Statins abrogate gemcitabine-induced PD-L1 expression in pancreatic cancer-associated fibroblasts and cancer cells with improved therapeutic outcome.

A combination of chemotherapy with immunotherapy has been proposed to have better clinical outcomes in Pancreatic Ductal Adenocarcinoma (PDAC). On the other hand, chemotherapeutics is known to have certain unwanted effects on the tumor microenvironment that may mask the expected beneficial effects of immunotherapy. Here, we have investigated the effect of gemcitabine (GEM), on two immune checkpoint proteins (PD-L1 and PD-L2) expression in cancer associated fibroblasts (CAFs) and pancreatic cancer cells (PCCs). Findings of in vitro studies conducted by using in-culture activated mouse pancreatic stellate cells (mPSCs) and human PDAC patients derived CAFs demonstrated that GEM significantly induces PD-L1 and PD-L2 expression in these cells. Moreover, GEM induced phosphorylation of STAT1 and production of multiple known PD-L1-inducing secretory proteins including IFN-γ in CAFs. Upregulation of PD-L1 in PSCs/CAFs upon GEM treatment caused T cell inactivation and apoptosis in vitro. Importantly, Statins suppressed GEM-induced PD-L1 expression both in CAFs and PCCs while abrogating the inactivation of T-cells caused by GEM-treated PSCs/CAFs. Finally, in an immunocompetent syngeneic orthotopic mouse pancreatic tumor model, simvastatin and GEM combination therapy significantly reduced intra-tumor PD-L1 expression and noticeably reduced the overall tumor burden and metastasis incidence. Together, the findings of this study have provided experimental evidence that illustrates potential unwanted side effects of GEM that could hamper the effectiveness of this drug as mono and/or combination therapy. At the same time the findings also suggest use of statins along with GEM will help in overcoming these shortcomings and warrant further clinical investigation.

MIF confers survival advantage to pancreatic CAFs by suppressing interferon pathway-induced p53-dependent apoptosis.

The presence of activated pancreatic stellate cells (PSCs) in the pancreatic ductal adenocarcinoma (PDAC) microenvironment plays a significant role in cancer progression. Macrophage migration inhibitory factor (MIF) is overexpressed in PDAC tissues and expressed by both cancer and stromal cells. The pathophysiological role of MIF in PDAC-associated fibroblasts or PSCs is yet to be elucidated. Here we report that the PSCs of mouse or cancer-associated fibroblast cells (CAFs) of human expresses MIF and its receptors, whose expression gets upregulated upon LPS or TNF-α stimulation. In vitro functional experiments showed that MIF significantly conferred a survival advantage to CAFs/PSCs upon growth factor deprivation. Genetic or pharmacological inhibition of MIF also corroborated these findings. Further, co-injection of mouse pancreatic cancer cells with PSCs isolated from Mif-/- or Mif+/+ mice confirmed the pro-survival effect of MIF in PSCs and also demonstrated the pro-tumorigenic role of MIF expressed by CAFs in vivo. Differential gene expression analysis and in vitro mechanistic studies indicated that MIF expressed by activated CAFs/PSCs confers a survival advantage to these cells by suppression of interferon pathway induced p53 dependent apoptosis.

Biochemical, functional and genomic characterization of a new probiotic Ligilactobacillus salivarius F14 from the gut of tribes of Odisha.

Characterization of new potential probiotics is desirable in the field of research on probiotics for their extensive use in health and disease. Tribes could be an unusual source of probiotics due to their unique food habits and least dependence on medications and consumption of antibiotics. The aim of the present study is to isolate lactic acid bacteria from tribal fecal samples of Odisha, India, and characterize their genetic and probiotic attributes. In this context one of the catalase-negative and Gram-positive isolates, identified using 16S rRNA sequencing as Ligilactobacillus salivarius, was characterized in vitro for its acid and bile tolerance, cell adhesion and antimicrobial properties. The whole genome sequence was obtained and analyzed for strain level identification, presence of genomic determinants for probiotic-specific features, and safety. Genes responsible for its antimicrobial and immunomodulatory functions were detected. The secreted metabolites were analyzed using high resolution mass spectroscopy; the results indicated that the antimicrobial potential could be due to the presence of pyroglutamic acid, propionic acid, lactic acid, 2-hydroxyisocaproic acid, homoserine, and glutathione, and the immuno-modulating activity, contributed by the presence of short chain fatty acids such as acetate, propionate, and butyrate. So, to conclude we have successfully characterized a Ligilactobacillus salivarius species with potential antimicrobial and immunomodulatory ability. The health-promoting effects of this probiotic strain and/or its derivatives will be investigated in future.

Fluvastatin sensitizes pancreatic cancer cells toward radiation therapy and suppresses radiation- and/or TGF-ß-induced tumor-associated fibrosis.

Pancreatic cancer (PC) is highly resistant to chemo and radiotherapy. Radiation-induced fibrosis (RIF) is a major cause of clinical concern for various malignancies, including PC. In this study, we aimed to evaluate the radiosensitizing and anti-RIF potential of fluvastatin in PC. Short-term viability and clonogenic survival assays were used to evaluate the radiosensitizing potential of fluvastatin in multiple human and murine PC cell lines. The expression of different proteins was analyzed to understand the mechanisms of fluvastatin-mediated radiosensitization of PC cells and its anti-RIF effects in both mouse and human pancreatic stellate cells (PSCs). Finally, these effects of fluvastatin and/or radiation were assessed in an immune-competent syngeneic murine model of PC. Fluvastatin radiosensitized multiple PC cell lines, as well as radioresistant cell lines in vitro, by inhibiting radiation-induced DNA damage repair response. Nonmalignant cells, such as PSCs and NIH3T3 cells, were less sensitive to fluvastatin-mediated radiosensitization than PC cells. Interestingly, fluvastatin suppressed radiation and/or TGF-ß-induced activation of PSCs, as well as the fibrogenic properties of these cells in vitro. Fluvastatin considerably augmented the antitumor effect of external radiation therapy and also suppressed intra-tumor RIF in vivo. These findings suggested that along with radiation, fluvastatin co-treatment may be a potential therapeutic approach against PC.

Quantitative proteomics of hamster lung tissues infected with SARS-CoV-2 reveal host factors having implication in the disease pathogenesis and severity.

Syrian golden hamsters (Mesocricetus auratus) infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) manifests lung pathology. In this study, efforts were made to check the infectivity of a local SARS-CoV-2 isolate in a self-limiting and non-lethal hamster model and evaluate the differential expression of lung proteins during acute infection and convalescence. The findings of this study confirm the infectivity of this isolate in vivo. Analysis of clinical parameters and tissue samples show the pathophysiological manifestation of SARS-CoV-2 infection similar to that reported earlier in COVID-19 patients and hamsters infected with other isolates. However, diffuse alveolar damage (DAD), a common histopathological feature of human COVID-19 was only occasionally noticed. The lung-associated pathological changes were very prominent on the 4th day post-infection (dpi), mostly resolved by 14 dpi. Here, we carried out the quantitative proteomic analysis of the lung tissues from SARS-CoV-2-infected hamsters on day 4 and day 14 post-infection. This resulted in the identification of 1585 proteins of which 68 proteins were significantly altered between both the infected groups. Pathway analysis revealed complement and coagulation cascade, platelet activation, ferroptosis, and focal adhesion as the top enriched pathways. In addition, we also identified altered expression of two pulmonary surfactant-associated proteins (Sftpd and Sftpb), known for their protective role in lung function. Together, these findings will aid in understanding the mechanism(s) involved in SARS-CoV-2 pathogenesis and progression of the disease.

Whole Genome Mining and Characterization of a New Probiotic Strain Levilactobacillus brevis ILSH3 from Handia: An Ethnic Fermented Beverage of Odisha, India.

Isolation and characterization of probiotics from traditional fermented food have contributed many beneficial strains to the field of health and nutritional sciences. Handia, a traditional fermented alcoholic beverage popular in different parts of Odisha, was our source of isolation. This study characterizes one such potential bacteria, Levilactobacillus brevis ILSH3 (H3) isolated from Handia. The investigation for the probiotic attributes as per ICMR-DBT guidelines qualified the checkpoint assays like acid and bile tolerance, bile salt hydrolase activity, antimicrobial properties, and pathogen exclusion ability. The whole genome sequence of H3 (2,460,966 bp in size with GC content of 45.62%) was subjected to comparative genome analysis for its taxonomic identification and validation of probiotic potential. Various genes pertaining to its probiotic potential were identified in the genome and it showed zero matches against any pathogenic families. Metabolite profiling of cell-free supernatant using liquid chromatography-mass spectrometry revealed the presence of essential amino acids, short-chain fatty acids, antimicrobial molecules, immunomodulatory molecules, and flavor/aroma-enhancing compounds. Immunomodulatory property investigation using Bioplex and qRT-PCR showed a reduction in the levels of pro-inflammatory cytokines in L. brevis ILSH3-treated Caco-2 cells. Collectively, the results demonstrate that this Handia-origin bacteria Levilactobacillus brevis ILSH3 possesses desirable attributes of a probiotic, which is now open for nutritional and health biologists to explore. This new probiotic strain may show promising results when utilized in healthcare or functional foods.

Therapeutic role of N-acetyl cysteine (NAC) for the treatment and/or management of SARS-CoV-2-induced lung damage in hamster model.

Oxidative stress by reactive oxygen species (ROS) has been hypothesized to be the major mediator of SARS-CoV-2-induced pathogenesis. During infection, the redox homeostasis of cells is altered as a consequence of virus-induced cellular stress and inflammation. In such scenario, high levels of ROS bring about the production of pro-inflammatory molecules like IL-6, IL-1ß, etc. that are believed to be the mediators of severe COVID-19 pathology. Based on the known antioxidant, anti-inflammatory, mucolytic and antiviral properties of NAC, it has been hypothesized that NAC will have beneficial effects in COVID-19 patients. In the current study efforts have been made to evaluate the protective effect of NAC in combination with remdesivir against SARS-CoV-2 induced lung damage in the hamster model. The SARS-CoV-2 infected animals were administered with high (500 mg/kg/day) and low (150 mg/kg/day) doses of NAC intraperitoneally with and without remdesivir. Lung viral load, pathology score and expression of inflammatory molecules were checked by using standard techniques. The findings of this study show that high doses of NAC alone can significantly suppress the SARS-CoV-2 mediated severe lung damage (2 fold), but on the contrary, it fails to restrict viral load. Moreover, high doses of NAC with and without remdesivir significantly suppressed the expression of pro-inflammatory genes including IL-6 (4.16 fold), IL-1ß (1.96 fold), and TNF-α (5.55 fold) in lung tissues. Together, results of this study may guide future preclinical and clinical attempts to evaluate the efficacy of different doses and routes of NAC administration with or without other drugs against SARS-CoV-2 infection.

Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors.

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features of a unique and convenient model system. As highly proliferative cells are more susceptible to radiation-induced DNA damage, zebrafish embryos are a front-line in vivo model in radiation research. In addition, this model projects the effect of radiation and different drugs within a short time, along with major biological events and associated responses. Several cancer studies have used zebrafish, and this protocol is based on the use of radiation modifiers in the context of radiotherapy and cancer. This method can be readily used to validate the effects of different drugs on irradiated and control (non-irradiated) embryos, thus identifying drugs as radio sensitizing or protective drugs. Although this methodology is used in most drug screening experiments, the details of the experiment and the toxicity assessment with the background of X-ray radiation exposure are limited or only briefly addressed, making it difficult to perform. This protocol addresses this issue and discusses the procedure and toxicity evaluation with a detailed illustration. The procedure describes a simple approach for using zebrafish embryos for radiation studies and radiation-based drug screening with much reliability and reproducibility.

Tissue Distribution of ACE2 Protein in Syrian Golden Hamster (Mesocricetus auratus) and Its Possible Implications in SARS-CoV-2 Related Studies.

The Syrian golden hamster (Mesocricetus auratus) has recently been demonstrated as a clinically relevant animal model for SARS-CoV-2 infection. However, lack of knowledge about the tissue-specific expression pattern of various proteins in these animals and the unavailability of reagents like antibodies against this species hampers these models' optimal use. The major objective of our current study was to analyze the tissue-specific expression pattern of angiotensin-converting enzyme 2, a proven functional receptor for SARS-CoV-2 in different organs of the hamster. Using two different antibodies (MA5-32307 and AF933), we have conducted immunoblotting, immunohistochemistry, and immunofluorescence analysis to evaluate the ACE2 expression in different tissues of the hamster. Further, at the mRNA level, the expression of Ace2 in tissues was evaluated through RT-qPCR analysis. Both the antibodies detected expression of ACE2 in kidney, small intestine, tongue, and liver. Epithelium of proximal tubules of kidney and surface epithelium of ileum expresses a very high amount of this protein. Surprisingly, analysis of stained tissue sections showed no detectable expression of ACE2 in the lung or tracheal epithelial cells. Similarly, all parts of the large intestine were negative for ACE2 expression. Analysis of tissues from different age groups and sex didn't show any obvious difference in ACE2 expression pattern or level. Together, our findings corroborate some of the earlier reports related to ACE2 expression patterns in human tissues and contradict others. We believe that this study's findings have provided evidence that demands further investigation to understand the predominant respiratory pathology of SARS-CoV-2 infection and disease.