Establishment of asymptomatic Leishmania donovani infection in Indian langurs (Presbytis entellus) through intradermal route.

Indian langurs, which were previously reported to be highly susceptible, were infected intradermally using variable numbers of promastigotes along with different doses, 1/2 pair, 5 pairs and 10 pairs respectively of salivary gland lysate (SGL). Although, all the monkeys developed mild infection and remained subclinically infected throughout the observation period, which later resolved, none of them could develop the classical disease. No marked antigen specific antibody or lymphoproliferative response was noticed throughout the experimental period. However, a late IFN-gamma response (by day 90 pi.) was demonstrated in monkeys infected with 2 x 10(6) promastigotes +10 pairs SGL. It seems that a single intradermal dose of promastigotes with or without SGLs had a vaccines like effect. Perhaps, multiple frequent inoculations, as happens in the natural situation, may be necessary for the development of full-blown disease.

Successful vaccination against Leishmania donovani infection in Indian langur using alum-precipitated autoclaved Leishmania major with BCG.

Autoclaved Leishmania major (ALM) along with BCG, presently undergoing phase II clinical trial by WHO for its vaccine potential against cutaneous leishmaniasis, has been successfully evaluated in single and triple dose schedules against L. donovani in Indian langurs (Presbytis entellus). Encouraged with the results, another formulation alum-precipitated ALM (provided by WHO) along with BCG has been evaluated in this system. Eight monkeys were vaccinated with alum-precipitated ALM + BCG (1 mg of each per animal) while four were kept as unvaccinated controls. All were challenged with 100 x 10(6) amastigotes i.v. on day 60 post vaccination. Parasitic assessment in splenic tissue was performed on day 45, 90 and 180 p.c. Initially, seven of the eight vaccinated monkeys developed infection (two to six amastigotes per 1000 cell nuclei), which resolved by day 180 p.c., while the eighth monkey had a parasite burden of 14 amastigotes per 1000 cell nuclei on day 45 p.c. and died on day 130 p.c. On the other hand, there was progressive infection in unvaccinated control animals and three out of four died between days 110 and 120 p.c., and one monkey, which had low parasite burden, died on day 178 p.c. Prior to challenge, there was an initial rise in antileishmanaial antibodies in the vaccinated group compared to the unvaccinated control group, which later came down to normal level, while it remained higher in the unvaccinated control group. An increasing pattern of antigen-specific proliferative responses and interferon-gamma level to the two antigens--autoclaved L. donovani (ALD) and ALM--was observed in vaccinated monkeys throughout the experiment. There was a good correlation between parasite burden and IFN-gamma level on days 90 and 180 p.c., indicating IFN-gamma response as a sensitive parameter of immune status. The findings suggest alum-precipitated ALM+BCG as a potential vaccine against visceral leishmaniasis and warrants clinical trials.

Stimulation of nonspecific resistance by thymopentin and its analogs against Leishmania donovani infection in hamsters.

Thymopentin and its analogs have been synthesized by the solution phase method of peptide synthesis and evaluated for their prophylactic efficacy against L. donovani infection in hamsters. Thymopentin and some of the analogs were found to stimulate nonspecific resistance of the host against Leishmania donovani infection in hamsters.

Leishmania donovani: cellular and humoral immune responses in Indian langur monkeys, Presbytis entellus.

We have previously reported that disease mimicking human visceral leishmaniasis can be established in Presbytis entellus, the Indian langur monkey, following a single intravenous challenge of 10(8) Leishmania donovani amastigotes. In the present report, infection was assessed in monkeys infected intravenously with a single dose of 10(8) amastigotes (HDA group), three weekly doses of 10(7) amastigotes (LDA group) and three weekly doses of 5 x 10(7) promastigotes (HDP group). Typical clinical infection was established in all three groups with significant parasite load. There was a gradual and sustained rise in anti-leishmania specific immunoglobulin G response, and a severe fall in the lymphoproliferative response to the T cell mitogens PHA and Con A by day 80 post infection (p.i.). The antibody level remained elevated until death in monkeys of the HDA and HDP groups; the T-cell responses showed a recovery prior to death. T-cell responses to leishmania antigen, however, could not be demonstrated in any of these monkeys prior to death. One monkey of the LDA group survived for 155 days and two monkeys spontaneously eradicated the infection. Surprisingly, one monkey of the HDA group also achieved spontaneous cure. In the three monkeys which eradicated infection spontaneously, the antibody level declined to baseline levels on day 180 p.i. with a well demonstrable antigen specific lymphoproliferative response; no parasites could be demonstrated in splenic aspirates by direct examination of culture. These data demonstrate that disease severity may be the function of the total inoculum dose rather than the stage of the parasite and that the immunological responses in the Indian langur model parallel the reported changes in human visceral leishmaniasis. This makes the langur a potentially useful model for the evaluation of candidate anti-leishmanial drugs and vaccines.

Synthesis and antileishmanial activity of alpha-cyano ethyl propenoates--a new class of antileishmanials.

A number of alpha-cyano-beta-substituted ethyl propenoates were synthesised by condensation of appropriate aldehydes with ethyl cyanoacetate in basic medium. These compounds have been studied for their antileishmanial activities in vivo in hamsters. Two compounds exhibited 70-77% activities both by intramuscular and oral routes.

The latex agglutination test: standardization and comparison with direct agglutination and dot-ELISA in the diagnosis of visceral leishmaniasis in India.

Laboratory diagnosis of visceral leishmaniasis (VL) is usually based on the detection of Leishmania amastigotes in samples of bone marrow or splenic aspirate obtained by invasive procedures. Serological tests serve as a useful adjunct and are especially valuable in early or highly immune cases where amastigotes may be too scanty to be seen easily. The direct agglutination test (DAT) is generally considered the most suitable of the four types of tests currently employed (IFAT, counter immuno-electrophoresis, ELISA and DAT). However, the latex agglutination test (LAT) was recently reported to be a rapid and sensitive screening tool for VL and one which could be carried out at the patient's bedside. Further standardization and evaluation of LAT has now revealed that although it is comparable with DAT and dot-ELISA in terms of sensitivity it is far inferior because of cross-reactivity with other infections. This lack of specificity makes LAT unsuitable for routine diagnosis of VL even though it is rapid and sensitive. DAT still appears to be the best choice as a diagnostic tool, as it is very specific and does not require expensive equipment or reagents or much technical competence and the result can be visually interpreted. These merits make DAT very suitable for the diagnosis of VL in endemic areas of India.

Vaccination of langur monkeys (Presbytis entellus) against Leishmania donovani with autoclaved L. major plus BCG.

The protective potential of killed Leishmania major (ALM) along with BCG was evaluated against L. donovani in Indian langur monkeys in single and triple dose schedules. A delayed protection was observed in monkeys after a single dose schedule of ALM (3 mg)+BCG (3 mg) given intradermally 2 months before intravenous challenge with L. donovani. Triple dose schedule each of 1 mg ALM + 1 mg BCG was more effective. The status remained unchanged until the end of the experiment (approximately 8 months). The study indicates that a combination of ALM + BCG may be a good candidate vaccine for exploiting against human Kala-azar.

Correlation between degree of protection and humoral antibody response in hamsters immunized with somatic and excretory secretory antigens of Ancylostoma ceylanicum.

Correlation between the degree of protection and induced serum antibody response in hamsters immunized with somatic and excretory-secretory (ES) antigens of adult. A. ceylanicum was investigated. Hamsters were immunized with non purified and Sephadex G-200 fractions (F1, F2 and F3) of somatic and ES antigens. The degree of protection was assessed in terms of percent worm reduction compared to controls against challenge infection. Induced humoral antibody response was determined by ELISA. Both somatic and ES antigens had shown moderate to significant protection but the latter was found more immunogenic as highest level of protection (67.02%) was achieved by these antigens. Humoral antibody was found highly elevated in animals immunized with protective doses of somatic and ES antigens. The maximum serum antibody titer i.e. 1:3200 was noticed in animals immunized with high protective dose (64.59% protection) of fraction F1 of ES antigens. Antibody titer correspond to the degree of protection and a positive correlation between induced humoral antibodies and protection level was established.

In-vitro cultivation of exoerythrocytic stages of Plasmodium cynomolgi in hepatocytes of Macaca radiata.

Hepatocytes from bonet monkey (Macaca radiata) obtained by perfusion of a liver biopsy were infected in-vitro with Plasmodium cynomolgi bastianellii sporozoites raised in Anopheles stephensi. The development of exoerythrocytic (EE) stages was seen under phase contrast microscope and by Giemsa staining. Multinucleated EE-stages were seen in the cultured hepatocytes on day 7-8 post-sporozoite inoculation.

Leishmania donovani in hamsters: stimulation of non-specific resistance by novel lipopeptides and their effect in antileishmanial therapy.

Several novel type of lipopeptides were synthesized and evaluated for their ability to stimulate non-specific resistance against Leishmania donovani infection. Peritoneal macrophages isolated from young male hamsters treated with muramyl dipeptide (MDP) and various synthetic lipopeptides (6 mg/kg i.p.) 7 days earlier, were cultured in vitro and challenged 24 h later with L. donovani promastigotes. One lipopeptide, Central Drug Research Institute (CDRI) compound 86/450, exhibited significantly higher immunostimulatory activity than MDP. Its prophylactic activity was further confirmed in hamsters by giving 2 split doses of 3 mg/kg of the compound spaced at 2 weeks, i.e. on day -7 and +7 of challenge with L. donovani amastigotes. The prophylactic effect lasted for 7 days following the last treatment with compound 86/450. The antileishmanial action of sodium stibogluconate (SAG) was also found to be enhanced by 16% in hamsters primed with compound 86/450.

Metabolic disposition of methyl [5-[[4-(2-pyridinyl)-1-piperazinyl] carbonyl]-1-H-benzimidazol-2-yl] carbamate in hamsters: a study to understand chemoprophylactic action against experimental Ancylostomiasis.

Methyl [5-[[4-(2-pyridinyl-1-piperazinyl] carbonyl]-1H- benzimidazol-2-yl] carbamate (CDRI Compound 81-470) exhibits a long prophylactic action against experimental ancylostomiasis, when given parenterally but not orally. To find out an explanation for such a behaviour, metabolic disposition studies were performed in hamsters using [3H] compound 81-470. Following intramuscular administration, the compound was found to form a depot at the site of injection and to remain there in substantial amount for more than 7 weeks. The compound was fairly distributed in all the organs studied and the presence of radioactivity could be easily detected up to 7 weeks of observation period. The compound was very slowly eliminated from the body and only 38% of the radioactivity could be recovered in the urine and faeces during 14 days. The oral dose, to the contrary, was poorly absorbed and more than 62.8% was excreted in the faeces within 48 hr. Consequently, this dose yielded lesser area under plasma curve. More than 95% of the oral dose was eliminated within a week and hardly and radioactivity could be detected in the tissues after day 14. In accord with this pattern, in blood also the im dose was detected up to 7 weeks while the orally given compound reached undetectable level within 6 days only. The lower clearance and prolonged stay in the body of the im dose compared to quick elimination of the oral dose may be responsible for the long chemoprophylactic action of compound 81-470 when given through im route.

Role of reactive oxygen species in the chemoprophylactic action of methyl [5-[[4-(2-pyridinyl)-1-piperazinyl]-carbonyl]-1H-benzimidazol-2 yl] carbamate in hamster against Ancylostoma ceylanicum.

To delineate mechanisms involved in the prophylactic action of methyl [5-[[4-(2-pyridinyl)-1-piperazinyl]-carbonyl]-1H-benzimidazol-2 yl] carbamate (compound 81/470) in hamster against Ancylostoma ceylanicum infection, plasma level of the compound and status of reactive oxygen metabolites in jejunum at different periods of the drug treatment were examined. The compound was found to enhance the generation of both O2- and H2O2 by the jejunum possibly by activating xanthine oxidase. This stimulation was found to be both time and dose dependent. At 100 mg/kg dose the increase in O2- production could be recorded at least upto 50 days, whereas at 25 mg/kg the stimulation remained effective upto 20 days only, and at 5 mg/kg there was no change in the activity. This correlated well with the reported prophylactic pattern of the compound i.e. upto 45 and 7 days by 100 and 25 mg/kg doses, respectively. Plasma level of the compound also exhibited dose dependent variation. The compound given at 100 mg/kg dose could be detected in significant concentration upto at least 42 days while that given in 25 and 5 mg/kg doses was present in equivalent concentration upto 14 days and 1 day, respectively. It is concluded that the activation of respiratory burst in the jejunum induced by the persistent presence of compound 81/470 may represent one of the important mechanisms for the chemoprophylactic activity of this anthelmintic.

Evaluation of enzyme-linked immunosorbent assay in the diagnosis of kala-azar in Malda district (West Bengal).

An immunological test based on indirect (plate) ELISA has been successfully standardized and modified using promastigote soluble antigen. The test carried out on 813 subjects from a kala-azar endemic area (including parasitologically confirmed patients, subjects presenting with clinical symptoms of visceral leishmaniasis and endemic controls) and a non-endemic area (with diseases other than kala-azar and apparently normal subjects) was found to detect, specifically, antileishmanial antibodies. The plate ELISA has been simplified to a more sensitive dot-ELISA where the results are read within 2-3 h. The antigen requirement is 250 ng per test. No cross-reactivity with sera from patients of malaria, tuberculosis, leprosy, amoebiasis and filariasis was observed. The follow up monitoring of antibodies in successfully treated kala-azar patients showed a decline of antibodies. A drop of blood taken on filter paper is sufficient to conduct the test. Dot ELISA therefore is a simple, inexpensive and stable test in serodiagnosis of visceral leishmaniasis.

Role of reactive oxygen species in expulsion of Nippostrongylus brasiliensis from rats.

To understand the mechanism for the expulsion of Nippostrongylus brasiliensis from rats, age-dependent variations in the metabolism of reactive oxygen species in the parasite and the host intestines were examined. N. brasiliensis showed an age-dependent increase in its susceptibility to xanthine-xanthine oxidase and t-butyl hydroperoxide generated oxidants as well as to H2O2. Protection obtained with several scavengers suggested that the worms were damaged by the combined action of oxidants generated by the in vitro systems employed. The level of superoxide dismutase in the nematode and its release into the surroundings exhibited a marked depression with advancement of age. No such alteration was, however, recorded for catalase and glutathione peroxidase. An appreciable decrease in the level of reduced glutathione in older N. brasiliensis appears to render them prone to oxidant attack. The rat intestines, on the other hand, exhibited an appreciable depression in catalase and a reduced glutathione content with progress of the infection. Vitamin E levels were elevated. The release of O2-. and H2O2 by the intestines was also found to be greater during later stages of the infection. The combined effect of the changes observed in N. brasiliensis and in the rat intestines may be at least partly responsible for expulsion of the nematode from the rats after day 10.

Ancylostoma ceylanicum infection in female hamsters: an observation on altered reproductive function.

Reproductive performances of female hamsters were investigated during Ancylostoma ceylanicum (hookworm) infection. Animals having the highest levels of infection (34.96 +/- 1.11 worms) showed degenerative changes in the reproductive system. Ovaries of infected animals contained a few primary or secondary follicles. On cocaging with males of proven fertility, only 7-8% (80% in controls) of the infected females mated but did not conceive as evidenced by the absence of corpora lutea or implantation sites on day 10 postcoitum. Animals with low worm burdens (5.94 +/- 0.65 worms), however, showed almost normal fertility. The uterine weight bioassay and compensatory ovarian hypertrophy suggest strong suppression of pituitary gonadotrophin contents in infected females. Resorptive effects on the pregnancy outcome of infected female hamsters were also recorded.

Host resistance assays as predictive models in styrene immunomodulation.

Three infection models namely an oncogenic virus Encephalomyocarditis (EMCV), a rodent strain of malaria, Plasmodium berghei, and a rodent hookworm parasite, Nippostrongylus brasiliensis, were used to confirm the in vivo immunotoxic potential of styrene reported in our previous communication. The altered host resistance to these challenge infections was evaluated in rodents pre-treated with 0, 0.02, 0.03 or 0.05 x LD50 dose of styrene (5 days/week) for 4 weeks. Significantly increased mortality in mice was observed at the various tested dose levels of styrene when challenged with EMCV. Similarly the results obtained in the malaria infection model indicated increased blood parasitaemia as well as significantly enhanced mortality in styrene-treated animals. Also the rejection of N. brasiliensis was also found to be significantly impaired in animals treated with a higher dose of styrene. These results indicate that the exposure of rodents to styrene can markedly impair host resistance which may have biological significance.

Effect of five-membered heterocycles against Leishmania donovani infection.

Impact of change of heteroatom in pentavalent heterocycles, viz., pyrroles, isoxazoles, imidazoles and crotonates on the profile of antileishmanial activity against amastigotes of L. donovani using in vivo test system and macrophage-amastigote culture system has been studied. Sixty-three compounds were tested. Nine imidazoles showed marginal activity in vivo, whereas 3 out of 10 compounds of isoxazolone series and 2 out of 4 substituted aminocrotonates exhibited antileishmanial activity. Of the 30 substituted pyrroles, except 8 all showed antileishmanial activity in vivo on day 7 post treatment.

Modulatory effects of metanil yellow on immunity in rodents.

Pathomorphological and immunological studies were carried out on rodents following oral administration of 0, 0.1, 0.25 and 0.5% (w/w) metanil yellow, mixed in diet, for 30 days. No significant change in hematologic parameters and histologic architecture of liver, kidney, mesenteric lymph node, thymus and urinary bladder was observed except for mild desquamation of intestinal villi and moderate changes in Peyer's patches of small intestine with higher doses. Among immunological parameters, significant enhancement in the primary humoral immune response (anti-SRBC IgM plaque forming cells of spleen) was observed with the lowest dose of metanil yellow while higher doses produced opposing effects. An elevated cutaneous delayed type hypersensitivity (DTH) reaction to SRBC was seen in 0.1% metanil yellow treated animals but higher doses did not influence the reaction. The treatment also caused changes in functional capabilities of macrophages. Although these immune alterations could hardly influence the local immunity of gut, as measured by the capacity of animals to cause rejection of Nippostrongylus brasiliensis parasite, the potential to modulate the immunity in general by metanil yellow however assumes considerable biological significance.