The effect of blood transfusion on serum hepcidin levels in chronically transfused patients of ß-thalassemia major: An observational study in a tertiary care centre in Western Maharashtra.

INTRODUCTION: Hepcidin is the key regulator of systemic iron homeostasis. In iron-loading anemias, hepcidin levels are regulated by opposite forces of erythropoiesis and iron overload. In ß-thalassemia major patients, transfusions are the predominant cause of iron overload; in such chronically transfused patients, hepcidin concentrations are significantly higher than nontransfused patients, due to both increased iron load of transfusions and the suppression of ineffective erythropoiesis. AIM: This study aims to evaluate the effect of blood transfusions on serum hepcidin levels in chronically transfused patients of ß-thalassemia major and correlate with hemoglobin and serum ferritin levels of pre- and posttransfusion. MATERIALS AND METHODS: Thirty-three ß-thalassemia major patients requiring monthly transfusions were included in the study. Blood samples, collected pretransfusion and 7 days posttransfusion, were evaluated for hemoglobin, serum ferritin, and serum hepcidin using enzyme immunoassay. STATISTICAL ANALYSIS: Data were statistically analyzed through SPSS software and P < 0.05 is considered statically significant. RESULTS: Posttransfusion levels of hemoglobin, serum ferritin, and serum hepcidin increased. Posttransfusion levels of hepcidin were near normal levels. Pre- and posttransfusion hepcidin concentrations were significantly associated with hemoglobin levels. CONCLUSION: Serum hepcidin concentrations vary depending on the degree of erythropoiesis drive and level of anemia. We found that the serum hepcidin levels decrease over the inter-transfusion interval and transfusions cause suppression of ineffective erythropoiesis by the increase in hemoglobin. Posttransfusion values of hepcidin in our study were closer to normal levels which may be due to lower erythropoietic drive posttransfusion. We suggest that the measurement of serum hepcidin in chronically transfused ß-thalassemia patients can be used as a follow-up investigation for better management of these patients.

Role of cytokine in malignant T-cell metabolism and subsequent alternation in T-cell tumor microenvironment.

T cells are an important component of adaptive immunity and T-cell-derived lymphomas are very complex due to many functional sub-types and functional elasticity of T-cells. As with other tumors, tissues specific factors are crucial in the development of T-cell lymphomas. In addition to neoplastic cells, T- cell lymphomas consist of a tumor micro-environment composed of normal cells and stroma. Numerous studies established the qualitative and quantitative differences between the tumor microenvironment and normal cell surroundings. Interaction between the various component of the tumor microenvironment is crucial since tumor cells can change the microenvironment and vice versa. In normal T-cell development, T-cells must respond to various stimulants deferentially and during these courses of adaptation. T-cells undergo various metabolic alterations. From the stage of quiescence to attention of fully active form T-cells undergoes various stage in terms of metabolic activity. Predominantly quiescent T-cells have ATP-generating metabolism while during the proliferative stage, their metabolism tilted towards the growth-promoting pathways. In addition to this, a functionally different subset of T-cells requires to activate the different metabolic pathways, and consequently, this regulation of the metabolic pathway control activation and function of T-cells. So, it is obvious that dynamic, and well-regulated metabolic pathways are important for the normal functioning of T-cells and their interaction with the microenvironment. There are various cell signaling mechanisms of metabolism are involved in this regulation and more and more studies have suggested the involvement of additional signaling in the development of the overall metabolic phenotype of T cells. These important signaling mediators include cytokines and hormones. The impact and role of these mediators especially the cytokines on the interplay between T-cell metabolism and the interaction of T-cells with their micro-environments in the context of T-cells lymphomas are discussed in this review article.

Diagnostic significance of dysregulated miRNAs in T-cell malignancies and their metabolic roles.

T-cell malignancy is a broad term used for a diverse group of disease subtypes representing dysfunctional malignant T cells transformed at various stages of their clonal evolution. Despite having similar clinical manifestations, these disease groups have different disease progressions and diagnostic parameters. The effective diagnosis and prognosis of such a diverse disease group demands testing of molecular entities that capture footprints of the disease physiology in its entirety. MicroRNAs (miRNAs) are a group of noncoding RNA molecules that regulate the expression of genes and, while doing so, leave behind specific miRNA signatures corresponding to cellular expression status in an altered stage of a disease. Using miRNAs as a diagnostic tool is justified, as they can effectively distinguish expressional diversity between various tumors and within subtypes of T-cell malignancies. As global attention for cancer diagnosis shifts toward liquid biopsy, diagnosis using miRNAs is more relevant in blood cancers than in solid tumors. We also lay forward the diagnostic significance of miRNAs that are indicative of subtype, progression, severity, therapy response, and relapse. This review discusses the potential use and the role of miRNAs, miRNA signatures, or classifiers in the diagnosis of major groups of T-cell malignancies like T-cell acute lymphoblastic lymphoma (T-ALL), peripheral T-cell lymphoma (PTCL), extranodal NK/T-cell lymphoma (ENKTCL), and cutaneous T-cell lymphoma (CTCL). The review also briefly discusses major diagnostic miRNAs having prominent metabolic roles in these malignancies to highlight their importance among other dysregulated miRNAs.

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.

Curcumin, a traditional spice component, can hold the promise against COVID-19?

The severity of the recent pandemic and the absence of any specific medication impelled the identification of existing drugs with potential in the treatment of Coronavirus disease-2019 (COVID-19), caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). Curcumin, known for its pharmacological abilities especially as an anti-inflammatory agent, can be hypothesized as a potential candidate in the therapeutic regimen. COVID-19 has an assorted range of pathophysiological consequences, including pulmonary damage, elevated inflammatory response, coagulopathy, and multi-organ damage. This review summarizes the several evidences for the pharmacological benefits of curcumin in COVID-19-associated clinical manifestations. Curcumin can be appraised to hinder cellular entry, replication of SARS-CoV-2, and to prevent and repair COVID-19-associated damage of pneumocytes, renal cells, cardiomyocytes, hematopoietic stem cells, etc. The modulation and protective effect of curcumin on cytokine storm-related disorders are also discussed. Collectively, this review provides grounds for its clinical evaluation in the therapeutic management of SARS-CoV-2 infection.

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.

Immunomodulation induced through ornithine decarboxylase DNA immunization in Balb/c mice infected with Leishmania donovani.

We report here a Leishmania donovani ornithine decarboxylase (Ld-ODC) gene used as a DNA vaccine against visceral leishmaniasis in a murine Balb/c mouse model. This study also evaluated the possible mechanism of action directed by this candidate. We found a Th1 immune response after immunization using an Ld-ODC DNA vaccine, with results based on the rearrangement of TCR-V-α-2, proliferation of Carboxy fluorescein Succinimidyle ester positive T cells, which were able to produce cytokines such as TNF-α, IFN-γ, IL-12 and IL-2, but not IL-4, IL-5, IL-6 and IL-10, and modulations of the STAT-1 and p38 MAP kinase signaling pathways. The results were corroborated with the reduction in the amastigote proliferation and parasite killing in spleens after infection in vitro. We conclude this study suggesting that the Ld-ODC DNA construct could be a new vaccine candidate against visceral leishmaniasis.

Mining the Proteome of Leishmania donovani for the Development of Novel MHC Class I Restricted Epitope for the Control of Visceral Leishmaniasis.

Although, the precise host defence mechanism(s) is not completely understood, T cell-mediated immune responses is believed to play a pivotal role in controlling parasite infection. Here we target the stage dependent over expressed gene. Here, the consensus based computational approach was adopted for the screening of potential major histocompatibility complex class I restricted epitopes. Based on the computational analysis and previously published report, a set 19 antigenic proteins derived from Leishmania donovani were screened for further characterization as vaccine candidates. A total of 49 epitopes were predicted, which revealed a comprehensive binding affinity to the 40 different MHC class I supertypes. Based on the population coverage and HLA cross presentation, nine highly promiscuous epitopes such as LTYDDVWTV (P1), FLFPQRTAL(P2), FLFSNGAVV (P3), YIYNFGIRV (P4), YMTAAFAAL (P5), KLLRPFAPL (P6), FMLGWIVTI (P7), SLFERNKRV (P8), and SVWNRIFTL (P9) which have either a high or an intermediate TAP binding affinity were selected for further analysis. Theoretical population coverage analysis of polytope vaccine (P1-P9) revealed more than 92% population. Stimulation with the cocktail of peptide revealed a proliferative CD8+ T cell response and increased IFN-γ production. An upregulated NF-κB activity is thought to be play a pivotal role in T cell proliferation against the selected peptide. The Th1-type cytokine profile (presence of IFN-γ and absence of IL-10) suggests the potentiality of the cocktail of epitope as a subunit vaccine against leishmaniasis. However, the efficiency of these epitopes to trigger other Th1 cytokines and chemokines in a humanized mice model could explore its plausibility as a vaccine candidate. J. Cell. Biochem. 119: 378-391, 2018. © 2017 Wiley Periodicals, Inc.

Identification of Clinical Immunological Determinants in Asymptomatic VL and Post Kala-azar Dermal Leishmaniasis Patients.

BACKGROUND: Visceral Leishmaniasis (VL) caused by protozoa belonging to the genus Leishmania, usually have anthroponotic mode of transmission and is issue of great public health importance in Indian subcontinent. Asymptomatic cases of VL and PKDL are subject of keen interest to find their role in the transmission of VL in epidemic areas. We evaluated the immunological cytokine determinants expressed in most clinical suspects of asymptomatic VL and PKDL (IL-10, IFN-γ, and TNF-α). METHODS: Eighty-four participants were included at RMRIMS, Patna, India in 2016-17 out of which 64 asymptomatic individual positive for rK-39, without sign and symptoms of VL; 15 PKDL patient's with past history of VL and 5 endemic healthy subjects were recruited from VL endemic areas. DAT and quantitative assessment of plasma cytokines was determined from the blood samples collected in a plain and sodium-EDTA vacutainer respectively from the subjects. RESULTS: The mean level of IL-10 in DATpos LOW of asymptomatic VL and PKDL was significantly higher than endemic healthy (P<0.05). The cytokine polarization index (IFN-γ versus IL-10) was significantly low in PKDL cases compared with asymptomatic VL cases in DATpos LOW titre (P<0.05). This index was low again but statistically not significant in PKDL than in asymptomatic VL when TNF-α was considered against IL-10. The ratio of IFN-γ: IL-10 and TNF-α: IL-10 was observed decreased both in asymptomatic VL and PKDL cases than in healthy from endemic areas. CONCLUSION: Collectively we surmise from our data that asymptomatic VL can also play an important role like PKDL in transmission of VL.

Immuno-informatics based approaches to identify CD8+ T cell epitopes within the Leishmania donovani 3-ectonucleotidase in cured visceral leishmaniasis subjects.

Leishmaniases are vector-borne diseases for which no vaccine exists. These diseases are caused by the Leishmania species complex. Activation of the CD8+ T cell is crucial for protection against intracellular pathogens, and peptide antigens are attractive strategies for the precise activation of CD8+ T in vaccine development against intracellular infections. The traditional approach to mine the epitopes is an arduous task. However, with the advent of immunoinformatics, in silico epitope prediction tools are available to expedite epitope identification. In this study, we employ different immunoinformatics tools to predict CD8+ T cell specific 9 mer epitopes presented by HLA-A*02 and HLA-B40 within the highly conserved 3'-ectonucleotidase of Leishmania donovani. We identify five promiscuous epitopes, which have no homologs in humans, theoretically cover 85% of the world's population and are highly conserved (100%) among Leishmania species. Presentation of selected peptides was confirmed by T2 cell line based HLA-stabilization assay, and three of them were found to be strong binders. The in vitro peptide stimulation of peripheral blood mononuclear cells (PBMC) from cured HLA-A02+ visceral leishmaniasis (VL) subjects produced significantly higher IFN-γ, IL-2 and IL-12 compared to no peptide control healthy subjects. Further, CD8+ cells from treated VL subjects produced significantly higher intracellular IFN-γ, lymphocyte proliferation and cytotoxic activity against selected peptides from the PBMCs of treated HLA-A02+ VL subjects. Thus, the CD8+ T cell specific epitopes shown in this study will speed up the development of polytope vaccines for leishmaniasis.

Adenosine generated by ectonucleotidases modulates the host immune system during visceral leishmaniasis.

Adenosine, an endogenous purine nucleoside is one such extracellular signaling molecule whose role in the regulation of anti-inflammatory cytokines and immune pathogenicity in visceral leishmaniasis is indeterminate. Here, we have evaluated the adenosine in the plasma of 20 visceral leishmaniasis (VL) patients during active disease and after successful treatment. We observed the elevated plasma adenosine during active VL disease (26.73±1.95µM) and the level subsides as the treatment progresses and falls to the normal level after successful treatment (4.32±0.45µM). We demonstrated a direct correlation between changes in the plasma adenosine level and the Th1/Th2 balance in VL patients and it was corroborated with in vitro experiment. Further, we delineated the molecular mechanism involved in the elevation of plasma adenosine during visceral leishmaniasis. Our results reveal that the elevated plasma adenosine level associated with pathogenicity and plays a critical role in skewing immune response from Th1 to Th2 type to influence the outcome of the disease.

Immunization with Leishmania donovani protein disulfide isomerase DNA construct induces Th1 and Th17 dependent immune response and protection against experimental visceral leishmaniasis in Balb/c mice.

In the present study, the efficacy of Leishmania donovani protein disulfide isomerase (LdPDI) as a DNA vaccine was evaluated in BALB/C mice. Mice immunized with the LdPDI-DNA construct were found to be the most immuno-reactive, as the construct induced higher T-cell proliferation. The increased T-cell proliferation was associated with a substantial rise in Th1 and Th17+ CD4 cell response and triggered a higher proportion of CD8+ T cells for the release of interferon-gamma along with a reduced splenic parasite load on Days20 and 60 post challenge (PC). Furthermore, the vaccine construct triggered increased interferon (IFN)-γ, interleukin(IL)-17A, and IL-22 release accompanied by decreased extracellular signal-regulated kinases (ERK) 1/2 signaling and increased mitogen-activated protein kinase (MAPK) signaling coinciding with an increase in the amount of nitrite and reactive oxygen species (ROS)in vaccinating the splenocyts. We summarize from our data that the PDI-DNA construct of Leishmania donovani has the potential to elicit protective immunity through the pro-inflammatory cytokines of CD8+ and CD4+(Th1 and Th17) following an intervention in the downstream signaling event of ERK1/2 (probably through p38MAPK signaling). Therefore, the study suggests a new control against visceral leishmaniasis in the future.

Immunomodulation mediated through Leishmania donovani protein disulfide isomerase by eliciting CD8+ T-cell in cured visceral leishmaniasis subjects and identification of its possible HLA class-1 restricted T-cell epitopes.

Protein disulphide isomerase (PDI) is one of the key enzymes essential for the survival of Leishmania donovani in the host. Our study suggested that PDI is associated with the generation of Th1-type of cellular responses in treated Visceral leishmaniasis (VL) subjects. The stimulation of Peripheral blood mononuclear cells (PBMCs) with recombinant Protein Disulphide Isomerase upregulated the reactive oxygen species generation, Nitric oxide release, IL12 and IFN-γ production indicating its pivotal role in protective immune response. Further, a pre-stimulation of PBMCs with Protein disulphide isomerase induced a strong IFN-γ response through CD8+ T cells in treated VL subjects. These findings also supported through the evidence that this antigen was processed and presented by major histocompatibility complex class I (MHC-1) dependent pathway and had an immunoprophylactic potential which can induce CD8+ T cell protective immune response in MHC class I dependent manner against VL. To find out the possible epitopes that might be responsible for CD8+ T cell specific IFN-γ response, computational approach was adopted. Six novel promiscuous epitopes were predicted to be highly immunogenic and can be presented by 32 different HLA allele to CD8+ T cells. Further investigation will explore more about their immunological relevance and usefulness as vaccine candidates.

Computational prediction and analysis of potential antigenic CTL epitopes in Zika virus: A first step towards vaccine development.

The Zika virus disease is an Aedes mosquito-borne disease caused by the ZIKA virus. The unavailability of vaccines or proper chemotherapeutic treatment emphasizes the need for the development of preventive and therapeutic vaccines. T cell specific epitopes have been used as vaccine candidates to generate desired immune responses against a variety of viral pathogens. Herein, the immune-informatics approach was used for the screening of potential major histocompatibility complex class I restricted epitopes, which may be competent to generate a cell-mediated immune response in humans. A total of 63 epitopes were identified, which revealed a comprehensive binding affinity to the 42 different human leukocyte antigen class I supertypes: A01, A02, A08, A23, A24, A25, A26, A29, A30, A32, A66, A68, A69, A80, B07, B08, B14, B15, B27, B35, B39, B40, B42, B45, B46, B48, B51, B53, B54, B57, B58, B83, C12, C03, C04, C05, C06, C07, C08, C12, C14, and C15, and which had no homologs in humans. By combining the human leukocyte antigen binding specificity and population coverage, nine promiscuous epitopes located in Capsid 1 Protein (MVLAILAFL(P1)), Envelop Protein (RLKGVSYSL (P2) and RLITANPVI (P3)), NS2A (AILAALTPL (P4)), NS4B (LLVAHYMYL (P5) and LVAHYMYLI (P6)) and NS5 (SLINGVVRL (P7), ALNTFTNLV (P8) and YLSTQVRYL (P9)) were shortlisted. Most of these consensus epitopes revealed 100% conservancy in all Zika virus strains and were very less conserved against the human proteome. The combination of the selected epitopes accounted for an optimal coverage in the world wide population (>99%) independent of ethnicity. Structural analysis of these selected epitopes by the PatchDock web server showed their preferential mode of presentation to the T cell receptor. All these results recommended the possibility of a combined epitope vaccine strategy and can therefore be further investigated for their immunological relevance and usefulness as vaccine candidates.

Elevated Serum ADA Activity as a Marker for Diagnosis and Prognosis of Visceral Leishmaniasis and Post Kala-Azar Dermal Leishmaniasis in Indian Patients.

Serum adenosine deaminase (ADA) activity increases in diseases where cellular immunity is involved. Since cell-mediated immune responses play a paramount role in the pathogenesis and healing of the visceral leishmaniasis, therefore, the present study was undertaken to evaluate the serum ADA activity in different pathological conditions. Adenosine deaminase was determined in sera of active visceral leishmaniasis (VL) patients (n = 39), active postkala-azar dermal leishmaniasis (PKDL) cases (n = 34) at the point of diagnosis and after treatment stages along with healthy controls (n = 30), endemic healthy subjects (n = 34) and endemic asymptomatic subjects (n = 34).Our in-vitro result revealed that monocytes secrete significant ADA level in response to Leishmania donovani (L.donovani) stimulation. The serum ADA activity in active VL and PKDL subjects were found to be significantly higher than that of respective treated cases and healthy controls. We also observed a marginal number (17.6%) of endemic asymptomatic subjects showed elevated serum ADA activity. Further, the ADA activity in PKDL was found to be decreased gradually during the different phases of treatment. Interestingly, 2 out of 32 treated VL cases found to have high serum ADA activity during follow up period were relapsed within few days. These results suggest the possibility of ADA as a marker of clinical pathogenesis and can be used as a surrogate marker in the diagnosis and prognosis of VL and PKDL.

Up regulation of A2B adenosine receptor on monocytes are crucially required for immune pathogenicity in Indian patients exposed to Leishmania donovani.

Adenosine, an endogenous purine nucleoside is one such extracellular signalling molecule whose role in regulation of anti-inflammatory cytokines and immune pathogenicity in visceral leishmaniasis is not fully understood. Here, we investigated the relationship between Leishmania donovani infection and expression of A2B receptor on monocytes in VL patients in their pre and post treatment stage. We also investigated the molecular mechanisms influencing the interaction between immunopathogenicity and infection by exposing Leishmania donovani pulsed macrophages to Adenosine. A direct correlation of up-regulated A2B expression on monocytes with increased parasite load was also observed. Our results also suggested that A2B receptor activation is critically required for the stimulatory effect of adenosine on IL-10 production and suppression of nitric oxide release. The stimulatory effect of adenosine on Leishmania donovani induced IL-10 production required ERK1/2 activation and is p-38 MAPK independent.

Computational elucidation of potential antigenic CTL epitopes in Ebola virus.

Cell-mediated immunity is important for the control of Ebola virus infection. We hypothesized that those HLA A0201 and HLA B40 restricted epitopes derived from Ebola virus proteins, would mount a good antigenic response. Here we employed an immunoinformatics approach to identify specific 9mer amino acid which may be capable of inducing a robust cell-mediated immune response in humans. We identified a set of 28 epitopes that had no homologs in humans. Specifically, the epitopes derived from NP, RdRp, GP and VP40 share population coverage of 93.40%, 84.15%, 74.94% and 77.12%, respectively. Based on the other HLA binding specificity and population coverage, seven novel promiscuous epitopes were identified. These 7 promiscuous epitopes from NP, RdRp and GP were found to have world-wide population coverage of more than 95% indicating their potential significance as useful candidates for vaccine design. Epitope conservancy analysis also suggested that most of the peptides are highly conserved (100%) in other virulent Ebola strain (Mayinga-76, Kikwit-95 and Makona-G3816- 2014) and can therefore be further investigated for their immunological relevance and usefulness as vaccine candidates.

Leishmania donovani: impairment of the cellular immune response against recombinant ornithine decarboxylase protein as a possible evasion strategy of Leishmania in visceral leishmaniasis.

Ornithine decarboxylase, the rate limiting enzyme of the polyamine biosynthesis pathway, is significant in the synthesis of trypanothione, T(SH)2, the major reduced thiol which is responsible for the modulation of the immune response and pathogenesis in visceral leishmaniasis. Data on the relationship between ornithine decarboxylase and the cellular immune response in VL patients are limited. Therefore, we purified a recombinant ornithine decarboxylase from Leishmania donovani (r-LdODC) of approximately 77kDa and examined its effects on the immunological responses in peripheral blood mononuclear cells of human visceral leishmaniasis cases. For these studies, α-difluoromethylornithine was tested as an inhibitor and was used in parallel in all experiments. The r-LdODC was identified as having a direct correlation with parasite growth and significantly increased the number of promastigotes as well as axenic amastigotes after 96h of culture. The stimulation of peripheral blood mononuclear cells with r-LdODC up-regulated IL-10 production but not IFN-γ production from CD4(+) T cells in active as well as cured visceral leishmaniasis cases, indicating a pivotal role for r-LdODC in causing strong immune suppression in a susceptible host. In addition, severe hindrance of the immune response and anti-leishmanial macrophage function due to r-LdODC, as revealed by decreased IL-12 and nitric oxide production, and down-regulation in mean fluorescence intensities of reactive oxygen species, occurred in visceral leishmaniasis patients. There was little impact of r-LdODC in the killing of L. donovani amastigotes in macrophages of visceral leishmaniasis patients. In contrast, when cultures of promastigotes and amastigotes, and patients' blood samples, were directed against α-difluoromethylornithine, parasite numbers significantly reduced in culture, whereas the levels of IFN-γ and IL-12, and the production of reactive oxygen species and nitric oxide, were significantly elevated. Therefore, we demonstrated cross-talk with the use of α-difluoromethylornithine which can reduce the activity of ornithine decarboxylase of L. donovani, eliminating the parasite-induced immune suppression and inducing collateral host protective responses in visceral leishmaniasis.

Leishmania donovani phosphoproteins pp41 and pp29 re-establishes host protective immune response in visceral leishmaniasis.

As phospho proteins are reported to be involved in virulence and survival, the ability of Leishmania to inhibit macrophage effector functions may result from a direct interference of leishmanial molecules with macrophage signal transduction pathways. Several such proteins such as pp63, pp41 and pp29 have also been identified as a Th1 stimulatory protein in the Leishmania donovani. In the present study, the immunogenicity of a cocktail of pp63+pp41+pp29 was assessed by estimation of serum antibody titre, nitric oxide(NO) production, estimation of Th1 cytokine(IFN-γ) as well as Th2 cytokines(IL-4), and determination of parasite load in L. donovani infected mice. In the group immunized with antigenic cocktail there was a sharp rise in antibody titer up to Day 20 which reduced considerably by Day 50. Groups of mice vaccinated with pp63, pp41, pp29 and the antigenic cocktail expressed 10-fold, 16-fold, 22-fold and 25-fold increase respectively in NO production by splenocytes. The animal groups immunized with pp63, pp41, pp29 and the antigenic cocktail showed reduced parasite load in the liver and spleen, as well as increased IFN-gamma production in the spleen. Furthermore immunized animals remained with a normal hematological profile, whereas L. donovani in unimmunized mice lead to significant anemia.