An immunoprophylactic evaluation of Ld-ODC derived HLA-A0201 restricted peptides against visceral leishmaniasis.

Five (5) HLA-A 0201 restricted epitopes of ornithine decarboxylase derived from Leishmania donovani (Ld-ODC) were examined by reverse vaccinology to develop prophylactics against visceral leishmaniasis (VL). These consensus epitopes comprising (P1: RLMPSAHAI, P2: LLDQYQIHL, P3: GLYHSFNCI, P4: AVLEVLSAL and P5: RLPASPAAL) were observed and presented by diverse HLA alleles screened by immune-informatics tools. These epitopes were also observed for strong stability for appropriate immune response in in silico screening and molecular dynamics. Top five selected epitopes filtered from population coverage analysis and TAP binding affinity were identified and evaluated against treated cases of VL subjects. Experiments were run individually with synthetic peptides or as the cocktail of peptides. A major population of CD8+ T cells were predominantly IFN-γ producers but not the IL-10 cytokines and shown with granzyme-B activity. Therefore, it can be concluded that the screened HLA-A0201 restricted epitope hotspots derived from Leishmania ODC can trigger CD8+ T cells, which can skew other immune cells functions toward protection. However, a detailed analysis can explore its potentiality as a vaccine candidate.Communicated by Ramaswamy H. Sarma.

Validating Immunomodulatory Responses of r-LdODC Protein and Its Derived HLA-DRB1 Restricted Epitopes against Visceral Leishmaniasis in BALB/c Mice.

Vaccination is considered the most appropriate way to control visceral leishmaniasis (VL). With this background, the r-LdODC protein as well as its derived HLA-DRB1-restricted synthetic peptides (P1: RLMPSAHAI, P2: LLDQYQIHL, P3: GLYHSFNCI, P4: AVLEVLSAL, and P5: RLPASPAAL) were validated in BALB/c mice against visceral leishmaniasis. The study was initiated by immunization of the r-LdODC protein as well as its derived peptides cocktail with adjuvants (r-CD2 and MPL-A) in different mice groups, separately. Splenocytes isolated from the challenged and differentially immunized mice group exhibited significantly higher IFN-γ secretion, which was evidenced by the increase in the expression profile of intracellular CD4+IFN-γ T cells. However, the IL-10 secretion did not show a significant increase against the protein and peptide cocktail. Subsequently, the study confirmed the ability of peptides as immunoprophylactic agents, as the IE-I/AD-I molecule overexpressed on monocytes and macrophages of the challenged mice group. The parasitic load in macrophages of the protein and peptides cocktail immunized mice groups, and T cell proliferation rate, further established immunoprophylactic efficacy of the r-LdODC protein and peptide cocktail. This study suggests that the r-LdODC protein, as well as its derived HLA-DRB1-restricted synthetic peptides, have immunoprophylactic potential and can activate other immune cells' functions towards protection against visceral leishmaniasis. However, a detailed study in a humanized mice model can explore its potential as a vaccine candidate.

A randomized, open-label study to evaluate the efficacy and safety of liposomal amphotericin B (AmBisome) versus miltefosine in patients with post-kala-azar dermal leishmaniasis.

BACKGROUND: Treatment of post-kala-azar dermal leishmaniasis cases is of paramount importance for kala-azar elimination; however, limited treatment regimens are available as of now. AIM: To compare the effectiveness of liposomal amphotericin B vs miltefosine in post-kala-azar dermal leishmaniasis patients. METHODOLOGY: This was a randomized, open-label, parallel-group study. A total of 100 patients of post kala azar dermal leishmaniasis, aged between 5 and 65 years were recruited, 50 patients in each group A (liposomal amphotericin B) and B (miltefosine). Patients were randomized to receive either liposomal amphotericin B (30 mg/kg), six doses each 5 mg/kg, biweekly for 3 weeks or miltefosine 2.5 mg/kg or 100 mg/day for 12 weeks. All the patients were followed at 3rd, 6th and 12th months after the end of the treatment. RESULTS: In the liposomal amphotericin B group, two patients were lost to follow-up, whereas four patients were lost to follow-up in the miltefosine group. The initial cure rate by "intention to treat analysis" was 98% and 100% in liposomal amphotericin B and miltefosine group, respectively. The final cure rate by "per protocol analysis" was 74.5% and 86.9% in liposomal amphotericin B and miltefosine, respectively. Twelve patients (25.5%) in the liposomal amphotericin B group and six patients (13%) in the miltefosine group relapsed. None of the patients in either group developed any serious adverse events. LIMITATIONS: Quantitative polymerase chain reaction was not performed at all the follow-up visits and sample sizes. CONCLUSION: Efficacy of miltefosine was found to be better than liposomal amphotericin B, hence, the use of miltefosine as first-line therapy for post-kala-azar dermal leishmaniasis needs to be continued. However, liposomal amphotericin B could be considered as one of the treatment options for the elimination of kala-azar from the Indian subcontinent.

Dendritic cell engineered cTXN as new vaccine prospect against L. donovani.

Dendritic cells (DCs), as antigen-presenting cells, can reportedly be infected withLeishmaniaparasites and hence provide a better option to trigger T-cell primary immune responses and immunological memory. We consistently primed DCs during culture with purified recombinant cytosolic tryparedoxin (rcTXN) and then evaluated the vaccine prospect of presentation of rcTXN against VL in BALB/c mice. We reported earlier the immunogenic properties of cTXN antigen derived fromL. donovani when anti-cTXN antibody was detected in the sera of kala-azar patients. It was observed that cTXN antigen, when used as an immunogen with murine DCs acting as a vehicle, was able to induce complete protection against VL in an infected group of immunized mice. This vaccination triggered splenic macrophages to produce more IL-12 and GM-CSF, and restricted IL-10 release to a minimum in an immunized group of infected animals. Concomitant changes in T-cell responses against cTXN antigen were also noticed, which increased the release of protective cytokine-like IFN-γ under the influence of NF-κß in the indicated vaccinated group of animals. All cTXN-DCs-vaccinated BALB/c mice survived during the experimental period of 120 days. The results obtained in our study suggest that DCs primed with cTXN can be used as a vaccine prospect for the control of visceral leishmaniasis.

Corrigendum: Leishmania-Specific Promiscuous Membrane Protein Tubulin Folding Cofactor D Divulges Th1/Th2 Polarization in the Host via ERK-1/2 and p38 MAPK Signaling Cascade.

[This corrects the article DOI: 10.3389/fimmu.2020.00817.].

Leishmania-Specific Promiscuous Membrane Protein Tubulin Folding Cofactor D Divulges Th1/Th2 Polarization in the Host via ERK-1/2 and p38 MAPK Signaling Cascade.

Visceral leishmaniasis (VL)-related mortality and morbidity imposes a great deal of health concern across the globe. The existing anti-leishmanial drug regimen generally fails to eliminate newly emerging resistant isolates of this dreadful parasite. In such circumstances, the development of a prophylactic strategy to impart protection against the disease is likely to take center stage. In order to develop a promising prophylactic vaccine, it is desirable to identify an adequately potential vaccine candidate. In silico analysis of Leishmania tubulin folding cofactor D protein predicted its potential to activate both B- and T-cell repertoires. Furthermore, the ELISA employing anti-peptide27 (a segment of tubulin folding cofactor D) antibody revealed its proficiency in VL diagnosis and treatment monitoring. The peptide27 and its cocktail with another Leishmania peptide (peptide23) prompted the up-regulation of pro-inflammatory cytokines, such as IFN-γ, TNF-α, IL-2, IL-17, etc., and the down-regulation of immune-regulatory cytokines, such as IL-10, in the immunized BALB/c mice. Coherent to the consequence of peptide-specific humoral immune response, peptide cocktail-based immunization ensued in the predominant amplification of pathogen-specific IgG2a over the IgG1 isotype, up-regulated proliferation of T lymphocytes, and enhanced production of nitric oxide, reactive oxygen species, etc. We also established that the peptide cocktail modulated host MAPK signaling to favor the amplification of Th1-dominated immune response in the host. The peptide cocktail mediated the activation of the host immune armory, which was eventually translated into a significant decline in parasitic load in the visceral organs of experimental animals challenged with Leishmania donovani.

Conversion of asymptomatic infection to symptomatic visceral leishmaniasis: A study of possible immunological markers.

INTRODUCTION: Presence of asymptomatic individuals in endemic areas is common. The possible biomarkers in asymptomatic individuals once they get exposed to infection as well as following conversion to symptomatic disease are yet to be identified.We identified asymptomatic Visceral leishmaniasis (VL) infection amongst rK39+sorted direct agglutination test positive (DAT+) endemic healthy population and confirmed it by quantitative PCR(qPCR).The immunological determinants such as Adenosine deaminase (ADA), Interferon gamma (IFN-γ), Tumour Necrosis Factor alpha (TNF-α) and Interleukin 10 (IL-10)were examined to predict probable biomarkers for conversion to symptomatic VL. METHODS: Sample size was 5794 healthy individuals from VL endemic region. Antibody tests(DAT &rK39) were performed and later a qPCR assay was employed using kDNA specific primers and probes. Immunological biomarkers examined were ADA level by ADA-MTP kit and quantitative cytokines(IFN-γ, IL-10 and TNF-α) by ELISA. RESULTS: 120 asymptomatic individuals of 308 rK39 sero-positives were DAT positive comprising of 56 with previous history and 64 with no history of VL. RT-PCR confirmed asymptomatic VL in 42 sero-positives. These were followed up through repeated qPCR and evaluation of immunological determinants. We observed10 symptomatic cases converted from a total of 42 asymptomatic individuals identified at base-line. The level of ADA, IL-10 and IFN-γ remained consistently high in asymptomatic cases and amongst these, ADA and IL-10 but not IFN-γ remained higher at the development of clinical symptoms into active VL. On the contrary, there was no significant change in the mean concentration of TNF-α at both stages of the disease. DISCUSSION: We surmise from our data that considerable proportion of asymptomatic cases can be a reservoir and may play a crucial role in transmission of visceral leishmaniasis in endemic areas. The data also suggests that ADA and IL-10 can serve as a potential biomarker during the conversion of asymptomatic into symptomatic VL.

Advanced case of PKDL due to delayed treatment: A rare case report.

Post-kala-azar dermal leishmaniasis (PKDL) is clinical outcome of visceral leishmaniasis (VL) and is thought to be the potential reservoir of parasite. Miltefosine (MF) is the only oral drug existing for treatment of post-kala-azar dermal leishmaniasis (PKDL). Increased miltefosine tolerance in clinical isolates of Leishmania donovani has been reported and is one of the major concerns in the treatment of PKDL. Here, we report a highly ulcerated PKDL case that was successfully cured after miltefosine treatment.

Evaluating the immunomodulatory responses of LdODC-derived MHC Class-II restricted peptides against VL.

In a bid to develop a novel immunoprophylactic measure against visceral leishmaniasis (VL), MHC class-II-restricted epitopes of LdODC were identified by reverse vaccinology approach. Five consensus HLA-DRB1*0101-restricted epitopes were screened. The analysis revealed that the set of epitopes was presented by at least 54 diverse MHC class-II alleles. Based on in silico screening, followed by molecular dynamics simulation, population coverage analysis, and HLA cross-presentation ability, five best epitopes were evaluated. PBMCs isolated from treated VL subjects, when stimulated with synthetic peptide alone or as a cocktail of peptides, triggered a secretory IFN-γ, but not the IL-10 level. Support in this notion came from intracellular cytokine level with a considerable up-regulated IFN-γ produced by CD4+ T cells. Also, the enhanced IFN-γ seemed to be augmented with the activation of macrophages with prominent IL-12 production. Therefore, it can be concluded that the screened MHC class-II-restricted epitope hotspots derived from Leishmania ODC can trigger CD4+ T cells, which can skew macrophage functions towards protection. However, a detailed analysis can explore its potentiality as a vaccine candidate.

The usefulness of trained field workers in diagnosis of post-kala-azar dermal leishmaniasis (PKDL) and clinico-epidemiological profile in highly endemic areas of Bihar.

BACKGROUND: Surveillance of post-kala-azar dermal leishmaniasis (PKDL) is critical to the elimination of visceral leishmaniasis (VL). In this study we assessed the feasibility of using trained field workers for detecting suspected PKDL cases. METHODS: A cross-sectional study using a multistage sampling technique was conducted in the Araria district of Bihar. Trained field workers were utilized for identification of suspected PKDL case. RESULTS: We investigated 57 099 individuals from 11 300 households. The trained field workers were useful in identifying 107 (18%) probable PKDL cases. The calculated PKDL prevalences were 18.7/10 000 and 9.7/10 000 for probable and confirmed PKDL cases, respectively. The median duration of onset of PKDL was 23 months (interquartile range 16.5-56.5). The younger age group developed PKDL significantly more often compared with the older age group (p=0.007). Of the 107 patients, 25 (55.5%) were positive by microscopy of slit skin smear and 42 (93.3%) by polymerase chain reaction. Of 45 patients, 33 (73%) PKDL cases were cured after full treatment. The risk of not being cured with incomplete treatment was three times higher than with complete treatment (relative risk 3.12 [95% confidence interval 1.23 to 8.67], p=0.004). CONCLUSIONS: We conclude that the prevalence of PKDL is high and the use of trained field workers may be feasible to actively detect PKDL cases in VL-endemic areas of Bihar, India.

Modulation of the immune response and infection pattern to Leishmania donovani in visceral leishmaniasis due to arsenic exposure: An in vitro study.

The arsenic contamination of ground water in visceral leishmaniasis (VL) endemic areas in Bihar, India leads to human exposure through drinking water. Possibly, the consumed arsenic (As) accumulates in the tissues of VL patients, who subsequently internalize intracellular amastigotes to confer resistance against chemotherapy to the parasite, leading to modulation in the host's immune response. This hypothesis appears to be consistent with the in vitro findings that in arsenic-exposed parasites, the mitochondrial membrane potential became depolarized, whereas the reduced thiol and lactate production was overexpressed with enhanced glucose consumption; therefore, the reduced thiol possibly supports an immunosuppressive state in the host cells. This observation was well supported by the down-regulated expression of pro-inflammatory cytokines (IL-2, IL-12, IFN-γ, and TNF-α) with a suppressed anti-leishmanial function of macrophage (NO, ROS). In contrast, the pathophysiological mechanism of VL has received ample support by the promotion of Th2 cytokines (IL-4 and IL-10) in the presence of arsenic-exposed Leishmania parasites (LdAS). Dysfunction of mitochondria and the overexpression of lactate production raise the possibility of the Warburg effect being operative through the up-regulation of glucose consumption by parasites to enhance the energy production, possibly augmenting virulence. Therefore, we surmise from our data that arsenic exposure to Leishmania donovani modulates the immune response and infection pattern by impairing parasite function, which may affect the anti-leishmanial effect in VL.

The potential HLA Class I-restricted epitopes derived from LeIF and TSA of Leishmania donovani evoke anti-leishmania CD8+ T lymphocyte response.

To explore new protective measure against visceral leishmaniasis, reverse vaccinology approach was employed to identify key immunogenic regions which can mediate long-term immunity. In-depth computational analysis revealed nine promiscuous epitopes which can possibly be presented by 46 human leukocyte antigen, thereby broadening the worldwide population up to 94.16%. This is of reasonable significance that most of the epitopes shared 100% sequence homology with other Leishmania species and could evoke a common pattern of protective immune response. Transporter associated with antigen processing binding affinity, molecular docking approach followed by dynamics simulation and human leukocyte antigen stabilization assay suggested that the best five optimal set of epitopes bind in between α1 and α2 binding groove with sufficient affinity and stability which allows the translocation of intact epitope to the cell surface. Fascinatingly, the human leukocyte antigen stabilization assay exhibited a modest correlation with the positive immunogenicity score predicted by class I pMHC immunogenicity predictor. A support for this notion came from ELISA and FACS analysis where the epitopes as a cocktail induced CD8+ IFN-γ and Granzyme B levels significantly in treated visceral leishmaniasis subject which suggests the immunogenic ability of the selected epitopes.

Knowledge, stigma, health seeking behaviour and its determinants among patients with post kalaazar dermal leishmaniasis, Bihar, India.

BACKGROUND: Lesishmaniasis is a neglected tropical disease endemic in Bihar, India. Inappropriate health seeking behaviour of post kala-azar dermal leishmaniasis (PKDL) patients may increase the disease duration, severity and transmissibility. Simultaneously, lack of knowledge and perceived stigma may also increase the length of delay in receiving treatment. This ultimately effects the kala-azar elimination program. METHODS: A cross sectional study was conducted in 120 confirmed PKDL patients, aged 18 years and older. Data related to knowledge and health seeking behaviour was collected by a pre-tested questionnaire. EMIC stigma scale was used for assessing the perceived stigma. Patients were personally interviewed after taking informed consent. Data analysis was done by using SPSS 16 software. RESULTS: The time between appearance of symptoms and first medical consultation (patient delay) ranged from 15 days to 5475 days (15 years) with a median of 285 days. The time between first medical consultations to onset of specific treatment (system delay) ranged from 2 to 5475 days with a median of 365 days. Many patients approached first to quacks (8.4%), homeopathic and ayurvedic practitioners (25.8%) upon recognition of symptoms. Majority of the patients (68.3%) had poor knowledge about PKDL and its vector. Type of skin lesions and gender had significant association with patient delay and system delay respectively (p<0.05). Distance to primary health centre (PHC) had significant association with patients delay as well as system delay (p<0.05). Patients with younger age, unmarried and polymorphic lesions had higher stigma (p<0.05). Patients with PKDL feel stigmatized in different areas. CONCLUSION: PKDL treatment delays were unacceptably high and patients had poor knowledge compounded with feelings of stigmatization. To reduce the delay, a system may be evolved to establish some sort of public-private collaboration, besides awareness programs should be tailored, and implemented for improving the patient education regarding the disease and its linkage with VL.

Exploring new immunological insight on SP15 (∼14 kDa) family protein in saliva of Indian sand-fly (Phlebotomus argentipes) in experimental visceral leishmaniasis.

Visceral leishmaniasis (VL) is a disease caused by protozoan species of the genus Leishmania and is transmitted through bites from the Phlebotomus sand fly; it is associated with considerable morbidity and mortality in many parts of world, including India. Reports on the protective role played by saliva proteins of Lutozomyia longipalpis, Phlebotomus papatasi and Phlebotomus duboscqi. are available. However, no studies have explored the salivary proteins of P. argentipes, which is the known proven vector for the transmission of VL in the Indian sub-continent. Herein we revealed the presence of two proteins of 14.2 and one protein of 13.6 kDa in Indian strain P. argentipes which is absolute identical to previously reported protein of SP15 family (PagSP01, PagSP02 and PagSP07) of P. argentipes of NIH colony, USA. In an experimental study on P. argentipes from Bihar, India, we demonstrated that a strong humoral and cellular immune response was triggered to reduce the concomitant Leishmania load in groups of immunized mice. The immunized group produced a considerable amount of IgG antibodies, and their splenocytes generated TH1 cytokines (IL-12, IFN-γ) with the support of delayed-type hypersensitivity (DTH) reactivity in such mice at the challenged site. We summarize from our data that some identical proteins to previous from SP15 family protein of 14.2 and 13.6 kDa molecular size, derived from Indian P. argentipes and reported its first time, can also be significant in resolution of VL infection after modulation of host protective T cell response in VL.

Arsenic trioxide induces apoptosis and inhibits the growth of human liver cancer cells.

AIMS: As a fifth most common cancer type, Hepatocellularcarcinoma (HCC) ranked third leading cause of cancer deaths worldwide. Arsenic trioxide (As2O3) is known as chemotherapeutic agent against few cancer including Acute promyelocyticleukemia and solid tumors. But its effect and possible associated mechanism in HCC is meager. Present study aimed to assess As2O3 modulatory effect on liver cancer by assessing cell growth and viability. METHODS: Liver normal (Chang liver) and cancerous cells (Hep3B) were exposed to different concentration's (0, 1, 5, 10 & 15 µM) of As2O3 at different intervals (24, 48 & 72 h). Cell growth was assessed microscopically, and Cytotoxicity assays were done through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Water-soluble tetrazolium salt (WST) growth inhibition assays. Cell viability was studied by trypan blue staining. Apoptosis was analyzed by Annexin V/PI assay, and expression of genes (Notch and anti-apoptotic) were determined through western blotting and Q-PCR method. KEY FINDINGS: A significant reduction in cell growth and viability was reported in liver cancerous cells as compare to normal cells at 5 µM As2O3. Consistently, As2O3 induced apoptosis along with down-regulation of anti-apoptotic protein Bcl-xL, and up regulates expression of Notch that leads towards apoptosis. SIGNIFICANCE: Results clearly suggest that As2O3 restricted growth and induces apoptosis more in liver cancer cells as compared to normal cells. This finding suggests that it could be a promising potential therapeutic agent against liver cancer which need further testing by in-vivo investigations.

Leishmania donovani mediated higher expression of CCL4 induces differential accumulation of CD4+CD56+NKT and CD8+CD56+NKT cells at infection site.

Sterile cure from visceralized Leishmania donovani (L. donovani) needs Th1 cell support along with the assistance from innate immune cells, NK cells and NKT cells. NKT cells play as a connecting link between innate and adaptive immune cell and support T helper cell function. Earlier, a categorical function of CD56 positive CD4+ or CD8+ NKT cells was reported in visceral leishmaniasis (VL). It was observed in in vitro that CD4+CD56+NKT cells, but not CD8+CD56+NKT cells, were accumulated at the L. donovani infection site. Therefore, in vitro experiments have been carried out to decipher the mechanism behind preferential accumulation of CD4+CD56+NKT cells at infection site. In this study, 1.89 fold higher expression of CCL4/MIP-1ß was noticed in infected macrophages. The higher expression of CCL4 was correlated with preferential accumulation of CCR5+CD4+CD56+NKT cells and apoptosis of CD8+CD56+NKT cells at in vitro infection site. The CD4+CD56+NKT cells were also observed expressing TGF-ß dominantly. Interaction of CCL4 chemotaxis was interrupted by blocking, which led to drift back the TGF-ß producing CD4+CD56+NKT cells and promoted CD8+CD56+NKT cells recruitment in in vitro infection site. CCR5 blockade also reduced CD25 and FoxP3 positive CD4+CD56+NKT cells in in vitro infection site. Therefore, it was concluded that Leishmania promotes strategic expression of CCL4, which alternately attracts CCR5+ cells, mostly expressing regulatory cytokines, at infection site. This reduces the CD8+CD56+NKT cells at infection site through Smad4 mediated TGF-ß expression and activation of caspases. Data indicates that L. donovani induces higher expression of CCL4 in host cell to attract CCR5+ cells under its strategic plan to downregulate host immune response.

CD8dim but not CD8bright cells positive to CD56 dominantly express KIR and are cytotoxic during visceral leishmaniasis.

This study reports a structural and functional heterogeneity of CD8+CD56+NKT cells, which usually decrease quantitatively during visceral leishmaniasis. Based on fluorescence intensity of CD8 receptors on CD56+NKT cells, two populations of CD8+CD56+NKT cells have been identified. These cells were recognized as CD8dimCD56+NKT and CD8brightCD56+NKT cells. We further analyzed the functional nature of CD8dim and CD8bright positive CD56+NKT cells. In comparison to CD8brightCD56+NKT cells, a significantly higher percentage of CD8dimCD56+NKT cells expressed KIR during VL. The percentage of CD8dimCD56+NKT cells expressing KIR was found 4 fold higher in VL as compared to healthy subjects. But, the difference was insignificant in case of CD8brightCD56+NKT cells. CD8+CD56+NKT cells release granzyme B to kill the infected cells. A categorical difference was also observed in the function of CD8dimCD56+NKT and CD8brightCD56+NKT cells during visceral leishmaniasis. The percentage of granzyme B expressing CD8dimCD56+NKT cells was 2.83 fold higher in VL compared to healthy subjects. But, there was no significant difference in granzyme B expressing CD8brightCD56+NKT cells in samples from healthy and VL subjects. However, within VL subject, the percentage of granzyme B expressing CD8dimCD56+NKT cells was 5.7 fold higher in comparison to CD8brightCD56+NKT cells. This study concludes that CD8dimCD56+NKT cells are more cytotoxic than CD8brightCD56+NKT cells during VL.

Cytokines and chemokines differentially regulate innate immune cell trafficking during post kala-azar dermal leishmaniasis.

Post kala-azar dermal leishmaniasis (PKDL) is often considered to be the anthroponotic reservoir of visceral leishmaniasis (VL) in India. A better understanding of the host immune-response in dermal lesions of PKDL patients is therefore of utmost significance to minimize such patients and to restrict VL transmission. Although the innate immune response is known to play an important role in parasite clearance from dermal lesions, the actual contribution of innate cells to the pathogenicity of PKDL is poorly understood. The present study explored the immune-pathogenesis of PKDL patients to understand the expression of CD62L, CD11b, CXCL8/IL-8, and MIP1-α and their contribution in signaling during innate cell trafficking. Twenty-five individuals were enrolled, who comprised eight active and untreated macular cases, seven active and untreated cases with papulo-nodular PKDL manifestations, five successfully treated post PKDL cases and five healthy individuals from a non-endemic region of Bihar, India. The immunological investigation was performed on biopsy specimens prepared with a disaggregation technique and blood samples. We observed that the PMNs in nodular patients displayed decreased L-selectin (CD62L) levels and increased integrin (CD11b) expression compared with those in macular patients. Further analysis showed that lower PMN extravasation in macular patients occurred because of inadequate CXCL8/ IL-8 release. In summary, Leishmania donovani (L. donovani) infection in macular PKDL patients decreased leucocyte rolling (L-selectin shedding) and induced up-regulation of the cellular signaling factors involved in pathogenesis (ERK1/2) as well as down regulated the signaling elements (p38 MAPK) involved in the Th1 response, especially in PMNs.

Cytosolic tryparedoxin of Leishmania donovani modulates host immune response in visceral leishmaniasis.

Leishmaniasis is a neglected tropical disease caused by the unicellular protozoan parasite of genus Leishmania. Tryparedoxin (TXN) is a low molecular mass dithiol protein belonging to oxidoreductases super-family; which function in concert with tryparedoxin peroxidase (TXNPx) as a system in protozoan parasites including Leishmania. Leishmanial hydroperoxides detoxification cascade uses trypanothione as electron donor to reduce hydroperoxide inside the macrophages during infection. However, the mechanism by which tryparedoxin can contribute in progression of visceral leishmaniasis (VL) and its impact on host's cellular immune response during infection in Indian VL patient is unknown. In this study, we purified a ∼17 kDa recombinant cytosolic tryparedoxin (cTXN) protein of Leishmania donovani (rLdcTXN) and investigated its immunological responses in peripheral blood monocytes (PBMC) isolated from VL patients. The protein significantly enhanced the promastigotes count after 96 h of culture showing a direct correlation with parasite growth. Furthermore, stimulation of PBMC isolated from VL patients with rLdcTXN resulted in up-regulation of IL-4 and IL-10 production whereas IL-12 and IFN-γ was significantly down-regulated suggesting a pivotal role of cTXN in provoking the immune suppression during VL. Our study demonstrates the importance of cTXN protein which can potentially modulate the outcome of disease through suppressing host protective Th1 response in VL patients.