Autologous neutralizing antibody responses after antiretroviral therapy in acute and early HIV-1.

BACKGROUNDEarly antiretroviral therapy initiation (ARTi) in HIV-1 restricts reservoir size and diversity while preserving immune function, potentially improving opportunities for immunotherapeutic cure strategies. For antibody-based cure approaches, the development of autologous neutralizing antibodies (anAbs) after acute/early ARTi is relevant but is poorly understood.METHODSWe characterized antibody responses in a cohort of 23 participants following ARTi in acute HIV (<60 days after acquisition) and early HIV (60-128 days after acquisition).RESULTSPlasma virus sequences at the time of ARTi revealed evidence of escape from anAbs after early, but not acute, ARTi. HIV-1 envelopes representing the transmitted/founder virus(es) (acute ARTi) or escape variants (early ARTi) were tested for sensitivity to longitudinal plasma IgG. After acute ARTi, no anAb responses developed over months to years of suppressive ART. In 2 of the 3 acute ARTi participants who experienced viremia after ARTi, however, anAbs arose shortly thereafter. After early ARTi, anAbs targeting those early variants developed between 12 and 42 weeks of ART and continued to increase in breadth and potency thereafter.CONCLUSIONResults indicate a threshold of virus replication (~60 days) required to induce anAbs, after which they continue to expand on suppressive ART to better target the range of reservoir variants.TRIAL REGISTRATIONClinicalTrials.gov NCT02656511.FUNDINGNIH grants U01AI169767, R01AI162646, UM1AI164570, UM1AI164560, U19AI096109, K23GM112526, T32AI118684, P30AI045008, P30AI027763, R24AI067039; Gilead Sciences grant INUS2361354; Viiv Healthcare grant A126326.

Lymphoid tissues contribute to plasma viral clonotypes early after antiretroviral therapy interruption in SIV-infected rhesus macaques.

The rebound-competent viral reservoir, composed of a virus that is able to persist during antiretroviral therapy (ART) and mediate reactivation of systemic viral replication and rebound viremia after ART interruption (ATI), remains the biggest obstacle to treating HIV infection. A better understanding of the cellular and tissue origins and the dynamics of viral populations that initiate rebound upon ATI could help develop therapeutic strategies for reducing the rebound-competent viral reservoir. In this study, barcoded simian immunodeficiency virus (SIV), SIVmac239M, was used to infect rhesus macaques to enable monitoring of viral barcode clonotypes contributing to virus detectable in plasma after ATI. Blood and tissues from secondary lymphoid organs (spleen, mesenteric lymph nodes, and inguinal lymph nodes) and from the colon, ileum, lung, liver, and brain were analyzed using viral barcode sequencing, intact proviral DNA assay, single-cell RNA sequencing, and combined CODEX and RNAscope in situ hybridization. Four of seven animals had viral barcodes detectable by deep sequencing of plasma at necropsy, although plasma viral RNA remained below 22 copies per milliliter. Among the tissues studied, mesenteric lymph nodes, inguinal lymph nodes, and spleen contained viral barcodes detected in plasma. CD4+ T cells were the main cell type harboring viral RNA after ATI. Furthermore, T cell zones in lymphoid tissues showed higher viral RNA abundance than B cell zones for most animals. These findings are consistent with lymphoid tissues contributing to the virus present in plasma early after ATI.

Transmitted/founder SHIV.D replicates in the brain, causes neuropathogenesis, and persists on combination antiretroviral therapy in rhesus macaques.

A biologically relevant non-human primate (NHP) model of HIV persistence in the central nervous system (CNS) is necessary. Most current NHP/SIV models of HIV infection fail to recapitulate viral persistence in the CNS without encephalitis or fail to employ viruses that authentically represent the ongoing HIV-1 pandemic. Here, we demonstrate viral replication in the brain and neuropathogenesis after combination antiretroviral therapy (ART) in rhesus macaques (RMs) using novel macrophage-tropic transmitted/founder (TF) simian-human immunodeficiency virus SHIV.D.191,859 (SHIV.D). Quantitative immunohistochemistry (IHC) and DNA/RNAscope in situ hybridization (ISH) were performed on three brain regions from six SHIV.D-infected RMs; two necropsied while viremic, two during analytical treatment interruptions, and two on suppressive ART. We demonstrated myeloid-mediated neuroinflammation, viral replication, and proviral DNA in the brain in all animals. These results demonstrate that TF SHIV.D models native HIV-1 CNS replication, pathogenesis, and persistence on ART in rhesus macaques.

Lymphoid tissues contribute to viral clonotypes present in plasma at early post-ATI in SIV-infected rhesus macaques.

The rebound-competent viral reservoir (RCVR), comprised of virus that is able to persist during antiretroviral therapy (ART) and mediate reactivation of systemic viral replication and rebound viremia after antiretroviral therapy interruption (ATI), remains the biggest obstacle to the eradication of HIV infection. A better understanding of the cellular and tissue origins and the dynamics of viral populations that initiate rebound upon ATI could help develop targeted therapeutic strategies for reducing the RCVR. In this study, barcoded SIVmac239M was used to infect rhesus macaques to enable monitoring of viral barcode clonotypes contributing to virus detectable in plasma after ATI. Blood, lymphoid tissues (LTs, spleen, mesenteric and inguinal lymph nodes), and non-lymphoid tissues (NLTs, colon, ileum, lung, liver, and brain) were analyzed using viral barcode sequencing, intact proviral DNA assay, single-cell RNA sequencing, and combined CODEX/RNAscope/ in situ hybridization. Four of seven animals had viral barcodes detectable by deep sequencing of plasma at necropsy although plasma viral RNA remained < 22 copies/mL. Among the tissues studied, mesenteric and inguinal lymph nodes, and spleen contained viral barcodes detected in plasma, and trended to have higher cell-associated viral loads, higher intact provirus levels, and greater diversity of viral barcodes. CD4+ T cells were the main cell type harboring viral RNA (vRNA) after ATI. Further, T cell zones in LTs showed higher vRNA levels than B cell zones for most animals. These findings are consistent with LTs contributing to virus present in plasma early after ATI. One Sentence Summary: The reemerging of SIV clonotypes at early post-ATI are likely from the secondary lymphoid tissues.

Adaptation of a transmitted/founder simian-human immunodeficiency virus for enhanced replication in rhesus macaques.

Transmitted/founder (TF) simian-human immunodeficiency viruses (SHIVs) express HIV-1 envelopes modified at position 375 to efficiently infect rhesus macaques while preserving authentic HIV-1 Env biology. SHIV.C.CH505 is an extensively characterized virus encoding the TF HIV-1 Env CH505 mutated at position 375 shown to recapitulate key features of HIV-1 immunobiology, including CCR5-tropism, a tier 2 neutralization profile, reproducible early viral kinetics, and authentic immune responses. SHIV.C.CH505 is used frequently in nonhuman primate studies of HIV, but viral loads after months of infection are variable and typically lower than those in people living with HIV. We hypothesized that additional mutations besides Δ375 might further enhance virus fitness without compromising essential components of CH505 Env biology. From sequence analysis of SHIV.C.CH505-infected macaques across multiple experiments, we identified a signature of envelope mutations associated with higher viremia. We then used short-term in vivo mutational selection and competition to identify a minimally adapted SHIV.C.CH505 with just five amino acid changes that substantially improve virus replication fitness in macaques. Next, we validated the performance of the adapted SHIV in vitro and in vivo and identified the mechanistic contributions of selected mutations. In vitro, the adapted SHIV shows improved virus entry, enhanced replication on primary rhesus cells, and preserved neutralization profiles. In vivo, the minimally adapted virus rapidly outcompetes the parental SHIV with an estimated growth advantage of 0.14 days-1 and persists through suppressive antiretroviral therapy to rebound at treatment interruption. Here, we report the successful generation of a well-characterized, minimally adapted virus, termed SHIV.C.CH505.v2, with enhanced replication fitness and preserved native Env properties that can serve as a new reagent for NHP studies of HIV-1 transmission, pathogenesis, and cure.

Potential Anti-leishmanial Activity of a Semi-purified Fraction Isolated from the Leaves of Parthenium hysterophorus.

PURPOSE: In the present perspective, emergence of resistant strains of Leishmania donovani and severe side effects resulting from the use of conventional anti-leishmanial therapies present an urgent need for developing novel agents against this parasite. We have explored the effectiveness of secondary plant metabolites as alternative choices in the treatment for visceral leishmaniasis (vl). METHODS: The plant Parthenium hysterophorus L. (Asteraceae) was collected from the West Bengal State University Campus, Barasat, West Bengal, India. The leaves of this plant were extracted by different solvents, such as ethyl acetate, water, petroleum ether and hexane. Gas chromatography-mass spectrometry (GC-MS) analysis was also carried out for the identification of compounds in the hexane soluble fraction (PHFd) with substantial anti-leishmanial activities. The antipromastigote activity and cytotoxicity of this fraction were evaluated by the tetrazolium MTT assay. Other biochemical and physiological parameters were studied by microscopic observation and flow cytometric analyses. RESULTS: PHFd showed considerable activity against L. donovani promastigotes (IC50: 20 µg/ml). The PHFd also inhibited in vitro growth of L. major LV39 promastigotes dose dependently with an IC50 of 40 µg/ml. The GC-MS studies of this particular fraction revealed the presence of four major compounds with different retention times (RT) of 26.08, 33.11, 36.41, and 41.20 min. In this study, we also established that PHFd could induce DNA damage and subsequent apoptosis of L. donovani promastigotes with a concomitant increase in generations of reactive oxygen species (ROS) in a time-dependent manner. This fraction was also found to be effective in nitric oxide-mediated inhibition of intracellular amastigotes (IC50:12.5 µg/ml) without any noticeable cytotoxicity towards murine splenocytes in vitro. CONCLUSION: This study provides the basis for additional phytochemical and pharmacological studies on the antiprotozoal applications of P. hysterophorus.

Valosin-containing protein/p97 plays critical roles in the Japanese encephalitis virus life cycle.

Host factors provide critical support for every aspect of the virus life cycle. We recently identified the valosin-containing protein (VCP)/p97, an abundant cellular ATPase with diverse cellular functions, as a host factor important for Japanese encephalitis virus (JEV) replication. In cultured cells, using siRNA-mediated protein depletion and pharmacological inhibitors, we show that VCP is crucial for replication of three flaviviruses: JEV, Dengue, and West Nile viruses. An FDA-approved VCP inhibitor, CB-5083, extended survival of mice in the animal model of JEV infection. While VCP depletion did not inhibit JEV attachment on cells, it delayed capsid degradation, potentially through the entrapment of the endocytosed virus in clathrin-coated vesicles (CCVs). Early during infection, VCP-depleted cells showed an increased colocalization of JEV capsid with clathrin, and also higher viral RNA levels in purified CCVs. We show that VCP interacts with the JEV nonstructural protein NS5 and is an essential component of the virus replication complex. The depletion of the major VCP cofactor UFD-1 also significantly inhibited JEV replication. Mechanistically, thus, VCP affected two crucial steps of the JEV life cycle - nucleocapsid release and RNA replication. Our study establishes VCP as a common host factor with a broad antiviral potential against flaviviruses.ImportanceJEV is the leading cause of viral encephalitis epidemics in South-east Asia, affecting majorly children with high morbidity and mortality. Identification of host factors is thus essential for the rational design of anti-virals that are urgently need as therapeutics. Here we have identified the VCP protein as one such host-factor. This protein is highly abundant in cells and engages in diverse functions and cellular pathways by its ability to interact with different co-factors. Using siRNA mediated protein knockdown, we show that this protein is essential for release of the viral RNA into the cell so that it can initiate replication. The protein plays a second crucial role for the formation of the JEV replication complex. FDA-approved drugs targeting VCP show enhanced mouse survival in JE model of disease, suggesting that this could be a druggable target for flavivirus infections.

Atorvastatin ameliorates viral burden and neural stem/ progenitor cell (NSPC) death in an experimental model of Japanese encephalitis.

Japanese encephalitis virus, a neurotropic flavivirus, causes sporadic encephalitis with nearly 25% fatal case reports. JEV infects neural stem/progenitor cells (NSPCs) and decreases their proliferation. Statin, a commonly used class of cholesterol lowering drug, has been shown to possess potent anti-inflammatory and neuroprotective effects in acute brain injury and chronic neurodegenerative conditions. Here, we aimed to check the efficacy of atorvastatin in alleviating the symptoms of Japanese encephalitis (JE). Using BALB/c mouse model of JEV infection, we observed that atorvastatin effectively reduces viral load in the subventricular zone (SVZ) of infected pups and decreases the resultant cell death. Furthermore, atorvastatin abrogates microglial activation and production of proinflammatory cyto/chemokine production post JEV infection in vivo. It also reduced interferon-ß response in the neurogenic environs. The neuroprotective role of atorvastatin is again evident from the rescued neurosphere size and decreased cell death in vitro. It has also been observed that upon atorvastatin administration, cell cycle regulatory proteins and cell survival proteins are also restored to their respective expression level as observed in uninfected animals. Thus the antiviral, immunomodulatory and neuroprotective roles of atorvastatin reflect in our experimental observations. Therefore, this drug broadens a path for future therapeutic measures against JEV infection.

Combination of Mycobacterium indicus pranii and Heat-Induced Promastigotes Cures Drug-Resistant Leishmania Infection: Critical Role of Interleukin-6-Producing Classical Dendritic Cells.

The major issues in available therapeutic modalities against leishmaniasis are cost, toxicity, and the emergence of drug resistance. The aim of this work was to develop a successful therapeutic adjuvant against drug-resistant Leishmania donovani infection by means of combining Mycobacterium indicus pranii with heat-induced promastigotes (HIP). One-month postinfected BALB/c mice were administered subcutaneously with M. indicus pranii (108 cells) and HIP (100 µg) for 5 days. Spleens were harvested for flow cytometric and reverse transcriptase PCR analysis. The antileishmanial effect of the combination strategy was associated with induction of a disease-resolving Th1 and Th17 response with simultaneous downregulation of CD4+ CD25+ Foxp3+ (nTreg) cells and CD4+ CD25- Foxp3- (Tr1) cells in the spleen. The significant expansion of CD4+ TCM (CD4+ CD44hi CD11ahi CD62Lhi) cells was a further interesting outcome of this therapeutic strategy in the context of long-term protection of hosts against secondary infection. Toll-like receptor 2 (TLR2) was also found instrumental in this antiparasitic therapy. Induced interleukin-6 (IL-6) production from expanded CD11c+ CD8α+ (cDC1) and CD11c+ CD11b+ (cDC2) dendritic cells (DCs) but not from the CD11b+ Ly6c+ inflammatory monocytes (iMOs), was found critical in the protective expansion of Th17 as evidenced by an in vivo IL-6 neutralization assay. It also promoted the hematopoietic conversion toward DC progenitors (pre-DCs) from common dendritic cell progenitors (CDPs), the immediate precursors, in bone marrow. This novel combinational strategy demonstrated that expansion of Th17 by IL-6 released from CD11c+ classical DCs is crucial, together with the conventional Th1 response, to control drug-resistant infection.

Selective in vitro inhibition of Leishmania donovani by a semi-purified fraction of wild mushroom Grifola frondosa.

The current study was designed to assess the anti-leishmanial effect of a semi-purified fraction of wild mushroom Grifola frondosa against Leishmania donovani, in vitro. A total of five extracts from three wild mushrooms [Grifola frondosa (family, Meripilaceae) Laetiporus sulphurous (family, Polyporaceae) and Meripilus giganteus (family, Meripilaceae) were explored for novel anti-leishmanial leads against promastigotes. The ethanol extract of G. frondosa was selected as the most efficient against L. donovani promastigotes (IC50: 93.9 µg/mL). A semi-purified fraction was obtained from an active ethanol extract of G. frondosa and found to inhibit the survival of promastigotes of L. donovani (MHOM/IN/83/AG83) significantly (IC50: 20.37 µg/mL) and it also had some effect against L. major LV39 (MRHO/Sv/59/P strain) and L. tropica WR683 (MHOM/SU/58/OD) strains at higher concentrations (IC50: 46.08 µg/mL and 53.79 µg/mL respectively). The semi-purified fraction also interfered in lipid biosynthesis, altered parasite morphology and induced apoptosis in L. donovani promastigotes. The semi-purified fraction was also effective against intracellular amastigotes in infected macrophages and enhanced the release of nitric oxide and pro-inflammatory cytokines, in vitro. Interestingly, the 50% inhibitory concentration of the semi-purified fraction against the intracellular amastigotes (IC50: 2.48 µg/mL) was much lower in comparison to promastigotes (IC50: 20.37 µg/mL). The semi-purified fraction was found to inhibit the intracellular amastigotes slightly more efficiently in comparison to conventional anti-leishmanial drugs; sodium antimony gluconate, amphotericin B, miltefosine and paromomycin and noticeably non-toxic towards host splenocytes. The findings of the present study established that G. frondosa might be a natural resource for development of a new anti-leishmanial lead.

Successful Therapy of Murine Visceral Leishmaniasis with Astrakurkurone, a Triterpene Isolated from the Mushroom Astraeus hygrometricus, Involves the Induction of Protective Cell-Mediated Immunity and TLR9.

In our previous report, we showed that astrakurkurone, a triterpene isolated from the Indian mushroom Astraeus hygrometricus (Pers.) Morgan, induced reactive oxygen species, leading to apoptosis in Leishmania donovani promastigotes, and also was effective in inhibiting intracellular amastigotes at the 50% inhibitory concentration of 2.5 µg/ml. The aim of the present study is to characterize the associated immunomodulatory potentials and cellular activation provided by astrakurkurone, leading to effective antileishmanial activity in vitro and in vivo Astrakurkurone-mediated antileishmanial activity was evaluated by real-time PCR and flow cytometry. The involvement of Toll-like receptor 9 (TLR9) was studied by in vitro assay in the presence of a TLR9 agonist and antagonist and by in silico modeling of a three-dimensional structure of the ectodomain of TLR9 and its interaction with astrakurkurone. Astrakurkurone caused a significant increase in TLR9 expression of L. donovani-infected macrophages along with the activation of proinflammatory responses. The involvement of TLR9 in astrakurkurone-mediated amastigote killing has been evidenced from the fact that a TLR9 agonist (CpG, ODN 1826) in combination with astrakurkurone enhanced the amastigote killing, while a TLR9 antagonist (bafilomycin A1) alone or in combination with astrakurkurone curbed the amastigote killing, which could be further justified by in silico evidence of docking between mouse TLR9 and astrakurkurone. Astrakurkurone was found to reduce the parasite burden in vivo by inducing protective cytokines, gamma interferon and interleukin 17. Moreover, astrakurkurone was nontoxic toward peripheral blood mononuclear cells of immunocompromised patients with visceral leishmaniasis. Astrakurkurone, a nontoxic antileishmanial, enhances the immune efficiency of host cells, leading to parasite clearance in vitro and in vivo.

Induction of apoptosis by zerumbone isolated from Zingiber zerumbet (L.) Smith in protozoan parasite Leishmania donovani due to oxidative stress

Abstract In the present context of emergence of resistance aligned with the conventional anti-leishmanial drugs and occasional treatment failure compelled us to continue the search for replaceable therapeutic leads against Leishmaniainfection. Various ginger spices of the Zingiberaceae family are widely used as spices, flavouring agents, and medicines in Southeast Asia because of their unique flavour as well as due to their medicinal properties. Zerumbone, a natural component of Zingiber zerumbet (L.) Smith, has been studied for its pharmacological potential as antiulcer, antioxidant, anticancer, and antimicrobial. In this study, we have shown that zerumbone could induce ROS mediated apoptosis in Leishmania donovani promastigotes and also found effective in reducing intracellular amastigotes in infected-macrophages. We emphasized the potential of zerumbone to be employed in the development of new therapeutic drugs against L. donovaniinfection and provided the basis for future research on the application of transitional medicinal plants.

Induction of apoptosis by zerumbone isolated from Zingiber zerumbet (L.) Smith in protozoan parasite Leishmania donovani due to oxidative stress.

In the present context of emergence of resistance aligned with the conventional anti-leishmanial drugs and occasional treatment failure compelled us to continue the search for replaceable therapeutic leads against Leishmania infection. Various ginger spices of the Zingiberaceae family are widely used as spices, flavouring agents, and medicines in Southeast Asia because of their unique flavour as well as due to their medicinal properties. Zerumbone, a natural component of Zingiber zerumbet (L.) Smith, has been studied for its pharmacological potential as antiulcer, antioxidant, anticancer, and antimicrobial. In this study, we have shown that zerumbone could induce ROS mediated apoptosis in Leishmania donovani promastigotes and also found effective in reducing intracellular amastigotes in infected-macrophages. We emphasized the potential of zerumbone to be employed in the development of new therapeutic drugs against L. donovani infection and provided the basis for future research on the application of transitional medicinal plants.

Prospective microglia and brain macrophage distribution pattern in normal rat brain shows age sensitive dispersal and stabilization with development.

The monocytic lineage cells in brain, generally speaking brain macrophage and/or microglia show some dissimilar distribution patterns and disagreement regarding their origin and onset in brain. Here, we investigated its onset and distribution/colonization pattern in normal brain with development. Primarily, early and late embryonic stages, neonate and adult brains were sectioned for routine H/E staining; a modified silver-gold staining was used for discriminating monocytic lineage cells in brain; and TEM to deliver ultramicroscopic details of these cells in brain. Immunofluorescence study with CD11b marker revealed the distribution of active microglia/macrophage like cells. Overall, in early embryonic day 12, the band of densely stained cells are found at the margin of developing ventricles and cells sprout from there dispersed towards the outer edge. However, with development, this band shrunk and the dispersion trend decreased. The deeply stained macrophage like cell population migration from outer cortex to ventricle observed highest in late embryonic days, continued with decreased amount in neonates and settled down in adult. In adult, a few blood borne macrophage like cells were observed through the vascular margins. TEM study depicted less distinguishable features of cells in brain in early embryo, whereas from late embryo to adult different neuroglial populations and microglia/macrophages showed distinctive features and organization in brain. CD11b expression showed some similarity, though not fully, with the distribution pattern depending on the differentiation/activation status of these macrophage lineage cells. This study provides some generalized spatial and temporal pattern of macrophage/microglia distribution in rat brain, and further indicates some intrigue areas that need to be addressed.

A novel triterpene from Astraeus hygrometricus induces reactive oxygen species leading to death in Leishmania donovani.

AIM: The effect of astrakurkurone, a novel triterpene, isolated from Indian mushroom Astraeus hygrometricus has been investigated to elucidate the mechanisms involved in selective cell death of Leishmania donovani. MATERIALS & METHODS: The hypotheses were investigated using flow-cytometry, scanning electron microscopy and confocal microscopy. RESULTS: The time dependent elevation of astrakurkurone-induced reactive oxygen species (ROS) was found intimately associated with apoptosis. The involvement of ROS in promastigote death was found confirmed as NAC and GSH could decrease the ROS level and restored the mitochondrial membrane potential (ΔΨ(m)). It also inhibited the intracellular amastigotes. CONCLUSION: We claim the present invention as substantial in depth evidences that mushroom derived active molecules can be exploited as target specific, comparatively nontoxic leads for antileishmanial therapy.

Protective effect of Croton caudatus Geisel leaf extract against experimental visceral leishmaniasis induces proinflammatory cytokines in vitro and in vivo.

In the present state of overwhelming emergence of drug-unresponsive phenotypes of Leishmania donovani and persistent severe toxicity in conventional anti-leishmanial therapy, in search for novel leads, the aim of this study has been fixed to identify the active extract(s) of Croton caudatus Geisel. var. tomentosus Hook effective against the parasitic protozoans in vitro and in vivo. C. caudatus Geisel. is often used by Chakma and Hmar community, the local tribes of north-east India for medicinal and veterinary purposes. Among the five semi-purified extracts tested, C. caudatus leaves, extracted in hexane and subsequently semi-purified in a column packed with silica gel (70-130 µM; mesh size 60 A°) using ethyl acetate-hexane solvent (9:1), was found to be the most effective growth inhibitor (JDHex) against the Leishmania promastigotes and amastigotes. JDHex significantly altered the biochemical parameters (protein, lipid and carbohydrates) in promastigotes followed by the morphological changes, DNA condensation and subsequent apoptosis in L. donovani. In consequent steps, it has been also proved that JDHex reduced the replication of intracellular amastigotes with concomitant release of nitric oxide and pro-inflammatory cytokines, IL-12 and TNF-α in vitro. Significantly, the 50% inhibitory concentration of JDHex was estimated much lower against the intracellular amastigotes (2.5 µg/mL) in comparison to promastigotes (10 µg/mL). JDHex was also found efficient in reducing parasite burden in spleen and liver when treated in vivo and increased the intracellular IFN-γ and decreased the IL-10 in CD4+ T cells in splenocytes of orally treated animals. The results of this study support the importance in exploration of novel anti-leishmanial leads from C. caudatus Geisel. var. tomentosus Hook. against the L. donovani (MHOM/IN/83/AG83) infection. Partial chemical characterization of JDHex revealed the presence of terpenoids. However, the further chemical investigation of JDHex is warranted.

Selective inhibition of Leishmania donovani by active extracts of wild mushrooms used by the tribal population of India: An in vitro exploration for new leads against parasitic protozoans.

The study was intended at evaluating the anti-proliferating effect of mushrooms used in traditional folklore of Santal tribal population in India against Leishmania donovani (MHOM/IN/83/AG83). A total of eighteen extracts, three estracts from each mushroom [(80% ethanol extracted; Fa), (water-soluble polysaccharide fraction; Fb), (polyphenolic fraction; Fc)], from six wild mushrooms were obtained. These extracts were tested against the promastigotes and amastigotes for their antileishmanial capacity. Fa fractions (250 µg/mL) of Astraeus hygrometricus and Tricholoma giganteum significantly inhibited the growth of L. donovani promastigotes and interfered in lipid biosynthesis. Moreover, both fractions induced apoptosis in promastigotes. Water soluble Fb fractions of A. hygrometricus, Russula laurocerasi, Russula albonigra, Termitomyces eurhizus, Russula delica and polyphenolic Fc fraction of R. laurocerasi were found to inhibit the replication of intracellular amastigotes in macrophages dose dependently. Significantly, 50% inhibitory concentration of the active extracts against intracellular amastigotes induced release of nitric oxide and IL-12 in murine macrophages and dendritic cells assay and also found considerably non-toxic on murine splenocytes. Results of this study can be used as a basis for further phytochemical and pharmacological investigations in the effort for search of novel anti-leishmanial leads.

Chromone linked nitrone derivative induces the expression of iNOS2 and Th1 cytokines but reduces the Th2 response in experimental visceral leishmaniasis.

In our previous work we have shown that the novel synthetic chromone derivative could effectively inhibit the Leishmania donovani replication in vitro and in vivo with less cytotoxicity on murine splenocytes. The aim of the present study is to explore the possible mechanism of anti-leishmanial effect of C-(6-methyl-4-oxo-4H-1-benzopyran-3-yl)-N-(p-tolyl) nitrone (designated as NP1) in vitro and in vivo in experimental visceral leishmaniasis caused by L. donovani. The cytotoxic effect of this derivative was studied in murine peritoneal macrophages by MTT method. NP1 at a dose of 17.06 µM showed 50% inhibition on L. donovani promastigotes but found less cytotoxic to the RAW 264.7 cells. Even the higher concentration of IC50 (up to four fold) did not exert much cytotoxic effect on RAW 264.7. Interestingly, NP1 at lower concentration (8.53 µM) could inhibit 50% of intracellular amastigotes in murine peritoneal macrophages. L. donovani is known to exert its pathogenic effects mainly by the suppression of NO generation and subversion of the cellular inflammatory responses in the macrophages. NP1 was found to induce a potent host-protective immune response by enhancing NO generation and iNOS2 expression at mRNA level and by up-regulating proinflammatory cytokines such as IL-12 and IFN-γ and limiting the expression of IL-10 in vivo. The NO dependent killing was further confirmed in iNOS(-/-) mice compared to wild type. In agreement with the fact, induced synthesis of IL-12 and IFN-γ and associated down-regulation of IL-10 by the treatment of NP1 clearly indicated the possibility of novel strategy of drug development against Leishmania infection.

Protective therapy with novel chromone derivative against Leishmania donovani infection induces Th1 response in vivo.

BACKGROUND: Visceral leishmaniasis is a chronic protozoan disease caused by Leishmania donovani, an obligatory intracellular parasite that resides and multiplies within macrophages of the reticuloendothelial system. The aim of this study was to evaluate the efficacy of nine novel synthetic chromone derivatives as antileishmanial molecules in experimental murine visceral leishmaniasis. METHODS: In vitro activity of the molecules (2, 5 and 10 µg/ml) was assessed against promastigotes of both pentavalent antimonial-responsive strain AG83 and pentavalent antimonial-resistant strain GE1F8R at days 2 (48 h), 4 (96 h) and 6 (144 h). The efficacy of the most efficient chromone derivative [C-(6-Methyl-4-oxo-4H-1-benzopyran-3-yl)-N-(p-tolyl) nitrone], designated here as NP1, was also tested against intracellular amastigotes in vitro and in vivo. RESULTS: NP1, 5 µg/ml, inhibited the growth of AG83 and GE1F8R promastigotes by 98.57% (day 4) and 75.75% (day 6), respectively, and also inhibited the growth of intracellular amastigotes by 85% (day 3), compared to DMSO control. Treatment of L. donovani-infected mice with NP1 resulted in a 70% significant decrease in parasite load in the spleen in the 7th week after infection (5 mice in each group), with associated induction of interferon-γ synthesis by dose 2 (37.5 mg/kg body weight) compared to DMSO control. Dose 2 was found efficient over dose 1 (25 mg/kg body weight). CONCLUSIONS: The novel synthetic chromone derivative is effective in the treatment of visceral leishmaniasis and induces the synthesis of interferon-γ in rodent models.