The Effects of Binary Clostridioides difficile Toxin CDT on the Epithelial Barrier

Einleitung: Clostridioides difficile ist die häufigste Ursache antibiotikaassoziierter Diarrhöen. Hypervirulente C. difficile-Stämme produzieren zusätzlich zu den Toxinen TcdA und TcdB das binäre Toxin CDT. Eine Infektion mit diesen CDT-produzierenden Stämmen ist mit einem erhöhten Risiko für einen schwerwiegenden Krankheitsverlauf assoziiert. Die epitheliale Barriere wird durch Tight-Junction-Proteine ausgebildet und ist häufig Zielstruktur bakterieller Toxine. Ziel dieser Arbeit ist daher die Untersuchung der Wirkung des CDT auf die epitheliale Barriere. Methode: HT-29/B6-GR/MR-Monolayer wurden mit subtoxischen Konzentrationen von CDT behandelt. Eine funktionelle Charakterisierung der epithelialen Barriere erfolgte durch die Messung des transepithelialen elektrischen Widerstands (TER) und der Permeabilität für die Makromoleküle Fluorescein (332 Da) und FITC-Dextran (4 kDa). Eine mögliche Induktion von Nekrosen wurde mit einem LDH-Release-Assay und eine mögliche Apoptoseinduktion mit einem TUNEL-Assay analysiert. Die Proteinexpression verschiedener Tight Junction (TJ)-Proteine wurde mittels Western Blot untersucht. Eine Analyse der Lokalisation und Zusammensetzung verschiedener TJ-Proteine erfolgte mittels konfokaler Laser-Scanning-Mikroskopie und hochauflösender STED-Mikroskopie. Ergebnisse: Subtoxische Konzentrationen des C. difficile-Toxins CDT bewirkten eine funktionelle Störung der epithelialen Barriere mit reduziertem TER und erhöhter Permeabilität für die Makromoleküle Fluorescein und FITC-Dextran. In den verwendeten Konzentrationen war keine relevante Induktion von Nekrose und Apoptose und keine veränderte Expression der untersuchten TJ-Proteine nachzuweisen. Molekulares Korrelat für die Barrierestörung hingegen war eine subzelluläre Umverteilung der TJ-Proteine Trizellulin, Occludin und Claudin-4 und eine perijunktionale Kondensation des Aktin-Zytoskeletts. Die Effekte des CDT auf die epitheliale Barriere konnten durch Inhibition der Myosin-leichte-Ketten-Kinase MLCK verhindert werden. Diskussion: Subtoxische Konzentrationen des CDT beeinträchtigen die funktionelle Integrität der epithelialen Barriere durch eine MLCK-vermittelte gestörte Lokalisation von barrierebildenden TJ-Proteinen. Ein veränderter Phosphorylierungsstatus und ein Calciumeinstrom stellen mögliche Ursachen für eine CDT-vermittelte Aktivierung der MLCK dar. Diese Barrierestörung, insbesondere die Öffnung des parazellulären Durchtrittsweges für Makromoleküle, ist ein Erklärungsansatz für die Hypervirulenz von CDT-produzierenden C. difficile-Stämmen. Als weitere mögliche Ursachen der Hypervirulenz werden die CDT-vermittelte Zytotoxizität, eine veränderte Expression der Toxine TcdA und TcdB, die Wirkung des CDTb als porenformendes Toxin, ein Durchtritt von luminalen Antigenen im Sinne des Leaky-Gut-Konzeptes ein erleichterter Rezeptorzugang für die Toxine TcdA und TcdB diskutiert.Introduction: Clostridioides difficile is the most common cause of antibiotic-associated diarrhea. Hypervirulent C. difficile strains produce, in addition to toxins TcdA and TcdB, the binary toxin CDT. An infection with these CDT-producing strains is associated with an increased risk of a severe disease course. The epithelial barrier is formed by tight junction proteins and is often the target structure of bacterial toxins. Therefore, the aim of this study is to investigate the effect of CDT on the epithelial barrier. Methods: HT-29/B6-GR/MR monolayers were treated with subtoxic concentrations of CDT. Functional characterization of the epithelial barrier was performed by measuring transepithelial electrical resistance (TER) and the permeability to the macromolecules fluorescein (332 Da) and FITC-dextran (4 kDa). Potential induction of necrosis was analyzed using an LDH release assay. Potential apoptosis induction was assessed using TUNEL assay. The expression level of various tight junction (TJ) proteins was examined by Western blotting. Analysis of the subcellular localization of different TJ proteins was performed using confocal laser-scanning microscopy and high-resolution STED microscopy. Results: Subtoxic concentrations of the C. difficile toxin CDT resulted in a functional disruption of the epithelial barrier, characterized by reduced TER and increased permeability to macromolecules such as fluorescein and FITC-Dextran. At the concentrations used, there was no relevant induction of necrosis and apoptosis, and no altered expression of the examined TJ proteins could be detected. The molecular correlate for the barrier disruption, however, was a subcellular redistribution of the TJ proteins tricellulin, occludin and claudin-4, as well as perijunctional condensation of the actin cytoskeleton. The effects of CDT on the epithelial barrier could be prevented by inhibiting the Myosin Light-Chain Kinase (MLCK). Discussion: Subtoxic concentrations of CDT impaired the functional integrity of the epithelial barrier through MLCK-mediated de-localization of barrier-forming TJ proteins. An altered phosphorylation status and calcium influx are potential causes for CDT-mediated MLCK activation. This barrier disruption, particularly the opening of the paracellular passage for macromolecules, provides an explanation for the hypervirulence of CDT-producing C. difficile strains. Other potential causes of hypervirulence discussed include CDT-mediated cytotoxicity, altered expression of the toxins TcdA and TcdB, the poreforming effect of CDTb, the passage of luminal antigens in line with the Leaky Gut concept, and facilitated receptor access for the toxins TcdA and TcdB

Towards the clinical implementation of pharmacogenetics in bipolar disorder.

BackgroundBipolar disorder (BD) is a psychiatric illness defined by pathological alterations between the mood states of mania and depression, causing disability, imposing healthcare costs and elevating the risk of suicide. Although effective treatments for BD exist, variability in outcomes leads to a large number of treatment failures, typically followed by a trial and error process of medication switches that can take years. Pharmacogenetic testing (PGT), by tailoring drug choice to an individual, may personalize and expedite treatment so as to identify more rapidly medications well suited to individual BD patients.DiscussionA number of associations have been made in BD between medication response phenotypes and specific genetic markers. However, to date clinical adoption of PGT has been limited, often citing questions that must be answered before it can be widely utilized. These include: What are the requirements of supporting evidence? How large is a clinically relevant effect? What degree of specificity and sensitivity are required? Does a given marker influence decision making and have clinical utility? In many cases, the answers to these questions remain unknown, and ultimately, the question of whether PGT is valid and useful must be determined empirically. Towards this aim, we have reviewed the literature and selected drug-genotype associations with the strongest evidence for utility in BD.SummaryBased upon these findings, we propose a preliminary panel for use in PGT, and a method by which the results of a PGT panel can be integrated for clinical interpretation. Finally, we argue that based on the sufficiency of accumulated evidence, PGT implementation studies are now warranted. We propose and discuss the design for a randomized clinical trial to test the use of PGT in the treatment of BD

Novel Primate Model of Serotonin Transporter Genetic Polymorphisms Associated with Gene Expression, Anxiety and Sensitivity to Antidepressants

This is the final version of the article. It first appeared from Nature Publishing Group via https://dx.doi.org/10.1038/npp.2016.41Genetic polymorphisms in the repeat upstream region of the serotonin transporter gene (SLC6A4) are associated with individual differences in stress reactivity, vulnerability to affective disorders and response to pharmacotherapy. However, the molecular, neurodevelopmental and psychopharmacological mechanisms underlying the link between SLC6A4 polymorphisms and the emotionally vulnerable phenotype are not fully understood. Thus, using the marmoset monkey Callithrix jacchus we characterize here a new neurobiological model to help to address these questions. We first sequenced the marmoset SLC6A4 promoter and identified a double nucleotide polymorphism (−2053AC/CT) and two single nucleotide polymorphisms (−2022C/T and −1592G/C) within the repeat upstream region. We showed their association with gene expression using in vivo quantitative PCR and with affective behavior using a primate test of anxiety (human intruder test). The low-expressing haplotype (AC/C/G) was linked with high anxiety whilst the high-expressing one (CT/T/C) was associated with an active coping strategy in response to threat. Pharmacological challenge with an acute dose of the selective serotonin reuptake inhibitor (SSRI), citalopram, revealed a genotype-dependent behavioral response. Whilst individuals homozygous for the high anxiety-related haplotype AC/C/G exhibited a dose-dependent, anxiogenic response, individuals homozygous for the low anxiety-related haplotype CT/T/C showed an opposing, dose-dependent anxiolytic effect. These findings provide a novel genetic and behavioral primate model to study the molecular, neurodevelopmental and psychopharmacological mechanisms that underlie genetic variation-associated complex behaviors, with specific implications for the understanding of normal and abnormal serotonin actions and the development of personalized pharmacological treatments for psychiatric disorders.Work was supported by an MRC Programme (ACR; G0901884) and performed within the Behavioural and Clinical Neuroscience Institute, University of Cambridge, funded jointly by the Wellcome Trust and MRC. AMS was supported by a McDonnell Foundation grant (PI’s: E. Phelps, T.W. Robbins; Co-Investigators: ACR and J. LeDoux; 22002015501) and currently supported by MRC; YS supported by the Long Term Student Support Program provided by Osaka University and the Ministry of Education, Culture, Sports, Science and Technology of Japan; HC supported by MRC Career Development Award and ACFS/MI supported by grants from the MRC and Wellcome Trust. GC supported by the Behavioural and Clinical Neuroscience Institute, Cambridge, United Kingdom. EHSS was self-funded

Gender differences in the genetic and environmental determinants of adolescent depression

Background: The well-documented gender differences in the risk for depression may be explained by genetic factors, by different responses to social context, or by a combination of both. We sought to assess whether there were gender differences in the longitudinal associations between serotonin transporter promoter ( 5-HTTLPR ) genotype and depressive symptoms in adolescents, and whether macrosocial context plays a role in explaining any observed differences. Methods: Using data from a nationally representative survey of adolescents, we applied multilevel mixed models to assess, separately for adolescent males and females (a) the relation between 5-HTTLPR genotype and depressive symptoms and (b) the interaction of county-level deprivation and 5- HTTLPR genotype in models predicting depressive symptoms. All models adjusted for age and other covariates. Results: Among females ( n =560), main effects models showed an association between the sl genotype and lowered risk of depressive symptoms ( b =−.18, P =.03). Among males ( n =524), interaction models showed an association between sl genotype and lowered risk of depressive symptoms in deprived counties only ( b =−.32, P =.04). Conclusions: In adolescent females, the 5-HTTLPR sl genotype confers protection against depressive symptoms independent of county-level social context, whereas in adolescent males, protection by the same genotype is conferred only within the context of county-level deprivation. Future work should aim to understand how genetic and macrosocial factors jointly shape risk for mental illness, and how these factors shape gender differences in mental illness. Depression and Anxiety, 2010.© 2010 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/77512/1/20692_ftp.pd

A colonic organoid model challenged with the large toxins of Clostridioides difficile TcdA and TcdB exhibit deregulated tight junction proteins

BACKGROUND: Clostridioides difficile toxins TcdA and TcdB are responsible for diarrhea and colitis. Lack of functional studies in organoid models of the gut prompted us to elucidate the toxin’s effects on epithelial barrier function and the molecular mechanisms for diarrhea and inflammation. METHODS: Human adult colon organoids were cultured on membrane inserts. Tight junction (TJ) proteins and actin cytoskeleton were analyzed for expression via Western blotting and via confocal laser-scanning microscopy for subcellular localization. RESULTS: Polarized intestinal organoid monolayers were established from stem cell-containing colon organoids to apply toxins from the apical side and to perform functional measurements in the organoid model. The toxins caused a reduction in transepithelial electrical resistance in human colonic organoid monolayers with sublethal concentrations. Concomitantly, we detected increased paracellular permeability fluorescein and FITC-dextran-4000. Human colonic organoid monolayers exposed to the toxins exhibited redistribution of barrier-forming TJ proteins claudin-1, -4 and tricellulin, whereas channel-forming claudin-2 expression was increased. Perijunctional F-actin cytoskeleton organization was affected. CONCLUSIONS: Adult stem cell-derived human colonic organoid monolayers were applicable as a colon infection model for electrophysiological measurements. The TJ changes noted can explain the epithelial barrier dysfunction and diarrhea in patients, as well as increased entry of luminal antigens triggering inflammation

Altered 5-hydroxytryptamine signaling in patients with constipation- and diarrhea-predominant irritable bowel syndrome

Background & Aims: Evidence suggests that postprandial platelet-depleted plasma 5-hydroxytryptamine (5-HT) concentrations may be abnormal in irritable bowel syndrome (IBS). However, interpretation of the data has been hampered by the variable methodology and rather small numbers used in previous studies. Therefore, the aim of this study was to measure concentrations of platelet-depleted plasma 5-HT and its metabolite 5-HIAA under fasting and fed conditions in a large group of patients with diarrhea-predominant (d-) and constipation-predominant (c-) IBS, compared with controls. The ratio of plasma 5-HIAA:5-HT and platelet stores was also assessed. Methods: Twenty-nine c-IBS patients (aged, 19-53 years), 55 d-IBS patients (aged, 19-52 years), and 35 healthy volunteers (aged, 18-46 years) had platelet-depleted plasma 5-HT/5-HIAA concentrations measured using reverse-phase, high-performance liquid chromatography with fluorimetric detection before and after a standard meal. Results: d-IBS patients had raised platelet-depleted plasma 5-HT concentrations under fasting and fed conditions (P <.05). However, the postprandial relative to fasting concentration was similar to controls. In contrast, c-IBS patients failed to show an increase in platelet-depleted plasma 5-HT concentration with meal ingestion compared with controls (P <.01). c-IBS was associated with decreased 5-HIAA (P <.01) but normal 5-HIAA:5-HT ratio and d-IBS with normal 5-HIAA concentrations but reduced 5-HIAA:5-HT ratio (P <.005). C-IBS but not d-IBS patients had increased platelet 5-HT. Conclusions: These results support the concept that d-IBS is characterized by reduced 5-HT reuptake, whereas impaired release may be a feature of c-IBS. These results also provide a rational basis for current pharmacologic approaches involving modulation of different 5-HT receptors in c- and d-IBS. © 2006 by the American Gastroenterological Association

Pilot study of Biomarkers for predicting effectiveness of ramosetron in diarrhea‐predominant irritable bowel syndrome: expression of S100A10 and polymorphisms of TPH

Background Serotonin type 3 receptor (5‐ HT 3 R) antagonists are potentially useful therapeutic agents for diarrhea‐predominant irritable bowel syndrome ( IBS ‐D). To identify biomarkers predicting effectiveness of the 5‐ HT 3 R antagonist (ramosetron) in IBS ‐D. Methods Irritable bowel syndrome‐D Japanese subjects received 2.5 or 5  μ g of ramosetron once daily for 4 weeks. Colonic mucosal S100A and tryptophan hydroxylase ( TPH ) mRNA expression levels were measured before treatment. Genomic DNA was extracted from blood and polymorphisms of TPH 1 and TPH 2 were analyzed. Key Results Forty‐two patients (27 men and 15 women, mean age 42 years) with IBS ‐D were included for analysis. Improvement of IBS symptoms was seen in 26 (61.9%). Baseline S100A10 ( p  = 0.02) and TPH 1 ( p  = 0.02) expression were significantly higher in the ramosetron responders than in the non‐responders. The frequencies of the TPH 1 rs4537731G allele in linkage disequilibrium with the TPH 1 rs7130929 T allele (11.5% vs 50%, p  = 0.003; OR : 12; 95% CI: 2.1–69) along with TPH 1 rs211105 C allele (3.8% vs 43.8%, p  = 0.0003; OR : 19; 95% CI: 2.1–181) were significantly lower in the responders than in the non‐responders. The mean scores of diarrhea at baseline were significantly higher (5.2 vs 3.7, p  = 0.005) in patients with TPH 1 rs211105 T/T than those with the G allele. Conclusions & Inferences TPH 1 gene polymorphisms and S100A10 expression, which correlate with 5‐ HT signaling were associated with ramosetron effectiveness in IBS ‐D, and may possibly lead to prospective identification of the resistance to treatment. Colonic mucosal S100A and TPH mRNA expression levels and TPH1 SNPs were analyzed in 42 treated patients. Increased S100A10 and TPH1 expression and TPH1 high producer SNPs appear to be associated with not only diarrhea symptoms, but also greater ramosetron effectiveness in IBS‐D patients.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/109926/1/nmo12473.pd

Neuroticism-related personality traits are related to symptom severity in patients with premenstrual dysphoric disorder and to the serotonin transporter gene-linked polymorphism 5-HTTPLPR

Neuroticism has been linked to a functional polymorphism in the serotonin transporter gene (5-HTTLPR), with short-allele carriers being overrepresented among high-scorers on neuroticism. Studies evaluating neuroticism-related personality traits in relation to the 5-HTTLPR polymorphism among patients with premenstrual dysphoric disorder (PMDD) and are lacking. The primary aim of this study was to evaluate the relationship between PMDD and neuroticism-related personality traits, and secondly, to relate the personality trait scores of PMDD patients to experienced symptom severity and to the 5-HTTLPR short allele. Thirty PMDD patients and 55 asymptomatic healthy controls were included in the study. The Swedish Universities Scale of Personality was used to evaluate personality traits. Genotype analyses were available in 27 PMDD patients and 18 healthy controls. Women with PMDD displayed higher levels of neuroticism-related personality traits (psychic trait anxiety, somatic trait anxiety, embitterment, stress susceptibility and mistrust) than healthy controls, and these effects were most prominent in women with more severe luteal phase symptoms. Furthermore, PMDD patients with at least one copy of the short allele of the 5-HTTLPR polymorphism scored higher on psychic trait anxiety and lack of assertiveness than PMDD patients who were homozygous for the long allele. PMDD patients who suffer from more severe luteal phase symptoms also display increased scores of neuroticism-related personality traits in comparison with healthy controls. Within the group of PMDD patients, differences in certain personality trait scores are associated with the short allele of the 5-HTTLPR polymorphism

Genetics of migraine in the age of genome-wide association studies

Genetic factors importantly contribute to migraine. However, unlike for rare monogenic forms of migraine, approaches to identify genes for common forms of migraine have been of limited success. Candidate gene association studies were often negative and positive results were often not replicated or replication failed. Further, the significance of positive results from linkage studies remains unclear owing to the inability to pinpoint the genes under the peaks that may be involved in migraine. Problems hampering these studies include limited sample sizes, methods of migraine ascertainment, and the heterogeneous clinical phenotype. Three genome-wide association studies are available now and have successfully identified four new genetic variants associated with migraine. One new variant (rs1835740) modulates glutamate homeostasis, thus integrates well with current concepts of neurotransmitter disturbances. This variant may be more specific for severe forms of migraine such as migraine with aura than migraine without aura. Another variant (rs11172113) implicates the lipoprotein receptor LRP1, which may interact with neuronal glutamate receptors, thus also providing a link to the glutamate pathway. In contrast, rs10166942 is in close proximity to TRPM8, which codes for a cold and pain sensor. For the first time this links a gene explicitly implicated in pain related pathways to migraine. The potential function of the fourth variant rs2651899 (PRDM16) in migraine is unclear. All these variants only confer a small to moderate change in risk for migraine, which concurs with migraine being a heterogeneous disorder. Ongoing large international collaborations will likely identify additional gene variants for migraine