'U.S.

DEFENSE RESEARCH
SOFFICE-LATIN AMERICA
ARMY ELEMENT
FOR "LIA FI' pi

J. . .. .*•• . ,

•- __.• .. ;. 7';..•

DOC
E NOV 22N5

Research Report
Kidney and Liver Pathology in Human and Experimental Leptospirosis
ANNUU.L REPORT
By: Dr. Thales de Brito
Army Proj. No. 2NO14501B71D 00 021 LA - Grant No. DA-ARO-49-092-65-G61
Universidade do Sao Paulo, Instituto de Medicina Tropical
Sao Paulo, S. P., Brazil
Rio de Janeiro, Brazil
Office of the Chief, Research & Development, Department of the Army
Washington, D. C. 20310
 REPORT NU!IBER 1

KJDff)~ :JD LIVI.R P .THOLOGY IN HUI.LJ ~JM F-XP,:.MItv[T XL

Liý.PTOSPIHOSIS

..NNU,.J REPORT

BY
T, do Brito
E. FroyrwillUr
H.S. Sintcs
D.O pann"I
S. Sr,-Lr03 do amuid,%

OCTOBER 1965

U.S. ,r!zW EJloant

Defense Resea~rch Office fcr Litin iAmerica
Ric. do Jmoinoro, Brnz:Ll
D.A'6-,-49- 092-65-C,61
U.S. Dcp'u'tzmnt of thu .'.rmy Project Number 2N014501B71D 00 021 Ll.

Universidr.-do do Sac. Paulo

Institutc do !4odicina Tropic~'t
Esc.-Aa PKIlitocnica - Sacq~c do Micrc-scopi'a Eletronicai
Hospit-Ll IlfiL.o Ribas"
S~r. P'~ulr - S.P. - Brazil

Distribution of This Documnort is Unlimited

1- SU. -- light and electron microscopy of human and experimental
loptospirosis of the guinea pig was done, The human study was carried
out in kidney biopsies, but a liver biopsy study is now in progrer,.
The experimental work, in guinea pigs, was done both in the kidney and
liver and histcehemical techniques were also performed.
The earliest lesion found was at the cell membrane, with
partial or total disappearence of the brush border of the cells of the
proximal tubuli 'as well as pnrtial disappearence and distortion of the
micrc'villi of the hepatic cells,, Intercellular spaces wore found to be
onlarged both in the liver and kidney. Capillaries of the experimental
animal showed endothelial cell 'tumefaction and, sometimes, disjunction
of the endothelial lining, a finding also in accordance with the basic
pathology of the disease.. Mitochondrial pathology, seen in humans, was
seen in the experimental animal only at the late phase of the&disease.
However, a definite increase of so called "dense bodies", whose origin
was discussed, was seen in both cases. ,lso described, a mild but de-
finite focal glomerular lesion, which provides anatomical basis for the
proteinuria seen in the disease.
The above described pathology is in accordance with the
possibility of a toxin as the main mechanism acting in leptospiral path
ogenicity.

2- FORORD - Previous work has shown that it is possible to safety per

formk1dey and liver (1,3,7,9) biopsies in patients with leptospirosis,
provided that a maximum of care be taken to avoid complications. New
possibilities in the study of the basic lesions of the disease were o -
penned, and techniques like the histochemical ones and those using the
electron microscope could now be usodt
In order to better understand the disease in humans a re
eva-luation of the experimental disease was in order.
In conducting the reseatch described in this report, the
investigators adhered to the Principles of Laboratory Animal Care as
established by the National Society for Medical Research.

3- BODY OF REPORT - The study of the human disease has been continued
and a first paper has been delivered recently (1). So far we have per -
formed 21 kidney biopsies in leptospirotic patients without complica -
tions. The light microscopy study of these biopsies showed that the le-
sions wore less marked but essentially similar to the ones seen in au-
topsy.
The electron microscopy study was done in six patients,
all of them, with the exception of a 12 year-old boy, being male adults,
ranging in age from 20 to 41 years. The biopsies were performed at dif-
ferent stages of the illness but they were always postponed until the
grossly hemorrhagic phase of the disease hAd been superseded.
Slices 0.3-0.5 mm thick wore cut with a raor blade knife
from threc different levels of the fragments and fixed 1.5 to 2 hours At
52C in 1 per cent osmium tetroxide buffered to pH 7.3 with voronal ace-
tatc buffer (P;LAIDE). '•tor being dehydrated in a graded series of as..;
cending alcohols, tissues wore embedded either in methacrylate or in
pon 812. Thin sections wore cut on a Porter-Blum microtome equipped
with glass knives. The sections were doubly staned, first in uranl &eq
 2.

tatc ,.nd then in lead citrA.te. Th,,. proparati( ns were examined in a Siv
mrins lniskop I uIcctrcn microscopu with a 50 cbjective aperture and
Sporating at 80 kv.
This electron microscpy study disclosed a definite glo-
mrnrul"r lesi: n in human leptospircsis, charactcrized by focal thickening
(f the basal membrane ind fusi n of the foot preccss of the glomerular
epithclial cull. This is in agreement with the proteinuria seen in the
dise-iso. •,orth while mentioning that previous light microscopy studies
by KOPPISCH and BOND (5) had also pointed out tQ a glcrnfrular lesion in
humaon lcpt spirosis.
Hcwv.xwr, tubular pathology was more prominent than the
glc,m. rular lesi-ns and was chnract rized by a total or partial brush
border loss, a finding in agreement with thu poor P,.S stain of the pro-
xim'l tubules b rdcr seen in light microscopy. The tubular cells showed
frequent densc bcdios in their cytoplasm, about thu size and shape cf
th n-dit-'chcndria. ,s a matter of fact few of them had remanescent aistor
tcd cristac which was in agreement with a first interprctation, that is,
that they wcre altered mit(chondria. However, in the discussion it can-
not loft :ut the possibility that thiy could be lysosomes and even pro-
tein drcplcts inside digestive vacuoles of the cell. This idea is in
acccrdancc with the proteinuria seen in the disease.
,, mitochondrial pathology was seen by us in our light mi
cr. scý-y' study (9) and confirmed in this electron microscopy approaci.
There wcre tubular cells with a definite mitochondrial deplction. Howe-
vw r no structur-.l alteration of these crgqnclles were seen, unless we
int-rpretcd some of the previously described dense bodies as altered
mitochondria.
.ncth,;r finding was a partial disjuncticn of the cellular
limits between adjacent tubular cells, giving rise to an enlarged inter-
cellular space. This cellular disjunctin, aracng other factors, could
c,.ntribute tc the tubular failure through a shunt mechanism between glo-
morular filtrate and the kidney interstitium. However, it iv necessary
tc ;cntic n that the juncti-nal complexes of the epithelial cells appeared
preserved. If this interpretation is correct we must postulate an in-
creased functi-nal permeability of those complexes which usually act as
a ',se-l" of the intercellular space. .mnother interpretation is th,%t the
enlargement of the intLrcellular spaces is due to interstitial edema
fluid which, originating from altered vessels went through-tht basal tu
bular mcmbrane and is now dissociating thv epithelial cells. Capillary
pathalogy was poor in th, human disease.
Liver biopsies arc available to us, obtained through a
micrc.laparotomry, a technique used by MONT.IAS (6) and which appears to
be a safe procedure in hum-in loptospirosis. However, before further
studying th,%human liver using histochemical and electron microscopy
procedures a reevaluaticn of the expurimental disease with sLmilar tech
niuec .,a• in order.
,, experimt.nt was carried out using forty-three guinea-
pigs, most of them weighing an average of 421 g. Five axperiwnts were
performed. The strains of Leptospira icterohaemorrhagiae used were ori-
ginally isolated from rats and cultivated from fragments of liver and
kidney in Fletcher's medium for 8 to 10 days at 282C. Virulence was
on~h-nc,.d thrcugh an initina1 incculxuii of 0.5 mil of cul.lur.. intc the p.24r
toneuji f' healthy guinea pigs vuighinp, an avcrnao 2.f 250 g. ,t the ter-_
minal phaso cf the ciieasu thesc amxvuds wore; killed with a blow in the
hon' -ind 1:5 liver..kidnjy hrgna. in saline were prupare-d . ,bcut
1 nil c£ thc suspensirn was EaZministe.ýr.d intraporitoncally in the animals
uscA, in th,- ,xpcri;.-,nt. .,niraals were s-,crificed in a initial phasce of
th,. diso-.se, usually arcund 3-4days -7rthe inocu2luim 'and iv.a terminal
ph'ýs,; of the disease, usually -,round th, 5-7 th day of' illiess. Two
hi-althy guinea pigs were, sim~ilarly mnt,-,puJ1-tod for each experiment.
Necrc~psy was pu-rfermud Urriediateliy %Iftrd&ath and be-
siukcs a light microsc( nv stu~lv o-f 'iir rv and 'iddn.'y- 7
iaic '.! studty was also car-ried out. Fr-ýpnnts ci' liver and kidncy were cut
7 cicra thick in A crvc stat micr'utcim.. either without or with 24 hrcurs
fi-xati n at 59C in 4% fc~rmr.in, pH 7.2, plus 7.,54 of sacarose. Ths, fixed
frirmc~nts befo-re cutting were tronsfcrvd tC. a miycturtu of sucrose and gum,
a-cnacia%for 24-48 h,-urs at 50C. Succi'.nodeLhidregunasu activity using as
substrat,- the ?%'itrc; B.T (dittetrazoliuxa chloriulo) anC the M.T.T. r3-(435-
K'i.IILthylthiazolil-2)-2,5 -iphonyl tetrazolium bromide) was stuc',-d ac-
cý-rding to tochniquvs describvd by FPJR5Z (8) in tho non fixed fragmen~.a
U~so, studied in the non fixed frag:n,-,nts ')ut only in two oxpor~ieints was
the cytuchr.c:.ie oxidaso activity using a3~ substrate thes paraamincodipheny-
1aii-,acc-rding tc. techninue of DVJRST%.N-- (2). In the fixed fragments
alkalyne (using as sub stratvs eithý.-r Goniori's modium or sodium -n %ph--
tyl phos')hatc) 2 ncifd phosphatac- (HOLT's techni-quc, 4) and inespecific
c.stcr-%so (using -ýs substrate the s-)diuz -naphtyl acotaýte.) were studied.
In 10 animals olectr-n rnicrc scopy was also carriud cut in a way similar
t,- that usu.4 ftor humans~ uxce-pt that the inclusirii was only in EPON 812.2
Tht,. light niicr boopy study showed findings which are in
agrecrncnt with pruvious descriptions. Histc chemistry revua2.ed that only
thý, alkalync ph' sphatasc activity, denwnstrated only in %he kidney tubu-
1Ls. ccrrclated well with the degreeý of the kidney lesicn0 ,.tthe early
ph-%sL. of the diseaso its activity wis seen to disappear fron groups of
n,-phrons mnd, at the late phase lrrgc- ar~a!- 'f enzyme activity (Rpletion
w~s -.bsorvr-d. Tho P...S prsitive zone: of thu orcxi~m-l tubules Ula" disap-
pu'din most of the proxirmal tubuli at thtu latu phase ,,f the diseabsh
Th,ýsc findings ccorrelite, well with the. eltectr,,n micrc.scopy d.ata which
shr-wud brush borh-,r alterition simil2.r to that soon in hurvans. as~c 3een,
th%. ccllul-i~r disjunction with th, appearonnG of enlargod int',rcollular
sp~ic.s and the presurvation c~f thu juncti n-.1 complexes.
The; r~.spiraitory enzymes, acid phosphatasu -and inespecifiC
cstceraso- dic' not show prominent nlt ratinn. However, -thobo findings m,.ust
bL, takecn carefully because only a qualitritivu and not ai quantitativu.
stud-y was carriod -ut in this expTcr-ti~nt.
Liver cells also. showed i de'pletion and/or Pn alt~.ration
if thc micrcvilli at thc, late phase of the disease. Bilia-ry ductulots
a7Lsc rt-vua.,le altcrW;( iicrovilli. Int.,.rcd;llular spaces appeare-d enla-rgod
Sin few c.ses, disippearenco c~f junctional complexes was seen. This
is particularly evident in cases where th,. light microscopy st~udy showad
th-, lack of nor~mal trabeculation of thG hepatic cells 1 a finding dos
cribudL in human autopsy examinatiorns (5).
 Regarding the hopr-tic and tubular cells as a whale, an in
crcase: numbar of sc-called "dcnsL b,.dios" was sown writh a morphological
aspect similar to the ones described in humans. Besidus the previous
intirpretations regarding their origin, the ones located in hepatic sells
ccul," bu regardcd as lipofuscin granules.
Gloricrular pathology was loss evident than that of human
cases. However, in few animals killnd at the. late phase of the disease
a btsal membrane thickening and focal foot process fusion of the epi -
thli-l ceils was observed.
Mitochondrial pathology was seen in only few cases and at
the 1-te phase of the diseasc. It was characterized by mitochondrial
d~p2.;ti.-. n d 3•wc~ll. niituchundria with altered cl-istac.
Capillarics of the kidney interstitium showed swollen on
c othclial cells and, sometimes, areas of disjunction between cells.
Kupffer's cells appearcd enlarged with many "dense bodies" in their cy-
te:plasm which could be inturpretod as engulfed debris.
Leptospir%. wore soon in this experimental study, both in
th,; liver -- %n kidney, made up by a ccntrpl axis with spirao around it.
They werieý located between liver cells, in the tubular and in the liver
sinusuidal lumina,
Both the hum.'n and the -xperimentcl data show that the
earliest lesion of loptcspirosis is it the :.ll membrane. 'lthough L.
icterohaemorrhagie, has not been conclusively demonstrated to possess
"a toxin, the clinical, histolAogical and cytological evidences suggests
"a t xin qs the. mechanism of leptospiral pathcgenicity. Our work is in
accor'Ance with a circulating toxin which, in the liver of the expe -
riinuntal animal would produce microvilli distortion and disappearence,
int,.rflring with the normal exchanges between the cell and the blood.
Both in the kidney of humans and experimental animals this so far hy -
poth .tical toxin action would be mild in the glomerulus and more de -
finite in the tubuli, chiefly proximal tubules, where it produces the
brush borlor pathclcgy through an enhanced action due to their con -
centration power. The enlsargd intercellular spact's and the capillary
lcsions of the experimental animal could also be explained by a similar
toxin iction. Only at the late phase of the disease that other organel-
los would deteriornte in a such way that in the more severe cases cel -
lular necrosis supervenes.
The above findings ,re in accord-nce with a low level of
serum tr-nsaminasc seen in the disease. More difficult to explain is the
high lc.v-l -,f mucoprcteins unLss we could admit that they were mainly
located at the cell mambrane, being then liberated through the toxin
acticn.
No conclusive explanation for the mechanism of the icte-
rus in lcptospirosis was found. The lesions in the biliary ductules are
incspecific and found bcth in intra-hepatic and cxtra-hopatic forms of
chc-l±stasis. On the other hind, hepatic cell disjunction might provide
a short cut betwcen biliary ductulos and liver sinusoidal lining. How-
ever, studies of the biopsiod liver in human leptospirosis, now in
progress, are showing marked ictorus with similar lesions regarding
the biliary ductulct microvilli but withcut an accentuated disjunction
of the liver cells. It is possible that the icturus had a genesis si -
 5.

milar to that seen in other forms of cholestasis, like the one proauced
by chlorpromazin.

BQB0OG-iAP!Y
.-..Liu, T. ; PE1NA, D.O. ; SA.TOS, I.S.; FREYM;ILR, E.; SOARLS DZ ALHEI
DA, S. ; AYROSA GALVAO, P.A. & PIIRMIRA, V. - Electron microscopy of
human loptospirosis (kidney biopsies). Amer. J. Trop. iled., lk:
397-403, 1965.

2- BuIuTONE, M.S. - Enzyme histocheriistry and its application in the stu-

dy of neoplasms. Nuw York, Academic Press, 1962.

3- DOTTI, F. & SABBIO?-I, G. - I1 quadro istomorfologico d&l fegato nelle

leptospirose benigna: studio bioptico con particolare riguardo alle
formc anicteriche. Arch. Pat. Olin. h'cd., L6: 81-101, T959.

4- iOLT, S.J. & HICKS, R.M. - The localization of acid phosphatase in rat
liver cells as revealcd by combinod c-tochemicpl staining and eloc-
tron microscopy. J. Biophys. Biochem. Cytol., 1: 47-66, 1961.

5- v,%PPISCH, E. & BOND, .1. - Thu morbid anatomy of human leptospirosis:

a report of thirteen fatal cases. Symposium on L~ptospirosis (11-
12 Deccember, 1952), Medical Science Publication NO 1. Washington,
U.S. Govt. Printing Office, 1952. pg. P3-105.

6- ,ONTAND, D, Unpublished data.

7- OSTERTAG, H. - Luptospirosis ict(rohaemorrhagica in Bulgarien. Ztschr.

Hyg., il: 482-500, 1950,

8- PEARSE, A.G.E. - Histochomistry: th(oretical and applied. Second

edition. London, J.&A. Churchill, 1961.

9- PFRNA, D.O.; BRITO, T. ; AGUIAR PUPO, A. ; MARCOQtD`S MACHADO, M. ; AY-

ROSA GALVXO, P.A. & AINEIDA, S.S. - Kidney biopsy in human lop -
tospirosis. Amer. J. Trop. Mod., L2: 896-901, 1963.
 Unclassified
Security Classification
DOCUMENT CONTROL DATA - R&D
(Security claseiflcation of title, body of abstrect and indexing annotation must be entered .whn the overall report in clamesIhed)
I ORIGINATING ACTIVITY (Corporate author) 20 REPORT SECURITY CLASSIFICATION

Inhtituto de Medicina TropicalJ .nclassifi4.:

niversidade de Wfo Paulo 2b GROUP

"3.REPORT TITLE

Kidney and Liver Pathology in Human and Experimental Leptospirosic. . .

T
4 DESCRIPTIVE NO ES (Type of report and inclusive date&) ..

Annual Report, 15 Oct64 - 15 Oct 65. _ _ _ _

5 AUTHOR(S) (Last name, first name, initial)

de Brito, Thales

6. REPORT DATE ?, O'AL. O -)F PAGes Tb NO OF REFS

oct 65 5 9
On CONTRACT OR GRANT NO. 98 ORIGINATOR'S REPORT NUMBER(S)

DA-ARo-49-092.-65-061 Annual Report No. 1

b. PROJECT NO

2N014501]ID
c 9b OTHER REPORT NO(S) (Any othernumbere thatmay be assigned
this report)

d.

10 A VA IL AB#Li TY/LIMITATION NOTICES

Unli.ited

I1 SUPPLEMENTARY NOTES 12 SPONSORING MILITARY ACTIVITY

U.S. Army Element

Defense Research Office for
Latin America
13 ABSTRACTA light and electron microscopy of human and experimental leptospirosis
the guinea pig was done. The human study was carried out in kidney biopsies,
b t a liver biopsy study is now in progress. The experimental work, in guinea pigs,
done both in the kidney and liver and histochemical techniques were also
ormed. The earliest lesion found vas at the cell membrane, with partial or
t al disappearence of the brush border of the cells of the proximal tubuli as well
partial disappearence and distortion of the microvilli of the hepatic cells.
trcellular spaces were found to be enlarged both in the liver and kidney.
w
pillaries of the experimental animal showed endothelial cell tumefaction and,
a times, disjunction of the endothelial lining, a finding also in accordance with
t e basic pathology of the disease. Mitochondrial pathology, seen in humans. was
s en in the experimental animal only at the late phase of the disease. However,
definite increase of so called "dense bodies," whose origin wAs discussed, was
a en in both cases. Also described, a mild but definite focal glmerular lesion,
ich provides anatomical basis for the proteinuris seen in the disease. The
described pathology is in accordance with the possibility of a toxin aS the
n •ahazd acting in leptospiral pathogenicity.

DD IoJAN4 1473 Tta3 ne

gecuinty •lassification
 ai
U~

14 KYWRSLWN6K A L '4 8C 9 LIN4K C

Pathology
L~ePtospirosis
EleCtrorinicroscopy
Kidney Biopsy,
Liver Biopsy
Parasites
Hiatoeheimistry
Pathogenesis

I Ns r i&ICTIONS
1. ORIGINATING ACTIVITY: Enter the name and address 10 AVAtLABILITY/LIMITATION NOTICES: Enter any lint-
(if 'he (ontr..0! .- "h ucontractor, grantee. Department of De- itations on further dissemination of the report, other than those
Iensie aot ti'itv or other organization (corpoia~rc &!shnr) issuing imposed by security classification, using standard statements
ther(-on. such ee.
2a. REPORT SECURITY CLASSIFICATION: Enter the ovr (1) "Qi-alified requesters may obtain copies of this
all %ecurity classification r~f the report. Indicate whether report from DDC'
"~Reiitrited Date" is included. Marking Is to be in accord-. 2 Frinanucmn n ismnto fti
ance with appropriate security regulations.(2 "Frinaoucmtaddseiainofhs
'V. CROUP: Automatic downgradi.,Ag is specified in DoD Di- rpr yDCI o uhrzd
rective S200. 10 and Armed Forces Industrial Manual. Enter (3) -U. S. Government agencies may obtain copies of
the. group number. Also. when applicable, show that optional this report directly from DDC. Other qualified DDC
m -r been used for Group 3 &Ad Group 4 as author-
-nshv sr hl eus iog
ized

Icapital
.3. REPORT TITLE: Enter the complete report title ini all
If a meaningful title cannot be mst-iete without cisssifca-
(4) 1"U. S. military agencies may obtain copies of this
letters. Titles in all casea should be unclassified.reotdeclfomDCOhrquiiduss
report dreuetl fhromugh Ote uaiid sr
tion, sahow title classification in all capitals in parenthesis shl eqetthog
immediately following the title. _____________________

4. DESCRIPTIVE NOTES- If appropriate, enter the type of (5) "All distribution of this report is controlled Quaal-
report. e.g., interim, progress, summary, annual, or final. ified DDC users shall request through
Give the inicluilve dates-when a specific reporting period is
covered.
~. ATHO(S):Entr
te nae~s of uthr~s as how on
S. ATHO(S):Entr
te nae~s of uthr~s as how on
If the report has been furnished to the Office of Technical
Services, Department'of Cbmaterce. for sale to the public. din"
or in the report. Enter aIsat name, first name, middle initial, Cats this fact ahd enter the price, if known,
If military, show rank and branch of service. The name of
the principal author in an absolute minimum requireiment. IL SUPPLEMENTARY NOTES. User for additional earplans-
tb. REPORT DATL_ Enter the date of the report as day, tory notes.
month, year; or month, year. If more than one date appears 12Z SPONSQRING MILITARY ACTIVITY: Enter the name of
on the report, use date of publication, the departmenital project office or l aboratory sponsorinag (par -
7&. TOTAL NUMBER OF PAGES: The total page count ing lot) the research and development. Include ~dbdess. '.
s;hould follww normal pagination procedures. L~e., enter the 13. ABiSTRACT: Enter an abstract giving a brief and (actual
nur~ber of pages containing information. summary of the document indicative of the report, even though
7h; it may also appear elsewhere in the body of the technical re-
Nb
1UMBER OF REFERENCES. Enter the total number of port. If additional space is required, a continuation sheet
references cited in the report. Ishall be attached.
8a. CONTRACT OR GRANT NUMBER: If appropriate, en r Iit is hilthly'destrable that the abstract of classified re-
the applicable number of the contract or grant under which ports be unclissified., Each paragraph of the abstract shall
the report was written. end with an indication of the military security classification
8b, &i,, & 8d. PROJECT NUMBER: Enter the appropriate of the information itt the paragraph, represented as (TS), (S),
military department identification, such as project number, j(c). or (U.).
subproject number, system numbers, task number. etc. There is no limitation on the lenith of the abstract. Howe
9av. ORIGINATOR'S REPORT NUMBER(S); Enter the offi. ever, the suggested length is from I1'50 to 225,words.
ciath reortnumerby
doumnt hic
illbe detifed 4. EY ORS: ey ord ae -echic llf 'n~ainel trm

and co'ntrolled by the originating activity. This number must or short phrases that characterize a report and may be used as
be unique to this report. index entries for cataloging the report. Key words must be
9b. OTIIiER REPORT NUMBER(S): If the report has been nelecteri so that no security classification is required. Iden-
assigned any other report numbers (either by the originator (iers, such a% equipment model designation, trade name, m~ili-
or bv the sponsor). also enter this number(s). tary project code name, geoigraphic location, may be used as
Icontext.
key words but will be followed by an indication of technical
optional.
The assignme.-It ot links, rules, and weights is