Martin J. Wolff

IL-22-producing CD4+ cells are depleted in actively inflamed colitis tissue.

T helper type (Th17) cytokines such as interleukin (IL)-17A and IL-22 are important in maintaining mucosal barrier function and may be important in the pathogenesis of inflammatory bowel diseases (IBDs). Here, we analyzed cells from the colon of IBD patients and show that Crohn’s disease (CD) patients had significantly elevated numbers of IL-17+, CD4+ cells compared with healthy controls and ulcerative colitis (UC) patients, but these numbers did not vary based on the inflammatory status of the mucosa. By contrast, UC patients had significantly reduced numbers of IL-22+ cells in actively inflamed tissues compared with both normal tissue and healthy controls. There was a selective increase in mono-IL-17-producing cells from the mucosa of UC patients with active inflammation together with increased expression of transforming growth factor (TGF)-β and c-Maf. Increasing concentrations of TGF-β in lamina propria mononuclear cell cultures significantly depleted Th22 cells, whereas anti-TGF-β antibodies increased IL-22 production. When mucosal microbiota was examined, depletion of Th22 cells in actively inflamed tissue was associated with reduced populations of Clostridiales and increased populations of Proteobacteria. These results suggest that increased TGF-β during active inflammation in UC may lead to the loss of Th22 cells in the human intestinal mucosa.

Preferential HIV Infection of CCR6+ Th17 Cells Is Associated with Higher Levels of Virus Receptor Expression and Lack of CCR5 Ligands

ABSTRACT Th17 cells are enriched in the gut mucosa and play a critical role in maintenance of the mucosal barrier and host defense against extracellular bacteria and fungal infections. During chronic human immunodeficiency virus (HIV) infection, Th17 cells were more depleted compared to Th1 cells, even when the patients had low or undetectable viremia. To investigate the differential effects of HIV infection on Th17 and Th1 cells, a culture system was used in which CCR6+ CD4+ T cells were sorted from healthy human peripheral blood and activated in the presence of interleukin 1β (IL-1β) and IL-23 to drive expansion of Th17 cells while maintaining Th1 cells. HIV infection of these cultures had minimal effects on Th1 cells but caused depletion of Th17 cells. Th17 loss correlated with greater levels of virus-infected cells and cell death. In identifying cellular factors contributing to higher susceptibility of Th17 cells to HIV, we compared Th17-enriched CCR6+ and Th17-depleted CCR6− CD4 T cell cultures and noted that Th17-enriched CCR6+ cells expressed higher levels of α4β7 and bound HIV envelope in an α4β7-dependent manner. The cells also had greater expression of CD4 and CXCR4, but not CCR5, than CCR6− cells. Moreover, unlike Th1 cells, Th17 cells produced little CCR5 ligand, and transfection with one of the CCR5 ligands, MIP-1β (CCL4), increased their resistance against HIV. These results indicate that features unique to Th17 cells, including higher expression of HIV receptors and lack of autocrine CCR5 ligands, are associated with enhanced permissiveness of these cells to HIV.

TH17, TH22 and TReg Cells Are Enriched in the Healthy Human Cecum

There is increasing evidence that dysregulation of CD4+ T cell populations leads to intestinal inflammation, but the regional distribution of these populations throughout the intestinal tract in healthy individuals remains unclear. Here, we show that TH17, TH22 and TReg cells are enriched in the healthy human cecum compared to the terminal ileum and sigmoid colon, whereas TH1 and TH2 cells do not significantly vary by location. Transcriptional profiling analysis of paired pinch biopsies from different regions of the intestine identified significant differences in the metabolic state of the terminal ileum, cecum, and sigmoid colon. An increased proportion of TH17 cells was positively associated with expression of resistin (RETN) and negatively associated with expression of trefoil factor 1 (TFF1). These results suggest that CD4+ T helper cells that are important in maintaining mucosal barrier function may be enriched in the cecum as a result of metabolic differences of the surrounding microenvironment.

TGF-β1 Induces Endothelial Cell Apoptosis by Shifting VEGF Activation of p38MAPK from the Prosurvival p38β to Proapoptotic p38α

TGF-β1 and VEGF, both angiogenesis inducers, have opposing effects on vascular endothelial cells. TGF-β1 induces apoptosis; VEGF induces survival. We have previously shown that TGF-β1 induces endothelial cell expression of VEGF, which mediates TGF-β1 induction of apoptosis through activation of p38 mitogen-activated protein kinase (MAPK). Because VEGF activates p38MAPK but protects the cells from apoptosis, this finding suggested that TGF-β1 converts p38MAPK signaling from prosurvival to proapoptotic. Four isoforms of p38MAPK —α, β, γ, and δ—have been identified. Therefore, we hypothesized that different p38MAPK isoforms control endothelial cell apoptosis or survival, and that TGF-β1 directs VEGF activation of p38MAPK from a prosurvival to a proapoptotic isoform. Here, we report that cultured endothelial cells express p38α, β, and γ. VEGF activates p38β, whereas TGF-β1 activates p38α. TGF-β1 treatment rapidly induces p38α activation and apoptosis. Subsequently, p38α activation is downregulated, p38β is activated, and the surviving cells become refractory to TGF-β1 induction of apoptosis and proliferate. Gene silencing of p38α blocks TGF-β1 induction of apoptosis, whereas downregulation of p38β or p38γ expression results in massive apoptosis. Thus, in endothelial cells p38α mediates apoptotic signaling, whereas p38β and p38γ transduce survival signaling. TGF-β1 activation of p38α is mediated by VEGF, which in the absence of TGF-β1 activates p38β. Therefore, these results show that TGF-β1 induces endothelial cell apoptosis by shifting VEGF signaling from the prosurvival p38β to the proapoptotic p38α. Mol Cancer Res; 10(5); 605–14. ©2012 AACR.

Fatal hepatitis B reactivation due to everolimus in metastatic breast cancer: case report and review of literature

Hepatitis B reactivation can occur with cytotoxic chemotherapy in patients with hepatitis B and cancer. Reactivation can occur in a patient with chronic hepatitis, an inactive carrier, or one with resolved hepatitis. Clinical presentation may range from subclinical elevation of liver enzymes to fatal fulminant hepatic failure. Mammalian target of rapamycin inhibitors, which include everolimus, are a new generation of targeted agents that are currently approved for many cancers (since March 2009) including advanced hormone receptor positive, human epidermal growth factor receptor 2-negative breast cancer, in conjunction with exemestane (as of July 2012). We are therefore still learning the various adverse events that occur with this new class of agents. Here, we present an unfortunate case of fatal hepatitis B reactivation in a woman with metastatic breast cancer treated with everolimus and exemestane. We have detailed the controversies around hepatitis B screening prior to immunosuppressive therapy. Clinicians and patients should be aware of this rare but fatal complication prior to everolimus use, and a detailed history, screening for hepatitis B and prophylactic antiviral treatment should be considered.

Inferred metagenomic comparison of mucosal and fecal microbiota from individuals undergoing routine screening colonoscopy reveals similar differences observed during active inflammation.

The mucosal microbiota lives in close proximity with the intestinal epithelium and may interact more directly with the host immune system than the luminal/fecal bacteria. The availability of nutrients in the mucus layer of the epithelium is also very different from the gut lumen environment. Inferred metagenomic analysis for microbial function of the mucosal microbiota is possible by PICRUSt. We recently found that by using this approach, actively inflamed tissue of ulcerative colitis (UC) patients have mucosal communities enriched for genes involved in lipid and amino acid metabolism, and reduced for carbohydrate and nucleotide metabolism. Here, we find that the same bacterial taxa (e.g. Acinetobacter) and predicted microbial pathways enriched in actively inflamed colitis tissue are also enriched in the mucosa of subjects undergoing routine screening colonoscopies, when compared with paired samples of luminal/fecal bacteria. These results suggest that the mucosa of healthy individuals may be a reservoir of aerotolerant microbial communities expanded during colitis.

Objective structured clinical examination as a novel tool in inflammatory bowel disease fellowship education.

Background:Experiential learning in medical education, as exemplified by objective structured clinical examinations (OSCEs), is a well-validated approach for improving trainee performance. Furthermore, the Accreditation Council for Graduate Medical Education has identified OSCEs as an ideal method for assessing the core competency of interpersonal and communication skills. Here, we describe a novel educational tool, the inflammatory bowel disease OSCE (IBD OSCE), to assess and improve this clinical skill set in Gastroenterology fellows. Methods:We developed a 4-station IBD OSCE that assessed shared decision making, physician–physician communication, and physician–patient consultative skills specifically related to the care of patients with IBD. Each station was videotaped and observed live by faculty gastroenterologists. Behaviorally anchored checklists were scored independently by a faculty observer and the standardized patient/physician, who both provided feedback to the fellow immediately after each case. Post-OSCE, fellows attended a debriefing session on patient communication and were surveyed to assess their perspective on the examinations educational value. Results:Twelve second-year gastroenterology fellows from 5 fellowship programs participated in the IBD OSCE. Fellows performed well in all measured domains and rated the experience highly for its educational value. Fellows cited IBD as an area of relative deficiency in their education compared with other knowledge areas within gastroenterology. Conclusions:To our knowledge, this is the first OSCE designed specifically for the evaluation of skills as they relate to IBD management. Using OSCEs for IBD education provides an opportunity to robustly assess core competencies and the role of the physician as an educator.

Isolation and cytokine analysis of lamina propria lymphocytes from mucosal biopsies of the human colon

Much of our understanding of gut-microbial interactions has come from mouse models. Intestinal immunity is complex and a combination of host genetics and environmental factors play a significant role in regulating intestinal immunity. Due to this complexity, no mouse model to date gives a complete and accurate representation of human intestinal diseases, such as inflammatory bowel diseases. However, intestinal tissue from patients undergoing bowel resection reflects a condition of severe disease that has failed treatment; hence a more dynamic perspective of varying inflammatory states in IBD could be obtained through the analyses of pinch biopsy material. Here we describe our protocol for analyzing mucosal pinch biopsies collected predominantly during colonoscopies. We have optimized flow cytometry panels to analyze up to 8 cytokines produced by CD4+ and CD8+ cells, as well as for characterizing nuclear proteins and transcription factors such as Ki67 and Foxp3. Furthermore, we have optimized approaches to analyze the production of cytokines, including TGF-beta from direct ex vivo cultures of pinch biopsies and LPMCs isolated from biopsies. These approaches are part of our workflow to try and understand the role of the gut microbiota in complex and dynamic human intestinal diseases.

Integrated Analysis of Biopsies from Inflammatory Bowel Disease Patients Identifies SAA1 as a Link Between Mucosal Microbes with TH17 and TH22 Cells

Background: Inflammatory bowel diseases (IBD) are believed to be driven by dysregulated interactions between the host and the gut microbiota. Our goal is to characterize and infer relationships between mucosal T cells, the host tissue environment, and microbial communities in patients with IBD who will serve as basis for mechanistic studies on human IBD. Methods: We characterized mucosal CD4+ T cells using flow cytometry, along with matching mucosal global gene expression and microbial communities data from 35 pinch biopsy samples from patients with IBD. We analyzed these data sets using an integrated framework to identify predictors of inflammatory states and then reproduced some of the putative relationships formed among these predictors by analyzing data from the pediatric RISK cohort. Results: We identified 26 predictors from our combined data set that were effective in distinguishing between regions of the intestine undergoing active inflammation and regions that were normal. Network analysis on these 26 predictors revealed SAA1 as the most connected node linking the abundance of the genus Bacteroides with the production of IL17 and IL22 by CD4+ T cells. These SAA1-linked microbial and transcriptome interactions were further reproduced with data from the pediatric IBD RISK cohort. Conclusions: This study identifies expression of SAA1 as an important link between mucosal T cells, microbial communities, and their tissue environment in patients with IBD. A combination of T cell effector function data, gene expression and microbial profiling can distinguish between intestinal inflammatory states in IBD regardless of disease types.

Using the Objective Structured Clinical Examination to Assess ACGME Competencies in Pediatric Gastroenterology Fellows

Background: The Accreditation Council for Graduate Medical Education has described 6 core competencies with which trainees should demonstrate proficiency. Using the Objective Structured Clinical Examination (OSCE), we aimed to assess 4 of these competencies among Pediatric Gastrointestinal (GI) fellows (PGs). Methods: Eight first-year PGs from 6 medical centers in the New York area participated in a 4-station OSCE with trained standardized patient (SP) actors. The cases included an emergency department (ED) consult, or “ED Consult” for lower gastrointestinal bleeding; “Breaking Bad News” focusing on CF nutritional complications; “Second Opinion” for abdominal pain; “Transition of Care” for inflammatory bowel disease. At each station, attending faculty observed the encounters behind a 1-way mirror. SPs and faculties provided immediate feedback to the examined fellows. Previously validated OSCE checklists were used to assess performance. On completion, fellows attended debriefing sessions and completed surveys about the educational value. Results: Median overall milestone competency scores were 6.9 (PC1), 4.8 (PC2), 5.9 (MK1), 5.7 (MK2), 6.4 (ICS1), 6.9 (Prof1), and 6.7 (Prof3). Overall, fellows score highest (7/9) on the inflammatory bowel disease “Transition of Care” case, found the “Breaking Bad News” Cystic Fibrosis OSCE to be the most challenging, and were most comfortable with the “ED Consult” OSCE, as a commonly encountered scenario. Overall, the fellows rated the educational value of the program highly. Conclusions: To our knowledge, although the OSCE has been validated in other medical fields, this is the first OSCE program developed for PGs fellows. These OSCEs have included Accreditation Council for Graduate Medical Education competencies, serving to assess fellows’ skills in these areas while exposing them to challenging medical and psychosocial cases that they may not frequently encounter.