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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is the causative agent of the COVID-19 disease. COVID-19 viral infection can affect many cell types, including epithelial cells of the lungs and airways. Extracellular vesicles (EVs) are released by virtually all cell types, and their packaged cargo allows for intercellular communication, cell differentiation, and signal transduction. Cargo from virus-infected cells may include virally derived metabolites, miRNAs, nucleic acids, and proteins. We hypothesized that COVID-19 plasma EVs can induce the formation of signaling platforms known as lipid rafts after uptake by normal human small airway epithelial cells (SAECs). Circulating EVs from patients with or without COVID-19 were characterized by nanoparticle tracking analysis, Western blotting using specific antibodies, and transmission electron microscopy. Primary cultures of normal human small airway epithelial cells were challenged with EVs from the two patient groups, and lipid raft formation was measured by fluorescence microscopy and assessed by sucrose density gradient analysis. Collectively, our data suggest that circulating EVs from COVID-19-infected patients can induce the formation of lipid rafts in normal human small airway epithelial cells. These results suggest the need for future studies aimed at investigating whether the increased density of lipid rafts in these cells promotes viral entry and alteration of specific signaling pathways in the recipient cells. [ABSTRACT FROM AUTHOR]

Introduction: Through routine respiratory samples surveillance among COVID-19 patients in the intensive care, three patients with aspergillus were identified in a newly opened general intensive care unit during the second wave of the pandemic. Methodology: As no previous cases of aspergillus had occurred since the unit had opened. An urgent multidisciplinary outbreak meeting was held. The possible sources of aspergillus infection were explored. The multidisciplinary approach enabled stakeholders from different skills to discuss possible sources and management strategies. Environmental precipitants like air handling units were considered and the overall clinical practice was reviewed. Settle plates were placed around the unit to identify the source. Reports of recent water leaks were also investigated. Results: Growth of aspergillus on a settle plate was identified the potential source above a nurse's station. This was the site of a historic water leak from the ceiling above, that resolved promptly and was not investigated further. Subsequent investigation above the ceiling tiles found pooling of water and mould due to a slow water leak from a pipe. Conclusion: Water leaks in patient areas should be promptly notified to infection prevention. Detailed investigation to ascertain the actual cause of the leak and ensure any remedial work could be carried out swiftly. Outbreak meetings that include diverse people with various expertises (clinical and non-clinical) can enable prompt identification and resolution of contaminated areas to minimise risk to patients and staff. During challenging pandemic periods hospitals must not lose focus on other clusters and outbreaks occurring simultaneously. [ABSTRACT FROM AUTHOR]

INTRODUCTION: Proton pump inhibitor (PPI) use was recently reported to be associated with increased severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and worse clinical outcomes. The underlying mechanism(s) for this association are unclear. METHODS: We performed a prospective study of hospitalized coronavirus disease 2019 (COVID-19) patients and COVID-negative controls to understand how PPI use may affect angiotensin-converting enzyme 2 (ACE2) expression and stool SARS-CoV-2 RNA. Analysis of a retrospective cohort of hospitalized patients with COVID-19 from March 15, 2020 to August 15, 2020 in 6 hospitals was performed to evaluate the association of PPI use and mortality. Covariates with clinical relevance to COVID-19 outcomes were included to determine predictors of in-hospital mortality. RESULTS: Control PPI users had higher salivary ACE2 mRNA levels than nonusers, 2.39 6 1.15 vs 1.22 6 0.92 (P 5 0.02), respectively. Salivary ACE2 levels and stool SARS-CoV-2 RNA detection rates were comparable between users and nonusers of PPI. In 694 hospitalized patients with COVID-19 (age558 years, 46% men, and 65% black), mortality rate in PPI users and nonusers was 30% (68/227) vs 12.1% (53/439), respectively. Predictors of mortality by logistic regression were PPI use (adjusted odds ratio [aOR]52.72, P < 0.001), age (aOR51.66 per decade, P < 0.001), race (aOR53.03, P5 0.002), cancer (aOR52.22, P50.008), and diabetes (aOR51.95, P50.003). The PPI-associated mortality risk was higher in black patients (aOR 5 4.16, 95% confidence interval: 2.28-7.59) than others (aOR 5 1.62, 95% confidence interval: 0.82-3.19, P 5 0.04 for interaction). DISCUSSION: COVID-negative PPI users had higher salivary ACE2 expression. PPI use was associated with increased mortality risk in patients with COVID-19, particularly African Americans. [ABSTRACT FROM AUTHOR]

Chemokines are small proteins that promote leukocyte migration during development, infection, and inflammation. We and others isolated the unique chemokine CCL21, a potent chemo-attractant for naïve T-cells, naïve B-cells, and immature dendritic cells. CCL21 has a 37 amino acid carboxy terminal extension that is distinct from the rest of the chemokine family, which is thought to anchor it to venule endothelium where the amino terminus can interact with its cognate receptor, CCR7. We and others have reported that venule endothelium expressing CCL21 plays a crucial role in attracting naïve immune cells to sites of antigen presentation. In this study we generated a series of monoclonal antibodies to the amino terminus of CCL21 in an attempt to generate an antibody that blocked the interaction of CCL21 with its receptor CCR7. We found one humanized clone that blocked naïve T-cell migration towards CCL21, while memory effector T-cells were less affected. Using this monoclonal antibody, we also demonstrated that CCL21 is expressed in the mucosal venule endothelium of the large majority of inflammatory bowel diseases (IBD), including Crohn's disease, ulcerative colitis, and also in celiac disease. This expression correlated with active IBD in 5 of 6 cases, whereas none of 6 normal bowel biopsies had CCL21 expression. This study raises the possibility that this monoclonal antibody could be used to diagnose initial or recurrent of IBD. Significantly, this antibody could also be used for therapeutic intervention in IBD by selectively interfering with recruitment of naïve immune effector cells to sites of antigen presentation, without harming overall memory immunity. [ABSTRACT FROM AUTHOR]

Background: Environmental organic dust exposures enriched in Toll-like receptor (TLR) agonists can reduce allergic asthma development but are associated with occupational asthma and chronic bronchitis. The TLR adaptor protein myeloid differentiation factor88 (MyD88) is fundamental in regulating acute inflammatory responses to organic dust extract (ODE), yet its role in repetitive exposures is unknown and could inform future strategies.Methods: Wild-type (WT) and MyD88 knockout (KO) mice were exposed intranasally to ODE or saline daily for 3 weeks (repetitive exposure). Repetitively exposed animals were also subsequently rested with no treatments for 4 weeks followed by single rechallenge with saline/ODE.Results: Repetitive ODE exposure induced neutrophil influx and release of pro-inflammatory cytokines and chemokines were profoundly reduced in MyD88 KO mice. In comparison, ODE-induced cellular aggregates, B cells, mast cell infiltrates and serum IgE levels remained elevated in KO mice and mucous cell metaplasia was increased. Expression of ODE-induced tight junction protein(s) was also MyD88-dependent. Following recovery and then rechallenge with ODE, inflammatory mediators, but not neutrophil influx, was reduced in WT mice pretreated with ODE coincident with increased expression of IL-33 and IL-10, suggesting an adaptation response. Repetitively exposed MyD88 KO mice lacked inflammatory responsiveness upon ODE rechallenge.Conclusions: MyD88 is essential in mediating the classic airway inflammatory response to repetitive ODE, but targeting MyD88 does not reduce mucous cell metaplasia, lymphocyte influx, or IgE responsiveness. TLR-enriched dust exposures induce a prolonged adaptation response that is largely MyD88-independent. These findings demonstrate the complex role of MyD88-dependent signaling during acute vs. chronic organic dust exposures. [ABSTRACT FROM AUTHOR]

Nodal-related protein (ndr2) is a member of the transforming growth factor type β superfamily of factors and is required for ventral midline patterning of the embryonic central nervous system in zebrafish. In humans, mutations in the gene encoding nodal cause holoprosencephaly and heterotaxy. Mutations in the ndr2 gene in the zebrafish (Danio rerio) lead to similar phenotypes, including loss of the medial floor plate, severe deficits in ventral forebrain development and cyclopia. Alleles of the ndr2 gene have been useful in studying patterning of ventral structures of the central nervous system. Fifteen different ndr2 alleles have been reported in zebrafish, of which eight were generated using chemical mutagenesis, four were radiation-induced and the remaining alleles were obtained via random insertion, gene targeting (TALEN) or unknown methods. Therefore, most mutation sites were random and could not be predicted a priori. Using the CRISPR-Cas9 system from Streptococcus pyogenes, we targeted distinct regions in all three exons of zebrafish ndr2 and observed cyclopia in the injected (G0) embryos. We show that the use of sgRNA-Cas9 ribonucleoprotein (RNP) complexes can cause penetrant cyclopic phenotypes in injected (G0) embryos. Targeted polymerase chain reaction amplicon analysis using Sanger sequencing showed that most of the alleles had small indels resulting in frameshifts. The sequence information correlates with the loss of ndr2 activity. In this study, we validate multiple CRISPR targets using an in vitro nuclease assay and in vivo analysis using embryos. We describe one specific mutant allele resulting in the loss of conserved terminal cysteine-coding sequences. This study is another demonstration of the utility of the CRISPR-Cas9 system in generating domain-specific mutations and provides further insights into the structure-function of the ndr2 gene. [ABSTRACT FROM AUTHOR]

Question: For parents of children and young people (CYP) with diagnosed mental health difficulties, what are the levels of parents' well-being and psychological need?, Study Selection and Analysis: Medline, PsycINFO, EMBASE, AMED, CINAHL, Web of Science and Cochrane Library of Registered Trials were searched from inception to June 2023., Inclusion Criteria: parents of CYP aged 5-18 years with formal mental health diagnosis. Data were extracted from validated measures of well-being or psychological needs with established cut-off points or from a controlled study., Findings: 32 of the 73 310 records screened were included. Pooled means showed clinical range scores for one measure of depression, and all included measures of anxiety, parenting stress and general stress. Meta-analyses showed greater depression (g=0.24, 95% CI 0.11 to 0.38) and parenting stress (g=0.34, 95% CI 0.20 to 0.49) in parents of CYP with mental health difficulties versus those without. Mothers reported greater depression (g=0.42, 95% CI 0.18 to 0.66) and anxiety (g=0.73, 95% CI 0.27 to 1.18) than fathers. Narrative synthesis found no clear patterns in relation to CYP condition. Rates of parents with clinically relevant levels of distress varied. Typically, anxiety, parenting stress and general stress scored above clinical threshold. Quality appraisal revealed few studies with a clearly defined control group, or attempts to control for important variables such as parent gender., Conclusions: The somewhat mixed results suggest clinical anxiety, parenting and general stress may be common, with sometimes high depression. Assessment and support for parents of CYP with mental health problems is required. Further controlled studies, with consideration of pre-existing parental mental health difficulties are required., Prospero Registration Number: CRD42022344453., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY. Published by BMJ.)

Mast cell activation syndrome (MCAS) is a term applied to several clinical entities that have gained increased attention from patients and medical providers. Although several descriptive publications about MCAS exist, there are many gaps in knowledge, resulting in confusion about this clinical syndrome. Whether MCAS is a primary syndrome or exists as a constellation of symptoms in the context of known inflammatory, allergic, or clonal disorders associated with systemic mast cell activation is not well understood. More importantly, the underlying mechanisms and pathways that lead to mast cell activation in MCAS patients remain to be elucidated. Here we summarize the known literature, identify gaps in knowledge, and highlight research needs. Covered topics include contextualization of MCAS and MCAS-like endotypes and related diagnostic evaluations; mechanistic research; management of typical and refractory symptoms; and MCAS-specific education for patients and health care providers., Competing Interests: Disclosure statement M.C.C. and D.D.M. were supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH). The content is solely the responsibility of the authors and does not represent the official views of the NIH. Disclosure of potential conflict of interest: M. Castells has received research funding and consulting fees from Blueprint Medicines and consulting fees from Cogent Biosciences. M. P. Giannetti has received research funding and consulting fees from Blueprint Medicines and consulting fees from Cogent Biosciences. M. J. Hamilton has received consulting fees from Blueprint Medicines. P. Novak has received funding from Mona Taliaferro/Bay Shore Recycling, the National Heart, Lung, and Blood Institute (NHLNI; 1OT2HL156812-01), and FBRI (2022A018462); is current or previous shareholder of Moderna, Edidas Medicine, Novavax, and Pfizer; and has received royalties from Oxford University Press. J. Nicoloro-SantaBarbara has received consulting fees from Cogent Biosciences and Blueprint Medicines. S. C. Glover received consulting fees from Blueprint Medicine, Janssen, BMS, AbbVie, and Takeda. S. J. Galli has received research funding from and is scientific advisor to Evommune; and is on the scientific advisory board of Jasper Therapeutics. A. White is on the speakers bureau at Blueprint Medicines; and received consulting fees from Cogent Biosciences and Blueprint Medicines. P. Valent received funding from BMS/Celgene and AOP Orphan; and consultancy fees from Novartis, BMS/Celgene, Blueprint, Pfizer, Cogent, and Stemline. J. H. Butterfield has received a fee for the licensing of HMC-1 cell lines. D. D. Metcalfe has received consulting fees from Visterra. J. D. Milner has received consulting fees from Blueprint Medicines. The rest of the authors declare that they have no relevant conflicts of interest., (Copyright © 2024 American Academy of Allergy, Asthma & Immunology. All rights reserved.)

Recent case reports and epidemiological data suggest that fungal infections represent an underappreciated complication among people with severe COVID-19. However, the frequency of fungal colonization in patients with COVID-19 and associations with specific immune responses in the airways remain incompletely defined. We previously generated a single-cell RNA-sequencing data set characterizing the upper respiratory microenvironment during COVID-19 and mapped the relationship between disease severity and the local behavior of nasal epithelial cells and infiltrating immune cells. Our previous study, in agreement with findings from related human cohorts, demonstrated that a profound deficiency in host immunity, particularly in type I and type III interferon signaling in the upper respiratory tract, is associated with rapid progression to severe disease and worse clinical outcomes. We have now performed further analysis of this cohort and identified a subset of participants with severe COVID-19 and concurrent detection of Candida species-derived transcripts within samples collected from the nasopharynx and trachea. Here, we present the clinical characteristics of these individuals. Using matched single-cell transcriptomic profiles of these individuals' respiratory mucosa, we identify epithelial immune signatures suggestive of IL17 stimulation and anti-fungal immunity. Further, we observe a significant expression of anti-fungal inflammatory cascades in the nasal and tracheal epithelium of all participants who went on to develop severe COVID-19, even among participants without detectable genetic material from fungal pathogens. Together, our data suggest that IL17 stimulation-in part driven by Candida colonization-and blunted interferon signaling represent a common feature of severe COVID-19 infection., Importance: In this paper, we present an analysis suggesting that symptomatic and asymptomatic fungal coinfections can impact patient disease progression during COVID-19 hospitalization. By looking into the presence of other pathogens and their effect on the host immune response during COVID-19 hospitalizations, we aim to offer insight into an underestimated scenario, furthering our current knowledge of determinants of severity that could be considered for future diagnostic and intervention strategies., Competing Interests: A.K.S. reports compensation for consulting and/or SAB membership from Merck, Honeycomb Biotechnologies, Cellarity, Repertoire Immune Medicines, Hovione, Ochre Bio, Third Rock Ventures, FL82, Empress Therapeutics, Senda Biosciences, IntrECate Biotherapeutics, Relation Therapeutics, and Dahlia Biosciences. J.O.-M. reports compensation for consulting services with Cellarity, Tessel Biosciences, and Radera Biotherapeutics.

Background: Most studies on the safety and efficacy of antitumor necrosis factor alpha (anti-TNF) agents in the treatment of inflammatory bowel disease have included few Black patients., Aims: We aimed to evaluate the therapeutic response rate in Black IBD patients compared with White patients., Methods: We conducted a retrospective review of IBD patients who were treated with anti-TNF agents and assessed those with therapeutic drug levels for clinical, endoscopic, and radiologic response to anti-TNF treatment., Results: We identified 118 patients who met the inclusion criteria. Black IBD patients had significantly higher prevalence of endoscopic and radiologic active disease compared with White patients (62% and 34%, respectively; P = .023), despite similar proportions reaching therapeutic titers (67% and 55%, respectively; P = .20). Moreover, Black patients had significantly higher rate of IBD-related hospitalizations than White patients (30% vs 13%, respectively; P = .025) while on anti-TNF agents., Conclusions: Black IBD patients on anti-TNF agents had a significantly higher prevalence of active disease and more IBD-related hospitalizations than White patients., (© The Author(s) 2023. Published by Oxford University Press on behalf of Crohn’s & Colitis Foundation. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introduction: Parent-carers of children and young people (CYP) with mental health problems are at greater risk of poor outcomes, such as poor physical and mental health. Peer interventions for parent-carers of CYP with disabilities may improve parent-carer outcomes. This qualitative study investigates parent-carer experiences of using Parental Minds (PM), a multi-component peer support service for parent-carers of CYP with disabilities., Methods: Twelve current service-users and four staff/volunteers at PM participated in one-to-one semi-structured interviews. All participants were white females, except for one service-user who was male. All interviews were recorded and transcribed verbatim. Thematic analysis of results was used to explore perceived benefits and disadvantages of PM and possible behaviour change mechanisms., Results: Three themes and eight subthemes were identified. Participants identified that internal and external factors influence their self-concept. The identification of themselves as a priority, and empowerment by reassurance and affirmation lead to improved parent-carer self-efficacy and agency to better care for their CYP. Participants described the difficulty of speaking honestly with friends and family about what they experience because it is perceived as different to what "normal" parents experience. From participant accounts, PM enables the construction of a support network and links external services to help manage family circumstances rather than offer curative treatment/intervention. Proactive and immediate advice which is constantly and consistently available was valued by participants. Participants expressed the need for a flexible range of service components which provide holistic support that encompasses both health and social care., Conclusions: PM was perceived to be beneficial as a multi-component peer support service which increases parenting self-efficacy and empowerment, reduces isolation, improves access to services, and is tailored to individual needs. Parent-carers reported benefits in parenting and wellbeing practices. The development of a refined logic model will inform a future study of the effectiveness of PM on parent-carer outcomes., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Introduction: Globally, 8%-14% of children and young people (CYP) have a diagnosable mental health condition, many of whom receive no formal interventions. Parents/carers of CYP experience stress and distress owing to the mental health difficulties encountered by their CYP due to the lack of resources and support. Currently, little is known about (1) the content of interventions developed to support parents/carers nor (2) how effective interventions are at improving parents'/carers' well-being. The planned review aims to address these two gaps., Method and Analysis: A systematic review will be conducted to identify any study that describes an intervention aiming at least in part to support parents/carers with the impact of CYP (5-18 years) mental health difficulties, and to review any randomised controlled trials (RCTs) of these interventions. The following databases will be searched: MEDLINE, PsycINFO, CINAHL, AMED, EMBASE, Web of Science Core Collection and Cochrane Library CENTRAL, without any limitations applied. Analysis of the content of interventions will be structured using the Template for Intervention Description and Replication checklist as a framework. The effect of any RCTs on parents'/carers' outcomes (including well-being, satisfaction with parenting, mental health) will be extracted and assessed using the Cochrane Risk-of-Bias Tool. Data will be synthesised narratively, with meta-analysis of RCT results, if appropriate., Ethical Consideration and Dissemination: The protocol is approved by Coventry University Ethical Committee (reference number: P139611). Results will be shared in academic publications and in accessible formats using social media and public webinars., Prospero Registration Number: CRD42022344453., Competing Interests: Competing interests: All authors have completed the ICMJE uniform disclosure form. SG is an author and the director of Parental Minds, which provide services to the parent of children with mental health. The authors have no competing interests to declare., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.)

Serum tryptase is a biomarker used to aid in the identification of certain myeloid neoplasms, most notably systemic mastocytosis, where basal serum tryptase (BST) levels >20 ng/mL are a minor criterion for diagnosis. Although clonal myeloid neoplasms are rare, the common cause for elevated BST levels is the genetic trait hereditary α-tryptasemia (HαT) caused by increased germline TPSAB1 copy number. To date, the precise structural variation and mechanism(s) underlying elevated BST in HαT and the general clinical utility of tryptase genotyping, remain undefined. Through cloning, long-read sequencing, and assembling of the human tryptase locus from an individual with HαT, and validating our findings in vitro and in silico, we demonstrate that BST elevations arise from overexpression of replicated TPSAB1 loci encoding canonical α-tryptase protein owing to coinheritance of a linked overactive promoter element. Modeling BST levels based on TPSAB1 replication number, we generate new individualized clinical reference values for the upper limit of normal. Using this personalized laboratory medicine approach, we demonstrate the clinical utility of tryptase genotyping, finding that in the absence of HαT, BST levels >11.4 ng/mL frequently identify indolent clonal mast cell disease. Moreover, substantial BST elevations (eg, >100 ng/mL), which would ordinarily prompt bone marrow biopsy, can result from TPSAB1 replications alone and thus be within normal limits for certain individuals with HαT., (Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution.)

Introduction: Having a child or young person (CYP) with mental health problems can be highly distressing for parents/carers. The impact can include parental/carer depression, anxiety, lost productivity and poor family relationships. Currently, there is no synthesis of this evidence, which is needed to provide clarity around what support parents/carers may need, to meet the needs of family mental health. This review aims to identify the needs of the parents/carers of CYP who are receiving mental health services., Methods and Analysis: A systematic review will be conducted to identify potentially relevant studies that provide evidence concerning the needs and impact on parents/carers linked to their CYP having mental health difficulties. CYP mental health conditions included are anxiety disorders, depression, psychoses, oppositional defiant and other externalising disorders, labels of emerging personality disorders, eating disorders and attention deficit (hyperactive) disorders. The following databases were searched on November 2022 with no date restriction applied: Medline; PsycINFO; CINAHL; AMED; EMBASE; Web of Science; Cochrane Library; WHO International Clinical Trials Registry Platform; Social Policy and Practice; Applied Social Sciences Index and Abstracts; and Open Grey. Only studies reported in English will be included. The quality of the included studies will be assessed using Joanna Briggs Institute Critical Appraisal Checklist for qualitative studies and the Newcastle Ottawa Scale for quantitative studies. Qualitative data will be analysed thematically and inductively., Ethics and Dissemination: This review was approved by the ethical committee at Coventry University, UK, reference number P139611. The findings from this systematic review will be disseminated across various key stakeholders and published in peer-reviewed journals., Competing Interests: Competing interests: All authors have completed the ICMJE uniform disclosure form. SG is an author and the director of Parental Minds, which provides services to parent of children with mental health. All other authors declare no competing interests., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.)

Research on the care of inflammatory bowel disease (IBD) patients has been primarily in populations of European ancestry. However, the incidence of IBD, which comprises Crohn's disease and ulcerative colitis, is increasing in different populations around the world. In this comprehensive review, we examine the epidemiology, clinical presentations, disease phenotypes, treatment outcomes, social determinants of health, and genetic and environmental factors in the pathogenesis of IBD in Black and Hispanic patients in the United States. To improve health equity of underserved minorities with IBD, we identified the following priority areas: access to care, accurate assessment of treatment outcomes, incorporation of Black and Hispanic patients in therapeutic clinical trials, and investigation of environmental factors that lead to the increase in disease incidence., (© The Author(s) 2022. Published by Oxford University Press on behalf of Crohn’s & Colitis Foundation. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Type 2 diabetes (T2D) patients face increased risk of heart failure (HF) as they age. Sodium-glucose cotransporter 2 inhibitors (SGLT-2i) have demonstrated effectiveness in reducing HF hospitalizations in patients with T2D and HF with reduced ejection fraction (HFrEF). Diabetes guidelines recommend SGLT-2i therapy for patients with HFrEF; however, SGLT-2i cost is high., Objective: Study objectives were to assess SGLT-2i utilization and HF hospitalization rates in commercially insured adults (age <65) with T2D and heart failure with reduced ejection fraction (HFrEF) taking metformin with/without SGLT-2i use and conduct a cost-benefit analysis of SGLT-2i use from payer and societal perspectives., Methods: Economic models included HF hospitalization rates from real-world data (RWD) and hospitalization rate reductions from RWD and SGLT-2i clinical trials. Real-world HF hospitalization rates were obtained from claims data (MarketScan Commercial Database, years 2013-2018). Societal perspective analyses included indirect costs. Sensitivity analyses were conducted on key parameters., Results: Among adults with T2D and HFrEF age 30-64, SGLT-2i use increased (1.1% to 17.4%) between 2013 and 2018. The HF hospitalization rate without SGLT-2i use vs with was 15.5% vs 11.0% (absolute risk reduction of 4.5%). Base case scenario net-benefit was negative across all payer perspective models, while positive for societal-perspective. Payer perspective overall net-benefit in 30-64 population: -$1,725,758 (-$4106 per person). Societal perspective net-benefit in 30-64 population: $5,996,851 ($14,269 per person). In sensitivity analyses, estimated per person base case societal net-benefit of $14,269 was most sensitive to changes in baseline HF hospitalization rates, post-discharge mortality rates, and readmission rates. Lowering SGLT-2i prescription costs 50% and 80% resulted in per person net-benefit increases of $1737 and $4004, respectively., Conclusion: SGLT-2i utilization has steadily increased, with lower HF hospitalization rates observed among SGLT-2i users. Societal benefits of SGLT-2i use in this population are substantive; payer benefits are negative unless SGLT-2i cost is drastically reduced., Competing Interests: Melissa H Roberts reports grants from Sunovion Pharmaceuticals and GlaxoSmithKline and non-financial medical writing support for a journal article from AstraZeneca, outside the submitted work. The authors report no other potential conflicts of interest in relation to this work., (© 2022 Glover et al.)

Background & Aims: Whether preoperative treatment of inflammatory bowel disease (IBD) with tumor necrosis factor inhibitors (TNFis) increases the risk of postoperative infectious complications remains controversial. The primary aim of this study was to determine whether preoperative exposure to TNFis is an independent risk factor for postoperative infectious complications within 30 days of surgery., Methods: We conducted a multicenter prospective observational study of patients with IBD undergoing intra-abdominal surgery across 17 sites from the Crohn's & Colitis Foundation Clinical Research Alliance. Infectious complications were categorized as surgical site infections (SSIs) or non-SSIs. Current TNFi exposure was defined as use within 12 weeks of surgery, and serum was collected for drug-level analyses. Multivariable models for occurrence of the primary outcome, any infection, or SSI were adjusted by predefined covariates (age, sex, preoperative steroid use, and disease type), baseline variables significantly associated (P < .05) with any infection or SSI separately, and TNFi exposure status. Exploratory models used TNFi exposure based on serum drug concentration., Results: A total of 947 patients were enrolled from September 2014 through June 2017. Current TNFi exposure was reported by 382 patients. Any infection (18.1% vs 20.2%, P = .469) and SSI (12.0% vs 12.6%, P = .889) rates were similar in patients currently exposed to TNFis and those unexposed. In multivariable analysis, current TNFi exposure was not associated with any infection (odds ratio, 1.050; 95% confidence interval, 0.716-1.535) or SSI (odds ratio, 1.249; 95% confidence interval, 0.793-1.960). Detectable TNFi drug concentration was not associated with any infection or SSI., Conclusions: Preoperative TNFi exposure was not associated with postoperative infectious complications in a large prospective multicenter cohort., (Copyright © 2022 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Mutations in the tetratricopeptide repeat domain 7A (TTC7A) gene are a rare cause of congenital enteropathy that can result in significant morbidity. TTC7A deficiency leads to disruption of the intestinal epithelium. The histopathology of this condition has been partly described in case reports and clinical studies. This manuscript describes an in-depth investigation of the pediatric gastrointestinal pathology of the largest histologically examined cohort with confirmed TTC7A mutations reported to date and, for the first time, compared the findings to age-matched and sex-matched control patients with intestinal atresia not thought to be associated with TTC7A mutations. Hematoxylin and eosin-stained slides of endoscopically obtained mucosal biopsies and surgical resection specimens from 7 patients with known TTC7A mutations were examined retrospectively. The microscopic findings were found to be on a spectrum from atresia-predominant to those with predominantly epithelial abnormalities. Several unique histopathologic characteristics were observed when compared with controls. These included neutrophilic colitis and prominent lamina propria eosinophilia throughout the gastrointestinal tract. Striking architectural abnormalities of the epithelium were observed in 4 of the 7 patients. The 5 patients with intestinal atresia demonstrated hypertrophy and disorganization of the colonic muscularis mucosae accompanied by bland spindle cell nodules within the intestinal wall. The components of the latter were further elucidated using immunohistochemistry, and we subsequently hypothesize that they represent obliterated mucosa with remnants of the muscularis mucosae. Finally, atrophic gastritis was noted in 4 patients. In conclusion, the unique histopathologic characteristics of TTC7A mutation-associated enteropathy described herein more fully describe this novel disease entity in infants who present with congenital enteropathy or enterocolitis., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Chronic obstructive pulmonary disease (COPD) is a heterogeneous inflammatory airway disease. The epithelial-derived IL-33 and its receptor ST2 have been implicated in airway inflammation and infection. We aimed to determine whether astegolimab, a selective ST2 IgG2 monoclonal antibody, reduces exacerbations in COPD., Methods: COPD-ST2OP was a single-centre, randomised, double-blinded, placebo-controlled phase 2a trial in moderate-to-very severe COPD. Participants were randomly assigned (1:1) with a web-based system to received 490 mg subcutaneous astegolimab or subcutaneous placebo, every 4 weeks for 44 weeks. The primary endpoint was exacerbation rate assessed for 48 weeks assessed with a negative binomial count model in the intention-to-treat population, with prespecified subgroup analysis by baseline blood eosinophil count. The model was the number of exacerbations over the 48-week treatment period, with treatment group as a covariate. Safety was assessed in the whole study population until week 60. Secondary endpoints included Saint George's Respiratory Questionnaire for COPD (SGRQ-C), FEV 1 , and blood and sputum cell counts. The trial was registered with ClinicalTrials.gov, NCT03615040., Findings: The exacerbation rate at 48 weeks in the intention-to-treat analysis was not significantly different between the astegolimab group (2·18 [95% CI 1·59 to 2·78]) and the placebo group (2·81 [2·05 to 3·58]; rate ratio 0·78 [95% CI 0·53 to 1·14]; p=0·19]). In the prespecified analysis stratifying patients by blood eosinophil count, patients with 170 or fewer cells per μL had 0·69 exacerbations (0·39 to 1·21), whereas those with more than 170 cells per μL had 0·83 exacerbations (0·49 to 1·40). For the secondary outcomes, the mean difference between the SGRQ-C in the astegolimab group versus placebo group was -3·3 (95% CI -6·4 to -0·2; p=0·039), and mean difference in FEV 1 between the two groups was 40 mL (-10 to 90; p=0·094). The difference in geometric mean ratios between the two groups for blood eosinophil counts was 0·59 (95% CI 0·51 to 0·69; p<0·001) and 0·25 (0·19 to 0·33; p<0·001) for sputum eosinophil counts. Incidence of treatment-emergent adverse events was similar between groups., Interpretation: In patients with moderate-to-very severe COPD, astegolimab did not significantly reduce exacerbation rate, but did improve health status compared with placebo., Funding: Funded by Genentech and National Institute for Health Research Biomedical Research Centres., Competing Interests: Declaration of interests CEB reports funding from Genentech to support this study; grants from AstraZeneca, GlaxoSmithKline, Roche/Genentech, Boehringer Ingelheim, Novartis, Chiesi, Merck Sharp & Dohme, Sanofi, Regeneron, 4DPharma, and Mologic; and consulting fee paid to institution from AstraZeneca, GlaxoSmithKline, Roche/Genentech, Boehringer Ingelheim, Novartis, Chiesi, Merck Sharp & Dohme, Sanofi, Regeneron, and 4DPharma. NJG reports an investigator led grant from GlaxoSmithKline; National Institute for Health Research fellowship; and lecture honoraria from GlaxoSmithKline and Chiesi. AW reports a research grant from Sanofi Genzyme. CJ reports a Medical Research Council clinical research training fellowship. MAG, DFC, and DC are employed by Genentech and receive stock options. All other authors declare no competing interests., (Crown Copyright © 2022 Published by Elsevier Ltd. All rights reserved.)

Background: Inflammatory bowel diseases (IBD), like ulcerative colitis (UC) and Crohn's disease (CD), are associated with urinary extra-intestinal manifestations, like urolithiasis and uncomplicated urinary tract infections (UTIs). The literature reviewed for this study identifies an increased association of CD and urolithiasis against the general population as well as UC. Furthermore, the rates in which urinary comorbidities manifest have not been well characterized in cross-race analyses. The purpose of this study is to establish the prevalence of common urinary extra-intestinal manifestations in CD and UC and to further determine at what rate these affect the African American and Caucasian populations., Methodology: This is a retrospective cohort study using de-identified data collected from a research data base that included 6 integrated facilities associated with one tertiary healthcare center from 2012 to 2019. The electronic chart records for 3104 Caucasian and African American IBD patients were reviewed for frequency of urolithiasis and uncomplicated UTI via diagnosed ICD-10 codes. Comparison between data groups was made using multivariate regressions, t-tests, and chi square tests., Results: Our study included 3104 patients of which 59% were female, 38% were African American, and 43% were diagnosed with UC. Similar proportions of UC and CD diagnosed patients developed urolithiasis (6.0% vs 6.7%, p = 0.46), as well as uncomplicated UTIs (15.6% vs. 14.9%, p = 0.56). Similar proportions of African American and Caucasian patients developed urolithiasis (5.4% vs 7.0%, p = 0.09), but a higher proportion of African Americans developed uncomplicated UTIs (19.4% vs 12.6%, p ≤ 0.001)., Conclusion: We found similar rates of urolithiasis formation in both UC and CD in this study. Furthermore, these rates were not significantly different between African American and Caucasian IBD populations. This suggests that UC patients have an elevated risk of urolithiasis formation as those patients with CD. Additionally, African Americans with IBD have a higher frequency of uncomplicated UTI as compared to their Caucasian counterparts., (© 2022. The Author(s).)

SARS-CoV-2 infection can cause severe respiratory COVID-19. However, many individuals present with isolated upper respiratory symptoms, suggesting potential to constrain viral pathology to the nasopharynx. Which cells SARS-CoV-2 primarily targets and how infection influences the respiratory epithelium remains incompletely understood. We performed scRNA-seq on nasopharyngeal swabs from 58 healthy and COVID-19 participants. During COVID-19, we observe expansion of secretory, loss of ciliated, and epithelial cell repopulation via deuterosomal cell expansion. In mild and moderate COVID-19, epithelial cells express anti-viral/interferon-responsive genes, while cells in severe COVID-19 have muted anti-viral responses despite equivalent viral loads. SARS-CoV-2 RNA + host-target cells are highly heterogenous, including developing ciliated, interferon-responsive ciliated, AZGP1 high goblet, and KRT13 + "hillock"-like cells, and we identify genes associated with susceptibility, resistance, or infection response. Our study defines protective and detrimental responses to SARS-CoV-2, the direct viral targets of infection, and suggests that failed nasal epithelial anti-viral immunity may underlie and precede severe COVID-19., Competing Interests: Declaration of interests A.K.S. reports compensation for consulting and/or SAB membership from Merck, Honeycomb Biotechnologies, Cellarity, Repertoire Immune Medicines, Hovione, Ochre Bio, Third Rock Ventures, Relation Therapeutics, and Dahlia Biosciences. J.O.-M. reports compensation for consulting services with Cellarity and Hovione. C.G.K.Z., V.N.M., A.H.O., A.W.N., Y.T., J.D.B., A.K.S., S.C.G., B.H.H., and J.O.-M. are co-inventors on a provisional patent application relating to methods of stratifying and treating viral infections., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Hereditary alpha-tryptasemia (HαT) is characterized by elevated basal serum tryptase due to increased copies of the TPSAB1 gene. Individuals with HαT frequently present with multisystem complaints, including anaphylaxis and seemingly functional gastrointestinal (GI) symptoms., Objective: We sought to determine the prevalence of HαT in an irritable bowel syndrome cohort and associated immunologic characteristics that may distinguish patients with HαT from patients without HαT., Methods: Tryptase genotyping by droplet digital PCR, flow cytometry, cytometry by time-of-flight, immunohistochemistry, and other molecular biology techniques was used., Results: HαT prevalence in a large irritable bowel syndrome cohort was 5% (N = 8/158). Immunophenotyping of HαT PBMCs (N ≥ 27) revealed increased total and class-switched memory B cells. In the small bowel, expansion of tissue mast cells with expression of CD203c, HLA-DR, and FcεRI, higher intestinal epithelial cell pyroptosis, and increased class-switched memory B cells were observed. IgG profiles in sera from individuals with HαT (N = 21) significantly differed from those in individuals with quiescent Crohn disease (N = 20) and non-HαT controls (N = 19), with increased antibodies directed against GI-associated proteins identified in individuals with HαT., Conclusions: Increased mast cell number and intestinal epithelial cell pyroptosis in the small intestine, and class-switched memory B cells in both the gut and peripheral blood associated with IgG reactive to GI-related proteins, distinguish HαT from functional GI disease. These innate and adaptive immunologic findings identified in association with HαT are suggestive of subclinical intestinal inflammation in symptomatic individuals., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Young adults, 18-35 years of age, account for nearly half of all inflammatory bowel disease emergency department visits annually, costing millions of healthcare dollars and signifying undue pain and suffering. To mitigate this sequela, the study aimed to characterize the relationships between transition readiness (self-management ability), stress, and patient-centered outcomes. Outcomes were defined as disease activity and inflammatory bowel disease-related healthcare utilization (emergency department visits and inpatient hospitalization). This was a descriptive, correlational design via online survey of young adults with inflammatory bowel disease. Participants (n = 284) utilized an estimated 2.77 million healthcare dollars in 12 months. Transition readiness decreased the odds of having consistently active disease and healthcare utilization, with adjusted odds ratio ranging from 6.4 to 10.9 (p < .05). Higher stress levels increased the odds of having consistently active disease and healthcare utilization, with adjusted odds ratio ranging from 9.5 to 10.5 (p < .0001). Twenty-five percent (24.7%) of the variation in transition readiness was explained by changes in stress (p < .0001). Transition readiness and stress impacted all patient-centered outcomes. Stress negatively impacted transition readiness. These results are powerful reminders for healthcare providers to assess and treat stress and support transition readiness in young adults with inflammatory bowel disease. The potential to decrease pain, suffering, and healthcare cost is enormous., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 Society of Gastroenterology Nurses and Associates.)

Mast cells (MCs) are important in intestinal homeostasis and pathogen defense but are also implicated in many of the clinical manifestations in disorders such as irritable bowel syndrome. The utility of specific staining for MCs to quantify and phenotype them in intestinal biopsies in patients with gastrointestinal (GI) symptoms is controversial and is not a widely adopted practice. Whether or not intestinal MCs are increased or have a unique phenotype in individuals with hereditary alpha-tryptasemia (HαT), who have extra copies of the MC tryptase gene TPSAB1 and typically elevated baseline serum tryptase levels >8 ng/mL is not known. We examined the duodenal biopsies of 17 patients with HαT and compared them to 15 patients with mast cell activation syndrome who had baseline serum tryptases <8 ng/mL (MCAS-NT) and 12 GI-controls. We determined that the HαT subjects had increased MCs in the duodenum compared with MCAS-NT and GI-controls (median=30.0; interquartile range [IQR]: 20.0 to 40.0 vs. median=15.0; IQR: 5.00 to 20.0; P=0.013 and median=15.0; IQR: 13.8 to 20.0; P=0.004, respectively). These MCs were significantly found in clusters (<15 MCs) and were located throughout the mucosa and submucosa including the superficial villi compared with MCAS-NT and GI-control patients. Spindle-shaped MCs were observed in all groups including controls. These data demonstrate that HαT is associated with increased small intestinal MCs that may contribute to the prevalent GI manifestations observed among individuals with this genetic trait., Competing Interests: Conflicts of Interest and Source of Funding: Supported in part by a combined grant from the Mastocytosis Society and American Academy of Allergy, Asthma, and Immunology (M.J.H.), the Gatorade Trust through funds distributed by the University of Florida, Department of Medicine (S.C.G.), extramural NIH fund 1R21TR002639-01A1 (M.J.H. and S.C.G.), and the Division of Intramural Research of the National Institute of Allergy and Infectious Diseases, NIH (J.J.L.). The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government. The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Objectives: Health apps are software programs that are designed to prevent, diagnose, monitor, or manage conditions. Inconsistent terminology for apps is used in research literature and bibliographic database subject headings. It can therefore be challenging to retrieve evidence about them in literature searches. Information specialists at the United Kingdom's National Institute for Health and Care Excellence (NICE) have developed novel validated search filters to retrieve evidence about apps from MEDLINE and Embase (Ovid)., Methods: A selection of medical informatics journals was hand searched to identify a "gold standard" (GS) set of references about apps. The GS set was divided into a development and validation set. The filters' search terms were derived from and tested against the development set. An external development set containing app references from published NICE products was also used to inform the development of the filters. The filters were then validated using the validation set. Target recall was >90 percent. The filters' overall recall, specificity, and precision were calculated using all the references identified from the hand search., Results: Both filters achieved 98.6 percent recall against their validation sets. Overall, the MEDLINE filter had 98.8 percent recall, 71.3 percent specificity, and 22.6 percent precision. The Embase filter had 98.6 percent recall, 74.9 percent specificity, and 24.5 percent precision., Conclusions: The NICE health apps search filters retrieve evidence about apps from MEDLINE and Embase with high recall. They can be applied to literature searches to retrieve evidence about the interventions by information professionals, researchers, and clinicians.

Objective: Mucosal barrier dysfunction plays a crucial role in intestinal inflammation in Crohn's disease (CD). Intestinal epithelial cell (IEC) death resulting from innate immune activation, termed pyroptosis, was recently found to be a cause of this barrier defect. The aim of this study was to determine the predictive value of pretreatment ileal biopsy pyroptosis as a biomarker for clinical response to vedolizumab in CD., Design: Crohn's disease patients ranging 18 to 80 years old from 5 IBD centers with pre-vedolizumab ileal biopsies during colonoscopy were enrolled. Biopsies were stained for activated caspases, and levels of ileal IEC pyroptosis levels were quantified. The primary outcome was clinical response 6 months after therapy, defined as a reduction of Harvey-Bradshaw Index (HBI) of ≥5 points from baseline. Secondary outcomes included clinical remission, defined as HBI <5, and endoscopic improvement, as measured by the Simple Endoscopic Score for Crohn's Disease (SES-CD)., Results: One hundred CD patients (45 male, 55 female), median age 47 (19, 78) years, were included; clinical response rate was 60%, and clinical remission was 36%. The response rate in patients with ileal pyroptosis <14 positive cells per 1000 IECs was significantly higher than those above the threshold: 89% (25 of 28) vs 49% (35 of 72), odds ratio (OR) 8.8 (95% CI, 2.3-48.6; P < 0.001). Corresponding remission rates were 54% (15 of 28) vs 29% (21 of 72; OR 2.8 [1.03-7.59; P = 0.036]). For endoscopic improvement, ileal pyroptosis of 22 positive cells per 1000 IECs was the optimal threshold that determines the magnitude SES-CD change., Conclusions: Ileal biopsy IEC pyroptosis was predictive of clinical response and endoscopic improvement to vedolizmab in CD patients., (© 2019 Crohn’s & Colitis Foundation. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background and Aims: Medication persistence, defined as the time from drug initiation to discontinuation of therapy, has been suggested as a proxy for real-world therapeutic benefit and safety. This study seeks to compare the persistence of biologic drugs among patients with inflammatory bowel disease (IBD)., Methods: Patients with newly diagnosed IBD were included in a retrospective study using Truven MarketScan database. Treatment persistence and switching was compared among biologic medications including infliximab, adalimumab, certolizumab, golimumab, and vedolizumab. Predictors for discontinuation and switching were evaluated using time-dependent proportional hazard regression., Results: In total, 5612 patients with Crohn's disease (CD) and 3533 patients with ulcerative colitis (UC) were included in this analysis. Less than half of the patients continued using their initial biologic treatment after 1 year (48.48% in CD cohort; 44.78% in UC cohort). In the first year, adalimumab had the highest persistence and lowest switching rates for both CD (median survival time: 1.04 years) and UC (median survival time: 0.84 years). In subsequent years, infliximab users were more likely to persist in the use of biologic. Combination therapy with immunomodulators significantly decreased the risk of discontinuation, especially when immunomodulator therapy was started more than 30 days before the biologic (hazard ratio [HR], 0.22; CI, 0.16, 0.32). The major predictors for noncompliance included infection and hospitalization., Conclusion: Overall, the persistence profiles of biologics suggest a high rate of dissatisfaction or adverse disease outcomes resulting in discontinuation and switching to a different agent. Early initiation of immunomodulators will substantially increase the persistence of biologic treatment., (© 2019 Crohn’s & Colitis Foundation. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

This article presents the most common gastrointestinal, hepatic, and pancreatic manifestations of the primary immunodeficiency diseases, including the appropriate laboratory testing, endoscopic evaluation, and recommendations for further management., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Pyogenic granuloma (PG) represents a lobular capillary proliferation commonly seen in the skin or oral mucosa. They are rarely reported in the gastrointestinal tract. The mechanism underlying PG pathogenesis is not well understood. Only one case of cutaneous PG associated with cytomegalovirus (CMV) infection has been reported in the English literature. Here, we report such a unique case of PG arising from the small bowel. A 67-year-old male, status post ileocolic resection, presented for follow-up colonoscopy because of Crohn's disease of the terminal ileum and the colon. Colonoscopy revealed inflammation at the ileocolic anastomosis as well as an 8-mm pedunculated lesion with an irregular surface in the neo-terminal ileum. Histological studies of the small bowel mucosa revealed chronic active ileitis with pyloric gland metaplasia, consistent with his clinical history of Crohn's disease. The lesion demonstrated a lobular architecture consisting of clusters of small capillaries of various sizes lined by a single layer of cytologically bland endothelial cells, and accompanied by acute and chronic inflammatory infiltrates and surface erosion/ulceration. The histological features supported the diagnosis of PG. Scattered viral inclusions with positive CMV immunoreactivity were present in the endothelial cells and glandular cells of pyloric gland metaplasia within the PG. To the best of our knowledge, this is the first documented case of PG with local CMV infection in patients with inflammatory bowel disease., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Nodal-related protein (ndr2) is amember of the transforming growth factor type β superfamily of factors and is required for ventral midline patterning of the embryonic central nervous system in zebrafish. In humans, mutations in the gene encoding nodal cause holoprosencephaly and heterotaxy. Mutations in the ndr2 gene in the zebrafish ( Danio rerio ) lead to similar phenotypes, including loss of the medial floor plate, severe deficits in ventral forebrain development and cyclopia. Alleles of the ndr2 gene have been useful in studying patterning of ventral structures of the central nervous system. Fifteen different ndr2 alleles have been reported in zebrafish, of which eight were generated using chemical mutagenesis, four were radiation-induced and the remaining alleles were obtained via random insertion, gene targeting (TALEN) or unknown methods. Therefore, most mutation sites were random and could not be predicted a priori. Using the CRISPR-Cas9 system from Streptococcus pyogenes, we targeted distinct regions in all three exons of zebrafish ndr2 and observed cyclopia in the injected (G 0 ) embryos.We show that the use of sgRNA-Cas9 ribonucleoprotein (RNP) complexes can cause penetrant cyclopic phenotypes in injected (G 0 ) embryos. Targeted polymerase chain reaction amplicon analysis using Sanger sequencing showed that most of the alleles had small indels resulting in frameshifts. The sequence information correlates with the loss of ndr2 activity. In this study, we validate multiple CRISPR targets using an in vitro nuclease assay and in vivo analysis using embryos. We describe one specific mutant allele resulting in the loss of conserved terminal cysteine-coding sequences. This study is another demonstration of the utility of the CRISPR-Cas9 system in generating domain-specific mutations and provides further insights into the structure-function of the ndr2 gene.

Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, is a complex chronic inflammatory condition of the human gut of unknown causes. Traditionally, dysregulated adaptive immune responses are thought to play a major role; however, accumulating evidence suggests that innate immunity also contributes to this process. Innate lymphoid cells (ILCs) are recently identified important components of innate immunity. They have critical roles in immunity, tissue development and remodeling. Numerous researchers have linked ILCs to the pathogenesis of IBD. In this review, we describe recent progress in our understanding about the phenotype and function alterations of ILCs as well as its interactions with other key mucosal cells in the gut of IBD patients. A better delineation of the ILCs' behavior in the human intestine will contribute to our understanding of ILCs biology and provide valuable insights for potential therapeutic target selection for IBD patients.

The development of gene editing technologies, especially the CRISPR-Cas9 system, has been pivotal for understanding the functional role of proteins. Rapid and efficient genotyping methods are necessary to screen for generated mutations and streamline the isolation of homozygotes. CRISPR-Cas9 system targeting a single site in the gene typically results in small indels. Many genotyping methods utilize the heteroduplex that is formed when wild-type and mutant amplicons with small indels anneal during PCR creating a bubble due to mismatched strands. These methods include T7 endonuclease/Cel-I assay, high resolution melting (HRM) analysis, and heteroduplex mobility assay (HMA). Our protocol explains a simple, two step method of a mixing HMA (mHMA) to identify homozygous mutants, a modification of the previously published HMA. We have utilized the mHMA for screening and genotyping numerous CRISPR generated models. The mHMA method to differentiate homozygous wild type from homozygous mutant animals eliminates - •DNA sequencing, even with small indels that can be difficult to discern on a gel.•additional enzymatic reaction steps, such as with the T7EI/Cel-I assay.•specialized equipment and analysis tools, such as with HRM analysis.

Background: Electronic health records (EHRs), despite their positive attributes, increase physician workload and decrease efficiency. The aim of this study was to evaluate the impact of scribes in the Inflammatory Bowel Disease Clinic on improvement of the physician-patient relationship, physician productivity, clinical efficiency, and achievement of some Physician Quality Reporting System (PQRS) metrics., Methods: We analyzed of pre- and postscribe data between fiscal years 2015 (FY15) and 2016 (FY16) using data from patients at the Inflammatory Bowel Clinic at the University of Florida. The main outcomes were patient satisfaction scores (PSS), qualitative physician interview, clinic appointment lengths, work relative value units (wRVUs), level of coding, revenue, and PQRS data on bone density screening and vaccination., Results: PSS increased from 6.8/10 to 9.2/10 (P < 0.01), clinic appointment length decreased by 13.5 minutes (P < 0.05), and documentation stress decreased. Clinic visits increased by 76, leading to an increase in work RVUs by 332.55, total charges billed by $71,439, and total charges collected by $27,387 between the first quarters of FY15 and FY16. The extra revenue for the first quarter was 536% higher than the salary of the scribe for the same period ($4302.84). There was a 1.8-fold increase in referrals for bone density scans and 2.9-fold and 4.8-fold increases in vaccination rates for influenza and pneumonia, respectively., Conclusions: The use of scribes improved the physician-patient relationship, clinical efficiency, physician productivity, bone density screening, and vaccinations for flu and pneumonia. If adopted by health systems, it may lead to significant cost savings and improved clinical outcomes.

Background: IL10 receptor (IL10R) deficiency causes severe infantile-onset inflammatory bowel disease. Intact IL10R-dependent signals have been shown to be important for innate and adaptive immune cell functions in mice. We have previously reported a key role of IL10 in the generation and function of human anti-inflammatory macrophages. Independent of innate immune cell defects, the aim of the current study was to determine the role of IL10R signaling in regulating human CD4 T-cell function., Methods: Peripheral blood mononuclear cells and intestinal biopsies cells were collected from IL10/IL10R-deficient patients and controls. Frequencies of CD4 T-cell subsets, naive T-cell proliferation, regulatory T cell (Treg)-mediated suppression, and Treg and TH17 generation were determined by flow cytometry. Transcriptional profiling was performed by NanoString and quantitative real-time polymerase chain reaction. RNA in situ hybridization was used to determine the quantities of various transcripts in intestinal mucosa., Results: Analysis of 16 IL10- and IL10R-deficient patients demonstrated similar frequencies of peripheral blood and intestinal Tregs, compared with control subjects. In addition, in vitro Treg suppression of CD4 T-cell proliferation and generation of Treg were not dependent on IL10R signaling. However, IL10R-deficient T naive cells exhibited higher proliferative capacity, a strong TH17 signature, and an increase in polarization toward TH17 cells, compared with controls. Moreover, the frequency of TH17 cells was increased in the colon and ileum of IL10R-deficient patients. Finally, we show that stimulation of IL10R-deficient Tregs in the presence of IL1β leads to enhanced production of IL17A., Conclusions: IL10R signaling regulates TH17 polarization and T-cell proliferation in humans but is not required for the generation and in vitro suppression of Tregs. Therapies targeting the TH17 axis might be beneficial for IL10- and IL10R-deficient patients as a bridge to allogeneic hematopoietic stem cell transplantation.

Innate lymphoid cells (ILCs) are emerging as important components of our immune system that have critical effector and regulatory functions in both innate and adaptive immune responses. They are enriched at mucosal surfaces, such as lung and intestine. Our previous work has shown that Lineage - CRTH2 - CD45 + NKp44 - CD117 - CD127 + ILC1s accumulated in the inflamed terminal ileum of patients with Crohn's disease (CD) at the expense of NKp44 + ILC3s. This phenotype conversion impairs the intestinal barrier integrity and contributes to the dysregulated immune responses of CD patients. Our next step was to search for pathways to modulate this phenotype switch. The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor. Initial studies of AHR concentrated on its role in the detoxification of xenobiotics. However, recent research has focused on the immune system. Especially, AHR pathway is proven to be essential for the maintenance of intestinal ILC3s in mouse models. We examined whether AHR pathway participated in the human intestinal ILC phenotype change in the inflamed terminal ileum of CD patients. As anticipated, NKp44 + ILC3s, NKp44 - ILC3s and ILC1s had differential AHR expression. This AHR signaling mediated CD117 expression on the surface of ILC3s. The conversion from ILC3 to ILC1 was accompanied by the downregulation of AHR expression. We further observed that there was a disparity between AHR protein expression and mRNA expression in the inflamed terminal ileum tissues of CD patients compared to unaffected areas. These findings suggest that AHR pathway is also important for human intestinal ILC phenotype regulation and impaired AHR signaling in the inflamed gut of CD patients possibly contributes to the ILC3/ILC1 conversion., Competing Interests: Conflicting interests The authors have declared that no conflict of interests exist.

Background: Colorectal cancer remains the second leading cause of death in the United States despite improvements in incidence rates and advancements in screening. The present study evaluated the prognostic value of two tumor markers, MET and ROCK I, which have been noted in other cancers to provide more accurate prognoses of patient outcomes than tumor staging alone., Materials and Methods: We constructed a tissue microarray from surgical specimens of adenocarcinomas from 108 colorectal cancer patients. Using immunohistochemistry, we examined the expression levels of tumor markers MET and ROCK I, with a pathologist blinded to patient identities and clinical outcomes providing the scoring of MET and ROCK I expression. We then used retrospective analysis of patients' survival data to provide correlations with expression levels of MET and ROCK I., Results: Both MET and ROCK I were significantly over-expressed in colorectal cancer tissues, relative to the unaffected adjacent mucosa. Kaplan-Meier survival analysis revealed that patients' 5-year survival was inversely correlated with levels of expression of ROCK I. In contrast, MET was less strongly correlated with five-year survival., Conclusion: ROCK I provides better efficacy in predicting patient outcomes, compared to either tumor staging or MET expression. As a result, ROCK I may provide a less invasive method of assessing patient prognoses and directing therapeutic interventions., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background: Measurement of CD4+ T-lymphocytes (CD4) is a crucial parameter in the management of HIV patients, particularly in determining eligibility to initiate antiretroviral treatment (ART). A number of technologies exist for CD4 enumeration, with considerable variation in cost, complexity, and operational requirements. We conducted a systematic review of the performance of technologies for CD4 enumeration., Methods and Findings: Studies were identified by searching electronic databases MEDLINE and EMBASE using a pre-defined search strategy. Data on test accuracy and precision included bias and limits of agreement with a reference standard, and misclassification probabilities around CD4 thresholds of 200 and 350 cells/μl over a clinically relevant range. The secondary outcome measure was test imprecision, expressed as % coefficient of variation. Thirty-two studies evaluating 15 CD4 technologies were included, of which less than half presented data on bias and misclassification compared to the same reference technology. At CD4 counts <350 cells/μl, bias ranged from -35.2 to +13.1 cells/μl while at counts >350 cells/μl, bias ranged from -70.7 to +47 cells/μl, compared to the BD FACSCount as a reference technology. Misclassification around the threshold of 350 cells/μl ranged from 1-29% for upward classification, resulting in under-treatment, and 7-68% for downward classification resulting in overtreatment. Less than half of these studies reported within laboratory precision or reproducibility of the CD4 values obtained., Conclusions: A wide range of bias and percent misclassification around treatment thresholds were reported on the CD4 enumeration technologies included in this review, with few studies reporting assay precision. The lack of standardised methodology on test evaluation, including the use of different reference standards, is a barrier to assessing relative assay performance and could hinder the introduction of new point-of-care assays in countries where they are most needed.

Intracellular Tau inclusions are a pathological hallmark of several neurodegenerative diseases, collectively known as the tauopathies. They include Alzheimer disease, tangle-only dementia, Pick disease, argyrophilic grain disease, chronic traumatic encephalopathy, progressive supranuclear palsy, and corticobasal degeneration. Tau pathology appears to spread through intercellular propagation, requiring the formation of assembled "prion-like" species. Several cell and animal models have been described that recapitulate aspects of this phenomenon. However, the molecular characteristics of seed-competent Tau remain unclear. Here, we have used a cell model to understand the relationships between Tau structure/phosphorylation and seeding by aggregated Tau species from the brains of mice transgenic for human mutant P301S Tau and full-length aggregated recombinant P301S Tau. Deletion of motifs (275)VQIINK(280) and (306)VQIVYK(311) abolished the seeding activity of recombinant full-length Tau, suggesting that its aggregation was necessary for seeding. We describe conformational differences between native and synthetic Tau aggregates that may account for the higher seeding activity of native assembled Tau. When added to aggregated Tau seeds from the brains of mice transgenic for P301S Tau, soluble recombinant Tau aggregated and acquired the molecular properties of aggregated Tau from transgenic mouse brain. We show that seeding is conferred by aggregated Tau that enters cells through macropinocytosis and seeds the assembly of endogenous Tau into filaments., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Ulcerative colitis (UC) is an idiopathic chronic inflammatory disorder that affects the colonic mucosa. One class among the drugs used for its treatment is the 5-aminosalicylates (5-ASAs). While highly efficacious in treating mild-to-moderate UC, 5-ASAs are associated with rare but potentially life-threatening side effects such as pericarditis, myocarditis and pneumonitis. These adverse events appear to be caused by a hypersensitivity reaction and resolve after cessation of 5-ASA drugs. This article presents a case report of febrile pleuropericarditis in a UC patient treated with balsalazide, and provides a thorough literature review of the rare side effects of 5-ASAs, their incidence, clinical presentation, differential diagnosis and treatment. In conclusion, the clinicians should be aware that this type of adverse events to 5-ASA compounds can be easily overlooked but it has significant morbidity if not promptly diagnosed.

Inflammatory bowel disease (IBD) with its two distinct entities, Crohn's disease and ulcerative colitis, and Type 1 diabetes mellitus (T1D) are autoimmune diseases. The prevalence of these diseases continues to rapidly rise in the industrialized world. Despite the identification of several genetic loci that are associated with both IBD and T1D, thus far, there is a paucity of epidemiological data to support a clinical overlap. In an effort to better understand the underlying pathogenic mechanisms of both IBD and T1D, this review summarizes the literature about these related autoimmune diseases, describes the most recent advances in their etiopathogenesis and emphasizes the genetic and nongenetic factors that exercise a differential influence. Genome-wide association studies have identified genetic loci with a role in immune response regulation that are linked to both IBD (particularly Crohn's disease) and T1D. Some of these genetic loci (e.g., IL-18RAP) have a divergent role, conferring risk for one disease and protection for the other. Recent evidence highlights an important role of gut microbiota and cellular responses (e.g., endoplasmic reticulum stress) in the pathogenesis of both IBD and T1D.