Heightened TLR7 signaling primes BCR-activated B cells in chronic graft-versus-host disease for effector functions.

ABSTRACT: Chronic graft-versus-host disease (cGVHD) is a debilitating, autoimmune-like syndrome that can occur after allogeneic hematopoietic stem cell transplantation. Constitutively activated B cells contribute to ongoing alloreactivity and autoreactivity in patients with cGVHD. Excessive tissue damage that occurs after transplantation exposes B cells to nucleic acids in the extracellular environment. Recognition of endogenous nucleic acids within B cells can promote pathogenic B-cell activation. Therefore, we hypothesized that cGVHD B cells aberrantly signal through RNA and DNA sensors such as Toll-like receptor 7 (TLR7) and TLR9. We found that B cells from patients and mice with cGVHD had higher expression of TLR7 than non-cGVHD B cells. Using ex vivo assays, we found that B cells from patients with cGVHD also demonstrated increased interleukin-6 production after TLR7 stimulation with R848. Low-dose B-cell receptor (BCR) stimulation augmented B-cell responses to TLR7 activation. TLR7 hyperresponsiveness in cGVHD B cells correlated with increased expression and activation of the downstream transcription factor interferon regulatory factor 5. Because RNA-containing immune complexes can activate B cells through TLR7, we used a protein microarray to identify RNA-containing antigen targets of potential pathological relevance in cGVHD. We found that many of the unique targets of active cGVHD immunoglobulin G (IgG) were nucleic acid-binding proteins. This unbiased assay identified the autoantigen and known cGVHD target Ro-52, and we found that RNA was required for IgG binding to Ro-52. Herein, we find that BCR-activated B cells have aberrant TLR7 signaling responses that promote potential effector responses in cGVHD.

Development and Assessment of Simulation-Based Point-of-Care Ultrasound Curriculum in Undergraduate Medical Education.

OBJECTIVES: Implementation barriers and lack of standardized point-of-care ultrasound (POCUS) curricula make the development of effective POCUS curricula and methods of assessment challenging. The authors aim to develop a longitudinal POCUS curriculum through staged intervention. In the first stage, the authors hypothesized that the use of high-fidelity ultrasound simulation during the Internal Medicine clerkship would improve POCUS confidence and knowledge among medical students, minimizing the need for trained faculty. METHODS: A quasi-experimental study of third-year students on the Internal Medicine clerkship at a large academic medical center in the United States was performed assessing the efficacy of ultrasound simulation use. The control group consisted of students who received baseline POCUS education during teaching rounds but did not have access to the ultrasound simulator. The experimental group consisted of students who, in addition to baseline POCUS education, had access to a high-fidelity ultrasound simulator throughout the clerkship for a minimum of 1 hour per week. Students in both the control and experimental groups completed a pre- and post-intervention confidence survey and knowledge-based examination. RESULTS: Eighty-two percent (50/61) of students completed pre- and post-tests, with the control group demonstrating no significant difference in POCUS confidence or knowledge. After exposure to the ultrasound simulator, the experimental group demonstrated statistically significant improvement in POCUS confidence and overall POCUS knowledge (p < .01). CONCLUSION: The use of high-fidelity ultrasound simulation can improve POCUS confidence and knowledge among medical students while addressing common barriers to the implementation of a POCUS curriculum. Despite showing statistically significant improvement in overall knowledge, the results did not appear to hold educational significance. Additional POCUS educational methods are necessary to overcome cognitive bias and potential overconfidence. The next stage of curriculum development will include resident-led POCUS workshops to supplement simulation.

Ultrasound-guided pleural biopsy following a non-diagnostic thoracentesis for non-small cell lung cancer.

We report a case of a 65-year-old man with a cavitary lung mass and parietal-based pleural nodules in which a pleural ultrasound-guided approach yielded a definitive diagnosis of stage IV non-small cell lung carcinoma. Computed tomography-guided biopsy is often preferred approach for the majority of United States hospitals for sampling pleural nodules as compared to US. The advantages of an US-guided approach include [1]: increased portability [2]; decreased procedure time [3]; reduced reliance on dedicated ancillary support staff [4]; need for local anesthesia only [5]; lack of ionizing radiation exposure; and [6] cost reduction.

Single-cell landscape analysis unravels molecular programming of the human B cell compartment in chronic GVHD.

Alloreactivity can drive autoimmune syndromes. After allogeneic hematopoietic stem cell transplantation (allo-HCT), chronic graft-versus-host disease (cGVHD), a B cell-associated autoimmune-like syndrome, commonly occurs. Because donor-derived B cells continually develop under selective pressure from host alloantigens, aberrant B cell receptor (BCR) activation and IgG production can emerge and contribute to cGVHD pathobiology. To better understand molecular programing of B cells in allo-HCT, we performed scRNA-Seq analysis on high numbers of purified B cells from patients. An unsupervised analysis revealed 10 clusters, distinguishable by signature genes for maturation, activation, and memory. Within the memory B cell compartment, we found striking transcriptional differences in allo-HCT patients compared with healthy or infected individuals, including potentially pathogenic atypical B cells (ABCs) that were expanded in active cGVHD. To identify intrinsic alterations in potentially pathological B cells, we interrogated all clusters for differentially expressed genes (DEGs) in active cGVHD versus patients who never had signs of immune tolerance loss (no cGVHD). Active cGVHD DEGs occurred in both naive and BCR-activated B cell clusters. Remarkably, some DEGs occurred across most clusters, suggesting common molecular programs that may promote B cell plasticity. Our study of human allo-HCT and cGVHD provides understanding of altered B cell memory during chronic alloantigen stimulation.

Why do two procedures when one will do? Thoracic ultrasound guiding management of complex pleural space infections.

Complicated parapneumonic effusions and empyemas are associated with significant morbidity and mortality. When managing a potentially infected pleural space, current guidelines support performing diagnostic thoracentesis prior to consideration of chest tube insertion. We present a case demonstrating our clinical approach to the management of complicated parapneumonic effusions and empyemas, with patient presentation and initial point-of-care thoracic ultrasound assessment guiding consideration of immediate insertion of small-bore (14 F) chest tube.

A Phase I Trial of SYK Inhibition with Fostamatinib in the Prevention and Treatment of Chronic Graft-Versus-Host Disease.

Despite the exciting advancement of novel therapies, chronic graft-versus-host disease (cGVHD) remains the most common cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation (HCT). Frontline treatment of cGVHD involves systemic steroids, which are associated with significant morbidities. We previously found that inhibition of spleen tyrosine kinase (SYK) with fostamatinib preferentially eradicated aberrantly activated B cells in both ex vivo studies of cGVHD patient B cells, as well as in vivo mouse studies. These and other preclinical studies implicated hyper-reactive B-cell receptor signaling and increased SYK expression in the pathogenesis of cGVHD and compelled this first in-human allogeneic HCT clinical trial. We investigated the safety and efficacy of the oral SYK inhibitor, fostamatinib, for both the prevention and treatment of cGVHD. The primary objective was to evaluate the safety of fostamatinib and determine its maximum tolerated dose in the post-HCT setting. Secondary objectives included assessing the efficacy of fostamatinib in preventing and treating cGVHD, as well as examining alterations in B-cell compartments with treatment. This was a single-institution phase I clinical trial that evaluated the use of fostamatinib in allogeneic HCT patients before the development of cGVHD or at the time of steroid-refractory cGVHD (SR-cGVHD). Patients received fostamatinib at one of three dose levels using a continual reassessment algorithm to determine the maximum tolerated dose. Multiparameter flow cytometry was used to evaluate changes in B cell subpopulations over the first year of treatment with fostamatinib. Nineteen patients were enrolled in this phase I trial, with 5 in the prophylaxis arm and 14 in the therapeutic arm. One patient (5%) required discontinuation of therapy for a dose-limiting toxicity. At a median follow-up of over 3 years, no patients had cancer relapse while on fostamatinib treatment, and recurrent malignancy was observed in 1 patient 2 years after the end of therapy. In the prophylaxis arm, 1 of 5 patients (20%) developed cGVHD while on fostamatinib. In the therapeutic arm, the overall response rate was 77%, with a complete response rate of 31%. The median duration of response was 19.3 months and the 12-month failure-free survival was 69% (95% confidence interval, 48-100). Patients were able to reduce their steroid dose by a median of 80%, with 73% remaining on a lower dose at 1 year compared to baseline. There was an early reduction in the proportion of IgD-CD38hi plasmablast-like cells with fostamatinib treatment, particularly in those SR-cGVHD patients who had an eventual response. B-cell reconstitution was not significantly impacted by fostamatinib therapy after allogeneic HCT. Fostamatinib featured a favorable safety profile in the post-HCT setting. Our data suggests an early efficacy signal that was associated with effects on expected cell targets in both the prophylaxis and treatment of cGVHD, providing rationale for a phase II investigation.

IL-17A Contributes to Lung Fibrosis in a Model of Chronic Pulmonary Graft-versus-host Disease.

BACKGROUND: Chronic pulmonary graft-versus-host disease (cpGVHD) after hematopoietic cell transplant (HCT) manifests as progressive airway and parenchymal lung fibrosis. On the basis of our prior data, mice that undergo allogeneic HCT with Tbet-knockout donors (AlloTbet) have increased lung Th17 cells and IL-17A and develop fibrosis resembling human cpGVHD. The role of IL-17A in posttransplant pulmonary fibrosis remains incompletely understood. We hypothesized that IL-17A is necessary for development of murine cpGVHD in this model. METHODS: AlloTbet mice received weekly intraperitoneal anti-IL-17A or IgG (200 µg/mouse) starting 2 weeks post-HCT and were sacrificed after week 5. Histologic airway and parenchymal fibrosis were semiquantitatively graded in a blinded fashion. Lung cells and proteins were measured by flow cytometry, ELISA, and multicytokine assays. RESULTS: Anti-IL-17A modestly decreased airway and parenchymal lung fibrosis, along with a striking reduction in pulmonary neutrophilia, IL-6, MIP-1α, MIP-1ß, CXCL1, and CXCL5 in AlloTbet mice. Additionally, anti-IL-17A decreased CCL2, inflammatory monocytes and macrophages, and Th17 cells. CONCLUSIONS: In the setting of murine AlloHCT with Tbet donors, IL-17A blockade decreases fibrotic features of cpGVHD. This may be mediated by the observed reduction in neutrophils or specific lung monocyte and macrophage populations or alternatively via a direct effect on fibroblasts. Collectively, our results further suggest that anti-IL-17A strategies could prove useful in preventing alloimmune-driven fibrotic lung diseases.

B cells in chronic graft-versus-host disease.

Allogeneic hematopoietic stem cell transplantation (alloHCT) is the definitive therapy for numerous otherwise incurable hematologic malignancies and non-malignant diseases. The genetic disparity between donor and recipient both underpins therapeutic effects and confers donor immune system-mediated damage in the recipient, called graft-versus-host disease (GVHD). Chronic GVHD (cGVHD) is a major cause of late post-transplant morbidity and mortality. B cells have a substantiated role in cGVHD pathogenesis, as first demonstrated by clinical response to the anti-CD20 monoclonal antibody, rituximab. Initiation of CD20 blockade is met at times with limited therapeutic success that has been associated with altered peripheral B cell homeostasis and excess B Cell Activating Factor of the TNF family (BAFF). Increased BAFF to B cell ratios are associated with the presence of circulating, constitutively activated B cells in patients with cGVHD. These cGVHD patient B cells have increased survival capacity and signal through both BAFF-associated and B Cell Receptor (BCR) signaling pathways. Proximal BCR signaling molecules, Syk and BTK, appear to be hyper-activated in cGVHD B cells and can be targeted with small molecule inhibitors. Murine studies have confirmed roles for Syk and BTK in development of cGVHD. Emerging evidence has prompted investigation of several small molecule inhibitors in an attempt to restore B cell homeostasis and potentially target rare, pathologic B cell populations.

Artificial Intelligence in Health Care: Insights From an Educational Forum.

Discussions surrounding the future of artificial intelligenc (AI) in healthcare often cause consternation among healthcare professionals. These feelings may stem from a lack of formal education on AI and how to be a leader of AI implementation in medical systems. To address this, our academic medical center hosted an educational summit exploring how to become a leader of AI in healthcare. This article presents three lessons learned from hosting this summit, thus providing guidance for developing medical curriculum on the topic of AI in healthcare.

Acute cellular and antibody-mediated allograft rejection.

Survival post-lung transplantation remains limited to ∼ 50% at 5 years, far below survival after other solid organ transplants. Allograft rejection is a major cause of this limited survival. At least a third of lung transplant recipients experience acute rejection within 1 year posttransplantation. Acute rejection, though rarely a direct cause of death, represents the principal risk factor for chronic rejection, which is the greatest obstacle to long-term post-lung transplant survival. This article reviews in detail the two major subtypes of acute rejection: acute cellular rejection (ACR) and antibody-mediated rejection (AMR). ACR is diagnosed primarily by bronchoscopic transbronchial biopsies and is defined as perivascular or peribronchiolar lymphocytic infiltrates in the absence of infection. AMR remains poorly defined but is thought to involve anti-donor antibodies, allograft dysfunction, and pathological evidence of lung tissue injury or deposition of complement. Pathophysiological mechanisms, clinical presentation, clinical significance, known risk factors, and treatment strategies are discussed. Additionally, the limitations of current diagnostic modalities for both ACR and AMR are explained. Emerging data on the importance of donor-specific and non-donor-specific anti-human leukocyte antigen (anti-HLA) antibodies as well as non-HLA antibodies are presented. Larger cohorts have improved statistical analyses in recent years, leading to a clearer understanding of important topics related to ACR and AMR. Further collaborative studies and multicenter trials will be key in further advancing lung transplantation knowledge and improving outcomes in years to come.