Sita Naik

Leishmania donovani infection down-regulates TLR2-stimulated IL-12p40 and activates IL-10 in cells of macrophage/monocytic lineage by modulating MAPK pathways through a contact-dependent mechanism

The failure of Leishmania, an intracellular pathogen, to stimulate a pro‐inflammatory response following entry into macrophages has been well reported. This occurs in spite of the fact that ligands for the toll‐like receptors (TLR) have been recently shown on the parasite surface and their role in disease protection well documented. The outcome of infection in leishmaniasis is determined by the Th1 versus Th2 nature of the effector response and the generation of IL‐12 and IL‐10 by the infected macrophages is important for this decision. We evaluated the effect of L. donovani infection of monocytes (cell line THP‐1, and monocytes derived from human peripheral blood) on Pam3cys (TLR2 ligand) and lipopolysaccharide (TLR4 ligand) stimulated production of IL‐12p40 and IL‐10. L. donovani infection caused suppression of TLR2 and TLR4‐stimulated IL‐12p40, with an increase in IL‐10 production. Parasites also modulated the TLR2‐stimulated mitogen‐activated protein kinase (MAPK) pathway by suppressing MAPK P38 phosphorylation and activating extracellular regulated kinase (ERK)1/2 phosphorylation. These effects could be reversed either by using a MAPK P38 activator, anisomycin, or ERK1/2 inhibitor, U0126. L. donovani caused modulation of TLR2‐stimulated MAPK pathways in a contact‐dependent mechanism. In addition parasite structural integrity but not viability was required for suppression of TLR2‐stimulated IL‐12p40 and activation of IL‐10. These observations suggest that L. donovani has evolved survival strategies that subvert the pro‐inflammatory response generated through TLRs.

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.

Immunostimulatory cellular responses of cured Leishmania -infected patients and hamsters against the integral membrane proteins and non-membranous soluble proteins of a recent clinical isolate of Leishmania donovani

Development of an effective immunoprophylactic agent for visceral leishmaniasis (VL) has become imperative due to the increasing number of cases of drug resistance and relapse. Live and killed whole parasites as well as fractionated and recombinant preparations have been evaluated for vaccine potential. However, a successful vaccine against the disease has been elusive. Because protective immunity in human and experimental leishmaniasis is predominantly of the Th1 type, immunogens with Th1 stimulatory potential would make good vaccine candidates. In the present study, the integral membrane proteins (IMPs) and non‐membranous soluble proteins (NSPs), purified from promastigotes of a recent field isolate, Leishmania donovani stain 2001, were evaluated for their ability to induce cellular responses in cured patients (nu2003=u20039), endemic controls (nu2003=u20035) of visceral leishmaniasis (VL) and treated hamsters (nu2003=u200310). IMPs and NSPs induced significant proliferative responses (SI 6·3u2003±u20034·1 and 5·6u2003±u20032·3, respectively; Pu2003<u20030·01) and IFN‐γ production (356·3u2003±u2003213·4 and 294·29u2003±u2003107·6u2003pg/ml, respectively) in lymphocytes isolated from cured VL patients. Significant lymphoproliferative responses against IMPs and NSPs were also noticed in cured Leishmania animals (SI 7·2u2003±u20034·7 & 6·4u2003±u20034·1, respectively; Pu2003<u20030·01). In addition, significant NO production in response both IMPs and NSPs was also noticed in macrophages of hamsters and different cell lines (J774A‐1 and THP1). These results suggest that protective, immunostimulatory molecules are present in the IMP and NSP fractions, which may be exploited for development of a subunit vaccine for VL.

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.

Association of tumor necrosis factor alpha and IL-10 promoter polymorphisms with Rheumatoid arthritis in North Indian population

Rheumatoid arthritis is a chronic autoimmune disorder associated with altered expression of pro- and anti-inflammatory cytokines in the affected tissues. The aim of this study was to investigate the association between promoter polymorphisms of TNFα and IL-10 gene with susceptibility, age of disease onset and disease severity in North Indian patients with rheumatoid arthritis (RA). SNPs at position −308 and −863 of TNF gene and −819/−592 and −1082 position of IL-10 gene were determined in 222 patients and 208 healthy controls using RFLP or ARMS method. Polymorphism TNF −308A was less prevalent among the patients (1.7%) than controls (4.9%; pxa0=xa00.01, OR: 0.32, 95% CI: 0.13–0.76). Among female patients, IL-10 −592A allele associated with higher baseline disease activity scores (5.77xa0±xa01.99) than −592C (5.57xa0±xa01.19; pxa0=xa00.04). Female patients carrying allele A of TNFα −863 had earlier age of onset of RA (33.99xa0±xa09.6xa0years) than those with allele C (36.15xa0±xa011.21xa0years; pxa0=xa00.043). In conclusion, allele A at TNFα −308 locus provides protection against RA in North Indian population while another TNF allele A at −863 position had weak association with earlier onset of disease in female patients. On the other hand promoter polymorphisms of IL-10 did not affect susceptibility but polymorphism at −819/−592A was associated with higher disease activity scores at baseline.

mTOR signaling pathway regulates the IL-12/IL-10 axis in Leishmania donovani infection

Leishmania-induced interleukin-12 (IL-12) expression is negatively regulated by the phosphatidylinositol 3-kinase (PI3K) and extracellular signal regulated kinase (ERK) 1/2 pathways in human monocyte derived macrophages (MDMs). To extend these studies, we examined the pathways downstream from PI3K in L. donovani-induced reciprocal regulation of IL-12/IL-10 axis in THP-1-derived macrophages. We show for the first time that in THP-1-derived macrophages and human monocytes, mTOR inhibition by rapamycin reversed L. donovani-induced IL-12 and IL-10 modulation. L. donovani-induced phosphorylation of P70S6K, a correlate of mTOR activity, in TLR-stimulated THP-1 derived macrophages. This increase in P70S6K phosphorylation was completely blocked by rapamycin (mTOR inhibitor) and partially by wortmannin (PI3K inhibitor). These observations suggest that a PI3K independent pathway is operative in the modulation of IL-12 and IL-10. Blocking of TLR2 significantly attenuated IL-10 induced by the parasite, but did not affect IL-12 production. Thus, our data suggests that intracellular network of PI3K and mTOR pathway control IL-12/IL-10 modulation by L. donovani. mTOR inhibitors may be attractive molecules to reverse this modulation and may result in control of disease.

Prophylactic potential of autoclaved Leishmania donovani with BCG against experimental visceral leishmaniasis.

The prophylactic efficacy of autoclaved Leishmania donovani (ALD) and autoclaved L. major (ALM)--a heterologous vaccine developed against cutaneous leishmaniasis (used as a reference vaccine), along with BCG--was evaluated against L. donovani in hamsters (Mesocricetus auratus). Animals were immunized with triple doses (21 days apart) of either ALD or ALM (1.0 mg) with or without BCG (0.1 mg) and challenged 21 days later with 1 x 10(6) L. donovani amastigotes intracardially. Animals immunized with ALM + BCG and ALD + BCG yielded 94.3% and 86.1% parasite inhibition respectively in comparison to the BCG only and unvaccinated controls. Fifty and 33.3% of the vaccinated animals (ALM + BCG and ALD + BCG respectively) were completely devoid of parasites when tested on day 45 post-challenge (p.c.) and survived till the experiment was terminated. The mean survival of ALM + BCG and ALD + BCG groups (animals harbouring parasites) was longest (168 and 139 days respectively). No significant increase in anti-leishmanial antibody level (ELISA) was noticed in ALD + BCG and ALM + BCG groups whereas it increased progressively in the rest of the experimental groups. The lymphoproliferative responses to PHA and Con A, of the 2 vaccinated groups were comparable to that of normal controls on day 45 p.c. The study suggests that ALD along with BCG can offer substantial protection against visceral leishmaniasis in hamsters.

Effect of lead exposure on serum immunoglobulins and reactive nitrogen and oxygen intermediate

Metal toxicants may affect immune regulation with an increased incidence of infectious diseases, cancer or autoimmune diseases. Lead is the leading environmental toxin among heavy metals and has aroused concern, as continuous low-level exposure leads to a variety of health problems. We compared serum immunoglobulins (Ig) and reactive oxygen and nitrogen intermediates (super oxide and nitric oxide (NO)) in culture supernatant of lead-exposed (blood lead; Pb-B > 10 μg/dL) individuals with that of unexposed healthy controls (blood lead <10 μg/dL). The serum IgA level was significantly increased in lead-exposed individuals in comparison to controls (1829±53 versus 1389±52 μg/dL; (P <0.05). Furthermore, lipopolysaccharide-induced NO production by mouse macrophage cells, RAW 264.7, showed significant suppression (P<0.05) after treatment with lead acetate (100 ppm). This study suggested that lead could modulate the immune system by targeting the humoral as well as innate immune cells.

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.

In vitro up-regulation of HLA-G using dexamethasone and hydrocortisone in first-trimester trophoblast cells of women experiencing recurrent miscarriage.

The trophoblast cells at the maternal-fetal interface express an unusual combination of human leukocyte antigen (HLA)-C, HLA-E and HLA-G. Altered expression of HLA-G on the extravillous cytotrophoblast has been implicated in the etiology of recurrent miscarriages (RMs). We have assessed HLA-G expression in extravillous cytotrophoblast in cell cultures prepared from RM patients and compared with those of first-trimester voluntarily terminated normal pregnancies (control). Glucocorticoids, dexamethasone and hydrocortisone were examined for their role in modulation of the HLA-G expression. HLA-G promoter and 3UTR variants were investigated for their effect on the transcription of HLA-G. Cultured cytotrophoblast cells from the first-trimester RM patients were treated with dexamethasone and hydrocortisone (dose concentration 0-1000 ng/ml). HLA-G gene transcription was determined by semiquantitative and quantitative real-time polymerase chain reaction (RT-PCR), while protein expression was determined by a specific enzyme-linked immunosorbent assay (ELISA), flow cytometry and western blot analyses. HLA-G polymorphisms were detected by PCR and/or sequence-based typing. Low level of HLA-G was observed in untreated trophoblast cells obtained from RM patients as compared with controls. Upon treatment with glucocorticoids, the expression of HLA-G in these cells was up-regulated in a dose-dependent manner (P < 0.05), with no change in cellular proliferation and viability. There was no significant association between HLA-G polymorphism in RM patients and controls. HLA-G is minimally expressed in cultured trophoblast cells of RM patients. It can be up-regulated upon exposure with both dexamethasone and hydrocortisone. Glucocorticoids have the potential to modulate HLA-G expression in vitro, and can be further examined for their therapeutic applicability in RM.