concanavalin-a and Leishmaniasis--Visceral

One obstacle faced in the effective control of visceral leishmaniasis (VL) is the limited number of available treatment options. Furthermore, control efforts have been hindered further by the emergence of Leishmania resistance to many of the available drugs. In this study, we investigated the anti-leishmanial properties of 30 medicinally important plants from the VL endemic area of Bihar, India and compared them to two available anti-leishmanial drugs (sodium antimony gluconate and amphotericin B) and two plant lectins (phytohemagglutinin and concanavalin A) on Leishmania donovani promastigotes in vitro at 24 and 48 h after initiation of culture. We identified eight plant extracts in addition to phytohemagglutinin and amphotericin B that significantly inhibited the growth of promastigotes (p < 0.03). We further studied the minimum effective concentrations as well as the effect on axenic amastigotes viability and the cell cytotoxicity on human peripheral blood of four (Agave americana, Azadirachta indica, Eclipta alba and Piper longum) of the eight plant extracts that induced significant promastigotes killing (p = 0.00098). Effect-based dose finding analysis revealed that the threshold concentration of A. americana required to eliminate L. donovani after 24h was 0.05 mg/ml. A. indica and P. longum plant extracts eliminated L. donovani promastigotes after 48 h at concentrations of 0.1 and 0.5mg/ml, respectively. E. alba eliminated the promastigotes at a concentration of 0.5mg/ml within 24h. The axenic amastigote killing response was 1.90-, 2.52- and 1.3-fold higher than the promastigote killing response with A. indica, A. americana and E. alba plant extracts, respectively. A. americana and A. indica, respectively, led to approximate 2.5- and 1.3-fold declines in mitochondrial dehydrogenase activity compared with control. E. alba stimulation resulted in an up-regulation of dehydrogenase activity (p = 0.00329). The CSA from P. longum was found to be least cytotoxic; the observed difference in mitochondrial activity was insignificant (p = 0.16314). Further studies may reveal the pharmacological significance of many of the plants with anti-leishmanial properties identified in the present study.

Topics: Amphotericin B; Antimony Sodium Gluconate; Antiprotozoal Agents; Concanavalin A; Humans; India; Leishmania donovani; Leishmaniasis, Visceral; Leukocytes, Mononuclear; Phytohemagglutinins; Plant Extracts

Opportunistic infections, due to disease-related immunosuppression, constitute the major cause of death in American visceral leishmaniasis (AVL). Sera from these patients (AVL sera) non-specifically inhibit the in vitro proliferative response of normal human lymphocytes to lectins or antigens. In the present work, the mediation of this inhibition by IgG, immune complexes and low- or very low-density lipoproteins was studied. AVL serum fractions containing proteins with the molecular weight of IgG, and IgG, purified from AVL sera by anion exchange chromatography, did not suppress the lymphoproliferation. Most of the suppressive activity of AVL sera was associated with a fraction containing molecules with molecular weights above 430 kDa. This would be compatible with it being due to immune complexes and/or lipoproteins, and not to soluble IL-2 receptors as reported previously. However, neither of the two possibilities seem to be the case, as (1) depletion of immune complexes by protein-A followed by protein-G chromatographies did not affect the serum suppressive activity, (2) no correlation between immune complex contents and suppressive activities in individual sera was observed, and (3) plasma lipoproteins (VLDL and LDL), purified from AVL patients and from healthy individuals, had the same degree of immunosuppressive activity.

Topics: Animals; Antigen-Antibody Complex; Concanavalin A; Humans; Immunocompromised Host; Immunoglobulin G; Leishmania infantum; Leishmaniasis, Visceral; Leukocytes, Mononuclear; Lipoproteins, LDL; Lipoproteins, VLDL; Lymphocyte Activation

The antileishmanial property of a Benzyl derivative of a new antibiotic MT81 (Bz2MT81), isolated and purified from a fungal strain of Penicillium nigricans NRRL 917 was tested in free, liposome intercalated and mannose coated liposome intercalated forms in vivo against visceral leishmaniasis in hamsters. Mannose grafted liposome intercalated Bz2MT81 eliminated intracellular amastigotes of Leishmania donovani within splenic macrophages more efficiently than the liposome intercalated Bz2MT81 or free Bz2MT81. At a dose equivalent to 7.5 microg/Kg body weight when injected subcutaneously (s.c) in mannose grafted liposome intercalated form for 15 days in an interval of three days, the splenic parasitic load decreased to the extent of 79.1% of the total parasite present in infected control animals. Whereas, an identical amount (7.5 mug/Kg body weight) of Bz2MT81 in free or liposome intercalated form was found less effective in controlling the parasite in spleen (in free Bz2MT81 form, suppression of parasitic load is 49.8% and in liposome intercalated form, it is 55.1%). Both mannosylated liposomes and Bz2MT81 were noted non-toxic to the host peritoneal macrophages. Histological examinations of spleen and liver, kidney function tests (SGPT, alkaline phosphatase, creatinine and urea in blood plasma) showed that the toxicity of Bz2MT81 was reduced up to normal level when mannose grafted liposomal Bz2MT81 were administered.

Topics: Animals; Anthraquinones; Antiprotozoal Agents; Benzyl Compounds; Concanavalin A; Cricetinae; Drug Carriers; Drug Delivery Systems; Excipients; Fluoresceins; Fluorescent Dyes; Intercalating Agents; Kidney Function Tests; Leishmaniasis, Visceral; Liposomes; Liver Function Tests; Macrophages; Mannose; Mesocricetus; Mice; Mice, Inbred BALB C; Mononuclear Phagocyte System; Particle Size; Penicillium; Trinitrobenzenesulfonic Acid; Trypan Blue

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.

Topics: Alum Compounds; Animals; Antibodies, Protozoan; Antigens, Protozoan; BCG Vaccine; Cercopithecidae; Chemical Precipitation; Concanavalin A; Humans; Hypersensitivity, Delayed; Interferon-gamma; Leishmania donovani; Leishmania major; Leishmaniasis, Visceral; Lymphocyte Activation; Male; Protozoan Vaccines; Vaccines, Inactivated

Human visceral leishmaniosis is endemic in Southern Italy, where the dog is the main reservoir of viscerotropic strains of Leishmania infantum. The release of nitric oxide (NO) by interferon (IFN)-gamma-activated macrophages is an important leishmanicidal mechanism in several animal species. In this work NO production, phagocytosis and killing capacity of monocyte-derived dog macrophages were evaluated in vitro before and after administration of a vaccine composed of killed Leishmania infantum promastigotes. Moreover, IFN-gamma content was measured in concanavalin A-activated dog peripheral blood mononuclear cell (PBMC) supernatants employed for macrophage stimulation. Phagocytosis, killing capacity and NO production by canine macrophages increased significantly 1 month after vaccine administration, and the increase also persisted 5 months later. In addition, the amount of IFN-gamma in PBMC supernatants was significantly higher after vaccination. Overall, our results suggest the usefulness of evaluating the in vivo protective role of this promastigote preparation in dogs.

Topics: Animals; Concanavalin A; Cytotoxicity, Immunologic; Disease Reservoirs; Dog Diseases; Dogs; Female; Humans; Interferon-gamma; Italy; Leishmania infantum; Leishmaniasis, Visceral; Macrophage Activation; Macrophages; Male; Nitric Oxide; Phagocytosis; Protozoan Vaccines; Vaccination

The progressive visceral infection caused in golden hamsters by Leishmania donovani amastigotes led to gradual impairment of the proliferative response of their splenic (SPMC) or peripheral blood (PBMC) mononuclear cells to in vitro stimulation with leishmanial antigen, with mitogen (concanavalin A), and even with a combination of phorbol myristate acetate (PMA) and ionomycin (Io). Removal of macrophage-like adherent cells from SPMC or PBMC of infected animals, however, almost completely restored their proliferative response to PMA + Io, thus ruling out the possibility of any intrinsic defect in the signal-transduction pathways of lymphocyte activation and proliferation. Subsequent studies demonstrated that the generation of soluble mediators such as nitric oxide by these adherent cells is responsible, albeit partially, for the down-regulation of the lymphoproliferative response in hamsters with visceral leishmaniasis.

Topics: Animals; Antigens, Protozoan; Concanavalin A; Cricetinae; Dinoprostone; Immune Tolerance; Leishmania donovani; Leishmaniasis, Visceral; Leukocytes, Mononuclear; Lymphocyte Activation; Macrophages; Mesocricetus; Nitric Oxide; Spleen; T-Lymphocytes; Tetradecanoylphorbol Acetate

Dogs are the domestic reservoirs of zoonotic visceral leishmaniasis caused by Leishmania infantum. Early detection of canine infections evolving to clinically patent disease may be important to leishmaniasis control. In this study we firstly investigated the peripheral blood mononuclear cell (PBMC) response to leishmanial antigens and to polyclonal activators concanavalin A, phytohemagglutinin and pokeweed mitogen, of mixed-breed dogs with natural L. infantum infection, either in presymptomatic or in patent disease condition, compared to healthy animals. Leishmania antigens did not induce a clear proliferative response in any of the animals examined. Furthermore, mitogen-induced lymphocyte proliferation was found strongly reduced not only in symptomatic, but also in presymptomatic dogs suggesting that the cell-mediated immunity is suppressed in progressive canine leishmaniasis. To test this finding, naive Beagle dogs were exposed to natural L. infantum infection in a highly endemic area of southern Italy. Two to 10 months after exposure all dogs were found to be infected by Leishmania, and on month 2 of exposure they all showed a significant reduction in PBMC activation by mitogens. Our results indicate that suppression of the lymphoproliferative response is a common occurrence in dogs already at the beginning of an established leishmanial infection.

Topics: Animals; Antigens, Protozoan; Concanavalin A; Dog Diseases; Dogs; Enzyme-Linked Immunosorbent Assay; Female; Leishmania infantum; Leishmaniasis, Visceral; Lymphocyte Activation; Male; Phytohemagglutinins; Pokeweed Mitogens

Protozoa of the genus Leishmania infect reticuloendothelial cells of several mammalian species, including dogs, in which they often give rise to a chronic, not self-healing visceral disease. The parasitocidal mechanism of peripheral blood monocytes towards Leishmania in the dog has not been investigated in detail. Consequently, Leishmania infantum-infected monocyte cultures of healthy dogs were evaluated using the following parameters: (1) phagocytosis and killing capacities; (2) oxidative burst, in terms of superoxide anion (O2-) release, and (3) nitric oxide (NO) activity, in terms of nitrite (NO2-) production in the presence or absence of the NO synthase inhibitor NG-monomethyl-L-arginine (NGMMLA). Parallel experiments were performed on monocytes stimulated with supernatants of concanavalin A-activated PBMC and on unstimulated monocytes. The amount of IFN-gamma in PBMC supernatants used for monocyte activation was determined by a biological assay on a canine Madin Darby cell line. Results demonstrated that phagocytosis, killing capacity and O2- production significantly increased in monocytes stimulated with supernatants, in comparison with unstimulated cells. A positive correlation was observed between the killing capacity, the O2- production and the amount of IFN-gamma in PBMC supernatants employed for monocyte activation. No significant differences were observed in NO production between unstimulated and stimulated cultures, or between the same cultures with and without NGMMLA. Finally, the killing percentage was similar in the presence or absence of NGMMLA, suggesting that in this experimental model peripheral blood dog monocytes lack NO-mediated killing.

Topics: Animals; Concanavalin A; Dog Diseases; Dogs; Interferon-gamma; Leishmania infantum; Leishmaniasis, Visceral; Monocytes; Nitric Oxide; Phagocytosis; Respiratory Burst; Superoxides

Visceral leishmaniasis, caused by Leishmania donovani, is a chronic disease with a high mortality rate. This protozoan induces a serious dysfunction of the immune system characterized by suppression of the cellular response to parasite antigens. We provide evidence for the involvement of lipids in the immunological alterations of experimental leishmaniasis. Sera obtained from 60-day-infected hamsters present increased triglyceride levels. Inhibition of cell proliferation was observed when splenocytes from normal hamsters were stimulated with concanavalin A in the presence of 3% infected hamster serum (IHS) (Control 50 +/- 3 (x10(3)) cpm; IHS 5 +/- 1 (x10(3)) cpm). This inhibition was reversed by the addition of 5 mg/ml of delipidated bovine serum albumin (BSA) to the cultures (Control 65 +/- 1 (x10(3)) cpm; IHS 75 +/- 3 (x10(3)) cpm). The inhibitory effect of IHS was demonstrable only when added to the culture simultaneously with the mitogen. This effect was not as intense on fresh, pre-activated cells or on the CTLL-2 cells. This cell line stimulated by IL-2 in the presence of IHS is only marginally inhibited (about 20% inhibition). The suppressor effect on CTLL-2 was not reversed by the addition of increasing doses of IL-2 (up to 100 U/ml) to cultures. The inhibition of the proliferative response of the CTLL-2 cells caused by IHS was also reversed by the addition of delipidated BSA. Our data suggest a role for fatty acids in the infected hamster serum-induced suppression of normal or L. donovani-infected cell proliferation.

Topics: Animals; Cells, Cultured; Concanavalin A; Cricetinae; Female; Humans; Interleukin-2; Leishmania donovani; Leishmaniasis, Visceral; Mesocricetus; Mitogens; Mitosis; Serum Albumin, Bovine; Spleen; Suppressor Factors, Immunologic

A glycoconjugate antigen of 27-39 kDa was isolated from a cell-free extract of Leishmania donovani by affinity chromatography using a Concanavalin-A sepharose-4B column and eluted with 0.5 M alpha-methylmannoside. The antigen was recognized specifically by sera from kala-azar (visceral leishmaniasis) patients and did not react with sera from tuberculosis, leprosy or malaria patients. The antigen may therefore be useful in developing a serodiagnostic assay for visceral leishmaniasis.

Topics: Animals; Antigens, Protozoan; Concanavalin A; Electrophoresis, Polyacrylamide Gel; Epitopes; Humans; Leishmania donovani; Leishmaniasis, Visceral; Serologic Tests

In visceral leishmaniasis (VL), patient's lymphocytes are not activated by leishmania Ag stimulation, and their sera exhibit a potent nonspecific suppressive effect on the responses of normal lymphocytes. Sera were obtained from 33 VL patients, eight patients with subclinical VL, and from 27 normal volunteers. Only sera from VL patients markedly reduced Con A-induced lymphocyte proliferative responses, as well as IL-2 or IFN-gamma production by normal lymphocytes. Addition of exogenous human rIL-2 to cultures containing VL patient sera partially reversed the normal lymphocyte proliferative capacity and restored IFN-gamma production. This phenomenon was consistent with the presence of greatly elevated levels of soluble IL-2R (sIL-2R) in VL patients' sera (4299 +/- 2351 U/ml), well above those of normal sera (180 +/- 94 U/ml), or of sera from patients with subclinical leishmania infection without immunosuppression (1002 +/- 281 U/ml). Furthermore, the removal of sIL-2R reduced VL serum suppressive activity as evaluated by effects on IL-2 and on IFN-gamma production. These data suggest the participation of high levels of sIL-2R in the serum-mediated suppression in VL.

Topics: Concanavalin A; Humans; Immune Tolerance; Interferon-gamma; Interleukin-2; Leishmaniasis, Visceral; Lymphocyte Activation; Lymphocytes; Receptors, Interleukin-2; Solubility

Leishmania donovani infection in golden hamsters was studied as a model for human kala-azar. After intradermal inoculation of L. donovani amastigotes, hamsters developed positive skin reactions (delayed-type hypersensitivity [DTH]) to parasite antigens and lymphoid cells from these hamsters proliferated to parasite antigens in vitro and transferred DTH reactivity to normal recipients. In contrast, hamsters infected by the intracardial route developed progressive visceral infections and failed to respond to skin test antigens. Spleen cells, lymph node cells, and peripheral blood lymphocytes (PBLs) from these hamsters were unresponsive to parasite antigens in vitro, and spleen cells failed to transfer DTH to normal recipients. Spleen cells, but not PBLs, displayed depressed responses to T-cell mitogens and also suppressed the proliferative response of cells from hamsters inoculated intradermally. Removal of adherent cells restored the capacity of spleen cells, but not PBLs, to respond to parasite antigens. The nonadherent population of these spleen cells also transferred DTH to normal recipients. The adherent suppressor cells, which have the characteristics of macrophages, appear to be localized to the spleen and are apparently not responsible for the failure of peripheral lymphoid cells to respond to antigen. These studies suggest that hamsters with visceral infections develop a population of antigen-reactive cells and that in the absence of suppression these cells may express functional activities, including the capacity to elicit DTH responses.

Topics: Animals; Antigens, Protozoan; Cell Adhesion; Concanavalin A; Cricetinae; Hypersensitivity, Delayed; Immunity, Cellular; Leishmania donovani; Leishmaniasis, Visceral; Lymphocyte Activation; Male; Mesocricetus; Skin Tests; T-Lymphocytes; T-Lymphocytes, Regulatory

Immunosuppression in Leishmania brasiliensis (LB) or L. donovani (LD) infected hamsters is correlated with the appearance of two serum protein bands found at 21, 60, 68 and 76 days post LB-infection and with eight bands at 21 days post-LD-infection probably of host origin. A protein band from LB-infected hamster serum isolated by electrofocusing, suppressed the blastogenic response of normal lymphocytes to T and B cell mitogens.

Topics: Animals; Blood Proteins; Concanavalin A; Cricetinae; Isoelectric Focusing; Leishmania braziliensis; Leishmania donovani; Leishmaniasis, Mucocutaneous; Leishmaniasis, Visceral; Lymphocyte Activation; Lymphokines; Male; Pokeweed Mitogens

We examined the effect of sera from 11 patients with American visceral leishmaniasis on mitogen-driven lymphocyte proliferative capacity. All sera inhibited lymphocyte proliferation of patients' peripheral blood mononuclear cells (PBMC) when stimulated by either phytohemagglutinin, Concanavalin A or pokeweed mitogen. Serum was also strongly inhibitory for Concanavalin A-pulsed normal volunteers' PMBC. The effect of the serum was not due to cytotoxicity, inadequate nutritional support or altered kinetics of DNA synthesis. High levels of IgM or IgG (both total and antiparasite) and high levels of triglycerides were found in patients' sera.

Topics: Adolescent; Adult; Cells, Cultured; Child; Child, Preschool; Concanavalin A; Cytotoxicity, Immunologic; Dose-Response Relationship, Immunologic; Female; Humans; Immune Tolerance; Immunoglobulin G; Immunoglobulin M; Kinetics; Leishmania donovani; Leishmaniasis, Visceral; Lymphocyte Activation; Lymphocytes; Male; Triglycerides

Studies were carried out on the mechanisms by which B lymphocytes are polyclonally activated to secrete antibodies during visceral leishmaniasis. Crude extracts of Leishmania donovani, the aetiological agent of this disease, of Leishmania mexicana amazonensis, the etiological agent of cutaneous leishmaniasis, and of Herpetomonas muscarum, a related non-pathogenic organism, all contain components which cause strong in vitro polyclonal activation of hamster spleen cells leading to the production of antibodies. However, in vivo, only hamsters infected with L. donovani develop hypergammaglobulinaemia due to B cell polyclonal activation. Hamsters injected with the crude extracts of leishmania or infected with L. mexicana amazonensis do not manifest these alterations in their B cell response. Furthermore spleen cells of hamsters infected with L. donovani became unresponsive to stimulation with the T cell mitogen phytohaemagglutinin (PHA) by day 10 of infection, whereas their response to concanavalin A (Con A) was preserved. The decreased lymphocyte response to PHA coincided with the augmentation of the PFC/spleen ratio. In contrast, spleen cells from hamsters infected with L. mexicana amazonensis, responded normally to both mitogens throughout the course of infection. These results suggest that the hypergammaglobulinaemia present in visceral leishmaniasis may be the consequence of an inbalance of regulatory T cells, possibly associated with a direct stimulation of hamster B cells by L. donovani components.

Topics: Animals; Antibody Formation; B-Lymphocytes; Concanavalin A; Cricetinae; Female; Hypergammaglobulinemia; Leishmaniasis, Visceral; Male; Mitosis; Phytohemagglutinins; Spleen; T-Lymphocytes; Time Factors; Trypanosoma

Highly susceptible mice were infected with Leishmania donovani chagasi and were treated with supernatants, free or encapsulated in liposomes, from concanavalin A-stimulated or unstimulated mouse spleen cell cultures. Treatment consisted of multiple i.v. injections beginning 2 days before to 2 days after infection. Mice treated with lymphokine-rich supernatants encapsulated in liposomes had significantly fewer liver parasites than the control groups, demonstrating in vivo activity of lymphokine against an infectious organism.

Topics: Animals; Concanavalin A; Immunotherapy; Leishmania; Leishmaniasis, Visceral; Liposomes; Liver Diseases, Parasitic; Lymphokines; Macrophages; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Spleen

Topics: Concanavalin A; DNA; Humans; Kenya; Leishmaniasis, Visceral; Lymphocyte Activation; Phytohemagglutinins; T-Lymphocytes, Regulatory

In vitro lymphocyte reactivity to phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) was evaluated in 11 patients with American visceral leishmaniasis (AVL) and 11 control subjects. The diagnosis of AVL was established by the demonstration of leishmania in bone marrow aspirates. Thymidine incorporation (cpm +/- SEM) of PHA-stimulated cultures was 27, 520 +/- 4,488 in the AVL patients and 56,531 +/- 8,787 in the controls (P less than 0.01). No significant difference was observed in the response to Con A and PWM between AVL and control patients. The restoration of the PHA response to levels similar to normal was observed when cells from five AVL patients were cultured in medium supplemented with standard AB serum rather than autologous serum. In this group of experiments the average suppressor activity of the PHA response present in sera from AVL patients was 46%. Lymphocyte reactivity of normal subjects to PHA was also suppressed by the AVL serum: PHA-stimulated lymphocytes cultured in standard AB serum were 49,122 +/- 9,345 vs 23,115 +/- 4,935 cpm in cultures supplemented with AVL serum. The demonstration that AVL serum suppressed the PHA response indicates that some of the cellular immunological abnormalities in AVL patients may be dependent on inhibitor factory present in AVl serum.

Topics: Adolescent; Adult; Child; Child, Preschool; Concanavalin A; Humans; In Vitro Techniques; Infant; Leishmaniasis, Visceral; Lymphocyte Activation; Lymphocytes; Phytohemagglutinins; Pokeweed Mitogens; Thymidine