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Host polymorphisms of immune regulatory genes as risk factors for gastric cancer

The infection of the stomach with the gramnegative bacterium Helicobacter pylori is the main risk factor for the development of gastric cancer (GC). This led to the classification of this germ as "definite carcinogen" by the World Health Organization in 1994. The current model of gastric carcinogenesis is based on the interaction of multiple risk factors including virulence factors of the bacterium (e.g. CagA, VacA), environmental factors (diet, smoking) and host factors (gene polymorphisms). The complex interplay among these factors determines the clinical outcome of the infection leading to at least one of three major diseases in 1 out of 7 infected persons, namely ulcer disease, GC and "mucosa-associated lymphoid tissue" lymphoma in 15 %, 1% and 0.1% of all persons infected with H. pylori, respectively. Recently, an increasing number of genomic polymorphisms, mostly single nucleotide polymorphisms have been identified as risk factors for gastric cancer. Among them are genes encoding for cytokines, pattern recognition receptors, cell cycle-regulators, proteases, HLA-molecules, and enzymes for detoxification. In the last years it has become clear that an uniform "genomic risk pattern" for all GC patients does not exist. Most of these host factors are restricted either to the histological type (intestinal vs. diffuse), ethnical background (particularly Caucasian vs. Asian) and tumor localization (non-cardia vs. cardia cancer). Here, we review the current knowledge about the role of host factors for the gastric carcinogenesis focusing on immune-regulatory genes, in particular on the cytokine interleukln-1beta.

Analysis of cytotoxic effects of chemotherapeutic agents on lung and small intestinal neuroendocrine cell lines

Aim: Survival rates for gastrointestinal and bronchopulmonary neuroendocrine tumors (NETs) have not significantly altered in the past thirty years, while the icidence of these tumors continues to rise. Treatment protocols are based on data from clinical trials of non-neuroendocrine malignancies and no systematic analysis of chemotherapeutic agents has been undertaken in NET cell lines. Methods: The cytotoxic effects of ten common chemotherapeutic agents were assessed by MTT uptake and cell cycle distribution on three NET cell lines, NCI-H720 (atypical lung carcinoid), NCI-H727 (bronchopulmonary - typical carcinoid) and KRJ-I (small intestinal - typical carcinoid). Results: All three cell lines were sensitive to topetecan, NCI-H720 was most potently inhibited by topotecan (IC50 2.3 nM). Cyclophosphamide, bortezomib and paclitaxel all inhibited the cell cycle (P < 0.05). NCI-H727 also responded to topotecan (IC50 4.9 nM) and was also sensitive to doxorubucin liposomal (IC50 9.6 nM), cyclophosphamide (IC50 9.3 nM) and cisplatin (IC50 9.3 nM)(all P < 0.05) but resistant to sirolimus, gefitinib, paclitaxel, and doxorubicin hydrochloride. Borterzomib caused NCI-H727 cell cycle arrest (P < 0.05). KRJ-I responded to paclitaxel, topotecan, bortezomib, and cyclophosphamide (IC50 2-10 nM)(all P < 0.05) and poorly to doxorubucin liposomal. The KRJ-1 cell cycle was only altered by topotecan (G,/S arrest). Conclusion: Differences were evident in anti-proliferative responses between lung and intestinal NETs and between atypical and typical lung/ bronchial NET cell lines. This study provides evidence that individual (site and cell specific) NETs will respond differently to specific agents indicating that the use of specific agents for individual lesions is necessary to effectively treat NETs. © 2008 MedUnion Press.

Early diagnosis and prevention of gastric cancer - At least for high risk patients endoscopic screening is mandatory

Gastric cancer at the first clinical presentation and diagnosis is often in advanced and uncurable stage. This underscores the need for effective means of early diagnosis and prevention. Although being the most effective technique for early diagnosis of gastric cancer, endoscopy is not recommended in Europe for a general mass-screening due to a poor cost-effectiveness. Cheap and easy applicable techniques are needed, e.g. serological tests, to preselect patients at high risk who would be candidates for a targeted endoscopic screening. Helicobacter pylori infection is the most relevant risk factor for the development of gastric cancer, which is underscored by a clear causal relationship. H. pylori eradication as strategy for an effective prevention has so far only be applied selectively for defined risk groups. COX-inhibitors and modulators of gastrin-dependent pathways are under evaluation of their potential to inhibit gastric carcinogenesis. Moreover, dietary and - more than that - host factors like mutations of the e-cadherin-gene have to be considered to generate an individual risk profile for each patient.

Biologic Therapy for Inflammatory Bowel Disease: Real-World Comparative Effectiveness and Impact of Drug Sequencing in 13 222 Patients within the UK IBD BioResource.

BACKGROUND AND AIMS: This study compares the effectiveness of different biologic therapies and sequences in patients with inflammatory bowel disease [IBD] using real-world data from a large cohort with long exposure. METHODS: Demographic, disease, treatment, and outcome data were retrieved for patients in the UK IBD BioResource. Effectiveness of treatment was based on persistence free of discontinuation or failure, analysed by Kaplan-Meier survival analysis with inverse probability of treatment weighting to adjust for differences between groups. RESULTS: In total, 13 222 evaluable patients received at least one biologic. In ulcerative colitis [UC] first-line vedolizumab [VDZ] demonstrated superior effectiveness over 5 years compared to anti-tumour necrosis factor [anti-TNF] agents [p = 0.006]. VDZ was superior to both infliximab [IFX] and adalimumab [ADA] after ADA and IFX failure respectively [p 

Baseline Expression of Immune Gene Modules in Blood is Associated With Primary Response to Anti-TNF Therapy in Crohn's Disease Patients.

BACKGROUND AND AIMS: Anti-tumour necrosis factor [anti-TNF] therapy is widely used for the treatment of inflammatory bowel disease, yet many patients are primary non-responders, failing to respond to induction therapy. We aimed to identify blood gene expression differences between primary responders and primary non-responders to anti-TNF monoclonal antibodies [infliximab and adalimumab], and to predict response status from blood gene expression and clinical data. METHODS: The Personalised Anti-TNF Therapy in Crohn's Disease [PANTS] study is a UK-wide prospective observational cohort study of anti-TNF therapy outcome in anti-TNF-naive Crohn's disease patients [ClinicalTrials.gov identifier: NCT03088449]. Blood gene expression in 324 unique patients was measured by RNA-sequencing at baseline [week 0], and at weeks 14, 30, and 54 after treatment initiation [total sample size = 814]. RESULTS: After adjusting for clinical covariates and estimated blood cell composition, baseline expression of major histocompatibility complex, antigen presentation, myeloid cell enriched receptor, and other innate immune gene modules was significantly higher in anti-TNF responders vs non-responders. Expression changes from baseline to week 14 were generally of consistent direction but greater magnitude [i.e. amplified] in responders, but interferon-related genes were upregulated uniquely in non-responders. Expression differences between responders and non-responders observed at week 14 were maintained at weeks 30 and 54. Prediction of response status from baseline clinical data, cell composition, and module expression was poor. CONCLUSIONS: Baseline gene module expression was associated with primary response to anti-TNF therapy in PANTS patients. However, these baseline expression differences did not predict response with sufficient sensitivity for clinical use.

HLA-DP on Epithelial Cells Enables Tissue Damage by NKp44+ Natural Killer Cells in Ulcerative Colitis.

BACKGROUND & AIMS: Ulcerative colitis (UC) is characterized by severe inflammation and destruction of the intestinal epithelium, and is associated with specific risk single nucleotide polymorphisms in HLA class II. Given the recently discovered interactions between subsets of HLA-DP molecules and the activating natural killer (NK) cell receptor NKp44, genetic associations of UC and HLA-DP haplotypes and their functional implications were investigated. METHODS: HLA-DP haplotype and UC risk association analyses were performed (UC: n = 13,927; control: n = 26,764). Expression levels of HLA-DP on intestinal epithelial cells (IECs) in individuals with and without UC were quantified. Human intestinal 3-dimensional (3D) organoid cocultures with human NK cells were used to determine functional consequences of interactions between HLA-DP and NKp44. RESULTS: These studies identified HLA-DPA1∗01:03-DPB1∗04:01 (HLA-DP401) as a risk haplotype and HLA-DPA1∗01:03-DPB1∗03:01 (HLA-DP301) as a protective haplotype for UC in European populations. HLA-DP expression was significantly higher on IECs of individuals with UC compared with controls. IECs in human intestinal 3D organoids derived from HLA-DP401pos individuals showed significantly stronger binding of NKp44 compared with HLA-DP301pos IECs. HLA-DP401pos IECs in organoids triggered increased degranulation and tumor necrosis factor production by NKp44+ NK cells in cocultures, resulting in enhanced epithelial cell death compared with HLA-DP301pos organoids. Blocking of HLA-DP401-NKp44 interactions (anti-NKp44) abrogated NK cell activity in cocultures. CONCLUSIONS: We identified an UC risk HLA-DP haplotype that engages NKp44 and activates NKp44+ NK cells, mediating damage to intestinal epithelial cells in an HLA-DP haplotype-dependent manner. The molecular interaction between NKp44 and HLA-DP401 in UC can be targeted by therapeutic interventions to reduce NKp44+ NK cell-mediated destruction of the intestinal epithelium in UC.

A Roadmap for the Human Gut Cell Atlas.

The number of studies investigating the human gastrointestinal tract using various single-cell profiling methods has increased substantially in the past few years. Although this increase provides a unique opportunity for the generation of the first comprehensive Human Gut Cell Atlas (HGCA), there remains a range of major challenges ahead. Above all, the ultimate success will largely depend on a structured and coordinated approach that aligns global efforts undertaken by a large number of research groups. In this Roadmap, we discuss a comprehensive forward-thinking direction for the generation of the HGCA on behalf of the Gut Biological Network of the Human Cell Atlas. Based on the consensus opinion of experts from across the globe, we outline the main requirements for the first complete HGCA by summarizing existing data sets and highlighting anatomical regions and/or tissues with limited coverage. We provide recommendations for future studies and discuss key methodologies and the importance of integrating the healthy gut atlas with related diseases and gut organoids. Importantly, we critically overview the computational tools available and provide recommendations to overcome key challenges.

Anti-TNF Biologicals Enhance the Anti-Inflammatory Properties of IgG N-Glycome in Crohn's Disease.

Crohn's disease (CD) is a chronic inflammation of the digestive tract that significantly impairs patients' quality of life and well-being. Anti-TNF biologicals revolutionised the treatment of CD, yet many patients do not adequately respond to such therapy. Previous studies have demonstrated a pro-inflammatory pattern in the composition of CD patients' immunoglobulin G (IgG) N-glycome compared to healthy individuals. Here, we utilised the high-throughput UHPLC method for N-glycan analysis to explore the longitudinal effect of the anti-TNF drugs infliximab and adalimumab on N-glycome composition of total serum IgG in 198 patients, as well as the predictive potential of IgG N-glycans at baseline to detect primary non-responders to anti-TNF therapy in 1315 patients. We discovered a significant decrease in IgG agalactosylation and an increase in monogalactosylation, digalactosylation and sialylation during the 14 weeks of anti-TNF treatment, regardless of therapy response, all of which suggested a diminished inflammatory environment in CD patients treated with anti-TNF therapy. Furthermore, we observed that IgG N-glycome might contain certain information regarding the anti-TNF therapy outcome before initiating the treatment. However, it is impossible to predict future primary non-responders to anti-TNF therapy based solely on IgG N-glycome composition at baseline.

Phospho-dependent functional modulation of GABA(B) receptors by the metabolic sensor AMP-dependent protein kinase.

GABA(B) receptors are heterodimeric G protein-coupled receptors composed of R1 and R2 subunits that mediate slow synaptic inhibition in the brain by activating inwardly rectifying K(+) channels (GIRKs) and inhibiting Ca(2+) channels. We demonstrate here that GABA(B) receptors are intimately associated with 5'AMP-dependent protein kinase (AMPK). AMPK acts as a metabolic sensor that is potently activated by increases in 5'AMP concentration that are caused by enhanced metabolic activity, anoxia, or ischemia. AMPK binds the R1 subunit and directly phosphorylates S783 in the R2 subunit to enhance GABA(B) receptor activation of GIRKs. Phosphorylation of S783 is evident in many brain regions, and is increased dramatically after ischemic injury. Finally, we also reveal that S783 plays a critical role in enhancing neuronal survival after ischemia. Together our results provide evidence of a neuroprotective mechanism, which, under conditions of metabolic stress or after ischemia, increases GABA(B) receptor function to reduce excitotoxicity and thereby promotes neuronal survival.

Studying the Localization, Surface Stability and Endocytosis of Neurotransmitter Receptors by Antibody Labeling and Biotinylation Approaches

Alterations in the activity of post-synaptic neurotransmitter receptors including α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, N-methyl-D-aspartate (NMDA) receptors, kainate receptors and gamma hydroxybutric acid type A (GABAA) receptors make an important contribution to modulation of synapse strength, neuronal excitability and the plasticity of synapses. Changes in the strength of neurotransmitter receptor signalling are thought to be achieved, in part, by the modulation of channel gating and conductance or by regulating the number or location of receptors expressed on cell surface and synaptic membranes. Neurotransmitter receptor trafficking is now recognized as a key mechanism for altering the strength of synapses during synaptic plasticity by changing synaptic receptor number [1]. Here, we focus on receptor membrane trafficking and, in particular, receptor endocytosis as a mechanism to regulate the number of surface and synaptic neurotransmitter receptors. We briefly review some general cellular mechanisms that underlie surface membrane protein internalization and then discuss in detail some of the cell biological approaches that have been important for studies of neurotransmitter receptor trafficking.

Calcium insensitivity of FA-6, a cell line derived from a pancreatic cancer associated with humoral hypercalcemia, is mediated by the significantly reduced expression of the Calcium Sensitive Receptor transduction component p38 MAPK.

The Calcium-Sensing Receptor is a key component of Calcium/Parathyroid hormone homeostatic system that helps maintain appropriate plasma Ca2+ concentrations. It also has a number of non-homeostatic functions, including cell cycle regulation through the p38 MAPK pathway, and recent studies have indicated that it is required for Ca2+ mediated growth arrest in pancreatic carcinoma cells. Some pancreatic cancers produce pathogenic amounts of parathyroid like hormones, however, which significantly increase Ca2+ plasma concentrations and might be expected to block further cell growth. In this study we have investigated the expression and function of the p38 MAPK signaling pathway in Ca2+ sensitive (T3M-4) and insensitive (FA6) pancreatic cancer cell lines. FA-6 cells, which are derived from a pancreatic adenocarcinoma that secretes a parathyroid hormone related peptide, exhibit only very low levels of p38 MAPK expression, relative to T3M-4 cells. Transfecting FA-6 cells with a p38 MAPK expression construct greatly increases their sensitivity to Ca2+. Furthermore, the reduction of p38 MAPK in T3M-4 cells significantly reduces the extent to which high levels of Ca2+ inhibit proliferation. These results suggest that the low levels of p38 MAPK expression in FA-6 cells may serve to reduce their sensitivity to high concentrations of external Ca2+ that would otherwise block proliferation.

Unravelling the unusual signalling properties of the GABA(B) receptor.

GABA(B) receptors are the cornerstone receptors in the modulation of inhibitory signalling in the central nervous system and continue to be targets for the amelioration of a number of neuropsychiatric and neurological disorders. Unravelling the molecular identity of this receptor has spurred much research over the past five or so years and generated a renewed interest and excitement in the field. Many questions are being answered and lessons learnt, not only about GABA(B) receptor function but also about general mechanisms of G-protein-coupled receptor signalling. However, as questions are being answered as many new questions are being raised and many GABA(B)-related conundrums continue to remain unanswered. In this report, we review some of the most recent work in the area of GABA(B) receptor research. In particular, we focus our attentions on the emerging mechanisms thought to be important in GABA(B) receptor signalling and the growing complex of associated proteins that we consider to be part of the GABA(B) receptor "signalosome."

Phosphorylation and chronic agonist treatment atypically modulate GABAB receptor cell surface stability.

GABA(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. The dynamic control of the cell surface stability of GABA(B) receptors is likely to be of fundamental importance in the modulation of receptor signaling. Presently, however, this process is poorly understood. Here we demonstrate that GABA(B) receptors are remarkably stable at the plasma membrane showing little basal endocytosis in cultured cortical and hippocampal neurons. In addition, we show that exposure to baclofen, a well characterized GABA(B) receptor agonist, fails to enhance GABA(B) receptor endocytosis. Lack of receptor internalization in neurons correlates with an absence of agonist-induced phosphorylation and lack of arrestin recruitment in heterologous systems. We also demonstrate that chronic exposure to baclofen selectively promotes endocytosis-independent GABA(B) receptor degradation. The effect of baclofen can be attenuated by activation of cAMP-dependent protein kinase or co-stimulation of beta-adrenergic receptors. Furthermore, we show that increased degradation rates are correlated with reduced receptor phosphorylation at serine 892 in GABA(B)R2. Our results support a model in which GABA(B)R2 phosphorylation specifically stabilizes surface GABA(B) receptors in neurons. We propose that signaling pathways that regulate cAMP levels in neurons may have profound effects on the tonic synaptic inhibition by modulating the availability of GABA(B) receptors.

Identification of new candidate drugs in myelodysplastic syndromes with splicing factor mutations by transcriptional profiling and connectivity map analysis.

We sought to identify new candidate drugs for repurposing to myelodysplastic syndromes (MDS). Connectivity map analysis was performed on gene expression signatures generated from bone marrow CD34+ cells of splicing factor mutant MDS patients. Celastrol and Withaferin A (WA), two top-ranking compounds identified, markedly inhibited proliferation, arrested the cell cycle and induced apoptosis in leukaemia cells. These compounds also inhibited the viability of primary bone marrow MDS cells. We showed that Celastrol and WA inhibit interleukin-1 receptor-associated kinase 4-mediated nuclear factor kappa-light-chain-enhancer of activated B cells signalling activation in splicing factor mutant MDS and leukaemia cells. Celastrol and WA may represent novel candidate drugs for the treatment of MDS.

Distinct immune cell infiltration patterns in pancreatic ductal adenocarcinoma (PDAC) exhibit divergent immune cell selection and immunosuppressive mechanisms.

Pancreatic ductal adenocarcinoma has a dismal prognosis. A comprehensive analysis of single-cell multi-omic data from matched tumour-infiltrated CD45+ cells and peripheral blood in 12 patients, and two published datasets, reveals a complex immune infiltrate. Patients have either a myeloid-enriched or adaptive-enriched tumour microenvironment. Adaptive immune cell-enriched is intrinsically linked with highly distinct B and T cell clonal selection, diversification, and differentiation. Using TCR data, we see the largest clonal expansions in CD8 effector memory, senescent cells, and highly activated regulatory T cells which are induced within the tumour from naïve cells. We identify pathways that potentially lead to a suppressive microenvironment, including investigational targets TIGIT/PVR and SIRPA/CD47. Analysis of patients from the APACT clinical trial shows that myeloid enrichment had a shorter overall survival compared to those with adaptive cell enrichment. Strategies for rationale therapeutic development in this disease include boosting of B cell responses, targeting immunosuppressive macrophages, and specific Treg cell depletion approaches.

Genetic variation at IL7 provides mechanistic insights into toxicity to immune checkpoint blockade

SCALABLE MULTIPLE NETWORK INFERENCE WITH THE JOINT GRAPHICAL HORSESHOE

Network models are useful tools for modelling complex associations. In statistical omics such models are increasingly popular for identifying and assessing functional relationships and pathways. If a Gaussian graphical model is assumed, conditional independence is determined by the nonzero entries of the inverse covariance (precision) matrix of the data. The Bayesian graphical horseshoe estimator provides a robust and flexible framework for precision matrix inference, as it introduces local, edge-specific parameters which prevent over-shrinkage of nonzero off-diagonal elements. However, its applicability is currently limited in statistical omics settings, which often involve high-dimensional data from multiple conditions that might share common structures. We propose: (i) a scalable expectation conditional maximisation (ECM) algorithm for the original graphical horseshoe and (ii) a novel joint graphical horseshoe estimator, which borrows information across multiple related networks to improve estimation. We show numerically that our single-network ECM approach is more scalable than the existing graphical horseshoe Gibbs implementation, while achieving the same level of accuracy. We also show that our joint-network proposal successfully leverages shared edge-specific information between networks while still retaining differences, outperforming state-of-the-art methods at any level of network similarity. Finally, we leverage our approach to clarify gene regulation activity within and across immune stimulation conditions in monocytes, and formulate hypotheses on the pathogenesis of immune-mediated diseases.

An isoform quantitative trait locus in SBNO2 links genetic susceptibility to Crohn's disease with defective antimicrobial activity.

Despite major advances in linking single genetic variants to single causal genes, the significance of genetic variation on transcript-level regulation of expression, transcript-specific functions, and relevance to human disease has been poorly investigated. Strawberry notch homolog 2 (SBNO2) is a candidate gene in a susceptibility locus with different variants associated with Crohn's disease and bone mineral density. The SBNO2 locus is also differentially methylated in Crohn's disease but the functional mechanisms are unknown. Here we show that the isoforms of SBNO2 are differentially regulated by lipopolysaccharide and IL-10. We identify Crohn's disease associated isoform quantitative trait loci that negatively regulate the expression of the noncanonical isoform 2 corresponding with the methylation signals at the isoform 2 promoter in IBD and CD. The two isoforms of SBNO2 drive differential gene networks with isoform 2 dominantly impacting antimicrobial activity in macrophages. Our data highlight the role of isoform quantitative trait loci to understand disease susceptibility and resolve underlying mechanisms of disease.

Unraveling the polygenic architecture of complex traits using blood eQTL metaanalysis

Summary While many disease-associated variants have been identified through genome-wide association studies, their downstream molecular consequences remain unclear. To identify these effects, we performed cis- and trans-expression quantitative trait locus (eQTL) analysis in blood from 31,684 individuals through the eQTLGen Consortium. We observed that cis -eQTLs can be detected for 88% of the studied genes, but that they have a different genetic architecture compared to disease-associated variants, limiting our ability to use cis -eQTLs to pinpoint causal genes within susceptibility loci. In contrast, trans-eQTLs (detected for 37% of 10,317 studied trait-associated variants) were more informative. Multiple unlinked variants, associated to the same complex trait, often converged on trans-genes that are known to play central roles in disease etiology. We observed the same when ascertaining the effect of polygenic scores calculated for 1,263 genome-wide association study (GWAS) traits. Expression levels of 13% of the studied genes correlated with polygenic scores, and many resulting genes are known to drive these traits.

A common haplotype lowers PU.1 expression in myeloid cells and delays onset of Alzheimer’s disease

A genome-wide survival analysis of 14,406 Alzheimer’s disease (AD) cases and 25,849 controls identified eight previously reported AD risk loci and fourteen novel loci associated with age at onset. LD score regression of 220 cell types implicated regulation of myeloid gene expression in AD risk. In particular, the minor allele of rs1057233 (G), within the previously reported CELF1 AD risk locus, showed association with delayed AD onset and lower expression of SPI1 in monocytes and macrophages. SPI1 encodes PU.1, a transcription factor critical for myeloid cell development and function. AD heritability is enriched within the PU.1 cistrome, implicating a myeloid PU.1 target gene network in AD. Finally, experimentally altered PU.1 levels affect the expression of mouse orthologs of many AD risk genes and the phagocytic activity of mouse microglial cells. Our results suggest that lower SPI1 expression reduces AD risk by regulating myeloid gene expression and cell function.