Gheorghe Hundorfean

Functional Relevance of T Helper 17 (Th17) Cells and the IL-17 Cytokine Family in Inflammatory Bowel Disease

&NA; The recent discovery and characterization of T helper 17 cells (Th17) and their signature cytokines (IL‐17) represents a hallmark in T‐cell immunobiology by providing a new distinctive pathway for the communication between adaptive and innate immunity. From the six members of the IL‐17 cytokine family presently known, at least two have evident proinflammatory qualities and are involved in several chronic inflammatory disorders, including inflammatory bowel disease (IBD). IL‐17A and IL‐17F are abundantly found in inflamed IBD mucosa, suggesting their pivotal role in IBD. However, the precise implication of IL‐17 cytokine family members in IBD pathogenesis and the mechanisms regulating their secretion are incompletely understood. Importantly, recent findings suggest that beyond IL‐17 production‐Th17 cells may secret a plethora of other effector cytokines such as IL‐21, IL‐22, and IL‐9‐ which is in part induced by its own IL‐9 production. However, the use of anti‐IL‐17 therapeutic strategies in experimental models of chronic inflammation results in disease‐ameliorating effects suggesting their potential use in IBD patients. In this review article we discuss the latest findings on the role of Th17 cells and IL‐17 family members in IBD immunopathology, as well as research perspectives. (Inflamm Bowel Dis 2011;)

IL-9 and its receptor are predominantly involved in the pathogenesis of UC

Objective Several pathogenic roles attributed over the past two decades to either T helper (Th)1 or Th2 cells are increasingly becoming associated with interleukin (IL)-17 and most recently IL-9 signalling. However, the implication of IL-9 in IBD has not been addressed so far. Design We investigated the expression of IL-9 and IL-9R by using peripheral blood, biopsies and surgical samples. We addressed the functional role of IL-9 signalling by analysis of downstream effector proteins. Using Caco-2 cell monolayers we followed the effect of IL-9 on wound healing. Results IL-9 mRNA expression was significantly increased in inflamed samples from patients with UC as compared with controls. CD3+ T cells were major IL-9-expressing cells and some polymorphonuclear leucocytes (PMN) also expressed IL-9. IL-9 was co-localised with the key Th9 transcription factors interferon regulatory factor 4 and PU.1. Systemically, IL-9 was abundantly produced by activated peripheral blood lymphocytes, whereas its receptor was overexpressed on gut resident and circulating PMN. IL-9 stimulation of the latter induced IL-8 production in a dose-dependent manner and rendered PMN resistant to apoptosis suggesting a functional role for IL-9R signalling in the propagation of gut inflammation. Furthermore, IL-9R was overexpressed on gut epithelial cells and IL-9 induced STAT5 activation in these cells. Moreover, IL-9 inhibited the growth of Caco-2 epithelial cell monolayers in wound healing experiments. Conclusions Our results provide evidence that IL-9 is predominantly involved in the pathogenesis of UC suggesting that targeting IL-9 might become a therapeutic option for patients with UC.

Prevalence of collagen VII-specific autoantibodies in patients with autoimmune and inflammatory diseases

BackgroundAutoimmunity to collagen VII is typically associated with the skin blistering disease epidermolysis bullosa acquisita (EBA), but also occurs occasionally in patients with systemic lupus erythematosus or inflammatory bowel disease. The aim of our present study was to develop an accurate immunoassay for assessing the presence of autoantibodies against collagen VII in large cohorts of patients and healthy donors.MethodsBased on in silico antigenic analysis and previous wetlab epitope mapping data, we designed a chimeric collagen VII construct containing all collagen VII epitopes with higher antigenicity. ELISA was performed with sera from patients with EBA (n = 50), Crohns disease (CD, n = 50), ulcerative colitis (UC, n = 50), bullous pemphigoid (BP, n = 76), and pemphigus vulgaris (PV, n = 42) and healthy donors (n = 245).ResultsBy ELISA, the receiver operating characteristics analysis yielded an area under the curve of 0.98 (95% CI: 0.9638-1.005), allowing to set the cut-off at 0.32 OD at a calculated specificity of 98% and a sensitivity of 94%. Running the optimized test showed that serum IgG autoantibodies from 47 EBA (94%; 95% CI: 87.41%-100%), 2 CD (4%; 95% CI: 0%-9.43%), 8 UC (16%; 95% CI: 5.8%-26%), 2 BP (2.63%; 95% CI: 0%-6.23%), and 4 PV (9.52%; 95% CI: 0%-18.4%) patients as well as from 4 (1.63%; 95% CI: 0%-3.21%) healthy donors reacted with the chimeric protein. Further analysis revealed that in 34%, 37%, 16% and 100% of sera autoantibodies of IgG1, IgG2, IgG3, and IgG4 isotype, respectively, recognized the recombinant autoantigen.ConclusionsUsing a chimeric protein, we developed a new sensitive and specific ELISA to detect collagen specific antibodies. Our results show a low prevalence of collagen VII-specific autoantibodies in inflammatory bowel disease, pemphigus and bullous pemphigoid. Furthermore, we show that the autoimmune response against collagen VII is dominated by IgG4 autoantibodies. The new immunoassay should prove a useful tool for clinical and translational research and should improve the routine diagnosis and disease monitoring in diseases associated with collagen VII-specific autoimmunity.

Autoimmunity against type VII collagen in inflammatory bowel disease.

•  Introduction •  The autoantigen: type VII collagen •  Autoimmunity to type VII collagen •  Inflammatory bowel disease •  Autoimmune phenomena in IBD •  Association of IBD with autoimmune diseases •  Autoimmunity to type VII collagen in IBD •  Autoimmunity against type VII collagen and EBA show a strong association with IBD •  Sequence of occurrence of skin blistering and bowel disease in IBD associated with EBA •  Initiation of the type VII collagen‐specific autoimmune response in IBD •  Response of EBA and IBD to treatment •  Implications for the practical management of IBD and EBA •  Concluding remarks and perspectives

Autoimmune Colitis and Subsequent CMV-induced Hepatitis After Treatment With Ipilimumab.

Ipilimumab, a humanized CTLA-4 antibody, improves overall survival in patients with metastatic melanoma. However, immune-related adverse effects occur in about 65% of ipilimumab-treated patients and have to be adequately managed. A 55-year-old patient developed grade 3 autoimmune colitis 7 weeks after initiation of ipilimumab treatment and subsequently hepatitis with grade 3 elevation of transaminases and γ-glutamyl transferase. Colitis manifested with up to 18 watery and bloody stools per day and severe attacks of abdominal pain. After exclusion of infectious causes, immunosuppression with corticosteroids was initiated. Because of recurrent abdominal pain, spontaneous perforation of the colon had to be excluded. Elevated liver function tests (grade 3 CTCAE) occurred and differential diagnosis included immune-mediated, toxic, and viral hepatitis. It is interesting to note that, not an immune-mediated but a cytomegalovirus-induced hepatitis was diagnosed by serum blood tests and liver biopsy and was subsequently successfully treated. Careful elaboration of the patient under immunotherapy was essential as further immunosuppression mandatory for autoimmune hepatitis would have worsened the viral hepatitis. In conclusion, cytomegalovirus reactivation should be included in the differential in patients under immunotherapy with checkpoint inhibitors and has to be considered as a cause for morbidity.

Anti-TNF-refractory colitis after checkpoint inhibitor therapy: Possible role of CMV-mediated immunopathogenesis

ABSTRACT Immune-related adverse events (irAEs) induced by checkpoint inhibitors are well known. Since fatal outcomes have been reported early detection and adequate management are crucial. In particular, colitis is frequently observed and can result in intestinal perforation. This is the first report of an autoimmune colitis that was treated according to algorithms but became resistant due to a CMV reactivation. The 32-y-old male patient with metastatic melanoma treated within an anti-PD-1/ipilimumab combination study developed severe immune-mediated colitis (CTCAE grade 3) with up to 18 watery stools per day starting 2 weeks after treatment initiation. After improving upon therapy with immunosuppressive treatment (high dose steroids and infliximab) combined with parenteral nutrition diarrhea again exacerbated. Additionally, the patient had asymptomatic grade 3 CTCAE amylase and lipase elevation. Colitis was monitored by weekly endoscopies and colon biopsies were analyzed histologically with CMV staining, multi-epitope ligand cartography (MELC) and qRT-PCR for inflammatory genes. In the course, CMV reactivation was detected in the colon and treated with antiviral medication in parallel to a reduction of corticosteroids. Subsequently, symptoms improved. The patient showed a complete response for 2 y now including regression of bone metastases. CMV reactivation under checkpoint inhibitor therapy in combination with immunosuppressive treatment for autoimmune side effects has to be considered in these patients and if present treated. Potentially, CMV reactivation is underdiagnosed. Treatment algorithms should include CMV diagnostics.

Confocal laser endomicroscopy provides potential differentiation criteria between Crohn's disease and ulcerative colitis.

To the Editor: The diagnostic differentiation between the two complex entities of inflammatory bowel disease (IBD), Crohn’s disease (CD) and ulcerative colitis (UC), remains challenging in contemporary gut pathology. This delicate differentiation is of crucial importance for treatment strategy, surveillance, and surgical management. It implies a complex diagnostic orchestration and evaluation of clinical, endoscopic, radiologic, and histopathologic criteria. Among all these criteria, histological delineation needs careful assessment of multiple endoscopic biopsies from many areas of the colon and the terminal ileum. A nonharmful alternative in clarifying the disease entity as well as borderline cases could be confocal laser endomicroscopy (CLE), which is a new and sophisticated endoscopic imaging technique within the armamentarium of modern endoscopy. By enabling real-time and in vivo visualization of a plethora of novel cellular and subcellular details (at magnifications up to 1000-fold), which correlate with conventional histology, CLE has had a revolutionary impact on endoscopic diagnosis. Classically, the delimitation between CD and UC has been based on several endoscopic and histopathologic differentiation criteria that were considered together, whereas the use of endomicrosopic differentiation criteria per se has not been addressed yet. Beyond discussing possible endomicroscopy-based differential diagnostic criteria in IBD, the essential question remains whether in strictly defined disorders or patient collectives (e.g., impaired coagulation or high perforation risk) the optical biopsy provided by newly available endoscopic imaging and magnification techniques could counterbalance and eventually replace in the near future the more or less riskburdened bioptical sampling and subsequent conventional histopathological evaluation. The answer to this provocative question is not easy because it also combines the conventional confrontation between old and new or progress versus conservation of established systems. Surely, replacing conventional histology with optical histology in certain situations should not be confounded with a radical renouncement of histopathology and white light endoscopy in favor of the new and highly selective imaging techniques. Endoscopists in the last few years have gradually accumulated more and more ‘‘diagnostic autonomy’’ by gathering endopathological knowledge from conventional histopathology. Moreover, the advantages of a real-time nonharmful and easily repeatable optical biopsy over conventional biopsy with the given risks of bleeding, infections, and/or perforation (especially in a highly inflamed and friable colon tissue) entailing higher costs and time delay are indisputable. Several endomicroscopic diagnostic advances and imaging studies reporting high sensitivity and specificity (>95%) have been published in the past few years. Still the partial replacement of conventional biopsy by optical biopsy has to be addressed in prospective randomized studies in the years to come. Nevertheless, endomicroscopy could be the reliable modern answer in situations such as the TABLE 1. Proposed CLE-based Criteria for the Real-time and In Vivo Differentiation Between CD and UC.

Development and Validation of a Confocal Laser Endomicroscopy-Based Score for In Vivo Assessment of Mucosal Healing in Ulcerative Colitis Patients

Background Endoscopic monitoring is fundamental for evaluating the therapeutic response in IBD, but a validated endomicroscopic mucosal healing (MH) score is not available to date. However, confocal laser endomicroscopy (CLE) might define MH more precisely than conventional endoscopy. The major aim was to establish and validate an MH score for ulcerative colitis (UC), based on CLE. Methods In an initial pilot study (n = 10), various CLE changes were analyzed for identification of reproducible criteria for establishing a CLE score. Four reproducible CLE criteria were implemented in a following validation study. Subsequently, active UC patients (n = 23, Mayo score ≥6) were prospectively included and underwent colonoscopy with CLE before and after 3 anti-TNF applications. Patients were clinically followed over a period of 3 years. The endomicroscopic MH score (eMHs; range, 0-4) was compared with histopathology and endoscopy scores from the same colonic location. Results The eMHs showed high sensitivity, specificity, and accuracy values (100% with 95% confidence interval [CI] of 15.81%-100%; 93.75% with 95% CI of 69.77%-99.84%, and 94.44%, respectively). The eMHs showed a good correlation with the histological Gupta score (rs = 0.82, P < 0.0001) and the endoscopic Mayo subscore (rs = 0.81%, P < 0.0001). Sixty percent of therapy responders presented an eMHs <1, which translated into long-lasting clinical remission and reduced hospitalization, steroid, and surgery need. Conclusions CLE can accurately assess MH based on the newly developed and statistically validated eMHs in UC, and it is superior in predicting the long-lasting clinical outcome based on both descriptive and functional barrier imaging (NCT01417728).

High-definition endoscopic imaging with i-Scan for the detection and characterization of duodenal Crohn's disease.

Dear Sir, Virtual (dye-less) chromoendoscopy with i -Scan (Pentax, Japan) is a sophisticated, advanced imaging technique within contemporary endoscopy which relies on a computer-based color program that enhances the contrast of mucosal details.1 In certain diagnostic settings, high-definition endoscopy with i -Scan has been proved superior to standard video endoscopy.2 Recent publications from our group regarding advanced endoscopic imaging techniques have addressed the utility of i -Scan for the diagnosis of gastric Crohns disease3 as well …

In vivo detection of mucosal healing-involved histiocytes by confocal laser endomicroscopy

Histiocytes have a pivotal role in wound repair and intestinal epithelial recovery - the most important goal to sustain gut functionality. Yet, an in vivo description of colonic histiocytes by confocal laser endomicroscopy (CLE) is missing. Here, we report the case of a 45-years-old male patient who was referred to our clinic with weight loss and a history of two consecutive Clostridium difficile colitis episodes, the latter cured 3 wk before present admission. Stool microbiology was negative. Conventional colonoscopy showed atrophy and a light mucosal oedema in the distal colon. During on-going endoscopy, we performed a fluorescein-aided CLE which revealed large polygonal (histiocytes-like) cells with copious cytoplasm and large nuclei in the lamina propria of the sigmoid colon as well as regenerative epithelial changes. Histopathological assessment of biopsies from the same areas confirmed the endomicroscopical findings: Periodic acid-Schiff- and CD68-positive foamy histiocytes in the colonic lamina propria and an advanced epithelial recovery. Since stool microbiology was repeatedly negative and polymerase chain reaction-analysis from colonic biopsies could not detect any mRNA for Thropheryma whippleii and common pathogens, we interpreted this particular setting as a mucosal healing process after consecutive Clostridium difficile infections. In conclusion, by describing these colonic histiocytes, we highlight the clinical usefulness of CLE in describing the entity of histiocytes in vivo and in real-time during the process of post-infectious mucosal healing in the colon.