Saraca asoca seed extract treatment recovers the trace elements imbalances in experimental murine visceral leishmaniasis.

Saraca asoca is an important plant species of India having variety of medicinal activity such as antiviral, anti-diabetic, antimicrobial, anti-inflammatory, anti-cancer etc. Indian Kala-azar (KA) or visceral leishmaniasis (VL) is a protozoan parasitic disease caused by Leishmania sp and is endemic in Indian subcontinent. VL mainly targets the poorest people who have been suffering from deficiency in protein, nutrients and essential trace elements which ultimately leads to immunodeficiency. Essential trace element, Zinc (Zn) controls multiple aspects of innate and adaptive immunity while Iron (Fe) is required for various cellular activities. Bromine (Br) is important for assembly of collagen IV scaffolds in tissue development and helps in signalling and Copper (Cu) performs several functions related to immune system. Intra-cardiac blood was collected from the experimental BALB/c mice groups including (a) healthy control, (b) infected control, (c) Saraca asoca seed extract (Sa-SE) treated groups. The trace elements level in blood of mice was measured by Energy Dispersive X-Ray Fluorescence technique. Interestingly, the decreased level of Zn, Fe and Br as well as increased level of Cu in diseased state came back to almost normal range upon treatment with Sa-SE. The trace elements imbalances thus were almost restored to normalcy by treating the experimental BALB/c mice with ethanolic seed extract of Saraca asoca.

Genetic markers for antimony resistant clinical isolates differentiation from Indian Kala-azar.

Visceral Leishmaniasis or Kala-azar is caused by the protozoan parasites belonging to the Genus Leishmania. Once thought eradicated from the Indian subcontinent, the disease came back with drug resistance to almost all prevalent drugs. Molecular epidemiological studies revealed the polymorphic nature of the population of the main player of the disease, Leishmania donovani and involvement of other species (L. tropica) and other genus (Leptomonas) with the disease. This makes control measures almost futile. It also strongly demands the characterization of each and every isolate mandatory which is not done. In this background, the present study has been carried out to assess the genetic attributes of each clinical isolates (n=26) of KA and PKDL patients from India and Bangladesh. All the isolates were characterized through Restriction Fragment Length Polymorphism (RFLP) analysis to ascertain their species identity. 46.2% of the isolates were found to be Sodium Stibogluconate (SSG) resistant by amastigote-macrophage model. When the clinical isolates were subjected to Single Stranded Conformation Polymorphism (SSCP) of Internal Transcribed Spacer 1 (ITS1), Internal Transcribed Spacer 2 (ITS2) and some anonymous markers, the drug resistant Leishmania isolates of SSG can be distinguished from the sensitive isolates distinctly. This study showed for the first time, the genetic markers for SSG drug resistance of Indian Kala-azar clinical isolates.

Evaluation of s.c. route of immunization by homologous radio attenuated live vaccine in experimental murine model of visceral leishmaniasis.

Our previous studies in BALB/c mice showed substantial protection against the experimental murine visceral leishmaniasis (MVL) when the animals were immunized with γ-irradiated live Leishmania donovani parasites through intra peritoneal (i.p.) and intra muscular (i.m.) routes respectively. The observations encouraged us to check the prophylactic efficacy of subcutaneous (s.c.) route as it is better alternative for human trial. The mice immunized with two subsequent doses of the radio attenuated homologous vaccine were challenged with virulent L. donovani parasites. Seventy-five days post infection, the animals were sacrificed. The extent of protection against the disease was evaluated by assessing the reduction of parasite burden in spleen and liver, the generation of free radicals (NO & ROS) and release of the cytokines from T-lymphocyte helper 1 (Th 1) and T-lymphocyte helper 2 (Th 2) along with the measurement of the serum immunoglobulins. The reductions in parasitic burden were observed up to 21 and 24 % in spleen and liver of the immunized groups with NO and ROS productions 27 and 34 % respectively. Whereas the increase in IFN gamma releases was between 19 and 34 %, the decrease in IL-10 release was not more than 22 %. This indicates the failure of the establishment of pronounced Th1 ambience which was further corroborated by the observed IgG2a and IgG1 ratio. The present study when compared with our previous observations with i.m. and i.p. routes revealed that s.c. route may not be a good choice for the use of radio attenuated vaccine.

Therapy with radio-attenuated vaccine in experimental murine visceral leishmaniasis showed enhanced T cell and inducible nitric oxide synthase levels, suppressed tumor growth factor-beta production with higher expression of some signaling molecules

Background: Visceral leishmaniasis (VL) or Kala-Azar (KA) is one of the most deadly forms of disease among all neglected tropical diseases. There are no satisfactory drugs or vaccine candidates available for this dreaded disease. Our previous studies showed promising therapeutic and prophylactic efficacy of the live, radio-attenuated parasites through intramuscular (I.M.) and intraperitoneal (I.P.) route in BALB/c mice model. Methods: The T-cell proliferation level, the mRNA expression level of inducible nitric oxide synthase (iNOS) and tumor growth factor-beta (TGF-β) genes and finally the phosphorylation levels of phosphoinositide dependent kinase 1 (PDK1), phosphoinositide 3 kinase (PI3K) and p38 mitogen activated protein kinase (p38MAPK) molecules were checked in BALB/c mice model immunized with radio-attenuated Leishmania donovani parasites through I.M. route. Results: Higher T-cell proliferation, increased iNOS level, and suppressed TGF-β level were found in treated infected animal groups (100 and 150 Gy) in relation to untreated infected animals. Likewise, phosphorylation levels of PDK1, PI3K and p38MAPK of these two groups were increased when compared to untreated infected controls. Conclusion: The clearance of the parasites from treated infected groups of animals may be mediated by the restoration of T-cell due to therapy with radio-attenuated L. donovani parasites. The killing of parasites was mediated by increase in nitric oxide release through PDK1, PI3K and p38MAPK signaling pathways. A lower TGF-β expression has augmented the restored Th1 ambience in the 100 and 150 Gy treated animal groups proving further the efficacy of the candidate vaccine. .

Therapy with radio-attenuated vaccine in experimental murine visceral leishmaniasis showed enhanced T cell and inducible nitric oxide synthase levels, suppressed tumor growth factor-beta production with higher expression of some signaling molecules.

BACKGROUND: Visceral leishmaniasis (VL) or Kala-Azar (KA) is one of the most deadly forms of disease among all neglected tropical diseases. There are no satisfactory drugs or vaccine candidates available for this dreaded disease. Our previous studies showed promising therapeutic and prophylactic efficacy of the live, radio-attenuated parasites through intramuscular (I.M.) and intraperitoneal (I.P.) route in BALB/c mice model. METHODS: The T-cell proliferation level, the mRNA expression level of inducible nitric oxide synthase (iNOS) and tumor growth factor-beta (TGF-ß) genes and finally the phosphorylation levels of phosphoinositide dependent kinase 1 (PDK1), phosphoinositide 3 kinase (PI3K) and p38 mitogen activated protein kinase (p38MAPK) molecules were checked in BALB/c mice model immunized with radio-attenuated Leishmania donovani parasites through I.M. route. RESULTS: Higher T-cell proliferation, increased iNOS level, and suppressed TGF-ß level were found in treated infected animal groups (100 and 150Gy) in relation to untreated infected animals. Likewise, phosphorylation levels of PDK1, PI3K and p38MAPK of these two groups were increased when compared to untreated infected controls. CONCLUSION: The clearance of the parasites from treated infected groups of animals may be mediated by the restoration of T-cell due to therapy with radio-attenuated L. donovani parasites. The killing of parasites was mediated by increase in nitric oxide release through PDK1, PI3K and p38MAPK signaling pathways. A lower TGF-ß expression has augmented the restored Th1 ambience in the 100 and 150Gy treated animal groups proving further the efficacy of the candidate vaccine.

Therapeutic immunization with radio-attenuated Leishmania parasites through i.m. route revealed protection against the experimental murine visceral leishmaniasis.

After our promising results from prophylactic and therapeutic study (i.p. route) with the radio-attenuated Leishmania donovani parasites against experimental murine visceral leishmaniasis, we prompted to check their therapeutic efficacy through i.m route. BALB/c mice were infected with highly virulent L. donovani parasites. After 75 days, mice were treated with gamma (γ)-irradiated parasites. A second therapeutic immunization was given after 15 days of first immunization. The protection against kala-azar was estimated with the reduction of Leishman-Donovan unit from spleen and liver that scored up to 80% and 93%, respectively, while a twofold increase in nitric oxide (NO) and reactive oxygen species (ROS) productions has been observed in the immunized groups of animals. These groups of mice also showed disease regression by skewing Th2 cytokines (IL-10) towards Th1 cytokine (IFN-γ) bias along with the increased generation of NO and ROS, while the infected control group of mice without such treatment surrendered to the disease. Establishment of Th1 ambience in the treated groups has also been supported from the measured antileishmanial antibody IgG subsets (IgG2a and IgG1) with higher anti-soluble Leishmania antigen-specific IgG2a titer. As seen in our previous studies, doses of attenuation by γ-radiation should be taken into serious consideration. Attenuation of parasites at 50 Gy of absorbed dose of gamma rays has not worked well. Thus, therapeutic use of L. donovani parasites radio-attenuated at particular doses can be exploited as a promising vaccine agent. Absence of any adjuvant may increase its acceptability as vaccine candidate further.

Radio-attenuated leishmanial parasites as immunoprophylactic agent against experimental murine visceral leishmaniasis.

The present study intends to evaluate the role of radio-attenuated leishmania parasites as immunoprophylactic agents for experimental murine visceral leishmaniasis. BALB/c mice were immunized with gamma (γ)-irradiated Leishmania donovani. A second immunization was given after 15 days of first immunization. After two immunizations, mice were infected with virulent L. donovani promastigotes. Protection against Kala-azar (KA) was estimated from spleen and liver parasitic burden along with the measurement of nitrite and superoxide anion generation by isolation of splenocytes and also by T-lymphocyte helper 1(Th1) and T-lymphocyte helper 2(Th2) cytokines release from the experimental groups. It was observed that BALB/c mice having prior immunization with radio-attenuated parasites showed protection against L. donovani infection through higher expression of Th1 cytokines and suppression of Th2 cytokines along with the generation of protective free radicals. The group of mice without prior priming with radio-attenuated parasites surrendered to the disease. Thus it can be concluded that radio-attenuated L. donovani may be used for.

The molecular characterization of clinical isolates from Indian Kala-azar patients by MLEE and RAPD-PCR.

BACKGROUND: Kala-azar is a serious health problem in India. The situation has worsened further due to the occurrence of cases unresponsive to antimonials. About 30-50% patients do not respond to the prevailing regimen of antimonials. The etiological agent for Indian kala-azar has long been known to be Leishmania donovani. Recently, in a somewhat startling report, it was claimed that L. Tropica causes nearly 25% of current kala-azar cases in India. It was also suggested that this might be in some way related to the unresponsiveness to pentavalent antimonials in the field. MATERIAL/METHODS: Two independent molecular characterization techniques, multilocus enzyme electrophoresis (MLEE) and RAPD-PCR, were employed to analyze 15 clinical isolates from confirmed Indian kala-azar patients collected from the eastern part of the country over a period of nearly 20 years. The collection included six Sb5+-unresponsive and one PKDL case. RESULTS: Our observations strongly suggest that all the clinical isolates, including the antimony Sb5+-unresponsive and PKDL ones, we studied were identical to the WHO reference strain (DD8) for Leishmania donovani by both the above methods and no strain variation might have occurred in two major epidemic and inter-epidemic periods. We also observed that none of the Sb5+-unresponsive stains we analyzed was related to L. Tropica. CONCLUSIONS: We conclude that L. Donovani may be the causal agent for Indian kala-azar and that L. Tropica is most likely not an etiological agent for Indian Kala-azar cases that are unresponsive to antimonials.