Leukocyte Transendothelial Migration Research Articles

Abstract TP242: The Suppression Of Leukocyte Rolling Via Blocking P-selectin After Stroke Is Not Sustained In Mice With Type 2 Diabetes

Introduction: Diabetes is associated with worse stroke outcome, yet the mechanism contributing to a more severe ischemic brain injury is not well understood. Neutrophils invade the cerebral veins as early as 30 minutes after stroke and play a major role in acute ischemic brain injury and thrombosis. Leukocyte rolling on the activated endothelium is an early event of transendothelial migration and parenchymal infiltration of leukocytes including neutrophils, which involves p-selectin. The current study sought to determine the effect of blocking p-selectin in reducing leukocyte rolling in the cerebral vessels of diabetic mice after stroke. Methods: Stroke was induced in 4-month old diabetic db/db and control db/+ mice via middle cerebral artery occlusion (MCAO). P-selectin blocking antibody (P-aby) was administered intravenously 30 min after MCAO at 20 mg/kg. Adherent or rolling leukocytes was quantified at baseline before MCAO and after ischemic reperfusion by repeated 2-photon microscope imaging with fluorescent tracers. Infiltrating neutrophils and plasma soluble p-selectin were determined via FACS and ELISA, respectively. Results: Leukocyte rolling was immediate detected in the veins 1 hr after reperfusion and significantly increased 1d after MCAO in both genotypes of mice. Rolling tended to last days longer in the db/db compared to db/+ mice, and was often seen in the arterioles of the former but not in the latter mice. Increased infiltration of neutrophils was detected in db/db compared to db/+ brain parenchyma 1-3 ds after MCAO by FACS. Three to 5 fold-reduction in rolling by was detected 1 hr after reperfusion in both genotypes following P-aby treatment. However, rolling resumed in db/db mice 1 d after MCAO and antibody treatment, compared to a sustained suppression of rolling seen in the db/+ mice till days later. Elevated soluble p-selectin was also detected in the plasma of db/db compared to db/+ mice. Conclusions: Stroke-induced leukocyte rolling was only temporarily suppressed by P-selectin blocking antibody in db/db mice. Ongoing studies will determine the effect of P-selectin blocking on microthrombi formation, cerebral blood flow, neuroprotection and the role of soluble p-selectin in dampening the treatment effect.

Gene Expression in Embryos From Norwegian Red Bulls With High or Low Non Return Rate: An RNA-Seq Study of in vivo-Produced Single Embryos.

During the last decade, paternal effects on embryo development have been found to have greater importance than previously believed. In domestic cattle, embryo mortality is an issue of concern, causing huge economical losses for the dairy cattle industry. In attempts to reveal the paternal influence on embryo death, recent approaches have used transcriptome profiling of the embryo to find genes and pathways affected by different phenotypes in the bull. For practical and economic reasons, most such studies have used in vitro produced embryos. The aim of the present study was to investigate the differences in the global transcriptome of in vivo produced embryos, derived from sires with either high or low field fertility measured as the non-return rate (NRR) on day 56 after first AI of the inseminated cows. Superovulated heifers (n = 14) in the age span of 12–15 months were artificially inseminated with semen from either high fertility (n = 6) or low fertility (n = 6) bulls. On day seven after insemination, embryos were retrieved through uterine flushing. Embryos with first grade quality and IETS stage 5 (early blastocyst), 6 (blastocyst) or 7 (expanded blastocyst) were selected for further processing. In total, RNA extracted from 24 embryos was sequenced using Illumina sequencing, followed by differential expression analysis and gene set enrichment analysis. We found 62 genes differentially expressed between the two groups (adj.p-value<0.05), of which several genes and their linked pathways could explain the different developmental capacity. Transcripts highly expressed in the embryos from low fertility bulls were related to sterol metabolism and terpenoid backbone synthesis, while transcripts highly expressed in the high fertility embryos were linked to anti-apoptosis and the regulation of cytokine signaling. The leukocyte transendothelial migration and insulin signaling pathways were associated with enrichments in both groups. We also found some highly expressed transcripts in both groups which can be considered as new candidates in the regulation of embryo development. The present study is an important step in defining the paternal influence in embryonic development. Our results suggest that the sire’s genetic contribution affects several important processes linked to pre-and peri implantation regulation in the developing embryo.

Analysis of the Increase of Vascular Cell Adhesion Molecule-1 (VCAM-1) Expression and the Effect of Exposure in a Hyperbaric Chamber on VCAM-1 in Human Blood Serum: A Cross-Sectional Study.

Background and Objectives: Vascular cell adhesion molecule-1 (VCAM-1) was identified as a cell adhesion molecule that helps to regulate inflammation-associated vascular adhesion and the transendothelial migration of leukocytes, such as macrophages and T cells. VCAM-1 is expressed by the vascular system and can be induced by reactive oxygen species, interleukin 1 beta (IL-1β) or tumor necrosis factor alpha (TNFα), which are produced by many cell types. The newest data suggest that VCAM-1 is associated with the progression of numerous immunological disorders, such as rheumatoid arthritis, asthma, transplant rejection and cancer. The aim of this study was to analyze the increase in VCAM-1 expression and the impact of exposure in a hyperbaric chamber to VCAM-1 levels in human blood serum. Materials and Methods: The study included 92 volunteers. Blood for the tests was taken in the morning, from the basilic vein of fasting individuals, in accordance with the applicable procedure for blood collection for morphological tests. In both groups of volunteers, blood was collected before and after exposure, in heparinized tubes to obtain plasma and hemolysate, and in clot tubes to obtain serum. The level of VCAM-1 was determined using the immunoenzymatic ELISA method. Results: The study showed that the difference between the distribution of VCAM-1 before and after exposure corresponding to diving at a depth of 30 m was at the limit of statistical significance in the divers group and that, in most people, VCAM-1 was higher after exposure. Diving to a greater depth had a much more pronounced impact on changes in VCAM-1 values, as the changes observed in the VCAM-1 level as a result of diving to a depth of 60 m were statistically highly significant (p = 0.0002). The study showed an increase in VCAM-1 in relation to the baseline value, which reached as much as 80%, i.e., VCAM-1 after diving was almost twice as high in some people. There were statistically significant differences between the results obtained after exposure to diving conditions at a depth of 60 m and the values measured for the non-divers group. The leukocyte level increased statistically after exposure to 60 m. In contrast, hemoglobin levels decreased in most divers after exposure to diving at a depth of 30 m (p = 0.0098). Conclusions: Exposure in the hyperbaric chamber had an effect on serum VCAM-1 in the divers group and non-divers group. There is a correlation between the tested morphological parameters and the VCAM-1 level before and after exposure in the divers group and the non-divers group. Exposure may result in activation of the endothelium.

Identification of Candidate Gene Signatures and Regulatory Networks in Endometriosis and its Related Infertility by Integrated Analysis.

Endometriosis is a common gynecological disease associated with infertility, and it represents an economic burden worldwide. However, the molecular mechanisms underlying endometriosis development have not yet been fully elucidated. Here, we aimed to identify reliable key genes and the related regulatory network that may be involved in endometriosis. Differentially expressed genes (DEGs) were identified through integrated analysis of four expression datasets of endometriosis from Gene Expression Omnibus. Gene functional analysis and protein-protein interaction network construction were performed to reveal the potential function of DEGs. Subsequently, candidate hub genes were defined and validated in GSE105764 dataset, and the associated regulatory networks were constructed. Additionally, GSE120103 dataset was applied to identify the differential expression between the infertile and fertile groups of patients with stage IV endometriosis. Finally, real-time quantitative polymerase chain reaction analysis was performed to identify the differential expression of hub genes in the collected clinical specimens. Robust rank aggregation integrated analysis determined 158 DEGs. Epithelial cell differentiation was the most significantly enriched biological process, and leukocyte transendothelial migration was the most significantly enriched pathway. Eight hub genes including CLDN3, CLDN5, CLDN7, CLDN11, HOXC8, HOXC6, HOXB6, and HOXB7 were identified, and most of these were validated as abnormally expressed genes in both the infertile group and patients with endometriosis. Transcriptional factors and microRNAs related to these genes were identified. Altogether, our integrated analysis identified critical gene signatures, involved pathways, and regulatory networks, which could provide clinically significant insights into the molecular mechanisms underlying endometriosis and its related infertility.

Integrative transcriptomic and proteomic analysis reveals mechanisms of silica-induced pulmonary fibrosis in rats

BackgroundSilicosis is a systemic disease characterized by persistent inflammation and incurable pulmonary fibrosis. Although great effort has been made to understand the pathogenesis of the disease, molecular mechanism underlying silicosis is not fully elucidated. This study was aimed to explore proteomic and transcriptomic changes in rat model of silicosis.MethodsTwenty male Wistar rats were randomly divided into two groups with 10 rats in each group. Rats in the model group were intratracheally instilled with 50 mg/mL silicon dioxide (1 mL per rat) and rats in the control group were treated with 1.0 mL saline (1 mL per rat). Twenty-eight days later, transcriptomic analysis by microarray and tandem mass tags (TMT)-based proteomic analysis were performed to reveal the expression of mRNAs and proteins in lung tissues. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were applied to analyze the altered genes and proteins. The integrated analysis was performed between transcriptome and proteome. The data were further verified by RT-qPCR and parallel reaction monitoring (PRM).ResultsIn total, 1769 differentially expressed genes (DEGs) and 650 differentially expressed proteins (DEPs) were identified between the silicosis model and control groups. The integrated analysis showed 250 DEPs were correlated to the corresponding DEGs (cor-DEPs-DEGs), which were mainly enriched in phagosome, leukocyte transendothelial migration, complement and coagulation cascades and cellular adhesion molecule (CAM). These pathways are interrelated and converged at common points to produce an effect. GM2a, CHI3L1, LCN2 and GNAI1 are involved in the extracellular matrix (ECM) and inflammation contributing to fibrosis.ConclusionOur comprehensive transcriptome and proteome data provide new insights into the mechanisms of silicosis and helpful information for more targeted prevention and treatment of silicosis.

Monitoring Leukocyte Migration During Atherosclerosis In Vivo.

Transendothelial leukocyte migration is an early event in the progression of vascular inflammation, the underlying molecular mechanism of atherosclerosis. Inflammatory mediators such as adhesion molecules and chemokines are essential in this process. Leukocyte migration into the vascular wall can be monitored by the detection of CD11b-positive immune cells in animal models of atherosclerosis. This chapter will describe an immunohistochemical technique used to evaluate leukocyte migration in vivo.

Gene expression profile analysis reveals the effect of metformin treatment on HepG2 cells

Metformin is a first-line drug in the fight against type 2 diabetes. In recent years, studies have shown that metformin has some preventive and therapeutic effects on liver cancer, but the effects of metformin on the gene expression of liver cancer cells are not fully known. This study focused on the differences in the gene expression profiles in liver cancer cells treated with or without metformin. A total of 153 differentially expressed genes (DEGs) (FC > 2 and q-values < 0.001) were found, including 77 upregulated genes and 76 downregulated genes. These DEGs are involved in mitogen-activated protein kinase (MAPK), nuclear factor-kappa B (NF-κB), cell adhesion molecules (CAMs), and leukocyte transendothelial migration signaling pathways. These findings reveal the effects of metformin treatment on gene expression profiles in liver cancer cells and provide new clues for unveiling the mechanism of the antitumor effects of metformin.

Tertiary lymphoid structure stratifies glioma into three distinct tumor subtypes.

Objective: Tertiary lymphoid structure (TLS), also known as ectopic lymphoid organs, are found in cancer, chronic inflammation, and autoimmune diseases. However, the heterogeneity of TLS in gliomas is unclear. Therefore, it is necessary to identify TLS differences and define TLS subtypes.Methods: The TLS gene profile of 697 gliomas from The Cancer Genome Atlas (TCGA) was used for consensus clustering to identify robust clusters, and the reproducibility of the stratification method was assessed in Chinese Glioma Genome Atlas (CGGA) cohort1, CGGA_cohort2, and GSE16011. Analyses of clinical characteristics, immune infiltration, and potential biological functions were performed for each subtype.Results: Three resulting clusters (A, B, and C) were identified based on consensus clustering on the gene expression profile of TLS genes. There was a significant prognostic difference among the clusters, with a shorter survival for C than B and A. In comparison with the A and B subtypes, the C subtype was significantly enriched in primary immunodeficiency, intestinal immune network for lgG production, antigen processing and presentation, natural killer cell-mediated cytotoxicity, complement and coagulation cascades, cytokine-cytokine receptor interaction, leukocyte transendothelial migration, and some immune-related diseases. The levels of 23 immune cell types were higher in the C subtype than in the A and B subtypes. Finally, we developed and validated a riskscore based on TLS subtypes with better performance of prognosis prediction.Conclusions: This study presents a new stratification method according to the TLS gene profile and highlights TLS heterogeneity in gliomas.

Atomic Level Dissection of the Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1) Homophilic Binding Interface: Implications for Endothelial Cell Barrier Function.

PECAM-1 (platelet endothelial cell adhesion molecule 1) is a 130 kDa member of the immunoglobulin (Ig) gene superfamily that is expressed on the surfaces of platelets and leukocytes and concentrated at the intercellular junctions of confluent endothelial cell monolayers. PECAM-1 Ig domains 1 and 2 (IgD1 and IgD2) engage in homophilic interactions that support a host of vascular functions, including support of leukocyte transendothelial migration and the maintenance of endothelial junctional integrity. The recently solved crystal structure of PECAM-1 IgD1 and IgD2 revealed a number of intermolecular interfaces predicted to play important roles in stabilizing PECAM-1/PECAM-1 homophilic interactions and in formation and maintenance of endothelial cell-cell contacts. We sought to determine whether the protein interfaces implicated in the crystal structure reflect physiologically important interactions. Approach and Results: We assessed the impact of single amino acid substitutions at the interfaces between opposing PECAM-1 molecules on homophilic binding and endothelial cell function. Substitution of key residues within the IgD1-IgD1 and IgD1-IgD2 interfaces but not those within the smaller IgD2-IgD2 interface, markedly disrupted PECAM-1 homophilic binding and its downstream effector functions, including the ability of PECAM-1 to localize at endothelial cell-cell borders, mediate the formation of endothelial tubes, and restore endothelial barrier integrity. Taken together, these results validate the recently described PECAM-1 IgD1/IgD2 crystal structure by demonstrating that specific residues visualized within the IgD1-IgD1 and IgD1-IgD2 interfaces of opposing molecules in the crystal are required for functionally important homophilic interactions. This information can now be exploited to modulate functions of PECAM-1 in vivo.

CD146 as a Prognostic-Related Biomarker in ccRCC Correlating With Immune Infiltrates.

BackgroundsCD146 is highly expressed in various malignant tumors and associated with the poor prognosis. However, the role of CD146 in clear cell renal cell carcinoma (ccRCC) is still unknown. This study aimed to identify the role of CD146 in ccRCC by integrated bioinformatics analysis.MethodsCD146 mRNA expression and methylation data in ccRCC was examined using the TIMER, UALCAN, and MethSurv databases. CD146 expression in paraffin-embedded tissues (140 cancer samples and 140 paracancer tissues) from our cohort were examined by immunohistochemistry assay. The LinkedOmics database was used to study the signaling pathways related to CD146 expression. TIMER and TISIDB were used to analyze the correlations among CD146, CD146-coexpressed genes, tumor-infiltrating immune cells, and immunomodulators. The relationship between CD146 and drug response in renal cancer cell lines was analyzed by the CTRP and CCLE databases.ResultsThe mRNA and protein levels of CD146 were elevated in ccRCC tissues than that in paracancer tissues. The DNA methylation of CD146 in ccRCC tissues were lower than that in normal tissues. Importantly, high CD146 expression was associated with poor prognosis in patients with ccRCC. Furthermore, multivariate Cox regression analysis showed that CD146 was an independent prognostic factor in ccRCC. GO and KEGG pathway analyses indicated the co-expressed genes of CD146 were mainly related to a variety of immune-related pathways, including Th1 and Th2 cell differentiation, Th17 cell differentiation, and leukocyte transendothelial migration. Our data demonstrated that the expression and methylation status of CD146 were strongly correlated with immune infiltration levels, immunomodulators, and chemokines. Further, the sensitivity and resistance of renal cancer cell lines to some drugs were related to CD146 expression.ConclusionsOur study highlights the clinical significance of CD146 in ccRCC and provides novel insights into the immune function of CD146 in the tumor microenvironment.

Proteomics Analysis of Tears and Saliva From Sjogren's Syndrome Patients.

Sjogren’s syndrome (SS) is characterized by dysfunctional mucous membranes and dysregulated moisture-secreting glands resulting in various symptoms, including dry mouth and dry eyes. Here, we wanted to profile and compare the tear and saliva proteomes of SS patients to healthy controls. Tear and saliva samples were collected and subjected to an isotopic dimethylation labeling shotgun proteomics workflow to identify alterations in protein levels. In tear samples, we identified 83 upregulated and 112 downregulated proteins. Pathway enrichment analysis of the changing proteins by Metascape identified leukocyte transendothelial migration, neutrophil degranulation, and post-translation protein phosphorylation in tears of SS patients. In healthy controls’ tears, an enrichment for proteins related to glycolysis, amino acid metabolism and apoptotic signaling pathway were identified. In saliva, we identified 108 upregulated and 45 downregulated proteins. Altered pathways in SS patients’ saliva included cornification, sensory perception to taste and neutrophil degranulation. In healthy controls’ saliva, an enrichment for proteins related to JAK-STAT signaling after interleukin-12 stimulation, phagocytosis and glycolysis in senescence were identified. Dysregulated protease activity is implicated in the initiation of inflammation and immune cell recruitment in SS. We identified 20 proteases and protease inhibitors in tears and 18 in saliva which are differentially expressed between SS patients and healthy controls. Next, we quantified endogenous proteoglycan 4 (PRG4), a mucin-like glycoprotein, in tear wash and saliva samples via a bead-based immune assay. We identified decreased levels of PRG4 in SS patients’ tear wash compared to normal samples. Conversely, in saliva, we found elevated levels of PRG4 concentration and visualized PRG4 expression in human parotid gland via immunohistological staining. These findings will improve our mechanistic understanding of the disease and changes in SS patients’ protein expression will help identify new potential drug targets. PRG4 is among the promising targets, which we identified here, in saliva, for the first time.

Identification of hub genes and transcription factors involved in periodontitis on the basis of multiple microarray analysis.

To identify the differentially expressed genes (DEGs) during the pathogenesis of periodontitis by bioinformatics analysis. GEO2R was used to screen DEGs in GSE10334 and GSE16134. Then, the overlapped DEGs were used for further analysis. g:Profiler was used to perform Gene Ontology analysis and pathway analysis for upregulated and downregulated DEGs. The STRING database was used to construct the protein-protein interaction (PPI) network, which was further visua-lized and analyzed by Cytoscape software. Hub genes and key modules were identified by cytoHubba and MCODE plug-ins, respectively. Finally, transcription factors were predicted via iRegulon plug-in. A total of 196 DEGs were identified, including 139 upregulated and 57 downregulated DEGs. Functional enrichment analysis showed that the upregulated DEGs were mainly enriched in immune-related pathways including immune system, viral protein interaction with cytokine and cytokine receptor, cytokine-cytokine receptor interaction, leukocyte transendothelial migration, and chemokine receptors bind chemokines. On the contrary, the downregulated DEGs were mainly related to the formation of the cornified envelope and keratinization. The identified hub genes in the PPI network were CXCL8, CXCL1, CXCR4, SEL, CD19, and IKZF1. The top three modules were involved in chemokine response, B cell receptor signaling pathway, and interleukin response, respectively. iRegulon analysis revealed that IRF4 scored the highest. The pathogenesis of periodontitis was closely associated with the expression levels of the identified hub genes including CXCL8, CXCL1, CXCR4, SELL, CD19, and IKZF1. IRF4, the predicted transcription factor, might serve as a dominant upstream regulator.

Appearance of claudin-5+ leukocyte subtypes in the blood and CNS during progression of EAE

BackgroundTight junctions (TJs) are membrane specializations characteristic of barrier-forming membranes, which function to seal the aqueous pathway between endothelial cells or epithelial cells and, thereby, obstruct intercellular solute and cellular movement. However, previous work from our laboratory found that claudin-5 (CLN-5), a TJ protein prominent at the blood–brain barrier (BBB), was also detected, ectopically, on leukocytes (CLN-5+) in the blood and central nervous system (CNS) of mice with experimental autoimmune encephalomyelitis (EAE), a neuroinflammatory, demyelinating disease that is a model for multiple sclerosis. CLN-5 was further shown to be transferred from endothelial cells to circulating leukocytes during disease, prompting consideration this action is coupled to leukocyte transendothelial migration (TEM) into the CNS by fostering transient interactions between corresponding leukocyte and endothelial junctional proteins at the BBB.MethodsTo begin clarifying the significance of CLN-5+ leukocytes, flow cytometry was used to determine their appearance in the blood and CNS during EAE.ResultsFlow cytometric analysis revealed CLN-5+ populations among CD4 and CD8 T cells, B cells, monocytes and neutrophils, and these appeared with varying kinetics and to different extents in both blood and CNS. CLN-5 levels on circulating T cells further correlated highly with activation state. And, the percentage of CLN-5+ cells among each of the subtypes analyzed was considerably higher in CNS tissue than in blood, consistent with the interpretation that CLN-5+ leukocytes gain preferred access to the CNS.ConclusionSeveral leukocyte subtypes variably acquire CLN-5 in blood before they enter the CNS, an event that may represent a novel mechanism to guide leukocytes to sites for paracellular diapedesis across the BBB.

Pathway Association Studies Reveal Gene Loci and Pathway Networks that Associated With Plasma Cystatin C Levels

As a marker for glomerular filtration, plasma cystatin C level is used to evaluate kidney function. To decipher genetic factors that control the plasma cystatin C level, we performed genome-wide association and pathway association studies using United Kingdom Biobank data. One hundred fifteen loci yielded p values less than 1 × 10−100, three genes (clusters) showed the most significant associations, including the CST8-CST9 cluster on chromosome 20, the SH2B3-ATXN2 gene region on chromosome 12, and the SHROOM3-CCDC158 gene region on chromosome 4. In pathway association studies, forty significant pathways had FDR (false discovery rate) and or FWER (family-wise error rate) ≤ 0.001: spermatogenesis, leukocyte trans-endothelial migration, cell adhesion, glycoprotein, membrane lipid, steroid metabolic process, and insulin signaling pathways were among the most significant pathways that associated with the plasma cystatin C levels. We also performed Genome-wide association studies for eGFR, top associated genes were largely overlapped with those for cystatin C.

High fat diet induced gut dysbiosis alters corneal epithelial injury response in mice

PurposeCommensal microbiome secretes various metabolites that can exert important effects on the host immunity and inflammation and can alter cellular functions. However, little is known regarding the effect of microbiome on corneal immunity and genetic expression. The purpose of this study is to describe the effect of diet-induced gut dysbiosis on corneal immunity and corneal gene expression after wounding. MethodsThis study is approved by the Animal Care and Use of the University of Illinois. Six-week-old female C57BL6 mice were fed on a normal chow diet (ND), isocaloric low-fat control diet (LFD), or a 21% milk high-fat diet (HFD) for six weeks. 2 mm corneal epithelial debridement was performed (n = 10). Fecal samples from mice were used for microbial diversity analysis (n > 3). Immunofluorescence staining of corneal wholemount tissue post-debridement was used to visualize immune cell distribution. RNA Seq was performed on tissue samples from corneas following debridement. ResultsMice fed differing diets had significant alterations in gut microbial diversities. After corneal debridement, HFD mice experienced delayed wound healing in comparison to LFD mice and ND mice groups. However, fecal transplantation led to normalization of wound closure rates. Increased γδTCR staining was observed in the LFD group, and decreased LY6G was observed in HFD group (p < 0.05). Gene Ontology terms of differentially expressed genes included response to external stimulus, cell proliferation, migration, adhesion, defense response and leukocyte migration. Top over-represented pathways included ECM-receptor interaction, Cytokine-cytokine receptor interaction, Focal adhesion and Leukocyte trans-endothelial migration. ConclusionsGut microbial dysbiosis alters corneal immune cell distribution, corneal response to injury, and genes related to epithelial function and corneal immunity.

Prognostic Potential of Secreted Modular Calcium-Binding Protein 1 in Low-Grade Glioma.

Background: Secreted modular calcium-binding protein 1 (SMOC1) belongs to a family of matricellular proteins; it was involved in embryo development, endothelial cell proliferation, angiogenesis, integrin–matrix interactions, cell adhesion, and regulation of glucose metabolism. Previous studies showed that the expression of SMOC1 was increased in some tumors. However, the prognostic value and the biological function of SMOC1 in tumor remain unclear. Methods: In this study, we explored the expression profile and prognostic value of SMOC1 in pan-cancers, especially glioma, via multiple databases, including Oncomine, Gene Expression Profiling Interactive 2, PrognoScan, Kaplan–Meier plotter, and the Chinese Glioma Genome Atlas database. Furthermore, LinkedOmics was used to identify the genes coexpressed with SMOC1 and to perform Kyoto Encyclopedia of Genes and Genomes pathways and Gene Ontology analysis in low-grade glioma (LGG). Also, the Cancer Single-Cell State Atlas database was used to evaluate the correlation between SMOC1 expression and functional state activities in glioma cells. In addition, the Tumor Immune Estimation Resource and TISIDB databases were used to evaluate the correlations between SMOC1 expression and tumor-infiltrating immune cells in the tumor microenvironment. Results: Compared with normal brain tissues, the expression of SMOC1 was increased in LGG tissues. The higher expression of SMOC1 was significantly correlated with better survival of LGG patients. Additionally, functional analyses showed that the SMOC1 coexpressed genes were inhibited in processes such as response to type I interferon and interferon-gamma, lymphocyte-mediated immunity, leukocyte migration, adaptive immune response, neutrophil-mediated immunity, T cell activation, and pathways including EMC–receptor interaction, Th17 cell differentiation, and leukocyte trans-endothelial migration in LGG. Moreover, the expression of SMOC1 was correlated with stemness, hypoxia, EMT, and metastasis of glioma cells. Additionally, the expression of SMOC1 expression was negatively correlated with levels of infiltrating B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils and dendritic cells, and gene markers of most immune cells in LGG. Conclusion: Our results suggest that SMOC1 could be a potential biomarker to determine prognosis and might play a specific role in the tumor microenvironment of glioma, thereby influencing the development and progression of glioma. These findings provide some new insights for further investigation.

High-dose dexamethasone injection disordered metabolism and multiple protein kinases expression in the mouse kidney.

Glucocorticoids (GCs) have been widely used in clinical treatment as anti-inflammatory, anti-shock and immunosuppressive medicines. However, the effect of excessive GCs on immune response and metabolism of kidney remains unclear. Here, we profiled the gene expression of kidney from mice with high-dose dexamethasone (DEX) treatment. A total of 1193 differentially expressed genes (DEGs) were screened in DEX treatment group compared with the saline group, including 715 down- regulated and 478 up-regulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of these DEGs showed extracellular matrix (ECM)–receptor interaction, cell adhesion molecules signaling pathway were significantly enriched, and that the vast majority of DEGs were involved in monocarboxylic acid metabolism, leukocyte cell–cell adhesion and fatty acid metabolism. Gene set enrichment analysis (GSEA) revealed that DEGs were strongly associated with immune-response and cell adhesion gene sets, such as Fc γ R-mediated phagocytosis, leukocyte transendothelial migration, T-cell receptor signaling pathway, cell adhesion, ECM–receptor interaction and focal adhesion-associated pathways. KEGG pathway analysis of differentially expressed kinases (DEKs) showed T-cell receptor and forkhead box class O signaling pathway were enriched. Furthermore, we found multiple protein kinases expression were dysregulated greatly after dexamethasone treatment, including classical effector of GCs stimulation-serum and GC-regulated kinase. These protein kinases are involved in multiple signaling pathways in mice kidney, such as mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. We profiled the gene expression of the kidney from high-dose dexamethasone-treated mice and provided important information for further study the mechanism of side effects of GCs in clinical therapy.

Maternal nematode infection upregulates expression of Th2/Treg and diapedesis related genes in the neonatal brain

Intestinal nematode infections common during pregnancy have recently been shown to have impacts that extend to their uninfected offspring including altered brain gene expression. If maternal immune signals reach the neonatal brain, they might alter neuroimmune development. We explored expression of genes associated with four distinct types of T cells (Th1, Th2, Th17, Treg) and with leukocyte transendothelial migration and endocytosis transport across the blood–brain barrier (BBB) in the postnatal brain of offspring of nematode-infected mice, through secondary analysis of a whole brain gene expression database. Th1/Th17 expression was lowered by maternal infection as evidenced by down-regulated expression of IL1β, Th1 receptors and related proteins, and of IL22 and several Th17 genes associated with immunopathology. In contrast, Th2/Treg related pathways were upregulated as shown by higher expression of IL4 and TGF-β family genes. Maternal infection also upregulated expression of pathways and integrin genes involved in transport of leukocytes in between endothelial cells but downregulated endosome vesicle formation related genes that are necessary for endocytosis of immunoglobulins across the BBB. Taken together, pup brain gene expression indicates that maternal nematode infection enhanced movement of leukocytes across the neonatal BBB and promoted a Th2/Treg environment that presumably minimizes the proinflammatory Th1 response in the pup brain.

Characterisation of the Tumour Proteome in Primary Extramedullary Multiple Myeloma Identifies Key Proteins Associated with Transendothelial Migration

Introduction: Extramedullary multiple myeloma (EMM) refers to the spread of clonal plasma cells to tissues extending outside of the bone marrow microenvironment. EMM is present at the time of diagnosis in 6-10% of patients, however, this increases to 13-26% in patients with disease progression and relapse. Cancer cells are suspected to spread to new tissues and organs via the circulatory system as a result of molecular changes that allow malignant cells to escape the bone marrow (BM). For example, downregulation of CXCR4 (C-X-C Motif Chemokine Receptor 4), an important factor in cellular homing to the BM, has frequently been reported to be linked to the EMM phenotype. In addition, the majority of patients presenting with EMM have highly complex cytogenetic abnormalities and high-risk cytogenetic markers such as t(14;16). As EMM is an indicator of a more aggressive disease, more intensive treatment, including combination chemotherapy, is often recommended. Many of the underlying molecular mechanisms accompanying EMM are yet to be characterised. Our mass-spectrometry (MS)-based proteomic study provides insight into the unique molecular mechanisms associated with EMM, identifying key proteins linked to the progression of medullary multiple myeloma (MM) to EMM.Methods: Label-free liquid chromatography mass spectrometric analysis of age and gender matched medullary MM (n=8) and EMM (n=9) bone-marrow derived mononuclear cells (MNCs) was carried out using a Thermo Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific). Proteome Discoverer 2.2 using Sequest HT (Thermo Fisher Scientific) and a percolator were employed for the identification of peptides and proteins. For protein identification, the following search parameters were used: (i) 0.02 Da for MS/MS mass tolerance, (ii) 10 ppm for peptide mass tolerance, (iii) variable modification settings for methionine oxidation, (iv) fixed modification settings in relation to carbamido-methylation and (v) tolerance for up to two missed cleavages. Peptide probability was set to high confidence. Datasets were imported into Progenesis QI (version 2.0) software for further analysis. Data was filtered based on an ANOVA p-value of ≤0.05, fold change >1.5 between experimental groups, and proteins with ≥1 unique peptides contributing to the identification. Proteins with less than 70% valid values were removed from the analysis. G:profiler and STRING were utilized for functional enrichment and the characterisation of protein interaction patterns.Results: Our quantitative MS-based proteomic analysis identified a total of 492 proteins with significantly altered abundances between EMM and MM bone marrow MNC. Of these significant proteins, 275 were found to be increased in EMM compared to medullary MM and 217 were found to be decreased in EMM compared to medullary MM. Hierarchical clustering was performed to highlight the proteomic profile associated with extramedullary disease (Figure 1A). KEGG pathway analysis and gene ontology (GO) analysis of proteins found to be increased in EMM indicated an increase in proteins associated with cell adhesion, invasion, and migration pathways (Figure 1B). Interestingly, several proteins involved in leukocyte transendothelial migration were significantly increased in EMM indicating their potential involvement in the dissemination of MM cells from the bone marrow microenvironment to distal tissues (Figure 1C). Among the proteins found to be involved in this biological pathway was junctional adhesion molecule-A (F11R), a protein previously reported to play a role in EMD pathophysiology [1]. Other proteins involved in MM invasion and migration including Rho-associated protein kinase 2 (ROCK2), Ras-related C3 botulinum toxin substrate 1 (Rac1) and platelet endothelial cell adhesion molecule (PECAM-1) were significantly increased in EMM.Conclusion: Using high-resolution mass spectrometry to characterise the tumour proteome of MM patients with extramedullary disease, we have identified a significant increase in the abundance of proteins associated with leukocyte transendothelial invasion in primary EMM samples. Our study provides further insight into the molecular mechanisms within EMM and thus holds potential to enhance current efforts to provide a more personalised therapeutic approach for EMM patients.

Screening of characteristic genes in ulcerative colitis by integrating gene expression profiles

BackgroundThis study aimed to screen the feature modules and characteristic genes related to ulcerative colitis (UC) and construct a support vector machine (SVM) classifier to distinguish UC patients.MethodsFour datasets that contained UC and control samples were obtained from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) with consistency were screened via the MetaDE method. The weighted gene coexpression network (WGCNA) was used to distinguish significant modules based on the four datasets. The protein–protein interaction network was established based on intersection genes. Enrichment analysis of Gene Ontology (GO) biological processes (BPs) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were established based on DAVID. An SVM combined with recursive feature elimination was also applied to construct a disease classifier for the disease diagnosis of UC patients. The efficacy of the SVM classifier was evaluated through receiver operating characteristic curves.ResultsTwelve highly preserved modules were obtained using the WGCNA, and 2009 DEGs with significant consistency were selected using the MetaDE method. Sixteen significantly related GO BPs and 12 KEGG pathways were obtained, such as cytokine-cytokine receptor interaction, cell adhesion molecules, and leukocyte transendothelial migration. Subsequently, 41 genes were used to construct an SVM classifier, such as CXCL1, CCR2, IL1B, and IL1A. The area under the curve (AUC) was 0.999 in the training dataset, whereas the AUC was 0.886, 0.790, and 0.819 in the validation set (GSE65114, GSE37283, and GSE36807, respectively).ConclusionsAn SVM classifier based on feature genes might correctly identify healthy people or UC patients.