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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.

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- 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 seven 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 to controls. These included neutrophilic colitis and prominent lamina propria eosinophilia throughout the gastrointestinal tract. Striking architectural abnormalities of the epithelium were observed in four of the seven patients. The five 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 four 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.

Objective To describe our current understanding of hereditary α-tryptasemia (HαT), how HαT fits into the evolutionary context of tryptases and contemporary framework of mast cell–associated disorders, and to discuss the future clinical and therapeutic landscape for symptomatic individuals with HαT. Data Sources Primary peer-reviewed literature. Study Selections Basic, clinical, and translational studies describing tryptase gene composition, generation, secretion, and elevation and the associated clinical impacts of HαT and treatment of such individuals were reviewed. Results HαT is a common autosomal dominant genetic trait caused by increased TPSAB1 copy number encoding α-tryptase. Approximately 1 in 20 White individuals have HαT, making it by far the most common cause for elevated basal serum tryptase levels. Although many individuals with HαT may not manifest associated symptoms, the prevalence of HαT is increased in patients with clonal and nonclonal mast cell–associated disorders wherein it is linked to more prevalent and/or severe anaphylaxis and increased mast cell mediator-associated symptoms. Increased generation of mature α/β-tryptase heterotetramers, and their unique physiochemical properties, may be responsible for some of these clinical findings. Conclusion HαT is a common modifier of mast cell–associated disorders and reactions. Nevertheless, whether HαT may be an independent cause of clinical phenotypes with which it has been associated remains unproven. Correct identification of HαT is critical to accurate interpretation of serum tryptase levels in the clinical evaluation of patients. Beyond HαT, we foresee tryptase genotyping as an important parameter in the standard workup of patients with mast cell–associated disorders and development of therapeutic modalities targeting these patients and associated clinical phenotypes.

BackgroundMost 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.AimsWe aimed to evaluate the therapeutic response rate in Black IBD patients compared with White patients.MethodsWe 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.ResultsWe 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.ConclusionsBlack IBD patients on anti-TNF agents had a significantly higher prevalence of active disease and more IBD-related hospitalizations than White patients.

Recent case reports and epidemiological data suggest fungal infections represent an under-appreciated 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 (scRNA-seq) dataset 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 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 ofCandidaspecies-derived transcripts within samples collected from the nasopharynx and trachea. Here, we present the clinical characteristics of these individuals, including confirmatory diagnostic testing demonstrating elevated serum (1, 3)-β-D-glucan and/or confirmed fungal culture of the predicted pathogen. Using matched single-cell transcriptomic profiles of these individuals’ respiratory mucosa, we identify epithelial immune signatures suggestive of IL-17 stimulation and anti-fungal immunity. Further, we observe 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 suggests that IL-17 stimulation – in part driven byCandidacolonization – and blunted type I/III interferon signaling represents a common feature of severe COVID-19 infection.

Serum tryptase is a biomarker used to aid in the identification of certain myeloid neoplasms, most notably systemic mastocytosis, where baseline (BST) levels >20 ng/mL are a minor criterion for diagnosis. Whereas clonal myeloid neoplasms are rare, the common cause for elevated BST is the genetic trait hereditary alpha-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 over-expression of replicated TPSAB1 loci encoding wild-type α-tryptase due to co-inheritance of a linked over-active promoter element. Modeling BST levels based upon 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 (e.g., >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.

Background Therapeutic efficacy of biologics has remained at about 50% for 2 decades. In Crohn’s disease (CD) patients, we examined the predictive value of an epithelial cell biomarker, ileal microvillar length (MVL), for clinical response to ustekinumab (UST) and vedolizumab (VDZ) and its relationship to another biomarker, intestinal epithelial cell (IEC) pyroptosis, with respect to response to VDZ. Method Ileal biopsies from the UNITI-2 randomized controlled trial were analyzed for MVL as a predictor of clinical response to UST. In a 5-center academic retrospective cohort of CD patients, ileal MVL was analyzed to determine its predictive value for response to VDZ. Correlation between ileal MVL and IEC pyroptosis was determined, and the discriminant ability of the combination of 2 biomarkers to VDZ was examined. Results Clinical response in UST was significantly higher than placebo (65% vs 39%; P = 0.03), with patients with normal MVL (>1.7 µm) having the greatest therapeutic effect: 85% vs 20% (P = 0.02). For VDZ, clinical response with MVL of 1.35 to 1.55 µm was 82% vs 44% (1.55 µm; P = 0.038). There was no correlation between ileal MVL and IEC pyroptosis. The combination criteria of ileal pyroptosis Conclusion Ileal MVL was predictive of response to UST and VDZ in prospective and retrospective CD cohorts. It was independent of ileal IEC pyroptosis, and combination of the 2 biomarkers enhanced the discriminate ability of responders from nonresponders to VDZ.

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.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which presents with respiratory symptoms including fever, sore throat, cough and congestion, suggesting that infection of the oral cavity and nasopharynx (ONP) is an obligate step in its pathogenesis Consistent with this role in the initial infection, oral and nasopharyngeal epithelial cells express the SARS-CoV-2 spike protein receptor, angiotensin-converting enzyme 2 (ACE2) (1), and RT-PCR based testing can routinely detect SARS-CoV-2 RNA within both the nasal and buccal tissues Local innate and adaptive immune responses in the oral epithelia are almost certainly involved in the defense against SARS-CoV-2 infection, yet little is known about these defenses

Exosomes are nano‐sized vesicles that are involved in various biological processes including cell differentiation, proliferation, signaling, and intercellular communication. Urinary exosomes were isolated from a cohort of hereditary α‐tryptasemia (HαT) patients and from healthy volunteers. There was a greater number of exosomes isolated from the urine in the HαT group compared to the control volunteers. Here, we investigated the differences in both lipid classes and lipid species within urinary exosomes of the two groups. Lipids were extracted from urinary exosomes and subjected to liquid chromatography mass spectrometry using a targeted approach. Various molecular species of glycerophospholipids, glycerolipids, and sterols were significantly reduced in HαT patients. Out of a possible 1127 lipids, 521 lipid species were detected, and relative quantities were calculated. Sixty‐four lipids were significantly reduced in urinary exosomes of HαT patients compared to controls. All significantly reduced sphingolipids and most of the phospholipids were saturated or mono‐unsaturated lipids. These results suggest exosome secretion is augmented in HαT patients and the lipids within these exosomes may be involved in various biological processes. The unique lipid composition of urinary exosomes from HαT patients will contribute to our understanding of the biochemistry of this disease.

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 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 Conclusions Ileal biopsy IEC pyroptosis was predictive of clinical response and endoscopic improvement to vedolizmab in CD patients.

Background Very early onset inflammatory bowel disease (VEO-IBD) secondary to IL-10RA mutations have aggressive disease courses with increased nutritional needs compared to other monogenic forms of IBD. Presentation A 28-year old male was hospitalized at 18-days old for bloody diarrhea diagnosed as Crohn's disease at 6-months old. He showed failure to thrive and worsening inflammation while maintained on enteral nutrition (EN) and standard IBD treatment. He was hospitalized in 2016, at 28 years old, for a Chron's flare when sequencing confirmed a heterozygous mutation in IL10-RA. His weight and plasma micronutrient levels improved when transitioned to parenteral nutrition (PN). He was initiated on anakinra while awaiting hematopoietic stem cell transplant (HSCT) with substantial decrease in inflammation. He was able to gain weight, initiate an oral diet and decrease his PN requirement. Conclusions Our patient experienced progressive failure to thrive on EN. VEO-IBD incidence is rising and its diagnosis is often delayed. Therefore, prompt recognition with treatment initiation is essential to improving nutritional outcomes in this patient population. Further investigation is warranted to determine if these patients would benefit from early initiation of PN. This article is protected by copyright. All rights reserved.

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.

To investigate the relationship between intestinal microbiota and SARS-CoV-2-mediated pathogenicity in a United States, majority African American cohort. We prospectively collected fecal samples from 50 SARS-CoV-2 infected patients, 9 SARS-CoV-2 recovered patients, and 34 uninfected subjects seen by the hospital with unrelated respiratory medical conditions (controls). 16S rRNA sequencing and qPCR analysis was performed on fecal DNA/RNA. The fecal microbial composition was found to be significantly different between SARS-CoV-2 patients and controls (PERMANOVA FDR-P = .004), independent of antibiotic exposure. Peptoniphilus, Corynebacterium and Campylobacter were identified as the three most significantly enriched genera in COVID-19 patients compared to controls. Actively infected patients were also found to have a different gut microbiota than recovered patients (PERMANOVA FDR-P = .003), and the most enriched genus in infected patients was Campylobacter, with Agathobacter and Faecalibacterium being enriched in the recovered patients. No difference in microbial community structure between recovered patients and uninfected controls was observed, nor a difference in alpha diversity between the three groups. 24 of the 50 COVID-19 patients (48%) tested positive via RT-qPCR for fecal SARS-CoV-2 RNA. A significant difference in gut microbial composition between SARS-CoV-2 positive and negative samples was observed, with Klebsiella and Agathobacter being enriched in the positive cohort. No significant associations between microbiome composition and disease severity was found. The intestinal microbiota is sensitive to the presence of SARS-CoV-2, with increased relative abundance of genera (Campylobacter, Klebsiella) associated with gastrointestinal (GI) disease. Further studies are needed to investigate the functional impact of SARS-CoV-2 on GI health.

Background Acute increases of ≥20% + 2 ng/mL (20 + 2 rule) over basal serum tryptase (BST) is the recommended threshold supporting a clinical diagnosis of anaphylaxis. Prospective studies have demonstrated high sensitivity for this algorithm after parenteral exposure, but specificity has not been evaluated. Objective We sought to define a serum tryptase change that distinguishes baseline variability from anaphylaxis on the basis of intraindividual variation in BST. Methods Ninety-three total subjects with atopy (n = 62) or hereditary α-tryptasemia (HαT) (n = 31) and ≥2 BST measurements were identified. Sequential BST variability measurements were modeled and threshold ratios that optimized sensitivity and/or specificity determined. Models were tested in 22 individuals with physician-diagnosed anaphylaxis and validated in independent cohorts of individuals with HαT (n = 33), indolent systemic mastocytosis (ISM) (n = 52), and ISM + HαT (n = 12). Mature tryptase levels were measured in HαT (n = 19) and ISM (n = 20). An online application was developed for clinical use. Results As a result of BST variability, 9.7% (9/93) of primary cohort patients, and 18% (6/33) of HαT, 30% (16/53) of ISM, and 25% (3/12) of ISM + HαT patients from validation cohorts met the 20 + 2 rule despite absent immediate hypersensitivity symptoms; mature tryptase was noncontributory among individuals with HαT or ISM at baseline. A ratio of acute tryptase/BST exceeding 1.685 provided the optimized diagnostic rule for jointly maximizing sensitivity and specificity. Statistically significant improvement in specificity relative to the 20 + 2 rule was observed among individuals with elevated BST caused by HαT and ISM. Conclusions Using an acute tryptase/BST ratio of 1.685 improves specificity of measured changes among individuals with HαT and ISM while maintaining high sensitivity for confirmation of anaphylaxis.

Introduction: Clinical trials often have low enrollment of minorities, particularly African-Americans (AAs), which may limit the generalizability of research findings. Previously identified barriers to AAs recruitment include historical abuses leading to mistrust, communication issues with providers, socio-economic factors, and a lack of access to clinical trials. In a Historically Black College and University (HBCU) serving a primarily AA population at a large safe-net hospital, we evaluated the enrollment of eligible AA patients for a colorectal cancer (CRC) screening clinical trial. This was compared to the enrollment rates across other study sites. Methods: A large, prospective, multi-centered clinical trial to validate a blood-based test for early detection of CRC (PREEMPT-CRC) was initiated at a HBCU, where 84% of patients are AAs. To maximize study recruitment, culturally sensitive methods were employed including racially congruent recruitment staff as well as synchronized timing of consent/study procedures with preendoscopy COVID testing/clinic visits. Detailed information for all eligible subjects was recorded. Demographic and socio-economic data including census information for enrolled and not enrolled subjects were compared. The enrollment rate (defined as enrolled/eligible patients) over the first 6 weeks at the HBCU and that of the other study sites providing screening logs was analyzed. Results: The enrollment rate at the HBCU was 55% (44 out of 80 eligible patients;95% CI 43.5- 66.2%), compared to 49.8% (258 out of 518 eligible patients;95% CI 45.4- 54.2%) at the other 26 study sites. While age and gender of enrolled patients at the HBCU were comparable to other sites, the main difference was race: at the HBCU the study participants were 79.5% AAs and 9.1% whites, while at the other sites the participants were 11.5% AAs and 82.8% whites (p< 0.001). At the HBCU, the demographic characteristics and socio-demographic data including income, marital status insurance status/type, and census tract median household income of the 44 enrolled and 36 notenrolled subjects were similar (Table 1). Conclusion: Contrary to conventional belief that AAs do not want to be involved in clinical trials, we find their enrollment is similar to a predominant white study population when offered the opportunity in a culturally sensitive setting. Future trials should consider including HBCU sites in order to attain adequate AA enrollment to improve the generalizability of research findings. (Table Presented).

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, AZGP1high 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., Graphical abstract, A study of nasopharyngeal swabs from healthy and COVID-19-infected individuals shows how infection leads to compositional changes in the respiratory epithelium, with early dampened antiviral responses in the nasal epithelia likely underlying and preceding severe disease.

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 specifically staining for MCs in order 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 mast cell tryptase gene TPSAB1 and typically elevated baseline serum tryptase levels >8ng/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

[Abstract]In late 2019 and through 2020, the COVID-19 pandemic swept the world, presenting both scientific and medical challenges associated with understanding and treating a previously unknown disease. To help address the need for great understanding of COVID-19, the scientific community mobilized and banded together rapidly to characterize SARS-CoV-2 infection, pathogenesis and its distinct disease trajectories. The urgency of COVID-19 provided a pressing use-case for leveraging relatively new tools, technologies, and nascent collaborative networks. Single-cell biology is one such example that has emerged over the last decade as a powerful approach that provides unprecedented resolution to the cellular and molecular underpinnings of biological processes. Early foundational work within the single-cell community, including the Human Cell Atlas, utilized published and unpublished data to characterize the putative target cells of SARS-CoV-2 sampled from diverse organs based on expression of the viral receptor ACE2 and associated entry factors TMPRSS2 and CTSL (Muus et al., 2020; Sungnak et al., 2020; Ziegler et al., 2020). This initial characterization of reference data provided an important foundation for framing infection and pathology in the airway as well as other organs. However, initial community analysis was limited to samples derived from uninfected donors and other previously-sampled disease indications. This report provides an overview of a single-cell data resource derived from samples from COVID-19 patients along with initial observations and guidance on data reuse and exploration.

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 FceRI, 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.

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 +/- 1 15 vs 1 22 +/- 0 92 (P = 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 (age = 58 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] = 2 72, P < 0 001), age (aOR = 1 66 per decade, P < 0 001), race (aOR = 3 03, P = 0 002), cancer (aOR = 2 22, P = 0 008), and diabetes (aOR = 1 95, P = 0 003) The PPI-associated mortality risk was higher in black patients (aOR = 4 16, 95% confidence interval: 2 28-7 59) than others (aOR = 1 62, 95% confidence interval: 0 82-3 19, P = 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

A protocol for recovering viable single cell suspensions from cryopreserved human nasopharyngeal swabs for downstream applications, such as single-cell RNA-seq. The illustrated schematic below details the process.

Background Copper is a mineral that is absorbed in the stomach, duodenum, and jejunum. Gastric bypass surgery, gastrectomy, and short-bowel syndrome commonly lead to copper malabsorption. Copper deficiency primarily presents with hematological and neurological sequelae, including macrocytic anemia and myelopathy. Although hematological disturbances often correct with copper supplementation, neurological manifestations of copper deficiency may be irreversible. We present the case of copper deficiency secondary to malabsorption and management strategies to prevent irreversible neurological sequelae. Presentation A 48-year-old female with a history of hypothyroidism, ischemic stroke, and Crohn's disease, complicated by subtotal colectomy and small-bowel resections, was admitted for fatigue and progressive neurological deficiencies. Her vital signs were stable, and physical examination was remarkable for weakness of both upper and lower extremities, ataxia, and upper extremities paresthesia. Computed tomography scan of the head without contrast was unremarkable. Magnetic resonance imaging enterography revealed a focal area of narrowing of the remaining small bowel. Copper level was low at 39 µg/dL. After 5 days of intravenous replacement using trace element within parenteral nutrition, her copper level corrected to 81 µg/dL. Her ataxia improved after intravenous copper supplementation and did not recur. Conclusions Our patient presented with copper deficiency secondary to malabsoprtion. This case highlights the importance of copper testing in the bariatric surgery population and in patients with short-bowel syndrome. Given the irreversible nature of neurological symptoms when compared with the expense of nutrition supplements, routine copper testing should be considered in patients with malabsorptive states or altered anatomy, regardless of initial presentation.

ObjectiveTherapeutic efficacy of biologics has remained at about 50% for 2 decades. In Crohn’s disease (CD) patients, we examined the predictive value of an epithelial cell biomarker, ileal microvillar length (MVL), for clinical response to ustekinumab (UST) and vedolizumab (VDZ), and its relationship to another biomarker, intestinal epithelial cell (IEC) pyroptosis with respect to response to VDZ.DesignIleal biopsies from the UNITI-2 randomized controlled trial were analyzed for MVL as a predictor of clinical response to UST. In a 5-center academic retrospective cohort of CD patients, ileal MVL was analyzed to determine its predictive value for response to VDZ. Correlation between ileal MVL and IEC pyroptosis was determined, and the discriminant ability of the combination of two biomarkers to VDZ was examined.ResultsClinical response in UST was significantly higher than placebo (65% vs. 39%, p=0.03), with patients with normal MVL (>1.7 µm) having the greatest therapeutic effect: 85% vs. 20% (p=0.02). For VDZ, clinical response with MVL of 1.35-1.55 µm was 82% vs. 44% (1.55 µm) (p=0.038). There was no correlation between ileal MVL and IEC pyroptosis. The combination criteria of ileal pyroptosis < 14 positive cells/1000 IECs or MVL of 1.35-1.55 µm could identify 84% of responders and 67% of non-responders (p=0.001).ConclusionsIleal MVL was predictive of response to UST and VDZ in prospective and retrospective CD cohorts. It was independent of ileal IEC pyroptosis, combination of the two biomarkers enhanced the discriminate ability of responders from non-responders to VDZ.

Severe COVID-19 disease is associated with acute kidney injury in up to 57% of infected patients. The etiology of this is likely multifactorial. Extracellular vesicles (EVs) contain unique molecular cargo that contributes to health and disease in many different organ systems and can contain useful surrogate biomarkers. To gain insight into SARS-CoV2’s impact on the human kidney, we prospectively collected blood and urine from individuals hospitalized with severe COVID-19 infection and isolated EVs. Using these EVs, we investigated the impact of circulating EVs released during COVID-19 infection on healthy human glomerular mesangial cells. Nanoparticle tracking analysis and electron microscopy showed that EVs isolated from the plasma of COVID-19 patients were larger in size compared to those isolated from the plasma of non-COVID-19 patients, suggesting the presence of unique cargo within circulating EVs in the setting of COVID-19. The plasma derived EV’s from COVID-19 patients also increased glomerular cell proliferation, STAT3 activation, and cathepsin B protein expression in normal human glomerular cells when compared to EVs isolated from the plasma of non-COVID-19 patients. These data suggest systemic EVs during COVID-19 infection alter normal human mesangial cell function. and provide insight into the effects of SARS-CoV2 on the kidney. This work was supported by the University of Florida College of Medicine and the University of Mississippi Medical Center.

Alpha-defensins (AD) are antimicrobial peptides (AMP) that are found on mucosal surfaces, providing innate immune defense against pathogens. Human defensin-5 (HD-5) and HD-6 are AD produced only by Paneth cells of small intestinal crypts. SARS-CoV2 virus has been shown to infect the small intestinal cells via the ACE2 receptor and suppress inflammation in the gut. HD-5 and HD-6 bind ACE2 to prevent entry to intestinal cells. Based on the observation that human beta defensin-1 (hBD-1) is downregulated by influenza virus in airway epithelial cells, we hypothesized that HD-5 and HD-6 could be similarly downregulated by SARS-CoV2. To examine this in situ, we sampled patient cohorts with similar co-morbidities, with and without SARS-CoV2 positivity, for HD-5 and HD-6 gene expression via qRT-PCR. Stool samples were collected from all patients, diluted in Qiagen RNA Later and frozen at −80°C prior to total RNA extraction. Results revealed a significant decrease in both HD-5 (p=0.000006) and HD-6 (p=0.002) of SARS-CoV2 positive patients compared with non-Covid19 patients negative for SARS-CoV2. The results were consistent in fecal swabs or fecal streams. Gene expression in other significant markers of inflammation were also blunted: sPLA-2 (p=0.006), alpha-1-anti-trypsin (p=0.0001) and trypsin 2 (p=0.005). Other gut inflammatory markers PSTI, HIP/PAP, trypsin 3 and IL-8 were unchanged. There was a trend in IL-8 upregulation in Covid19 patients. This is the first demonstration of defensin mRNA detection in stool samples. The results suggest that decreasing AMPs may be a mechanism by which the virus increases its chance for survival. Supported by R21DE028378

Recent case reports and epidemiological data suggest fungal infections represent an under-appreciated respiratory co-infection among people with severe COVID-19, however the frequency and clinical impact is currently unknown. We have previously generated a single-cell RNA-sequencing (scRNA-seq) dataset characterizing the upper respiratory microenvironment during COVID-19 and mapped the relationship between disease severity with the local behavior of nasal epithelial and immune cells. In agreement with other human cohorts, this prior study suggested that a deficiency in host immunity, particularly in type I/ III interferon signaling in the upper respiratory tract, is associated with rapid progression to severe disease and worse clinical outcomes. Here, we have performed further analysis of this cohort and identified a subset of participants with severe COVID-19 and concurrent nasal and/or endotracheal infection with Candida spp. We present the clinical characteristics of these individuals, including confirmatory testing demonstrating elevated serum (1,3)-β-D-glucan and/or a positive fungal culture. Finally, using the matched single-cell transcriptomic profiles of these individuals’ respiratory mucosa, in addition to blunted type I/III interferon signaling previously reported, we identified epithelial and mucosal immune signatures consistent with augmented IL-17 responses, suggestive of activated anti-fungal immunity. These results indicate that a subset of patients with severe COVID-19 exhibit increased frequency of concurrent fungal infections associated with augmented IL-17 responses and suggest that concurrent fungal infection may be contributing to SARS-C0V-2 mediated inflammatory pathology. This project has been made possible in part by grant number 2020-216949 from the Chan Zuckerberg Initiative DAF, an advised fund of Silicon Valley Community Foundation to Alex K. Shalek and Jose Ordovas-Montanes. Carly G. K. Ziegler was supported by T32GM007753 from the NIH. Jose Ordovas-Montanes is a New York Stem Cell Foundation – Robertson Investigator. Jose Ordovas-Montanes was supported by the Richard and Susan Smith Family Foundation, the AGA Research Foundation’s AGA-Takeda Pharmaceuticals Research Scholar Award in IBD – AGA2020-13-01, the Food Allergy Science Initiative, and The New York Stem Cell Foundation. Bruce H. Horwitz was supported by DK122532-01A1, NIH; 12019PG-CD002, The Leona M. and Harry B. Helmsley Charitable Trust; SRA #54518, and Crohn’s and Colitis Foundation. P.L. was supported by 5T32AG000222 from the NIH. Andrew W. Navia was supported by the Ludwig Center for Molecular Oncology at MIT. Alex K. Shalek was supported by the Bill and Melinda Gates Foundation, Sloan Fellowship in Chemistry, the NIH (5U24AI118672), and the Ragon Institute of MGH, MIT and Harvard.

Although the effects of biologic agents on postoperative outcomes in Crohn's disease patients have been extensively studied, the effects on intraoperative outcomes, including blood loss, operative time, and length of small bowel resection, remain to be determined. This was a retrospective cohort study at a single tertiary referral center. Crohn's disease (CD) patients who underwent major abdominal surgery were identified. Patients receiving preoperative biologic agents were compared with controls. We compare operative outcomes between groups. A total of 144 patients who underwent major abdominal surgery at the University of Florida between March 2007 and March 2017 were included. One hundred and ten patients (76%) who received pre-operative biologic therapy were compared with 34 controls. On univariate analysis, preoperative biologic use was associated with a significantly shorter length of small bowel resection (21.2 cm in biologic group vs 34.5 cm, P = 0.01). There were no significant differences in intraoperative blood loss (100 vs 87.5 mL, P = 0.40) or total operative time (142 vs 154 minutes, P = 0.39) between groups. On multivariate analysis controlling for variables reflecting severity of disease and malnutrition, biologic use remained significantly associated with shorter length of bowel resection (incident rate ratio 0.58, P = 0.04). Preoperative biologic use is associated with a significantly shorter length of bowel resection in CD patients undergoing major abdominal surgery. No negative effects were noted on operative blood loss or total operative time. Our findings allow improved preoperative planning for surgeons and informed decision-making for CD patients undergoing major abdominal surgery.

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.