer, 281 [64], 293
Mitchell, S., 207 [93), 218
Mobraaten, L. 207 [95], 218,
281 [57], 292
Mogensen, 22 [71], 26 [43],
28 [15],29 [15),33 [42], 38
[15],41 [44], 42 [71], 43 [44),
44 [44), 46 [60], 47 [15], 48
[70], 51, 53, 54, 55, 58 [6,16],
59 [25], 60 [25], 63 [32], 70
[54], 73 [54], 79 [16],81 [54],
83, 84, 85, 100 [88,89,91], 110
Mogoc, J., 274 [30], 291
Mohr, S. J., 207 [103], 219, 240
[39], 247 [78], 248 [78,79], 250
[39,84],256 [84), 258, 260
Monaco, 240 [26,27], 250
[26,27],252 [26,27],254 [26],
255 281 [59], 292
Monno, R., 207 [108], 219
Moore, D. 90 [29], 108
Moore, S. 174 [39], 185
Moorhead, S., 94 [63], 109
Morahan, S., 209 [129], 220,
240 [1,14,15,16,17,24,25,36,
38],245 [70], 246 [1,38,70,72,
74],248 [24,25],250 [36,38,70],
251 [24,25,38,85,86,87,89],252
[1,14,15,16,17,24,25,70,74],
253 [1,15,16,17,24,36],254 [1,
14,15,16,17,24,70,89,95],255
[14,16,89],256 [16,24,70),
257, 258, 260
Morgan, 94 [61], 109
Morgan, W., 59 [61],74 [61],
..., 117 [14], 140
Morgan, R. 0., 120 [33], 141
Morel-Maroger, L., 119 [30],
Morell, G., 79 [75], 81 [75],
86
Mori, N., 244 [61], 259
Morinaga, N., 91 [45,46], 105 [45,
46], 108
Morris, 209 [120,121], 220
Morser, J., 46 [61], 54
Mortelrnans, J., 100 [87], 110
Mosher, 207 [93], 218
Mosier, D. 277 [49], 292
Mosny, S. 82 [83], .1, 133
[67], 142
Moulton-:-R., 274 [33], 291
Mozes, L. W., 35 [26], 52, 58 [7],
83
Mukainaka, 105 [115], 111
Miiller, 0., 121 [42], 129 [57],
135 [57], 141, 142
Mundy, R. L., 201 [45], 217
Munno, 1., 207 [108], 219
Munson, 187 [5],206 [75],
207 [5], 215, 218, 240 [1,35],
245 [70,71],
249 [35],250 [35,70],252 [1,70,
74],253 [1],254 [1,70,73],256
[70], 257, 258, 260
Munson, J. 187 [5],206 [75],
207 [5],209 [129], 215, 218, 220,
245 [71], 250 [36],253 [36], 258,
260
Murphy, R., 13 [145], 20
Murphy, 2 [9], 15
Murphy, F. 7 [67], 17
J. J., 240 [6,13,19],
252 [6,13,19],253 [6],254 [6],
257, 258
N
Nagai, 94 [54], 109
Nagano, 146 [2], 162, 167 [2],
182
Nagata, 1.,149 [39],153 [39], 154
[39], 164,244 [61], 259
Nahmias, J., 256 [98], 261
INIH;X
27:> [3tJ], 292
Nakoil1z, 1., 279 l52], 292
Namba, 26tJ [15], 2tJtJ [15],
291
Nash, 59 [61], 74 [61], 86,
117 [14], 140
Naylor, R., 169 [17], 183
Nei1, G., 95 [66], 109
Nelson, D. S., 275 [37], 291
Nemes, 5 [38], 149
[32,33], 160 [32,33], 164, 169
[9,12,15], 183 -
Neri, 6 [55], 17,287 [72],
293
Nesburn, 121 [40], 141
D., 72 [88],
87, 117 [16,17], 121 [42,43,44,
45], 140, 141
Nevanlinl1ia, R., 21 [1], 24 [1],
50
Newberg, N. R., 241 [49], 245
[49], 246 [49], 252 [49], 254
[49], 256 [49], 259
Newberl1e, J. W., 161 [63], 165,
207 [87], 218
Ng, 170 [23],
Ngan, J., 269 [19], 273 [19], 281
[56],288 [19], 291, 292
Niblack, J. F., 146 [17], 147 [17],
163,224 [3],225 [3],226 [3],
236, 237
Nichol, F. R., 146 [19], 147 [19],
163
Nikolaeva, D. V., 198 [25], 216
Nilsonne, U., 96 [74,75],97 [76],
109, 110
Nissen, 133 [68], 142
Nolan, J. 136 [80], 143
Nomura, 94 [54,62], 109
Nordlund, J. J., 172 [37], 185
Notkins, L., 269 [20], 291
Novokhatsky, S., 153 [52], 164
Nuwer, R., 67 [49], 68 [49],
85
))1, )N)jJ':X

Odenwald, V., 246 [75,76], 260
Ogburn, 28 [15],29 [15,16,
17],38 [15], 44 [50], 46 [17,66],
47 [15,66],48 [66,68], jU, 54, 55,
58 [6,8,9,13], 83, 84, 265 [11],
291
Ogburn, 3 [28], 16
Oh, J. 0., 82 [84], 87
Ohsawa, N., 94 [62], 109
Ohwaki, 90 [12], 107
Olivie, 14 [154], 20
Olsen, G. 116 [10], 140
Olson, 245 [103],256 [103],
261
Olson, w. 212 [140], 220
J. 27 [9,10],36 [9,
10], 51, 58 [15], 84, 99 [81],101
[99], 110, 160 [62], 165, 181 [53,
54], 186
R. J., 82 [81], 86, 129
[56,57], 135 [57], 142
Orfeo, 92 [47],93 [47],94
[47], 96 [47], 108
Osborn, J. 155 [59], 164
Osborne, L. 265 274
[33], 291
O'Shaughnessy, V., 90 [9], 91
[34], 107, 108
Osther, 24 [3], 50
Ottenbrite, R., 241 [50,51], 259
Outzen, 94 [55], 109
Ovejerra, 95 [66], 109
Overall, J. Jr., 82 [85], .1,
116 [10], 140, 199 [76], 200 [76],
201 [41], 216, 218
Oxman, N., 9 [104,105,106],
10 [106], 19, 118 [19], 140
Ozzello, L., 94 [53], 109

Pacini, 79 [72,73], 86
Pahwa, R., 129 [56], 142
Pahwa, s., 129 [5(;], 142
Palmer, 177[44], 185
103 [102], 111
Pannier, W. L., 174 [41], 185,
201 [44,45], 217
Panusarn, 229 [10],230 [10],
236
PapamatheakiS, J. D., 11 [131],
20, 240 [37], 250 [37], 251 [37],
253 [37], 258, 275 [35], 291
Papas, S., 253 [93], 260
Park, J. 170 [24], 184
Pascale, 240 [12], 241 [12],
244 [12], 245 [12], 252 [12], 257
Path, F. R. 106 [122], 111
Paucker, 3 [28], 16, 26 [43],
28 [15J, 29 [15,16,17], 38 [15J,
44 [50], 46 [17,65,66], 47 [15,
66], 48 [66,68], 51, 53, 54, 55,
58 [4,6,8,9,13,19],63 [33],64
[33], 83, 84, 85, 89 [1],90 [1],
107, 218, 265 [4,11], 269 [17],
290, 291
Paulidis, N. 11 [132], 20
Pazin, G. J., 133 [59], 142
Pearson, J. W., 104 [11F. 111
207 [104], 219, 240 [23], 250
[23], 252 [23], 253 [23], 258
D. L., 103 [105], 111
Perkins, J. 224 [2], 236
Person, D. 276 [42], 292
Pessina, G. 79 [72,73], 86
Petersen, 24 [3], 50
Petricevic, 1., 120 [37], 141
Phair, J. 211 [136], 220
Picciano, 91 [35], 108
Pierce, W., 273 [28], 274 [31],
279 [51], 288 [31,73], 291, 292
293
Pindak, F. F., 154 [54], 164, 201
[40],214 [40], 216, 240 [2,3,
42,43],249 [42,43],252 [2,3],
253[2,3],254 [2],255 [3], 257,
259
Pinkerton, 201 62], 209
62], 217
Piperno, 149 [33], 160 [33],
164
Pitha, 6 [46],10 [122], 17,
19, 59 [20], 60 [20], 62 [20], 84,
149 [29], 163, 181 [52], 186
Podgore, J. 256 [99], 261
Pollard, 209 [130], 220
Pollard, R. 75 [67], 76 [67],
81 [78], 86, 114 [3],125 [3],133
[3], 136 [3], 139, 123 [49], 142
Pontillon, }<'., 119 [30], 141
Popper, 177 [44], 185
Portnoy, J., 69 [87], 87, 149 [30],
163,201 [47,48], 217
Posner, J., 95 [68], 109
Postie, 59 [61],74 [61], 86,
117 [14], 140,200 [34], 216, 244
[59], 259
Povlsen, 0.,94 [51,52,56,57,
59], 95 [59,65], 108, 109
Prazie, 121 [47], 142
Prehn, R. 94 [55], 109
Priee, R., 106 [122], 111
Proehownik, V., 7 [68], 11
Proffitt, R., 282 [67], 293
Pry, W., 253 [93], 260
Pryor, J. W., 240 [39],250 [39],
258
Pugliese, 6 [42], 16
Pureell, R. 177 144), 185
L., 22 [71], 46 [60], 54,
55, 58 [16], 69 [56,57], 70 [54,
56,57], 71 [55,56], 73 [53,54,55,
56,57],75 [57],76 [55],79 [16],
81 [54], 84, 85, 100 [88], 110,
136 [74,75], 143
R
Rabinoviteh, 207 [106], 219
Rabson, S., 172 [26], 184
Rafajko, R. R., 199 [78], 200 [78],
218
lNIJI';X
120 IJH],
141
Ralpll, 279 [52], 292
Ramseur, J. 119 [22], 125
[22], 140
Ralla, W., 201 [61,62], 209
[61,62], 217
Ralld, 75 [67], 76 [67], 86,
123 [49], 142
Rankin, 214 [154,155], 221
Rapoza, N. 274 [30], 291
Ratller, L., 9 [95], 18
Ray, 100 [87], 110, 114
[2], 127 [2], 139
Rayehaudhuri, 187 [7], 197
[20], 205 ['71], 206 [71,74], 207
[20,71,86,91,96], 215, 218, 219
Reed, S., 74 [62], 77 [62,69], 78
[62,69], 86,122 [1], 139
Reed, S. 224 [1], 236
Reeder, W. J., 276 [43], 292
Regelson, W., 187 [5],206 [75],
207 [5], 209 [124,129], 210 [124],
211 [133,134,135], 212 [133,139],
215, 218, 220, 240 [1,22,35,36,
45],241 [52,53],245 [1,73,103],
246 [1,72,73,74],249 [35],250
[35,36],251 [90],252 [1,22,74],
253 [1,36],254 [1,73],256
[103], 257, 258, 259, 260, 261
Reizill, F. N., 6 [52],11
Remingtoll, J. S., 146 [7], 163,
240 [41],249 [41], 259
Revel, 6 [56],9 [93,94], 17,
18,120 [34], 141
Reynolds, F. 45 [51], 54
Rheins, S., 198 [29], 201 [56,
57,60,63],209 [56,57,60,63],
216, 217
Rhoads, 209 [120,121], 220
Riee, J., 117 [11], 140, 149 [35],
164, 172 [38], 174 [40], 185
Rieh, R. R., 274 [31], 279 [51],
288 [31,73], 291, 292, 293
Riehards, 129 [55], 135
[70], 142, 143

lticJlIllOnd, J. l55],
[9], [9], [96], [96,
101], [101], 261
Richter, 212 [142], 220
Hickse, J"., 169 [7], 183
Riley, F., 172 [33], 184
Ringborg, 97 [77], 98 [77], 110
Rita, G., 198 [30], 199 [30], 216
Ritter, W., 187 [6], 205 [6], 215
Ritts, R. 276 [42], 292
Riviere, 119 141
Roberts, 191 [8], 215, 201
[8]
Roberts, W. 288 [74], 293
Robinson, R. 99 [85], 110, 172
[36], 185
Robinson, W., 81 [78], 86, 114 [3],
125 [3],133 [3],136 [3,82], 139,
143
Hodin, I. 201 [54], 217
Rohovsky, W., 161 [63], 165
Roikhel, 6 [52], 17
Romball, G., 276 [44], 292
Rossman, G., 7 [62], 17, 65
[39], 85
Rostrick, J. 273 [29], 291
Roth, F. 227 [5],231 [13], 236
Rotte, 210 [132],211 [135], 220
Rousset, S., 7 [59], 17
Roye, D., 209 [120,121], 220
Rozee, R., 90 [9], 91 [34], 107,
108, 281 [56], 292
Rubel1is, 229 [10],230 [10,12],
232 [12], 234 [12], 236
Rubin, 240 [34], 249 [34],
258
Rubin, R. 137 [84], 143
Ruegg, 21 [2], 31 [2], 33
[2], 34 [2], 45 [56], 46 [56], 50,
54,58 [14], 84,100 [92], 110
124 [51],
Huiz, 240 [37], 250 [37], 251
[37], 253 [37], 258
Ruiz-Gomez, J., 13 [150], 20
Russell, S., 137 [84], 143
79 7;)],
llusso, 207 [106], 219
Rygaard, J., 94 [50,51,52,56,57,
59], 95 [59], 108, 109
Rytel, W., 146 [6], 163, 169
[8], 183
S
Saavedra, G., 212 [138], 220
Sacks, S., 136 [82], 143
Salb, J. 9 [86,87,88], 18
Salvin, S. 3 [30], 16,47 [67],
55, 58 [5], 83, 103 [107], 111,
265 [7,13], 290, 291
Sammol1s, L., 174 [41],180
[49], 186
Samuel, 8 [76], 18
Samuelsson, 12 [135],
Sancean, J., 38 [75],39 [75], 55
Sandberg, J., 240 [32], 249 [32],
250 [32], 258
Santiano, 12 [140], 20
Sarangi, F., 103 [100], 110
Sarl11a, S., 172 [28], 184
Savy, L., 13 [146], 20
Schafer, W., 69 [50], 73 [50],
85
Schall11, s. W., 125 [52,53,54],
13(; 142, 143
Schellekel1s, 125
(68], [77,79,83], 142,
143
Scheve, J., 191 [15], 201 [15],
215
Schiff, G. 210 [132], 211
[134,135,136], 220
Schlesinger, 94 [63], 109
Schmidt, J. 154 [54], 164,
201 [40],214 [40], 216, 240 [2,
3,43], 249 [43], 252 [2,3], 253
[2,3],254 [2],255 [3], 257,
259
Schl11idtke, J. R., 275 [39,40],
Schonne, 45 [55], 54,
244 [60], 259
Schryer, J., 3 [22], 16,59 [21],
60 [21], 84
Schuller, G. 240 [24,25],248
[24,25],251 [24,25,89],252 [24,
25],253 [24],254 [24,89}, 255
[89}, 256 [24}, 258, 260
Schultz, 9 [98,99], 18
Schultz, G., 246 [75,76], 260
Schultz, R. 11 [131,132], 20,
240 [37], 247 [78], 248 [78], 250
[37], 251 [37}, 253 [37], 258,
260, 275 [35,36], 291
Schultz, W. W., 2 [13], 15
Schwartz, S., 129 [56J, 142
Schwenteck, 38 [79], 56
Sclair, 41 [62],46 [62], Q.1
Scott, G. 77[69], 78 [69J, 86,
129 [55], 135 [70], 136 [78], 142,
143
Scott, 251 [88], 260
Scott, W. D., 213[147], 221
Scriba, 28[56],45 [56], 46
[56] ,54, 79 [76], 86
Scullard, G., 136 [82], 143
Sedmak, J. J., 59 [26],61 [26],
62 [29,30,31],63 [26,30,31],64
[30,31],67 84
Sehgal, 7 [61,65,66], 17
Sekellick, J., 9 [96,100], l..
Selhorst, J. 212 [139], 220
Sellers, R. F., 240 [10], 252 [10],
257
Semenov, F., 201[67], 206[67],
217
Sen, 9 [95],10 [107,108,
109], 18, 19
Senussi, 45 [54], 54, 62 [28],
63 [28], 64 [36], 84, 85
Shaddock, J. 174 [39], 185
Shaila, S., 9 [95],10 [107,108], 18
Shapiro, S. Z., 119 [24], 125 [24],
140
W., 95 [68], 109
lNl)I';;>;'
Shari, Z. D., [55],
Shearer, [6tJ], 293
Shemano, 1., 187 [7], 205 [ 71],
206 [71,74], 207 [71], 215, 218
Sher, N. 207 [104], 219
Sblmonaski, 240 [12], 241
[12],244 [12}, 245 [12],252
[12], 257
Shimosato, 94 [54,62], 109
Shiv, G., 172 [28], 184
Shnider, 1., 245 [103], 256
[103], 261
Shope, R. 169 [5,8], 182, 183
Shrestha, 72 [88], 87,117
[16], 140
Shuster, J. J . 121 [40], 141
Sieber, S. 209 [126], 220
Siegal, F. 129 [56}, 142
D., 147 [21], 163, 191
[8,9,10,12,15,16],201 [9,10,
12,15,16,39], 215, 216
Silver, G. 27 [11], 51
Siminoff, 149 [38], 150 [38],
164
Simons, J. 212 [140], 220
Simons, R. L., 231 [13], 236
Simpson, R. W., 9 [101], 18
Siraganian, R. 269 [20], 291
Sivilich, 179 [48],180 [48],
186
Skoda, R., 117 [17],121 [45],
140, 141
Skreko, F., 71 [60],74 [60], 86
Slattery, 9 [95],10 [109], 18,
19
Slavina, G., 105 [119], 111
J. 2 [12], 15
Smadel, S. 146 [5], 163
Smerdel, S., 120 [37,38,39], 121
[39,46,47], 141, 142
Smith, G., 270 [24], 271 [25],
276 [48], 277 [48], 279 [53],
291, 292
Smith, 240 [34], 249 [34],
258
IIt INIH:X
Srnitll, U., 248 [79], 250 [84], 256
[84], 260
Smith, J., 3 [21], 16, 245 [65],
259
Smith, w. R., 194 [19], 215
Smithwick, 129 [56], 142
Snodgra88, 240 [24,25], 248
[24,25],251 [24,25,85],252 [24,
25], 253 [24], 254 [24], 256 [24],
258, 260
Snyder, R. 119 [25], 140
SObl8, 10 [114], 119 [21],
125 [21], 140
Soderberg, G., 96 [74,75], 109
Soldo, 1., 120 [37], 141
Song, W., 250 [83], 260
Sonnabend, J. 8 [74], 18
Sonnenfeld, G., 269 [21], 291
S008, 120 [37,38,39], 121 [39,
46,47], 141, 142
Sopori, L., 9 [92,103], 18, 19
Sordat, 94 [53,64], 109
S08a-Martinez, J., 13 [150], 20
Sowin8ki, R., 161 [65], 165, 206
[79], 207 [85], 218
Spahn, G. F., 208 [111,112], 209
[111], 219, 248 [81],256 [81],
260
Speck, 133 [68], 142
Spertul, R. 0.,174 [41], 185
Spertzel, R. D., 201 [45], 217
Stancek, D., 58 [19], 84
Stanley, D., 228 [9], 229 [9,10],
230 [10,12],232 [12],233 [9],
234 [12], 236
Stanton, G. J., 265 [10], 274 [10],
279 [10],280 288 [10],
290
Starr, S. 256 [98], 261
Stehlin, J. S., 94 [58,60,61],95
[60], 109
Steinberg, D., 208 [111,112],
209 [111], 219, 248 [81], 256
[81], 260
Steinberg, D., 115 [7], 118 [7],
140
:11'(
Stepllen, 1<:. L., 174 [41], 179 [16,
47],180 [49], 185, 186,201[44,
45],207 [102], 217, 219
Stern, R., 169 [17], 183
Steven8, L. 207 [97,98,99,
100], 219
Stewart, J. 137 [84], 143
Stewart, W. 11,3 [26],8 [78],
16, 18, 31 [19,20,21,72], 44
[45,49,50],46 [73], 52, 53, 54,
55,58 [11,12],63 [11,33],64
[33], 84, 85, 213 [147], 221
Stinebring, W. R., 2 [7,11], 4 [7],
15,44 [46], 53,146 [3,8],147
[8], 152 [3], 162, 163, 201 [36],
216, 240 [48], 244 [48,58], 259
Stineburg, W. R., 234 [15], 235
[15], 237
Stitz, L., 136 [77,79], 143
Stobo, J., 103 [109], 111, 265 [8],
279 [8], 290
Stoger, 1., 146 [18], 163
Stollar, D., 10 [122], 19
Strand, 119 [24], 125 [24], 140
Strander, 21 [1], 22 [5,6,39,
40],24 [1,5,6], 33 [42], 50, 51,
53,69 [57], 70 [57], 73 [57J,
75 [57,64J, 76 [64],81 [64J, 85
86,91 [37,41,44],92 [41],94
[37,41],96 [70,71,72,73,74,75J,
97 [41,7:3J, 98 [73],100 [91J,
10:3 [44,7:3], 108, 109, 110, 1:33
[60, 62J, 142
Strannegard, 0., 281 [64J, 293
Stringfellow, D. 6 [57], 7 [58,
69A,69BJ, 8 [57,70,71,72], 17,
99 [78J, 100 [86],104 [78J, 110,
149 [36,37J, 150 [37J, 152 [55J,
153 [53], 154 [55], 155 [36,55,
58,60J, 157 [61], 161 [66,67],
164,165, 192 [17], 194 [17],
198 [17J, 199 [33,77], 200 [33,
77],201 [33,41],207 [107],2011
[109], 215, 216, 218, 219
L., 99 [1:>1], 110
Sweet, W., 191[8,9,10,15,16],
201[8,9,10,15,16,39], 215, 216
Swift, 120 [33], 141
Stylos, W. 11 [132], 20
Subrahmanytan, 67 [48], 68
[48], 85
Sugar, J., 11 7 [18], 140
Sulkin, S. 213 [147], 221
Sulkowski, 27 [9,10,11,12,13],
29 [9,10], 31 [38], 36 [29,31,32,
34,35,36],41 [62],45 [30,59],
46 [62], 48 [59], 51, 52, 53, 54,
58 [15], 84,101 [95,96,97,98,99],
110
Sundmacher, R., 117 [16,17], 121
[42,43,44,45], 140, 141
Surarez, 10 [117],
Sutherland, D. J. 103 [100],
110
Svet-Moldavsky, G. J., 105 [118,
119], 111
Szolgay, 146 [18], 163

Tadokuma, 274 [31], 288 [31],
291
Taira, 9 [95], 18
Talas, 146 [18],153 [51], 163,
164,200 [35],201 [64], 216, 217
Talmage, D. W., 288 [74], 293
1.,7 [61,65,66], 17
35 [28], 38 [28], 41 [28],
52
7 [64], 17,35 [28],
38 [28], 41 [28], 52, 90 [20], 91
[20], 92 [20], 107
Tanford, 64 [37], 85
Tarr, G. 8 [73],11, 133 [59J,
142
Taylor, J., 9 [85], 18
Taylor-Papadimitriou, J., 6 [45],
17
I NI >I':X
Tazaia, 181 t ;;2J,
Tazania, 1., 181 [52], 11:>(j
Tazawa, 1., 149 [29], 16:.!
Tazawa, S., 149 [29], 163
Tazulakhova, 153 [52], 164
tenKate, F. J. 136 [83], 143
Thang, N., 6 [48], 11
Thomas, D. W., 288 [74], 293
Thomas, J. 212 [139], 220
Thomas, L. L., 207 [96], 219
Thomas, 11 [126], 19, 90
[4,5,6,10],91 [30], 107, 108,
111
Tibor, 103 [105], 111
Tiernan, L., 191 [16], 201
[16], 215
Tikhomirova-Sidorova, N. S., 201
[54], 217
J. G., 3 [22], 9 [97], 16,
18,59 [21],60 [21],65 [40,41],
84, 85
201 [52], 217
Tolkoff-Rubin, N. 137 [84],
143
Tomeh, 256 [98], 261
Torell, D. 9 18
26 [43],46 [60,68],
53, 54, 55, 58 [9,13,16],79 [16],
84
Torrence, F., 99 [80], 110,
149 [26,27,28], 163
G., 11 [129], 20, 31
[20,22],46 [64], 52, 55, 90 [17,
21],91 [21],92 [21], 105 [112],
107, 111, 117 [13],119 [26,29,
30,31,32],288 [75], 293
S. 6 [44], 16
Trajer, D., 120 [37],121 [46],
141
Triche, J., 10 [115], 19
Trimble, S., 210 [132],211 [135],
220
Trivers, G., 274 [32], 291
Trukhmandva, L. 201 [54],
217
INI)j':X
1'. 149 129], 160 162],
163, 165, 181 [53,54], 186
Tsukimoto, 1., 211 [134], 220
Tumuraya, 94 [62], 109
Turner, V., 209 [125], 220
W., 172 [32], 184, 240
[23],250 [23],252 [23],253 [23],
258
Tyan, L., 282 [66], 293
Tyer, J. 169 [10], 183
Tyrell, D. 224 [1], 236
Tyrrell, D. Jr., 2 [6], 15, 74
[62],77 [62,69],78 [62,69], 86,
122 [1], 135 [70], 139, 143
Tytell, 3 [15], 5 [15,38],
16, 146 [14], 147 [14], 149 [31,
32], 160 [31,32], 163, 164, 169
[9], 183,265 [3], 290
U
Uhlendorf, 118 [20], 140, 169
[18,19], 183
Underwood, G. 146 [19],147
[19], 153 [52], 163, 164
Urbaniak, S. J., 81 [79], 86
V
Valle, J., 38 [76], 55, 59[22],
61 [22], 84
Van Damme, J., 34 [25], 52
Vandeputte, 240 [29],241 [29],
250 [29], 252 [29], 258, 281160],
Vanderberg, 161 [67], 165
Vanderhaeghe, 240 [7], 241 [7],
245 [7], 252 [7], 257
Van Dijck, J., 240 [44], 249
[44], 259
Vankirk, J. 172 [34], 184, 224
[2], 236
: I I
1'uLLcll, 1 . 21 I ;Hj,
[31], 258
Van't HuH, 133 [69], 143
VaraceHi, F., 7 [62], 8 [79], 17,
18
Vargin, V. V., 201 [67], 206 167],
217
Varnell, 117 [18], 140
Vengris, V. 10 [122], 19
Ver-Hodge, 181 [54], 186
Viano, 1., 12 [140], 20
Vickenstedt, 201 [42], 216
Vilcek, J., 3 [28,39],7 [62,63,
65,66],8 [79,82],10 [116], 16,
17, 18, 19,34 [24],35 [26],45
[52],48 [69], 52, 54, 55, 58 [7,
8,10], 59 [24], 60 [24], 62 [24],
65 [38,39], 83, 84, 100 [94], 110,
170 [23], 184,265 [11], 291
Vilner, L. 201 [54], 217
Virelizier, 290 [76], 293
Virelizier, J.-L., 290 [76], 293
Visfeldt, J., 94 [56], 109
Visintine, 256 [98], 261
Vladutiu, 136 [80,81], 143
Von Muenchhausen, W., 36 [29,32],
52, 101 [97], 110
W
Wackcr, 3[18], 16,2141150],
221
Wagner, 246175,76], 260
Wagner, J. G., 83 [86], 87, 177
[44], 185
Wagner, R. R., 3 [21], 5 [35,36],
8 [83], 16, 18, 119 [25], 140,
245 [65], 259
Waksman, 268 [15],288
[15], 291
Waldman, R. 229 [11], 230
[11], 232 [14], 233 [14],
[11], 236
Walker, S. 208 l110] , 209 l128]
219, 220
Wallace, J. 209 [125], 220
Wallen, W. 103 [108], 111
Waltman, S. R., 121 [40], 141
Wampler, G. L., 209 [124], 210
[124], 211 [133], 212 [133,142],
213 [146], 220, 221
Watson, J., 279 [52], 292
Weed, S. D., 146 [19],147 [19],
149 [36], 153 [53], 161 [66,67],
163, 164, 165
Weideli, 9 [103], 19
Weigle, W. 0.,276 [44], 292
Weil, R., 28 [56], 45 [56], 46 [56],
54, 79 [76], 86
Weimar, W., 125 [52,53,54], 131
[58],133 [58],136 [77,79,83],
142, 143
Weinman, 117 [15], 140
Weinstein, 279 [54], 282 [68],
292, 293
Weiszfeiler, G., 201[64], 217
Wenstrup, D. L., 191 [9,10,14],
201[9,10,14,39], 215, 216
West, W., 209 [120,121], 220
Wheelock, }'., 2 [10], 15, 38
[77], 55, 103 [101], 110, 146 [10],
163, 254 [94], 260, 265 [5], 279
[5], 288 [5], 290
Wiernick, 179 [48],180 [48],
186
Wildstein" 207 [97,98,99,100],
219
Will, G., 214 [153], 221
Williams, L. J., Jr., 94 [58,60,
61], 95 [60J, 109
Wilson, 198 [29], 201 [56,
57,60,63],209 [56,57,60,63],
216, 217
Winer, 201 [50],203 [68], 217
Winkle, S., 137 [84], 143
Wiranowska-Stewart, 3 [26],
16, 31 [21], 46 [63], 52, 55
i\t/TIIOlt
1\., 12(;J, .:ud
S. 172 l37], 185
Wolmark, N., 255 [97J,
Woltersdorf, 147 l22], 163
Wong, D. 0., 177 [44], 185
Wood, L., 240 [26,27], 250
[26,27],252 [26,27],254 [26],
255 [26,27], 258, 281 [59], 292
Woods, W. 247 [78], 248 [78],
260
Wooles, W. R., 240 [36], 246
[73],250 [36],253 [36],254
[73], 258, 260
Worthington, 117 [11], 119
[28], 140, 141, 170 [25], 172
[34], 184, 224 [2], 236
Wright, L. L., 240 [17], 252 [17],
253 [17], 254 [17], 258
Wright, R., 129 [55], 135 [70],
142, 143

Yajima, 240 [45], 259, 275
[38], 292
Yamazaki, S., 45 [52], 54, 58
[10],63 [10], 84
S. 209 [127], 220
Yaron, I., 6 [56], 17, 120 [34],
141
Yaron, 6 [56], 17, 120 [34],
141
Yasui, 245 [68], 259
Yeager, S., 77 [80],82 [80],
86
Yershov, F. I., 153 [52], 164
Yim, S. D., 94 [60,61], 109
Yin, F. 6 [44], 16
Yip, 3 [29], 16,48 [69],
55, 100 [94], 110
Yokota, 105 [115], 111
Yoshimura, S., 187 [3], 197 [22],
198 [31],201 [22,31,46], 215,
216
II!. I NI ,I,:X
W., 99[82], 110, 149
160 [32], 164, 172[36], 185
Youngner, J. S., 2 [7,11],3 [30],
4 [7], 15, 16,44[46],47 [67],
53, 55, 58 [5], 83, 103 [107], 111,
146 [3,8],147 [8],152[3], 162,
163, 201 [36], 216, 234 [15], 235
[15, 237, 240 [48],244 [48,58],
259, 265 [7,13], 290, 291
Z
Zaccara, 214 [150,151],221
Zajac, 1., 71 [60], 74 [60], 86
Zajdela, F., 90 [26], 108
Zapikan, Z., 224 [2], 236
Zbar, 13., 240 [40], 251 [40], 259
Zbinden, G., 207 [88], 218
Zeleznick, L. D., 172 [29], 184
Zilberstein, 9 [93], 18
Zinner, 120 [33], 141
Zoom, 21 [2], 31 [2], 33 [2],
34 [2], 50
Zoon, 100 [92,93], llO
Zor, U., 6 [56], 17, 120 [34],
141
Zschiesche, W., 201 [67], 206
[67], 217
Zucca, 6 [55], 12 [140], 11,
20, 287 [72], 293
Zunino, F., 214 [149,150,151,152,
153], 221
SUBJECT INDEX

(2-amino-5-bromo-6-methyl-
4-pyrimidinol), 149, 157
Acenaphthene, 191
Actinomycin D, 8, 100, 103
Adenovirus, 121
Adenylate cyclase, 12
Albumin-sepharose columns, inter-
feron purification, 36
Amnion, human, 150
Amphotericin effect interferon
response, 6
Amyotrophic lateral sclerosis,
tilorone therapy, 212
Animal models, antiviral chemo-
therapy, 116-120
Anthraquinones, 147, 191
Antibody
production affected intcr feron,
263-290
productioll eahallced polYl1uclco-
tides, 179
productiol1 pyraa, 246
Antibody affillity chromatography
of interferOllS, 28, 37
qualitative data, 30
Antibody columas, of, 28
Antibody res poase, effect of iater-
feroa 269-275
Antigellic properties
humaa illterferoas, 46
masking of, 29
Anti-iaterferoll aatibody, 277-279
absorptioa of, 29
323
Antiproliferatioll, interferons,
89, 90, 91, 93, 94
Antitumor
chemotherapy, 90
polyanioas, 249
Antiviral
chemotherapy, 8-11, 114-140, 151
polyallions, 251
Aativiral proteia, 8

Carboxylic polyaaions, illterferoa
illducers, 245
Catecholamiaes, effect iaterferon
inductioa, 156
growth, iaterferon,
89
Chemotherapy, combiaatioa witll
interfcron, 104
virus, 158
CJlOlcra
c[[cct 10
immuac 285-287
Chromatography
hydrophobic, 27
of il1terferOl1S, 22-50
Chromosome 21, 92
Chrol1ic relapsing l1europathy,
treatment with poly ICLC, 180
Ciba-crol1
purificatiol1 of il1terferOl1S, 36
trials, il1terferol1s, 96-99
Clil1ical use, il1terferOl1S, 114-137
(cl1lorite-oxblizelt
10se), 248-250
Sepharose, purification of
interferons, 28, 36
Concanavalin 275
Condy10mata accuminata, 120
Controlled pore glass beads,
of interferons, 37
961, 225
Crude leukocyte interferon (CIF) , 23
6, 12, 156, 158, 282-
288
Cyc10heximide, 7, 100, 103, 197,
201
Cytomegalovirus, chemotherapy of,
8, 131
D
Dane partic1e, interferon effect, 125
DEAE-dextran, 198
Denominator princip1e, 38
Dermatology and interferon, 120
Dibenzothiophene, 191
DL-thiocytic 64
Double-stranded RNA, 10, 147, 167-
182

Encephalomyocarditis virus, 7, 148,
157
Endotoxin
immune response, 269-272
interferon inducers, 197, 244
Escherichia 272
infections, interferon therapy,
121
F
Fever, side effect of interferon, 133
Fibroblast interferon production, 34
SUII,JIo:CT I NI
l''lU()l'antllOI1C, 191
147, 191
Friend leukemia virus, 157
effect of pyral1, 251
G
Gel filtration of crude interferon, 2(;
Gingivostomatitis, herpetic, 121
Graft-vs.-host reaction, 281-282
Guanidine hydrochloride, 64

Helenine, 169
Hemagglutination titration, 24, 29
Hemophilus influenzae al1tibody
vaccine enhanced poly ICLC,
179
Hepatitis, viral, 124
Hepatitis, chronic, treatment in
chimpanzees, 174
Herpes, 120
Hodgkin' s disease, interferon
therapy, 97, 98
Host defense, 13-15, 96, 105
impairment of, 13-14
Host resistance, virus infectiol1,
12-15
cells (il1 vitl'o)
il1terferon respol1se, 150
F form, 47, 48, 49
L form, 47, 48, 49
interferon, serum interferon
response to poly ICLC, 179
Hydrophobic chromatography of
il1terferons, 27, 36
Hyporeactivity, 152-160, 244
inducer dependel1t, 152
to interferol1 il1duction, 199, 201,
203
reversiol1, 159, 160
serum factor, 201
treatment regimel1s, 153
Ilyporesponsiveness, 8, 99, 100,
231, 233
1
Immune interferon, 226, 282-288
production, 38
separation of, 38
Immune modulation, 263-290
interferon, 12, 161, 162, 269
polyanions, 246
Immunosuppress ion, 269- 275
Inducers of interferon, 2, 4-7
antiviral effect, 225-226, 234
canine, 198
chemical structures, 147
classes of, 146
cost of, 182
experimental therapy of tumors,
177
fe1ine, 198
ferret, 198
hamster, 198
hepatitis therapy, 174
human cells (in vitro), 150
humans, 179
hyporeactiVity, 152-160
immune adjuvants, 182
immune modulation, 246-248
in organs, 194
in vitro, 198
low-molecular-weight, 147, 187
monkey, 198
nonviral, 168
oral, 192
Penicillium funiculosum, 169
peritoneal cells in V'itro, 245
phagocytosis in vivo, 246
polyanions, 239- 25 7
Poly poly U, 169
polynucleotides, 167-182
primates, 172
propanediamine, 224-236
pyrimidines, 147-160
rabblt, 198
rabies virus, 174
lInducers
renal toxicity, 161
species variation, 149
therapy of keratoconjunctivitis,
170
toxicity, 160, 161, 180, 245
yellow fever, therapy, 174
Induction, of interferon, 2, 4-7
Influenza, chemotherapy of, 2, 122,
157, 225-231
Interferon
antibody, 10, 14
antibody response, effect 269-
275
antigenicity, 3, 46
antiviral, 8-11, 92, 114-138, 116-
120
blological properties, 3
regulation, 11
cel1ular immunity, 281
classification of, 265
clearance, 79-81
clinical trials, 96-99
clinical use, antineoplastic, 89-
106
clinical use, antiviral, 114-137
clinical use, dermatology, 120
clinical use, res piratory, 122
control protein, 7
denaturation, 40
discovery, 2-4
infections, 121
fever, 133
fibroblast, 39, 58, 93, 103
glycoproteins, 45, 46
immune modulation, 263-290, 288-
290
immune production, 38
immunosuppression 269-275
leukocyte, 22, 58
lymphoblastoid, 33
molecular weight, 3, 197
mltNA dCj!;L'adation, 9-10
cHccts, 11-12

pll sla})ilily,
pl1ysical cl1aeaclcrislics, 2:\:\
l Inter
production, control 0[, 7-11
production methods, 21-50
properties, 2-4
purification methods, 21-50, 101
quality control, 101-102
regulation 0[, 7-11
ribosomes, effect 9
Sendai virus induced, 24
sens iti 12-13
side effects, 24, 81-83, 133-135
sodium dodecyl sulfate, 63
stability, 58-67
stomatological, 121
systemic, 122-131
T-cells, 279
therapy malig!la!lcies, 89-106
titration, 151
transcription, effect 9
tumor cells, 101
Isocytosille, 147-160
J
Japanese encephalitis, therapy of,
174

Keratitis, vaccinia, herpes, 121
Keratoconjunctivitis, 170
L
Leukocyte pro-
duction, 22
Lewis
effect of pyran, 250
Lipopolysaccharide structure, 243
Liposomes as delivery system, 40
LSTRA leukemia, effect, 250
!)2, !J;"
100
Lymphoblastoid
33
11, 268

Macrophages, 11, 272
246
248
Madison carcinoma,
antitumor effect, 250
89-106
melanoma, 98, 99
effects, 6, 10
receptors, of
264
Methyl xallthine, 285-287
Microdispersed preparation (pro-
panediamine), 229
Multifocalleukoencephalopathy,
therapy, 212
Multiple myeloma, 97, 98
leukemia virus, 10
N
Namalva cells, 33, 100
Namalva illterferoll, of,
33, 34
Neoplasia, 210-213
Newcastle disease virus, illducer of
149
11-12
Nuclease activatioll illterferon, 10
Nude mice, 94, 95, 96, 102

Osteosarcoma, 92, 93, 96, 103

Partially purified interferon (PIF) ,
23, 24
Peritoneal cells, interferon in vitro,
159, 245
Phagocytosis, stimulated poly-
anions, 246
Pharmacokinetics of interferon, 67-
80, 136
mouse, 67-74
rabbit, 73
Phenyl-sepharose purification of
interferons, 28
Phlebotomus fly, 211
Phosphodiesterase, 287
Phytohemagglutinin, 275
Pichinde virus, 174
Plant viruses, effect of polyanions,
251
Polyacrylic acid, interferon inducer,
240-250
Polyanions, (see pyran)
antifungal activity, 240
antimicrobial activity, 249
antiprotosal activity, 240
antitumor effect, 249
antiviral activity, 251
carboxylic, 245
immunomodulators, 246
interferon inducers, 239-257
mode of antiviral effect, 253
molecular weight, effect as in-
ducer, 245
against plant viruses, 251
of activity, 252
toxicity, 245
Poly 275
Polycarboxylate inducers, 14 7
Poly ICLC, 149, 167-182
168
immunoadjuvant effects, 177
leukopenia, 180
180
thermal denaturation, 172
toxicity, 180
l Poly ICLC]
of hepatitis chimps,
174
Poly 1: poly 157, 167-182
mismatched analogs, 181
stabilized against hydrolysis, 172
treatment of animal 175
toxicity, 180
172
effect of molecular weight, 130
Polymethacrylic acid, 240-250
Polynucleotides, 167-182, 265
hydrolysis, effect induction, 172
in 179
structure, 243
structure for of
feron, 169
thermal 172
toxicity, 160
Polyphosphates, interferon inducers,
240-250
147, 224-236
effect of interferon,
6-8, 100, 156, 157, 159, 160
Proteolytic inhititors, interfero!l
41
(see
mechanism, 253-255
antiviral spectrum, 252
maleic anhydride-divinyl ether,
240-250
Pyrimidines, 147-160
Pyrog"enicity
interferon, 133-135
Poly ICLC, 180
Q
control, 101-102
R
ltabies, poly ICLC therapy, 17'1
131
H,esurrection interferon, 44
Respiratory viral infections,
therapy, 228
Reverse affinity chromatography, 48
Rhinovirus, 122, 226-232
RNA, double-stranded, 5-6
Rubella virus, 211
Russian spring-summer encepha-
litis, 174
S
Sandfly fever, 211
SDS-PAGE, human interferon, 31,
44, 45
Semliki forest virus, 151, 157
Sendai virus, 24
Sequential antibody affinity chroma-
tography, 31
Sialic acid, in interferon, 79
Side effects of interferon, 81, 133-
135
Simian hemorrhagic fever virus, 174
Soluble immune response suppressor
(sms), 288
Species specificity of interferon, 2
Splenectomy, effect interferon
production, 244

chaotropic salts, 63- 64
human interferons, 40
mechanical, 62-65
thermal, 59-62
Staphylococcal enterotoxin 283-
287
Subacute sclerosing panencephalitis,
134,212
Superinduction of interferon, 7
Swine flu vaccine, antibody enhanced
poly ICLC, 179
'l'
Tacharibe virus, 174
mitogen, 279
156
Therapy, interferon, malignancy,
89-106
149, 157, 187-215
anti- inflammatory, 208
antitumor, 209
antiviral properties, 201
clinical studies, 210
complement, 207, 208
immune response, 250, 207
interferon induction, 190
in vitro, 203
LD
so
, 188
lymphopenia, 206
rubella virus, 211
sandfly fever, 211
toxicity, 206
transplantation, 207
analogs
RMI 10,024, 188, 191-194, 197,
202, 205, 206-210
10,874, 188, 191-194, 199,
202, 205-208
RMI 11,002, 188, 191-194, 197-
199, 202-214
RMI 11,513, 189-210
RMI 11,567, 189-214
11,645, 189-207
RMI 11,877, 190-214
RMI 12,358, 190-210
Tonsi1s, human, 150
Translation
interferon effect mRNA, 9
inhititory protein, 9
Trigeminal neuralgia, 133
Trinucleotide, 10
Tween 80, 64
V
Vaccinia virus, 120, 174, 225
Varicella zoster, 123
Virus replication, 12
W
Warts, genital, 120

Yellow fevel', inter feron, tJ1Ceap'y
of, 174
Z
Zinc chelate affinity
of interferon, 37