Antimony resistance mechanism in genetically different clinical isolates of Indian Kala-azar patients.

The central theme of this enterprise is to find common features, if any, displayed by genetically different antimony (Sb)-resistant viscerotropic Leishmania parasites to impart Sb resistance. In a limited number of clinical isolates (n = 3), we studied the breadth of variation in the following dimensions: (a) intracellular thiol content, (b) cell surface expression of glycan having N-acetyl-D-galactosaminyl residue as the terminal sugar, and (c) gene expression of thiol-synthesizing enzymes (CBS, MST, gamma-GCS, ODC, and TR), antimony-reducing enzymes (TDR and ACR2), and antimonial transporter genes (AQP1, MRPA, and PRP1). One of the isolates, T5, that was genotypically characterized as Leishmania tropica, caused Indian Kala-azar and was phenotypically Sb resistant (T5-LT-SSG-R), while the other two were Leishmania donovani, out of which one isolate, AG83, is antimony sensitive (AG83-LD-SSG-S) and the other isolate, T8, is Sb resistant (T8-LD-SSG-R). Our study showed that the Sb-resistant parasites, regardless of their genotype, showed significantly higher intracellular thiol compared with Sb-sensitive AG83-LD-SSG-S. Seemingly, T5-LT-SSG-R showed about 1.9-fold higher thiol content compared with T8-LD-SSG-R which essentially mirrored cell surface N-acetyl-D-galactosaminyl expression. Except TR, the expression of the remaining thiol-synthesizing genes was significantly higher in T8-LD-SSG-R and T5-LT-SSG-R than the sensitive one, and between the Sb-resistant parasites, the latter showed a significantly higher expression. Furthermore, the genes for Sb-reducing enzymes increased significantly in resistant parasites regardless of genotype compared with the sensitive one, and between two resistant parasites, there was hardly any difference in expression. Out of three antimony transporters, AQP1 was decreased with the concurrent increase in MRPA and PRP1 in resistant isolates when compared with the sensitive counterpart. Interestingly, no difference in expression of the above-mentioned transporters was noted between two Sb-resistant isolates. The enduring image that resonated from our study is that the genetically diverse Sb-resistant parasites showed enhanced thiol-synthesizing and antimony transporter gene expression than the sensitive counterpart to confer a resistant phenotype.

Synthesis, anticancer activity, SAR and binding mode of interaction studies of substituted pentanoic acids: part II.

Aim: Our previous results suggest that phenyl/naphthylacetyl pentanoic acid derivatives may exhibit dual MMP-2 and HDAC8 inhibitory activities and show effective cytotoxic properties. Methodology: Here, 13 new compounds (C1-C13) were synthesized and characterized. Along with these new compounds, 16 previously reported phenyl/napthylacetyl pentanoic acid derivatives (C14-C29) were biologically evaluated. Results: Compounds C6 and C27 showed good cytotoxicity against leukemia cell line Jurkat E6.1. The mechanisms of cytotoxicity of these compounds were confirmed by DNA deformation assay and reactive oxygen species assay. MMP-2 and HDAC8 expression assays suggested the dual inhibiting property of these two compounds. These findings were supported by results of molecular docking studies. In silico pharmacokinetic properties showed compounds C6 and C27 have high gastrointestinal absorption. Conclusion: This study highlights the action of phenyl/naphthylacetyl pentanoic acid derivatives as anticancer agents.

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.

RFLPs of ITS, ITS1 and hsp70 amplicons and sequencing of ITS1 of recent clinical isolates of Kala-azar from India and Bangladesh confirms the association of L. tropica with the disease.

Visceral Leishmaniasis or Kala-azar (KA) is a serious health concern in India. In the present study, Restriction Fragment Length Polymorphism (RFLP) of three genetic markers viz., Internal Transcribed Spacer (ITS), ITS1 and heat shock protein 70 (hsp70) have been employed for typing the clinical isolates [n=15] of KA and post Kala-azar Dermal Leishmaniosis (PKDL) collected from India and Bangladesh in the period of 2006-2010. Experimentally, ITS, ITS1 and hsp70 regions of genomes of all the clinical isolates were separately amplified by PCR and then digested with restriction enzymes: ITS with Alu1, EcoR1 and Msp1, ITS1 with Hae III and Rsa1 and hsp70 with Hae III. The resultant fragments were analyzed by agarose gel electrophoresis and the RFLP profiles of the clinical isolates were compared with that of the WHO reference strains for Leishmania donovani (DD8) and Leishmania tropica (K27), respectively. Also, the ITS1 regions of all the clinical isolates along with the two WHO reference strains were sequenced and a phylogram was constructed to ascertain the extent of similarity or dissimilarity. Interestingly, the RFLP profiles of one of the isolates showed a significant homology with K27 and the phylogram revealed its closeness with the same putting credence to our earlier typing of isolates by RAPD method. This observation also supported an earlier report claiming that both the species are responsible for KA in India and thus, emphasizes urgent need for thorough systematic characterization of the clinical isolates of Indian KA as appropriate treatment regime relies primarily on proper diagnosis.

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.

Characterization of the recent clinical isolates of Indian Kala-azar patients by RAPD-PCR method.

Leishmaniasis is one of the most important vector borne diseases caused by kinetoplastid protozoa Leishmania sp. Among all forms of Leishmaniasis, Visceral leishmaniasis (VL) or Kala-azar is the severest form of the illness. VL is characterized by fever, hepatosplenomegaly, anaemia, edema, weight loss and invariably fatal if left untreated. Characterization of Leishmania sp. is extremely necessary to understand the epidemiology, taxonomy and population genetics of the parasites which ultimately helps in designing appropriate drug regimen to combat the disease. In this study, we aimed to type the clinical isolates of Leishmania species collected in the period 2006-2010 from patients (n = 9) diagnosed with Kala-azar and post Kala-azar dermal leishmaniasis (PKDL) by RAPD-PCR method using eight selected primers. Genome of the clinical isolates were amplified and electrophoresed in agarose gel. These were compared with the RAPD PCR profiles of WHO reference strains for L. donovani (DD8) and L. tropica (K27) respectively. We calculated the Jaccard's Similarity Coefficient and found one (study code T5) out of nine isolates as L. tropica while the rest were L. donovani. This pilot study supports the earlier single report claiming that both the species are responsible for Kala-azar in India and it also emphasizes the need for more systematic typing of clinical isolates of Indian Kala-azar.

Designing therapies against experimental visceral leishmaniasis by modulating the membrane fluidity of antigen-presenting cells.

The membrane fluidity of antigen-presenting cells (APCs) has a significant bearing on T-cell-stimulating ability and is dependent on the cholesterol content of the membrane. The relationship, if any, between membrane fluidity and defective cell-mediated immunity in visceral leishmaniasis has been investigated. Systemic administration of cholesterol by liposome delivery (cholesterol liposomes) in Leishmania donovani-infected hamsters was found to cure the infection. Splenic macrophages as a prototype of APCs in infected hamsters had decreased membrane cholesterol and an inability to drive T cells, which was corrected by cholesterol liposome treatment. The effect was cholesterol specific because liposomes made up of the analogue 4-cholesten-3-one provided almost no protection. Infection led to increases in interleukin-10 (IL-10), transforming growth factor beta, and IL-4 signals and concomitant decreases in gamma interferon (IFN-gamma), tumor necrosis factor alpha, and inducible NO synthase signals, which reverted upon cholesterol liposome treatment. The antileishmanial T-cell repertoire, whose expansion appeared to be associated with protection, was presumably type Th1, as shown by enhanced IFN-gamma signals and the predominance of the immunoglobulin G2 isotype. The protected group produced significantly more reactive oxygen species and NO than the infected groups, which culminated in killing of L. donovani parasites. Therefore, cholesterol liposome treatment may be yet another simple strategy to enhance the cell-mediated immune response to L. donovani infection. To our knowledge, this is the first report on the therapeutic effect of cholesterol liposomes in any form of the disease.