Absence of specific alternatively spliced exon of CD44 in macrophages prevents colitis.

CD44 is a transmembrane molecule appearing in numerous isoforms generated by insertions of alternatively spliced variant exons (CD44v) and having various binding partners. CD44v7 on T cells was proposed to promote colitis by preventing T-cell apoptosis. Here we demonstrate that Cd44v7-deficient T cells - like Cd44 wild-type (Cd44WT) T cells - provoked disease in two different colitis models: the model induced by CD4+CD45RBhigh T-cell transfer into Rag2-deficient mice and a new model based on ovalbumin (OVA)-specific T-cell transfer into Rag-sufficient, OVA-challenged mice. In contrast, CD44v7 absence on macrophages in recipient mice prevented colitis. Prevention was associated with the downregulation of signal transducer and activator of transcription 3 (STAT3)-activating and Foxp3-counteracting interleukin-6 (IL-6), lower numbers of phospho-STAT3-containing lymphocytes, and higher Foxp3+ T-cell counts in the colon. Consequently, the protected colons showed lower IL-12, IL-1ß expression, and decreased interferon-γ levels. Importantly, stimulation of T cells by Cd44v7-deficient macrophages induced upregulation of Foxp3 in vitro, while cotransfer of Cd44WT macrophages into Cd44v7-deficient mice reduced Foxp3+ T-cell counts and caused colitis. Accordingly, the CD44v7 ligand osteopontin, whose levels were elevated in Crohn's disease, specifically induced IL-6 in human monocytes, a cytokine also increased in these patients. We suggest macrophage-specific targeting of the CD44v7 pathway as a novel therapeutic option for Crohn's disease.

Preventive Anti-CD2 Treatment does not Impair Parasite Control in a Murine Toxoplasmosis Model.

Targeting human CD2 with the monoclonal antibody (mAb) CB.219 reduces intestinal inflammation in a colitis model where T cells carry human CD2. Here, we asked whether this mAb has adverse effects on infection control. Mice expressing human CD2 on T cells (huCD2tg) were orally infected with Toxoplasma (T.) gondii and treated with the human CD2-specific mAb CB.219 in a preventive setting. The intestinal T. gondii loads in CB.219 treated mice did not differ from the control group. Histologically, huCD2tg mice showed moderate ileal inflammation that did not change with CB.219 treatment. In the ileum, CB.219 treatment reduced the protein levels of interferon-γ, transforming growth factor ß and interleukin-6, whereas interleukin-18 mRNA was slightly increased. The infiltration of neutrophils, macrophages, and T cells into the ileum was unaffected by CB.219 treatment. However, CB.219 treatment decreased the numbers of forkhead box P3(+) regulatory T cells (Treg) in ileum and liver of huCD2tg mice. This was confirmed in vitro using human peripheral blood mononuclear cells. Taken together, targeting CD2(+) T cells by the human CD2 mAb CB.219 does not prevent beneficial immune reactions necessary for pathogen control. Further experiments will address gut specificity, underlying mechanisms, and general applicability of CB.219 treatment.

Permissive expansion and homing of adoptively transferred T cells in tumor-bearing hosts.

Activated T cells expressing endogenous or transduced TCRs are two cell types currently used in clinical adoptive T-cell therapy. The ability of these cells to recognize their antigen, expand and traffic to the tumor site are the initial steps necessary for successful therapy. In this study, we used in vivo bioluminescent imaging (BLI) of Renilla luciferase (RLuc) expressing T cells to evaluate the ability of adoptively transferred T cells to survive, expand and home to tumor site in vivo. Using this method, termed RT-Rack (Rluc T cell tracking), we followed T-cell response against tumors in vivo. Expansion and homing of adoptively transferred T cells were antigen dependent, but independent of the host immune status. Moreover, we successfully detected T-cell response to small and large tumors, including autochthonous liver tumors. The adoptively transferred T cells were not ignorant or excluded in a partially tolerant host, which expressed low level of the target in the periphery. Using T cell receptor (TCR)-engineered T cells, we showed the ability of these cells to respond in tumor-bearing hosts by expanding and homing to the tumor site. In all these models, the host immune status, the nature of the tumor or of the antigen, the tumor size and the presence of the targeted antigen in the periphery did not prevent the adoptively transferred T cells from responding by expanding and homing to the tumor. However, T cells had higher expression of the inhibitory receptor PD1 and reduced functional activity when a self-antigen was targeted.

[Cutaneous Rosai-Dorfman syndrome. Successful therapy with intrralesional corticosteroids]. / Kutanes Rosai-Dorfman-Syndrom. Erfolgreiche Therapie mit intraläsional applizierten Steroiden.

BACKGROUND: Cutaneous Rosai-Dorfman disease is a rare disorder belonging to the spectrum of non-Langerhans cell histiocytoses. It is characterized by dermal and subcutaneous infiltrates of histiocytes as well as accompanying lymphocytes, plasma cells and granulocytes. Because it is so rare, standard therapies have not been established. CASE REPORT: A 27-year-old man showed an excellent response to intralesional corticosteroids after unsuccessful prior treatment with methotrexate, systemic steroids and surgery as well as laser therapy.

Mechanisms and rescue strategies of calcineurin inhibitor mediated tolerance abrogation induced by anti-CD4 mAb treatment.

To ensure safety tolerance induction protocols are accompanied by conventional immunosuppressive drugs (IS). But IS such as calcineurin inhibitors (CNI), for example, cyclosporin A (CsA), can interfere with tolerance induction. We investigated the effect of an additional transient CsA treatment on anti-CD4mAb-induced tolerance induction upon rat kidney transplantation. Additional CsA treatment induced deteriorated graft function, resulting in chronic rejection characterized by glomerulosclerosis, interstitial fibrosis, tubular atrophy and vascular changes. Microarray analysis revealed enhanced intragraft expression of the B cell attracting chemokine CXCL13 early during CsA treatment. Increase in CXCL13 expression is accompanied by enhanced B cell infiltration with local and systemic IgG production and C3d deposition as early as 5 days upon CsA withdrawal. Adding different CNIs to cultures of primary mesangial cells isolated from glomeruli resulted in a concentration-dependent increase in CXCL13 transcription. CsA in synergy with TNF-α can enhance the B cell attracting and activating potential of mesangial cells. Transient B cell depletion or transfer of splenocytes from tolerant recipients 3 weeks after transplantation could rescue tolerance induction and did inhibit intragraft B cell accumulation, alloantibody production and ameliorate chronic rejection.

Mesenteric fat - control site for bacterial translocation in colitis?

In Crohn's disease bacteria could be detected in the adjacent mesenteric fat characterized by hypertrophy of unknown function. This study aimed to define effector responses of this compartment induced by bacterial translocation during intestinal inflammation. Dextran sulfate sodium-induced colitis served as a model of intestinal inflammation. Translocation of peptides and bacteria into mesenteric fat was evaluated. Innate functions of mesenteric fat and epithelium were characterized at whole tissue, cellular, and effector molecule levels. Orally applied peptides translocated in healthy wild-type (WT) mice. Bacterial translocation was not detected in healthy and acute but increased in chronic colitis. Mesenteric fat from colitic mice released elevated levels of cytokines and was infiltrated by immune cells. In MyD88(-/-) mice bacterial translocation occurred in health and increased in colitis. The exaggerated cytokine production in mesenteric fat accompanying colonic inflammation in WT mice was less distinct in MyD88(-/-) mice. In vitro studies revealed that fat not only increases cytokine production following contact with bacterial products, but also that preadipocytes are potent phagocytes. Colonic inflammation is accompanied by massive cytokine production and immune cell infiltration in adjacent adipose tissue. These effects can be considered as protective mechanisms of the mesenteric fat in the defense of bacterial translocation.

UDCA slows down intestinal cell proliferation by inducing high and sustained ERK phosphorylation.

Ursodeoxycholic acid (UDCA) attenuates colon carcinogenesis in humans and in animal models by an unknown mechanism. We investigated UDCA effects on normal intestinal epithelium in vivo and in vitro to identify the potential chemopreventive mechanism. Feeding of mice with 0.4% UDCA reduced cell proliferation to 50% and suppressed several potential proproliferatory genes including insulin receptor substrate 1 (Irs-1). A similar transcriptional response was observed in the rat intestinal cell line IEC-6 which was then used as an in vitro model. UDCA slowed down the proliferation of IEC-6 cells and induced sustained hyperphosphorylation of ERK1/ERK2 kinases which completely inhibited the proproliferatory effects of EGF and IGF-1. The hyperphosphorylation of ERK1 led to a transcriptional suppression of the Irs-1 gene. Both, the hyperphosphorylation of ERK as well as the suppression of Irs-1 were sufficient to inhibit proliferation of IEC-6 cells. ERK1/ERK2 inhibition in vitro or ERK1 elimination in vitro or in vivo abrogated the antiproliferatory effects of UDCA. We show that UDCA inhibits proliferation of nontransformed intestinal epithelial cells by inducing a sustained hyperphosphorylation of ERK1 kinase which slows down the cell cycle and reduces expression of Irs-1 protein. These data extend our understanding of the physiological and potentially chemopreventive effects of UDCA and identify new targets for chemoprevention.

[Tropheryma whipplei infection. Colonization, self-limiting infection and Whipple's disease]. / Infektionen mit Tropheryma whipplei. Besiedlung, selbstlimitierte Infektion und Morbus Whipple.

Whipple's disease is a multisystemic infection caused by the ubiquitous bacterium Tropheryma whipplei. Immunological host factors enable classical Whipple's disease; however, T. whipplei can be found in three other clinical conditions: healthy colonization, self-limiting infections, and isolated endocarditis. The genetic predisposition of the host rather than the genotype of the bacterium influences the infection. Modern diagnostic methods elucidate the many facets of Whipple's disease. In particular, isolated T. whipplei-induced infective endocarditis can only be diagnosed after valve resection. The sole treatment of Whipple's disease evaluated prospectively comprises intravenous induction therapy with ceftriaxone or meropenem, followed by continuation therapy with oral TMP-SMX. In the case of Immune reconstitution inflammatory syndrome (IRIS) or inflammatory lesions of the CNS in the setting of Whipple's disease, additional treatment with corticosteroids should be considered to avoid severe tissue damage.

[Histopathology of graft-versus-host disease]. / Histopathologie der Graft-versus-Host-Erkrankung.

Graft-versus-host disease (GvHD) occurs as a major complication in 35%-50% of all patients undergoing allogeneic stem cell transplantation. A distinction is made between acute and chronic GvHD depending on the time of onset, type of clinical symptoms and histology. Both forms preferably show manifestations in the skin, mucosal membranes, gastrointestinal tract, liver and (less often) lung. As the clinical presentation is rather unspecific, particularly for the diagnosis of acute GvHD, histology is important to exclude infectious diseases or drug reactions. This review covers the diagnostic morphological criteria and differential diagnoses of GvHD in the skin, gastrointestinal tract and the liver.

Selective gelatinase blockage ameliorates acute DSS colitis.

In the experimental models of intestinal inflammation and humans with inflammatory bowel diseases (IBD), increased levels of the matrix metalloproteinases (MMPs), MMP-2 and -9 (also referred to as gelatinase A and B, respectively), in inflamed tissue sites can be detected. In the presented study, we investigated potential beneficial effects exerted by doxycycline nonselectively blocking MMPs and the selective gelatinase inhibitor RO28-2653 in acute DSS colitis. Treatment with either compound for 8 days ameliorated clinical colitis pathology with a superior outcome in RO28-2653-treated animals. As compared to placebo controls, histopathological changes in the colon were less distinct following MMP blockage and IL-6 secretion in ex vivo biopsies was downregulated, paralleled by a diminished influx of pro-inflammatory immune cells and lack of overgrowth of the colonic lumen by potentially pro-inflammatory Escherichia coli of the commensal colon flora. We conclude that selective gelatinase inhibition not only exerts beneficial effects by disrupting the vicious cycle of positive feedback between immune cell stimulation and MMP induction but also prevents overgrowth of the colonic lumen by pro-inflammatory E. coli despite a lack of direct anti-bacterial properties, thus unaffecting the commensal gut microbiota. These findings put RO28-2653 into a center stage for development of intervention strategies in human IBD.

What you eat is what you get: Novel Campylobacter models in the quadrangle relationship between nutrition, obesity, microbiota and susceptibility to infection.

Enterocolitis caused by Campylobacter jejuni-infections represents an important socioeconomic burden worldwide. Recent results from novel murine infection models reveal that the intestinal microbiota is essential for maintaining colonization resistance against C. jejuni. We extended these studies to investigate the role of nutrition and obesity in susceptibility to C. jejuni-infection. Gnotobiotic (GB) mice generated by antibiotic treatment, which were fed with a human cafeteria diet (CAF), as well as obese (ob/ob) mice with a conventional microbiota harbored higher Escherichia coli loads in their colon as compared to respective controls. Following oral infection, C. jejuni 43431 ATCC readily colonized the intestines of CAF and ob/ob mice, whereas GB mice fed with a standard chow (MUD) eradicated the pathogen within days. Furthermore, live C. jejuni translocated into mesenteric lymph nodes of CAF, but not MUD mice. Strikingly, stably infected animals developed enterocolitis as indicated by increased numbers of immune and apoptotic cells in the colon in situ. We conclude that a specific human diet and obesity render mice susceptible to C. jejuni infection. The corresponding murine models are excellently suited for the study of C. jejuni pathogenesis and will help to get further insights into interplays between C. jejuni, microbiota, diet, obesity and immunity.

The distinct roles of MMP-2 and MMP-9 in acute DSS colitis.

Expression of gelatinases A and B, also referred to matrixmetalloproteinases (MMP)-2 and -9, respectively, is increased in inflamed tissues of experimental intestinal inflammation and humans with inflammatory bowel disease (IBDs). Given that we recently reported that treatment with the selective gelatinase inhibitor RO28-2653 ameliorates acute dextrane sulfate sodium (DSS) colitis, we asked whether gelatinase A or B expression is pivotal in mediating large intestinal inflammation. Results from our study reveal that symptoms of acute DSS colitis as well as histopathological colonic changes were ameliorated in MMP-2-, but not MMP-9-deficient mice, and were paralleled by a diminished influx of immune cells. In MMP-2-deficient mice, we observed lower expression of pro-inflammatory cytokines including interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and IL-6 in colonic biopsies and less overgrowth of the colonic lumen by potentially pro-inflammatory enterobacteria from the commensal gut microbiota. We conclude that rather MMP-2 than MMP-9 is causative for the establishment of DSS colitis in mice. The discrepancy of these data to prior reports might be due to substantial differences in the intestinal microbiota composition of the mice bred at different animal facilities impacting susceptibility to inflammatory stimuli. Consequently, a detailed survey of the gut microbiota should be implemented in immunological/inflammatory studies in the future in order to allow comparison of data from different facilities.

The immune response to sporadic colorectal cancer in a novel mouse model.

Current mouse models do not reflect the sporadic nature of colon cancer and do not allow the analysis of antitumor immune response because of the lack of known tumor-specific antigens. Two transgenic mouse models with spontaneous tumor development were generated, directing the expression of SV40T antigen (Tag) either constitutively (Vil-Cre × LoxP-Tag-transgenic mice) or stochastically (Vil-Cre-ER(T2) × LoxP-Tag-transgenic mice) into the putative stem cell region of the crypt of Lieberkühn. Tumor development and antitumor immune response were monitored. Vil-Cre × LoxP-Tag mice developed multiple adenocarcinomas of the small intestine and colon at an average age of 6 months. During the tumor development, Tag-specific immunoglobulin G (IgG) antibodies were induced in half of the mice, although they had developed neonatal cytotoxic T lymphocyte (CTL) tolerance. This model shows similarity to hereditary colon cancer but not to the sporadic tumor development. Therefore, the conditional Vil-Cre-ER(T2) × LoxP-Tag mice were established, in which expression of the dormant Tag was induced by stochastic, tissue-specific activation of Cre recombinase. These mice spontaneously developed highly invasive, metastasizing colon carcinomas at an average age of 20 months. Colon carcinomas expressed epithelial and/or neuroendocrine markers depending on the grade of differentiation. Young Vil-Cre-ER(T2) × LoxP-Tag mice had retained CTL responses against epitope IV of Tag. The tumors induced strong anti-Tag IgG responses. We report, for the first time, a mouse model based on stochastic, tissue-specific activation of a dormant oncogene in the colon allowing the analysis of antitumor immune response against primary colorectal cancer.

Cellular senescence predicts treatment outcome in metastasised colorectal cancer.

BACKGROUND: Cellular senescence is a terminal cell-cycle arrest that occurs in response to activated oncogenes and DNA-damaging chemotherapy. Whether cancer cell senescence at diagnosis might be predictive for treatment outcome is unknown. METHODS: A senescence index (SI) was developed and used to retrospectively correlate the treatment outcome of 30 UICC stage IV colorectal cancer (CRC) patients with their SI at diagnosis. RESULTS: 5-Fluorouracil/leucovorin-treated CRC patients achieved a significantly longer progression-free survival when presenting with SI-positive tumours before therapy (median 12.0 vs 6.0 months; P=0.044). CONCLUSION: Cancer cell senescence predicts treatment outcome in metastasised CRC. Prospective analyses of larger patient cohorts are needed.

[Primary ductal adenocarcinoma near the orbit]. / Primäres duktales Adenokarzinom im Bereich der Orbita.

The case of a 59-year-old patient is described who presented with a left-sided pressure sensation in the left orbit and exophthalmus with a proud bulbus. The limited bulbus motility led to double vision. A preoperative MRI showed a space-occupying lesion in the left medial orbit. The morphological and immunohistochemical findings led to the diagnosis of a primary ductal carcinoma resembling salivary duct carcinoma (SDC). The tumor could be removed without compromising the eye. The patient suffers no side-effects or recurrences 12 months later.

Lipocalin2 protects against airway inflammation and hyperresponsiveness in a murine model of allergic airway disease.

BACKGROUND: Allergen-induced bronchial asthma is a chronic airway disease that involves the interplay of various genes with environmental factors triggering different inflammatory pathways. OBJECTIVE: The aim of this study was to identify possible mediators of airway inflammation (AI) in a model of allergic AI via microarray comparisons and to analyse one of these mediators, Lipocalin2 (Lcn2), for its role in a murine model of allergic airway disease. METHODS: Gene microarrays were used to identify genes with at least a twofold increase in gene expression in the lungs of two separate mouse strains with high and low allergic susceptibility, respectively. Validation of mRNA data was obtained by Western blotting, followed by functional analysis of one of the identified genes, Lcn2, in mice with targeted disruption of specific gene expression. Epithelial cell cultures were undertaken to define induction requirements and possible mechanistic basis of the results observed in the Lcn2 knock-out mice. RESULTS: Lcn2 was up-regulated upon allergen sensitization and airway challenges in lung tissues of both mouse strains and retraced on the protein level in bronchoalveolar lavage fluids. Functional relevance was assessed in mice genetically deficient for Lcn2, which showed enhanced airway resistance and increased AI associated with decreased apoptosis of lung inflammatory cells, compared with wild-type controls. Similarly, application of Lcn2-blocking antibodies before airway challenges resulted in increased inflammation and reduced apoptosis. CONCLUSION: These data indicate a protective role for Lcn2 in allergic airway disease, suggesting a pro-apoptotic effect as the underlying mechanism.

Diagnostic value of confocal endomicroscopy in celiac disease.

BACKGROUND: Improved endoscopic screening with targeted biopsies might enhance diagnostic yield in celiac disease. Confocal endomicroscopy (CEM) allows high-resolution in vivo histological analysis. We compared the endomicroscopic findings during ongoing endoscopy with the histological findings graded according to the Marsh classification. METHODS: Twenty-four patients with celiac disease and six patients with celiac disease that was refractory on a gluten-free diet were examined using CEM. The duodenal mucosa was evaluated by CEM and by conventional histological analysis in respect of villous atrophy, crypt hyperplasia, and increased numbers of intraepithelial lymphocytes (IELs > 40 / 100 enterocytes). The CEM results were assessed as to sensitivity, specificity, and interobserver variability. A Marsh classification score determined by CEM was compared to that obtained by histology. Thirty patients undergoing routine upper gastrointestinal endoscopy were used as controls. RESULTS: Conventional histology showed villous atrophy and crypt hyperplasia in 23 and increased numbers of IELs in 27 of the 30 patients with celiac disease. With CEM, villous atrophy, crypt hyperplasia, and increased IELs were respectively identified in 17, 12, and 22 of the 30 patients. The agreement of the findings on CEM with those of conventional histology was good in relation to villous atrophy (sensitivity 74 %) and increased numbers of IELs (sensitivity 81 %), but inadequate in relation to crypt hyperplasia (sensitivity 52 %). The kappa values for determination of interobserver variability were 0.90 for villous atrophy, 1.00 for crypt hyperplasia, and 0.84 for IEL detection. In the 30 control patients, normal duodenal architecture was found by both histology and endomicroscopy, indicating an overall specificity of 100 %. CONCLUSION: The assessment of duodenal histology by CEM in patients with celiac disease is sensitive and specific in determining increased numbers of IELs and villous atrophy, but insufficient in respect of crypt hyperplasia. For routine use of CEM in patients with celiac disease, the technique would need to be improved.

Long-term reduction in local inflammation by a lipid raft molecule in atopic dermatitis.

BACKGROUND: The complex pathogenesis of atopic dermatitis (AD) is guided by cell surface receptor-mediated signal transduction regulated in lipid rafts. Miltefosine is a raft-modulating molecule targeting cell membranes. With this controlled clinical study, the clinical and immunomodulatory efficacy of miltefosine was investigated in patients with AD in comparison with a topical corticosteroid treatment. METHODS: Sixteen patients with AD were treated topically with miltefosine and hydrocortisone localized on representative AD target lesions for 3 weeks. To assess the clinical efficacy, the three item severity (TIS) score was evaluated before, during and after treatment as well as after 4-week-follow-up period. To study the anti-inflammatory effect of miltefosine on the cellular T cell pattern, skin biopsies were analysed before and after treatment. RESULTS: The TIS score dropped in both groups significantly after treatment. A carry-over effect was exclusively seen for miltefosine after discontinuing the treatment. These findings were substantiated by thermographic imaging with a significant decrease in the maximum temperature (T(max)) after miltefosine application (P = 0.034, DeltaT(max) = 1.7 degrees C [2.1-3.9]). Immunohistochemically, a reduction in lesional CD4(+)-infiltrating T cells was observed in both treatments. Moreover, increased FoxP3(+) cells were present in the skin after miltefosine treatment (before 5.4% [1.9-9.8], after 6.2% [3.5-9.5]). CONCLUSION: We demonstrate that miltefosine is locally active in patients with AD and led to a sustained clinical improvement in local skin inflammation. Moreover, the increased frequency of FoxP3(+) cells in the skin of patients with AD suggests its immunomodulatory properties.