Proteomic profiling of the mesenteric lymph after hemorrhagic shock: Differential gel electrophoresis and mass spectrometry analysis

Experiments show that upon traumatic injury the composition of mesenteric lymph changes such that it initiates an immune response that can ultimately result in multiple organ dysfunction syndrome (MODS). To identify candidate protein mediators of this process we carried out a quantitative proteomic study on mesenteric lymph from a well characterized rat shock model. We analyzed three animals using analytical 2D differential gel electrophoresis. Intra-animal variation for the majority of protein spots was minor. Functional clustering of proteins revealed changes arising from several global classes that give novel insight into fundamental mechanisms of MODS. Mass spectrometry based proteomic analysis of proteins in mesenteric lymph can effectively be used to identify candidate mediators and loss of protective agents in shock models

Cross-talk between red blood cells and plasma influences blood flow and omics phenotypes in severe COVID-19

Coronavirus disease 2019 (COVID-19) is caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and can affect multiple organs, among which is the circulatory system. Inflammation and mortality risk markers were previously detected in COVID-19 plasma and red blood cells (RBCs) metabolic and proteomic profiles. Additionally, biophysical properties, such as deformability, were found to be changed during the infection. Based on such data, we aim to better characterize RBC functions in COVID-19. We evaluate the flow properties of RBCs in severe COVID-19 patients admitted to the intensive care unit by using microfluidic techniques and automated methods, including artificial neural networks, for an unbiased RBC analysis. We find strong flow and RBC shape impairment in COVID-19 samples and demonstrate that such changes are reversible upon suspension of COVID-19 RBCs in healthy plasma. Vice versa, healthy RBCs resemble COVID-19 RBCs when suspended in COVID-19 plasma. Proteomics and metabolomics analyses allow us to detect the effect of plasma exchanges on both plasma and RBCs and demonstrate a new role of RBCs in maintaining plasma equilibria at the expense of their flow properties. Our findings provide a framework for further investigations of clinical relevance for therapies against COVID-19 and possibly other infectious diseases

Cross-talk between red blood cells and plasma influences blood flow and omics phenotypes in severe COVID-19

Coronavirus disease 2019 (COVID-19) is caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and can affect multiple organs, among which is the circulatory system. Inflammation and mortality risk markers were previously detected in COVID-19 plasma and red blood cells (RBCs) metabolic and proteomic profiles. Additionally, biophysical properties, such as deformability, were found to be changed during the infection. Based on such data, we aim to better characterize RBC functions in COVID-19. We evaluate the flow properties of RBCs in severe COVID-19 patients admitted to the intensive care unit by using microfluidic techniques and automated methods, including artificial neural networks, for an unbiased RBC analysis. We find strong flow and RBC shape impairment in COVID-19 samples and demonstrate that such changes are reversible upon suspension of COVID-19 RBCs in healthy plasma. Vice versa, healthy RBCs resemble COVID-19 RBCs when suspended in COVID-19 plasma. Proteomics and metabolomics analyses allow us to detect the effect of plasma exchanges on both plasma and RBCs and demonstrate a new role of RBCs in maintaining plasma equilibria at the expense of their flow properties. Our findings provide a framework for further investigations of clinical relevance for therapies against COVID-19 and possibly other infectious diseases

Interleukin-1 homologues IL-1F7b and IL-18 contain functional mRNA instability elements within the coding region responsive to lipopolysaccharide

IL-1F7b, a novel homologue of the IL-1 (interleukin 1) family, was discovered by computational cloning. We demonstrated that IL-1F7b shares critical amino acid residues with IL-18 and binds to the IL-18-binding protein enhancing its ability to inhibit IL-18-induced interferon-γ. We also showed that low levels of IL-1F7b are constitutively present intracellularly in human blood monocytes. In this study, we demonstrate that similar to IL-18, both mRNA and intracellular protein expression of IL-1F7b are up-regulated by LPS (lipopolysaccharide) in human monocytes. In stable transfectants of murine RAW264.7 macrophage cells, there was no IL-1F7b protein expression despite a highly active CMV promoter. We found that IL-1F7b-specific mRNA was rapidly degraded in transfected cells, via a 3′-UTR (untranslated region)-independent control of IL-1F7b transcript stability. After LPS stimulation, there was a rapid transient increase in IL-1F7b-specific mRNA and concomitant protein levels. Using sequence alignment, we found a conserved ten-nucleotide homology box within the open reading frame of IL-F7b, which is flanking the coding region instability elements of some selective genes. In-frame deletion of downstream exon 5 from the full-length IL-1F7b cDNA markedly increased the levels of IL-1F7b mRNA. A similar coding region element is located in IL-18. When transfected into RAW264.7 macrophages, IL-18 mRNA was also unstable unless treated with LPS. These results indicate that both IL-1F7b and IL-18 mRNA contain functional instability determinants within their coding region, which influence mRNA decay as a novel mechanism to regulate the expression of IL-1 family members

Red Blood Cells from Individuals with Lesch–Nyhan Syndrome: Multi-Omics Insights into a Novel S162N Mutation Causing Hypoxanthine-Guanine Phosphoribosyltransferase Deficiency

Lesch–Nyhan syndrome (LN) is an is an X-linked recessive inborn error of metabolism that arises from a deficiency of purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). The disease manifests severely, causing intellectual deficits and other neural abnormalities, hypercoagulability, uncontrolled self-injury, and gout. While allopurinol is used to alleviate gout, other symptoms are less understood, impeding treatment. Herein, we present a high-throughput multi-omics analysis of red blood cells (RBCs) from three pediatric siblings carrying a novel S162N HPRT1 mutation. RBCs from both parents—the mother, a heterozygous carrier, and the father, a clinically healthy control—were also analyzed. Global metabolite analysis of LN RBCs shows accumulation of glycolytic intermediates upstream of pyruvate kinase, unsaturated fatty acids, and long chain acylcarnitines. Similarly, highly unsaturated phosphatidylcholines are also elevated in LN RBCs, while free choline is decreased. Intracellular iron, zinc, selenium, and potassium are also decreased in LN RBCs. Global proteomics documented changes in RBC membrane proteins, hemoglobin, redox homeostasis proteins, and the enrichment of coagulation proteins. These changes were accompanied by elevation in protein glutamine deamidation and methylation in the LN children and carrier mother. Treatment with allopurinol incompletely reversed the observed phenotypes in the two older siblings currently on this treatment. This unique data set provides novel opportunities for investigations aimed at potential therapies for LN-associated sequelae

OLT1177, a beta-sulfonyl nitrile compound, safe in humans, inhibits the NLRP3 inflammasome and reverses the metabolic cost of inflammation

Contains fulltext : 183973.pdf (publisher's version ) (Open Access

Hypertonic saline attenuates the cytokine-induced pro-inflammatory signature in primary human lung epithelia.

Trauma/hemorrhagic shock is a complex physiological phenomenon that leads to dysregulation of many molecular pathways. For over a decade, hypertonic saline (HTS) has been used as an alternative resuscitation fluid in the setting of trauma/hemorrhagic shock. In addition to restoring circulating volume within the vascular space, studies have shown a positive immunomodulatory effect of HTS. Targeted studies have shown that HTS affects the transcription of several pro-inflammatory cytokines by inhibiting the NF-κB-IκB pathway in model cell lines and rats. However, few studies have been undertaken to assess the unbiased effects of HTS on the whole transcriptome. This study was designed to interrogate the global transcriptional responses induced by HTS and provides insight into the underlying molecular mechanisms and pathways affected by HTS. In this study, RNA sequencing was employed to explore early changes in transcriptional response, identify key mediators, signaling pathways, and transcriptional modules that are affected by HTS in the presence of a strong inflammatory stimulus. Our results suggest that primary human small airway lung epithelial cells (SAECS) exposed to HTS in the presence and absence of a strong pro-inflammatory stimulus exhibit very distinct effects on cellular response, where HTS is highly effective in attenuating cytokine-induced pro-inflammatory responses via mechanisms that involve transcriptional regulation of inflammation which is cell type and stimulus specific. HTS is a highly effective anti-inflammatory agent that inhibits the chemotaxis of leucocytes towards a pro-inflammatory gradient and may attenuate the progression of both the innate and adaptive immune response

Secretory phospholipase A2 is required to produce histologic changes associated with gastroduodenal reflux in a murine model

ObjectiveThe earliest response of esophageal mucosa to gastric reflux is the development of oxidative damage and inflammation. These processes contribute to the development of metaplasia known as Barrett's esophagus, as well as the progression to malignancy. Secretory phospholipase A2 is a mediator of inflammation with levels that are increased in Barrett's metaplasia and carcinoma when compared with levels in normal samples. Our goal is to determine the role of secretory phospholipase A2 in the development of reflux-associated changes in the esophageal mucosa.MethodsSecretory phospholipase A2–deficient mice (C57BL/6, n = 5) and mice known to express high levels of secretory phospholipase A2 (BALB/c, n = 5) underwent side-to-side surgical anastomosis of the first portion of the duodenum and gastroesophageal junction, allowing exposure of esophageal mucosa to duodenal and gastric contents duodeno-gastroesophageal anastomosis. Control animals (n = 5) of each strain underwent laparotomy with esophagotomy and repair. Tissue was frozen in embedding medium. Hematoxylin and eosin staining and Ki67 and secretory phospholipase A2 immunohistochemistry were used to evaluate esophageal tissue and its response to duodeno-gastroesophageal anastomosis.ResultsImmunofluorescent staining confirmed the absence of secretory phospholipase A2 in C57BL/6 mice and its presence in BALB/c mice. Hematoxylin and eosin staining demonstrated significant thickening of the esophageal mucosa in response to gastroesophageal reflux in the presence of secretory phospholipase A2. Mice known to express high levels of secretory phospholipase A2 also demonstrated increased numbers of proliferating cells. Secretory phospholipase A2–deficient mice were immune to the early changes induced by mixed reflux.ConclusionsThe presence of secretory phospholipase A2 appears necessary for early histologic changes produced by exposure of the esophagus to gastroduodenal contents. This enzyme is identified as a promising target for evaluation of mechanisms of carcinogenesis and chemoprevention of esophageal carcinoma