Lung CD11c+ cells from mice deficient in Epstein-Barr virus-induced gene 3 (EBI-3) prevent airway hyper-responsiveness in experimental asthma

Epstein-Barr virus-induced gene (EBI)-3 codes for a soluble type 1 cytokine receptor homologous to the p40 subunit of IL-12 that is expressed by antigen-presenting cells following activation. Here, we analyzed the functional role of EBI-3 in a murine model of asthma associated with airway hyper-responsiveness (AHR) in ovalbumin-sensitized mice. Upon allergen challenge, EBI-3-/- mice showed less severe AHR, decreased numbers and degranulation of eosinophils and a significantly reduced number of VCAM-1+ cells in the lungs as compared to wild-type littermates. We thus analyzed lung CD11c+ cells before and after allergen challenge in these mice and found that before allergen challenge, lung CD1…

IL-12 family members in experimental colitis

Interleukin (IL)-12 p35/p40 is a heterodimeric cytokine that plays an important role in T helper (Th) cell polarization and Th1 T-cell differentiation. Recent findings have shown that both p35 and p40 can form other cytokines with different proteins (IL-23: p19/p40; IL-35: p35/EBI3). Furthermore, the cytokine IL-27 (EBI3/p28) has been identified as a member of the IL-12 family. Here, we discuss the recent findings on the role of IL-12 family members in experimental colitis. In particular, the role of IL-23 as a master regulator of effector T-cell activation is highlighted. These findings have important implications for the design of new therapeutic approaches in chronic intestinal inflammat…

The role of signal transducers and activators of transcription in T inflammatory bowel diseases.

Signal transducers and activators of transcription (STAT) proteins are intracellular effector molecules of cytokine-modulated signaling. On the one hand, they play an important role in hematopoiesis and the development of the human immune system. STAT transcription factors are necessary for embryogenesis and the maintenance of the mammalian immune response. In the adult, STAT signaling is responsible for T-cell polarization toward interferon gamma-secreting Th1 T cells or interleukin 4-producing Th2 cells. On the other hand, these proteins are involved in the regulation of T-cell survival. STAT activation is strongly associated with tyrosine phosphorylation by tyrosine kinases, namely Jak1,…

RAGE Mediates a Novel Proinflammatory Axis

S100/calgranulin polypeptides are present at sites of inflammation, likely released by inflammatory cells targeted to such loci by a range of environmental cues. We report here that receptor for AGE (RAGE) is a central cell surface receptor for EN-RAGE (extracellular newly identified RAGE-binding protein) and related members of the S100/calgranulin superfamily. Interaction of EN-RAGEs with cellular RAGE on endothelium, mononuclear phagocytes, and lymphocytes triggers cellular activation, with generation of key proinflammatory mediators. Blockade of EN-RAGE/RAGE quenches delayed-type hypersensitivity and inflammatory colitis in murine models by arresting activation of central signaling pathw…

Azathioprine, Mycophenolate Mofetil, and Methotrexate Specifically Modulate Cytokine Production by T Cells

Chromoendoscopy in Inflammatory Bowel Disease

Chromoendoscopy with methylene blue or indigo carmine significantly increases the diagnostic yield of finding intraepithelial neoplasia in patients with longstanding colitis. The number needed to treat is 14 for panchromoendoscopy to identify 1 additional patient with dysplasia. Chromoendoscopy can greatly facilitate the identification of flat lesions harboring intraepithelial neoplasia. Chromoendoscopy can guide biopsies and clearly reduces the amount of biopsies that are needed per patient. Magnifying endoscopy or CLE are additional techniques, which can be used in conjunction with chromoendoscopy to further reduce the amount of biopsies and to further increase the diagnostic yield. Chrom…

Noninvasive assessment of Crohn's disease activity: a comparison of 18F-fluorodeoxyglucose positron emission tomography, hydromagnetic resonance imaging, and granulocyte scintigraphy with labeled antibodies.

Detection of disease activity in Crohn's disease (CD) is of crucial importance for diagnosis and management of the disease. Noninvasive methods for monitoring are desirable and comprise hydromagnetic resonance imaging (hydro-MRI) and leukocyte scintigraphy. In addition, a recent case report indicated the potential of 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) to assess CD activity. However, comparative prospective studies are lacking.Between February, 1999 and August, 2000, 59 patients with CD were enrolled in a prospective study to assess disease activity by FDG-PET, hydro-MRI, and immunoscintigraphy with anti-nonspecific cross-reacting antigen 95 antigranulocyte antibod…

A new model of chronic colitis in SCID mice induced by adoptive transfer of CD62L+ CD4+ T cells: insights into the regulatory role of interleukin-6 on apoptosis.

<i>Objective:</i> The proinflammatory cytokine interleukin (IL)-6 is involved in various chronic inflammatory processes. IL-6 is a predominant cytokine produced by lamina propria T cells in Crohn’s disease and experimental colitis. This study was designed to examine the effect of a neutralizing IL-6-receptor (IL-6R) antibody on the programmed cell death of mucosal T cells in the CD62L+ CD4+ SCID transfer model of chronic experimental colitis in mice and to gain more insight into the pathogenesis of this transfer colitis model. <i>Methods:</i> For adoptive transfer, we isolated CD62L+ CD4+ double-positive T cells from wild-type BALB/c mice followed by intraperitoneal …

Collagen ultrastructure in ruptured cruciate ligaments

The ultrastructure of collagen fibrils was investigated in normal (n 39) and ruptured (n 23) human anterior cruciate ligaments. The normal ligament had a complex three-dimensional structure. Collagen fibrils predominantly had a unidirectional course with parallel arrangement and a mean diameter of 75 (20-185) nm. Four days after anterior cruciate ligament rupture, the mean fibril diameter was increased; it later decreased, probably due to synthesis of young, thin 30-40 nm fibrils. Interfibrillar dysplastic collagen fibrils were detected in the extracellular matrix of ruptured ligaments. They were more frequently found later than 3 days after rupture and were seen also at a distance of 2-3 c…

Is chromoendoscopy the new standard for cancer surveillance in patients with ulcerative colitis?

Chromoendoscopy was introduced in 2003 as a novel 'red flag' technique that aimed to increase the sensitivity of identifying flat, neoplastic lesions in patients with ulcerative colitis. The improved sensitivity of chromoendoscopy over standard white-light endoscopy has been confirmed in European and Asian centers. This commentary discusses the findings from a prospective, controlled study from the Mount Sinai Hospital in New York. The findings of this study provide unequivocal evidence that chromoendoscopy is superior to white-light endoscopy in the detection of neoplasias in patients with IBD. The authors of this study identified a greater number of lesions and a higher number of patients…

Mucosal T cells: mediators or guardians of inflammatory bowel disease?

Because the mucosal immune system is continuously exposed to a myriad of potentially harmful environmental antigens, it frequently reacts with antiinflammatory/regulatory T cell responses driven by TGF-β-producing T H 3 cells and IL-10-producing regulatory T cells. Intestinal inflammation in patients with inflammatory bowel diseases is thought to result from an overwhelming uncontrolled activation of the mucosal immune system induced by antigens of the normal luminal flora in genetically susceptible individuals. Inflammatory bowel disease appears to be mediated by subsets of CD4 + T lymphocytes or NK T cells secreting high levels of proinflammatory cytokines such as TNF-α. The increased exp…

Apoptosis of T cells and the control of inflammatory bowel disease: therapeutic implications.

Inflammatory bowel diseases (IBDs) such as Crohn’s disease and ulcerative colitis are the result of an imbalanced mucosal T cell response. Despite the identification of a genetic susceptibility region in the NOD2/CARD15 (nucleotide-binding oligomerisation domain 2/caspase recruitment domain 15) gene, the aetiology is still unclear. Thus, the hunt for disease-initiating factors such as defects in the mucosal barrier or pathogenic microorganisms is ongoing. By contrast, the immunopathogenesis in IBDs is better understood. The identification of cytokines that are involved in T cell and monocyte signalling led to specific therapeutic concepts. Recent data have clearly shown that the most powerf…

Differential TNF-Signaling in Chronic Inflammatory Disorders

TNF-alpha is a pleiotropic cytokine with strong proinflammatory and immunomodulatory properties. TNF-alpha plays a critical role in many acute or chronic inflammatory diseases and anti-TNF-strategies have proven to be clinically effective. Two TNF-specific cell surface receptors TNF-R1 and TNF-R2 have been identified and the function of these receptors and the downstream intracellular signal transduction pathways have been extensively studied in vitro. For a long time TNF-R1 was considered to be the predominant mediator of TNF-signaling, whereas TNF-R2 was ascribed only auxilliary function. However, there is increasing clinical and experimental evidence for an important independent role of …

Posttranslationally modified proteins as mediators of sustained intestinal inflammation.

Oxidative and carbonyl stress leads to generation of N(epsilon)-carboxymethyllysine-modified proteins (CML-mps), which are known to bind the receptor for advanced glycation end products (RAGE) and induce nuclear factor (NF)-kappaB-dependent proinflammatory gene expression. To determine the impact of CML-mps in vivo, RAGE-dependent sustained NF-kappaB activation was studied in resection gut specimens from patients with inflammatory bowel disease. Inflamed gut biopsy tissue demonstrated a significant up-regulation of RAGE and increased NF-kappaB activation. Protein extracts from the inflamed zones, but not from noninflamed resection borders, caused perpetuated NF-kappaB activation in cultured…

Azathioprine suppresses ezrin-radixin-moesin-dependent T cell-APC conjugation through inhibition of Vav guanosine exchange activity on rac proteins

Abstract We have shown recently that the azathioprine metabolite 6-Thio-GTP causes immunosuppression by blockade of GTPase activation in T lymphocytes. In the present study, we describe a new molecular mechanism by which 6-Thio-GTP blocks GTPase activation. Although 6-Thio-GTP could bind to various small GTPases, it specifically blocked activation of Rac1 and Rac2 but not of closely related Rho family members such as Cdc42 and RhoA in primary T cells upon stimulation with αCD28 or fibronectin. Binding of 6-Thio-GTP to Rac1 did not suppress Rac effector coupling directly but blocked Vav1 exchange activity upon 6-Thio-GTP hydrolysis, suggesting that 6-Thio-GTP loading leads to accumulation of…

Anti-TNF-Antikörper zur Therapie von entzündlichen Darmstenosen bei Morbus Crohn

Stenoses are a frequent complication in patients with Crohn's disease and represent a major diagnostic and therapeutic challenge. The proper assessment of the nature of a stenosis as inflammatory or fibrotic is critical for appropriate treatment, since symptomatic fibrotic stenoses require surgical resection. Standard diagnostic procedures to assess the nature of a stenosis include endoscopy, conventional contrast radiography and magnetic resonance tomography. Recent data suggest, that the positron-emission-tomography possesses a high sensitivity and specificity to confirm inflammatory activity in the bowel. The recombinant monoclonal anti-TNF-antibody Infliximab (Remicade) has been approve…

Chromoscopy-Guided Endomicroscopy Increases the Diagnostic Yield of Intraepithelial Neoplasia in Ulcerative Colitis

Background & Aims: Because of the large number of biopsy specimens, surveillance colonoscopy in ulcerative colitis (UC) is currently time consuming and significant flat lesions still may be missed. In this study we assessed the value of combined chromoscopy and endomicroscopy for the diagnosis of intraepithelial neoplasias in a randomized controlled trial. Methods: A total of 161 patients with long-term UC in clinical remission were randomized at a 1:1 ratio to undergo conventional colonoscopy or chromoscopy with endomicroscopy. Eight patients were excluded because of insufficient bowel preparation. In the conventional colonoscopic group (n = 73), random biopsy examinations and targeted bio…

Local barrier dysfunction identified by confocal laser endomicroscopy predicts relapse in inflammatory bowel disease

Objectives: Loss of intestinal barrier function plays an important role in the pathogenesis of inflammatory bowel disease (IBD). Shedding of intestinal epithelial cells is a potential cause of barrier loss during inflammation. The objectives of the study were (1) to determine whether cell shedding and barrier loss in humans can be detected by confocal endomicroscopy and (2) whether these parameters predict relapse of IBD. Methods: Confocal endomicroscopy was performed in IBD and control patients using intravenous fluorescein to determine the relationship between cell shedding and local barrier dysfunction. A grading system based on appearances at confocal endomicroscopy in humans was devise…

Mucosal immunoregulation: transcription factors as possible therapeutic targets.

Much progress has been recently made with regard to our understanding of the mucosal immune system in health and disease. In particular, it has been shown that uncontrolled mucosal immune responses driven by lymphocytes or non-lymphoid cells may lead to immunological diseases such as allergy, hypersensitivity and inflammation. Thus, a more detailed understanding of mucosal immune regulation and decision making at mucosal surfaces is essential for a better understanding of mucosal immune responses in health and disease. Antigen presenting cells and T lymphocytes play a key role in controlling mucosal immune responses. To deal with this key task, T helper cells differentiate into functionally…

Cutting Edge: IL-23 Cross-Regulates IL-12 Production in T Cell-Dependent Experimental Colitis

Abstract Although IL-12 and IL-23 share the common p40 subunit, IL-23, rather than IL-12, seems to drive the pathogenesis of experimental autoimmune encephalomyelitis and arthritis, because IL-23/p19 knockout mice are protected from disease. In contrast, we describe in this study that newly created LacZ knockin mice deficient for IL-23 p19 were highly susceptible for the development of experimental T cell-mediated TNBS colitis and showed even more severe colitis than wild-type mice by endoscopic and histologic criteria. Subsequent studies revealed that dendritic cells from p19-deficient mice produce elevated levels of IL-12, and that IL-23 down-regulates IL-12 expression upon TLR ligation. …

Endoscopic detection of early lower gastrointestinal cancer

The prognosis for patients with malignancies of the lower gastrointestinal tract is strictly dependent on early detection of premalignant and malignant lesions. What should an ideal screening and surveillance colonoscopy be able to accomplish? The technique should allow detection of large but also discrete mucosal alterations. Ideally, endoscopic discrimination between neoplastic and non-neoplastic lesions would be possible during the ongoing procedure. At present, endoscopy can be performed with powerful new endoscopes. Comparable to the rapid development in chip technology, the optical features of the newly designed endoscopes offer resolutions, which allow new surface details to be seen.…

Context-dependent Pax-5 repression of a PU.1/NF-κB regulated reporter gene in B lineage cells

Enhancers located in the 3' end of the locus in part regulate immunoglobulin heavy chain (IgH) gene expression. One of these enhancers, HS 1,2, is developmentally regulated by DNA binding proteins like NF-kappaB, Pax-5 and the protein complex NF-alphaP in B lineage cells. Here we report that NF-alphaP is the ets protein PU.1. A glutathione-S-transferase (GST)-pulldown assay demonstrated that PU.1 can physically interact with NF-kappaB in solution. Experiments in COS cells showed that PU.1 and NF-kappaB (p50/c-Rel) can activate transcription of an enhancer linked reporter gene. The paired domain protein Pax-5 has previously been shown to repress enhancer-dependent transcription. Additional c…

Tonnenkonkremente im Ductus choledochus bei einer 4 Jahre vergessenen, impaktierten Endoprothese

We report on a 67-year-old female patient who presented in July 2005 with sudden onset of pain in the right upper abdomen. The patient had undergone cholecystectomy in 1987. Because of recurrrent complaints in the right upper abdomen, a pigtail stent was placed into the common hepatic duct in 2001. When the patients presented now, the laboratory tests including liver enzymes were within normal ranges. Endoscopic retrograde cholangiogi aphy, however, revealed a remaining 10-French, impacted double pigtail endoprothesis that was obstructed by sludge as well as multiple giant bile duct stones of 20 to 30 mm in size. The giant stones could be finally removed by the combined use of ESWL and endo…

Programming of Intestinal Epithelial Differentiation by IL-33 Derived from Pericryptal Fibroblasts in Response to Systemic Infection.

SummaryThe intestinal epithelium constitutes an efficient barrier against the microbial flora. Here, we demonstrate an unexpected function of IL-33 as a regulator of epithelial barrier functions. Mice lacking IL-33 showed decreased Paneth cell numbers and lethal systemic infection in response to Salmonella typhimurium. IL-33 was produced upon microbial challenge by a distinct population of pericryptal fibroblasts neighboring the intestinal stem cell niche. IL-33 programmed the differentiation of epithelial progenitors toward secretory IEC including Paneth and goblet cells. Finally, IL-33 suppressed Notch signaling in epithelial cells and induced expression of transcription factors governing…

Tolerance towards resident intestinal flora in mice is abrogated in experimental colitis and restored by treatment with interleukin-10 or antibodies to interleukin-12

There is now increasing evidence that hyperresponsiveness towards intestinal flora is a crucial event in the pathogenesis of inflammatory bowel disease (IBD). In support of this hypothesis, we recently described in humans that tolerance exists towards indigenous intestinal flora but is broken in active IBD lesions. In the present study, we have attempted to transfer this model into mice from different genetic backgrounds (BALB/c, SJL/J, C3H/HeJ). We found that mononuclear cells from spleen, small bowel and large bowel of mice do not proliferate, i.e. are tolerant when exposed to bacterial sonicates derived from autologous intestine (BsA) but do proliferate, i.e. are immune when exposed to b…

Anti-PR-3 antibodies induce endothelial IL-8 release

Background It has been shown that interaction of anti-PR-3 antibodies with human endothelial cells (EC) leads to an activation of EC in vitro, i.e. induction of adhesion molecules like E-selectin, VCAM-1 and tissue factor. The aim of this study was to investigate the effect of anti-PR-3 antibodies on endothelial IL-8 expression. Materials and methods EC were cultured in 96-well plates and stimulated with TNF-α and IL-1β for 1 h to induce membrane expression of endothelial PR-3. Anti-PR-3 antibodies were purified from sera from patients with clinically active Wegener's granulomatosis. Purified anti-Ro, anti-centromere, anti-dsDNA antibodies and a monoclonal anti-PR-3 antibody (WGM2) served a…

From Immunogenic Mechanisms to Novel Therapeutic Approaches in Inflammatory Bowel Disease

Crohn’s disease (CD) and ulcerative colitis (UC) are the two most common forms of chronic inflammatory bowel disease (IBD). The etiology of IBD is still unclear and should be considered as multi-factorial according to recent studies.1 Genetic factors seem to play a pathogenetic role as well as environmental, infectious and immulogical factors. Substantial progress, however, has been made in the understanding of the pathogenesis of IBD during the past years persuing the view, that IBD could result from disturbances of the intestinal barrier and a pathologic activation of the intestinal immune response towards luminal, bacterial antigens. This paradigm has led to the identification of key pla…

6-thio-GTP Derivatives

Animal models of intestinal inflammation: new insights into the molecular pathogenesis and immunotherapy of inflammatory bowel disease

Inflammatory bowel diseases (IBDs) in humans are complex chronic inflammatory disorders of largely unknown cause. Several mouse models that in some respects resemble human IBDs have recently been developed and have provided new insights into immunoregulatory processes in the gut. Both genetic and environmental factors have been shown to be involved in chronic intestinal inflammation. In most of the models CD4+ T lymphocytes have been identified as central mediators of inflammation. Inappropriate activation of T(H)1-dominated cytokine pathways upon contact with luminal bacterial antigens and lack of tolerance appear to be crucial for intestinal pathology. We present a brief overview of impor…

Cellular ultrastructure of the ruptured anterior cruciate ligament. A transmission electron microscopic and immunohistochemical study in 55 cases.

To evaluate the cellular ultrastructure following injury, we examined the anterior cruciate ligaments in 55 patients with complete tears in different phases after the injury and compared them to a control group of 39 cadaver knees. Samples were analyzed by electron microscopy, immunofluorescence, and ultramorphometry. After an invasion of inflammatory cells into the stumps of the ruptured ligaments, a marked proliferation of fibroblasts was found at the end of Phase 1 (2-3 days after the ligament injury), that was even more pronounced at the beginning of Phase II (4-17 days). These cells were initially highly metabolically active and secreted Type III collagen precursors. In Phase III (4-45…

Regulation of IL-12 p40 Promoter Activity in Primary Human Monocytes: Roles of NF-κB, CCAAT/Enhancer-Binding Protein β, and PU.1 and Identification of a Novel Repressor Element (GA-12) That Responds to IL-4 and Prostaglandin E2

Abstract Appropriate regulation of IL-12 expression is critical for cell-mediated immune responses. In the present study, we have analyzed the regulation of IL-12 p40 promoter activity in primary human monocytes in vivo. Accordingly, we analyzed the p40 promoter by in vivo footprinting in resting and activated primary human blood CD14+ monocytes. Interestingly, footprints at binding sites for trans-activating proteins such as C/EBP, NF-κB, and ETS were only found upon stimulation with LPS and IFN-γ. In contrast, a footprint over a purine-rich sequence at −155, termed GA-12 (GATA sequence in the IL-12 promoter), was observed in resting, but not activated, cells. Further characterization of t…

Surveillance colonoscopy in ulcerative colitis: magnifying chromoendoscopy in the spotlight

Chromoendoscopy in ulcerative colitis may offer the possibility of detecting dysplastic or neoplastic changes at a curable stage, and thus expand the indication for chromoendoscopy from screening to surveillance colonoscopy Colonoscopy is the gold standard for colorectal cancer screening.1–3 Detection and complete removal of adenomas disrupt the adenoma-carcinoma sequence and thus prevent the development of colorectal cancer. However, endoscopists still fear that they may have overlooked relevant lesions despite the availability of modern videoendoscopes. This problem is underlined by a relatively high rate of adenomas missed by conventional endoscopy (up to 27%), as determined by back to b…

Targeting of the transcription factor STAT4 by antisense phosphorothioate oligonucleotides suppresses collagen-induced arthritis

Abstract The transcription factor STAT4 mediates signals of various proinflammatory cytokines, such as IL-12, IL-15, and IL-23, that initiate and stabilize Th1 cytokine production. Although Th1 cytokine production has been suggested to play a major pathogenic role in rheumatoid arthritis, the role of STAT4 in this disease is poorly understood. In this study, we demonstrate a key functional role of STAT4 in murine collagen-induced arthritis (CIA). In initial studies we found that STAT4 expression is strongly induced in CD4+ T cells and to a lesser extent in CD11b+ APCs during CIA. To analyze the role of STAT4 for arthritis manifestation, we next investigated the outcome of interfering with S…

Protection from graft-versus-host disease by HIV-1 envelope protein gp120-mediated activation of human CD4+CD25+ regulatory T cells.

AbstractNaturally occurring CD4+CD25+ regulatory T cells (Tregs) represent a unique T-cell lineage that is endowed with the ability to actively suppress immune responses. Therefore, approaches to modulate Treg function in vivo could provide ways to enhance or reduce immune responses and lead to novel therapies. Here we show that the CD4 binding human immunodeficiency virus-1 envelope glycoprotein gp120 is a useful and potent tool for functional activation of human Tregs in vitro and in vivo. Gp120 activates human Tregs by binding and signaling through CD4. Upon stimulation with gp120, human Tregs accumulate cyclic adenosine monophosphate (cAMP) in their cytosol. Inhibition of endogeneous cA…

Review article: in vivo imaging by endocytoscopy

Aliment Pharmacol Ther 2011; 33: 1183–1193 Summary Background  Endocytoscopy (EC) enables in vivo microscopic imaging at 1400-fold magnification, thereby allowing the analysis of mucosal structures at the cellular level. In contrast to fluorescence imaging with confocal laser endomicroscopy which allows analysis of mucosal structures up to 250 μm in depth, EC is based on the principle of contact light microscopy and only allows visualisation of the very superficial mucosal layer. Aim  To systematically review the feasibility and diagnostic yield of EC for in vivo diagnosis of diseases. Methods  A systematic search of the literature on diagnostic interventions in the gastrointestinal tract u…

Both IL-12p70 and IL-23 are synthesized during active Crohnʼs disease and are down-regulated by treatment with anti-IL-12 p40 monoclonal antibody

Background: Interleukin (IL)-12p70 and IL-23 are key T helper-1 (TH1) cytokines that drive the inflammation seen in numerous models of intestinal inflammation. These molecules contain an identical p40 chain that is bound to a p35 chain in IL-12 and a p19 chain in IL-23, making both potentially susceptible to modulation by an anti-IL-12p40 monoclonal antibody (mAb). Methods: In the present study, we sought to determine whether active inflammation in Crohn's disease (CD) is associated with the increased synthesis of both of these cytokines and whether patients treated with an anti-IL-12p40 mAb down-regulate IL-23 as well as IL-12p70 as previous reported. Results: To this end we initially dete…

High resolution colonoscopy in live mice.

Endoscopy in humans is a powerful method for physicians to examine the gut for inflammatory or neoplastic changes. In medical and immunological research, animal models of intestinal diseases are established key tools to investigate the mucosal immune system, colitis and cancer development in the gut. Moreover, such models represent valid systems for testing of novel drugs. In the past, mice had to be killed in order to analyze colitis activity and tumor development. The following protocol describes a method to perform high resolution endoscopic monitoring of live mice. Mice developing colitis or colonic tumors are anesthetized and examined with a miniendoscope. The endoscope is introduced v…

IL-6 trans-Signaling

The molecular consequence of the fever response has been illuminated by a recent study showing that a temperature shift to 40 degrees C resulted in increased leukocyte adhesion to tissue sections, which was mediated by L-selectin activation in lymphocytes. This L-selectin activation during heat responses was dependent on IL-6 trans-signaling via the soluble IL-6R.

Control Of Organ Transplant-Associated Graft-versus-Host Disease By Activated Host Lymphocyte Infusions

Background Prolonged persistence of donor-derived T cells after organ transplantation has been proposed to improve long-term allograft survival. However, surviving transplant-derived T cells are also able to mediate devastating graft-versus-host disease (GvHD). Currently, GvHD after organ transplantation is usually refractory to conventional therapy and the disease outcome fatal. Methods Graft-reactive host T cells were generated ex vivo from a patient suffering from a severe and refractory liver-transplant-associated GvHD. To control GvHD, activated alloreactive host T cells were repetitively retransferred into the patient (activated host lymphocyte infusion [aHLI]). Results Adoptive trans…

A Key Regulatory Role of the Transcription Factor NFATc2 in Bronchial Adenocarcinoma via CD8+ T Lymphocytes

AbstractThe Ca2+-regulated calcineurin/nuclear factor of activated T cells (NFAT) cascade controls alternative pathways of T-cell activation and peripheral tolerance. Here, we describe reduction of NFATc2 mRNA expression in the lungs of patients with bronchial adenocarcinoma. In a murine model of bronchoalveolar adenocarcinoma, mice lacking NFATc2 developed more and larger solid tumors than wild-type littermates. The extent of central tumor necrosis was decreased in the tumors in NFATc2(−/−) mice, and this finding was associated with reduced tumor necrosis factor-α and interleukin-2 (IL-2) production by CD8+ T cells. Adoptive transfer of CD8+ T cells of NFATc2(−/−) mice induced transforming…

Assessment of Tumor Development and Wound Healing Using Endoscopic Techniques in Mice

Mouse models of intestinal inflammation and colon cancer are valuable tools to gain insights into the pathogenesis of the corresponding human diseases. Recently, in vivo mouse endoscopy has been developed, allowing not only the high-resolution monitoring and scoring of experimental disease development, but also enables the investigator to perform manipulations, including local injection of reagents or the taking of biopsies for molecular and histopathologic analyses. Chromoendoscopic staining with methylene blue enables visualization of the crypt structure and allows discrimination between inflammatory and neoplastic changes. The development of endoscopic techniques in live mice opened new …

Novel cytokine-targeted therapies and intestinal inflammation

Several cytokines have been identified as critical mediators of chronic inflammation in inflammatory bowel disease (IBD) and biological therapies that target these molecules have been developed during recent years. Thereby, anti-TNF agents have noticeably improved the treatment of patients with IBD in comparison to conventional therapy. Furthermore, initial clinical trials showed promising results with anti-IL-6 and anti-IL-12/IL-23 agents. In addition to these well-known mediators of IBD, various novel cytokines have been described as critical during the pathogenesis of IBD in recent experimental studies and therapeutic targeting of these cytokines could provide new strategies for human di…

Molecular imaging of VEGF in gastrointestinal cancer in vivo using confocal laser endomicroscopy

Vascular endothelial growth factor (VEGF) is a therapeutic target in gastrointestinal cancer (GiC). However, its in vivo visualisation could not be achieved to date with endoscopic techniques. Confocal laser endomicroscopy (CLE) is a novel imaging technique for gastrointestinal endoscopy providing in vivo microscopy at subcellular resolution. The aim of the study was to evaluate CLE for in vivo molecular imaging of VEGF in GiC.Molecular imaging of tumours in APCmin mice, in xenograft models and in surgical specimens of patients with colorectal cancer (CRC) was achieved after application of labelled antibodies. The tumour sites were scanned with the probe for the strongest specific fluoresce…

Correlative histologic and arthroscopic evaluation in rheumatoid knee joints.

The correlation between arthroscopic observations and histologic changes in rheumatoid arthritis is still controversial. Synovial samples of 21 knee joints in rheumatoid arthritis patients were comparatively investigated by endoscopy and histology. Biopsies were scored by an endoscopist and subsequently dissected. Different histochemical and immunocytochemical staining techniques were used to define inflammatory activity. Arthroscopic and histological values were compared by rating scales and variance analysis. Our study indicates that synovial biopsy is of diagnostic value in rheumatoid arthritis. However, its usefulness depends on the histochemical methods used. The results revealed highl…

RORgamma-expressing Th17 cells induce murine chronic intestinal inflammation via redundant effects of IL-17A and IL-17F.

Background and Aims IL-17–producing CD4 + T-helper cells (Th17) contribute to chronic autoimmune inflammation in the brain, and levels of Th17-derived cytokines increase in patients with colitis, suggesting a role in pathogenesis. We analyzed the roles of Th17 cells and the transcription factor retinoic acid receptor-related organ receptor (ROR)γ, which regulates Th17 differentiation, in chronic intestinal inflammation. Methods Using an adoptive transfer model of colitis, we compared the colitogenic potential of wild-type, interleukin-17A (IL-17A)–, IL-17F–, IL-22–, and RORγ-deficient CD4 + CD25 − T cells in RAG1-null mice. Results Adoptive transfer of IL-17A–, IL-17F–, or IL-22–deficient T…

Gene therapy using IL 12 family members in infection, auto immunity, and cancer.

Interleukin-12 (IL-12) is known for several years to have an essential role in inflammatory responses and innate resistance to infection and cancer. This has been largely attributed to its ability to initiate the differentiation of T-helper-1 (Th1) cells producing interferon-gamma. Recently, two new cytokines, IL-23 and IL-27, with homology to IL-12 were discovered and assigned to the IL-12 family of cytokines. Growing evidence supports a role for IL-23 as key mediator of autoimmune disease regulating the new Th17 subset of CD4+ T cells. IL-27 can have pro- and anti-inflammatory effects, which increase Th1 differentiation, suppress Th2 proliferation, or stimulate cytotoxic T cell activity. …

A Critical Regulatory Role of Leucin Zipper Transcription Factor c-Maf in Th1-Mediated Experimental Colitis

Abstract In this study, we investigated the role of c-Maf, a transcription factor known to induce IL-4 production, in inflammatory bowel diseases and experimental colitis. Although Crohn′s disease (CD) is associated with low IL-4 production by T-bet-expressing Th1 cells in the lamina propria, surprisingly a higher expression of c-Maf in these cells was found as compared with control patients. The relevance of this finding was further evaluated in an animal model of CD induced by adoptive transfer of CD4+CD62L+ T cells in RAG-deficient mice. In this Th1-mediated model, an increase of c-Maf-expressing T lymphocytes in the lamina propria over time was observed. Interestingly, adoptive transfer…

The role of Th1/Th2 polarization in mucosal immunity

Mucosal immunity relies on the delicate balance between antigen responsiveness and tolerance. The polarization of T helper cells plays a key role in maintaining or disrupting this equilibrium.

Dépistage et diagnostic précoce du cancer colorectal

Qu’est-on en droit d’attendre d’une methode ideale de depistage et de surveillance par coloscopie? La technique doit permettre la detection d’importantes lesions mais aussi de discretes alterations muqueuses. Idealement, la distinction endoscopique entre lesion neoplasique et non neoplasique devrait etre possible en cours d’examen. Actuellement, l’endoscopie peut etre realisee avec de nouveaux endoscopes plus puissants. Parallelement au developpement rapide des puces electroniques, les caracteristiques optiques des nouveaux endoscopes offrent des resolutions qui permettent de voir de nouveaux details de la surface muqueuse. En complement de la chromo-endoscopie, les nouveaux videocoloscopes…

Regulation of T-cell apoptosis in inflammatory bowel disease: to die or not to die, that is the mucosal question.

T-cell resistance against apoptosis contributes to inappropriate T-cell accumulation and the perpetuation of chronic mucosal inflammation in inflammatory bowel diseases (IBDs). Anti-interleukin-12 (IL-12) and anti-IL-6 receptor antibodies suppress colitis activity by the induction of T-cell apoptosis. These findings have important implications for the design of effective treatment regimens in IBD.

Confocal laser endomicroscopy.

A miniaturized confocal microscope was developed that could be integrated in the distal tip of a conventional colonoscope. With this technique, denoted confocal endomicroscopy, subsurface analysis of the gut mucosa and in-vivo histology during ongoing endoscopy are possible in full resolution by point scanning laser analysis. The diagnostic spectrum of confocal endomicroscopy is expanding from screening and surveillance for colorectal cancer to Barrett's esophagus, Helicobacter pylori-associated gastritis, and gastric cancer. The new detailed images seen with confocal laser endomicroscopy allow a unique look on cellular structures at and below the surface of the gut. This review describes t…

Methylene blue–aided cholangioscopy unravels the endoscopic features of ischemic-type biliary lesions after liver transplantation

Chromoendoscopy in Barrett's oesophagus: is cresyl violet the magic bullet?

The endoscopic detection of Barrett’s epithelium remains challenging even for modern endoscopy. This is mainly due to the fact that Barrett’s epithelium is often patchy and can easily be overlooked by conventional endoscopy with random biopsies. Thus, chromoendoscopy and magnifying endoscopy were introduced to facilitate diagnosis of Barrett’s epithelium and Barrett’s associated neoplasias. Methylene blue-aided chromoendoscopy was firstly introduced by Canto et al. [1]. The authors could show that methylene blue selectively stains specialised columnar epithelium, which is pathognomonic for Barrett’s epithelium. In contrast, dysplastic areas revealed no or weaker staining due to changes in t…

Isolation and subsequent analysis of murine lamina propria mononuclear cells from colonic tissue

Studies on colonic cells in the lamina propria (LP) of mice are important for understanding the cellular and immune responses in the gut, especially in inflammatory bowel diseases (such as morbus crohn and colitis ulcerosa). This protocol details a method to isolate LP cells and characterize freshly isolated cells by quality control experiments to obtain cells that can be used for further investigations. After different steps of digestion of the tissue using collagenase, DNase and dispase, the resulting cells are purified using Percoll gradient. The success of the isolation can be analyzed by cell viability test (Trypan Blue exclusion test) and by flow cytometric analysis to assess apoptosi…

Animal Models of Inflammatory Bowel Diseases

Cytokine Gene Transcription By NF-kappaB Family Members in Patients with Inflammatory Bowel Disease

We examined the expression of the transcription factor NF-kappa B, a nuclear trans-acting factor known to play a key role in cytokine gene regulation, in patients with inflammatory bowel disease (IBD). It was found that LP macrophages in Crohn's disease (CD) and ulcerative colitis (UC) display high levels of NF-kappa B DNA-binding activity accompanied by an increased production of interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF) alpha. Western blot studies showed an increased expression of the p50 and c-rel subunits of NF-kappa B; however, the most striking finding was an increased expression level of NF-kappa B p65 in patients with CD and UC. Selective downregulation of p65 in IBD…

Regulation of Protein-DNA Interactions at the Interferon-gamma Gene Promoter by Corticosteroids: Implications for Inflammatory Bowel Diseases

T cell specificity and cross reactivity towards enterobacteria,Bacteroides,Bifidobacterium, and antigens from resident intestinal flora in humans

BACKGROUNDT cell responses to normal intestinal bacteria or their products may be important in the immunopathogenesis of chronic enterocolitis.AIMSTo investigate the T cell specificity and cross reactivity towards intestinal bacteria.PATIENTS/METHODST cell clones were isolated with phytohaemagglutinin from peripheral blood and biopsy specimens of inflamed and non-inflamed colon from five patients with inflammatory bowel disease (IBD) and two controls. T cell clones were restimulated with anaerobicBacteroides andBifidobacteria species, enterobacteria, and direct isolates of aerobic intestinal flora. T cell phenotype was analysed by single-cell immunocyte assay.RESULTSAnalysis of 96 T cell cl…

6-thioguanosine diphosphate and triphosphate levels in red blood cells and response to azathioprine therapy in Crohn's disease.

Background & Aims: Azathioprine is the gold standard for immunosuppressive therapy in Crohn's disease (CD) and its molecular mechanism of action is caused by the metabolite 6-thioguanosine triphosphate (TGTP). In this study we assessed the impact of TGTP levels for monitoring of azathioprine therapy. Methods: A novel, highly sensitive assay was established to measure levels of TGTP and its precursors 6-thioguanosine monophosphates and 6-thioguanosine diphosphates (TGDP) in red blood cells from 50 CD patients. The results were correlated with clinical outcome. Results: TGTP levels could be quantified in 47 patients and a subgroup of these patients showed significantly high levels of TGDP. 6-…

Soluble gp130 is the natural inhibitor of soluble interleukin-6 receptor transsignaling responses

Signal transduction in response to interleukin-6 (IL-6) requires binding of the cytokine to its receptor (IL-6R) and subsequent homodimerization of the signal transducer gp130. The complex of IL-6 and soluble IL-6R (sIL-6R) triggers dimerization of gp130 and induces responses on cells that do not express membrane bound IL-6R. Naturally occurring soluble gp130 (sgp130) can be found in a ternary complex with IL-6 and sIL-6R. We created recombinant sgp130 proteins that showed binding to IL-6 in complex with sIL-6R and inhibited IL-6/sIL-6R induced proliferation of BAF/3 cells expressing gp130. Surprisingly, sgp130 proteins did not affect IL-6 stimulated proliferation of BAF/3 cells expressing …

Chemically induced mouse models of colitis.

Crohn's disease (CD) and ulcerative colitis (UC), both of which are referred to as inflammatory bowel disease (IBD), are chronic inflammatory disorders of the gastrointestinal tract that have characteristic clinical, pathological, endoscopic, and radiologic features. Knowledge about the pathogenesis of IBD has dramatically increased in recent years based in part on the use of experimental models of IBD. Although none of these models exactly mimics the human disorder, they have proven to be useful for studying many important aspects of these conditions. Detailed in this unit is a description of the most commonly used chemically induced mouse models of IBD. These include trinitrobenzene sulfo…

Mongersen, an oral SMAD7 antisense oligonucleotide, and crohn's disease

Crohn's disease-related inflammation is characterized by reduced activity of the immunosuppressive cytokine transforming growth factor β1 (TGF-β1) due to high levels of SMAD7, an inhibitor of TGF-β1 signaling. Preclinical studies and a phase 1 study have shown that an oral SMAD7 antisense oligonucleotide, mongersen, targets ileal and colonic SMAD7.In a double-blind, placebo-controlled, phase 2 trial, we evaluated the efficacy of mongersen for the treatment of persons with active Crohn's disease. Patients were randomly assigned to receive 10, 40, or 160 mg of mongersen or placebo per day for 2 weeks. The primary outcomes were clinical remission at day 15, defined as a Crohn's Disease Activit…

TGF-β Dependent T-Cell Regulation in Colitis and Colon Cancer

Transforming growth factor-β (TGF-β) is a potent growth inhibitor endowed with tumor-suppressing activity. Unfortunately, cancers are often resistant to such growth inhibition. This evasion frequently results from a genetic loss of functional TGF-β signaling components. On the other hand, cancer cells often produce high amounts TGF-β1 by themselves and sometimes respond to it with invasion and metastasis. Much effort is being done to develop therapeutic approaches to modulate TGF-β signaling in cancer cells either to inhibit the TGF-β-induced invasive phenotype or to reestablish its growth-inhibitory activities. However, TGF-β is a pleiotropic cytokine with important functions not only in c…

A genetic basis for IFN-gamma production and T-bet expression in humans.

Abstract Th1 and Th2 cytokines secreted by polarized effector T cells play a pivotal role in the development of autoimmune and allergic diseases. However, the genetic basis of cytokine production by T lymphocytes in humans is poorly understood. In this study, we investigated the genetic contribution to cytokine production and regulation of T cell-specific transcription factors in a prospective twin study. We found a substantial genetic contribution to the production of Th1 cytokines such as IFN-γ and TNF-α with heritabilities of 0.85 (95% confidence intervals, 0.74–0.95) and 0.72 (0.50–0.93), respectively, whereas no genetic influence on production of the Th2 signature cytokine IL-4 was obs…

TGF-beta as a T cell regulator in colitis and colon cancer

TGF-beta is a pleiotropic cytokine with powerful immunosuppressive functions. Mice deficient for TGF-beta1 show a dramatic phenotype with severe multiorgan inflammation and die shortly after birth. Recent investigations have highlighted the role of TGF-beta in suppression of T cell mediated autoimmune inflammation and anti-tumor immunity. In addition to its direct anti-inflammatory effects on T cells, TGF-beta has been implicated as central regulator of regulatory T cells. TGF-beta not only mediates the suppression of effector T cells by Tregs, recent evidence also reveals a role for TGF-beta along with TCR stimulation in the peripheral induction of regulatory T cells from naïve CD4+CD25- c…

CD4+CD25+ Regulatory T Cells and TGF-Beta in Mucosal Inflammation

Transforming growth factor-beta (TGF-beta) is an anti-inflammatory cytokine which plays a key role in the maintenance of the immune system homeostasis. Indeed the abrogation of the TGF-beta signaling in immune cells leads to autoimmunity and inflammation in several organs including the gut. TGF-beta acts at multiple levels to maintain the immune system in check. However, TGF-beta has been recently shown to play a key role in the peripheral generation and function of CD4+CD25+ regulatory T cells, a subset of suppressive lymphocytes involved in the control of effector T cell activation and proliferation. Consistently abrogation of Tregs maturation as observed in different systems leads to a p…

T-bet as a possible therapeutic target in autoimmune disease

The prominent role of pro-inflammatory cytokines produced by T helper-1 (T(H1)) cells in regulating autoimmune responses in vitro and in vivo has been demonstrated. Recent observations of T cell polarisation by regulatory transcription factors--especially T-bet (T-box expressed in T cells)--raise the question of their influence in controlling autoimmune diseases. Here, the authors summarise recent observations of the role of T-bet in controlling chronic inflammatory and autoimmune diseases and discuss the implications of these findings for future therapeutic approaches.

Methotrexate specifically modulates cytokine production by T cells and macrophages in murine collagen-induced arthritis (CIA): a mechanism for methotrexate-mediated immunosuppression

SUMMARYImmunosuppressive therapy with methotrexate (MTX) has been established as effective treatment for patients with rheumatoid arthritis. To analyse the therapeutic potential and mechanisms of action of MTX, we determined serum cytokine levels and cytokine production by splenic T cells and macrophages in untreated and MTX-treated mice. Furthermore, we assessed the role of MTX in a murine model of experimental arthritis induced by collagen type II (CIA). MTX reduced spontaneous and IL-15-induced tumour necrosis factor (TNF) production by splenic T cells but not by macrophages from healthy mice in vitro in a dose-dependent manner. In contrast, interferon-gamma (IFN-γ) production was less s…

IL-27 controls the development of inducible regulatory T cells and Th17 cells via differential effects on STAT1

IL-27 is an IL-12-related cytokine frequently present at sites of inflammation that can promote both anti- and pro-inflammatory immune responses. Here, we have analyzed the mechanisms how IL-27 may drive such divergent immune responses. While IL-27 suppressed the development of proinflammatory Th17 cells, a novel role for this cytokine in inhibiting the development of anti-inflammatory, inducible regulatory T cells (iTreg) was identified. In fact, IL-27 suppressed the development of adaptive, TGF-beta-induced Forkhead box transcription factor p3-positive (Foxp3(+)) Treg. Whereas the blockade of Th17 development was dependent on the transcription factor STAT1, the suppression of iTreg develo…

Magnifying chromoendoscopy: Effective diagnostic tool for screening colonoscopy

The transcription factor NFATc2 controls IL-6–dependent T cell activation in experimental colitis

The nuclear factor of activated T cells (NFAT) family of transcription factors controls calcium signaling in T lymphocytes. In this study, we have identified a crucial regulatory role of the transcription factor NFATc2 in T cell–dependent experimental colitis. Similar to ulcerative colitis in humans, the expression of NFATc2 was up-regulated in oxazolone-induced chronic intestinal inflammation. Furthermore, NFATc2 deficiency suppressed colitis induced by oxazolone administration. This finding was associated with enhanced T cell apoptosis in the lamina propria and strikingly reduced production of IL-6, -13, and -17 by mucosal T lymphocytes. Further studies using knockout mice showed that IL-…

Cutting Edge: Trans-Signaling via the Soluble IL-6R Abrogates the Induction of FoxP3 in Naive CD4+CD25− T Cells

Abstract Chronic inflammatory diseases may develop when regulatory T cells (Tregs) fail to control the balance between tolerance and immunity. Alternatively, activated immune cells might prevent the induction or activation of Tregs in such diseases. In this study, we demonstrate that trans-signaling into T cells via the soluble IL-6 receptor completely abrogates the de novo induction of adaptive Tregs. Mechanistically, IL-6 trans-signaling augmented the expression of the TGF-β signaling inhibitor SMAD7. Consequently, SMAD7 overexpression in T cells using newly created transgenic mice rendered CD4+CD25− T cells resistant to the induction of FoxP3. Finally, IL-6 trans-signaling inhibited Treg…

A new treatment for severe malabsorption due to radiation enteritis.

Specific Regulation of T Helper Cell 1–mediated Murine Colitis by CEACAM1

Carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) is a cell surface molecule that has been proposed to negatively regulate T cell function. We have shown that CEACAM1 is associated with specific regulation of T helper cell (Th)1 pathways, T-bet–mediated Th1 cytokine signaling, and Th1-mediated immunopathology in vivo. Mice treated with anti–mouse CEACAM1-specific monoclonal antibody (mAb) CC1 during the effector phase exhibited a reduced severity of trinitrobenzene sulfonic acid colitis in association with decreased interferon (IFN)-γ production. Although oxazolone colitis has been reported as Th2 mediated, mice treated with the CC1 mAb or a CEACAM1-Fc chimeric protein…

T-bet and mucosal Th1 responses in the gastrointestinal tract

T cells play an essential role in regulating mucosal immune responses in the gastrointestinal tract. Recent observations on T helper cell differentiation and activation by regulatory transcription factors-especially T-bet-in chronic inflammatory diseases have provided new perspectives for understanding mucosal immunity. Here we summarise recent advances in the field of transcription factors and discuss the implications of these findings for future therapeutic approaches in inflammatory bowel diseases. In particular, we have focused on the role of T-bet in controlling mucosal Th1 responses in the gastrointestinal tract.

Interleukin-33-Dependent Innate Lymphoid Cells Mediate Hepatic Fibrosis

SummaryLiver fibrosis is a consequence of chronic liver diseases and thus a major cause of mortality and morbidity. Clinical evidence and animal studies suggest that local tissue homeostasis is disturbed due to immunological responses to chronic hepatocellular stress. Poorly defined stress-associated inflammatory networks are thought to mediate gradual accumulation of extracellular-matrix components, ultimately leading to fibrosis and liver failure. Here we have reported that hepatic expression of interleukin-33 (IL-33) was both required and sufficient for severe hepatic fibrosis in vivo. We have demonstrated that IL-33’s profibrotic effects related to activation and expansion of liver resi…

Diagnosing Helicobacter pylori in vivo by confocal laser endoscopy.

Background & Aims: Confocal laser endomicroscopy enables subsurface microscopic imaging of living tissue during ongoing endoscopy. This case report describes the in vivo detection of Helicobacter pylori by endomicroscopy.Methods: Endomicroscopy (Pentax, Tokyo, EC-3870CIFK) was performed by using two different contrast stains: Topical Acriflavine in addition to intravenously applied fluorescein netted the surface and allowed identification of focal accumulation of Helicobacter pylori at the surface and in deeper layer of the gastric epithelium. Biopsies were performed at the antrum and corpus for urease testing and histology. In addition, biopsies were cultured for Helicobacter pylori. Cultu…

Antibodies against tumor necrosis factor (TNF) induce T-cell apoptosis in patients with inflammatory bowel diseases via TNF receptor 2 and intestinal CD14⁺ macrophages.

Background & Aims The anti–tumor necrosis factor (TNF) antibodies infliximab, adalimumab, and certolizumab pegol have proven clinical efficacy in Crohn's disease. Here, we assessed the effects of anti-TNF antibodies on apoptosis in inflammatory bowel disease (IBD). Methods CD14 + macrophages and CD4 + T cells were isolated from peripheral blood and lamina propria mononuclear cells from patients with IBD and control patients. Cell surface markers and apoptosis were assessed by immunohistology and fluorescence-activated cell sorting techniques. Results Lamina propria CD14 + macrophages showed significantly more frequent and higher membrane-bound TNF (mTNF) expression than CD4 + T cells in IBD…

Histologie in vivo avec endomicroscopie confocale dans le diagnostic des cancers colorectaux

L’endomicroscopie confocale constitue un nouvel outil diagnostique destine au diagnostic in vivo des modifications epitheliales constatees en cours de coloscopie. Apres l’application d’agents fluorescents, la technique permet d’obtenir en temps reel, une imagerie de la surface muqueuse et de la couche sous-muqueuse sous microscopie a haute resolution.

Chronic intestinal inflammation in mice expressing viral Flip in epithelial cells

Viruses are present in the intestinal microflora and are currently discussed as a potential causative mechanism for the development of inflammatory bowel disease. A number of viruses, such as Human Herpesvirus-8, express homologs to cellular FLIPs, which are major contributors for the regulation of epithelial cell death. In this study we analyzed the consequences of constitutive expression of HHV8-viral FLIP in intestinal epithelial cells (IECs) in mice. Surprisingly, expression of vFlip disrupts tissue homeostasis and induces severe intestinal inflammation. Moreover vFlip(IEC-tg) mice showed reduced Paneth cell numbers, associated with excessive necrotic cell death. On a molecular level vF…

The transcription factor IFN regulatory factor–4 controls experimental colitis in mice via T cell–derived IL-6

The proinflammatory cytokine IL-6 seems to have an important role in the intestinal inflammation that characterizes inflammatory bowel diseases (IBDs) such as Crohn disease and ulcerative colitis. However, little is known about the molecular mechanisms regulating IL-6 production in IBD. Here, we assessed the role of the transcriptional regulator IFN regulatory factor-4 (IRF4) in this process. Patients with either Crohn disease or ulcerative colitis exhibited increased IRF4 expression in lamina propria CD3+ T cells as compared with control patients. Consistent with IRF4 having a regulatory function in T cells, in a mouse model of IBD whereby colitis is induced in RAG-deficient mice by transp…

Chromoendoscopy and Magnifying Endoscopy in Patients with Gastroesophageal Reflux Disease

Gastroesophageal reflux disease (GERD) is common in the Western world. Upper endoscopy is needed to characterize the disease. Barrett’s esophagus as a complication of GERD is an established precancerous condition which can lead to adenocarcinoma in the distal esophagus. This review summarizes recent advances in the endoscopic characterization of Barrett’s esophagus using magnification endoscopy and chromoendoscopy. Methylene blue, indigo carmine and acetic acid are commonly used dyes to facilitate diagnosis of Barrett’s esophagus. Methylene blue is absorbed in the specialized columnar epithelium, which is pathognomonic for Barrett’s esophagus. Indigo carmine and acetic acid are used as cont…

Endoscopic confocal imaging.

In vivo fluorescence endomicroscopy is a newly developed diagnostic tool enabling virtual in vivo histology of the mucosal layer during ongoing endoscopy. This review summarizes currently available data about the technique and clinical use of confocal endomicroscopy. Indications discussed include colorectal cancer evaluation, ulcerative colitis and surveillance, Barrett's esophagus, and detection of Helicobacter pylori infection in vivo.

An inducible mouse model of colon carcinogenesis for the analysis of sporadic and inflammation-driven tumor progression.

Colorectal cancer is a life-threatening disease that can develop spontaneously or as a complication of inflammatory bowel diseases. Mouse models are essential tools for the preclinical testing of novel therapeutic options in vivo. Here, we provide a highly reliable protocol for an experimental mouse model to study the development of colon cancers. It is based on the mutagenic agent azoxymethane (AOM), which exerts colonotropic carcinogenicity. Repeated intraperitoneal administration of AOM results in the development of spontaneous tumors within 30 weeks. As an alternative option, inflammation-dependent tumor growth can be investigated by combining the administration of AOM with the inflamma…

Normal interleukin-12 production in individuals with antibodies toHelicobacter pylori

It is increasingly recognized that the inability of the immune system to clear H. pylori infection is caused by an inadequate immune response and is associated with chronic gastric inflammation. To further investigate the cellular immune response to H. pylori, we studied PBMC from 31 H. pylori antibody-negative and 16 H. pylori antibody-positive individuals for H. pylori-induced DNA synthesis, secretion of the Th1-type cytokine IFN-gamma and secretion of IL-12, a cytokine produced by bacteria-stimulated monocyte/macrophages and a potent inducer of antibacterial immune responses and Th1-type T cells. All experiments were performed using Y. enterocolitica 03 as control. Our results demonstrat…

Endoscopic mucosal resection: an evolving therapeutic strategy for non-polypoid colorectal neoplasia

Endoscopic management for laterally spreading tumours of the colorectum is a safe and effective treatment and may be an alternative to surgery in selected patients Colorectal cancer develops in approximately 5–6% of the adult population and is one of the leading causes of cancer death in Europe and the USA.1,2 Screening colonoscopy is the widely accepted gold standard for early diagnosis of colorectal cancer and should be offered to patients older than 50 years.3–5 However, colonoscopy is increasingly in competition with computed tomographic virtual endoscopy.6 Therefore, it is essential to understand whether total colonoscopy rather than virtual endoscopy enables the detection of all relev…

New therapies for sepsis: focus on the interleukin (IL)12 family member IL27

Sepsis is a severe complication of abdominal infections such as peritonitis and is associated with high mortality. Unfortunately, the molecular mechanisms controlling the development of sepsis are still incompletely understood. Interestingly, the interleukin (IL) 12 family member IL27 seems to play a key role in sepsis. In a murine model of septic peritonitis induced by caecal ligation and puncture (CLP), IL27 levels were found to be strongly induced. Furthermore, mice deficient for the EBI3 subunit of IL27 were resistant to CLP-induced septic peritonitis as compared to wild-type controls. This effect could be suppressed by injection of recombinant IL27. Further studies demonstrated that IL…

Randomised trial of mycophenolate mofetil versus azathioprine for treatment of chronic active Crohn’s disease

BACKGROUNDCrohn’s disease is a chronic inflammatory disease of the alimentary tract. Azathioprine is an effective agent in the management of chronic active Crohn’s disease leading to long term remission of disease activity. Such treatment leads to limited efficacy or side effects in a small subset of patients.AIMSTo compare efficacy and side effects of treatment with azathioprine plus corticosteroids versus mycophenolate mofetil (MMF) plus corticosteroids in patients with chronic active Crohn’s disease.METHODSSeventy patients with chronic active Crohn’s disease (Crohn’s disease activity index (CDAI) greater than 150) were randomised for treatment with azathioprine/cortisone or MMF/cortisone…

Constitutive and inducible protein/DNA interactions of the interferon-γ promoter inin vivo CD45RA and CD45RO T helper subsets

Interferon-gamma (IFN-gamma) is a key cytokine of T lymphocytes with major regulatory functions in the immune system. To determine and compare protein/DNA interactions at the native IFN-gamma locus in T cells, we analyzed the human IFN-gamma promoter by ligation-mediated polymerase chain reaction (LM-PCR) techniques. Accordingly, Jurkat T cells and primary CD45RA and CD45R0 CD4+ T cell subsets isolated from peripheral blood using immunomagnetic beads were cultured and analyzed by LM-PCR. Constitutive and inducible protein/DNA interactions of the IFN-gamma promoter in vivo were detected in all T cells tested. Interestingly, an inducible footprint between -183 and -196 was consistently observ…

Anti-neutrophil cytoplasmic antibodies in a rat model of trinitrobenzenesulphonic acid-induced liver injury

Background In sera from patients with autoimmune liver diseases, e.g. primary sclerosing cholangitis (PSC) and autoimmune hepatitis, anti-neutrophil cytoplasmic antibodies (ANCAs) can be found. Until now, no animal model of ANCA induction in liver disease has been described. In this study, we describe a novel rat model of acute liver injury and subsequent ANCA production. Materials and methods The hapten reagent 2,4,6-trinitrobenzenesulphonic acid (TNBS) was injected into the portal vein of female Lewis rats. Two experimental groups were studied: group A (TNBS/ethanol) received different TNBS concentrations; control animals of group B (ethanol) were injected with 10% (v/v) ethanol/0.9% (w/v…

Struktur und Funktion von kollagenen und nichtkollagenen Matrixkomponenten in der Knochen-Band-Übergangszone — eine immunhistochemische und immunelektronenmikroskopische Studie des Knochen- und Bandgewebes

Nach Alm und Stromberg (1974) kann die Knochen-Band-Ubergangszone in 4 verschiedene Zonen unterteilt werden (Abb. 1). Uber die Zusammensetzung und Komposition dieser zonalen Gruppen ist jedoch nur wenig bekannt. Ziel unserer Studie war daher die Detektion verschiedener Matrixproteine am Knoehen-Band-Ubergang.

Identification of epithelial gaps in human small and large intestine by confocal endomicroscopy.

Background & Aims: Confocal endomicroscopy is an emerging technology that poses the endoscopist with challenges for identifying epithelial structures in the human intestine. We have shown previously that the murine intestinal epithelium is punctuated by gaps caused by cell shedding. The goals of this study were to determine if confocal endomicroscopy could resolve the presence of human epithelial gaps and whether a proinflammatory cytokine could increase cell shedding. Methods: Intestinal mucosa was imaged after staining with acriflavine. Confocal endomicroscopy of 17 patients yielded 6277 images from the human terminal ileum and rectum. Results were validated by parallel studies of anesthe…

In vivo molecular and morphological imaging by real time confocal mini-microscopy

We evaluated a newly developed miniaturized confocal laser microscopy probe for real-time in vivo molecular and morphological imaging of normal, inflammatory, and malignant tissue in rodents. In the rigid mini-microscopy probe (diameter 7 mm), a single line laser delivers an excitation wavelength of 488 nm. Optical slice thickness is 7 μm, lateral resolution 0.7 μm. The range of the z-axis is 0 - 250 μm below the tissue surface. Organ systems were examined in vivo in rodent models of human diseases. FITC-labeled Lycopersion esculentum lectin was injected or selected cell populations stained for molecular targeting. Morphological imaging was performed using fluorescein sodium, FITC-labeled d…

Smad7 in T cells drives T helper 1 responses in multiple sclerosis and experimental autoimmune encephalomyelitis

Autoreactive CD4+ T lymphocytes play a vital role in the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Since the discovery of T helper 17 cells, there is an ongoing debate whether T helper 1, T helper 17 or both subtypes of T lymphocytes are important for the initiation of autoimmune neuroinflammation. We examined peripheral blood CD4+ cells from patients with active and stable relapsing-remitting multiple sclerosis, and used mice with conditional deletion or over-expression of the transforming growth factor-beta inhibitor Smad7, to delineate the role of Smad7 in T cell differentiation and autoimmune neuroinflammation. We found that Smad…

Signaling molecules: the pathogenic role of the IL-6/STAT-3 trans signaling pathway in intestinal inflammation and in colonic cancer.

Although the precise etiology of inflammatory bowel diseases (IBD) still remains unclear, considerable progress has been made in the identification of novel signal transduction pathways that elucidate the immunopathogenesis involved in the perpetuation of the inflammatory process. As both ulcerative colitis and Crohn's disease are associated with an increased risk for developing colorectal cancer (CRC) and precancerous dysplastic epithelial changes, further studies have concentrated on finding a common signaling pathway that could serve as a mechanistic link between inflammation and associated colonic cancer in IBD. This review presents the current data concerning the pathogenic role of the…

IL-23: a master regulator in Crohn disease

Three studies should shift thinking about the causes of inflammatory bowel disease. It seems that researchers have been focusing on the wrong cytokine as a driving force.

IRF4 regulates IL-17A promoter activity and controls RORγt-dependent Th17 colitis in vivo

The transcription factor IRF4 is involved in several T-cell-dependent chronic inflammatory diseases. To elucidate the mechanisms for pathological cytokine production in colitis, we addressed the role of the IRF transcription factors in human inflammatory bowel disease (IBD) and experimental colitis.IRF levels and cytokine production in IBD patients were studied as well as the effects of IRF4 deficiency in experimental colitis.In contrast to IRF1, IRF5, and IRF8, IRF4 expression in IBD was augmented in the presence of active inflammation. Furthermore, IRF4 levels significantly correlated with IL-6 and IL-17 mRNA expression and to a lesser extent with IL-22 mRNA expression in IBD. To further …

Antisense Phosphorothioate Oligonucleotides to the p65 Subunit of NF-κB Abrogate Fulminant Septic Shock Induced byS. typhimuriumin Mice

The aim of this study was to characterize the functional relevance of the transcription factor NF-κB in the pathogenesis of septic shock. BALB/c mice were infected with two wild-type (WT 1, WT 2) strains of S. typhimurium that induce NF-κB or an escape variant that lacks this ability (P21) at a dose of 1 × 109/animal, respectively. Furthermore, wild-type infected mice were treated with antisense oligonucleotides directed against NF-κB 24 h before and 3 or 6 h after infection, while mismatched oligonucleotides were used as controls. Subsequently, the clinical course, histological and immunological alterations were monitored. Infection with WT 1 and WT 2 strains led to lethal septic shock wit…

Alternative Splice Forms of CYLD Mediate Ubiquitination of SMAD7 to Prevent TGFB Signaling and Promote Colitis

Background & Aims The CYLD lysine 63 deubiquitinase gene (CYLD) encodes tumor suppressor protein that is mutated in familial cylindromatosus, and variants have been associated with Crohn disease (CD). Splice forms of CYLD that lack exons 7 and 8 regulate transcription factors and functions of immune cells. We examined the expression of splice forms of CYLD in colon tissues from patients with CD and their effects in mice. Methods We performed immunohistochemical analyses of colon tissues from patients with untreated CD and patients without inflammatory bowel diseases (controls). We obtained mice that expressed splice forms of CYLD (sCYLD mice) without or with SMAD7 (sCYLD/SMAD7 mice) from tr…

The Transcription Factor T-bet Regulates Mucosal T Cell Activation in Experimental Colitis and Crohn's Disease

The balance between pro and antiinflammatory cytokines secreted by T cells regulates both the initiation and perpetuation of inflammatory bowel diseases (IBD). In particular, the balance between interferon (IFN)-gamma/interleukin (IL)-4 and transforming growth factor (TGF)-beta activity controls chronic intestinal inflammation. However, the molecular pathways that evoke these responses are not well understood. Here, we describe a critical role for the transcription factor T-bet in controlling the mucosal cytokine balance and clinical disease. We studied the expression and function of T-bet in patients with IBD and in mucosal T cells in various T helper (Th)1- and Th2-mediated animal models …

Perforin deficiency attenuates inflammation and tumor growth in colitis-associated cancer

Background: Patients with inflammatory bowel disease (IBD) have a markedly increased risk to develop colon cancer, but there are only limited data about the host antitumor response in such colitis-associated cancer. In the present study we aimed at assessing the role of perforin-dependent effector mechanisms in the immune response in a murine model of colitis-associated colon cancer. Methods: Wildtype and perforin-deficient mice were analyzed in a mouse model of colitis-associated colon cancer using azoxymethane (AOM) and dextran sodium sulfate (DSS). Results: Tumors of wildtype mice showed infiltration of CD4+, CD8+ T cells, natural killer (NK) cells, high numbers of apoptotic cells, and e…

Confocal Endomicroscopy Identifies Loss of Local Barrier Function in the Duodenum of Patients with Crohnʼs Disease and Ulcerative Colitis

Background: Increased cell shedding with gap formation and local barrier dysfunction can be identified endomicroscopically in the terminal ileum of patients with inflammatory bowel disease. We aim to evaluate whether these changes are also present in the duodenum of patients with inflammatory bowel disease. Methods: Fifteen patients with Crohn's disease (CD), 10 patients with ulcerative colitis (UC), and 10 controls underwent fluorescein-aided confocal laser endomicroscopy (CLE). CLE was performed on macroscopically normal antral and duodenal (D1, D2, D3, D4) mucosa. Representative CLE images were prospectively analyzed. Images were scored for the number of epithelial gaps, cell shedding, a…

Endoscopic surveillance in ulcerative colitis: smart biopsies do it better.

Patients with ulcerative colitis (UC) have a significantly higher risk for the development of colitisassociated colorectal cancer. Endoscopic surveillance is recommended. The growth pattern of dysplastic tissue is often multifocal and flat. Thus, significant lesions might be overlooked. Multiple random biopsies are recommended, but cannot eliminate the possibility of missed cancers with severe consequences for the patient. Chromoendoscopy with methylene blue or indigo carmine can unmask flat lesions and may solve this problem. In addition, confocal laser endomicroscopy enables in vivo histology with subcellular resolution during ongoing colonoscopy and leads to highly selected mucosal biops…

Cutting Edge: TGF-β Signaling Is Required for the In Vivo Expansion and Immunosuppressive Capacity of Regulatory CD4+CD25+ T Cells

Abstract Data regarding the role of TGF-β for the in vivo function of regulatory CD4+CD25+ T cells (Treg) are controversial. A transgenic mouse model with impaired TGF-β signaling specifically in T cells was used to assess the role of endogenous TGF-β for the in vivo function of CD4+CD25+ Treg in a murine model of colitis induced by dextran sulfate. Transfer of wild-type, but not transgenic CD4+CD25+ Treg was found to suppress colitis in wild-type mice. In addition, by transferring CFSE-labeled CD4+CD25+ Treg we could demonstrate that endogenous TGF-β promotes the expansion of CD4+CD25+ Treg in vivo. Transgenic mice themselves developed reduced numbers of peripheral CD4+CD25+ Treg and were …

Pathogenesis of inflammatory bowel disease: transcription factors in the spotlight.

See article on page 477 Dysregulated cytokine production by mucosal lymphocytes and macrophages has been implicated in the pathogenesis of both Crohn’s disease and ulcerative colitis, the two major forms of human inflammatory bowel disease (IBD).1 Over the past few years, various murine models of chronic intestinal inflammation resembling IBD have been discovered which have provided important clues as to the nature of this dysregulation and to its possible treatment with cytokines.2 Thus, in studies of several of the models most closely resembling Crohn’s disease it has been shown that production of large amounts of Th1-type cytokines—for example, interferon γ, by T cells is a major and ess…

Treatment of T Cell-Dependent Experimental Colitis in SCID Mice by Local Administration of an Adenovirus Expressing IL-18 Antisense mRNA

Abstract Recent studies have shown that IL-18, a pleiotropic cytokine that augments IFN-γ production, is produced by intestinal epithelial cells and lamina propria cells from patients with Crohn’s disease. In this study, we show that IL-18 is strongly expressed by intestinal epithelial cells in a murine model of Crohn’s disease induced by transfer of CD62L+CD4+ T cells into SCID mice. To specifically down-regulate IL-18 expression in this model, we constructed an E1/E3-deleted adenovirus expressing IL-18 antisense mRNA, denoted Ad-asIL-18, and demonstrated the capacity of such a vector to down-regulate IL-18 expression in colon-derived DLD-1 cells and RAW264.7 macrophages. Local administrat…

Cutting Edge: TGF-β Induces a Regulatory Phenotype in CD4+CD25− T Cells through Foxp3 Induction and Down-Regulation of Smad7

Abstract CD4+CD25+ regulatory cells are a subpopulation of T lymphocytes of thymic origin. However, recent data suggest an alternative commitment of regulatory T cells in the periphery, although the precise mechanism is unknown. In the present work, we demonstrate that TGF-β is able to induce Foxp3 expression and subsequently a regulatory phenotype in CD4+CD25− peripheral murine T cells. Similarly, TGF-β induced Foxp3 in human CD4+CD25− T cells. Moreover, we show that the inhibitory Smad7 protein that is normally induced by TGF-β and limits TGF-β signaling, is strongly down-regulated by Foxp3 at the transcriptional level. Foxp3-mediated down-regulation of Smad7 subsequently rendered CD4+CD2…

TGF-β Suppresses Tumor Progression in Colon Cancer by Inhibition of IL-6 trans-Signaling

Alterations of TGF-beta signaling have been described in colorectal cancer, although the molecular consequences are largely unknown. By using transgenic mice overexpressing TGF-beta or a dominant-negative TGF-betaRII, we demonstrate that TGF-beta signaling in tumor infiltrating T lymphocytes controls the growth of dysplastic epithelial cells in experimental colorectal cancer, as determined by histology and a novel system for high-resolution chromoendoscopy. At the molecular level, TGF-beta signaling in T cells regulated STAT-3 activation in tumor cells via IL-6. IL-6 signaling required tumor cell-derived soluble IL-6R rather than membrane bound IL-6R and suppression of such TGF-beta-depende…

Fragebogen zur Messung der psychosozialen Belastungen bei chronisch-entzündlichen Darmerkrankungen (FBCED): Konstruktion und Evaluation

The "Inflammatory Bowel Disease Stress Inventory (IBD-SI)" measures a broad spectrum of disease-specific every day stress factors in patients with inflammatory bowel disease (IBD). Based on a multistage factor analysis on 300 CED patients, the final form comprises 32 items assigned to 8 different domains: depressed mood, doctor-patient relationship, anxiety about loss of bowel control, occupational impairment, worries about deteriovation, stress from medical treatment, impairment of sexuality/partnership and physical complaints. In addition, a global stress score can be obtained. The reliability values of the 8 subscales with 4 items each indicate a good internal consistency (alpha = 0.76 -…

Colonoscopy for screening of colorectal cancer. What are the news? Review

The goal of every routine endoscopy in the gut is the early diagnosis of malignant and premalignant changes of the mucosa. Chromo- and magnifying endoscopes are exciting new tools and offer detailed analysis of the colonic mucosal surface and pit pattern architecture. This review summarizes recent advances in endoscopic characterization of colorectal lesions using magnification endoscopy and chromoendoscopy. Surface analysis of the colon using chromoendoscopy allows a prediction between nonneoplastic and neoplastic lesions with high specificity. The precise delineation of the borders and a more detailed macroscopic analysis of the lesions are further advantages. In particular, flat adenomas…

Angiogenesis, immune system and growth factors: new targets in colorectal cancer therapy.

Colorectal cancer is the second most common malignant human neoplasia. Over recent years, many efforts have been performed in order to develop and improve therapeutic protocols, and many advances have been accomplished in both the field of adjuvant and palliative therapy. Most of the chemotherapic agents currently used in the clinical setting are the products of decades of research aimed at inhibiting the uncontrolled growth of dysplastic cells. However, new frontiers in this field have recently been opened, with the identification of key molecules involved in physiologic mechanisms that are of fundamental importance for cancer development and progression. Tumor-induced angiogenesis, the ca…

Involvement of IL-6 in the Pathogenesis of Inflammatory Bowel Disease and Colon Cancer

Inflammatory bowel disease (IBD), which consists of Crohn’s disease and ulcerative colitis, is defined as a chronic inflammation of the gastrointestinbal tract. The etiopathogenetic mechanisms underlying the development of IBD are still not completely understood, and the therapeutic strategies used thus far have been limited to mostly evidence-based principles. There is growing evidence that the pro-inflammatory cytokine interleukin (IL)-6 plays a crucial part in the uncontrolled intestinal inflammatory process, which is a main characteristic of IBD. There is elevated production of IL-6 and its soluble receptor (sIL-6R) by intestinal macrophages and CD4+ T-cells. The increased formation of …

Functional and Molecular Imaging with Confocal Laser Endomicroscopy

In vivo functional and molecular imaging is an emerging new field in gastroenterology. Ex vivo histopathological examination of tissue specimens offers a snapshot view into the tissue, capturing the moment at which the biopsy has been taken. The specimen is subjected to the fixation and staining process, making it prone to artefact. In vivo imaging with confocal endomicroscopy, however, offers the possibility of dynamic monitoring of the living tissue without the need for fixation. To our current knowledge, staining with intravenous fluorescein sodium or topical acriflavine hydrochloride does not alter tissue properties in a way that could influence biological processes. Therefore, with con…

Mycophenolate mofetil for treatment of active inflammatory bowel disease. Clinical and immunological studies.

Novel Signal Transduction Pathways: Analysis of STAT-3 and Rac-1 Signaling in Inflammatory Bowel Disease

Although the precise etiology of inflammatory bowel disease still remains unclear, considerable progress has been made in the identification of novel signal transduction pathways that elucidate the immunopathogenesis involved in the perpetuation of the inflammatory process. Augmented T cell resistance against apoptosis is regarded as a pivotal factor in the pathogenesis, as it impairs mucosal homeostasis and leads to unrestrained accumulation of activated T cells, which subsequently lead to the amplification of the inflammatory response. Therefore novel therapeutic strategies aim at restoring mucosal T cell susceptibility to apoptosis through targeting of signal transduction pathways that a…

Zur Immunpathogenese der chronisch entz�ndlichen Darmerkrankungen

Die chronisch entzundlichen Darmerkrankungen (CED: Morbus Crohn, Colitis ulcerosa) sind durch schubartig verlaufende destruierende Entzundungsreaktionen der Darmschleimhaut gekennzeichnet. In den letzten Jahren konnte gezeigt werden, das bei diesen Erkrankungen eine pathologische Aktivierung des intestinalen Immunsystems durch mucosale Antigene auftritt. In der chronischen Phase der Erkankungen sowie bei der Entwicklung postoperativer Rezidive sind Anderungen in der Zellmigration sowie der Cytokinproduktion intestinaler Zellen wahrscheinlich von entscheidender Bedeutung. Basierend auf diesen neuen pathogenetischen Erkenntnissen werden zur Zeit innovative klinische Behandlungsstrategien gete…

In vivo confocal laser laparoscopy allows real time subsurface microscopy in animal models of liver disease.

Background/Aims Histopathology is essential in the diagnostic workup of most liver diseases. However, biopsy sampling might carry risks, is subject to sampling error, and does not provide dynamic tissue imaging. Therefore a newly developed miniaturised confocal probe was evaluated for in vivo microscopic imaging in rodent models of human liver diseases. Methods The handheld laparoscopy probe used a 488nm single line laser for fluorophore excitation. Optical slice thickness was 7μm, lateral resolution 0.7μm. Imaging depth was 0–250μm below the tissue surface. Imaging using different fluorescent staining protocols was performed in healthy mice, IFNγ- and IL-12-induced hepatitis, after bile du…

Methylene blue-aided chromoendoscopy for the detection of intraepithelial neoplasia and colon cancer in ulcerative colitis.

Timely diagnosis of intraepithelial neoplasias (IN) and colitis-associated colon carcinomas (CRC) is crucially important for the treatment of ulcerative colitis (UC). We performed a randomized, controlled trial to test whether chromoendoscopy (CE) might facilitate early detection of IN and CRC in UC.A total of 263 patients with long-standing UC (or=8 years) were screened for potential inclusion in the study, 165 of whom were randomized at a 1:1 ratio to undergo conventional colonoscopy or colonoscopy with CE using 0.1% methylene blue. Five mucosal biopsy specimens were taken every 10 cm between the rectum and cecum. Circumscript lesions in the colon were evaluated according to a modified pi…

Protection from lethal septic peritonitis by neutralizing the biological function of interleukin 27

The immune response to bacterial infections must be tightly controlled to guarantee pathogen elimination while preventing tissue damage by uncontrolled inflammation. Here, we demonstrate a key role of interleukin (IL)-27 in regulating this critical balance. IL-27 was rapidly induced during murine experimental peritonitis induced by cecal ligation and puncture (CLP). Furthermore, mice deficient for the EBI3 subunit of IL-27 were resistant to CLP-induced septic peritonitis as compared with wild-type controls, and this effect could be suppressed by injection of recombinant single-chain IL-27. EBI3−/− mice displayed significantly enhanced neutrophil migration and oxidative burst capacity during…

STAT3 links IL-22 signaling in intestinal epithelial cells to mucosal wound healing.

Signal transducer and activator of transcription (STAT) 3 is a pleiotropic transcription factor with important functions in cytokine signaling in a variety of tissues. However, the role of STAT3 in the intestinal epithelium is not well understood. We demonstrate that development of colonic inflammation is associated with the induction of STAT3 activity in intestinal epithelial cells (IECs). Studies in genetically engineered mice showed that epithelial STAT3 activation in dextran sodium sulfate colitis is dependent on interleukin (IL)-22 rather than IL-6. IL-22 was secreted by colonic CD11c+ cells in response to Toll-like receptor stimulation. Conditional knockout mice with an IEC-specific d…

Détection du cancer colorectal et des lésions prémalignes par chromoscopie et endoscopie à optique grossissante

En Europe, le cancer colorectal (CCC) est la seconde cause de mortalite consecutive a une pathologie tumorale [1]. En depit des techniques chirurgicales eprouvees et des nouvelles formes de chimiotherapie, les tumeurs invasives, a l’exception des stades du debut, conservent encore a 5 ans des resultats de survie decevants, de l’ordre de 70 % avec envahissement metastatique proximal et seulement 6% en cas de metastase a distance [1]. Les taux de survie et de mortalite du CCR peuvent etre ameliores par une detection de cancer au debut a un stade preclinique. Par consequent, l’incidence du CCR peut etre reduite grâce a une detection endoscopique de lesions premalignes ou de cancers au debut. L…

Tumor necrosis factor-receptor 2 is up-regulated on lamina propria T cells in Crohn's disease and promotes experimental colitis in vivo

Tumor necrosis factor (TNF) plays a pivotal role in the pathogenesis of Crohn's disease (CD). However, little is known about the role of TNF receptors (TNF-R) in this disease. Here, we found that TNF-R2 (in contrast to TNF-R1) was significantly up-regulated on lamina propria and peripheral blood T cells in CD compared to control patients. To directly test the functional role of TNF-R2 in Th1-mediated experimental colitis in vivo, we took advantage of transgenic animals overexpressing TNF-R2 in T cells. Reconstitution of SCID mice with CD4+ CD62L+ T cells from TNF-R2 transgenic mice led to an earlier wasting syndrome, a more severe colitis and augmented Th1 cytokine production than reconstit…

Chromoendoscopy: An Evolving Standard in Surveillance for Ulcerative Colitis

EBV-Induced Gene 3 Transcription Is Induced by TLR Signaling in Primary Dendritic Cells via NF-κB Activation

Abstract The EBV-induced gene 3 (EBI3) is expressed in dendritic cells (DCs) and part of the cytokine IL-27 that controls Th cell development. However, its regulated expression in DCs is poorly understood. In the present study we demonstrate that EBI3 is expressed in splenic CD8−, CD8+, and plasmacytoid DC subsets and is induced upon TLR signaling. Cloning and functional analysis of the EBI3 promoter using in vivo footprinting and mutagenesis showed that stimulation via TLR2, TLR4, and TLR9 transactivated the promoter in primary DCs via NF-κB and Ets binding sites at −90 and −73 bp upstream of the transcriptional start site, respectively. Furthermore, we observed that NF-κB p50/p65 and PU.1…

IL-21 regulates experimental colitis by modulating the balance between Treg and Th17 cells

Regulatory T (T(reg)) cells play a key role in the maintenance of the immune system homeostasis. T(reg) cells can be generated in the periphery under control of TGF-beta, a cytokine involved in the negative control of the immune system. However, TGF-beta cooperates with IL-6 in the generation of Th17 cells, a novel class of effector cells involved in numerous inflammatory diseases, including colitis. Therefore, TGF-beta emerges as a mediator of both anti-inflammatory and pro-inflammatory processes, depending on the local cytokine milieu. Here we demonstrate that IL-21, a type-1 cytokine produced by T cells and involved in the pathogenesis of immune-mediated diseases, prevents the TGF-beta-d…

The T-box transcription factor eomesodermin controls CD8 T cell activity and lymph node metastasis in human colorectal cancer.

An efficient cytolytic T cell function is essential for immune mediated rejection of colorectal cancer. However, the molecular mechanisms driving T cell mediated cancer rejection are still poorly understood. Here, we assessed the relevance of the T-box transcription factor eomesodermin in colorectal cancer. METHODS/ RESULTS: By analysing tissue probes from 88 different colorectal tumours, a significant (p0.02) inverse correlation between eomesodermin expression in colorectal cancers and the presence of lymph node metastases could be shown, whereas no such correlation was noted for the master transcription factor of regulatory T cells, FoxP3 and CD8 alpha expression. To evaluate whether this…

NF-κB in inflammatory bowel disease

Apart from genetic and environmental factors, the mucosal immune system of the gut plays a central role in the pathogenesis of inflammatory bowel disease (IBD). In the healthy gut, the mucosal immune system ensures the balance between pro- and anti-inflammatory mediators and thereby allows an effective defence against luminal pathogens but at the same time prevents an overwhelming immune reaction directed against the huge amount of harmless luminal antigens (for example, components of food or nonpathological bacteria). In both entities of IBD (Crohn's disease and ulcerative colitis) this immunological balance is severely impaired and shifted towards the pro-inflammatory side. The chronic mu…

Five cases of de novo inflammatory bowel disease after orthotopic liver transplantation.

Immunosuppression is currently the treatment of choice for severe inflammatory bowel disease (IBD). Thus, it was anticipated that the course of preexisting IBD should improve after orthotopic liver transplantation (OLT). Despite sufficient allograft immunosuppressive therapy, however, exacerbation of IBD or the development of de novo IBD after OLT were described in some cases, primarily in patients transplanted for end-stage primary sclerosing cholangitis (PSC). In addition, the development of de novo IBD in patients undergoing OLT for indications other than PSC was described. Evaluating our collective of 314 liver transplanted patients we found five patients transplanted for various indica…

Externalized decondensed neutrophil chromatin occludes pancreatic ducts and drives pancreatitis

Ductal occlusion has been postulated to precipitate focal pancreatic inflammation, while the nature of the primary occluding agents has remained elusive. Neutrophils make use of histone citrullination by peptidyl arginine deiminase-4 (PADI4) in contact to particulate agents to extrude decondensed chromatin as neutrophil extracellular traps (NETs). In high cellular density, NETs form macroscopically visible aggregates. Here we show that such aggregates form inside pancreatic ducts in humans and mice occluding pancreatic ducts and thereby driving pancreatic inflammation. Experimental models indicate that PADI4 is critical for intraductal aggregate formation and that PADI4-deficiency abrogates…

Confocal laser endoscopy: new approach to the early diagnosis of tumors of the esophagus and stomach

The prognosis of malignancies of the upper gastrointestinal tract is poor if early diagnosis is missed. Therefore, a rapid in vivo diagnosis of early cancer and premalignant lesions is highly important for succesful therapy. Confocal laser endomicroscopy allows in vivo cellular and subcellular imaging at high resolutions during ongoing endoscopy. The first studies and clinical observations suggest a major role of this novel technique in the in vivo diagnosis of cancer of the esophagus and stomach and of precursor conditions, such as Barrett’s esophagus, intestinal metaplasia of the stomach and Helicobacter pylori infection. Based on recent experiences from animal models, future studies wil…

Confocal laser endomicroscopy for gastrointestinal diseases.

Confocal laser endomicroscopy enables in vivo microscopy of the mucosal layer of the gastrointestinal tract with subcellular resolution during ongoing endoscopy. Endomicroscopy opens the door to immediate tissue and vessel analysis. Different types of diseases can be diagnosed with optical surface and subsurface analysis. Analysis of the in vivo microarchitecture can be used for targeting biopsies to relevant areas, and subsurface imaging can unmask microscopic diseases or bacterial infection. Molecular imaging is becoming feasible, which will enable new indications in gastrointestinal endoscopy. This article reviews the current and rapidly expanding clinical data on endomicroscopy and give…

VEGF receptor signaling links inflammation and tumorigenesis in colitis-associated cancer.

Inflammation drives expression of VEGFR2, which is expressed on and drives growth of tumor cells in colitis-associated cancer.

Designer Thiopurine-analogues for Optimised Immunosuppression in Inflammatory Bowel Diseases.

Background and Aims: The clinical use of azathioprine and 6-mercaptopurine is limited by their delayed onset of action and potential side effects such as myelosuppression and hepatotoxicity. As these drugs specifically target the Vav1/Rac1 signalling pathway in T lamina propria lymphocytes via their metabolite 6-thio-GTP, we studied expression and optimised suppression of this pathway in inflammatory bowel diseases [IBD]. Methods: Rac1 and Vav1 expressions were analysed in mucosal immune cells in IBD patients. Targeted molecular modelling of the 6-thio-GTP molecule was performed to optimise Rac1 blockade; 44 modified designer thiopurine-analogues were tested for apoptosis induction, potenti…

IL-6 Signaling Promotes Tumor Growth in Colorectal Cancer

Recent investigations support an important role for TGF-beta in the development of colorectal cancer. However, the molecular consequences of TGF-beta signaling in the colon remains incompletely understood. In a recent study in Immunity, we analyzed the role of TGF-beta in a murine model of colon cancer. Using transgenic mice overexpressing TGF-beta or a dominant negative TGF-beta receptor II under control of the CD2 minigene, we show that TGF-beta signaling in tumor infiltrating T lymphocytes regulates the growth of dysplastic colon epithelial cells, as determined by histology and a novel system for high resolution chromoendoscopy in vivo. At the molecular level, TGF-beta signaling in T cel…

Identification and characterization of a novel Ets-2-related nuclear complex implicated in the activation of the human interleukin-12 p40 gene promoter.

Interleukin-12 (IL-12) is a proinflammatory cytokine produced by antigen-presenting cells in response to many microbial infections. IL-12 plays an important role in the generation of T helper type-1 cells, which favor cell-mediated immune response. IL-12 is composed of two different subunits, p40 and p35, whose expression can be regulated concomitantly or differentially. Monocytic cells, the major producers of IL-12, can be primed by interferon-gamma (IFN-gamma) to produce optimal amounts of IL-12 in response to LPS stimulation as a consequence of bacterial infection. The priming effect is exerted primarily at the transcriptional level on the p40 promoter in conjunction with the effects of …

Exacerbation of ulcerative colitis after rituximab salvage therapy

Background: B-cells are considered to play a pathogenic role in human ulcerative colitis (UC) by producing autoantibodies that cause epithelial cell damage. Here we report on a patient with intractable UC who suffered from a severe exacerbation of UC after salvage therapy with rituximab, a B-cell-depleting anti-CD20-antibody. Methods: A 58-year-old patient with active long-standing UC and unresponsiveness or adverse events to mesalamine, corticosteroids, azathioprine, methotrexate, infliximab, leukapheresis, mycophenolate mofetil, and adalimumab received 375 mg/m2 rituximab. Results: A severe exacerbation of UC activity was noted upon therapy that required hospitalization. Subsequent studie…

Predominant pathogenic role of tumor necrosis factor in experimental colitis in mice

Antibodies to tumor necrosis factor (TNF)-alpha have been recently proposed as effective treatment for patients with Crohn's disease. Here, we analyze the functional role of TNF-alpha in a mouse model of chronic intestinal inflammation induced by the hapten reagent 2,4,6,-trinitrobenzene sulfonic acid (TNBS) that mimics some characteristics of Crohn's disease in humans. Macrophage-enriched lamina propria (LP) mononuclear cells from mice with TNBS-induced colitis produced 10-30-fold higher levels of TNF-alpha mRNA and protein than cells from control mice. When mice with chronic colitis were treated by intraperitoneal injection of antibodies to TNF-alpha, an improvement of both the clinical a…

Chromoendoscopy and Other Novel Imaging Techniques

The newly developed high-resolution and magnification endoscopes offer features that allow more and new mucosal details to be seen. They are commonly used in conjunction with chromoendoscopy. The analysis of mucosal surface details is beginning to resemble histologic examination. More accurate recognition of small flat and depressed neoplastic lesions is possible. Endoscopic prediction of neoplastic and nonneoplastic tissue is possible by analysis of surface architecture of the mucosa, which influences the endoscopic management. For the diagnosis of flat adenomas, chromoendoscopy should be a part of the endoscopist's armamentarium. In inflammatory bowel disease, chromoendoscopy can be used …

Zur Immunpathogenese des Asthma bronchiale

Allergic asthma is a chronic pulmonary disease associated with bronchoconstriction and inflammation. Recent studies have shown that mediator substances and proinflammatory cytokines produced by mast cells, eosinophils and T-lymphocytes appear to be important for the pathogenesis of asthma. These substances contribute both to the initiation and perpetuation of the disease. In particular, it has been shown that allergic asthma is associated with increased TH2 (IL-4, IL-5, IL-13) cytokine production that causes activation of eosinophils and T-cells and production of chemokines (e.g. eotaxin) by pulmonary fibroblasts. Based on recent advances in our understanding of the immunopathogenesis of as…

Acetic acid-guided biopsies after magnifying endoscopy compared with random biopsies in the detection of Barrett's esophagus: a prospective randomized trial with crossover design.

Background In contrast to standard video endoscopy, magnifying endoscopy after local acetic acid application enables recognition of mucosal surface architecture. Objective To investigate the diagnostic yield of magnifying endoscopy with acetic acid–targeted biopsies compared to random, 4-quadrant biopsies. Design Prospective randomized trial (ratio 1:1) with crossover design. Setting Two referral hospitals in Germany. Patients Thirty-one patients with Barrett's esophagus or visible columnar-lined lower esophagus. Interventions Patients were randomized to undergo either standard video endoscopy with 4-quadrant biopsies or magnifying endoscopy in conjunction with acetic acid application. All …

Role of NF-kappaB in immune and inflammatory responses in the gut.

NF-kappaB is a pleiotropic transcription factor with key functions in the intestinal immune system. NF-kappaB family members control transcriptional activity of various promoters of proinflammatory cytokines, cell surface receptors, transcription factors, and adhesion molecules that are involved in intestinal inflammation. The perpetuated activation of NF-kappaB in patients with active inflammatory bowel disease suggests that regulation of NF-kappaB activity is a very attractive target for therapeutic intervention. Such strategies include antioxidants, proteasome inhibitors, inhibition of NF-kappaB by adenoviral I kappaB alpha expression vectors, and antisense DNA targeting of NF-kappaB. Th…

Drug insight: novel small molecules and drugs for immunosuppression.

Gastrointestinal diseases can result from the inadequate or excessive response of the immune system to self or innocuous antigens. Moreover, the physiologic activation of the immune system against non-self antigens is a major clinical problem in liver organ transplantation. At present, many drugs are available that suppress the activation of the immune system, although most of the currently used immunosuppressive drugs lack specificity in terms of their molecular targets and, therefore, have the potential to generate numerous side effects. The advances that have been made in understanding the molecular events that underlie the activation of the immune system have led to the development of a…

Physiological activation of the IgH 3' enhancer in B lineage cells is not blocked by Pax-5.

The mouse 3' enhancer contains a high-affinity binding site for the paired box protein Pax-5. Here, we demonstrate by genomic footprinting that the rat 3' enhancer contains a low-affinity binding site for Pax-5, which is occupied in activated splenic B cells. Thus, binding of Pax-5 to the IgH 3' enhancer appears to be evolutionarily conserved in rodents. Analysis of Pax-5 expression in primary B cells demonstrates that Pax-5 remains expressed after 4 days of lipopolysaccharide (LPS) induction, but is down-regulated in 5-day stimulated cells. Similarly, the expression of Pax-5 is down-regulated in vivo in activated large splenocytes, in contrast to small resting cells. Multimerization of the…

Long-Term Evaluation of a Rat Model of Chronic Cholangitis Resembling Human Primary Sclerosing Cholangitis

Primary sclerosing cholangitis (PSC) is a chronic cholestatic disorder with a presumed autoimmune aetiopathogenesis. We have recently described a novel organ-specific rat model of fibrosing cholangitis induced by intrabiliary administration of the hapten-reagent 2,4,6-trinitrobenzenesulfonic acid (TNBS) with similarities to human PSC. In the present report, we have evaluated the long-term outcome of TNBS-induced cholangitis in this model. Mild stenosis of the common bile duct of female Lewis rats (n = 18) was achieved by subtotal ligation and cholangitis induced by TNBS injection (50 mg/kg) into the dilated bile duct after a second laparotomy. After 8 and 12 months, we found no evidence of …

Epithelial NEMO links innate immunity to chronic intestinal inflammation

Deregulation of intestinal immune responses seems to have a principal function in the pathogenesis of inflammatory bowel disease(1-4). The gut epithelium is critically involved in the maintenance of intestinal immune homeostasis-acting as a physical barrier separating luminal bacteria and immune cells, and also expressing antimicrobial peptides(3,5,6). However, the molecular mechanisms that control this function of gut epithelial cells are poorly understood. Here we show that the transcription factor NF kappa B, a master regulator of pro-inflammatory responses(7,8), functions in gut epithelial cells to control epithelial integrity and the interaction between the mucosal immune system and gu…

Kombiniertes Simulationstraining: Ein neues Kurskonzept trainiert und verbessert das Krisenmanagement der gastroenterologischen Endoskopie

Introduction Crisis management as well as realistic emergency situations can be trained in the new developed simulation workshop "Gastrointestinal Endoscopy and Crisis Resource Management" by combining a full-scale simulator and the Erlanger Endoscopy Trainer. The aim of the current study was to evaluate the efficiency of the newly developed simulation workshop. Methods Endoscopists with more than 12 months experience can train their endoscopic skills and crisis resource management with the help of different simulators. In addition, two different scenarios (GI bleeding with significant blood loss and sedation overdoses) embedded in a realistic surrounding (emergency room) have to be managed…

Liver-primed memory T cells generated under noninflammatory conditions provide anti-infectious immunity.

SummaryDevelopment of CD8+ T cell (CTL) immunity or tolerance is linked to the conditions during T cell priming. Dendritic cells (DCs) matured during inflammation generate effector/memory T cells, whereas immature DCs cause T cell deletion/anergy. We identify a third outcome of T cell priming in absence of inflammation enabled by cross-presenting liver sinusoidal endothelial cells. Such priming generated memory T cells that were spared from deletion by immature DCs. Similar to central memory T cells, liver-primed T cells differentiated into effector CTLs upon antigen re-encounter on matured DCs even after prolonged absence of antigen. Their reactivation required combinatorial signaling thro…

IL-28A Is a Key Regulator of T-Cell–Mediated Liver Injury via the T-Box Transcription Factor T-Bet

Background & Aims: T-cell–mediated fulminant hepatitis is a potentially life-threatening event for which the underlying pathogenic mechanisms are not fully understood. Here, we demonstrate a key regulatory role of IL-28A in T-cell–mediated hepatitis. Methods: We cloned the murine IL-28A gene by reverse-transcription polymerase chain reaction, assessed the effects of recombinant IL-28A, and generated IL-28A–transgenic mice. Results: IL-28A induced TH1 cytokine production by CD4+ T lymphocytes in a T-bet–dependent manner and was up-regulated in a murine model of T-cell–mediated hepatitis upon Con A administration. In vivo, CD4+ T cells from newly created IL-28A–transgenic animals revealed an …

What new endoscopic imaging modalities will become important in the diagnosis of IBD?

Understanding the delayed onset of action of azathioprine in IBD: are we there yet?

In this issue of Gut , Ben-Horin et al propose an innovative explanation for the well known phenomenon of the delayed onset of action of thiopurines ( see page 396 ). They thereby contribute to an improved insight into the exact mode of action of the classic immunosuppressive drug azathioprine.1 Developing azathioprine as an innovative immunosuppressive drug in 1957, Gertrude Elion and George Hitchings laid the basis for the currently utilised concept of steroid-sparing treatment strategies in inflammatory bowel diseases (IBD).2 With regard to the evidence-based immunosuppressive capacity and clinical efficacy of azathioprine in the context of IBD as well as considering the proven long-term…

Collagenous colitis: new diagnostic possibilities with endomicroscopy

Collagenous colitis is a kind of microscopic colitis. It is characterized by chronic watery diarrhea and abdominal pain. The etiology is still unknown. So far, for the diagnose a histological evaluation was necessary with the presence of thickened subepithelial collagneous bands in the lamina propria. A new developed endoscope with a confocal laser allows analysing cellular and subcellular details of the mucosal layer at high resolution in vivo. In this case report we describe for the first time to diagnose collagenous colitis during ongoing colonoscopy by using this confocal endomicroscopy. In a 67 year old female patient with typical symptoms the characteristic histological changes could …

Protein�A immunoadsorption therapy for refractory, mitomycin�C?associated thrombotic microangiopathy

BACKGROUND: Mitomycin C–associated thrombotic microangiopathy (TMA) has a poor prognosis with limited therapeutic options. Most patients die within 4 months of diagnosis due to pulmonary or renal failure. Here, a patient resistant to total plasma exchange (TPE) and immunosuppressive therapy with glucocorticoids, rituximab, vincristine, and splenectomy who was successfully treated with protein A immunoadsorption is described. CASE REPORT: A 29-year-old woman developed a TMA after chemotherapy with mitomycin C. She presented with thrombocytopenia, pulmonary edema, hemolytic anemia with presence of schistocytes, and renal failure. Immediate TPE (>120 times) and immunosuppressive therapy with g…

Mycophenolate mofetil versus azathioprine in patients with chronic active ulcerative colitis: a 12-month pilot study.

Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) of unknown etiology frequently requiring long-term therapy for control of symptoms and prevention of relapse. Azathioprine (AZA) has been shown to be effective and safe in the treatment of chronic active UC. However, the alternatives to treatment with AZA are limited. Our aim was to compare the efficacy and safety of treatment with mycophenolate mofetil (MMF)/prednisolone versus standard immunosuppressive treatment with azathioprine (AZA)/prednisolone in patients with chronic active UC.The study was designed as an open comparison of MMF versus AZA. Twenty-four patients with active UC (Rachmilewitz scoreor =6 points) were randoml…

Blockade of interleukin 6 trans signaling suppresses T-cell resistance against apoptosis in chronic intestinal inflammation: Evidence in Crohn disease and experimental colitis in vivo

The pro-inflammatory cytokine interleukin (IL)-6 (refs. 1-5) can bind to cells lacking the IL-6 receptor (IL-6R) when it forms a complex with the soluble IL-6R (sIL-6R) (trans signaling). Here, we have assessed the contribution of this system to the increased resistance of mucosal T cells against apoptosis in Crohn disease (CD), a chronic inflammatory disease of the gastrointestinal tract. A neutralizing antibody against IL-6R suppressed established experimental colitis in various animal models of CD mediated by type 1 T-helper cells, by inducing apoptosis of lamina propria T cells. Similarly, specific neutralization of sIL-6R in vivo by a newly designed gp130-Fc fusion protein caused suppr…

Ulcerative colitis - How can we improve surveillance colonoscopy?

In vivo imaging of colitis and colon cancer development in mice using high resolution chromoendoscopy

Background: Mouse models of colitis and cancer are indispensable for our understanding of the pathogenesis of these diseases. In the past, mice had to be sacrificed in order to analyse colitis activity and tumour development. We have developed a safe method for high resolution endoscopic monitoring of living mice. Methods: Mice developing colitis or colonic tumours were anaesthetised using avertine and repeatedly examined by endoscopy. A novel miniendoscope (1.9 mm outer diameter), denoted Coloview, was introduced via the anus and the colon was carefully insufflated with an air pump before analysis of the colonic mucosa. An extra working channel allowed the introduction of biopsy forceps or…

Zytokine bei chronisch-entzündlichen Darmerkrankungen

In Vivo Molecular Imaging of Somatostatin Receptors in Pancreatic Islet Cells and Neuroendocrine Tumors by Miniaturized Confocal Laser-Scanning Fluorescence Microscopy

The aim of the study was to evaluate real time in vivo molecular imaging of somatostatin receptors (sstrs) using a handheld miniaturized confocal laser scan microscope (CLM) in conjunction with fluorescein-labeled octreotate (OcF) in healthy mice and murine models of neuroendocrine tumors. For CLM a small rigid probe (diameter 7 mm) with an integrated single line laser (488 nm) was used (optical slice thickness 7 μm; lateral resolution 0.7 μm). OcF was synthesized via Fmoc solid-phase peptide synthesis and purified by HPLC showing high-affinity binding to the sstr2 (IC50 6.2 nmol). For in vitro evaluation, rat and human pancreatic cancer cells were used and characterized with respect to its…

Local blockade of IL-6R signaling induces lung CD4+ T cell apoptosis in a murine model of asthma via regulatory T cells.

We previously reported high levels of the soluble form of the IL-6R (sIL-6R) in the airways of asthmatic subjects. Here, we analyzed the IL-6R effects on Th2 cell survival in the lung by locally antagonizing sIL-6R-mediated trans-signaling with a designer fusion protein (gp130-Fc) as well as IL-6R signaling with an antibody against the gp80 unit of the IL-6R (alphaIL-6R) in a murine model of asthma after ovalbumin peptide (OVA) sensitization and challenge. Blockade of the sIL-6R led to a significant decrease in inflammatory cells by an apoptosis-independent mechanism. In contrast, local treatment with alphaIL-6R antibodies that also block signaling via the membrane-bound IL-6R (mIL-6R) led …

IL-2 receptor beta-chain signaling controls immunosuppressive CD4+ T cells in the draining lymph nodes and lung during allergic airway inflammation in vivo.

Abstract IL-2 influences both survival and differentiation of CD4+ T effector and regulatory T cells. We studied the effect of i.n. administration of Abs against the α- and the β-chains of the IL-2R in a murine model of allergic asthma. Blockade of the β- but not the α-chain of the IL-2R after allergen challenge led to a significant reduction of airway hyperresponsiveness. Although both treatments led to reduction of lung inflammation, IL-2 signaling, STAT-5 phosphorylation, and Th2-type cytokine production (IL-4 and IL-5) by lung T cells, IL-13 production and CD4+ T cell survival were solely inhibited by the blockade of the IL-2R β-chain. Moreover, local blockade of the common IL-2R/IL-15R…

Perspectives of Chromo and Magnifying Endoscopy

The goal of every routine endoscopy in the gut is the early diagnosis of malignant and premalignant changes of the mucosa. Chromo- and magnifying endoscopes are exciting new tools and offer detailed analysis of the colonic mucosal surface and pit pattern architecture. This review summarizes recent advances in endoscopic characterization of colorectal lesions using magnification endoscopy and chromoendoscopy. Surface analysis of the colon using chromoendoscopy allows a prediction between non-neoplastic and neoplastic lesions with high specificity. The precise delineation of the borders and a more detailed macroscopic analysis of the lesions are further advantages. In particular, flat adenoma…

Immunotherapeutic approaches to inflammatory bowel diseases

For a long time corticosteroids, aminosalicylic acid preparations and antibiotics have represented the principal approaches in evidence-based drug therapy for chronic inflammatory bowel diseases (IBD), e.g., Crohn's disease (CD) and ulcerative colitis (UC), and are able to suppress disease activity in most cases. However, there are cases that do not respond to conventional drug therapy or remain dependent on high doses of steroids associated with severe side effects in the long run. It is generally accepted now that IBD has an immunological basis and results from a hyperresponsive state of the intestinal immune system. Although the primary etiological defect respectively immunogenic agent s…

Local administration of antisense phosphorothioate oligonucleotides to the p65 subunit of NF-kappa B abrogates established experimental colitis in mice.

Chronic intestinal inflammation induced by 2,4,6,-trinitrobenzene sulfonic acid (TNBS) is characterized by a transmural granulomatous colitis that mimics some characteristics of human Crohn's disease. Here, we show that the transcription factor NF-kappa B p65 was strongly activated in TNBS-induced colitis and in colitis of interleukin-10-deficient mice. Local administration of p65 antisense phosphorothioate oligonucleotides abrogated clinical and histological signs of colitis and was more effective in treating TNBS-induced colitis than single or daily administration of glucocorticoids. The data provide direct evidence for the central importance of p65 in chronic intestinal inflammation and …

Reciprocal IFN-gamma and TGF-beta responses regulate the occurrence of mucosal inflammation.

The above new findings concerning the immunological mechanisms governing mucosa, immune responses and oral tolerance in TCR-transgenic mice, as well as those operative in mice with experimental colitis, greatly expand our understanding of the processes that normally control mucosal inflammation and possibly other types of inflammation as well (Fig. 1). They indicate that, in the nondiseased mouse, ingested proteins evoke a Th1-cell (IFN-gamma) response in the mucosal follicles that is quickly counter-regulated by induction of T-cell anergy/deletion, if this Th1-cell response is inhibited (experimentally by anti-IL-12), TGF beta-producing cells appear, and these are capable of active immune …

Disruption of T helper 2-immune responses in Epstein–Barr virus-induced gene 3-deficient mice

Epstein–Barr virus-induced gene 3 (EBI3) is a widely expressed IL-12p40-related protein that associates as a heterodimer with either IL-12p35 or an IL-12p35 homologue, p28, to create a new cytokine (IL-27). To define the function of EBI3in vivo, we generated knockout mice in which theebi3gene was targeted by homologous recombination. EBI3−/−mice exhibited normal numbers of both naive and mature CD4+and CD8+T cells and B cells, but markedly decreased numbers of invariant natural killer T cells (iNKT) as defined by staining with an α-galactosylceramide (αGalCer)-loaded CD1d-tetramer. iNKT cells from EBI3−/−mice exhibited decreased IL-4 and, to a lesser extent, IFN-γ production after αGalCer s…

Simultaneous confocal laser endomicroscopy and chromoendoscopy with topical cresyl violet

Background Confocal laser endomicroscopy (CLE) has been shown to reliably predict histology during ongoing endoscopy. To unmask lesions for CLE, chromoendoscopy has been mandated. Usually fluorescein then serves as a contrast agent for CLE, but it does not allow direct nuclear visualization, must be injected, leads to a transient skin discoloration, and may have allergic side effects. Objective To establish a single topical dye, cresyl violet (CV), for simultaneous chromoendoscopy and in vivo CLE of the lower GI tract. Design Animal preclinical study, prospective clinical trial. Setting Mainz University Clinic (tertiary care center). Patients, Methods, and Interventions To establish the sta…

Stepwise Regulation of TH1 Responses in Autoimmunity: Il-12-Related Cytokines and Their Receptors

Interleukin (IL)-12 is a key cytokine of cell-mediated immune responses. Until recently, IL-12 was believed to be unique in its ability to induce the differentiation of naive T cells toward the TH1 phenotype and in its pathogenic activity, as shown in various disease models including inflammatory bowel disease. However, recently, 2 additional cytokines closely related to IL-12, IL-23 and IL-27, were discovered. Until then, the role of IL-12 was overestimated because it was believed that the p40 subunit was unique to IL-12. The discovery that IL-12 shares p40 with IL-23 and that IL-23 but not IL-12 is essential in models of chronic inflammation and autoimmunity led to a model in which IL-12 …

Impaired Th1 responses in mice deficient in Epstein-Barr virus-induced gene 3 and challenged with physiological doses of Leishmania major.

Protection against Leishmania major is dependent on IL-12 release from L. major-infected dendritic cells (DC) that induce IFN-gamma-producing Th1/Tc1 cells. IL-27, a novel member of the IL-12 family, is a heterodimer composed of p28 and IL-12p40-related Epstein-Barr virus-induced gene 3 (EBI3), and was shown to be produced by DC. In this study, we utilized EBI3-deficient mice to investigate the role of IL-27 in leishmaniasis using physiological low-dose infections that mimic natural transmissions. Lesions in EBI3(-/-) mice were significantly larger between weeks 3 and 10 post infection, reaching up to approximately threefold increased lesion volumes compared to wild types. In parallel, derm…

Akutes Lungenversagen bei Malaria tropica in der Schwangerschaft: Erfolgreiche Behandlung durch extrakorporale CO2-Elimination

Eine 32jahrige Schwangere (26. Schwangerschaftswoche) erkrankte am 6. Tag nach der Ruckkehr von einem Indonesienaufenthalt mit Fieber bis 42°C, hamolytischer Anamie (Hamoglobin 7,6 g/dl) und Thrombozytopenie (7000/µl). Es wurde eine Malaria diagnostiziert; eine Prophylaxe hatte sie nicht durchgefuhrt. Unter antiparasitarer Chemotherapie mit Chloroquin, Chinin und Pyrimethamin kam es zur Elimination der Parasiten (Makrogametozyten von Plasmodium falciparum) aus dem peripheren Blut. Im weiteren Verlauf entwickelte sich jedoch ein progredientes Lungenversagen und eine therapierefraktare Pneumonie, die zu einem »adult respiratory distress syndrome« (ARDS; Morel-Stadium 4) fuhrten. Bei drohendem…

Cutting Edge: A Key Pathogenic Role of IL-27 in T Cell- Mediated Hepatitis

Abstract The signals driving T cell activation in T cell-mediated fulminant hepatitis are not fully understood. In this study, we identify the cytokine IL-27p28/EBI3 as a major pathogenic factor in the ConA model of T cell-mediated hepatitis. We found an up-regulation of hepatic EBI3 and p28 expression and augmented levels of IL-27 in wild-type mice after ConA administration, suggesting a potential pathogenic role of this cytokine in ConA hepatitis. Consistently, IL-27 EBI3-deficient mice were almost completely protected from ConA-induced liver damage. Such protection was associated with reduced levels of IFN-γ and its signaling proteins pSTAT-1 and T-bet. Finally, in vivo blockade of IL-27…

Endomicroscopy is born—do we still need the pathologist?

Confocal laser endoscopy for diagnosing intraepithelial neoplasias and colorectal cancer in vivo

Background & Aims: Aconfocal laser endoscopy system has recently been developed that may allow subsurface imaging of living cells in colonic tissue in vivo. The aim of the present study was to assess its potential for prediction of histology during screening colonoscopy for colorectal cancer. Methods: Twenty-seven patients underwent colonoscopy with the confocal endoscope using acriflavine hydrochloride or fluorescein sodium with blue laser illumination. Furthermore, 42 patients underwent colonoscopy with this system using fluorescein sodium. Standardized locations and circumscript lesions were examined by confocal imaging before taking biopsy specimens. Confocal images were graded accordin…

Local administration of antisense phosphorothioate oligonucleotides to the c-kit ligand, stem cell factor, suppresses airway inflammation and IL-4 production in a murine model of asthma.

Abstract Background: The c-kit ligand, stem cell factor (SCF), is an important activating and chemotactic factor for both mast cells and eosinophils. These cells are known to play a fundamental role in the pathogenesis of asthma. Objective: Our goal was to analyze the functional role of SCF in the pathogenesis of asthma. Methods: The expression of SCF was targeted in fibroblasts, epithelial cells, and locally in a murine model of asthma in mice induced by ovalbumin sensitization with an antisense DNA strategy. Results: We could suppress SCF expression in NIH 3T3 fibroblasts and SP1 epithelial cells by a specific antisense phosphorothioate oligonucleotide overlapping the translation start si…

Transforming growth factor   induced FoxP3+ regulatory T cells suppress Th1 mediated experimental colitis

Background and aims: The imbalance between effector and regulatory T cells plays a central role in the pathogenesis of inflammatory bowel diseases. In addition to the thymus, CD4+CD25+ regulatory T cells can be induced in the periphery from a population of CD25− T cells by treatment with transforming growth factor β (TGF-β). Here, we analysed the in vivo function of TGF-β induced regulatory T (Ti-Treg) cells in experimental colitis. Methods: Ti-Treg cells were generated in cell culture in the presence or absence of TGF-β and tested for their regulatory potential in experimental colitis using the CD4+CD62L+ T cell transfer model. Results: Ti-Treg cells significantly suppressed Th1 mediated c…

Selective targeting of activated T cells in chronic intestinal inflammation

Programmed cell death (apoptosis) has been implicated in normal biological processes as well as in the pathology of human diseases.1 The characterisation of genes involved in apoptosis has been pursued intensively and led to the identification of two major classes of genes: the bcl-2 family and the caspase family. Caspases are proteases that cleave their target substrates at specific peptide sequences and during apoptosis the activation of caspases takes place in a cascade fashion, leading to nuclear engulfment and cell death. Thus, caspases represent key functional components of the apoptosis pathway in human cells. Resistance against apoptosis is a key phenomenon in various chronic inflam…

Mouse models of inflammatory bowel disease.

Animal models of intestinal inflammation are indispensable for our understanding of the pathogenesis of Crohn disease and Ulcerative colitis, the idiopathic forms of inflammatory bowel disease in humans. The clinical appearance of human IBD is heterogeneous, a fact that is also reflected by the steadily increasing number of mouse strains displaying IBD like intestinal alterations. The analysis of these models together with genetic studies in humans greatly enhanced our insights into immunoregulatory processes in the gut and led to the generally accepted hypothesis that a deregulated immune response against components of the intestinal microbiota is critically involved in IBD pathophysiology…

Allergen-induced IgE-dependent gut inflammation in a human PBMC-engrafted murine model of allergy.

Background Humanized murine models comprise a new tool to analyze novel therapeutic strategies for allergic diseases of the intestine. Objective In this study we developed a human PBMC–engrafted murine model of allergen-driven gut inflammation and analyzed the underlying immunologic mechanisms. Methods Nonobese diabetic (NOD)– scid -γc −/− mice were injected intraperitoneally with human PBMCs from allergic donors together with the respective allergen or not. Three weeks later, mice were challenged with the allergen orally or rectally, and gut inflammation was monitored with a high-resolution video miniendoscopic system, as well as histologically. Results Using the aeroallergens birch or gra…

Azathioprine in inflammatory bowel disease: improved molecular insights and resulting clinical implications

Azathioprine and 6-mercaptopurine represent important first-line immunosuppressive drugs in the treatment of inflammatory bowel disease. Owing to 45 years of clinical experience with thiopurines in inflammatory bowel disease, there currently exist strong data from numerous clinical trials and meta-analyses, which clearly document the therapeutic efficacy of azathioprine and 6-mercaptopurine in the treatment of inflammatory bowel disease. However, the exact molecular mechanism of action of these drugs was insufficiently understood for a long time. During the last few years, important new insights into the intracellular effects of azathioprine have been gained and thiopurines have been identi…

Responses to self and non-self intestinal microflora in health and inflammatory bowel disease.

Confocal laser endomicroscopy is a new imaging modality for recognition of intramucosal bacteria in inflammatory bowel disease in vivo.

International audience; BACKGROUND AND OBJECTIVES: Interaction of bacteria with the immune system within the intestinal mucosa plays a key role in the pathogenesis of inflammatory bowel disease (IBD). The aim of the current study was to develop a fluorescein-aided confocal laser endomicroscopy (CLE) method to visualise intramucosal enteric bacteria in vivo and to determine the involved mucosal area in the colon and ileum in patients with ulcerative colitis (UC) and Crohn's disease (CD). METHODS: Initially, E coli strains expressing enhanced green fluorescent protein (pEGFP) were endomicroscopically imaged in mice. In addition, ex vivo and in vivo imaging of fluorescent human enteric bacteri…

Ultrastructural causes of rupture of hand tendons in patients with rheumatoid arthritis. A transmission and scanning electron microscopic study.

To identify the cause of rupture of hand tendons in patients with rheumatoid arthritis, we studied the underlying ultrastructural changes of the collagenous fibril systems. Samples of the flexor digitorum superficialis (n = 12) and the extensor digitorum communis (n = 20) were taken during tenosynovectomy. Tendons dissected at necropsy (n = 30) served as controls. Specimens were analysed by transmission and scanning electron microscopy. Interfibrillar dysplastic fibrils, "Luse bodies", and intracellular collagen were found in rheumatoid tissues. The diameters of collagen fibrils were significantly reduced compared with the control group (p0.01). The duration of the disease usually correlate…

Predominant role of NF-kappa B p65 in the pathogenesis of chronic intestinal inflammation.

NF-kappa B is a key transcription factor of lymphocytes and macrophages with important regulatory functions in the immune system and inflammatory processes. These functions are at least partially based on its ability to regulate the promoters of a variety of genes whose products, such as cytokines, adhesion molecules and acute phase proteins, are critical for inflammatory processes. In the present study, we describe a method to treat established intestinal inflammation by local or systemic application of antisense phosphorothioate oligonucleotides targeting the translation start site of the p65 subunit of NF-kappa B. Specific downregulation of p65 by administration of antisense phosphorothi…

Translating Inflammatory Bowel Disease Research into Clinical Medicine

Recent studies have provided important insights into the pathogenesis of inflammatory bowel disease (IBD). The development of new therapeutic agents has been triggered by basic research and studies in mouse models of IBD. It is expected that improved translational research will lead to optimized therapy and new individualized treatment options.

Mucosal immunity in Crohn's disease.

Confocal Laser Endomicroscopy: Technical Advances and Clinical Applications

Since its introduction in 2004, confocal laser endomicroscopy (CLE) has emerged as a valuable tool for gastrointestinal endoscopic imaging. Endomicroscopy enables the endoscopist to obtain real time in vivo histology during ongoing endoscopy thereby creating “optical biopsies.” To date, numerous studies have shown potential applications of endomicroscopy in the clinical setting, including in vivo diagnosis of esophageal squamous cell carcinoma, Barrett’s esophagus, celiac disease, and colonic polyps. Moreover, recent data suggest the potential application of endomicroscopy in the field of molecular imaging. Additionally, in recent months new applications and developments in the field of con…

Tumour necrosis factor (TNF) production by T cell receptor-primed T lymphocytes is a target for low dose methotrexate in rheumatoid arthritis

SUMMARYMethotrexate (MTX) is an effective immunosuppressive agent in various chronic inflammatory diseases such as rheumatoid arthritis (RA). However, its mechanisms of action are only partially understood. In this study, we assessed the effects of MTX on the differentiation of peripheral blood (PB) CD4+CD45RA ‘naive’ and CD4+CD45RO ‘memory’ T cells from healthy controls and patients with RA. Accordingly, purified T cells were primed and restimulated in vitro via the T cell receptor (TCR) in the presence of IL-2 to generate effector T cells secreting large amounts of Th1 and Th2 cytokines. We observed that low doses of MTX strongly suppress TNF and to a lesser extent interferon-gamma (IFN-γ…

Activation and methotrexate-mediated suppression of the TNF alpha promoter in T cells and macrophages.

Transplanting the genetic susceptibility to Crohn’s disease

Susceptibility to Crohn’s disease may be transferred via haematopoietic stem cells, highlighting the pivotal role of genetic factors in the pathogenesis of Crohn’s disease Crohn’s disease (CD) is one of the two most common forms of inflammatory bowel disease (IBD). The prevalence of CD has increased in Western countries over the past decades and mainly young patients are affected, with a peak incidence between 15 and 35 years.1 The aetiology of IBD is still unclear and should be considered as multifactorial according to recent studies.2 Genetic factors seem to play a pathogenic role as well as environmental, infectious, and immunological factors. All of these different aetiological aspects …

Functional relevance of soluble TNF-alpha, transmembrane TNF-alpha and TNF-signal transduction in gastrointestinal diseases with special reference to inflammatory bowel diseases.

As a result of extensive clinical and basic research, the pivotal role of tumour necrosis factor (TNF) in the pathogenesis of chronic inflammatory diseases such as inflammatory bowel disease (IBD) has now generally been acknowledged. This has led to promising clinically effective anti-TNF-strategies. Of note, there is more and more evidence that TNF seems to play a key role in other gastrointestinal diseases including Helicobacter pylori infection, pancreatitis, viral hepatitis and toxic liver damage, too. The action of TNF at the cellular level is mediated by two cell surface receptors, TNF-R1 (p60) and TNF-R2 (p80). The function of these receptors and the downstream intracellular signal t…

CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes

Azathioprine and its metabolite 6-mercaptopurine (6-MP) are immunosuppressive drugs that are used in organ transplantation and autoimmune and chronic inflammatory diseases such as Crohn disease. However, their molecular mechanism of action is unknown. In the present study, we have identified a unique and unexpected role for azathioprine and its metabolites in the control of T cell apoptosis by modulation of Rac1 activation upon CD28 costimulation. We found that azathioprine and its metabolites induced apoptosis of T cells from patients with Crohn disease and control patients. Apoptosis induction required costimulation with CD28 and was mediated by specific block- ade of Rac1 activation thro…

Activation pattern of signal transducers and activators of transcription (STAT) factors in inflammatory bowel diseases.

Cytokine signaling pathways involving transcription factors of the signal transducers and activators of transcription (STAT) family play a key role in the pathogenesis of inflammatory bowel diseases (IBD). STAT proteins are latent cytoplasmic transcription factors that induce transcription upon phosphorylation, dimerization, and nuclear translocation. However, their activation pattern in IBD is poorly understood. The aim of our study was to characterize STAT-expression in IBD.Mononuclear cells were isolated from 36 colonic specimens of Crohn's disease, ulcerative colitis, or from control patients. Cells were stimulated overnight with antibodies against human CD2 and CD28 and mononuclear cel…

Recognition and characterization of small colonic neoplasia with high-definition colonoscopy using i-Scan is as precise as chromoendoscopy

Abstract Background The EPKi system (Pentax, Japan) enables resolution above HDTV. Aim of the study was to test the efficacy of HD+ alone and with the new post-processing digital filter i-Scan or chromoendoscopy (Methylene blue 0.1%) in screening for colorectal cancer. We focused on lesions less than 5 mm as a surrogate marker for the optical possibilities of the EPKi system. Methods The last 30 cm of the colon in a screening population were inspected with HD+ alone, in combination with i-Scan (2:1 randomisation) and subsequently with chromoendoscopy. All lesions were characterized and targeted biopsies were performed. Results i-Scan augmented in 69 patients the identification of lesions fr…

Das mukosale Immunsystem

Untersuchungen der letzten 10 Jahre haben zu einem verbesserten Verstandnis der Pathophysiologie von chronisch entzundlichen Darmerkrankungen gefuhrt. Neuere Studien belegen hierbei v. a. die funktionelle Bedeutung einer Aktivierung des mukosalen Immunsystems durch bakterielle Antigene fur die Pathogenese der chronisch entzundlichen Darmerkrankungen (CED). Neben Chemokinen und Adhasionsmolekulen scheinen vor allem Zytokine, die von antigenprasentierenden Zellen und T-Lymphozyten produziert werden, pathogenetisch fur die CED von entscheidender Bedeutung zu sein. So konnte tierexperimentell gezeigt werden, dass Antikorper gegen proinflammatorische Zytokine zu einer Suppression von chronisch i…

New therapeutic strategies for treatment of inflammatory bowel disease

Although the precise etiology of inflammatory bowel disease (IBD) still remains unclear, considerable progress has been made in the identification of cytokine-mediated signaling pathways involved in the inflammatory process. Recent data have clearly shown that these pathways induce augmented intestinal T-cell activation and thus resistance to apoptosis, which is a central process in disease pathogenesis, as it impairs mucosal homeostasis. Therefore, novel therapeutic strategies aim at restoring activated effector T-cell susceptibility to apoptosis in the gut, based on a pathophysiological rationale. This development is best exemplified by the emergence of agents that target the TNF pathway,…

Overexpression of STAT-1 by adenoviral gene transfer does not inhibit hepatitis B virus replication.

Objectives Interferons are known to inhibit the replication of hepatitis B viruses (HBV) in several animal models in vitro and in vivo as well in humans. The STAT-1 protein plays a central role in the biological activity of both type I and type II interferons. The lack of functional STAT-1 renders cells and organisms susceptible to bacterial and viral infectious agents. We analysed whether the overexpression of STAT-1 protein enhances the biological interferon response and whether it elicits antiviral acitivity against HBV in vitro. Methods To achieve an efficient STAT-1 overexpression in primary liver cells and hepatoma cells, we generated a recombinant, replication-deficient adenovirus ex…

Efficient gene delivery to the inflamed colon by local administration of recombinant adenoviruses with normal or modified fibre structure

BACKGROUND/AIMSReplication deficient recombinant adenoviruses represent an efficient means of transferring genes in vivo into a wide variety of dividing and quiescent cells from many different organs. Although the gastrointestinal tract is a potentially attractive target for gene therapy approaches, only a few studies on the use of viral gene transfer vehicles in the gut have been reported. The prospects of using recombinant adenoviruses for gene delivery into epithelial and subepithelial cells of the normal and inflamed colon are here analysed.METHODSAn E1/E3 deleted recombinant adenovirus (denoted AdCMVβGal) and an adenovirus with modified fibre structure (denoted AdZ.F(pk7)) both express…

Revue : Possibilités des nouvelles techniques endoscopiques : coloration vitale et endomicroscopie confocalein vivo dans la détection des lésions précancéreuses et du cancer précoce chez les patients porteurs de colite ulcéreuse

L’enjeu de la surveillance endoscopique des patients souffrant de colite ulcereuse de longue duree est le diagnostic precoce des lesions malignes et des modifications precancereuses de la muqueuse.

Magnifying chromoendoscopy for the detection of premalignant gastrointestinal lesions

The prognosis for patients with malignancies of the gastrointestinal tract is strictly dependent on the early detection of premalignant and malignant lesions. At present, endoscopy can be performed with new, powerful high-resolution or magnifying endoscopes. Comparable to the rapid development in chip technology, the optic features of the newly designed endoscopes offer resolutions that allow new mucosal surface details to be seen. In conjunction with chromoendoscopy, the newly discovered tool of video endoscopy is much easier to use and more impressive than previously used fibreoptic endoscopy. This review summarises the value of magnifying endoscopy in the upper and lower gastrointestinal…

The many roads to inflammatory bowel diseases.

Two independent studies by Rakoff-Nahoum et al. (2006) and Uhlig et al. (2006) in this issue of Immunity have illuminated a unique pathogenic role of innate immunity via Toll-like receptor and interleukin-23 signaling, respectively, in intestinal inflammation. These data define new roads to gut inflammation and future avenues for therapy.

In vivo subsurface morphological and functional cellular and subcellular imaging of the gastrointestinal tract with confocal mini-microscopy.

AIM: To evaluate a newly developed hand-held confocal probe for in vivo microscopic imaging of the complete gastrointestinal tract in rodents. METHODS: A novel rigid confocal probe (diameter 7 mm) was designed with optical features similar to the flexible endomicroscopy system for use in humans using a 488 nm single line laser for fluorophore excitation. Light emission was detected at 505 to 750 nm. The field of view was 475 μm × 475 μm. Optical slice thickness was 7 μm with a lateral resolution of 0.7 μm. Subsurface serial images at different depths (surface to 250 μm) were generated in real time at 1024 × 1024 pixels (0.8 frames/s) by placing the probe onto the tissue in gentle, stable co…

Il-6 signaling in inflammatory bowel disease: pathophysiological role and clinical relevance.

Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory bowel diseases (IBDs) occurring in the gut of genetically susceptible individuals independent of a specific pathogen. The interaction between antigen-presenting cells and the local bacterial flora contributes to an uncontrolled activation of mucosal CD4+ T lymphocytes with the consecutive release of proinflammatory cytokines such as tumor necrosis factor (TNF), interleukin (IL)-6, IL-12, IL-23, IL-27, and also IL-17, which is attributed to a specific, differentiated CD4+ lineage called Th17 (TH-IL17, THi). Recent data suggest that IL-6 contributes to Th17 differentiation. However, to clarify the importance of Th17 cel…

Technology Insight: confocal laser endoscopy for in vivo diagnosis of colorectal cancer

Recent studies on a novel technology, denoted confocal laser endomicroscopy (CLE), have altered thinking about the possibilities of endoscopy in the diagnosis and treatment of colorectal cancer. CLE is a new endoscopic tool that allows in vivo histology at subcellular resolution during ongoing endoscopy, and permits subsurface imaging of normal and neoplastic human mucosa. This new technique has unequivocal major implications for the diagnosis and clinical management of patients scheduled for screening or surveillance colonoscopy for colorectal cancer. For instance, CLE allows immediate diagnosis of colonic neoplasias, and the detection of neoplastic cells helps to target endoscopic interve…

Smad7 controls resistance of colitogenic T cells to regulatory T cell-mediated suppression.

Background & Aims Foxp3-expressing regulatory T cells (Tregs) play a key role in the maintenance of the gut immune homeostasis, and an intact transforming growth factor (TGF)-β signaling is required for their function. In inflammatory bowel disease (IBD), the TGF-β signaling is impaired because of high expression of the inhibitory molecule Smad7. Although no intrinsic defects in Tregs function have been shown in IBD, it is still unknown whether colitogenic T cells are susceptible to Treg-mediated suppression. In this study, we have investigated whether IBD mucosal CD4+ T cells are resistant to Tregs and whether Smad7 is involved in this process. Methods IBD lamina propria mononuclear cells …

A key pathogenic role for the STAT1/T-bet signaling pathway in T-cell-mediated liver inflammation.

TH1 cytokines have been suggested to contribute to the pathogenesis of T-cell-mediated liver injury and inflammation. However, the molecular signaling pathways involved in such injury are still poorly understood. In the present study, we investigated the role of the STAT1/T-bet signaling pathway in a murine model of T-cell-mediated liver inflammation induced by the application of concanavalin A (Con A) using newly created STAT1 transgenic mice as well as STAT1- and T-bet-deficient mice. Liver injury induced by Con A was associated with an increase of both pSTAT1 and T-bet levels in the liver. Furthermore, functional studies suggested a pathogenic role for STAT1 in Con A-induced liver injury…

In vitro generation of CD4+CD25+ regulatory cells from murine naive T cells

CD4+ CD25+ regulatory T cells (Tregs) are crucial for the maintenance of immunological tolerance. Recent data indicate that Tregs not only develop in the thymus during ontogeny but can also differentiate from naive T cells in the periphery. The following protocol describes a method by which Tregs are generated in vitro by stimulation of naive T cells in the presence of transforming growth factor beta (Ti-Tregs). In vitro-induced regulatory T cells express markers of conventional Treg such as CD25 and the genetic program committing transcription factor FoxP3. Functionally the in vitro-generated Ti-Tregs suppress T-cell activation and proliferation while in vivo these cells have been proven t…

Zur Heterogenität der Epiphysenfuge — eine morphologische Studie zur enchondralen Ossifikation der Tibia

Bei der chondralen Ossifikation des Epiphysenknorpels sezernieren die Chondrocyten zahlreiche differente Strukturmolekule, wie Kollagene, Lipide und Proteoglykane in die sie umgebende Extrazellularmatrix (Hinchcliffe u. Johnson 1983). Diese sollen in komplexer Weise an der Knochenmineralisation beteiligt sein (Eggli et al. 1985). Die sechs Zonen der Wachstumsfuge sind durch eine spezifische Morphologie charakterisiert. Bisher liegen nur wenige Untersuchungen vor, die die Verteilung von spezifischen Glykosaminoglykanstrukturen diesen Bereichen zuordnen. In der vorliegenden Arbeit wurde versucht, das Bindungsverhalten von Neoglykoproteinen und Lektinen mit den konsekutiven Stadien der Knochen…

In vivo histology of Barrett's esophagus and associated neoplasia by confocal laser endomicroscopy.

Background & Aims: Confocal laser endomicroscopy allows subsurface analysis of the intestinal mucosa and in vivo histology during ongoing endoscopy. Here, we have applied this technique to the in vivo diagnosis of Barrett's epithelium and associated neoplasia. Methods: Fluorescein-aided endomicroscopy was performed by applying the endomicroscope over the whole columnar-lined lower esophagus. Images obtained within 1 cm of the columnar-lined lower esophagus were stored digitally and a targeted biopsy examination or endoscopic mucosal resection of the examined areas was performed. In vivo histology was compared with the histologic specimens. All digitally stored images were re-assessed by a b…

A variant of Smurf2 protects mice against colitis-associated colon cancer by inducing transforming growth factor β signaling.

Background & Aims Transforming growth factor (TGF)-β signaling, which is down-regulated by the E3 ubiquitin ligase Smad ubiquitin regulating factor 2 (Smurf2), promotes development of cancer. We identified a splice variant of Smurf2 (ΔE2Smurf2) and investigated its role in colon carcinogenesis in mice. Methods Colitis-associated colon cancer was induced in mice by administration of azoxymethane, followed by 3 cycles of oral administration of dextran sodium sulfate. Messenger RNA levels of Smurf2 in colon tumors and control tissue were measured by quantitative polymerase chain reaction; lymphocyte and cytokine levels were measured in tumor and tissue samples. Results Tumor-infiltrating CD4 +…

Chromo- and magnifying endoscopy for colorectal lesions

It is essential to identify patients with premalignant or early malignant changes during colonoscopy. Thus, curative resection can be offered. At present, endoscopy can be performed with new powerful high-resolution or magnifying endoscopes. Comparably to the rapid development in chip technology, the optic features of the newly designed endoscopes offer resolutions which allow new mucosal surface details to be seen. In conjunction with chromoendoscopy, the newly discovered tool video endoscopy is much easier and more impressive than with conventional fibre optics. This review summarizes the value of magnifying endoscopy in the lower gastrointestinal tract and focuses on colorectal lesions.

T helper cell polarisation in coeliac disease: any (T-)bet ?

Recent data strongly support the view that coeliac disease is a Th1 mediated inflammatory disease as both interferon γ production and T-bet levels in gut infiltrating cells are upregulated The puzzling observation on high interferon γ (IFN-γ) but low interleukin (IL)-12 levels in coeliac disease (CD) has resulted in questions about the underlying principles of T helper cell polarisation. In this issue of G ut ,1 the molecular basis of T helper cell polarisation in CD has been illuminated by the finding that T-bet, the master transcription factor of T helper cell type 1 (Th1) cells, is upregulated in this disease [see page 1090] . The past decade has witnessed a dramatic improvement in our p…

Caspase-8 regulates TNF-alpha induced epithelial necroptosis and terminal ileitis

Two groups identify the regulation of death-receptor-induced necroptosis as an epithelial intrinsic mechanism that is important for the maintenance of immune homeostasis and the prevention of intestinal inflammation in mice. Welz et al. describe an unexpected physiological function for FADD (Fas-associated protein with death domain), an adaptor protein required for death-receptor-induced apoptosis. Mice with intestinal epithelial specific knockout of FADD develop severe colon inflammation due to increased death of FADD-deficient colonic epithelial cells. Gunther et al. report a novel and unexpected function of caspase-8 in maintaining immune homeostasis in the gut. Caspase-8 expression by g…

Development of spontaneous airway changes consistent with human asthma in mice lacking T-bet.

Human asthma is associated with airway infiltration by T helper 2 (TH2) lymphocytes. We observed reduced expression of the TH1 transcription factor, T-bet, in T cells from airways of patients with asthma compared with that in T cells from airways of nonasthmatic patients, suggesting that loss of T-bet might be associated with asthma. Mice with a targeted deletion of the T-bet gene and severe combined immunodeficient mice receiving CD4+cells from T-bet knockout mice spontaneously demonstrated multiple physiological and inflammatory features characteristic of asthma. Thus, T-bet deficiency, in the absence of allergen exposure, induces a murine phenotype reminiscent of both acute and chronic h…

Future Trends in Confocal Laser Endomicroscopy: Improved Imaging Quality and Immunoendoscopy

Confocal laser endomicroscopy (CLE) is a powerful new technique that permits subsurface imaging at high resolution during ongoing endoscopy in the upper and lower GI tract [1]. Here, we will discuss future trends in CLE and their potential application in clinical practice. In particular, we will focus on molecular imaging using labelled antibodies for immunoendoscopy.

Gene transfer approaches for the treatment of inflammatory bowel disease.

The pathogenesis of Crohn's disease and ulcerative colitis, the two major forms of inflammatory bowel disease, involves a complex interplay between certain genetic, environmental and immunological factors. Considerable research progress in the last decade defined key inflammatory pathways in the inflamed gut and identified new potential therapeutic targets. Since the current medical treatment with corticosteroids and anti-inflammatory drugs is often associated with undesired side effects and cannot completely cure IBD, these current advances in our understanding of intestinal pathology may now allow the development of new biologic treatment strategies including gene therapy. In this review,…

Minimal Change Esophagitis: Prospective Comparison of Endoscopic and Histological Markers between Patients with Non-Erosive Reflux Disease and Normal Controls Using Magnifying Endoscopy

<i>Introduction:</i> More than half the patients with gastroesophageal reflux disease (GERD) show no endoscopic abnormality or minimal change esophagitis (non-erosive reflux disease, NERD). We investigated the value of endoscopic and histological markers for the prediction of NERD before and after treatment with 20 mg esomeprazole. <i>Methods:</i> Between July and October 2002, consecutive patients presenting for upper endoscopy were stratified into GERD and non-reflux patients (control group) with the help of a questionnaire. The endoscopist was blind to the presence of reflux symptoms. Using magnifying endoscopes minimal change esophagitis was defined by the presen…

IL-28A (IFN-λ2) modulates lung DC function to promote Th1 immune skewing and suppress allergic airway disease.

IL-28 (IFN-λ) cytokines exhibit potent antiviral and antitumor function but their full spectrum of activities remains largely unknown. Recently, IL-28 cytokine family members were found to be profoundly down-regulated in allergic asthma. We now reveal a novel role of IL-28 cytokines in inducing type 1 immunity and protection from allergic airway disease. Treatment of wild-type mice with recombinant or adenovirally expressed IL-28A ameliorated allergic airway disease, suppressed Th2 and Th17 responses and induced IFN-γ. Moreover, abrogation of endogenous IL-28 cytokine function in IL-28Rα(-/-) mice exacerbated allergic airway inflammation by augmenting Th2 and Th17 responses, and IgE levels.…

Elevated levels of Bcl-3 inhibits Treg development and function resulting in spontaneous colitis

Bcl-3 is an atypical NF-κB family member that regulates NF-κB-dependent gene expression in effector T cells, but a cell-intrinsic function in regulatory T (Treg) cells and colitis is not clear. Here we show that Bcl-3 expression levels in colonic T cells correlate with disease manifestation in patients with inflammatory bowel disease. Mice with T-cell-specific overexpression of Bcl-3 develop severe colitis that can be attributed to defective Treg cell development and function, leading to the infiltration of immune cells such as pro-inflammatory γδT cells, but not αβ T cells. In Treg cells, Bcl-3 associates directly with NF-κB p50 to inhibit DNA binding of p50/p50 and p50/p65 NF-κB dimers, t…

Confocal laser endomicroscopy: technical status and current indications.

Confocal laser endomicroscopy is a newly introduced endoscopic tool that makes it possible to carry out confocal microscopic examination of the mucosal layer during ongoing endoscopy. Different types of tissue and diseases can be diagnosed immediately, facilitating early diagnosis of gastrointestinal cancer. Analysis of the in vivo microarchitecture is helpful in targeting biopsies to relevant areas. In addition, subsurface imaging can unmask microscopic diseases - (microscopic colitis) or bacterial infection ( HELICOBACTER PYLORI), for example. Molecular imaging is becoming feasible, and this will shortly open the door to new indications in gastrointestinal endoscopy (e.g., in vivo recepto…

Endomicroscopy for in vivo Diagnosis of Colorectal Cancer

Activation of Intestinal Epithelial Stat3 Orchestrates Tissue Defense during Gastrointestinal Infection

Gastrointestinal infections with EHEC and EPEC are responsible for outbreaks of diarrheal diseases and represent a global health problem. Innate first-line-defense mechanisms such as production of mucus and antimicrobial peptides by intestinal epithelial cells are of utmost importance for host control of gastrointestinal infections. For the first time, we directly demonstrate a critical role for Stat3 activation in intestinal epithelial cells upon infection of mice with Citrobacter rodentium - a murine pathogen that mimics human infections with attaching and effacing Escherichia coli. C. rodentium induced transcription of IL-6 and IL-22 in gut samples of mice and was associated with activat…

In-vivo confocal real-time mini-microscopy in animal models of human inflammatory and neoplastic diseases

Background and study aims Although various improvements in tissue imaging modalities have recently been achieved, in-vivo molecular and subsurface imaging in the field of gastroenterology remains a technical challenge. In this study we evaluated a newly developed, handheld, miniaturized confocal laser microscopy probe for real-time in-vivo molecular and subsurface imaging in rodent models of human disease. Materials and methods The minimicroscope uses a 488-nm, single line laser for fluorophore excitation. The optical slice thickness is 7 microm, the lateral resolution 0.7 microm. The range of the z-axis is 0-250 microm below the tissue surface. Imaging was performed using different fluores…

An innate cell-mediated, murine ulcerative colitis-like syndrome in the absence of nuclear factor of activated T cells.

Abstract Background & Aims: Nuclear factor of activated T cells transcription factors plays a central role in immunity by regulating the expression of multiple cytokines and other regulatory molecules, many of which have been heavily implicated in the pathogenesis of inflammatory bowel disease. However, few studies have directly investigated the nuclear factor of activated T cells proteins in inflammatory bowel disease. We describe here a specific role for nuclear factor of activated T cells c2 in the pathogenesis of murine inflammatory bowel disease. Methods: Mice deficient for nuclear factor of activated T cells c2, recombinase activating gene-2, or both and transgenic or nontransgenic fo…

Chemically induced mouse models of intestinal inflammation

Animal models of intestinal inflammation are indispensable for our understanding of the pathogenesis of Crohn disease and ulcerative colitis, the two major forms of inflammatory bowel disease in humans. Here, we provide protocols for establishing murine 2,4,6-trinitro benzene sulfonic acid (TNBS)-, oxazolone- and both acute and chronic dextran sodium sulfate (DSS) colitis, the most widely used chemically induced models of intestinal inflammation. In the former two models, colitis is induced by intrarectal administration of the covalently reactive reagents TNBS/oxazolone, which are believed to induce a T-cell-mediated response against hapten-modified autologous proteins/luminal antigens. In …

Treatment of allergic airway inflammation and hyperresponsiveness by antisense-induced local blockade of GATA-3 expression.

Recent studies in transgenic mice have revealed that expression of a dominant negative form of the transcription factor GATA-3 in T cells can prevent T helper cell type 2 (Th2)-mediated allergic airway inflammation in mice. However, it remains unclear whether GATA-3 plays a role in the effector phase of allergic airway inflammation and whether antagonizing the expression and/or function of GATA-3 can be used for the therapy of allergic airway inflammation and hyperresponsiveness. Here, we analyzed the effects of locally antagonizing GATA-3 function in a murine model of asthma. We could suppress GATA-3 expression in interleukin (IL)-4–producing T cells in vitro and in vivo by an antisense ph…

Immune cells in colorectal cancer: prognostic relevance and therapeutic strategies.

During the last two decades, considerable efforts have been made to improve the prevention, early diagnosis and therapy of colorectal cancer by gaining enhanced insights into disease-specific pathogenesis. Along these lines, tumor-infiltrating immune cells turned out to be critical indicators for an efficient antitumor immune response and the number and type of tumor-infiltrating immune cells determined the resulting tumor prognosis. This review aims to describe the prognostic relevance of the different subsets of tumor-infiltrating immune cells and highlights their specific function in the complex process of immune system-mediated rejection of colorectal cancer cells. Considering the clini…

Endomicroscopy of Colon Pathology

Colorectal cancer is still one of the leading causes of cancer-related death in the Western world. Screening colonoscopy is widely accepted as the gold standard for early diagnosis of cancer. The prognosis for patients with colonic neoplasms is strictly dependent on the depth of infiltration and therefore depends on early detection of pre-invasive and neoplastic changes. Early detection makes it possible to cure the patient by means of immediate endoscopic resection.

A novel rat model of chronic fibrosing cholangitis induced by local administration of a hapten reagent into the dilated bile duct is associated with increased TNF-alpha production and autoantibodies.

Abstract Background/Aim: The cholangiopathies represent hepatobiliary diseases in which bile-duct epithelial cells are targets for destructive processes, including immune-mediated damage. We describe a novel rat model of chronic fibrosing cholangitis induced by administration of the hapten reagent 2,4,6-trinitrobenzenesulfonic acid (TNBS) into the dilated bile duct. Methods: The common bile duct was dilated due to a mild stenosis in 8-week-old female Lewis rats. TNBS (50 mg/kg) was injected during a second laparotomy. Results: TNBS-treatment reproducibly resulted in chronic fibrosing cholangitis. In retrograde cholangiography the bile ducts showed irregularities, beading and strictures. Alk…

The yopJ locus is required for Yersinia-mediated inhibition of NF-kappaB activation and cytokine expression: YopJ contains a eukaryotic SH2-like domain that is essential for its repressive activity.

Upon exposure to bacteria, eukaryotic cells activate signalling pathways that result in the increased expression of several defence-related genes. Here, we report that the yopJ locus of the enteropathogen Yersinia pseudotuberculosis encodes a protein that inhibits the activation of NF-kappaB transcription factors by a mechanism(s), which prevents the phosphorylation and subsequent degradation of the inhibitor protein IkappaB. Consequently, eukaryotic cells infected with YopJ-expressing Yersinia become impaired in NF-kappaB-dependent cytokine expression. In addition, the blockage of inducible cytokine production coincides with yopJ-dependent induction of apoptosis. Interestingly, the YopJ pr…