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.

Visceral leishmaniasis: A novel nuclear envelope protein 'nucleoporins-93 (NUP-93)' from Leishmania donovani prompts macrophage signaling for T-cell activation towards host protective immune response.

The shift of macrophage and T-cell repertoires towards proinflammatory cytokine signalling ensures the generation of host-protective machinery that is otherwise compromised in cases of the intracellular Leishmania parasite. Different groups have attempted to restore host protective immunity. These vaccine candidates showed good responses and protective effects in murine models, but they generally failed during human trials. In the present study, we evaluated the effect of 97 kDa recombinant nucleoporin-93 of Leishmania donovani (rLd-NUP93) on mononuclear cells in healthy and treated visceral leishmaniasis (VL) patients and on THP-1 cell lines. rLd-NUP93 stimulation increased the expression of the early lymphocyte activation marker CD69 on CD4+ and CD8+ T cells. The expression of the host protective pro-inflammatory cytokines IFN-γ, IL-12 and TNF-α was increased, with a corresponding down-regulation of IL-10 and TGF-ß upon rLd-NUP93 stimulation. This immune polarization resulted in the up-regulation of NF-κB p50 with scant expression of SMAD-4. Augmenting lymphocyte proliferation upon priming with rLd-NUP93 ensured its potential for activation and generation of strong T-cell mediated immune responses. This stimulation extended the leishmanicidal activity of macrophages by releasing high amounts of reactive oxygen species (ROS). Further, the leishmanicidal activity of macrophages was intensified by the elevated production of nitric oxide (NO). The fact that this antigen was earlier reported in circulating immune complexes of VL patients highlights its antigenic importance. In addition, in silico analysis suggested the presence of MHC class I and II-restricted epitopes that proficiently trigger CD8+ and CD4+ T-cells, respectively. This study reported that rLd-NUP93 was an effective immunoprophylactic agent that can be explored in future vaccine design.

Iron oxide nanoparticles based antiviral activity of H1N1 influenza A virus.

Influenza virus is a common human pathogenic agent that has caused serious respiratory illness and death over the past century and in recent year. Treatment options against pandemic influenza strain A/H1N1 are very limited and unsatisfactory. Therefore we have developed iron oxide nanoparticles (IO-NPs) with particle size in the range of 10-15 nm against pandemic influenza strain A/H1N1/Eastern India/66/PR8-H1N1. Cell viability and anti-influenza activity was measured by MTT assay, plaque inhibition and quantifying viral transcripts using quantitative real-time PCR with Iron oxide nanoparticles in a dose- and time-dependent manner. 50% cell viability (TD50) was observed at 4.25 pg ± .2 pg of Iron oxide nanoparticles. The percentage of plaque inhibition relative to the infection and the IC50 (50% virus reduction) of PR8-H1N1strain (0.5 moi) were measured in vitro by the plate forming unit (pfu) in MA104 cells. Finding were observed at 01 pg after 72 h. The Antiviral activity determined by change in viral RNA transcripts within 24 h of virus infection by RT-PCR, 08 fold reductions in virus found when treated with Iron oxide nanoparticles Thus; it opens a new avenue for use of IP-NPs against virus infections.

Prevalence, molecular characterization, genetic heterogeneity and antimicrobial resistance of Listeria monocytogenes associated with fish and fishery environment in Kerala, India.

The prevalence of Listeria monocytogenes in the retail fish markets of the Kerala, India was investigated by screening 227 samples comprising of marine finfish (n = 97) shellfish (n = 19), ready-to-cook fish products (n = 47), ready-to-eat fish products (n = 10), dried fish (n = 11) and retail ice (n = 43). The prevalence of L. monocytogenes and L. innocua was 2·7% and 17·2% respectively. Sample category wise, prevalence of L. monocytogenes was higher in marine finfish (1·8%) and retail ice (0·9%). All the L. monocytogenes isolates carried virulent genes namely inlA, inlC, inlJ, hlyA, iap, plcA, prfA genes and majority (82%) belonged to 1/2a, 3a serogroups. L. monocytogenes isolates were multidrug-resistant and showed resistance to ampicillin, penicillin, erythromycin, tetracycline and clindamycin. Enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) delineated 58% genetic heterogeneity among the L. monocytogenes strains. The study reports that genetic similarities of the isolates were interlinked to their serogroup and sample origin. SIGNIFICANCE AND IMPACT OF THE STUDY: Prevalence of Listeria monocytogenes, in the retail fish markets of Kerala, India was low but their relatively higher presence in marine finfish and retail ice and virulent nature of the isolates signifies food safety concerns. Moreover, multidrug-resistant nature of these isolates may potentially lead to spread of antimicrobial resistance. This study identified retail ice as a vehicle for entry of L. monocytogenes in retail fish and hence, there is a need to ensure quality of retail ice used for maintaining the cold-chain.

Magnitude of unreported kala-azar cases in a highly endemic district of Bihar, India: A positive impact of Indian elimination programme.

BACKGROUND & OBJECTIVES: In India, kala-azar surveillance is weak and no public-private partnership exists for disease containment. Estimate of disease burden is not reliably available and still cases are going to private providers for the treatment. The present study aimed to assess the magnitude of kala-azar cases actually detected and managed at private set-up and unreported to existing health management information system. METHODS: Institution based cross-sectional prospective pilot study was conducted. List of facilities was created with the help of key informants. The information about incidence of kala-azar cases were captured on monthly basis from July 2010 to June 2011. Rapid diagnostic strip test (rk-39) or bone marrow/splenic puncture were applied as laboratory methods for the diagnosis of kala-azar. Descriptive statistics as well as chi-square test for comparison between proportions was conducted. RESULTS: Overall availability of private practitioners (PPs) was 4.59/1,00,000 population and maximum PPs (46; 93.9%) were from qualified category. The median years of medical practice was 25 yr (inter quartile-range [18, 28]). Interestingly, only a small proportion (240; 19%) of cases was managed by PPs. Amongst the PPs, only low proportion (32; 18.2%) managed >2 cases per month. The mean number of kala-azar suspects and cases identified varied significantly between different PPs' professions with p <0.048 and p <0.032, respectively. A highly significant difference (p <0.0001) was observed for kala-azar case load between qualified and unqualified practitioners. A small proportion (38; 15.8%) of kala-azar cases was not present in the public health system record. INTERPRETATION & CONCLUSION: Still sizeable proportions of cases are going to PPs and unrecorded into government surveillance system. A mechanism need to be devised to involve at least qualified PPs in order to reduce treatment delay and increase case detection in the region.

Co-factor-independent phosphoglycerate mutase of Leishmania donovani modulates macrophage signalling and promotes T-cell repertoires bearing epitopes for both MHC-I and MHC-II.

Immunoactivation depends upon the antigen potential to modulate T-cell repertoires. The present study has enumerated the effect of 61 kDa recombinant Leishmania donovani co-factor-independent phosphoglycerate mutase (rLd-iPGAM) on mononuclear cells of healthy and treated visceral leishmaniasis subjects as well as on THP-1 cell line. rLd-iPGAM stimulation induced higher expression of interleukin-1ß (IL-1ß) in the phagocytic cell, its receptor and CD69 on T-cell subsets. These cellular activations resulted in upregulation of host-protective cytokines IL-2, IL-12, IL-17, tumour necrosis factor-α and interferon-γ, and downregulation of IL-4, IL-10 and tumour growth factor-ß. This immune polarization was also evidenced by upregulation of nuclear factor-κ light-chain enhancer of activated B cells p50 and regulated expression of suppressor of mother against decapentaplegic protein-4. rLd-iPGAM stimulation also promoted lymphocyte proliferation and boosted the leishmaniacidal activity of macrophages by upregulating reactive oxygen species. It also induced 1·8-fold higher release of nitric oxide (NO) by promoting the transcription of inducible nitric oxide synthase gene. Besides, in silico analysis suggested the presence of major histocompatibility complex class I and II restricted epitopes, which can proficiently trigger CD8+ and CD4+ cells, respectively. This study reports rLd-iPGAM as an effective immunoprophylactic agent, which can be used in future vaccine design.

The Leprosy Post-Exposure Prophylaxis (LPEP) programme: update and interim analysis.

Innovative approaches are required to further enhance leprosy control, reduce the number of people developing leprosy, and curb transmission. Early case detection, contact screening, and chemoprophylaxis currently is the most promising approach to achieve this goal. The Leprosy Post-Exposure Prophylaxis (LPEP) programme generates evidence on the feasibility of integrating contact tracing and single-dose rifampicin (SDR) administration into routine leprosy control activities in different settings. The LPEP programme is implemented within the leprosy control programmes of Brazil, Cambodia, India, Indonesia, Myanmar, Nepal, Sri Lanka and Tanzania. Focus is on three key interventions: tracing the contacts of newly diagnosed leprosy patients; screening the contacts for leprosy; and administering SDR to eligible contacts. Country-specific protocol adaptations refer to contact definition, minimal age for SDR, and staff involved. Central coordination, detailed documentation and rigorous supervision ensure quality evidence. Around 2 years of field work had been completed in seven countries by July 2017. The 5,941 enrolled index patients (89·4% of the registered) identified a total of 123,311 contacts, of which 99·1% were traced and screened. Among them, 406 new leprosy patients were identified (329/100,000), and 10,883 (8·9%) were excluded from SDR for various reasons. Also, 785 contacts (0·7%) refused the prophylactic treatment with SDR. Overall, SDR was administered to 89·0% of the listed contacts. Post-exposure prophylaxis with SDR is safe; can be integrated into the routines of different leprosy control programmes; and is generally well accepted by index patients, their contacts and the health workforce. The programme has also invigorated local leprosy control.

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.

Vaccine potential of HLA-A2 epitopes from Leishmania Cysteine Protease Type III (CPC).

Although the precise host-defence mechanisms are not completely understood, T-cell-mediated immune responses are believed to play a pivotal role in controlling parasite infection. In this study, the potential HLA*A2 restricted peptides were predicted and the ability of peptides to bind HLA-A*02 was confirmed by a MHC stabilization assay. Two of the peptides tested stabilized HLA-A*02: (a) LLATTVSGL (P1) and (b) LMTNGPLEV (P3). The potential of the peptides to generate protective immune response was evaluated in patients with treated visceral leishmaniasis as well as in healthy control subjects. Our data suggest that CD8+ T-cell proliferation against the selected peptide was significantly higher compared to unstimulated culture conditions. The stimulation of peripheral blood mononuclear cells with epitopes individually or as a cocktail upregulated IFN-γ production, which indicates its pivotal role in protective immune response. The IFN-γ production was mainly in a CD8+ T-cells-dependent manner, which suggested that these epitopes had an immunoprophylactic potential in a MHC class I-dependent manner. Moreover, no role of the CD3+ T cell was observed in the IL-10 production against the selected peptides, and no role was found in disease pathogenesis. Further studies on the role of these synthetic peptides may contribute significantly to developing a polytope vaccine idea towards leishmaniasis.

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.

Utility and limitations of multiplex ligation-dependent probe amplification technique in the detection of cytogenetic abnormalities in products of conception.

Background and Introduction: Chromosomal abnormality is found in about half of first-trimester abortions. Karyotype is the gold standard to detect chromosomal abnormalities. Multiplex ligation-dependent probe amplification (MLPA) offers advantage over karyotype in terms of lower failure rate, faster turnaround time, and much higher resolution than conventional karyotyping and found to be 98% concordant with conventional karyotype. AIM: We performed this study to look for the utility of MLPA in diagnosing chromosomal abnormalities in first-trimester abortions. MATERIALS AND METHODS: MLPA using subtelomeric SALSA probe sets (P036 and P070) was used to detect cytogenetic abnormalities in products of conception in missed/spontaneous abortions. RESULTS: A total of ninety abortus samples were analyzed by MLPA. Successful results were provided in (67) 74.4% of the cases while no conclusion could be drawn in 25.6% (23) of the cases. Fifty-five (82.1%) cases were cytogenetically normal and 17.9% (12) had some abnormality. Aneuploidy was detected in 8 (66.7%) cases, 3 (25%) had double-segment imbalance, and one (8.3%) had partial aneuploidy. CONCLUSION: We suggest that MLPA is a good substitute to traditional karyotype.

Leishmania donovani skews the CD56(+) Natural Killer T cell response during human visceral leishmaniasis.

The objective of this study was to understand the categorical function of CD4(+)CD56(+) and CD8(+)CD56(+) NKT cells in human visceral leishmaniasis. These cell populations were significantly deregulated in human peripheral blood during VL. The in vitro experiments showed that CD4(+)NKT cells, but not CD8(+)NKT cells, migrated towards the Leishmania donovani infection site. Additionally, CD4(+)NKT cells from VL subjects primarily expressed CD25(+)Foxp3 and IL-10 compared with healthy subjects. However, CD8(+)NKT cells expressed primarily IFN-γ and killer cell immunoglobulin receptor compared with healthy subjects. Because the ratio of CD4(+) and CD8(+)NKT cells was 1:10, adoptive transfer of CD3(+)CD56(+) NKT cells effectively reduced the L. donovani burden in infected macrophages. This study concludes that although CD4(+)NKT cells are pathogenic and accumulate at the infection site, CD8(+)NKT cells may be protective in contact with target cells.

Evaluation of Leishmania donovani disulfide isomerase as a potential target of cellular immunity against visceral leishmaniasis.

In Leishmania species, protein disulfide isomerase (PDI) - a redox chaperone is primarily associated with virulence and survival. The precise mechanism, especially in relation to redox changes and its effects on immunological responses in visceral leishmaniasis (VL) is not completely understood as yet. Therefore, we purified a recombinant PDI from Leishmania donovani (r-LdPDI) which was of ∼15 kDa molecular size and examined its effects on immunological responses in peripheral blood (PBMC) of human VL cases. For these studies, alanine was tested as an inhibitor and was used in parallel to all experiments. This protein was identified to have a direct correlation with parasite growth which significantly increased number of promastigotes as well as axenic amastigotes after 96 h of culture. Our experiments examining the immunological response against r-LdPDI also indicate the activation of pro-L. donovani dictated immunological responses in VL. The stimulation of PBMC with r-LdPDI induced lactate dehydrogenase (LDH) activities and up regulated interleukin-10 (IL-10) production but not the HLA-DR expression, Nitric oxide (NO) release and IFN-γ production indicating a pivoted role for r-LdPDI in causing a strong immunosuppression in a susceptible host. Further, we observed that an addition of alanine in L. donovani culture offers a significant inhibition in growth of parasite and helps in reconstitution of protective immune response in VL cases. Therefore, we demonstrate a future cross talk on use of alanine which can reduce the activities of PDI of L. donovani, eliminating the parasite induced immunosuppression and inducing collateral host protective response in VL.

Oral miltefosine for Indian post-kala-azar dermal leishmaniasis: a randomised trial.

OBJECTIVE: Standard treatment of Indian post-kala-azar dermal leishmaniasis (PKDL) is unsatisfactory because to achieve therapeutic effectiveness, heroic courses of parenteral and toxic agents have to be administered. Our objective was to evaluate oral miltefosine for its potential to provide effective as well as tolerable treatment for this disease. METHOD: Open-label, randomised, parallel-group multicentric trial. Miltefosine, 100 mg/day to all but one patient, was administered for 12 weeks or 8 weeks, with a target of 18 patients in each treatment group. Key endpoints were tolerance during treatment and efficacy at 12 months of follow-up. RESULTS: The ITT and per-protocol cure rates after 12 months of follow-up for patients receiving 12 weeks of therapy were 78% (14 of 18 patients: 95% CI = 61-88%) and 93% (14 of 15 patients: 95% CI = 71-95%), respectively, after 12 months of follow-up. The ITT and per-protocol cure rates for patients receiving 8 weeks of therapy were 76% (13 of 17 patients: 95% CI = 53-90%) and 81% (13 of 16 patients: 95% CI = 57-93%), respectively. Gastrointestinal and other adverse events were rare. CONCLUSIONS: This study suggests that oral miltefosine for 2-3 months can be considered a treatment of choice for Indian PKDL.

Degree of anemia correlates with increased utilization of heme by Leishmania donovani parasites in visceral leishmaniasis.

Anemia in kala-azar patients is a serious problem. The present study has addressed this problem with the hypothesis that as the Leishmania parasite is completely devoid of heme biosynthetic pathway, therefore the excessive use of heme by the parasites in the human patients can be one of the possible reason of anemia. We investigated that whether, the inability of Leishmania donovani to synthesize heme, can enforce Leishmania parasite to utilize heme derived from host sources in Indian KA patients. Patients had higher tendency of their macrophages to bind with Hb which was pronounced after sensitization with drug resistant Leishmania strain compared to sensitive.

Fatal acute pancreatitis in a patient with visceral leishmaniasis during miltefosine treatment.

Pancreatitis is a known side effect of the once commonly used drug, sodium stibogluconate, for treatment of visceral leishmaniasis (VL). In India, miltefosine has recently been introduced as the first-line drug. Its side effects include loose motions, vomiting, and teratogenicity. We report here a case of a 41-year-old parasitologically confirmed male case of VL, who developed acute pancreatitis during treatment with miltefosine. On the 13 th day of treatment, he presented with abdominal pain and vomiting. The biochemical, hematological, and radiological features were suggestive of acute pancreatitis. The patient was put on conservative treatment for pancreatitis at the specialized center but succumbed to renal failure and septicaemia.

Development of post-kala-azar dermal leishmaniasis in AmBisome treated visceral leishmaniasis: a possible challenge to elimination program in India.

We report two cases, one male (33 years) and a female (14 years), that developed Post-Kala-azar Dermal Leishmaniasis (PKDL) after successful treatment for visceral leishmaniasis (VL) or Kala-azar with AmBisome, the lipid complex of Amphotericin B. Both cases presented with hypo-pigmented macular lesions all over the body. The patients responded well to AmBisome after treatment with three courses. This first ever case report from India indicates that possibly there is no effective drug for VL until date, which can prevent post-treatment development of PKDL.

Post-kala-azar dermal leishmaniasis in a patient treated with injectable paromomycin for visceral leishmaniasis in India.

Post kala-azar dermal leishmaniasis (PKDL) is a skin manifestation that usually develops after treatment of visceral leishmaniasis (VL), a major public health problem in India. The diagnosis and management of PKDL is complex. This is the first case report from India in which PKDL occurred after paromomycin treatment for VL in an Indian patient.

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 3'UTR 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.