The receptor DNGR-1 signals for phagosomal rupture to promote cross-presentation of dead-cell-associated antigens.

Type 1 conventional dendritic (cDC1) cells are necessary for cross-presentation of many viral and tumor antigens to CD8+ T cells. cDC1 cells can be identified in mice and humans by high expression of DNGR-1 (also known as CLEC9A), a receptor that binds dead-cell debris and facilitates XP of corpse-associated antigens. Here, we show that DNGR-1 is a dedicated XP receptor that signals upon ligand engagement to promote phagosomal rupture. This allows escape of phagosomal contents into the cytosol, where they access the endogenous major histocompatibility complex class I antigen processing pathway. The activity of DNGR-1 maps to its signaling domain, which activates SYK and NADPH oxidase to cause phagosomal damage even when spliced into a heterologous receptor and expressed in heterologous cells. Our data reveal the existence of innate immune receptors that couple ligand binding to endocytic vesicle damage to permit MHC class I antigen presentation of exogenous antigens and to regulate adaptive immunity.

Synthesis of 15N-Pyridines and Higher Mass Isotopologs via Zincke Imine Intermediates.

Methods to incorporate stable radioisotopes are integral to pharmaceutical and agrochemical development. However, despite the prevalence of pyridines in candidate compounds, methods to incorporate 15N atoms within their structures are limited. Here, we present a general approach to pyridine 15N-labeling that proceeds via ring-opening to NTf-Zincke imines and then ring-closure with commercially available 15NH4Cl salts. This process functions on a range of substituted pyridines, from simple building block-type compounds to late-stage labeling of complex pharmaceuticals, and 15N-incorporation is >95% in most cases. The reactivity of the Zincke imine intermediates also enables deuteration of the pyridine C3- and C5-positions, resulting in higher mass isotopologs required for LCMS analysis of biological fluids during drug development.

Evaluation of patient health outcomes of a student-run free clinic in East Harlem.

BACKGROUND: Most United States medical schools have affiliated student-run free clinics, but the quality of services provided in such contexts compared to national metrics is unknown. This study determines whether a student-run, attending-supervised free clinic servicing a low-income and minority race patient population in New York City can meet national metrics of care. METHODS: Through chart review from January 1, 2020 to December 31, 2020, patient outcomes and service utilization in the Healthcare Effectiveness Data and Information Set were examined and compared to national rates of patients using Medicaid HMO or Medicare. Patients are ≥ 21 years of age, residents of East Harlem, and ineligible for health insurance because of legal residency requirements. The majority identify as Hispanic and speak Spanish as their primary language. All patients who were seen in the clinic during the 2020 calendar year were included. The primary study outcome is the number of Healthcare Effectiveness Data and Information Set measures in which patients, seen in a student-run free clinic, meet or exceed national comparisons. RESULTS: The healthcare outcomes of 238 patients, mean age 47.8 years and 54.6% female, were examined in 18 Healthcare Effectiveness Data and Information Set measures. The student-run free clinic met or exceeded national metrics in 16 out of 18 categories. CONCLUSIONS: The student-run free clinic met or exceeded the national standard of care according to national metrics. Evidence-based priorities have been clarified for future improvement. Other student-run free clinics should similarly evaluate the quality of their services.

A novel mutation in EROS (CYBC1) causes chronic granulomatous disease.

Chronic Granulomatous Disease (CGD) is an inborn error of immunity characterised by opportunistic infection and sterile granulomatous inflammation. CGD is caused by a failure of reactive oxygen species (ROS) production by the phagocyte NADPH oxidase. Mutations in the genes encoding phagocyte NADPH oxidase subunits cause CGD. We and others have described a novel form of CGD (CGD5) secondary to lack of EROS (CYBC1), a highly selective chaperone for gp91phox. EROS-deficient cells express minimal levels of gp91phox and its binding partner p22phox, but EROS also controls the expression of other proteins such as P2X7. The full nature of CGD5 is currently unknown. We describe a homozygous frameshift mutation in CYBC1 leading to CGD. Individuals who are heterozygous for this mutation are found in South Asian populations (allele frequency = 0.00006545), thus it is not a private mutation. Therefore, it is likely to be the underlying cause of other cases of CGD.

Lentiviral gene therapy rescues p47phox chronic granulomatous disease and the ability to fight Salmonella infection in mice.

Chronic granulomatous disease (CGD) is an inherited primary immunodeficiency disorder characterised by recurrent and often life-threatening infections and hyperinflammation. It is caused by defects of the phagocytic NADPH oxidase, a multicomponent enzyme system responsible for effective pathogen killing. A phase I/II clinical trial of lentiviral gene therapy is underway for the most common form of CGD, X-linked, caused by mutations in the gp91phox subunit of the NADPH oxidase. We propose to use a similar strategy to tackle p47phox-deficient CGD, caused by mutations in NCF1, which encodes the p47phox cytosolic component of the enzymatic complex. We generated a pCCLCHIM-p47phox lentiviral vector, containing the chimeric Cathepsin G/FES myeloid promoter and a codon-optimised version of the human NCF1 cDNA. Here we show that transduction with the pCCLCHIM-p47phox vector efficiently restores p47phox expression and biochemical NADPH oxidase function in p47phox-deficient human and murine cells. We also tested the ability of our gene therapy approach to control infection by challenging p47phox-null mice with Salmonella Typhimurium, a leading cause of sepsis in CGD patients, and found that mice reconstituted with lentivirus-transduced hematopoietic stem cells had a reduced bacterial load compared with untreated mice. Overall, our results potentially support the clinical development of a gene therapy approach using the pCCLCHIM-p47phox vector.

Exploring Antidepressant Adherence at a Student-Run Free Mental Health Clinic.

Minority groups experience higher depression but lower treatment rates. Student-run free mental health (MH) clinics, such as the East Harlem Health Outreach Partnership (EHHOP) MH clinic, address this disparity. This study scrutinized EHHOP MH's depression treatment by measuring adherence to antidepressants. Pharmacy data from seventy-nine patients were reviewed according to HEDIS criteria. Results compare EHHOP MH to New York State (NYS) Medicaid and NYS commercial insurance providers. In the acute treatment phase, EHHOP MH performed similarly to NYS Medicaid. In all other comparisons, EHHOP MH had lower adherence rates. Physician notes were reviewed to identify reasons for low adherence.

Antibody repertoire analysis in polygenic autoimmune diseases.

High-throughput sequencing of the DNA/RNA encoding antibody heavy- and light-chains is rapidly transforming the field of adaptive immunity. It can address key questions, including: (i) how the B-cell repertoire differs in health and disease; and (ii) if it does differ, the point(s) in B-cell development at which this occurs. The advent of technologies, such as whole-genome sequencing, offers the chance to link abnormalities in the B-cell antibody repertoire to specific genomic variants and polymorphisms. Here, we discuss the current research using B-cell antibody repertoire sequencing in three polygenic autoimmune diseases where there is good evidence for a pathological role for B-cells, namely systemic lupus erythematosus, multiple sclerosis and rheumatoid arthritis. These autoimmune diseases exhibit significantly skewed B-cell receptor repertoires compared with healthy controls. Interestingly, some common repertoire defects are shared between diseases, such as elevated IGHV4-34 gene usage. B-cell clones have effectively been characterized and tracked between different tissues and blood in autoimmune disease. It has been hypothesized that these differences may signify differences in B-cell tolerance; however, the mechanisms and implications of these defects are not clear.

A model for integrating the assessment and management of geriatric syndromes into internal medicine continuity practice: 5-year report.

A geriatric ambulatory curriculum was created to improve internal medicine residents' care of geriatric patients. Second-year residents met for a 3-hour session weekly for 4 consecutive weeks during a block rotation with faculty geriatricians for a curriculum focused on dementia, falls, and urinary incontinence. After a 1-hour case-based didactic session, residents applied learned content and concepts to patient consultations. Consultative encounters were precepted by faculty and shared with the team. After completing our curriculum, residents reported knowledge acquired and enhanced evaluation and management skills of these three syndromes and were more likely to use all recommended screening tests in future practice. This article describes the process and strategies guiding development of a successful ambulatory geriatric curriculum model that can be embedded into preexisting internal medicine clinics to help future internists to better manage these and other common geriatric syndromes.

Interpreter training for medical students: pilot implementation and assessment in a student-run clinic.

BACKGROUND: Trained medical interpreters are instrumental to patient satisfaction and quality of care. They are especially important in student-run clinics, where many patients have limited English proficiency. Because student-run clinics have ties to their medical schools, they have access to bilingual students who may volunteer to interpret, but are not necessarily formally trained. METHODS: To study the feasibility and efficacy of leveraging medical student volunteers to improve interpretation services, we performed a pilot study at the student-run clinic at the Icahn School of Medicine at Mount Sinai. In each fall semester in 2012-2015, we implemented a 6-h course providing didactic and interactive training on medical Spanish interpreting techniques and language skills to bilingual students. We then assessed the impact of the course on interpreter abilities. RESULTS: Participants' comfort levels, understanding of their roles, and understanding of terminology significantly increased after the course (p < 0.05), and these gains remained several months later (p < 0.05) and were repeated in an independent cohort. Patients and student clinicians also rated participants highly (averages above 4.5 out of 5) on these measures in real clinical encounters. CONCLUSIONS: These findings suggest that a formal interpreter training course tailored for medical students in the setting of a student-run clinic is feasible and effective. This program for training qualified student interpreters can serve as a model for other settings where medical students serve as interpreters.

MiR-210 is induced by Oct-2, regulates B cells, and inhibits autoantibody production.

MicroRNAs (MiRs) are small, noncoding RNAs that regulate gene expression posttranscriptionally. In this study, we show that MiR-210 is induced by Oct-2, a key transcriptional mediator of B cell activation. Germline deletion of MiR-210 results in the development of autoantibodies from 5 mo of age. Overexpression of MiR-210 in vivo resulted in cell autonomous expansion of the B1 lineage and impaired fitness of B2 cells. Mice overexpressing MiR-210 exhibited impaired class-switched Ab responses, a finding confirmed in wild-type B cells transfected with a MiR-210 mimic. In vitro studies demonstrated defects in cellular proliferation and cell cycle entry, which were consistent with the transcriptomic analysis demonstrating downregulation of genes involved in cellular proliferation and B cell activation. These findings indicate that Oct-2 induction of MiR-210 provides a novel inhibitory mechanism for the control of B cells and autoantibody production.

An essential role for EROS in redox-dependent endothelial signal transduction.

The chaperone protein EROS ("Essential for Reactive Oxygen Species") was recently discovered in phagocytes. EROS was shown to regulate the abundance of the ROS-producing enzyme NADPH oxidase isoform 2 (NOX2) and to control ROS-mediated cell killing. Reactive oxygen species are important not only in immune surveillance, but also modulate physiological signaling responses in multiple tissues. The roles of EROS have not been previously explored in the context of oxidant-modulated cell signaling. Here we show that EROS plays a key role in ROS-dependent signal transduction in vascular endothelial cells. We used siRNA-mediated knockdown and developed CRISPR/Cas9 knockout of EROS in human umbilical vein endothelial cells (HUVEC), both of which cause a significant decrease in the abundance of NOX2 protein, associated with a marked decrease in RAC1, a small G protein that activates NOX2. Loss of EROS also attenuates receptor-mediated hydrogen peroxide (H2O2) and Ca2+ signaling, disrupts cytoskeleton organization, decreases cell migration, and promotes cellular senescence. EROS knockdown blocks agonist-modulated eNOS phosphorylation and nitric oxide (NO●) generation. These effects of EROS knockdown are strikingly similar to the alterations in endothelial cell responses that we previously observed following RAC1 knockdown. Proteomic analyses following EROS or RAC1 knockdown in endothelial cells showed that reduced abundance of these two distinct proteins led to largely overlapping effects on endothelial biological processes, including oxidoreductase, protein phosphorylation, and endothelial nitric oxide synthase (eNOS) pathways. These studies demonstrate that EROS plays a central role in oxidant-modulated endothelial cell signaling by modulating NOX2 and RAC1.

Resident satisfaction with continuity clinic and career choice in general internal medicine.

BACKGROUND: The quality of the continuity clinic experience for internal medicine (IM) residents may influence their choice to enter general internal medicine (GIM), yet few data exist to support this hypothesis. OBJECTIVE: To assess the relationship between IM residents' satisfaction with continuity clinic and interest in GIM careers. DESIGN: Cross-sectional survey assessing satisfaction with elements of continuity clinic and residents' likelihood of career choice in GIM. PARTICIPANTS: IM residents at three urban medical centers. MAIN MEASURES: Bivariate and multivariate associations between satisfaction with 32 elements of outpatient clinic in 6 domains (clinical preceptors, educational environment, ancillary staff, time management, administrative, personal experience) and likelihood of considering a GIM career. KEY RESULTS: Of the 225 (90 %) residents who completed surveys, 48 % planned to enter GIM before beginning their continuity clinic, whereas only 38 % did as a result of continuity clinic. Comparing residents' likelihood to enter GIM as a result of clinic to likelihood to enter a career in GIM before clinic showed that 59 % of residents had no difference in likelihood, 28 % reported a lower likelihood as a result of clinic, and 11 % reported higher likelihood as a result of clinic. Most residents were very satisfied or satisfied with all clinic elements. Significantly more residents (p ≤ 0.002) were likely vs. unlikely to enter GIM if they were very satisfied with faculty mentorship (76 % vs. 53 %), time for appointments (28 % vs. 11 %), number of patients seen (33 % vs. 15 %), personal reward from work (51 % vs. 23 %), relationship with patients (64 % vs. 42 %), and continuity with patients (57 % vs. 33 %). In the multivariate analysis, being likely to enter GIM before clinic (OR 29.0, 95 % CI 24.0-34.8) and being very satisfied with the continuity of relationships with patients (OR 4.08, 95 % CI 2.50-6.64) were the strongest independent predictors of likelihood to enter GIM as a result of clinic. CONCLUSIONS: Resident satisfaction with most aspects of continuity clinic was high; yet, continuity clinic had an overall negative influence on residents' attitudes toward GIM careers. Targeting resources toward improving ambulatory patient continuity, workflow efficiency and increasing pre-residency interest in primary care may help build the primary care workforce.

Integrating service learning into the curriculum: lessons from the field.

The authors, representing two of the "signature" community service learning (CSL) programs in the 2010 Flexner Centenary volume of Academic Medicine, provide details of their programs--Frontera de Salud, a community-based program at the University of Texas Medical Branch, and the East Harlem Health Outreach Partnership, a clinic-based program at the Mount Sinai School of Medicine--specific to the task of integrating CSL into the medical school curriculum. They explain the nature and purpose of CSL, note gaps in the present curriculum which CSL aims to fill and highlight elements of CSL that are highly pertinent to Association of American Medical Colleges, Accreditation Council for Graduate Medical Education and Liaison Committee on Medical Education guidelines for undergraduate and graduate medical education. They also discuss barriers to the integration of CSL into the medical school curriculum and detail ways to overcome the logistic and fiscal challenges involved in making this highly effective and rewarding educational experience available to students of medicine.

Trainee Love and Breakup Letters to NephSIM: A Free, Mobile-Optimized, Nephrology Teaching Tool.

BACKGROUND: It is not known how learners feel about free open access medical education (FOAMed) as they progress through their training from medical school to fellowship. Love and breakup letter methodology (LBM) is a technique that has been used extensively in user experience technology-based research but has not previously been used in evaluating medical education tools. LBM asks participants to creatively write a "love" or "breakup" letter to a product under study to capture their thoughts and emotions when engaging with it. We conducted qualitative analysis of data from focus groups to explore how attitudes toward a learning platform change at various training stages and to broaden our understanding of how we meet learners' needs through a nephrology FOAMed tool, NephSIM. METHODS: Three virtual, recorded focus groups were conducted with second-year medical students, internal medicine residents, and nephrology fellows ( N =18). At the start of the focus group, participants composed and read their love and breakup letters. Semistructured discussions were then led by facilitator-driven questions and peer comments. After transcription, inductive data analysis was conducted using Braun and Clarke's six-step thematic analysis. RESULTS: Four main themes were seen across all groups: attitudes toward teaching tool, perception of nephrology, learning needs and approach, and application to practice. Preclinical students positively viewed the opportunity to simulate the clinical setting and unanimously wrote love letters. Reactions from residents and fellows were mixed. Residents were interested in brevity and speed of learning, preferring algorithms and succinct approaches to meet their practice-based learning needs. Fellows' learning needs were driven by a desire to prepare for the nephrology board examination and review cases uncommonly seen in practice. CONCLUSIONS: LBM provided a valuable methodology through which to identify trainee reactions to a FOAMed tool and highlighted the challenges of meeting learning needs of a continuum of trainees with a single learning platform.

Learning in Tension: A Case Study Examining What Internal Medicine Residents Learn in the Ambulatory Care Setting.

Introduction: Medical care of patients with complex conditions has shifted to the ambulatory setting, whereas current knowledge of resident learning is primarily based on studies from inpatient settings. Preparing trainees to adapt to this shift necessitates an understanding of what internal medicine (IM) residents currently learn during ambulatory rotations. The aim of this study is to identify what residents learn during their ambulatory care experience. Methods: Using a qualitative instrumental case study design, the authors conducted separate focus groups with IM trainees (n = 15), supervisors (n = 16), and program directors (n = 5) from two IM programs in New York City, USA in 2019. Participants were invited via email, and focus group sessions were complemented by document analysis of ambulatory syllabi. Results: Based on focus group commentary and document analysis, content learned in the ambulatory setting encompassed three domains; 1) patient needs, 2) the resident's role within a healthcare team, and 3) health system opportunities and limitations. Residents also learned about tensions within and between these domains including the skills needed to care for patients versus the skills acquired, a desire for ownership of patient care versus fragmented care, and time allotted versus time required. Discussion: This study revealed two outcomes about what residents learn during their ambulatory care experience. First, learning content largely fell into three domains. Second, residents learned about the tensions between ideal care delivery and the realities of practice. These results highlight the imperative to better align curricula with clinical environments to meet the learning needs of residents.

Multi-omics identify falling LRRC15 as a COVID-19 severity marker and persistent pro-thrombotic signals in convalescence.

Patients with end-stage kidney disease (ESKD) are at high risk of severe COVID-19. Here, we perform longitudinal blood sampling of ESKD haemodialysis patients with COVID-19, collecting samples pre-infection, serially during infection, and after clinical recovery. Using plasma proteomics, and RNA-sequencing and flow cytometry of immune cells, we identify transcriptomic and proteomic signatures of COVID-19 severity, and find distinct temporal molecular profiles in patients with severe disease. Supervised learning reveals that the plasma proteome is a superior indicator of clinical severity than the PBMC transcriptome. We show that a decreasing trajectory of plasma LRRC15, a proposed co-receptor for SARS-CoV-2, is associated with a more severe clinical course. We observe that two months after the acute infection, patients still display dysregulated gene expression related to vascular, platelet and coagulation pathways, including PF4 (platelet factor 4), which may explain the prolonged thrombotic risk following COVID-19.

EROS is a selective chaperone regulating the phagocyte NADPH oxidase and purinergic signalling.

EROS (essential for reactive oxygen species) protein is indispensable for expression of gp91phox, the catalytic core of the phagocyte NADPH oxidase. EROS deficiency in humans is a novel cause of the severe immunodeficiency, chronic granulomatous disease, but its mechanism of action was unknown until now. We elucidate the role of EROS, showing it acts at the earliest stages of gp91phox maturation. It binds the immature 58 kDa gp91phox directly, preventing gp91phox degradation and allowing glycosylation via the oligosaccharyltransferase machinery and the incorporation of the heme prosthetic groups essential for catalysis. EROS also regulates the purine receptors P2X7 and P2X1 through direct interactions, and P2X7 is almost absent in EROS-deficient mouse and human primary cells. Accordingly, lack of murine EROS results in markedly abnormal P2X7 signalling, inflammasome activation, and T cell responses. The loss of both ROS and P2X7 signalling leads to resistance to influenza infection in mice. Our work identifies EROS as a highly selective chaperone for key proteins in innate and adaptive immunity and a rheostat for immunity to infection. It has profound implications for our understanding of immune physiology, ROS dysregulation, and possibly gene therapy.

Immunodeficiency, autoimmunity, and increased risk of B cell malignancy in humans with TRAF3 mutations.

Tumor necrosis factor receptor-associated factor 3 (TRAF3) is a central regulator of immunity. TRAF3 is often somatically mutated in B cell malignancies, but its role in human immunity is not defined. Here, in five unrelated families, we describe an immune dysregulation syndrome of recurrent bacterial infections, autoimmunity, systemic inflammation, B cell lymphoproliferation, and hypergammaglobulinemia. Affected individuals each had monoallelic mutations in TRAF3 that reduced TRAF3 expression. Immunophenotyping showed that patients' B cells were dysregulated, exhibiting increased nuclear factor-κB 2 activation, elevated mitochondrial respiration, and heightened inflammatory responses. Patients had mild CD4+ T cell lymphopenia, with a reduced proportion of naïve T cells but increased regulatory T cells and circulating T follicular helper cells. Guided by this clinical phenotype, targeted analyses demonstrated that common genetic variants, which also reduce TRAF3 expression, are associated with an increased risk of B cell malignancies, systemic lupus erythematosus, higher immunoglobulin levels, and bacterial infections in the wider population. Reduced TRAF3 conveys disease risks by driving B cell hyperactivity via intrinsic activation of multiple intracellular proinflammatory pathways and increased mitochondrial respiration, with a likely contribution from dysregulated T cell help. Thus, we define monogenic TRAF3 haploinsufficiency syndrome and demonstrate how common TRAF3 variants affect a range of human diseases.