Endocrine disruptive toxicity of cypermethrin in Labeo catla: Involvement of genes and proteins related to the HPG axis.

Cypermethrin (CYP) is a synthetic pyrethroid abundantly used in agriculture and aquaculture. It is an established potent endocrine disruptor to fish, yet the molecular mechanism behind its reproductive toxicity remains unclear. In this study, fish Labeo catla (Catla) was exposed to environmentally relevant concentration of CYP (0.7 µg/L) and 0.14 µg/L for 30 days. The changes in circulating sex steroids, genes, and hormones linked to the hypothalamic-pituitary-gonadal (HPG) axis, stress response and associated histological alterations were studied. Significant decline (P < 0.05) in serum 17 beta (ß) estradiol (E2), 11 ketotestosterone (11-KT), and brain (FSH and GnRH) were observed in 0.7 µg/L dose of CYP. These effects may be due to the down-regulated expression of the upstream genes of the HPG axis i.e. Kiss 1 and Kiss 2, which further downregulates the expression of the GnRH gene. The decreased level of E2 and 11-KT also affects the vitellogenin (Vtg) gene expression, reducing the production of Vtg, a crucial protein for ovarian development. Principal component analysis (PCA) revealed the relationship between CYP and the biosynthesis of sex steroids. The toxic effect of CYP was also visible in antioxidant enzyme assay and related histological alterations. Overall, the study elucidated that long-term exposure to CYP, even at an environmentally relevant dose, may affect reproductive potential and fish recruitment. The study provides important insights into molecular mechanisms underlying CYP-induced endocrine disruption in fish, and it also raises questions about CYP's potential toxicity at environmentally relevant concentration in terms of understanding ecological risk.

Emerging contaminant triclosan incites endocrine disruption, reproductive impairments and oxidative stress in the commercially important carp, Catla (Labeo catla): An insight through molecular, histopathological and bioinformatic approach.

Triclosan (TCS), a broad-spectrum antimicrobial agent is ubiquitous in aquatic ecosystems; however, the mechanisms regarding TCS-induced reproductive toxicity in the teleost still remains uncertain. In this context, Labeo catla were subjected to sub-lethal doses of TCS for 30 days and variations in expression of genes and hormones comprising the hypothalamic-pituitary-gonadal (HPG) axis along with alterations in sex steroids were evaluated. Moreover, manifestation of oxidative stress, histopathological alterations, in silico docking and the potential to bioaccumulate were also investigated. Exposure to TCS may lead to an inevitable onset of the steroidogenic pathway through its interaction at several loci along the reproductive axis: TCS stimulated synthesis of kisspeptin 2 (Kiss 2) mRNAs which in turn prompts the hypothalamus to secrete gonadotropin-releasing hormone (GnRH), resulting in elevated serum 17ß-estradiol (E2) as a consequence; TCS exposure increased aromatase synthesis by brain, which by converting androgens to oestrogens may raise E2 levels; Moreover, TCS treatment resulted in elevated production of GnRH and gonadotropins by the hypothalamus and pituitary, respectively resulting in the induction of E2. The elevation in serum E2 may be linked to abnormally elevated levels of vitellogenin (Vtg) with harmful consequences evident as hypertrophy of hepatocytes and increment in hepatosomatic indices. Additionally, molecular docking studies revealed potential interactions with multiple targets viz. Vtg and luteinizing hormone (LH). Furthermore, TCS exposure induced oxidative stress and caused extensive damage to tissue architecture. This study elucidated molecular mechanisms underlying TCS-induced reproductive toxicity and the need for regulated use and efficient alternatives which could suffice for TCS.

Hepatocyte SHP-1 is a Critical Modulator of Inflammation During Endotoxemia.

Liver hepatocytes (Hep) are known to be central players during the inflammatory response to systemic infection. Interestingly, the protein tyrosine phosphatases (PTP) SHP-1, has been recognized as a major regulator of inflammation; however their implication in the control of Hep-mediated inflammatory response is still unknown. To study its implication in the regulation of the Hep-mediated inflammatory response during endotoxemia, Cre-Lox mice with a Hep-specific Ptpn6 deletion (Ptpn6 H-KO ) were injected with LPS. In contrast to the wild-type mice (Ptpn6 f/f ) that started to die by 24 hrs post-inoculation, the Ptpn6 H-KO mice exhibited mortality by 6 hrs. In parallel, higher amounts of metabolic markers, pro-inflammatory mediators and circulating cytokines were detected in Ptpn6 H-KO mice. Primary Hep obtained from Ptpn6 H-KO , also showed increased secretion of pro-inflammatory cytokines and nitric oxide (NO) comparatively to its wild type (Ptpn6 f/f ) counterpart. Pharmacological approaches to block TNF-α and NO production protected both the Ptpn6 f/f and the Ptpn6 H-KO mice against deadly LPS-mediated endotoxemia. Collectively, these results establish hepatocyte SHP-1 is a critical player regulating systemic inflammation. Our findings further suggest that SHP-1 activation could represent a new therapeutic avenue to better control inflammatory-related pathologies.

Leishmania exosomes and other virulence factors: Impact on innate immune response and macrophage functions.

Leishmania parasites are the causative agents of the leishmaniases, a collection of vector-borne diseases that range from simple cutaneous to fatal visceral forms. Employing potent immune modulation mechanisms, Leishmania is able to render the host macrophage inactive and persist inside its phagolysosome. In the last few years, the role of exosomes in Leishmania-host interactions has been increasingly investigated. For instance, it was reported that Leishmania exosome release is augmented following temperature shift, a condition mimicking parasite's entry into its mammalian host. Leishmania exosomes were found to strongly affect macrophage cell signaling and functions, similarly to whole parasites. Importantly, these vesicles were shown to be pro-inflammatory, capable to recruit neutrophils at their inoculation site exacerbating the pathology. In this review, we provide the most recent insights on the role of exosomes and other virulence factors, especially the surface protease GP63, in Leishmania-host interactions, deepening our knowledge on leishmaniasis and paving the way for the development of new therapeutics.

Mycobacterium indicus pranii (Mw)-mediated protection against visceral leishmaniasis: involvement of TLR4 signalling.

OBJECTIVES: The aim of this study was to characterize the antileishmanial activity of heat-killed Mycobacterium indicus pranii (Mw) alone or in combination with a subtoxic dose of amphotericin B [AMB(st)]. METHODS: Mw- and Mw + AMB(st)-mediated antileishmanial activity was evaluated by microscopic counting of intracellular amastigotes in Giemsa-stained macrophages and real-time PCR analysis of inducible nitric oxide synthase (iNOS) expression and measurement of nitric oxide generation by Griess reagent. The relationship between Mw and Toll-like receptor 4 (TLR4) signalling was studied by fluorescence-activated cell sorting, western blot and confocal microscopy. The effect of Mw alone or in combination with AMB(st) on the expression and production of interleukin (IL)-12, tumour necrosis factor-α, IL-10 and transforming growth factor-ß was analysed by real-time PCR and ELISA, respectively. RESULTS: Mw treatment alone or with AMB(st) caused a significant increase in TLR4 expression of L. donovani-infected macrophages along with the activation of TLR4 downstream signalling, facilitating active nuclear translocation of nuclear factor κB (NF-κB). These events culminated in the up-regulation of the proinflammatory response, which was abrogated by treatment with TLR4-specific small-interfering RNA. In addition, this study demonstrates that this chemoimmunotherapeutic strategy confers protection against leishmanial pathogenesis via TLR4-dependent counter-regulation of inducible nitric oxide synthase (iNOS) and arginase1 activity. CONCLUSIONS: These results provide a mechanistic understanding of Mw- or Mw + AMB(st)-mediated protection against leishmanial parasites within host macrophages.

Mycobacterium indicus pranii (Mw) re-establishes host protective immune response in Leishmania donovani infected macrophages: critical role of IL-12.

Leishmania donovani, a protozoan parasite, causes a strong immunosuppression in a susceptible host and inflicts the fatal disease visceral leishmaniasis. Relatively high toxicity, low therapeutic index, and failure in reinstating host-protective anti-leishmanial immune responses have made anti-leishmanial drugs patient non-compliant and an immuno-modulatory treatment a necessity. Therefore, we have tested the anti-leishmanial efficacy of a combination of a novel immunomodulator, Mycobacterium indicus pranii (Mw), and an anti-leishmanial drug, Amphotericin B (AmpB). We observe that Mw alone or with a suboptimal dose of AmpB offers significant protection against L. donovani infection by activating the macrophages. Our experiments examining the anti-leishmanial activity of Mw alone or with AmpB also indicate a p38MAPK and ERK-1/2 regulated pro-inflammatory responses. The Mw-AmpB combination induced nitric oxide production, restored Th1 response, and significantly reduced parasite burden in wild type macrophages but not in IL-12-deficient macrophages indicating a pivotal role for IL-12 in the induction of host-protection by Mw and AmpB treatments. In addition, we observed that Mw alone or in combination with suboptimal dose of AmpB render protection against L. donovani infection in susceptible BALB/c mice. However, these treatments failed to render protection in IL-12-deficient mice in vivo which added further support that IL-12 played a central role in this chemo immunotherapeutic approach. Thus, we demonstrate a novel chemo-immunotherapeutic approach- Mw and AmpB crosstalk eliminating the parasite-induced immunosuppression and inducing collateral host-protective effects.

Miltefosine triggers a strong proinflammatory cytokine response during visceral leishmaniasis: role of TLR4 and TLR9.

Visceral leishmaniasis (VL) caused by the protozoan parasite, Leishmania donovani, is associated with irregular fever, weight loss, hepatosplenomegaly and anemia. The therapeutic arsenal against VL is limited and the recent advent of a novel immunomodulatory drug, Miltefosine has shown promising results for effective treatment of VL but its dependence on Toll like receptors (TLR) has not been explored. In this study, we have shown that the non-cytotoxic dose (5 µM) of Miltefosine could render significant protection corresponding to 88% and 95% reduction in intracellular parasite load at 24 h and 48 h in L. donovani infected THP1 cells. This was accompanied by a strong proinflammatory cytokine response in the form of IFN-γ, IL-12 and TNF-α as evident by enzyme linked immunosorbent assay (ELISA) and real time PCR (RT-PCR). This Miltefosine induced proinflammatory cytokine response in infected THP1 cells was also accompanied by simultaneous 10- and 12-fold increase in TLR4 mRNA and TLR9 mRNA. These changes in cytokine response and TLR expression were also studied in peripheral blood mononuclear cells (PBMC) of VL patients treated with Miltefosine by RT-PCR which showed similar results as in THP1 cells. Thereby, suggesting a probable dependence of Miltefosine on TLR4 and TLR9 in triggering a proinflammatory response.

Arabinosylated lipoarabinomannan skews Th2 phenotype towards Th1 during Leishmania infection by chromatin modification: involvement of MAPK signaling.

The parasitic protozoan Leishmania donovani is the causative organism for visceral leishmaniasis (VL) which persists in the host macrophages by deactivating its signaling machinery resulting in a critical shift from proinflammatory (Th1) to an anti-inflammatory (Th2) response. The severity of this disease is mainly determined by the production of IL-12 and IL-10 which could be reversed by use of effective immunoprophylactics. In this study we have evaluated the potential of Arabinosylated Lipoarabinomannan (Ara-LAM), a cell wall glycolipid isolated from non pathogenic Mycobacterium smegmatis, in regulating the host effector response via effective regulation of mitogen-activated protein kinases (MAPK) signaling cascades in Leishmania donovani infected macrophages isolated from BALB/C mice. Ara-LAM, a Toll-like receptor 2 (TLR2) specific ligand, was found to activate p38 MAPK signaling along with subsequent abrogation of extracellular signal-regulated kinase (ERKs) signaling. The use of pharmacological inhibitors of p38MAPK and ERK signaling showed the importance of these signaling pathways in the regulation of IL-10 and IL-12 in Ara-LAM pretreated parasitized macrophages. Molecular characterization of this regulation of IL-10 and IL-12 was revealed by chromatin immunoprecipitation assay (CHIP) which showed that in Ara-LAM pretreated parasitized murine macrophages there was a significant induction of IL-12 by selective phosphorylation and acetylation of histone H3 residues at its promoter region. While, IL-10 production was attenuated by Ara-LAM pretreatment via abrogation of histone H3 phosphorylation and acetylation at its promoter region. This Ara-LAM mediated antagonistic regulations in the induction of IL-10 and IL-12 genes were further correlated to changes in the transcriptional regulators Signal transducer and activator of transcription 3 (STAT3) and Suppressor of cytokine signaling 3 (SOCS3). These results demonstrate the crucial role played by Ara-LAM in regulating the MAPK signaling pathway along with subsequent changes in host effector response during VL which might provide crucial clues in understanding the Ara-LAM mediated protection during Leishmania induced pathogenesis.

Treatment with IP-10 induces host-protective immune response by regulating the T regulatory cell functioning in Leishmania donovani-infected mice.

Visceral leishmaniasis (VL), caused by the protozoan parasite, Leishmania donovani, is characterized by an infection in the liver and spleen. The failure of the first-line drugs has led to the development of new strategies for combating VL. Recently, our group has shown that interferon-γ-inducible protein (IP)-10, a CXC chemokine, renders protection against VL. In the present study, we have elucidated the mechanism by which IP-10 renders protection in in vivo L. donovani infection. We observed that IP-10-treated parasitized BALB/c mice showed a strong host-protective T helper cell (Th) 1 immune response along with marked decrease in immunosuppressive cytokines, tumor growth factor (TGF)-ß, and interleukin (IL)-10 secreting CD4(+) T cells. This IP-10-mediated decrease in immunosuppressive cytokines was correlated with the reduction in the elevated frequency of CD4(+)CD25(+) T regulatory (Treg) cells along with the reduced TFG-ß production from these Treg cells in Leishmania-infected mice. This reduction in TGF-ß production was due to effective modulation of TGF-ß signaling by IP-10, which reduced the immunosuppressive activity of Treg cells. Thus, these findings put forward a detailed mechanistic insight into IP-10-mediated regulation of the Treg cell functioning during experimental VL, which might be helpful in combating Leishmania-induced pathogenesis.

Amphotericin B regulates the host immune response in visceral leishmaniasis: reciprocal regulation of protein kinase C isoforms.

OBJECTIVES: Treatment of visceral leishmaniasis (VL) is marked by the failure of pentavalent antimonials which has brought amphotericin B (AmpB) to the forefront. In this study we have focused on signaling pathway regulating AmpB triggered effector response. METHODS: AmpB triggered effector response in the form of free radicals and proinflammatory cytokines was determined by FACS, colorimetric estimation or Real-Time PCR (RT-PCR). Specific peptide inhibitors for classical and atypical protein kinase C (PKC) were used to investigate the role of PKC isoforms in the functioning of AmpB during VL. RESULTS: In vitro studies with THP1 cells showed that 2 microg/ml dose of AmpB could mediate effective parasite clearance due to strong induction of free radicals and proinflammatory cytokines. This induction of proinflammatory response paralleled with antagonistic regulation of classical and atypical PKC. Further confirmation was provided by RT-PCR of (peripheral blood mononuclear cells) PBMC isolated from VL infected patients undergoing AmpB treatment. CONCLUSIONS: Overall, our results suggest that classical and atypical PKC signaling pathways are involved in the modulation of proinflammatory response triggered by AmpB against Leishmania donovani. These observations may contribute to the understanding of the mechanism responsible for the initiation of protective response induced by AmpB during VL.

Arabinosylated lipoarabinomannan-mediated protection in visceral leishmaniasis through up-regulation of toll-like receptor 2 signaling: an immunoprophylactic approach.

Visceral leishmaniasis is characterized by severe immunosuppression of the host cell, resulting in loss of the proinflammatory response. Toll-like receptor 2 (TLR2) is involved in myriad disease forms, including visceral leishmaniasis. During Leishmania donovani infection, the parasite modulates TLR2 to suppress interleukin 12 production, indicating the possible involvement of TLR2 in regulation of the immune response against L. donovani infection. Arabinosylated lipoarabinomannan (Ara-LAM) possesses immunomodulatory properties and induces proinflammatory responses via induction of TLR2-mediated signaling. Here, we found that pretreatment of L. donovani-infected macrophages with Ara-LAM caused a significant increase in TLR2 expression along with the activation of TLR2-mediated downstream signaling, facilitating active nuclear translocation of nuclear factor kappaB. These events culminated in up-regulation of the proinflammatory response, which was abrogated by treatment with TLR2-specific small interfering RNA. In vivo experiments were also suggestive of Ara-LAM playing a long-term protective role. This study demonstrates that Ara-LAM confers protection against leishmanial pathogenesis via TLR2 signaling-mediated induction of the proinflammatory response.

CXC chemokine-mediated protection against visceral leishmaniasis: involvement of the proinflammatory response.

Visceral leishmaniasis, caused by the protozoan parasite Leishmania donovani, is characterized by the loss of ability of the host to generate an effective immune response. In the present study, the comparative potential of CXC chemokines, interferon-gamma-inducible protein-10 (IP-10) and interleukin-8 (IL-8) in restricting Leishmania donovani infection via the release of nitric oxide and proinflammatory cytokines was studied in an in vitro model. Nitric oxide, a crucial mediator for IP-10-mediated leishmanicidal activity, was found to be dependent on inducible nitric oxide synthase 2 (iNOS2) expression and was linked to the mitogen-activated protein kinases (MAPK) signaling pathway. Further, IP-10 was also able to abrogate the survival of Leishmania in an in vivo model of visceral leishmaniasis by restoration of Th1 cytokines and nitric oxide. Thus, this study strongly demonstrates that IP-10, like CC chemokines, is involved in rendering a protective response in visceral leishmaniasis via up-regulation of proinflammatory mediators.

Anti-iL-10 mAb protection against experimental visceral leishmaniasis via induction of Th1 cytokines and nitric oxide.

Visceral leishmaniasis is characterized by severe immune suppression of the host. This suppression of the host immune system is primarily mediated by the immunosuppressive cytokine Interleukin-10 (IL-10), whose levels are significantly upregulated during leishmaniasis. This immune suppression is reflected at the level of T-cell dysfunction and abrogation of leishmaniacidal molecules along with a dampened Th1 cytokine response. In the present study, we showed in vivo neutralization of IL-10 by administration of anti IL-10 monoclonal antibodies (mAb) could confer protection against leishmanial pathogenesis. This protective response was primarily mediated by a strong induction of T cell proliferation along with a Th1 biased cytokine response which was further aided by the generation of, leishmanicidal molecules, nitric oxide.

Darwin's manufactory hypothesis is confirmed and predicts the extinction risk of extant birds.

In the Origin of Species Darwin hypothesized that the "manufactory" of species operates at different rates in different lineages and that the richness of taxonomic units is autocorrelated across levels of the taxonomic hierarchy. We confirm the manufactory hypothesis using a database of all the world's extant avian subspecies, species and genera. The hypothesis is confirmed both in correlations across all genera and in paired comparisons controlling for phylogeny. We also find that the modern risk of extinction, as measured by "Red List" classifications, differs across the different categories of genera identified by Darwin. Specifically, species in "manufactory" genera are less likely to be threatened, endangered or recently extinct than are "weak manufactory" genera. Therefore, although Darwin used his hypothesis to investigate past evolutionary processes, we find that the hypothesis also foreshadows future changes to the evolutionary tree.