Targeted Proteomics Reveals Quantitative Differences in Low-Abundance Glycosyltransferases of Patients with Congenital Disorders of Glycosylation.

Protein glycosylation is an essential post-translational modification in all domains of life. Its impairment in humans can result in severe diseases named congenital disorders of glycosylation (CDGs). Most of the glycosyltransferases (GTs) responsible for proper glycosylation are polytopic membrane proteins that represent challenging targets in proteomics. We established a multiple reaction monitoring (MRM) assay to comprehensively quantify GTs involved in the processes of N-glycosylation and O- and C-mannosylation in the endoplasmic reticulum. High robustness was achieved by using an enriched membrane protein fraction of isotopically labeled HEK 293T cells as an internal protein standard. The analysis of primary skin fibroblasts from eight CDG type I patients with impaired ALG1, ALG2, and ALG11 genes, respectively, revealed a substantial reduction in the corresponding protein levels. The abundance of the other GTs, however, remained unchanged at the transcript and protein levels, indicating that there is no fail-safe mechanism for the early steps of glycosylation in the endoplasmic reticulum. The established MRM assay was shared with the scientific community via the commonly used open source Skyline software environment, including Skyline Batch for automated data analysis. We demonstrate that another research group could easily reproduce all analysis steps, even while using different LC-MS hardware.

A complementary study approach unravels novel players in the pathoetiology of Hirschsprung disease.

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.

A Nonviable Probiotic in Irritable Bowel Syndrome: A Randomized, Double-Blind, Placebo-Controlled, Multicenter Study.

BACKGROUND & AIMS: The aim of this study was to investigate the effectiveness of oral treatment with a nonviable 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, multicenter (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 to either placebo or BL after a 2-week baseline period. The primary outcome was based on the European Medicines 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 dropout rates. Overall, the response was similar between BL and placebo for IBS-GAI (17.4% and 14.4%, respectively; P = ·4787) and abdominal pain (42.0% and 35.4%, respectively; P = ·1419). Some secondary outcome measures and sensitivity analyses pointed toward potentially higher sensitivity of the abdominal pain measures in diarrhea-predominant IBS (IBS-D) but not the other subtypes. For the GAI, no subgroup differences were detected. For IBS-D, post hoc analyses for abdominal pain response over time and stool consistency showed potentially promising effects of BL. Finally, the treatment with BL was well-tolerated. CONCLUSIONS: BL is not effective across all IBS subtypes. However, BL may offer a treatment option for IBS-D that needs verification by an adequately powered drug trial; EudraCT-No.: 2012-002741-38.

Gastrointestinal dysfunction in autism displayed by altered motility and achalasia in Foxp1+/- mice.

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.

Interaction of ERα and NRF2 Impacts Survival in Ovarian Cancer Patients.

Nuclear factor erythroid 2-related factor 2 (NRF2) regulates cytoprotective antioxidant processes. In this study, the prognostic potential of NRF2 and its interactions with the estrogen receptor α (ERα) in ovarian cancer cells was investigated. NRF2 and ERα protein expression in ovarian cancer tissue was analyzed as well as mRNA expression of NRF2 (NFE2L2) and ERα (ESR1) in four ovarian cancer and one benign cell line. NFE2L2 silencing was carried out to evaluate a potential interplay between NRF2 and ERα. Cytoplasmic NRF2 expression as inactive form had significantly higher expression in patients with low-grade histology (p = 0.03). In the serous cancer subtype, high cytoplasmic NRF2 expression (overall survival (OS), median 50.6 vs. 29.3 months; p = 0.04) and high ERα expression (OS, median 74.5 vs. 27.1 months; p = 0.002) was associated with longer overall survival as well as combined expression of both inactive cytoplasmic NRF2 and ERα in the whole cohort (median 74.5 vs. 37.7 months; p = 0.04). Cytoplasmic NRF2 expression showed a positive correlation with ERα expression (p = 0.004). NFE2L2 was found to be highly expressed in the ovarian cancer cell lines OVCAR3, UWB1.289, and TOV112D. Compared with the benign cell line HOSEpiC, ESR1 expression was reduced in all ovary cancer cell lines (all p < 0.001). Silencing of NFE2L2 induced a higher mRNA expression of ESR1 in the NFE2L2 downregulated cancer cell lines OVCAR3 (p = 0.003) and ES2 (p < 0.001), confirming genetic interactions of NRF2 and ERα. In this study, both inactive cytoplasmic NRF2 and high ERα expression were demonstrated to be associated with improved survival in ovarian cancer patients. Further understanding of interactions within the estradiol⁻ERα⁻NRF2 pathway could better predict the impact of endocrine therapy in ovarian cancer.

miR-16 and miR-125b are involved in barrier function dysregulation through the modulation of claudin-2 and cingulin expression in the jejunum in IBS with diarrhoea.

OBJECTIVE: Micro-RNAs (miRNAs) play a crucial role in controlling intestinal epithelial barrier function partly by modulating the expression of tight junction (TJ) proteins. We have previously shown differential messenger RNA (mRNA) expression correlated with ultrastructural abnormalities of the epithelial barrier in patients with diarrhoea-predominant IBS (IBS-D). However, the participation of miRNAs in these differential mRNA-associated findings remains to be established. Our aims were (1) to identify miRNAs differentially expressed in the small bowel mucosa of patients with IBS-D and (2) to explore putative target genes specifically involved in epithelial barrier function that are controlled by specific dysregulated IBS-D miRNAs. DESIGN: Healthy controls and patients meeting Rome III IBS-D criteria were studied. Intestinal tissue samples were analysed to identify potential candidates by: (a) miRNA-mRNA profiling; (b) miRNA-mRNA pairing analysis to assess the co-expression profile of miRNA-mRNA pairs; (c) pathway analysis and upstream regulator identification; (d) miRNA and target mRNA validation. Candidate miRNA-mRNA pairs were functionally assessed in intestinal epithelial cells. RESULTS: IBS-D samples showed distinct miRNA and mRNA profiles compared with healthy controls. TJ signalling was associated with the IBS-D transcriptional profile. Further validation of selected genes showed consistent upregulation in 75% of genes involved in epithelial barrier function. Bioinformatic analysis of putative miRNA binding sites identified hsa-miR-125b-5p and hsa-miR-16 as regulating expression of the TJ genes CGN (cingulin) and CLDN2 (claudin-2), respectively. Consistently, protein expression of CGN and CLDN2 was upregulated in IBS-D, while the respective targeting miRNAs were downregulated. In addition, bowel dysfunction, perceived stress and depression and number of mast cells correlated with the expression of hsa-miR-125b-5p and hsa-miR-16 and their respective target proteins. CONCLUSIONS: Modulation of the intestinal epithelial barrier function in IBS-D involves both transcriptional and post-transcriptional mechanisms. These molecular mechanisms include miRNAs as master regulators in controlling the expression of TJ proteins and are associated with major clinical symptoms.

The Human Serotonin Type 3 Receptor Gene (HTR3A-E) Allelic Variant Database.

Serotonin type 3 (5-HT3 ) receptors are ligand-gated ion channels formed by five subunits (5-HT3A-E), which are encoded by the HTR3A, HTR3B, HTR3C, HTR3D, and HTR3E genes. Functional receptors are pentameric complexes of diverse composition. Different receptor subtypes confer a predisposition to nausea and vomiting during chemotherapy, pregnancy, and following surgery. In addition, different subtypes contribute to neurogastroenterologic disorders such irritable bowel syndrome (IBS) and eating disorders as well as comorbid psychiatric conditions. 5-HT3 receptor antagonists are established treatments for emesis and IBS and are beneficial in the treatment of psychiatric diseases. Several case-control and pharmacogenetic studies have demonstrated an association between HTR3 variants and psychiatric and neurogastroenterologic phenotypes. Recently, their potential as predictors of nausea and vomiting and treatment of psychiatric disorders became evident. This information is now available in the serotonin receptor 3 HTR3 gene allelic variant database (www.htr3.uni-hd.de), which contains five sub-databases, one for each of the five different serotonin receptor genes HTR3A-E. Information on HTR3 variants, their functional relevance, associated phenotypes, and pharmacogenetic data such as drug response and side effects are available. This central information pool should help clinicians as well as scientists to evaluate their findings and to use the relevant information for subsequent genotype-phenotype correlation studies and pharmacogenetic approaches.

Impact of Altered WNT2B Expression on Bladder Wall Fibroblasts: Implications for Apoptosis Regulation in the Stroma of the Lower Urinary Tract.

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.

SHOX triggers the lysosomal pathway of apoptosis via oxidative stress.

The SHOX gene encodes for a transcription factor important for normal bone development. Mutations in the gene are associated with idiopathic short stature and are responsible for the growth failure and skeletal defects found in the majority of patients with Léri-Weill dyschondrosteosis (LWD) and Langer mesomelic dysplasia. SHOX is expressed in growth plate chondrocytes where it is supposed to modulate the proliferation, differentiation and cell death of these cells. Supporting this hypothesis, in vitro studies have shown that SHOX expression induces cell cycle arrest and apoptosis in both transformed and primary cells. In this study, we further characterized the cell death mechanisms triggered by SHOX and compared them with the effects induced by one clinically relevant mutant form of SHOX, detected in LWD patients (SHOX R153L) and a SHOX C-terminally truncated version (L185X). We show that SHOX expression in U2OS osteosarcoma cells leads to oxidative stress that, in turn, induces lysosomal membrane rupture with release of active cathepsin B to the cytosol and subsequent activation of the intrinsic apoptotic pathway characterized by mitochondrial membrane permeabilization and caspase activation. Importantly, cells expressing SHOX R153L or L185X did not display any of these features. Given the fact that many of the events observed in SHOX-expressing cells also characterize the complex cell death process occurring in the growth plate during endochondral ossification, our findings further support the hypothesis that SHOX may play a central role in the regulation of the cell death pathways activated during long bone development.

Exploring the genetics of irritable bowel syndrome: a GWA study in the general population and replication in multinational case-control cohorts.

OBJECTIVE: IBS shows genetic predisposition, but adequately powered gene-hunting efforts have been scarce so far. We sought to identify true IBS genetic risk factors by means of genome-wide association (GWA) and independent replication studies. DESIGN: We conducted a GWA study (GWAS) of IBS in a general population sample of 11,326 Swedish twins. IBS cases (N=534) and asymptomatic controls (N=4932) were identified based on questionnaire data. Suggestive association signals were followed-up in 3511 individuals from six case-control cohorts. We sought genotype-gene expression correlations through single nucleotide polymorphism (SNP)-expression quantitative trait loci interactions testing, and performed in silico prediction of gene function. We compared candidate gene expression by real-time qPCR in rectal mucosal biopsies of patients with IBS and controls. RESULTS: One locus at 7p22.1, which includes the genes KDELR2 (KDEL endoplasmic reticulum protein retention receptor 2) and GRID2IP (glutamate receptor, ionotropic, delta 2 (Grid2) interacting protein), showed consistent IBS risk effects in the index GWAS and all replication cohorts and reached p=9.31×10(-6) in a meta-analysis of all datasets. Several SNPs in this region are associated with cis effects on KDELR2 expression, and a trend for increased mucosal KDLER2 mRNA expression was observed in IBS cases compared with controls. CONCLUSIONS: Our results demonstrate that general population-based studies combined with analyses of patient cohorts provide good opportunities for gene discovery in IBS. The 7p22.1 and other risk signals detected in this study constitute a good starting platform for hypothesis testing in future functional investigations.

No association between the common calcium-sensing receptor polymorphism rs1801725 and irritable bowel syndrome.

BACKGROUND: The calcium-sensing receptor (CaSR) is a calcium (Ca(2+)) sensitive G protein-coupled receptor implicated in various biological processes. In particular, it regulates Ca(2+)/Mg(2+)- homeostasis and senses interstitial Ca(2+) levels and thereby controls downstream signalling cascades. Due to its expression in the gut epithelium, the enteric nervous system and smooth muscles and its key function in regulation and coordination of muscular contraction and secretion, it represents an excellent candidate gene to be investigated in the pathophysiology of irritable bowel syndrome (IBS). Disturbed CaSR structure and function may impact gastrointestinal regulation of muscular contraction, neuronal excitation and secretion and consequently contribute to symptoms seen in IBS, such as disordered defecation as well as disturbed gut motility and visceral sensitivity. METHODS: We have therefore genotyped the functional CASR SNP rs1801725 in three case control samples from the UK, Belgium and the USA. RESULTS: Genotype frequencies showed no association in the three genotyped case-control samples, neither with IBS nor with IBS subtypes. CONCLUSIONS: Although we could not associate the SNP to any of the established bowel symptom based IBS subtypes we cannot rule out association to altered Ca(2+) levels and disturbed secretion and gut motility which were unfortunately not assessed in the patients genotyped. This underlines the necessity of a more detailed phenotyping of IBS patients and control individuals in future studies.

Relationship between genome and epigenome--challenges and requirements for future research.

Understanding the links between genetic, epigenetic and non-genetic factors throughout the lifespan and across generations and their role in disease susceptibility and disease progression offer entirely new avenues and solutions to major problems in our society. To overcome the numerous challenges, we have come up with nine major conclusions to set the vision for future policies and research agendas at the European level.

A common microdeletion affecting a hippocampus- and amygdala-specific isoform of tryptophan hydroxylase 2 is not associated with affective disorders.

OBJECTIVES: Copy number variants (CNVs) have been shown to affect susceptibility for neuropsychiatric disorders. To date, studies implicating the serotonergic system in complex conditions have just focused on single nucleotide polymorphisms (SNPs). We therefore sought to identify novel common genetic copy number polymorphisms affecting genes of the serotonergic system, and to assess their putative role in bipolar affective disorder (BPAD) and major depressive disorder (MDD). METHODS: A selection of 41 genes of the serotonergic system encoding receptors, the serotonin transporter, metabolic enzymes and chaperones were investigated using a paired-end mapping (PEM) approach on next-generation sequencing data from the pilot project of the 1000 Genomes Project. For association testing, 593 patients with MDD, 1,145 patients with BPAD, and 1,738 healthy controls were included in the study. RESULTS: PEM led to the identification of a microdeletion in the gene encoding tryptophan hydroxylase 2 (TPH2), affecting an amygdala- and hippocampus-specific isoform. It was not associated with BPAD or MDD using a case-control association approach. CONCLUSIONS: We did not find evidence for a role of the TPH2 microdeletion in the pathoetiology of affective disorders. Further studies examining its putative role in behavioral traits regulated by the limbic system are warranted.

Comparative analysis of whole plant, flower and root extracts of Chamomilla recutita L. and characteristic pure compounds reveals differential anti-inflammatory effects on human T cells.

Introduction: Chronic inflammation is a hallmark of chronic wounds and inflammatory skin diseases. Due to a hyperactive and prolonged inflammation triggered by proinflammatory immune cells, transitioning to the repair and healing phase is halted. T cells may exacerbate the proinflammatory milieu by secreting proinflammatory cytokines. Chamomilla recutita L. (chamomile) has been suggested for use in several inflammatory diseases, implying a capability to modulate T cells. Here, we have characterized and compared the effects of differently prepared chamomile extracts and characteristic pure compounds on the T cell redox milieu as well as on the migration, activation, proliferation, and cytokine production of primary human T cells. Methods: Phytochemical analysis of the extracts was carried out by LC-MS/MS. Primary human T cells from peripheral blood (PBTs) were pretreated with aqueous or hydroethanolic chamomile extracts or pure compounds. Subsequently, the effects on intracellular ROS levels, SDF-1α induced T cell migration, T cell activation, proliferation, and cytokine production after TCR/CD3 and CD28 costimulation were determined. Gene expression profiling was performed using nCounter analysis, followed by ingenuity pathway analysis, and validation at protein levels. Results: The tested chamomile extracts and pure compounds differentially affected intracellular ROS levels, migration, and activation of T cells. Three out of five differently prepared extracts and two out of three pure compounds diminished T cell proliferation. In line with these findings, LC-MS/MS analysis revealed high heterogeneity of phytochemicals among the different extracts. nCounter based gene expression profiling identified several genes related to T cell functions associated with activation and differentiation to be downregulated. Most prominently, apigenin significantly reduced granzyme B induction and cytotoxic T cell activity. Conclusion: Our results demonstrate an anti-inflammatory effect of chamomile- derived products on primary human T cells. These findings provide molecular explanations for the observed anti-inflammatory action of chamomile and imply a broader use of chamomile extracts in T cell driven chronic inflammatory diseases such as chronic wounds and inflammatory skin diseases. Importantly, the mode of extract preparation needs to be considered as the resulting different phytochemicals can result in differential effects on T cells.

Curricular representation of neurogastroenterology: A survey among medical students in Germany.

BACKGROUND: Neurogastroenterological disorders (NGDs) are highly prevalent and substantially impact patients' quality of life. Effective treatment of NGDs depends on the competence and training of medical caregivers. Students' perceived competence in neurogastroenterology and its place in medical school curricula are assessed in this study. METHODS: A multi-center digital survey among medical students was conducted at five universities. Self-ratings of competence regarding basic mechanisms, diagnosis, and treatment of six chronic medical conditions were assessed. These included irritable bowel syndrome (IBS), gastroesophageal reflux disease, and achalasia. Ulcerative colitis, hypertension, and migraine were included as references. KEY RESULTS: Of 231 participants, 38% remembered that neurogastroenterology was covered in their curriculum. Highest competence ratings were stated for hypertension and the lowest for IBS. These findings were identical for all institutions irrespective of their curricular model and demographic parameters. Students who remembered neurogastroenterology as a part of their curriculum reported higher competence ratings. According to 72% of students, NGDs should be highlighted more prominently in the curriculum. CONCLUSIONS & INFERENCES: Despite its epidemiological relevance, neurogastroenterology is only weakly represented in medical curricula. Students report low levels of subjective competence in handling NGDs. In general, assessing the learners' perspective on an empirical basis may enrichen the process of national standardization of medical school curricula.

Associations of Mucosal Nerve Fiber Innervation Density with Hirschsprung-Associated Enterocolitis: A Retrospective Three-Center Cohort Study.

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 ßIII-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 HTR3A polymorphism c. -42C>T is associated with amygdala responsiveness in patients with irritable bowel syndrome.

BACKGROUND & AIMS: 5-Hydroxytryptamine (5-HT)3 receptor (5-HT3R) antagonists are effective in treating patients with irritable bowel syndrome (IBS) and have anxiolytic effects. Their therapeutic effects are related, in part, to reducing amygdala engagement during expected visceral pain. A single nucleotide polymorphism in HTR3A, c.-42C>T;(C178T; rs1062613), is associated with altered reactivity of the amygdala during emotional face processing in healthy subjects (controls). We evaluated the influence of this single nucleotide polymorphism on amygdala reactivity to emotional faces and nonemotional stimuli in female patients with IBS and controls. METHODS: We measured brain responses during an affect-matching paradigm in 54 women (26 with IBS, 29 controls) using functional magnetic resonance imaging. We examined associations between HTR3A c.-42C>T genotype (C/C vs T carrier) and responses in amygdala and other regions of brain that expressed high levels of 5-HT3R. RESULTS: The C/C genotype was associated with greater anxiety symptoms in patients with IBS and controls and increased activation of the amygdala under emotional and nonemotional conditions. Among patients with IBS, C/C genotype was associated with greater symptom ratings. A subset of IBS patients with the C/C genotype had increased amygdala responses to nonemotional stimuli, compared with other subjects with C/C genotype. CONCLUSIONS: Regardless of diagnosis, the C/C genotype of the c.-42C>T polymorphism in HTR3A, compared with T carrier status, is associated with increased anxiety and amygdala responsiveness during emotional and nonemotional tasks. This polymorphism was associated with severity of IBS symptoms. Although this genotype is not sufficient for diagnosis of IBS, it is associated with severity of symptoms.

Keratinocyte-induced costimulation of human T cells through CD6 - but not CD2 - activates mTOR and prevents oxidative stress.

In psoriasis and other inflammatory skin diseases, keratinocytes (KCs) secrete chemokines that attract T cells, which, in turn, cause epidermal hyperplasia by secreting proinflammatory cytokines. To date, it remains unclear whether skin-homing T cells, particularly memory T cells, can also be activated by direct cell contact with KCs. In this study, we demonstrated the ability of primary human KCs to activate human memory T cells directly by transmitting costimulatory signals through the CD6/CD166/CD318 axis. Interestingly, despite being negative for CD80/CD86, KCs initiate a metabolic shift within T cells. Blockade of the CD6/CD166/CD318 axis prevents mammalian target of rapamycin activation and T cell proliferation but promotes oxidative stress and aerobic glycolysis. In addition, it diminishes formation of central memory T cells. Importantly, although KC-mediated costimulation by CD2/CD58 also activates T cells, it cannot compensate for the lack of CD6 costimulation. Therefore, KCs likely differentially regulate T cell functions in the skin through two distinct costimulatory receptors: CD6 and CD2. This may at least in part explain the divergent effects observed when treating inflammatory skin diseases with antibodies to CD6 versus CD2. Moreover, our findings may provide a molecular basis for selective interference with either CD6/CD166/CD318, or CD2/CD58, or both to specifically treat different types of inflammatory skin diseases.