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Immunopharmacology and Immunotoxicology, 2012; 34(3): 477–483 © 2012 Informa Healthcare USA, Inc. ISSN 0892-3973 print/ISSN 1532-2513 online DOI: 10.3109/08923973.2011.630008

ReseaRch aRtIcle

online DOI: 10.3109/08923973.2011.630008 ReseaRch aRtIcle Immunomodulatory activity of diethylcarbamazine on humoral,

Immunomodulatory activity of diethylcarbamazine on humoral, cellular cytokine response and respiratory burst in BALB/c mice

Carlos E. Medina-De la Garza 1,2 , Graciela Guerrero-Ramírez 1 , Marisela García-Hernández 1,2 , M. Angeles Castro-Corona 1,2 , Ernesto Torres-López 1,2 , Norbert W. Brattig 3 , and Mario C. Salinas-Carmona 1

, Norbert W. Brattig 3 , and Mario C. Salinas-Carmona 1 Immunology Department, School of Medicine,

Immunology Department, School of Medicine, 2 Center of Research and Development in Health Science, Universidad Autónoma de Nuevo León (UANL), Monterrey, México, and 3 Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany

1

abstract Diethylcarbamazine (DEC) is an anthelmintic piperazine derivative drug with putative immunomodulating properties, including increased platelet and granulocyte adhesion to parasites and enhanced production of cytokines. To further analyse these properties in a well-established animal model, we evaluated the effect of DEC on antibody, cellular cytokine response and respiratory burst in BALB/c mice. Animals were challenged with a thymus-dependent (tetanus toxoid, (TT)) and with a thymus-independent (lipopolysaccharide, (LPS)) antigen and treated with DEC for seven days with two different doses (50 mg/day and 500 mg/day). Serum was assessed for antibody production at 0, 4, 7, 14, 21 and 28 days after stimulation and at 0, 24 and 48 h for IL-2, IFN-γ, IL-10 and IL-12 release. Respiratory burst of neutrophils and monocytes from peripheral blood was measured by flow cytometry. We found low-dose treatment with DEC enhanced cytokine production vs. TT and antibody production vs. LPS, whereas a higher dose enhanced significantly the respiratory burst of both polymorphonuclear leukocytes and monocytes, with a significant higher effect on the former. Our results suggest a stimulating, dose-dependent immunomodulatory effect of DEC with a higher effect on the phagocytic cells. Keywords: Diethylcarbamazine, immunomodulation, cytokines, antibody production, respiratory burst

Introduction

The piperazine derivative diethylcarbamazine citrate (DEC, N,N-dyethyl-4-methylpiperazine-1-carboxamide) is a synthetic organic antifilarial drug which has been extensively used since 1947 for the treatment for onchocerciasis and is currently used against Wuchereria, Brugia malayi and B. timori. The drug is effective in vivo against microfilaria (mf) of the parasites. Nevertheless, its mechanisms of antiparasitic action remain poorly understood. (1) Interference with the metabolism of arachidonic acid and breakdown of mf membranes, organelle damage and apoptosis among others, have been proposed. (24) DEC inhibits leukotriene production via 5-lipoxigenase and mice infected with W. bancrofti

and deficient on nitric oxide synthase showed a lesser effect of the drug on the parasite as is also observed with the inhibition of cyclooxygenase-1. (5,6) The involve- ment of phagocytes and immunologic effectors in the increased in vitro adherence and killing of Onchocerca mf by eosinophils, increased activation of neutrophils and enhanced adherence of platelets to parasites, sug- gested DEC would be effective through the immune system of infected host. (79) Based on the evidence of DEC interaction with the humoral and cellular effec- tors, putative immunomodulatory properties have been investigated: an increased antibody response and 10-fold increased peripheral monocyte counts in cats after a vaccination with feline leukemia virus vaccine has been

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478 C. E. Medina-De la Garza et al.

Table 1. Experimental groups of BALB/c mice.

Group

Treatment

1

Control (untreated)

2

TT 0.03 IU/mouse

3

LPS 10 µg/mouse

4

Control 25 µg DEC

5

TT 0.03 IU/mouse + 25 µg DEC

6

LPS 10 µg/mouse + 25 µg DEC

7

TT 0.03 IU/mouse + 250 µg DEC

8

LPS 10 µg/mouse + 250 µg DEC

DEC, diethylcarbamazine; LPS, lipopolysaccharides; TT, tetanus toxoid.

reported. (10) Similarly, kittens infected with the virus and treated with DEC were prevented or delayed to develop lymphopenia. (11) Some other studies with variant meth- odology have reported diverse degree of putative immu- nomodulatory effects, including the increased clearance of bacteria and fungi in mice under treatment with the drug and lack of growth of Mycobacterium tuberculosis- infected human blood supplemented with the drug. (12) So far, different animal models and notably variant experi- mental approaches have been used for the assessment of DEC as a potential immunomodulator. Therefore, a definite evaluation of DEC as immuno- modulator deserves a methodological continuity that allows a comprehensive assessment of the drug’s poten- tial. In this study we investigated the effect of DEC in an immunologically well-defined model of healthy BALB/c, analysing the antibody production, cytokine response and oxidative metabolism of phagocytes.

Materials and methods

Agents

DEC citrate was purchased from Sigma (Sigma, St Louis, Mo., USA). Antigens used to inoculate mice were: as thy- mus-dependent antigen, tetanus toxoid (TT) in solution (Behring-Aventis, Mexico) and as thymus-independent antigen, lipopolysaccharides (LPS) from Escherichia coli (serotype 0111:B4) (Sigma L2630). For the standardization of an ELISA-test to determine antibodies against these antigens, we used anti-mouse peroxidase conjugated anti- bodies of isotypes IgG (Sigma A2554) and anti-mouse poly- valent immunoglobulins (G,A, and M) (Sigma A0412).

Mice, immunization and treatments

This study was performed in accordance with the Studies Division Posgrade and Ethical Committee of School of Medicine, University Autonomous of Nuevo León (U.A.N.L.). All animal protocols were approved by the rules and regulation of our Institution for animal care. Experiments were performed on BALB/c female and male mice, between 6 and 12 weeks old. We used eight groups of seven BALB/c mice each, for the different experimental conditions: Group 1, non-immunized and untreated mice as control; Group 2, mice immunized with TT 0.03 IU/mouse and Group 3: mice immunized

with LPS 10 µg/mouse, both subcutaneously (s.c.) on the leg as appropriate as antigen control. Group 4, pharma- cological control, mice treated with DEC in a daily dose of 25 µg/mouse for 7 days. Group 5, mice immunized with 0.03 IU of TT/mouse and treated with a daily dose of DEC 25 µg/mouse (equivalent of a 50 mg/day human dose) for seven days; Group 6, mice immunized with 10 µg of LPS/mouse and treated with a daily dose of DEC

25 µg/mouse. Group 7: mice immunized with 0.03 IU of

TT/mouse and treated with a daily dose of DEC 250 µg/ mouse (equivalent to the highest human dose, 500 mg/ day) for 7 days. Group 8: mice immunized with 10 µg/ mouse of LPS, and treated with a daily dose of DEC 250 µg/mouse for 7 days (Table 1). For stimulation, all antigens were injected subcutane-

ously without additional adjuvant. However, mice of TT group were also immunized 7 days prior to start of trial (treatment) with a dose of 0.03 IU. For oral administra- tion of DEC, we used 1 mL syringes and a gastrointesti- nal tube. DEC solutions were prepared of 0.25 mg/mL, 0.85 mg/mL, 2.5 mg/mL and 1.7 mg/mL in sterile saline solution 0.9% PiSA) for the administration to mice. DEC was administered on a dose of 25 µg/mouse/day (Groups

3 and 4), and with 250 µg/mouse a day (Group 5). This treatment was given for 7 days.

Sera for antibody and cytokine determination

All groups of mice were bled with Pasteur pipettes through

puncture of retro-orbital plexus on days 0, 7, 10, 14, 21 and

28 for serum to determine antibodies, and on days 0, 24

and 48 h for serum to determine concentration of cytok- ines. An amount of approximately 500 µL of whole blood from each mouse was retrieved; blood samples were immediately placed in a centrifuge (Beckman, Allegra 64R) with refrigeration at 4°C and 12,000 rpm/10 min. Those for determination of antibodies were stored at −20°C and for determination of cytokines were aliquoted and stored at −70°C.

Control sera

For negative and positive control sera vs. LPS, five mice were immunized subcutaneously with 10 µg/mouse LPS with Freund’s complete adjuvant (Sigma F5881) on day 0, and with incomplete Freund’s adjuvant (Sigma F5506) on days 7, 14, 21 and 28. The mice were bled on days

0 and 28. The pool of serum from day 0 was labeled as

negative, and serum pools from day 28 were labeled as positive controls. For control sera to TT, five mice were immunized by subcutaneous injection of 0.3 IU/mL of TT with Freund’s complete adjuvant (Sigma F5881) on day 0, and with incomplete Freund’s adjuvant (Sigma F5506) on days 7, 14, 21 and 28. The mice were bled on days 0, and 28. The pool of serum from day 0 was labeled as negative control and sera from day 28 as positive control.

Determination of antibodies to TT by ELISA

ELISA plates and assays for IgG and total antibodies vs. TT by ELISA were prepared as described elsewhere. (13)

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total antibodies vs. TT by ELISA were prepared as described elsewhere. ( 1 3 ) Immunopharmacology

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Microplates were coated with antigen adding 50 µL of a solution of 15 IU of TT diluted in PBS buffer pH 7.4 and to a final concentration of 0.03 IU per well and incu- bated overnight at 2–8°C. The content was discarded and washed thrice with PBS–Tween Buffer 1:1000 (PBS–T). After washing, 50 µL/well blocking solution of PBS–T 5% skim milk was added, and incubated 1 h. After washing thrice, the microplate was stored at 2–8°C until later use. For determining total antibodies, 50 µL of the antibody conjugate were added (Anti-mouse polyvalent immuno- globulins (G,A, and M) antibody conjugated with peroxi- dase, Sigma A0412) diluted 1:10 000 in blocking solution PBS−T and 1% skim milk. For IgG determination, 50 µL of conjugate were added (anti-mouse IgG-antibody conjugated with peroxidase, Sigma A2554) diluted 1:70 000 in blocking solution and incubated for 1 h/37°C and washed. 50 µL substrate o-phenyldiamine dihydrochlo- ride (OPD, Sigma P9187) was added and incubated in darkness at room temperature for 30 min. Reaction was stopped with 0.1 M H 2 SO 4 and proceeded immediately to determine the absorbance at 495 nm wavelength.

Determination of antibodies to LPS by ELISA

Briefly, for IgG and total antibodies vs. LPS, antigen was fixed as 200 µL of a solution of 50 mg of LPS in 1 mL of carbonate buffer pH 9, with a final concentration of 0.3 µg/well. Subsequently the microplate was incubated at 2–8°C overnight. The content was discarded and the plate washed thrice with wash solution (PBS−T). Once washed, it was added 200 µL/well of blocking solution (PBS–T and 5% skim milk), and incubated 1 h. It was washed again as described and stored at −20°C. For total antibodies determination, 200 µL of antibody conjugate were added (anti-mouse polyvalent immunoglobulins (G,A,M) anti- body conjugated with peroxidase, Sigma A0412) 1:10 000 diluted in blocking solution (PBS–T and 1% skim milk). For IgG antibodies determination, 200 µL of anti-mouse IgG-antibody conjugated with peroxidase (Sigma A2554) diluted 1:70 000 in blocking solution was added and incubated for 1 h at 37°C. After incubation and wash- ing, 100 µL substrate (OPD) was added and incubated in darkness at room temperature for 30 min. Reaction was stopped with 0.1 M H 2 SO 4 and proceeded immediately to determine the absorbance at 495 nm wavelength propor- tional to antibodies amount.

Determination of cytokines

Measurement of cytokines was made after the inocula- tion of TT or LPS and concomitant treatment with DEC at 25 µg/mouse/day (low-dose) and 250 µg/mouse/ day (high-dose) throughout the 48 h. Sera from mice so treated were obtained at 0, 24, and 48 h after the inocula- tion and start of treatment, and stored at 70°C. These sera were thawed for determination of following cytokines IL-2, IL-10, IL-12 and IFN- γ by a commercial ELISA kit accordingly to the manufacturer’s specifications for each cytokine. The kits used were: IL-2 (mouse Interleukin-2, EMIL22, Pierce, USA), IL-10 (mouse interleukin-10

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Immunomodulatory activity of diethylcarbamazine 479

ELISA, EM2IL10, Pierce), IL-12 (Mouse Interleukin12 P70 ELISA, EMIL122, Pierce), IFN- γ (Mouse IFN-γ gamma ELISA, EM10012, Pierce).

Determination of respiratory burst in neutrophils and monocytes

To determine respiratory burst, two control groups and four test groups of 5 mice each were formed. The first group was untreated mice and second group mice were treated with 0.1 mL of saline. To conform the test groups, two variables were considered: (1) doses of DEC and (2) time of testing after DEC intake. Therefore, two groups were set using a treatment dose of 85 µg/mouse (200 mg/day) of DEC in 0.1 mL of saline solution, where one was measured at 1 h and the other at 2.5 h. Other two groups were set using a treatment dose of 170 µg/mouse (400 mg/day) of DEC in 0.1 mL of saline solution, where one was measured at 1 h and the other at 2.5 h. Doses used for respiratory burst experiments were based on pharmacologic studies using the range corresponding to a one-dose scheme (14) and are different from those used as stimulant for humoral and cellular response, where DEC is administered upon several days. The timing of measurement was based upon reported serum high- est peak on rodents (1 h) (15,16) and human (2.5 h). (14,17) Mice leukocytes were obtained using 700 µL of blood with EDTA. The blood was placed into tubes and mixed with 7 mL of lysis solution, previously incubated at room temperature (FACS, Becton Dickinson). The tubes mixed with the lysis solution were incubated for 10 min at 37°C and centrifuged at 1300 rpm at 4°C by 5 min. (centrifuge, Eppendorf 5403). The supernatant was discarded, and the pellet was washed and centrifuged twice with PBS pH 7.4 at the same conditions. Finally, the cells were resuspended in 7 mL of PBS pH 7.4 for each tube (approximately 2.5 × 10 6 cells/mL), and ready to use in the assay. The cells were analysed under dif- ferent conditions with the dyhydrorhodamine 123 tech- nique (123-DHR, Sigma D1054) using zymosan (ZYM; Zymosan A Saccharomyces cerevisiae, Sigma Z4250) and PMA (Phorbol-myristate acetate, Sigma P8139) as stimulus. (18) Measurement of mean fluorescence intensity (MFI) of neutrophils was conducted by flow cytometry (Becton Dickinson FACsort) using Cellquest software. We analysed the region for granulocytes and for monocytes. The results were obtained by printing histograms containing values and statistical analysis of the MFI and percentage of cells that were detected for both cell populations.

Statistical analysis

For the comparison of the results obtained and deter- mine significant differences among studied groups tested for antibodies, cytokines and respiratory burst, a nonparametric Wilcoxon test for multiple comparisons was applied, using the statistical packages SPSS 10.0, R software for statistical computing and graphs version 2.10.0 and Excel.

using the statistical packages SPSS 10.0, R software for statistical computing and graphs version 2.10.0 and

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480 C. E. Medina-De la Garza et al.

Results

Effect of in vivo DEC treatment on the production of antibodies to TT and LPS in BALB/c mice

The effect of a DEC treatment on the production of IgG antibodies to TT in mice is shown in Figure 1. A pronounced antibody response (OD = 0.54 ± 0.07) was obtained with a high-dose DEC (500 mg/day) treatment when compared with the untreated control group at day 28. DEC at low dose (50 mg/day) presented values similar to untreated control at day 28 (OD = 0.34 ± 0.15). The dif- ferences, however, were not statistically significant. The determination of total antibody response to TT (data not shown) had a similar shape as the IgG production. Regarding antibody response to LPS, only the group with low-dose DEC (50 mg/day) showed a higher value constantly from day 14 on 28 days after immunization. The antibody response to LPS is significantly higher in animals treated with low concentration of DEC (OD = 0.62 ± 0.1) compared with the untreated control (OD = 0.23 ± 0.05) (p ≤ 0.05) (Figure 2). The OD values for the total anti-LPS antibodies were similar to those of IgG anti-LPS antibodies (data not shown).

Effect of DEC on in vivo cytokine response to TT and LPS

To evaluate the effect of DEC on a Th1 cytokine response, we analysed the release
To evaluate the effect of DEC on a Th1 cytokine response,
we analysed the release of IL-2, IFN- γ , and IL-12 at day
0, 1 (24 h) and 2 (48 h) after stimulus. Concerning the
release of IL-2 in response to TT, the mice treated with the
low-dose of DEC showed a higher cytokine production at
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Figure 1. Production of IgG antibodies induced by tetanus toxoid (TT). Five groups of BALB/c mice (n = 7) were used in this study. Blood was collected at days 0, 7, 10, 14, 21 up to 28. Serum was separated and used to the estimation of antibody production using TT as antigen. Values represent mean ODs ± S.D. There was no significant difference.

24 h (267 ± 170 pg/mL; p ≤ 0.05) when compared to nega- tive control (Figure 3). The other test groups showed very low cytokine concentrations at both, 24 and 48 h with almost no differences. Production of IFN-gamma was not

with almost no differences. Production of IFN-gamma was not Figure 2. Production of IgG antibodies induced

Figure 2. Production of IgG antibodies induced by lipopolysac­ charides (LPS). Five groups of BALB/c mice (n = 7) were used in this study. Blood was collected at days 0, 7, 10, 14, 21 up to 28. Serum was separated and used to the estimation of antibody production using LPS as antigen. OD values represent mean ± S.D. There was a significant difference at day 28 (*p ≤ 0.05) comparing the low dose of diethylcarbamazine (DEC) to antigen alone and to high­dose DEC.

(DEC) to antigen alone and to high­dose DEC. Figure 3. Effect on IL­2 production induced by

Figure 3. Effect on IL­2 production induced by TT. Cytokine expression was followed up to 48 h (n = 5). Values represent mean ± S.D. There was a significant difference at 24 h (*p ≤ 0.05) comparing the low dose of DEC to antigen alone and to high­dose DEC.

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p ≤ 0.05) comparing the low dose of DEC to antigen alone and to high­dose DEC.

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stimulated in any group and any time (data not shown). IL-10 was slightly stimulated, but again there were no significant differences between any group at any time or doses. In difference, the production of IL-12 was signifi- cantly enhanced at 24 h by low-dose DEC (379 ± 13 pg/mL) compared to the untreated control (281 ± 63 pg/mL) and the high-dose of DEC (235 ± 62; p ≤ 0.05) (Figure 4). Equally to the response to TT, after stimulation with LPS, all groups showed similar IL-2 and IFN-gamma lev- els. Interleukin-10, however, showed at 24 h significantly higher concentrations after treatment with both DEC doses than the control (p ≤ 0.05); at 48 h, the production of the cytokine was decreased (Figure 5). The levels of IL-12 were significantly higher in the group treated with low-dose DEC (p ≤ 0.05) than the antigen control, DEC alone and high-dose DEC at 48 h (Figure 6).

In vivo effect of DEC on the respiratory burst of neutrophilic granulocytes and monocytes

One hour after oral intake of DEC at high-dose (170 µg/ mouse), peripheral neutrophilic granulocytes (neu- trophils) reacted with a more pronounced respiratory burst stimulated with PMA, ZYM and both PMA/ZYM and expressed by mean fluorescence intensity (MFI) (Figure 7A). A comparatively much lower response was observed when measured 2.5 h after intake of the drug. The MFI of peripheral monocytes was much lower (Figure 7B). The high-dose DEC effect measured at 1 h showed a significant difference to low-dose DEC and unstimulated controls (p ≤ 0.05). MFI measurements with high-dose DEC at 2.5 h were lower than that of 1 h,

with high-dose DEC at 2.5 h were lower than that of 1 h, Figure 4. Effect

Figure 4. Effect on IL­12 production induced by TT. Cytokine expression was followed up to 48 h (n = 5). Values represent mean ± S.D. There was a significant difference at 24 h ( * p ≤ 0.05) comparing the low dose of DEC to antigen alone and to high­dose DEC.

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Immunomodulatory activity of diethylcarbamazine

481

but significantly higher than the DEC-low-dose at both time conditions, using PMA and PMA/ZYM (p ≤ 0.05). In all cases, both, neutrophils and monocytes showed dose- dependent reactions with a highest effect recorded at 1 h after intake of DEC.

with a highest effect recorded at 1 h after intake of DEC. Figure 5. Effect on

Figure 5. Effect on IL­10 production induced by LPS with DEC treatment. The cytokine expression was followed up to 48 h (n = 5). Values represent mean ± S.D. There was a significant difference at 24 h ( * p ≤ 0.05) comparing both doses of DEC to antigen alone.

p ≤ 0.05) comparing both doses of DEC to antigen alone. Figure 6. Effect on IL­12

Figure 6. Effect on IL­12 production induced by LPS with DEC treatment. The cytokine expression was followed up to 48 h (n = 5). Values represent mean ± S.D. There was a significant difference at 48 h ( * p ≤ 0.05) only vs. antigen alone, DEC alone and high­dose DEC.

S.D. There was a significant difference at 48 h ( * p ≤ 0.05) only vs.

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Figure 7. Effect of DEC on the respiratory burst of phagocytes from peripheral blood of mice. (A) Neutrophils, the expression of MFI with high­dose (H) of DEC at 1 h showed a significant difference compared to control groups and low­dose (L) DEC, and to DEC (H) at 2.5 h in PMA + DHR and ZYM + DHR ( * p ≤ 0.05). (B) Monocytes, the expression of MFI with high­dose (H) of DEC at 1 h showed a significant difference compared to control groups and low dose (L) DEC ( * p ≤ 0.05) but not to DEC (H) at 2.5 h. All values represents mean ± S.D.

Discussion

DEC is a chemically well-characterized drug whose therapeutic doses and effects have been extensively studied. (17) DEC functions as antifilarial drug and has been proposed as candidate for evaluation as immuno- modulatory drug with therapeutic potential. However, from our view, some studies to investigate this effect

have been performed in a non-consistent fashion with varying drug doses, methodologies and experimental designs. In the present study, we investigated the in vivo effect of DEC on humoral and cellular cytokine responses in BALB/c mice immunized with thymus- dependent and -independent antigens, and on phago- cytic effectors. The doses used in antibody and cytokine production experiments reflected the human thera- peutic dose (low-dose DEC) and the highest human dose allowed (high-dose DEC). The respiratory burst experiments used doses of DEC calculated for a single- dose scheme. We deliberately avoided experimental conditions using infra- or supra-therapeutic doses. (10,19) Positive effects on the antibody production of other reports was corroborated by our results. (8) The increase of total antibody classes and IgG anti-TT titers with high-dose of DEC was demonstrated. The induction of antibodies against LPS with low-dose DEC produced an evident effect. These interesting results suggest a poten- tiating DEC effect on humoral responses that depends on nature of antigen. A possible adjuvant effect of DEC must be further investigated. Kitchen et al. observed a more pronounced effect on the antibody response using animal model with different vaccines. (19) The increment of the antibody response against LPS, in particular, sug- gests a potential use for DEC against LPS-containing microbes. Effects on the cytokine production by DEC have been reported. (20) DEC enhanced the generation of pro- inflammatory cytokines as IL-6 and IL-2 in mononuclear cells in vitro. (21) In our study, DEC showed its in vivo effect on pro- and anti-inflammatory cytokine responses represented by IL-2, IFN- γ , IL-12 and IL-10. Mice treated with high-dose DEC and immunized with TT expressed enhanced IL-2 and IL-12 which relates to the increase in the antibody response to this antigen. In addition, we observed a decrease of serum IL-10 level. In contrast, in sera of the group receiving low-dose DEC and immunized with LPS, IL-2 was not detected but high levels of IL-10 that correlates with the increasing response of antibodies to LPS. We further investigated the phagocytic activity through burst oxidative. Evident differences of MFI occurred among all groups. Although intermediary oxidative species production by phagocytes has been shown to be enhanced by DEC, (22) the present report represents the first on the assessment of burst oxida- tive of phagocytic cells stimulated by DEC treatment in vivo. Further, these results appear to correlate with morphological studies, an increase of antifilarial activ- ity by respiratory burst derivatives from granulocytes in tissues and ex vivo (2) and also an increase of some other granulocyte enzymes as myeloperoxidase, elastase, lactoferrin, and lysosyme. (23) One feature observed was the time-depending effect observed in the respiratory burst. Highest DEC concentration has been shown 1 h after application in cotton rats (16) whereas, for man it ranges between 2.2 and 2.7 h. (17) The highest effect on

Immunopharmacology and Immunotoxicology

for man it ranges between 2.2 and 2.7 h. ( 1 7 ) The highest effect

Immunomodulatory activity of diethylcarbamazine

483

   

phagocytes at 1 h in BALB/c correlated well with the pharmacokinetic data from rodents.

 

8. Medina­De la Garza, C.E., Brattig, N.W., Tischendorf, F.W., Jarrett, J.M. Serum­dependent interaction of granulocytes with Onchocerca volvulus microfilariae in generalized and chronic hyper­reactive diethylcarbamazine. Trans R Soc Trop Med Hyg

   

conclusion

 

1990, 84, 701–706.

       

9. Cesbron, J.Y., Capron, A., Vargaftig, B.B., Lagarde, M., Pincemail,

   

The presented results show that DEC could act directly on phagocytic response and affect the humoral and

 

J., Braquet, P., Taelman, H., Joseph, M. Platelets mediate the action of diethylcarbamazine on microfilariae. Nature 1987, 325,

   

cytokine responses. Thus, experimental evidence of DEC

533–536.

   

as potential immunomodulator or adjuvant appears jus-

10.

11.

Kitchen, L.W. Effect of diethylcarbamazine on cats given feline leukaemia virus vaccine. Vaccine 1987, 5, 266–267.

179–181.

   

tified for its further assessment as an immunologic modi-

 

Kitchen, L.W., Mather, F.J., Cotter, S.M. Effect of continuous oral

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fier for potential use in the treatment of human diseases, e.g. in chronic pyogenic and insidious infections.

diethylcarbamazine treatment on lymphocyte counts of feline leukemia virus­infected cats. J Clin Lab Immunol 1988, 27,

 

acknowledgments

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

Kitchen, L.W., Weston, C.M., Day, S.P. Diethylcarbamazine­related antimicrobial activity in Mycobacterium tuberculosis­infected blood. J Antimicrob Chemother 1998, 42, 241–243.

 

We thank head-librarian Martina-Christine Koschwitz at Bernhard Nocht Institute, Hamburg, Germany, and Dr Gloria Molina-Salinas from CIBIN-IMSS, Monterrey, Mexico, for specific bibliographical material. We thank

Salinas­Carmona, M.C., Welsh, O., Casillas, S.M. Enzyme­linked immunosorbent assay for serological diagnosis of Nocardia brasiliensis and clinical correlation with mycetoma infections. J Clin Microbiol 1993, 31, 2901–2906.

   

Rèe, G.H., Hall, A.P. Plasma levels of diehytlcarbamazine in man.

 

the staff of CIMAT, Monterrey-Unit, for support on orga- nizing data.

 

Transactions of the Royal Society of Tropical Medicine and Hygiene 1977, 71, 542–543. Horii, Y., Aoki, Y. Plasma levels of diethylcarbamazine and their effects on implanted microfilariae of Brugia pahangi in Rats. J Vet

 

Declaration of interest

Med Sci 5910:961–963.

     

Roy, T.K., Satyavan, S., Srivastava, V.M. Comparative tissue

 

This work was supported by grant PAICyT SA-611-01 from Universidad Autónoma de Nuevo León, and by the

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