IL1B Induced Smad 7 Negatively Regulates Gastrin Expression

BACKGROUND: Helicobacter pylori elicited IL1B is one of the various modulators responsible for perturbation of acid secretion in gut. We have earlier reported that IL1B activated NFkB downregulates gastrin, a major modulator of acid secretion. However, we hypothesized that regulation of gastrin by IL1B would depend on the cell's ability to integrate inputs from multiple signaling pathways to generate appropriate biological response. PRINCIPAL FINDING: In this study, we report that IL1B induces Smad 7 expression by about 4.5 fold in gastric carcinoma cell line, AGS. Smad 7 resulted in transcriptional repression of gastrin promoter by about 6.5 fold when co-transfected with Smad 7 expression vector and gastrin-promoter luciferase in AGS cells. IL1B inhibited phosphorylation of Smad 3 and subsequently interfered with nuclear translocation of the positive Smad complex, thus occluding it off the gastrin promoter. IL1B promoter polymorphisms (-511T/-31C IL1B) are known to be associated with H. pylori associated gastro-duodenal ulcer. We observed that IL1B expressed from -31T promoter driven IL1B cDNA elicited 3.5 fold more Smad 7 than that expressed from the IL1B-31C variant in AGS cells. This differential activation of Smad 7 by IL1B promoter variants translated into differential downregulation of gastrin expression. We further analyzed Smad 7, NFkB, IL1B and gastrin expression in antral gut biopsy samples of patients with H. pylori associated duodenal ulcer and normal individuals. We observed that individuals with duodenal ulcer had significantly lower levels of IL1B, Smad 7, NFkB and corresponding higher level of gastrin expression. CONCLUSION: Pro-inflammatory cytokine IL1B repress gastrin expression by activating Smad 7 and subsequent inhibition of nuclear localization of Smad 3/4 complex. Polymorphic promoter variants of IL1B gene can modulate the IL1B expression which resulted in differential activation Smad 7 and consequent repression of gastrin expression, respectively. Analysis of H. pylori infected duodenal ulcer patient's gut biopsy samples also supported this observation

Dietary and nutritional change in India: implications for strategies, policies, and interventions

Despite the global transition to overnutrition, stunting affected approximately 159 million children worldwide in 2014, while an estimated 50 million children were wasted. India is an important front in the fight against malnutrition and is grappling with the coexistence of undernutrition, overnutrition, and micronutrient deficiencies. This report summarizes discussions on trends in malnutrition in India, its evolution in the context of economic growth, intrahousehold aspects, infant and young child feeding practices, women's status, maternal nutrition, and nutrition policymaking. The discussion focuses on a review of trends in malnutrition and dietary intakes in India in the context of economic change over the past four decades, identification of household dynamics affecting food choices and their consequences for family nutritional status in India, and effective malnutrition prevention and treatment interventions and programs in India and associated policy challenges

SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion

Abstract: The B.1.617.2 (Delta) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha)1. In vitro, B.1.617.2 is sixfold less sensitive to serum neutralizing antibodies from recovered individuals, and eightfold less sensitive to vaccine-elicited antibodies, compared with wild-type Wuhan-1 bearing D614G. Serum neutralizing titres against B.1.617.2 were lower in ChAdOx1 vaccinees than in BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies to the receptor-binding domain and the amino-terminal domain. B.1.617.2 demonstrated higher replication efficiency than B.1.1.7 in both airway organoid and human airway epithelial systems, associated with B.1.617.2 spike being in a predominantly cleaved state compared with B.1.1.7 spike. The B.1.617.2 spike protein was able to mediate highly efficient syncytium formation that was less sensitive to inhibition by neutralizing antibody, compared with that of wild-type spike. We also observed that B.1.617.2 had higher replication and spike-mediated entry than B.1.617.1, potentially explaining the B.1.617.2 dominance. In an analysis of more than 130 SARS-CoV-2-infected health care workers across three centres in India during a period of mixed lineage circulation, we observed reduced ChAdOx1 vaccine effectiveness against B.1.617.2 relative to non-B.1.617.2, with the caveat of possible residual confounding. Compromised vaccine efficacy against the highly fit and immune-evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era

A FACS-based novel isolation technique identifies heterogeneous CTCs in oral squamous cell carcinoma

PurposeIsolating circulating tumour cells (CTCs) from the blood is challenging due to their low abundance and heterogeneity. Limitations of conventional CTC detection methods highlight the need for improved strategies to detect and isolate CTCs. Currently, the Food and Drug Administration (FDA)-approved CellSearch™ and other RUO techniques are not available in India. Therefore, we wanted to develop a flexible CTC detection/isolation technique that addresses the limitation(s) of currently available techniques and is suitable for various downstream applications.MethodsWe developed a novel, efficient, user-friendly CTC isolation strategy combining density gradient centrifugation and immuno-magnetic hematogenous cell depletion with fluorescence-activated cell sorting (FACS)-based positive selection using multiple CTC-specific cell-surface markers. For FACS, a stringent gating strategy was optimised to exclude debris and doublets by side scatter/forward scatter (SSC/FSC) discriminator, remove dead cells by 4′,6-diamidino-2-phenylindole (DAPI) staining, and eliminate non-specific fluorescence using a “dump” channel. APC-labelled anti-CD45mAB was used to gate remaining hematogenous cells, while multiple epithelial markers (EpCAM, EGFR, and Pan-Cytokeratin) and an epithelial–mesenchymal transition (EMT) marker (Vimentin) labelled with fluorescein isothiocyanate (FITC) were used to sort cancer cells. The technique was initially developed by spiking Cal 27 cancer cells into the blood of healthy donors and then validated in 95 biopsy-proven oral squamous cell carcinoma (OSCC) patients. CTCs isolated from patients were reconfirmed by Giemsa staining, immuno-staining, and whole transcriptome amplification (WTA), followed by qRT-PCR. In vitro culture and RNA sequencing (RNA-Seq) were also performed to confirm their suitability for various downstream applications.ResultsThe mean detection efficiency for the Cal 27 tongue cancer cells spiked in the whole blood of healthy donors was 32.82% ± 12.71%. While ~75% of our patients (71/95) had detectable CTCs, the CTC positivity was independent of the TNM staging. The isolated potential cancer cells from OSCC patients were heterogeneous in size. They expressed different CTC-specific markers in various combinations as identified by qRT-PCR after WTA in different patients. Isolated CTCs were also found to be suitable for downstream applications like short-term CTC culture and RNA-Seq.ConclusionWe developed a sensitive, specific, flexible, and affordable CTC detection/isolation technique, which is scalable to larger patient cohorts, provides a snapshot of CTC heterogeneity, isolates live CTCs ready for downstream molecular analysis, and, most importantly, is suitable for developing countries

Expression profile of Smad 7, NFkB, IL1B and gastrin in <i>H. pylori</i> infected duodenal ulcer patients (HP+U+) and asymptomatic individuals (HP+U−).

<p>(A) Smad 7 expression is significantly lower in <i>H. pylori</i> infected duodenal ulcer patients (HP+U+) compared to infected asymptomatic individuals (HP+U−): RNA was extracted from gut biopsy samples of <i>H. pylori</i> infected duodenal ulcer patients (HP+U+ = 12) and <i>H. pylori</i> infected asymptomatic individuals (HP+U− = 8). Real time PCR analysis for Smad 7 mRNA was performed from cDNA prepared from those samples. Beta-actin was taken as endogenous control. The mean relative quantification value from each of the group is represented in the graph. (B) NFkB expression is significantly lower HP+U+ group compared to HP+U-: Gut biopsy samples from both the groups were homogenized and lysed for immuno blot with NFKB p50 antibody. Beta-actin was used as input control. Bradford assay was used to quantify the protein and 50 µg was loaded in each well. The mean normalized value from each of the group is represented in the graph. (C) IL1B expression is significantly lower in HP+U+ group compared to HP+U−: RNA was extracted from gut biopsy samples of both the groups. Real time PCR analysis for IL1B was performed from cDNA prepared from those samples. Beta-actin was taken as the endogenous control. The mean relative quantification value from each of the group is represented in the graph. (D) Gastrin expression is moderately higher in HP+U+ group compared to HP+U−: Samples were homogenized and lysed to measure Gastrin-17 levels by ELISA. Bradford assay was used to quantify the protein. The normalized mean value obtained from each group is graphically plotted.</p

Dose dependent effect of IL1B upon Smad7 expression.

<p>(A) Quantitative analysis of IL1B induced <i>Smad7</i> mRNA expression. RNA was extracted from AGS cells treated with variable concentration of recombinant IL1B (0, 2.5, 5, 10 ng/ml) for two hours. Real time PCR analysis for <i>Smad</i> 7 was performed from cDNA prepared from those samples. Beta-actin was taken as the endogenous control. The graph represents the mean of relative quantification measured from three different experiments +_{_} SD. (B) Analysis of IL1B induced Smad 7 protein expression. AGS cells were treated with increasing concentration of recombinant IL1B protein for two hours and then lysed and immunoblotted with Smad7 antibody. The bands were scanned by Image J software and normalized band intensity of Smad7 from three different experiments +_{_} SD was plotted as a histogram. A representative blot is shown.</p

Effect of IL1B promoter polymorphism on Smad 7 expression.

<p>(A) <i>IL1B</i> promoter polymorphisms differentially regulate Smad 7 protein expression in AGS cells. AGS cells were transfected with the IL1B promoter-driven luciferase construct having either the C or T allele at the −31 position (the constructs are represented as the line diagram) and cells were harvested after 48 hours for immuno blot with Smad7 antibody. The band intensity was scanned and normalized with Beta- actin. The normalized band intensity of three different experiments was plotted. (B) <i>IL1B</i> promoter polymorphisms differentially downregulate gastrin via Smad 7, independent of NFkB pathaway. AGS cells were co-transfected with the <i>IL1B</i> promoter-driven luciferase construct having either the C or T allele at the −31 position along with pGas-Luc and after 24 hours of transfection cells were either treated with NBD or left untreated. Beta-Gal plasmid was used as transfection control. Cells were harvested after 48 hours of transfection for luciferase assay. Protein in each case was normalized by Bradford assay. The normalized mean RLU/µg protein +_ SD of three different experiments was plotted. The same lysates were also subjected to immunoblot analysis with Smad 7 and NFkB antibody. Beta-actin was used as input control.</p

Deletion mapping of 240bp human gastrin promoter for IL1B responsive elements.

<p>(A) Schematic diagram of serial deletion of putative gastrin promoter cloned upstream of luciferase reporter gene. The boxes and circle represent different transcription factor binding sites within the 240 bp gastrin promoter. The line diagrams depict the different deletion clones used for the study. (B) The fold inhibition in luciferase activity of different deletion constructs of the gastrin promoter in AGS cells in presence and absence of recombinant IL1B (10 ng/ml). AGS cells were transfected with respective gastrin promoter deletion constructs. Forty-six hours after transfection, cells were either treated or left untreated with 10 ng/ml of IL1B for two hours and then harvested. Luciferase activity was measured and the average fold differences between the treated and untreated sets were plotted from three different experiments.</p

IL1B inhibits Smad 3 phosphorylation thus perturbing its nuclear localization.

<p>AGS cells were treated with increasing concentration of recombinant IL1B protein for two hours and then lysed and immuno-blotted with (A) phosphorylated Smad3 specific antibody (p-SMAD 3) and antibody against Smad 3 protein (SMAD 3). A representative blot is shown. (B) AGS cells were co-transfected with different concentrations pCMV-TAK1 and equal amount of pCMV-TAB1, the two immediate downstream mediator of IL1B, and harvested for immuno-blot analysis with p-SMAD3 antibody after forty eight hours. A representative blot is shown. (C) Nuclear localization of Smad 3 in AGS cells treated with or without IL1B (10 ng/ml). In each panel the left section represents FITC conjugated anti-SMAD 3 antibody and the right panel represents the overlay of DAPI/FITC images captured at that field. The experiment establishes that IL1B inhibits nuclear translocation of Smad 3.</p