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.

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.

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.

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.

Development of high efficacy peptide coated iron oxide nanoparticles encapsulated amphotericin B drug delivery system against visceral leishmaniasis.

Visceral leishmaniasis, a protozoan disease transmitted by phlebotomine sandflies which affect mostly in Indian sub-continent. The treatment for visceral leishmaniasis (VL) caused by Leishmania donovani are limited and unsatisfactory. Currently available drug against such as miltefosine and polymer based drugs amBisome has high efficacy against VL but found serious side effects and poor absorbance. To overcome this, we developed peptide (glycine) coated iron oxide (Fe3O4) nanoparticles (GINPs) encapsulated amphoterecin B (AmB) drug against visceral leishmaniasis. Synthesis of GINPs was carried out and different characterization technique used to confirm the synthesis and size of GINPs GINPs-AmB showed that particle size in the range of 10-15nm and closed to spherical in shaped. GINPs-AmB showed release rate of AmB is higher at lower pH. Significantly two fold higher efficacies of GINP encapsulated AmB during in vitro study. There was a substantial reduction in the total parasite burden in spleen in treated groups (GINPs encapsulate AmB), compare to AmB alone. The results obtained from this study revealed that AmB loaded GINPs is twofold effective than AmB and therefore, it opens a new avenue for use of AmB loaded GINPs against VL.

Antibiotics as Japanese Encephalitis virus inhibitors: a combinatorial computational approach.

Japanese encephalitis (JE) is a viral neurologic disease of global public health importance. JE is caused by an RNA virus - the Japanese encephalitis virus (JEV) belonging to genus Flavivirus in the family Flaviviridae. JE is endemic in many parts of Asia and western pacific. Annually, approximately 50,000 JE cases are reported with case fatality rates as high as 30-35% resulting in ~15,000 deaths. Presently, there are no successful drugs against JE. Docking of JEV NS3 helicase/NTPase helicase domain with 10 compounds was performed in iGEMDOCK v2.1. Integrated docking, screening, post- analysis and visualization were performed using RasMol software. The drug susceptibility was evaluated by virus yield reduction assay. Three ligands out of 10 antibiotic compounds studied showed highest binding affinity with receptor protein. Kanamycin, Rolitetracycline and Doxycycline showed better binding energy compared to two study standards- Ribavirin and Minocycline. Interacting bonds were formed in all three domains of NS3 helicase/NTPase. The interactions in motifs I, II and VI of helicase are important; these would have possibly inhibited viral replication. Biological assay showed that Kanamycin (Inhibitory concentration50, IC50 - 70 µg/ml), Rolitetracycline (IC50 - 76 µg/ml) and Doxycycline (IC50 - 22 µg/ml) inhibited plaque formation.

Genetic analyses of ribosomal loci of Anopheles minimus species from north east India.

Anopheles minimus is one of the major vectors for transmission of malaria disease in north eastern (NE) region of India. The minimus species complex of Minimus subgroup of Myzomyia series of anophelines were studied in malaria affected states--Assam and Arunachal Pradesh (AP) of NE India. Ribosomal DNA markers--second internal transcribed spacer (ITS2) and third domain (D3) of 28S gene were used to characterize An. minimus species. Sequence homogeneity was observed in D3 sequences of An.minimus specimens throughout both the states. However, a transversion in ITS2 sequence of single specimen collected from Assam-Meghalaya border areas illustrates possibility of intra population polymorphism in ITS2 sequence within the geographical region.

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.

Asymptomatic infection of visceral leishmaniasis in hyperendemic areas of Vaishali district, Bihar, India: a challenge to kala-azar elimination programmes.

A cohort of 91 asymptomatic individuals with visceral leishmaniasis (VL) were identified during base line screening using recombinant 39-aminoacid antigen (rk-39) and polymerase chain reaction (PCR) conducted from December 2005 to June 2006 involving 997 individuals of two highly endemic villages of Vaishali district, Bihar. The point prevalence of asymptomatic infection was 98 per 1000 persons at baseline. There was no statistically significant difference between rk-39 and PCR positivity rate (P>0.05), even though PCR positivity alone was found significantly higher (4.2%) than rk-39 positivity alone (2.6%). The monthly follow-up of the asymptomatic cohort revealed a disease conversion rate of 23.1 per 100 persons within a year. There was a statistically significant difference in conversion of disease when individuals were positive by both tests as compared to single tests by rk-39 and PCR (P<0.01). Disease conversion rate in the subjects residing in households with a history of VL (62%, 13/21) was higher than those residing in the households without a history of VL (38%, 8/21). Most of the identified asymptomatic individuals were from low socio-economic strata similar to that of VL cases in general. Apart from rk-39, PCR may be considered for screening of asymptomatic Leishmania donovani infection in large-scale epidemiological studies. Screening of asymptomatic cases and their close follow-up to ascertain early detection and treatment of VL may be considered in addition to the existing VL control strategies.

Wilson disease with visceral leishmaniasis: an extremely uncommon presentation.

Visceral leishmaniasis (VL), which is caused by the protozoa Leishmania donovani and transmitted by the bite of the female sand fly Phlebotomus argentipes, is common in Bihar, India. Wilson disease is an autosomal recessive disorder of copper metabolism in which copper is deposited in the brain and liver. We report a case of an extremely uncommon combination of these diseases in a patient. Treatment options for such a combination of diseases are limited and difficult.