Received: 24 October 2007 / Accepted: 31 October 2007 / Published online: 30 December 2007 Ó Birkhäuser Boston 2007

Transcription

1 Med Chem Res (28) 17: DOI 1.17/s ORIGINAL RESEARCH MEDICINAL CHEMISTRY RESEARCH Identification of Brugia malayi adult worm molecules immunoreactive with sera of infected animals treated with antifilarial and re-infected with homologous infection and their role in the establishment of infection in Mastomys coucha R. L. Gaur Æ M. K. Sahoo Æ S. Dixit Æ S. K. Joseph Æ P. K. Murthy Received: 24 October 27 / Accepted: 31 October 27 / Published online: 3 December 27 Ó Birkhäuser Boston 27 Abstract The present study was aimed at identifying molecules of Brugia malayi adult worm (BmA) that are immunoreactive with sera of B. malayi-infected Mastomys coucha treated with albendazole (ALB) or diethylcarbamazine (DEC) and reexposed to infection and the effect of immunization with the molecules on the establishment of infection in M. coucha. A*62 kda molecule showed strong reactivity with sera of infected ALB-treated reinfected animals whereas *32 kda and *45 kda molecules strongly reacted with sera of DEC-treated reinfected animals. Two molecules (*22 kda and *28 kda) reacted with sera of infected untreated and reinfected control animals. Immunization with *62 kda molecules reduced the adult worm recovery by 58% (P \.1) and microfilaremia by 32 54% (P \.5.1) of unimmunized controls. Animals immunized with the *32 kda molecule exhibited 63% recovery of adult worms with no effect on circulating microfilaraemia. L 3 inoculation in *62 kda-immunized animals upregulated cellular proliferation, interferon-c (IFN-c) and IgG responses (P \.1) but downregulated interleukin-1 (IL-1) response (P \.1). Animals immunized with *22, *28, *32 and *45 kda molecules and bearing L 3 - induced infection showed mixed responses. Lymph node cells of unimmunized animals exposed to *28, *32 and *62 kda molecules showed significant DNA damage (P \.1) as compared to untreated control; *62 kda molecule induced maximum damage. In conclusion, immunization with a *62 kda molecule suppressed the establishment of L 3 -induced infection in M. coucha and this correlated with enhanced cellular proliferation and IFN-c release, downregulation of IL-1, increased levels and DNA damage in lymph node cells. Keywords Brugia malayi Albendazole Diethylcarbamazine Mastomys coucha IFN-c IL-1 IgG DNA damage R. L. Gaur M. K. Sahoo S. Dixit S. K. Joseph P. K. Murthy (&) Division of Parasitology, Central Drug Research Institute, Lucknow 2261, India drpkmurthy@yahoo.com

2 446 Med Chem Res (28) 17: Introduction Human lymphatic filariasis, a mosquito-borne disease of approximately 8 tropical and subtropical countries, is caused by the nematode parasites Wuchereria bancrofti, Brugia malayi, and B. timori. A billion people are estimated to be at risk of infection, with 12 million already infected and 4 million seriously incapacitated or disfigured by the disease (Ottesen, 2). While the disease does not result directly in death, it is the second most common cause of long-term disability globally and responsible for huge economic loss. One third of people infected with lymphatic filariasis (LF) live in India, a third live in Africa and the remainder live in Papua New Guinea, Southeast Asia, the Pacific Islands, and the Americas. In India, more than 5 million people are exposed to infection with 45.5 million people harboring circulating microfilaraemia and another 22.5 million people suffering from chronic manifestations of the disease like hydrocele, lymphoedema, and elephantiasis. Around 5% of the infected persons suffer from acute episodic attacks of adenolymphangitis and fever (WHO, 21; Melrose, 24). Reduction of morbidity associated with the disease and interruption of transmission are the general strategies currently being used for the control of human filariasis employing chemotherapy and vector control methods. Despite considerable growth in the knowledge about the causative agents and manifestations of the disease as well as constant efforts to develop drug treatment using antifilarials alone or combination approaches (Gelband, 1994; Navaratnam, 1993; Ottesen et al., 1997) to free patients of infection, the global burden of this disease continues to be high, showing evidence of the reappearance of microfilariae (MF) in MF carriers after treatment. Therefore, to check reinfection remains a big challenge, the main problem being with the limited knowledge about the host parasite interaction after drug treatment. In this regard we recently demonstrated that, in the Mastomys coucha model of human brugian filariasis, ALB but not DEC treatment may help to prevent the development of reinfections. Encouraged by these results, the present study aimed to (i) identify functional molecule(s) of the parasites that reacted with sera of M. coucha harboring B. malayi infection that were treated with ALB or DEC and subsequently re-exposed to infective larvae (L 3 ) and (ii) evaluate the responses of M. coucha to the identified molecules in relation to establishment of L 3 -induced infection, specific IgG, cellular proliferation and cytokine release. Materials and methods Isolation of parasites and preparation of parasite extract B. malayi adult worms (BmA) were recovered from the peritoneal cavity of jirds (Meriones unguiculatus) experimentally infected with B. malayi (Murthy et al., 1997). These were incubated at 37 C for 1 h using phosphate buffered saline (PBS) in Petri dishes to remove host cells adhered to the parasite surface, washed thoroughly with sterile phosphate buffered saline (PBS,.1 M, ph 7.2). Soluble extract of BmA () was then prepared as described by Tandon et al. (1988). Briefly, parasites

3 Med Chem Res (28) 17: were washed with sterile PBS (ph 7.2) and homogenized in a Porter Elvejhm tissue grinder (A. Thomas Scientific, Philadelphia, PA) at 4 C. The homogenate was sonicated at 1 kc/sec for 1 min with 6 s intervals after every stroke of 6 s in cold conditions (4 C) followed by centrifugation at 1, g for 1 min at 4 C; the protein content of the supernatant measured by the method of Lowry et al.(1951). The parasite preparation was stored in.5 ml aliquots at -2 C until use. Fractionation and isolation of resolved protein molecules from sodium doedecyl polyacrylamide gel electrophoresis (SDS-PAGE) Extract of BmA isolated as above was prepared in sample buffer and resolved following broadly the method of Laemmli, et al. (197) with some modifications as described earlier (Murthy et al., 1999). Immunoblotting of resolved components of BmA with sera of M. coucha Resolved components in gel were transferred onto nitrocellulose paper (NCP) as described by Towbin et al. (1979). Immunoreactivity of NCP blotted components was analyzed using sera of M. coucha according to the method described by Murthy et al. (1999). Briefly, NCP was cut vertically into 2 mm wide pieces, washed with 1 mm Tris-buffered saline (TBS; ph 7.4) in a chambered incubation tray and blocked with 3% gelatin prepared in TBS for 2 h at 37 C. After washing with TBS-T2 (.5%), the strips were incubated with sera (1:5) at 37 C for 9 min followed by washing and incubation with antimouse IgG-conjugated with horse radish peroxidase (1:1) for the same time period. Substrate solution containing diaminobenzidine (DAB) prepared in TBS and H 2 O 2 was added and the bands were visualized. The reaction was stopped with water; the strips were then dried and analyzed. Isolation of identified molecules and protein elution Preparatory gel (1%) was used for isolation of molecules of interest. A measured amount of protein per run (approximately 5 lg) was used on each occasion so that amount of protein in each band of interest remained constant. The protein bands of interest were cut with the help of prestained molecular-weight protein marker (Sigma Chemical Co. USA) using a clean sharp scalpel. Proteins from the gel strips were electroeluted (Electroeluter, Millipore, India), concentrated, and estimated by the method of Lowry et al. (1951). These semipurified molecules were used in the present study. Isolation of L 3 B. malayi L 3 used for inoculation were freshly isolated from mosquitoes, which were fed on microfilaremic M. coucha 9 1 days before (Murthy et al., 1983).

4 448 Med Chem Res (28) 17: Immunization of animals and exposure to parasites Healthy 1- to 11-week-old male M. coucha were used in the study. All the experiments were conducted in compliance with the institutional animal ethics committee guidelines for the use and handling of animals. Throughout the study, the animals were housed in controlled climate (23 ± 2 C; relative humidity: 6%) and photoperiod (12 h light dark cycles). They were fed standard rodent chow supplemented with dried shrimps and drinking water was supplied ad libitum. The study included four groups (5 8 animals per group). Animals in groups 2 and 4 received three subcutaneous (s.c.) injections of protein molecules in gel minced with Freund s incomplete adjuvant (FIA). Each animal received lg proteins of the molecules at weekly intervals. Groups 1 and 3 received gel only (i.e., without the parasite molecules) in FIA and served as age-matched controls for groups 2 and 4, respectively. On day 21 post first injection of the protein molecules or gel only, groups 3 and 4 received 1 L 3 through the subcutaneous (s.c.) route. These animals were sacrificed on day 15-post L 3 inoculation (p.l.i.). Animals in groups 1 and 2 were sacrificed on day 21 post first dose of FIA. The animals were sacrificed by an overdose of ether anaesthesia. All the parameters were carried out on day 21 post first injection of FIA (group 1) or parasite molecules (group 2), or 15 p.l.i. (groups 3 and 4). Assessment of parasite burden Microfilaraemia was assessed on day 9 p.l.i. and thereafter at weekly interval until the termination of the experiment. Adult worms from various organs such as heart, lungs, testes, lymph nodes, and vessels of the animals were isolated and counted (Murthy et al., 1983). Cell-mediated immune response (CMI) The lymphocyte transformation test (LTT) using spleen cells was carried out to assess CMI broadly following the method of Klei et al. (199) with some modifications as described by Dixit et al. (26). The results were expressed as counts per minutes (cpm). For cytokine assay or LPS stimulated culture supernatants from 24-well plate were collected at 48 h post-stimulation (PS) and kept at -2 C until use. Cytokine determination IFN-c and IL-1 (Pharmingen) were estimated in culture supernatants as described earlier (Dixit et al., 26).

5 Med Chem Res (28) 17: IgG determination Filaria-specific IgG was detected in sera of animals following the method as described earlier (Murthy et al., 1995). Briefly, enzyme-linked immunosorbent assay (ELISA) plates/strips (Nunc, Denmark) were coated with (.1 lg/ml) prepared in carbonate buffer (.6 M; ph 9.6). Sera were used at a dilution of 1:5, and probed with rabbit anti-mouse-igg conjugated with horseradish peroxidase (Sigma Chem. Co, USA) at 1:1 dilutions. Absorbance was read at 492 nm in an ELISA reader (Power WaveX, BioTek, USA). Assessment of DNA damage Isolation of lymph node cells from animals The lymph nodes isolated from naive M. coucha in incomplete RPMI-164 medium containing antibiotics were teased. The cells isolated were passed through a 1-lm nylon filter, centrifuged, and suspended in complete medium (containing 1% FCS). The viability of cells was checked using.1% trypan blue exclusion method and the cell concentration (viable cells) was adjusted to /ml in complete RPMI-164 medium. The cell suspension ( cells in 2 ll/well) was dispensed in 96-well tissue culture plate (Nunc, Rosklide Denmark) in triplicate and exposed to optimum concentration of protein molecules (1 lg/ml). Unexposed cells served as control. Cells were collected after 2 h and 24 h PS. Comet assay The technique was performed using lymph nodes cells as described by Singh et al. (1988). Briefly, the cells were suspended in 11 ll of low melting agarose (.5% in phosphate-buffered saline) and layered on a partly frosted slide, precoated with 1 ll of normal melting point agarose (1% in tripple distilled water). Finally, 11 ll of low-melting-point agarose was layered on the top. The slides (in triplicate) were left for 2 h at 4 C in freshly prepared lysing solution [(2.5 M NaCl, 1 mm Na-ethylenediamineetraacetic acid (EDTA), 1 mm Tris, 1% Triton X- 1) and 1% dimethyl sulfoxide]. The cells were treated with alkaline buffer (1 mm Na 2 EDTA in 3 mm NaOH; ph [ 13.) for 3 min to allow for denaturing and unwinding of the DNA. This was followed by electrophoresis in the same buffer at 2 V (.8 V/cm) and 3 ma for 3 min to allow damaged DNA fragments to migrate towards the anode. The slides were neutralized with.4 M Tris buffer (ph 7.5), washed, dried, and stained with silver staining (Nadin et al., 21). DNA damage was quantified as tail length. For each treatment, 8 1 nuclei were measured in two slides.

6 45 Med Chem Res (28) 17: Statistical analysis Results were presented as mean ± standard deviation (SD) of two experiments and the data were analyzed in GraphPad Prism 3.3 using Newman Keuls or Student s t-test. Differences with P \.5 were considered significant. Results Immunoblot reactivity of resolved protein bands of BmA with sera of B. malayi infected M. coucha that were treated with ALB or DEC and subsequently reexposed to L 3 The components of parasite extract with sera of DEC- or ALB-treated and reinfected animals resolved by the SDS-PAGE immunoreactivity profile are illustrated in Fig. 1. Normal animal sera did not react with any of the resolved components of BmA except *29 kda. Sera of infected reinfected animals (control) reacted with a wide range of molecules (14.2 to [ 2 kda) of BmA but *22 and *28 kda bands were strongly reactive with sera of infected untreated and reinfected animals (control). Sera of infected DEC-treated reinfected animals reacted strongly with *32 and *45 kda but some of the molecules also showed weak reactivity with other molecules (*58, *6, *62, *64, *73, *79, *97.4 kda). However, in the case of sera of infected ALB-treated reinfected animals only *62 kda band was identified to be strongly reactive. Fig. 1 Immunoblot reactivity of SDS-PAGE resolved fractions of adult B. malayi parasite with pooled sera of infected M. coucha treated with antifilarial and reinfected with L 3. Lanes showing immunoreactivity with sera of A: ALB-treated reinfected animals; B: DEC-treated reinfected animals; C: infected untreated reinfected animals

7 Med Chem Res (28) 17: The molecular weights of the identified protein molecules isolated as mentioned in the Materials and Methods section were confirmed by SDS-PAGE (Fig. 2). Host responses to identified molecules Effect of identified molecules on the establishment of L 3 induced infection in animals Animals from all the immunized groups and those subsequently inoculated with L 3 (group 4) showed MF in the peripheral blood between days 9 and 92 (data not shown) compared to control animals (group 3). Microfilaraemia in *62 kdaimmunized animals was increasingly suppressed until the day of sacrifice compared to group 3 animals (Table 1; P \.5.1). Microfilaraemia in animals immunized with the rest of the molecules + L 3 was comparable to group 3 animals (control). On autopsy *62 kda- and *32 kda-immunized animals showed 58% and 63% less adult worm recovery (P \.1), respectively, compared to group 3 animals (Fig. 3). However, adult worms recovered from *45-, *28- and *22 kda-immunized animals were comparable to group 3 animals. In summary, the findings demonstrate that immunization with the *62 kda molecule affected the establishment of L 3 -induced infection. Cell proliferative response Experiments were performed to investigate the effect of immunization with parasite molecules and subsequent exposure to L 3 on the proliferative responses of Fig. 2 SDS-PAGE (1%) resolved fractions of adult Brugia malayi parasite. Lanes A: molecular weight marker; B: *62 kda, C: *45 kda, D: *32 kda, E: *28 kda, and F: *22 kda bands. Protein molecules were isolated by electroelution, concentrated, and molecular weights confirmed in SDS-PAGE

8 452 Med Chem Res (28) 17: Table 1 Pattern of microfilaraemia in L3-induced B. malayi infection in M. coucha immunized with semipurified parasite molecules # Parasite protein molecules(kda) Microfilariae count/1 ll of blood on day post L3 inoculation (mean ± SD) ± ± ± ± ± ± ± ± ± ± ± a 32. ± ± 1.41 b 8.67 ± ± ± ± 2.19 c 8.8 ± 4.66 c 26.2 ± ± ± ± ± ± ± ±.18 c ± ± ± c ± ± ± ± 5.75 a ± 8.45 a ± 11.9 a 38. ± 7. c 33. ± 17.1 c ± 2.74 c 55. ± 34. b 82.8 ± c (Control) 15. ± ± ± ± ± ± ± ± 2.99 # 22 and 28 kda = reactive with sera of infected untreated reinfected animals; 32 and 45 kda = reactive with sera of DEC-treated reinfected animals; 62 kda = reactive with sera of ALB-treated reinfected animals; Control = naïve animal. a P \.5, b P \.1, c P \.1 (P values indicate significance vs control of respective time point)

9 Med Chem Res (28) 17: worms recovered of No. ( Mean ± SEM ) P<.1 (vs control) Ctrl Parasite molecules (kda) 62 Fig. 3 Worm recovery 15 days after B. malayi L 3 exposure to M. coucha pre-immunized with semipurified molecules in gel + FIA. P \.1, unpaired t-test splenocytes of the animals to in vitro stimulation with or Con A and to correlate these effects with the survival of adult worms or the establishment of L 3 - induced infection. The results were compared between immunized alone and immunized + L 3 -inoculated groups of each molecule (Figs. 4A F). After inoculation of L 3 in nonimmunized animals, the filarial-specific proliferative responses of the cells decreased (L 3 exposure; P \.1.1; Fig. 4A). Animals immunized with the *22 and *28 kda molecules receiving L 3 showed increases in responses to both and Con A challenge in vitro (P \.1.1; Fig. 4B,C). The responses in *32 kda-immunized animals bearing L 3 decreased (: P \.1; Con A: P \.1; Fig. 4D). On the other hand, L 3 administration in *45 kdaimmuized animals upregulated the specific response (P \.1; Fig. 4E). Interestingly, cells from animals of *62 kda-immunized animals receiving L 3 exposure showed enhanced cellular proliferative responses to both (P \.1) and Con A (P \.1) challenge in vitro (Fig. 4F). Cytokine release Studies were carried out to investigate the effect of IFN-c and IL-1 release from cells of animals immunized with the molecules and subsequently exposed to B. malayi L 3 in response to or LPS stimulation in vitro and to correlate this with parasite burden. The results were compared between immunized alone and immunized + L 3 -inoculated groups of each molecule. L 3 inoculation in *62 kda-immunized animals upregulated -specific IFN-c release (P \.1; Fig. 5F) but the release was downregulated (*28 kda: P \.1; *45 kda: P \.5) or remained unaltered (*22 kda and *32 kda) in animals immunized with the rest of the molecules. Con A failed to stimulate the release of IFN-c in any of the immunized animals (Fig. 5A E). In contrast, IL-1 release from cells of *62 kda immunized animals was downregulated (P \.1; Fig. 6F). Cells of *28 kda- and *45 kda-

10 454 Med Chem Res (28) 17: A Counts per minute ( Mean ± SD ) Gr 1 Gr 3 ** B Counts per minute ( Mean ± SD ) Gr 2 Gr 4 Con A Con A C Counts per minute ( Mean ± SD ) D Counts per minute ( Mean ± SD ) ** Con A Con A E Counts per minute ( Mean ± SD ) Con A F Counts per minute ( Mean ± SD ) ** Con A Fig. 4 Proliferative response to or Con A of spleen cells from naïve or immunized M. coucha infected with L 3. Control (A) gel + FIA (group 1) or gel + FIA + L 3 (group 3) whereas immunized animals received molecules in gel + FIA (group 2) or molecules in gel + FIA + L 3 (group 4). (B) *22 kda and (C) *28 kda molecules reactive with sera of infected untreated and reinfected animals; (D) *32 kda and (E) *45 kda molecules reactive with sera of DEC-treated reinfected animals and (F) *62 kda reactive with sera of ALB-treated reinfected animals. ** P \.1; P \.1, unpaired t-test immunized + L 3 exposed animals released significantly higher IL-1 (P \.1.1; Figs. 6C,E) release but *22 kda- and *32 kda-immunized + L 3 exposed animals did not alter the IL-1 release as compared to their counterparts immunized alone (Fig. 6B,D). IgG response L 3 inoculation resulted elevated response of anti- IgG in all the immunized animals (P \.1), the highest level being observed in *22 kda- and *32 kdafollowed by *62 kda-immunized animals. However, the level of IgG in *28- and *45 kda-immunized animals bearing infection was comparatively low. Unimmunized + L 3 inoculated animals showed the lowest response (Fig. 7).

11 Med Chem Res (28) 17: A Gr 1 Gr 3 5 ** B I FN- γ ( pg/ml; Mea n ± SD ) I FN- γ ( pg/ml; Mea n ± SD ) Gr 2 Gr 4 LPS LPS C NS D I FN- γ ( pg/ml; Mea n ± SD ) I FN- γ ( pg/ml; Mea n ± SD ) NS NS LPS LPS E * NS LPS F I FN- γ ( pg/ml; Mea n ± SD ) I FN- γ ( pg/ml; Mea n ± SD ) ** NS LPS Fig. 5 IFN-c release in response to challenge in vitro of spleen cells from M. coucha injected with gel only + FIA (group 1) and given L 3 (group 3) or semipurified molecules in gel + FIA (group 2) and subsequently exposed to L 3 of B. malayi (group 4). LPS challenge served as a positive control. (A) Group 1 versus group 3, (B) *22 kda and (C) *28 kda molecules reactive with sera of infected untreated and reinfected animals; (D) *32 kda and (E) *45 kda molecules reactive with sera of DECtreated reinfected animals and (F) *62 kda reactive with sera of ALB-treated reinfected animals (group 2 versus group 4). * P \.5; ** P \.1, P \.1, unpaired t-test DNA Damage Results of DNA damage incurred by lymph node cells of naïve animals in response to different semipurified molecules are presented in Fig. 8. Cells exposed to *28, *32 and *62 kda molecules for 24 h showed significant DNA damage (P \.1) as compared to untreated control; the *62 kda molecule induced maximum damage. The other two molecules did not affect the DNA. The H 2 O 2

12 456 Med Chem Res (28) 17: A I L-1 (pg/ml; Mean ± SD ) Gr 1 Gr 3 Gr 2 Gr 4 B I L-1 (pg/ml; Mean ± SD ) * LPS LPS C I L-1 (pg/ml; Mean ± SD ) D I L-1 (pg/ml; Mean ± SD ) LPS LPS E I L-1 (pg/ml; Mean ± SD ) ** LPS F I L-1 (pg/ml; Mean ± SD ) LPS Fig. 6 IL-1 release in response to challenge in vitro of spleen cells from M. coucha injected with gel only + FIA (group 1) or semipurified molecules (group 2) in gel + FIA and subsequently exposed to L 3 of B. malayi (groups 3 and 4). LPS challenge served as a positive control. (A) Group 1 versus group 3, (B) *22 kda and (C) *28 kda molecules reactive with sera of infected untreated and reinfected animals; (D) *32 kda and E) *45 kda molecules reactive with sera of DEC-treated reinfected animals and (F) *62 kda reactive with sera of ALB treated reinfected animals (group 2 versus group 4). * P \.5; ** P \.1, P \.1, unpaired t-test (positive control) treatment resulted in much higher DNA damage (P \.1) in comparison to untreated cells as well as molecule-exposed cells. Discussion Lymphatic filariasis is worldwide problem and there are several approaches to reduce the disease manifestation, the principle one being drug treatment. Surveys have revealed that areas previously known to be free from filariasis show

13 Med Chem Res (28) 17: OD at 49nm (Mean±SD) ** Gr 1 Gr 3 Gr 2 Gr 4. Control Semipurified parasite molecules (~kda) 62 Fig. 7 Specific IgG levels in M. coucha injected with gel only + FIA (group 1) or semipurified molecules in gel + FIA (group 2), and that received L 3 infection (groups 3 and 4). P \.1, unpaired t-test 6 5 T ail length ( µ M ) Control 22kDa 28kDa 32kDa 45kDa 62kDa H 2 O 2 Fig. 8 Scattergram of DNA damage in lymph node cells of naïve M. coucha exposed to parasite molecules in vitro for 24 h. H 2 O 2 exposure served as a positive control. P \.1 over control, unpaired t-test unpredictable recurrence after treatment. The reasons for this are not clear. Our previous study showed that treatment with ALB inhibits the development of new incoming infection while treatment with DEC facilitates it (Khan et al., 24). These findings suggested that alterations in the specific immunological responses may play an important role in the development of reinfection. Roberts et al. (1993) while working with schistosomiasis suggested that treatment of infected persons would result in accelerated development of acquired immunity, which may be operative and help in the development of protective immunity against reinfection. Furthermore, there is no information on those molecules that have been identified in the sera of subjects treated with antifilarial drugs and then re-exposed to infection. Such an approach may be helpful in developing agents and devising strategies for control of this disease. The present study therefore aimed at identifying molecule(s)

14 458 Med Chem Res (28) 17: of the parasites that were reactive with sera of a B. malayi-infected antifilarialtreated (ALB or DEC) model and subsequently re-exposed to infection and evaluating the potential of these molecules to modulate the parasitological and immunological responses of the host. The analysis of the present study revealed three major findings. (i) Of the five immunoreactive bands identified, the *28 and *22 kda bands were immunoreactive with sera of reinfected animals. The *62 kda band was identified to be strongly reactive with sera of infected ALB-treated reinfected animals, and *32 and *45 kda molecules were reactive with sera of infected DEC-treated reinfected animals. (ii) The *62 kda molecule(s) suppressed the establishment of L 3 -induced infection with significant reduction in MF count over controls throughout the observation period. These findings were accompanied by the upregulation of specific IgG and cellular responses including IFN-c and downregulation of IL-1. On the other hand, L 3 inoculation in animals immunized with the *22, *28, *32, and *45 kda compounds exhibited mixed responses. (iii) Of the five identified semipurified molecules the *62 kda molecule induced significant damage to the DNA of lymph node cells. In humans it has been found that many DEC-treated patients appeared to reach a steady state of antibody levels (Lammie et al., 1988). In Western blot analysis 66 and 13 kda parasite antigen molecules appeared to be reactive with the sera of DEC-treated patients but did not shown reactivity with the sera of patients not treated with DEC. On the other hand, these molecules have also been demonstrated to be both dependent and independent of microfilaraemia. However, Fletcher et al. (1986) have shown *22 and *18.5 kda antigens present in adult and MF B. pahangi that were reactive with the sera of cats before the elimination of microfilaraemia, which became amicrofilaraemic thereafter. In the present study *22 kda and *28 kda antigens were found to be strongly reactive in infected and L 3 re-exposed animals. Balloul et al. (1987) reported that *28 kda and *22 kda antigens were protective in mice and rodents against schistosoma infection. Contrary to this *45 kda antigen band, which was identified with sera of infected DEC-treated L 3 re-exposed hosts, has been reported for use in monoclonal antibody production (Sutanto et al., 1985). In the present study the *62 kda antigen expressed strong reactivity with ALB-treated re-exposed animals sera and appeared to have a protective function in relation to the development of L 3. The same molecular weight antigen has been reported to be reactive and developed as Gib-13 monoclonal antibody specific by Lal et al. (1987). In our study we observed that the sera of infected DEC-treated reinfected animals reacted strongly with *45 kda (near to 43 kda). Freedman et al. (1989) identified the *43 kda molecule of B. malayi L 3 to be reactive with endemic normals and suggested that this molecule may have prophylactic potential. However, Singh et al. (1997) reported that the *43 kda compound of all three life stages of B. malayi (MF, L 3, and adult worms) was reactive with MF-positive subjects. Since the *45 kda molecule identified in the present study was from B. malayi, which is closely related to W. bancrofti (a human lymphatic-dwelling parasite) we expected that it might have protective potential, but surprisingly this was not observed in the present study. The reason at

15 Med Chem Res (28) 17: present is not clear. However animals immunized with the *32 kda molecule and then exposed to L 3 showed significantly lower worm burden. It is considered that parasites were directly responsible for the alterations in the cytokine/cellular proliferative response. The decreased L 3 -induced infection accompanied by the increase in specific IFN-c with a decrease in IL-1 release by cells of *62 kda-immunized + L 3 -exposed animals as observed in the present study are indicative of the development of protective immunity. Our earlier study demonstrated that protection against L 3 imparted by BmAFII was via proinflammatory responses (Dixit et al. 26). On the other hand an elevated level of IgG and mixed cellular immune responses of the animals immunized with the other molecules (*22, *28, *32, and *45 kda) + L 3 exposure support the parasitological findings. These mixed cellular responses could be largely due to progressive increase in microfilaraemia in these animals. Nevertheless, it is known that persistent release of microfilariae results in a Th2 response (Maizels and Lawrence, 1991). In the immune system, cell death is an important step to maintain lymphoid homeostasis and avoid disease. It can occur through apoptotic or necrotic pathways. There are two morphologically and biochemically distinct modes of death in nucleated eukaryotic cells (Searle et al., 1982; Arends and Wyllie, 1991). These cells do not die by a process of disordered dissolution but by two different sets of stereotyped reaction, i.e., necrosis and apoptosis. Necrosis is associated with major perturbation in the cellular environment and has been regarded as a pathological response, whilst apoptosis, which is an orderly process that proceeds through several morphological phrases (condensation and shrinkage of cells), has been observed in many instances where the death of the cell is not pathological but appears to be part of homeostatic regulation. Chronic exposure with any infectious agents or toxic substances is known to lead DNA damage in host s cells (Awara et al., 1998, Holzmuller et al., 22). Our result shows for the first time that cells exposed to *62, *28, and *32 kda molecules induced DNA damage in lymph node cells after 24 h exposure. However no such damage in DNA was observed in lymphocytes exposed to *22 and *45 kda molecules. On the other hand H 2 O 2 -mediated DNA damage in cells was observed after 2 h exposure. This indicates that, unlike the oxidative agent H 2 O 2, the proteins do not induce oxidative stress in cells. The DNA damage incurred by *62 kda-fraction-exposed cells might have been via some longer pathway of DNA damage. It is reported that the immune response generated to an external stimulus is not only limited by the lymphokines produced by the Th cell and their downstream effects on Th cell activation genes but may also be influenced by the availability of accessory signals required during activation (Copeland and Heeney, 1996). Th-cell activation may result in the coupling of the Fas receptor (CD95) with its ligand, and ligation of CD95 with the Fas ligand (FasL) causes rapid apoptosis in sensitive cells (Suda and Nagata, 1994). The preferential expression of FasL on Th1 cells (Ramsdell et al., 1994) arms these cells to kill other cell types or other Th1 cells by apoptosis. In the present study the expression of Th-1 cytokines and a significant suppression in worm burden appeared to correlate with this observation.

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