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BACKGROUND: Single-nucleotide polymorphisms (SNPs) of the serotonin type 3 receptor subunit (HTR3) genes have been associated with psychosomatic symptoms, but it is not clear whether these associations exist in irritable bowel syndrome (IBS). AIM: To assess the association of HTR3 polymorphisms with depressive, anxiety, and somatization symptoms in individuals with IBS. METHODS: In this retrospective study, 623 participants with IBS were recruited from five specialty centers in Germany, Sweden, the United States, the United Kingdom, and Ireland. Depressive, anxiety, and somatization symptoms and sociodemographic characteristics were collected. Four functional SNPs - HTR3A c.-42C>T, HTR3B c.386A>C, HTR3C c.489C>A, and HTR3E c.*76G>A - were genotyped and analyzed using the dominant and recessive models. We also performed separate analyses for sex and IBS subtypes. SNP scores were calculated as the number of minor alleles of the SNPs above. The impact of HTR3C c.489C>A was tested by radioligand-binding and calcium influx assays. RESULTS: Depressive and anxiety symptoms significantly worsened with increasing numbers of minor HTR3C c.489C>A alleles in the dominant model (F depressive = 7.475, P depressive = 0.006; F anxiety = 6.535, P anxiety = 0.011). A higher SNP score (range 0-6) was linked to a worsened depressive symptoms score (F = 7.710, P-linear trend = 0.006) in IBS. The potential relevance of the HTR3C SNP was corroborated, showing changes in the expression level of 5-HT3AC variant receptors. CONCLUSION: We have provided the first evidence that HTR3C c.489C>A is involved in depressive and anxiety symptoms in individuals with IBS. The SNP score indicated that an increasing number of minor alleles is linked to the worsening of depressive symptoms in IBS. ispartof: WORLD JOURNAL OF GASTROENTEROLOGY vol:28 issue:21 pages:2334-2349 ispartof: location:United States status: published

Objective Hirschsprung's disease (HSCR) is a congenital intestinal neurodevelopmental disorder characterized by the absence of enteric ganglion cells in the distal colon. Although Hirschsprung-associated enterocolitis (HAEC) is the most frequent life-threatening complication in HSCR, to date reliable biomarkers predicting the likelihood of HAEC are yet to be established. We established a three-center retrospective study including 104 HSCR patients surgically treated between 1998 and 2019. Materials and Methods Patient-derived cryopreserved or paraffin-preserved colonic tissue at surgery was analyzed via βIII-tubulin immunohistochemistry. We subsequently determined extrinsic mucosal nerve fiber density in resected rectosigmoid specimens and classified HSCR patients accordingly into nerve fiber-high or fiber-low groups. We compared the distribution of clinical parameters obtained from medical records between the fiber-high (n = 36) and fiber-low (n = 68) patient groups. We assessed the association between fiber phenotype and enterocolitis using univariate and multivariate logistic regression adjusted for age at operation. Results Enterocolitis was more prevalent in patients with sparse mucosal nerve fiber innervation (fiber-low phenotype, 87%) compared with the fiber-high phenotype (13%; p = 0.002). In addition, patients developing enterocolitis had a younger age at surgery (3 vs. 7 months; p = 0.016). In the univariate analysis, the odds for enterocolitis development in the fiber-low phenotype was 5.26 (95% confidence interval [CI], 1.67–16.59; p = 0.005) and 4.01 (95% CI, 1.22–13.17; p = 0.022) when adjusted for age. Conclusion Here, we showed that HSCR patients with a low mucosal nerve fiber innervation grade in the distal aganglionic colon have a higher risk of developing HAEC. Consequently, histopathologic analysis of the nerve fiber innervation grade could serve as a novel sensitive prognostic marker associated with the development of enterocolitis in HSCR patients.

The enteric nervous system (ENS) is the largest division of the peripheral nervous system and closely resembles components and functions of the central nervous system. Although the central role of the ENS in congenital enteric neuropathic disorders, including Hirschsprung disease and inflammatory and functional bowel diseases, is well acknowledged, its role in systemic diseases is less understood. Evidence of a disordered ENS has accumulated in neurodegenerative diseases ranging from amyotrophic lateral sclerosis, Alzheimer disease and multiple sclerosis to Parkinson disease as well as neurodevelopmental disorders such as autism. The ENS is a key modulator of gut barrier function and a regulator of enteric homeostasis. A 'leaky gut' represents the gateway for bacterial and toxin translocation that might initiate downstream processes. Data indicate that changes in the gut microbiome acting in concert with the individual genetic background can modify the ENS, central nervous system and the immune system, impair barrier function, and contribute to various disorders such as irritable bowel syndrome, inflammatory bowel disease or neurodegeneration. Here, we summarize the current knowledge on the role of the ENS in gastrointestinal and systemic diseases, highlighting its interaction with various key players involved in shaping the phenotypes. Finally, current flaws and pitfalls related to ENS research in addition to future perspectives are also addressed.

Background and Objectives: Pluripotent embryonic stem (ES) cells represent a perfect model system for the investigation of early developmental processes. Besides their differentiation into derivatives of the three primary germ layers, they can also be differentiated into derivatives of the ‘fourth’ germ layer, the neural crest (NC). Due to its multipotency, extensive migration and outstanding capacity to generate a remarkable number of different cell types, the NC plays a key role in early developmental processes. Cardiac neural crest (CNC) cells are a subpopulation of the NC, which are of crucial importance for precise cardiovascular and pharyngeal glands’ development. CNC-associated malformations are rare, but always severe and life-threatening. Appropriate cell models could help to unravel underlying pathomechanisms and to develop new therapeutic options for relevant heart malformations. Methods: Murine ES cells were differentiated according to a mesodermal-lineage promoting protocol. Expression profiles of ES cell-derived progeny at various differentiation stages were investigated on transcript and protein level. Results: Comparative expression profiling of murine ES cell multilineage progeny versus undifferentiated ES cells confirmed differentiation into known cell derivatives of the three primary germ layers and provided evidence that ES cells have the capacity to differentiate into NC/CNC-like cells. Applying the NC/CNC cell-specific marker, 4E9R, an unambiguous identification of ES cell-derived NC/CNC-like cells was achieved. Conclusions: Our findings will facilitate the establishment of an ES cell-derived CNC cell model for the investigation of molecular pathways during cardiac development in health and disease.

Cytokines released during chronic inflammatory diseases induce pro-inflammatory properties in polymorphonuclear neutrophils (PMNs). Here, we describe the development of a subgroup of human PMNs expressing CCR5, termed CCR5

The 5-HT3 receptor (nomenclature as agreed by the NC-IUPHAR Subcommittee on 5-Hydroxytryptamine (serotonin) receptors [69]) is a ligand-gated ion channel of the Cys-loop family that includes the zinc-activated channels, nicotinic acetylcholine, GABAA and strychnine-sensitive glycine receptors. The receptor exists as a pentamer of 4 transmembrane (TM) subunits that form an intrinsic cation selective channel [7]. Five human 5-HT3 receptor subunits have been cloned and homo-oligomeric assemblies of 5-HT3A and hetero-oligomeric assemblies of 5-HT3A and 5-HT3B subunits have been characterised in detail. The 5-HT3C (HTR3C, Q8WXA8), 5-HT3D (HTR3D, Q70Z44) and 5-HT3E (HTR3E, A5X5Y0) subunits [86, 125], like the 5-HT3B subunit, do not form functional homomers, but are reported to assemble with the 5-HT3A subunit to influence its functional expression rather than pharmacological profile [127, 66, 161]. 5-HT3A, -C, -D, and -E subunits also interact with the chaperone RIC-3 which predominantly enhances the surface expression of homomeric 5-HT3A receptor [161]. The co-expression of 5-HT3A and 5-HT3C-E subunits has been demonstrated in human colon [85]. A recombinant hetero-oligomeric 5-HT3AB receptor has been reported to contain two copies of the 5-HT3A subunit and three copies of the 5-HT3B subunit in the order B-B-A-B-A [9], but this is inconsistent with recent reports which show at least one A-A interface [99, 154]. The 5-HT3B subunit imparts distinctive biophysical properties upon hetero-oligomeric 5-HT3AB versus homo-oligomeric 5-HT3A recombinant receptors [35, 44, 59, 88, 143, 132, 82], influences the potency of channel blockers, but generally has only a modest effect upon the apparent affinity of agonists, or the affinity of antagonists ([19], but see [44, 33, 38]) which may be explained by the orthosteric binding site residing at an interface formed between 5-HT3A subunits [99, 154]. However, 5-HT3A and 5-HT3AB receptors differ in their allosteric regulation by some general anaesthetic agents, small alcohols and indoles [142, 139, 73]. The potential diversity of 5-HT3 receptors is increased by alternative splicing of the genes HTR3A and HTR3E [67, 21, 127, 126, 123]. In addition, the use of tissue-specific promoters driving expression from different transcriptional start sites has been reported for the HTR3A, HTR3B, HTR3D and HTR3E genes, which could result in 5-HT3 subunits harbouring different N-termini [156, 82, 123]. To date, inclusion of the 5-HT3A subunit appears imperative for 5-HT3 receptor function.

Cytokines released during chronic inflammatory diseases induce pro-inflammatory properties in polymorphonuclear neutrophils (PMN). Here we show that in vitro cytokine treatment leads to the development of a subgroup of human PMN expressing CCR5, termed CCR5+ cytokine-induced PMN (CCR5+ cPMN). Auto/paracrine TNF signaling increases intracellular neutrophil elastase (ELANE) abundance and induces NETosis in CCR5+ cPMN. Triggering of CCR5 amplifies NETosis. Membranous TNF (mTNF) outside-in signaling induces the formation of reactive oxygen species, a known activator of NETosis. In vivo, we find an increased number of CCR5+ cPMN in the peripheral blood and inflamed lamina propria of patients with ulcerative colitis (UC) but not Crohn’s disease (CD). Notably, failure of anti-TNF therapy is associated with higher frequencies of CCR5+ cPMN. In conclusion, we identify a phenotype of pro-NETotic, CCR5 positive PMN present in inflamed tissue in vivo and inducible in vitro. These cells may reflect an important component of tissue damage during chronic inflammation and could be of diagnostic value.

H-1 parvovirus (H-1PV) is a promising anticancer therapy. However, in-depth understanding of its life cycle, including the host cell factors needed for infectivity and oncolysis, is lacking. This understanding may guide the rational design of combination strategies, aid development of more effective viruses, and help identify biomarkers of susceptibility to H-1PV treatment. To identify the host cell factors involved, we carry out siRNA library screening using a druggable genome library. We identify one crucial modulator of H-1PV infection: laminin γ1 (LAMC1). Using loss- and gain-of-function studies, competition experiments, and ELISA, we validate LAMC1 and laminin family members as being essential to H-1PV cell attachment and entry. H-1PV binding to laminins is dependent on their sialic acid moieties and is inhibited by heparin. We show that laminins are differentially expressed in various tumour entities, including glioblastoma. We confirm the expression pattern of laminin γ1 in glioblastoma biopsies by immunohistochemistry. We also provide evidence of a direct correlation between LAMC1 expression levels and H-1PV oncolytic activity in 59 cancer cell lines and in 3D organotypic spheroid cultures with different sensitivities to H-1PV infection. These results support the idea that tumours with elevated levels of γ1 containing laminins are more susceptible to H-1PV-based therapies., Rat H-1 parvovirus (H-1PV) is in clinical development for oncolytic therapy. Here, Kulkarni et al. identify LAMC1 as a modulator of H-1PV cell attachment and entry and find that LAMC1 levels and H-1PV oncolytic activity correlate in 59 tested cancer cell lines.

Gastrointestinal dysfunctions in individuals with autism spectrum disorder are poorly understood, although they are common among this group of patients. FOXP1 haploinsufficiency is characterized by autistic behavior, language impairment, and intellectual disability, but feeding difficulties and gastrointestinal problems have also been reported. Whether these are primary impairments, the result of altered eating behavior, or side effects of psychotropic medication remains unclear. To address this question, we investigated Foxp1 +/− mice reflecting FOXP1 haploinsufficiency. These animals show decreased body weight and altered feeding behavior with reduced food and water intake. A pronounced muscular atrophy was detected in the esophagus and colon, caused by reduced muscle cell proliferation. Nitric oxide-induced relaxation of the lower esophageal sphincter was impaired and achalasia was confirmed in vivo by manometry. Foxp1 targets ( Nexn , Rbms3 , and Wls ) identified in the brain were dysregulated in the adult Foxp1 +/− esophagus. Total gastrointestinal transit was significantly prolonged due to impaired colonic contractility. Our results have uncovered a previously unknown dysfunction (achalasia and impaired gut motility) that explains the gastrointestinal disturbances in patients with FOXP1 syndrome, with potential wider relevance for autism.

Purpose This study aimed to investigate the potential prognostic impact of nuclear factor erythroid 2-related factor 2 (NRF2) and progesterone receptor A (PRA)/progesterone receptor B (PRB) in ovarian cancer patients which might be the rationale for putative new treatment strategies. Patients and methods The presence of NRF2 and PRA/PRB was investigated in 156 ovarian cancer samples using immunohistochemistry (IHC). Staining of NRF2 and PRA/PRB was rated using the semi-quantitative immunoreactive score (IR score, Remmele's score) and correlated to clinical and pathological data. NRF2 and PRA/PRB expression were compared with respect to the overall survival (OS). Results NRF2 staining was different in both, the cytoplasm and nucleus between the histological subtypes (p=0.001 and p=0.02, respectively). There was a significant difference in the PRA expression comparing all histological subtypes (p=0.02). Histological subtypes showed no significant differences in the PRB expression. A strong correlation of cytoplasmic NRF2 and PRA expression was detected (cc=0.247, p=0.003) as well as of cytoplasmic NRF2 and PRB expression (cc=0.25, p=0.003), confirmed by immunofluorescence double staining. Cytoplasmic NRF2 expression was associated with a longer OS (median 50.6 vs 32.5 months; p=0.1) as it was seen for PRA expression (median 63.4 vs 33.1 months; p=0.08), although not statistically significant. In addition, high PRB expression (median 80.4 vs 32.5 months; p=0.04) and concurrent expression of cytoplasmic NRF2 and PRA were associated with a significantly longer OS (median 109.7 vs 30.6 months; p=0.02). The same relationship was also noted for NRF2 and PRB with improved OS for patients expressing both cytoplasmic NRF2 and PRB (median 153.5 vs 30.6 months; p=0.009). Silencing of NFE2L2 induced higher mRNA expression of PGR in the cancer cell line OVCAR3 (p>0.05) confirming genetic interactions of NRF2 and PR. Conclusion In this study, the combination of cytoplasmic NRF2 and high PRA/PRB expression was demonstrated to be associated with improved overall survival in ovarian cancer patients. Further understanding of interactions within the NRF2/AKR1C1/PR pathway could open new additional therapeutic approaches.

The interplay between keratinocytes and immune cells, especially T cells, plays an important role in the pathogenesis of chronic inflammatory skin diseases. During psoriasis, keratinocytes attract T cells by releasing chemokines, while skin-infiltrating self-reactive T cells secrete proinflammatory cytokines, e.g., IFNγ and IL-17A, that cause epidermal hyperplasia. Similarly, in chronic graft-versus-host disease, allogenic IFNγ-producing Th1/Tc1 and IL-17-producing Th17/Tc17 cells are recruited by keratinocyte-derived chemokines and accumulate in the skin. However, whether keratinocytes act as nonprofessional antigen-presenting cells to directly activate naive human T cells in the epidermis remains unknown. Here, we demonstrate that under proinflammatory conditions, primary human keratinocytes indeed activate naive human T cells. This activation required cell contact and costimulatory signaling via CD58/CD2 and CD54/LFA-1. Naive T cells costimulated by keratinocytes selectively differentiated into Th1 and Th17 cells. In particular, keratinocyte-initiated Th1 differentiation was dependent on costimulation through CD58/CD2. The latter molecule initiated STAT1 signaling and IFNγ production in T cells. Costimulation of T cells by keratinocytes resulting in Th1 and Th17 differentiation represents a new explanation for the local enrichment of Th1 and Th17 cells in the skin of patients with a chronic inflammatory skin disease. Consequently, local interference with T cell–keratinocyte interactions may represent a novel strategy for the treatment of Th1 and Th17 cell-driven skin diseases.

Irritable bowel syndrome (IBS) is a functional gut brain gastrointestinal (GI) disorder, typically accompanied by constipation or diarrhea, usually without any organic evidence. The prevalence of IBS is rather high of about 10-15% (10, 1 % according to Rome III and 4, 1% according to Rome IV, Enck P. et al 2016, Sperber A.D. et al 2020, Black C.J. et al 2020) in the working population. Quality of life in patients with IBS is reduced and therefore a major obstacle to the normal physical and social wellbeing. In intensified clinical research worldwide new pathogenic mechanisms of IBS are suggested, including intestinal dysbiois one of the critical contributing factors to onset or further development of IBS. Intestinal microbiome represents a real ecosystem of microorganisms and human GI tract lining cells. The diversity and composition of the GI microbiome may differ significantly inter- and intra-individually, depending on sex, age or physiological conditions (pregnancy, disease, etc). Intestinal microbiome composition frequently changes in association with IBS symptoms, and the purpose of this study was to investigate if there is a clear relationship in microbial composition and relative abundance of microbial taxa in feces of persons diagnosed with IBS. Fecal microbiota profiling was done in a group of nine clinically confirmed IBS patients and 6 corresponding healthy controls, based on species specific 16s RNA gene. No statistically significant differences in Alpha and Beta diversity indices were found.

Still little is known about the nature of the gastrointestinal pathological alterations occurring in Parkinson’s disease (PD). Here, we used multiplexed mRNA profiling to measure the expression of a panel of 770 genes related to neuropathological processes in deep submucosal rectal biopsies of PD patients and healthy controls. Altered enteric neuropathological traits based on the expression of 22 genes related to neuroglial and mitochondrial functions, vesicle trafficking and inflammation was observed in 9 out of 12 PD patients in comparison to healthy controls. These results provide new evidences that intestinal neuropathological alterations may occur in a large proportion of PD patients.

Background & Aims The aim of this study was to investigate the effectiveness of oral treatment with a non-viable probiotic lysate (BL) of Escherichia coli (DSM 17252) and Enterococcus faecalis (DSM 16440) in patients with irritable bowel syndrome (IBS). Methods A phase IV, randomized, double-blind, placebo-controlled, multi-center (30 study sites), parallel group study was conducted in 389 patients of both sexes with IBS according to Rome III criteria. The treatment period was 26 weeks. The participants were allocated either to placebo or BL after a two week baseline period. The primary outcome was based on the European Medical Agency IBS guideline: Improvement in global assessment (GAI) and improvement in abdominal pain. Results Patients (BL n=191; placebo n=198) had similar baseline values and drop-out rates. Overall, the response was similar between BL and placebo for IBS-GAI (17.4% and 14.4%, resp., p=·4787) and abdominal pain (42.0% and 35.4%, resp.; p=·1419). Some secondary outcome measures and sensitivity analyses pointed towards potentially higher sensitivity of the abdominal pain measures in IBS-D, but not the other subtypes. For the GAI, no subgroup differences were detected. For diarrhea-predominant IBS (IBS-D), post-hoc analyses for abdominal pain response over time and stool consistency showed potentially promising effects of BL. Finally, the treatment with bacterial lysate was well tolerated. Conclusions BL is not effective across all IBS subtypes. However, BL may offer a treatment option for IBS-D which needs verification by an adequately powered drug-trial; EudraCT-No.: 2012-002741-38.

Parkinson’s disease (PD) usually has a late clinical onset. The lack of early biomarkers for the disease represents a major challenge for developing timely treatment interventions. Here, we use an α-synuclein-overexpressing transgenic (Th-1-SNCA-A30P) mouse model of PD to identify appropriate candidate markers in the gut for early stages of PD before hallmark symptoms begin to manifest. A30P mice did not show alterations in gait parameters at 2 months of age, and these mice were therefore defined as pre-symptomatic A30P mice (psA30P). We discovered early functional motility changes in the gut and early molecular dysregulations in the myenteric plexus of psA30P mice by comparative protein and miRNA profiling and cell culture experiments. We found that the proteins neurofilament light chain, vesicle-associated membrane protein 2 and calbindin 2, together with the miRNAs that regulate them, are potential biomarkers of early PD that may facilitate timely treatment and/or prevention of PD in men.

[Abstract] Hirschsprung disease (HSCR, OMIM 142623) involves congenital intestinal obstruction caused by dysfunction of neural crest cells and their progeny during enteric nervous system (ENS) development. HSCR is a multifactorial disorder; pathogenetic variants accounting for disease phenotype are identified only in a minority of cases, and the identification of novel disease-relevant genes remains challenging. In order to identify and to validate a potential disease-causing relevance of novel HSCR candidate genes, we established a complementary study approach, combining whole exome sequencing (WES) with transcriptome analysis of murine embryonic ENS-related tissues, literature and database searches, in silico network analyses, and functional readouts using candidate gene-specific genome-edited cell clones. WES datasets of two patients with HSCR and their non-affected parents were analysed, and four novel HSCR candidate genes could be identified: ATP7A, SREBF1, ABCD1 and PIAS2. Further rare variants in these genes were identified in additional HSCR patients, suggesting disease relevance. Transcriptomics revealed that these genes are expressed in embryonic and fetal gastrointestinal tissues. Knockout of these genes in neuronal cells demonstrated impaired cell differentiation, proliferation and/or survival. Our approach identified and validated candidate HSCR genes and provided further insight into the underlying pathomechanisms of HSCR., [Author summary] Hirschsprung disease (HSCR) is a rare developmental disorder. It leads to the absence of enteric nerve cells (aganglionosis) in the large intestine and is caused by functional defects of neuronal precursor cells during embryonic development of the gut nervous system. The aganglionosis manifests as a variety of symptoms including impaired peristalsis and the formation of a pathogenic dilatation of the intestine (megacolon). The etiology of HSCR is considered to be multifactorial. Variants in more than 20 genes have been reported to be overrepresented in HSCR and replicated in independent cohorts. However, variants in those risk genes account for only 30% of all cases, suggesting that many more genes have to be implicated in the development of HSCR. As the identification and the subsequent validation of novel gene variants to be disease-causing or not, still remains a major challenge, we established and applied a complementary study pipeline. This enabled us to identify four novel candidate genes in two HSCR patients and to validate their potential disease relevance. Our approach represents a suitable way to dissect the complex genetic architecture underlying HSCR., This study was supported by the Heidelberg Stiftung Chirurgie (https://www.stiftung-chirurgie.de) (P. Romero and B. Niesler), the Heinz and Heide Dürr Stiftung (https://www.heinzundheideduerrstiftung.de) (B. Niesler and P. Romero; 2017/2.2.1/04), the Heidelberg University Hospital (https://www.heidelberg-university-hospital.com) (G. Rappold), and the Dres. Majic/Majic Schlez Stiftung (T. Mederer). Tanja Mederer is a PhD fellow of HBIGS (http://www.hbigs.uni-heidelberg.de) and was funded by the Studienstiftung des Deutschen Volkes (https://www.studienstiftung.de). Cristina Martínez is supported by Instituto de Salud Carlos III, Subdirección General de Investigación Sanitaria, Ministerio de Ciencia, Innovación y Universidades (https://www.isciii.es) (CP18/00116). Salud Borrego was supported by Instituto de Salud Carlos III (https://www.isciii.es) through the project "PI16/0142" and "PI19/01550" (Co-funded by European Regional Development Fund/European Social Fund "A way to make Europe"/"Investing in your future").

Background A Giardia outbreak in Bergen, Norway, caused postinfectious functional gastrointestinal disorders (PI-FGIDs). Despite the devastating effects of this outbreak, it presented a unique chance to investigate the implication on the dysregulation of genetic pathways in PI-FGID. Methods We performed the first comparative expression profiling of miRNAs and their potential target genes in microdissected rectal biopsies from 20 Giardia-induced PI-FGID patients vs 18 healthy controls by nCounter analysis. Subsequently, candidates were validated on protein level by immunostaining. Key results miRNA profiling on rectal biopsy samples from 5 diarrhea-predominant PI-IBS cases compared to 10 healthy controls revealed differential expression in the epithelial layer. The top five regulated miRNAs were implicated in GI disease, inflammatory response, and immunological disease. Subsequently, these miRNAs and 100 potential mRNA targets were examined in 20 PI-FGID cases and 18 healthy controls in both the mucosal epithelium and the lamina propria. Although deregulation of the selected miRNAs could not be verified in the larger sample set, mRNAs involved in barrier function were downregulated in the epithelium. Pro-inflammatory genes and genes implicated in epigenetic modifications were upregulated in the lamina propria. Immunostaining for selected candidates on 17 PI-FGID cases and 16 healthy controls revealed increased tryptase levels as well as a decreased and aberrant subcellular expression of occludin. Conclusions and inferences Genes relevant to immune and barrier function as well as stress response and epigenetic modulation are differentially expressed in PI-FGIDs and may contribute to disease manifestation.

Aims CD40 ligand (CD40L) signaling controls vascular oxidative stress and related dysfunction in angiotensin-II-induced arterial hypertension by regulating vascular immune cell recruitment and platelet activation. Here we investigated the role of CD40L in experimental hyperlipidemia. Methods and results Male wild type and CD40L−/− mice (C57BL/6 background) were subjected to high fat diet for sixteen weeks. Weight, cholesterol, HDL, and LDL levels, endothelial function (isometric tension recording), oxidative stress (NADPH oxidase expression, dihydroethidium fluorescence) and inflammatory parameters (inducible nitric oxide synthase, interleukin-6 expression) were assessed. CD40L expression, weight, leptin and lipids were increased, and endothelial dysfunction, oxidative stress and inflammation were more pronounced in wild type mice on a high fat diet, all of which was almost normalized by CD40L deficiency. Similar results were obtained in diabetic db/db mice with CD40/TRAF6 inhibitor (6877002) therapy. In a small human study higher serum sCD40L levels and an inflammatory phenotype were detected in the blood and Aorta ascendens of obese patients (body mass index > 35) that underwent by-pass surgery. Conclusion CD40L controls obesity-associated vascular inflammation, oxidative stress and endothelial dysfunction in mice and potentially humans. Thus, CD40L represents a therapeutic target in lipid metabolic disorders which is a leading cause in cardiovascular disease.

Background: Little is known about the role of WNT signalling in pathological processes involving the urinary tract stroma. Here the impact of WNT signalling on bladder wall fibroblasts (BWFs) was studied using integrated expression profiling. Material and Methods: WNT ligand and downstream WNT pathway component expression was profiled in human BWFs using qRT-PCR. Highly expressed WNT2B was knocked down using siRNA in BWFs. The expression of 730 mRNAs and 800 miRNAs was analyzed on the nCounter MAX platform in #WNT2B and control transfected BWFs. qRT-PCR was used for validation in vitro and in matched scar and healthy bladder wall tissue samples of 12 patients with vesico-urethral anastomotic stricture (VUAS). Results: Thirteen genes and 9 miRNAs showed differential expression in #WNT2B cells. Among these were TNFSF10, a key apoptosis inductor, (0.22fold, p = 0.011) and miR-1246 (36.2fold, p = 0.031). miRNA target prediction indicated TNFSF10 to be regulated by miR-1246. qRT-PCR analysis confirmed differential expression of miR-1246 and TNFSF10 in #WNT2B BWFs. Furthermore, TNFSF10 was significantly underexpressed in VUAS tissue (p = 0.009). Conclusion: Perturbation of WNT signalling results in an altered expression of the apoptosis inductor TNFSF10. Similar changes are observed in VUAS. Further studies investigating the crosslink between WNT signalling and apoptosis regulation in the urinary tract stroma are warranted.

Intrinsic malignant brain tumors, such as glioblastomas are frequently resistant to immune checkpoint blockade (ICB) with few hypermutated glioblastomas showing response. Modeling patient-individual resistance is challenging due to the lack of predictive biomarkers and limited accessibility of tissue for serial biopsies. Here, we investigate resistance mechanisms to anti-PD-1 and anti-CTLA-4 therapy in syngeneic hypermutated experimental gliomas and show a clear dichotomy and acquired immune heterogeneity in ICB-responder and non-responder tumors. We made use of this dichotomy to establish a radiomic signature predicting tumor regression after pseudoprogression induced by ICB therapy based on serial magnetic resonance imaging. We provide evidence that macrophage-driven ICB resistance is established by CD4 T cell suppression and Treg expansion in the tumor microenvironment via the PD-L1/PD-1/CD80 axis. These findings uncover an unexpected heterogeneity of response to ICB in strictly syngeneic tumors and provide a rationale for targeting PD-L1-expressing tumor-associated macrophages to overcome resistance to ICB., Modeling patient-individual resistance to immunotherapy is challenging. Here, the authors use a syngeneic experimental hypermutated orthotopic glioma model to define radiological and biological features that can predict or explain the mechanistic differences between responders and non-responders to immunotherapy.

Gastrointestinal dysfunctions in individuals with autism spectrum disorder are poorly understood, although they are common among this group of patients. FOXP1 haploinsufficiency is characterized by autistic behavior, language impairment, and intellectual disability, but feeding difficulties and gastrointestinal problems have also been reported. Whether these are primary impairments, the result of altered eating behavior, or side effects of psychotropic medication remains unclear. To address this question, we investigated Foxp1(+/−) mice reflecting FOXP1 haploinsufficiency. These animals show decreased body weight and altered feeding behavior with reduced food and water intake. A pronounced muscular atrophy was detected in the esophagus and colon, caused by reduced muscle cell proliferation. Nitric oxide-induced relaxation of the lower esophageal sphincter was impaired and achalasia was confirmed in vivo by manometry. Foxp1 targets (Nexn, Rbms3, and Wls) identified in the brain were dysregulated in the adult Foxp1(+/−) esophagus. Total gastrointestinal transit was significantly prolonged due to impaired colonic contractility. Our results have uncovered a previously unknown dysfunction (achalasia and impaired gut motility) that explains the gastrointestinal disturbances in patients with FOXP1 syndrome, with potential wider relevance for autism.

Background The enteric nervous system (ENS), a complex network of neurons and glial cells, coordinates major gastrointestinal functions. Impaired development or secondary aberrations cause severe enteric neuropathies. Neural crest-derived stem cells as well as enteric neuronal progenitor cells, which form enteric neurospheres, represent a promising tool to unravel molecular pathomechanisms and to develop novel therapy options. However, so far little is known about the detailed cellular composition and the proportional distribution of enteric neurospheres. Comprehensive knowledge will not only be essential for basic research but also for prospective cell replacement therapies to restore or to improve enteric neuronal dysfunction. Methods Human enteric neurospheres were generated from three individuals with varying age. For detailed molecular characterization, nCounter target gene expression analyses focusing on stem, progenitor, neuronal, glial, muscular, and epithelial cell markers were performed. Corresponding archived paraffin-embedded individuals' specimens were analyzed accordingly. Key results Our data revealed a remarkable molecular complexity of enteric neurospheres and archived specimens. Amongst the expression of multipotent stem cell, progenitor cell, neuronal, glial, muscle and epithelial cell markers, moderate levels for the pluripotency marker POU5F1 were observed. Furthermore, besides the interindividual variability, we identified highly distinct intraindividual expression profiles. Conclusions & inferences Our results emphasize the assessment of molecular signatures to be essential for standardized use, optimization of experimental approaches, and elimination of potential risk factors, as the formation of tumors. Our study pipeline may serve as a blueprint implemented into the characterization procedure of enteric neurospheres for various future applications.

Intestinal epithelial cells (IECs) are exposed to the low-oxygen environment present in the lumen of the gut. These hypoxic conditions on one hand are fundamental for the survival of the commensal microbiota and, on the other hand, favor the formation of a selective semipermeable barrier, allowing IECs to transport essential nutrients/water while keeping the sterile internal compartments separated from the lumen containing commensals. The hypoxia-inducible factor (HIF) complex, which allows cells to respond and adapt to fluctuations in oxygen levels, has been described as a key regulator in maintaining IEC barrier function by regulating their tight junction integrity. In this study, we sought to better evaluate the mechanisms by which low oxygen conditions impact the barrier function of human IECs. By profiling miRNA expression in IECs under hypoxia, we identified microRNA 320a (miRNA-320a) as a novel barrier formation regulator. Using pharmacological inhibitors and short hairpin RNA-mediated silencing, we could demonstrate that expression of this microRNA (miRNA) was HIF dependent. Importantly, using overexpression and knockdown approaches of miRNA-320a, we could confirm its direct role in the regulation of barrier function in human IECs. These results reveal an important link between miRNA expression and barrier integrity, providing a novel insight into mechanisms of hypoxia-driven epithelial homeostasis.

BACKGROUND: Irritable bowel syndrome with diarrhoea (IBS-D) is a common condition, greatly reducing the quality of life with few effective treatment options available.AIM: To report the beneficial response shown in our trial with the 5-hydroyxtryptamine (5-HT) receptor 3 antagonist, ondansetron in IBS-D METHODS: A randomised, placebo-controlled, cross-over trial of 5 weeks of ondansetron versus placebo in 125 patients meeting modified Rome III criteria for IBS-D as previously described. Patients were compared to 21 healthy controls. 5-HT and 5-HIAA were measured in rectal biopsies. Whole gut transit time was assessed using a radio-opaque marker technique. Whole blood DNA was genotyped for an insertion polymorphism in the promoter region of the serotonin transporter gene SLC6A4, as well as single nucleotide polymorphisms (SNPs) of the tryptophan hydroxylase gene TPH1 and 5-HT3 receptor genes HTR3A, C and E.RESULTS: Patients' biopsies showed significantly higher 5-HIAA levels (2.1 (1.2-4.2) pmol/mg protein vs 1.1 (0.4-1.5) in controls, P < .0001). 39 patients used < 4 mg/d ("super-responders") while 55 required ≥ 4 mg/d. 5-HT concentrations in rectal biopsies were significantly lower in super-responders (21.3 (17.0-31.8) vs 37.7 (21.4-61.4), P = .0357) and the increase in transit time on ondansetron was significantly greater (15.6 (1.8-31) hours vs 3.9 (-5.1-17.9) hours). Stool consistency responders were more likely to carry the CC genotype of the SNP p.N163K rs6766410 of the HTR3C gene (33% vs 14%, P = .0066).CONCLUSION: IBS-D patients have significant abnormalities in mucosal 5-HT metabolism. Those with the lowest concentration of 5-HT in rectal biopsies showed the greatest responsiveness to ondansetron.