Regulatory role of Transcription factor-EB (TFEB) in parasite control through alteration of antigen presentation in visceral leishmaniasis.

Leishmania donovani, an obligate intracellular parasite, the causative agent of visceral leishmaniasis is known to subvert the host immune system for its own survival. Although the precise mechanism is still unknown, emerging evidences indicate that L. donovani efficiently suppress MHC I mediated antigen presentation, rendering inadequate CD8+T cell activation and weakening host defense against parasite. The role of transcription factor EB (TFEB) was recognized in modulating antigen presentation besides its role in lysosomal biogenesis and function. Here, we investigated the regulatory role of TFEB in the modulation of presentation of Leishmania antigen in host tissue. Our results showed an increased expression of TFEB after Leishmania infection both in vitro and in vivo and there was a decrease in the expression of Th-1 cytokine IFNγ along with MHC class I and CD8+T cells indicating attenuation of cell mediated immunity and possibly MHC I restricted antigen presentation. Silencing of TFEB resulted in increased expression of IFNγ and MHC I along with increased CD8+T cells population without any significant change in CD4+T cell number. We also observed a decreased parasite burden in TFEB silenced condition which indicates enhanced parasite clearance by alteration of immunological response possibly through induction of presentation of Leishmania antigen through MHC I. The present study explains the role of TFEB silencing in parasite clearance through regulating the antigen presentation of Leishmania antigen thereby promises to formulate a potential therapeutic strategy against visceral leishmaniasis.

Phosphorylated Proteins from Serum: A Promising Potential Diagnostic Biomarker of Cancer.

Cancer is a fatal disease worldwide. Each year ten million people are diagnosed around the world, and more than half of patients eventually die from it in many countries. A majority of cancer remains asymptomatic in the earlier stages, with specific symptoms appearing in the advanced stages when the chances of adequate treatment are low. Cancer screening is generally executed by different imaging techniques like ultrasonography (USG), mammography, CT-scan, and magnetic resonance imaging (MRI). Imaging techniques, however, fail to distinguish between cancerous and non-cancerous cells for early diagnosis. To confirm the imaging result, solid and liquid biopsies are done which have certain limitations such as invasive (in case of solid biopsy) or missed early diagnosis due to extremely low concentrations of circulating tumor DNA (in case of liquid biopsy). Therefore, it is essential to detect certain biomarkers by a noninvasive approach. One approach is a proteomic or glycoproteomic study which mostly identifies proteins and glycoproteins present in tissues and serum. Some of these studies are approved by the Food and Drug Administration (FDA). Another non-expensive and comparatively easier method to detect glycoprotein biomarkers is by ELISA, which uses lectins of diverse specificities. Several of the FDA approved proteins used as cancer biomarkers do not show optimal sensitivities for precise diagnosis of the diseases. In this regard, expression of phosphoproteins is associated with a more specific stage of a particular disease with high sensitivity and specificity. In this review, we discuss the expression of different serum phosphoproteins in various cancers. These phosphoproteins are detected either by phosphoprotein enrichment by immunoprecipitation using phosphospecific antibody and metal oxide affinity chromatography followed by LC-MS/MS or by 2D gel electrophoresis followed by MALDI-ToF/MS analysis. The updated knowledge on phosphorylated proteins in clinical samples from various cancer patients would help to develop these serum phophoproteins as potential diagnostic/prognostic biomarkers of cancer.

ß-Endosulfan-mediated induction of pro-fibrotic markers in renal (HK-2) cells in vitro: A new insight in the pathogenesis of chronic kidney disease of unknown etiology.

Chronic kidney disease of unknown etiology (CKDu), manifested clinically as tubulo interstitial fibrosis, has emerged as the second major cause of chronic kidney disease (CKD) in the Indian subcontinent and various agrochemicals have been implicated in its occurance. Among the agrochemicals organochlorine pesticides particularly endosulfan is well known for its toxicity and recent residue analysis have shown its presence in the blood samples of general population. In this present study, we have investigated the consequences of endosulfan exposure at a concentration (0.01 µM) equivalent to their highest reported presence in human blood sample of some CKDu patients, to human renal proximal tubular epithelial (HK-2) cell line with regard to ROS generation and expression of profibrotic and epithelial to mesenchymal (EMT) markers in order to find out endosulfan's ability to induce profibrotic changes in renal cell. We demonstrated a significant increase in intracellular ROS generation and increased expression of TGF-ß1 when cells were incubated with ß-endosulfan (0.01 µM) indicating occurrence of oxidative stress and fibrotic process. Again, decreased expression of epithelial marker E-cadherin and increase in the expression of mesenchymal marker α-smooth muscle actin (α-SMA) suggest possible onset of EMT process. Pre-treatment with 5 mM concentration of anti-oxidant N-acetyl cysteine partially attenuated the above process. In conclusion, these findings suggest possible involvement of ß-endosulfan in the development of CKDu through oxidative stress and profibrotic signaling.

Immunomodulator mediated changes in plasma membrane calcium ATPase in controlling visceral leishmaniasis.

Immunomodulation is an emerging concept to combat infection in recent years. Immunomodulators like arabinosylated-lipoarabinomannan (Ara-LAM) and glycyrrhizic-acid (GA) possess anti-leishmanial property, whereas sodium-antimony-gluconate (SAG) is still considered as the first choice for chemotherapy against leishmaniasis. During infection, invasion of Leishmania donovani needs the potential requirement of Ca2+, which is further responsible for apoptosis in intracellular amastigotes. However, suppression of elevated intracellular calcium by the activation of plasma-membrane-calcium-ATPase (PMCA4) facilitates survival of L. donovani in the host. In the present study, SAG, Ara-LAM, and GA were found to evoke significant increase in intracellular Ca2+ in L. donovani infected macrophages by inhibiting PMCA4. Moreover, PMCA4 inhibition by TFP or PMCA4 siRNA elevated the level of PKCß, whereas calcium-independent upregulation of PKCζ remained unchanged in infected macrophages. Furthermore, application of immunomodulators in infected macrophages resulted in down-regulation of PKCζ, conversion of anti-inflammatory to pro-inflammatory cytokines and inhibition of PMCA4. Plasma membrane-associated ceramide which is known to be elevated during leishmaniasis, triggered upregulation of PMCA4 via PKCζ activation. Interestingly, immunomodulators attenuated ceramide generation, which resulted into reduced PKCζ activation leading to the decreased PMCA expression in infected macrophages. Therefore, our study elucidated the efficacy of SAG, Ara-LAM, and GA in the reduction of parasite burden in macrophages by suppressing PMCA activation through inhibition of ceramide mediated upregulation of PKCζ.

Organochlorine pesticide level in patients with chronic kidney disease of unknown etiology and its association with renal function.

BACKGROUND: Involvement of agrochemicals have been suggested in the development of chronic kidney disease of unknown etiology (CKDu). The association between CKDu and blood level of organochlorine pesticides (OCPs) in CKDu patients has been examined in the present study. METHODS: All the recruited study subjects (n = 300) were divided in three groups, namely, healthy control (n = 100), patients with chronic kidney disease of unknown etiology (n = 100), and patients with chronic kidney disease of known etiology (CKDk) (n = 100). Blood OCP levels of all three study groups were analyzed by gas chromatography. RESULTS: Increased level of OCPs, namely α-HCH, aldrin, and ß-endosulfan, were observed in CKDu patients as compared to healthy control and CKD patients of known etiology. The levels of these pesticides significantly correlated negatively with the estimated glomerular filtration rate (eGFR) and positively with urinary albumin of CKD patients. Logistic regression analysis revealed association of γ-HCH, p, p'-DDE, and ß-endosulfan with CKDu on adjustment of age, sex, BMI, and total lipid content. CONCLUSIONS: Increased blood level of certain organochlorine pesticides is associated with the development of chronic kidney disease of unknown etiology.

Organochlorine Pesticide-Mediated Induction of NADPH Oxidase and Nitric-Oxide Synthase in Endothelial Cell.

INTRODUCTION: Organochlorine Pesticides (OCPs) are detected ubiquitously in human and have been shown to be associated with Cardiovascular Disease (CVD) and atherosclerosis. AIM: To find out the effect of organochlorine pesticides in endothelial cell with regard to oxidative stress and associated expression of enzymes producing superoxide and Nitric Oxide (NO). MATERIALS AND METHODS: Human Umbilical Vein Endothelial Cells (HUVEC) were cultured and treated with four OCPs which were found in human blood at a concentration of 0.1µM. The cells were tested for Reactive Oxygen Species (ROS) generation, NO production and mRNA expression of NAPDH oxidase (p47phox) and endothelial Nitric Oxide Synthase (eNOS). ROS generation was measured by using 2', 7'-dichlorodihydrofluorescein diacetate (H2DCFDA) method. NO was analysed by Bioxytech nitric oxide assay kit method and mRNA of NADPH oxidase and eNOS was quantified by real time PCR. Data were expressed as the mean±SEM. Comparison between the groups were made by student's t-test (2-tailed) or one-way ANOVA with Tukey's post-hoc analysis depending on number of groups. For all statistical tests, p< 0.05 was considered to be significant. RESULTS: All the four pesticides generated ROS accompanied by enhanced expression of NADPH oxidase. Maximum effect was observed with ß-endosulfan. Level of NO was found to be decreased significantly in endothelial cells treated with these pesticides accompanied by enhanced expression of eNOS. The antioxidant N-acetylcysteine (NAC) reduced ROS generation and enhanced NO formation. Pesticide-mediated ROS generation possibly reacts with NO forming peroxinitrite and thereby reducing the bioavailability of NO although eNOS expression is increased. CONCLUSION: OCPs induce endothelial dysfunction through increased ROS generation via NADPH oxidase expression and reduced bioavailability of nitric oxide.