Matthias Ross

CCR6 identifies lymphoid tissue inducer cells within cryptopatches

The chemokine receptor CCR6 is expressed by dendritic cells, B and T cells predominantly within the organized structures of the gut‐associated lymphatic tissue. Its ligand CCL20 is synthesized by the follicle‐associated epithelium and is crucial for the development of M cells within Peyers patches. In addition, lineage‐negative c‐kit positive lymphocytes within cryptopatches (CP) express CCR6. CCR6‐deficient mice exhibit an altered intestinal immune system containing increased amounts of intraepithelial lymphocytes and show smaller Peyers patches, while progression of cryptopatches to mature isolated lymphoid follicles (ILF) is inhibited. In this report, we show that lin‐ c‐kit+ lymphocytes express a variety of different chemokine receptors and that CCR6 identifies those cells located within CP. In contrast, cells found outside CP are positive for CXCR3 and exhibit a different surface marker profile, suggesting that at least two different populations of lin‐ c‐kit+ cells are present. The presence of CCR6 does not influence the expression of Notch molecules on lin‐ c‐kit+ cells, nor does it influence Notch ligand expression on bone marrow‐derived dendritic cells. In the human gut, CCR6 identifies clusters of lymphocytes resembling murine CP. CCR6 seems to have an important role for lin‐ c‐kit+ cells inside CP, is expressed in a regulated manner and identifies potential human CP.

Melanocortin‐derived tripeptide KPV has anti‐inflammatory potential in murine models of inflammatory bowel disease

Background: Despite some progress in recent years, the options for treating inflammatory bowel disease (IBD) are still dissatisfying, and surgery rates are still high. The anti‐inflammatory effects of melanocortin peptides such as alpha‐melanocyte‐stimulating hormone (&agr;‐MSH) have been described recently in, for example, dextran sodium sulfate (DSS) colitis in mice. The aim of this study was to investigate the therapeutic potential of the melanocortin‐derived tripeptide &agr;‐MSH(11–13) (KPV) and its mode of action in 2 models of intestinal inflammation. Methods: The anti‐inflammatory activity of KPV was analyzed in 2 well‐described models of IBD: DSS colitis, and CD45RBhi transfer colitis. Furthermore, animals expressing a nonfunctional melanocortin‐1 receptor (MC1Re/e) received DSS for induction of colitis and were treated with KPV. The course of inflammation was monitored by weight loss and histological changes in the colon as well as by myeloperoxidase (MPO) activity. Results: In the DSS‐colitis model, treatment with KPV led to earlier recovery and significantly stronger regain of body weight. Histologically, inflammatory infiltrates were significantly reduced in KPV‐treated mice, which was confirmed by the significant reduction of MPO activity in colonic tissue after KPV treatment. Supporting these findings, KPV treatment of transfer colitis led to recovery, regain of body weight, and reduced inflammatory changes histologically. In MC1Re/e mice, KPV treatment rescued all animals in the treatment group from death during DSS colitis. Conclusions: The melanocortin‐derived tripeptide KPV showed significant anti‐inflammatory effects in 2 murine models of colitis. These effects seem to be at least partially independent of MC1R signaling. In conclusion, our data suggest KPV as an interesting therapeutic option for the treatment of IBD.

Contrast-enhanced harmonic endoscopic ultrasound is able to discriminate benign submucosal lesions from gastrointestinal stromal tumors

Abstract Background. Although endoscopic ultrasound (EUS) has improved the diagnostic of potential malignancies, gastric lesions with suspicion of gastrointestinal stromal tumors (GIST) or benign lesions like lipoma or leiomyoma can often not be accurately differentiated by EUS, therefore, requiring tissue sampling with the risk of bleeding complications especially in GIST. As with the newest generation of EUS machines, contrast-enhanced harmonic endoscopic ultrasound (CEH-EUS) has become a new option to determine perfusion characteristics. The aim of this analysis was to evaluate whether CEH-EUS may help to discriminate various submucosal lesions. Methods. Data sets of 17 patients with suspicious gastric or esophageal lesions, who were investigated with CEH-EUS were analyzed. Perfusion characteristics were classified by the investigator immediately and statistically analyzed after investigation. Samples from EUS-fine needle aspirates, biopsy samples after needle cut or surgical specimen served as gold standard. Results. CEH-EUS showed nine lesions with reduced contrast enhancement (maximum intensity 6.2 ± 1.9 db) and eight lesions with hyperenhancement (47.3 ± 11.6 db). The latter eight lesions were all histologically identified as GIST, while the nine hypoenhanced lesions emerged to be four lipoma and five leiomyoma. Statistical analysis corresponded with initial perfusion classification in all cases. Conclusion. This is the first study showing that CEH-EUS can discriminate GIST from benign lesions with good accuracy. In the future, CEH-EUS-guided discrimination may lead to individualized diagnostic and therapeutic strategies in handling submucosal lesions.

Reprogramming of Monocytes by GM-CSF Contributes to Regulatory Immune Functions during Intestinal Inflammation

Human and murine studies showed that GM-CSF exerts beneficial effects in intestinal inflammation. To explore whether GM-CSF mediates its effects via monocytes, we analyzed effects of GM-CSF on monocytes in vitro and assessed the immunomodulatory potential of GM-CSF–activated monocytes (GMaMs) in vivo. We used microarray technology and functional assays to characterize GMaMs in vitro and used a mouse model of colitis to study GMaM functions in vivo. GM-CSF activates monocytes to increase adherence, migration, chemotaxis, and oxidative burst in vitro, and primes monocyte response to secondary microbial stimuli. In addition, GMaMs accelerate epithelial healing in vitro. Most important, in a mouse model of experimental T cell–induced colitis, GMaMs show therapeutic activity and protect mice from colitis. This is accompanied by increased production of IL-4, IL-10, and IL-13, and decreased production of IFN-γ in lamina propria mononuclear cells in vivo. Confirming this finding, GMaMs attract T cells and shape their differentiation toward Th2 by upregulating IL-4, IL-10, and IL-13 in T cells in vitro. Beneficial effects of GM-CSF in Crohn’s disease may possibly be mediated through reprogramming of monocytes to simultaneously improved bacterial clearance and induction of wound healing, as well as regulation of adaptive immunity to limit excessive inflammation.

Translational 18F-FDG PET/CT Imaging to Monitor Lesion Activity in Intestinal Inflammation

In patients with inflammatory bowel disease (IBD) and in murine IBD models, mucosal disease activity is routinely assessed by endoscopy and histologic evaluation. This information is valuable for monitoring treatment response, with mucosal healing being a major treatment goal. The aim of this study was to evaluate the translational potential of noninvasive 18F-FDG PET/CT for the assessment of mucosal damage in murine dextran sodium sulfate (DSS) colitis and human IBD. Methods: After induction of DSS colitis, 18F-FDG uptake was serially assessed from colonic volumes of interest defined on PET/CT scans and intraindividually correlated to histologic findings and to infiltrating cell types. In addition, 18F-FDG PET/CT scans of 25 Crohn disease patients were analyzed, and colonic 18F-FDG uptake was correlated to endoscopically assessed damage. Results: At days 4 and 7 after DSS induction, colonic 18F-FDG uptake was significantly increased, with a distinct peak in the medial colon. 18F-FDG uptake strongly correlated with histologic epithelial damage. Additionally, 18F-FDG uptake increased in the bone marrow in the course of the disease, correlating with an increase in intestinal 18F-FDG uptake. Histology and fluorescence-activated cell sorting analysis of the bone marrow of DSS mice revealed an increased number of immature neutrophils, whereas mucosal polymerase chain reaction suggested a correlation of 18F-FDG uptake to T cell infiltration. In accordance with the results of 18F-FDG PET/CT in DSS colitis, an increased 18F-FDG uptake was found in 87% of deep mucosal ulcerations in IBD patients, whereas mild endoscopic lesions were detected only by 18F-FDG PET/CT in about 50% of patients assessed. Conclusion: 18F-FDG PET/CT is a noninvasive method for evaluation of both experimental colitis and Crohn disease patients and thereby offers promising translational potential.

Immune Suppression via Glucocorticoid-Stimulated Monocytes: A Novel Mechanism To Cope with Inflammation

Glucocorticoids (GCs) are used as first-line therapies for generalized suppression of inflammation (e.g., allergies or autoimmune diseases), but their long-term use is limited by severe side effects. Our previous work revealed that GCs induced a stable anti-inflammatory phenotype in monocytes, the GC-stimulated monocytes (GCsMs) that we exploited for targeted GC-mediated therapeutic effects. We demonstrate that GCsMs interact with T cells in suppressing proliferation, as well as cytokine release of CD8+ and, especially, CD4+ T cells in vitro, and that they support generation of Foxp3+ cells. Therefore, we tested their immunosuppressive potential in CD4+ T cell–induced colitis in vivo. We found that injection of GCsMs into mice with severe colitis abolished the inflammation and resulted in significant clinical improvement within a few days. T cells recovered from GCsM-treated mice exhibited reduced secretion of proinflammatory cytokines IFN-γ and IL-17. Furthermore, clusters of Foxp3+ CD4+ T cells were detectable at local sites of inflammation in the colon. Thus, GCsMs are able to modify T cell responses in vitro and in vivo, as well as to downregulate and clinically cure severe T cell–mediated colitis.

Cytomegalovirus (CMV)-Specific Perforin and Granzyme B ELISPOT Assays Detect Reactivation of CMV Infection in Inflammatory Bowel Disease

The role of cytomegalovirus (CMV) infection in the pathogenesis and exacerbation of Inflammatory Bowel Disease (IBD) has been unresolved. Typically, the CMV genome remains dormant in infected cells, but a breakdown of immune surveillance can lead to re-activation of viral replication in the gut mucosa, which is not necessarily associated with viremia or changes in antibody titers. We hypothesized that the detection of CMV-specific CD8 effector T cells should permit the distinction between dormant and active CMV infection. As CD8 effector T cells, unlike memory CD8 T cells, have perforin (PFN) and granzyme B (GzB) preformed in their cytoplasmic granules, we employed single cell resolution ELISPOT assays to measure the CMV antigen-triggered release of these molecules by CD8 T cells isolated from subjects with IBD, and age-matched healthy controls. The frequencies of CMV-specific (GzB) and PFN-producing CD8 T cells were increased in IBD patients compared to healthy controls. Furthermore, the increased CMV reactivity was associated with active IBD disease and with longer disease duration. Notably, PCR on serum frequently failed to detect CMV DNA during flares. The data show that during active IBD there is a flare of CD8 T cell activity against CMV in a substantial proportion of IBD patients, suggesting CMV reactivation that serum PCR does not detect. While it remains open whether CMV reactivation is a cause or consequence of IBD, our data suggest that monitoring CMV antigen-specific effector CD8 T cells with GzB and PFN ELISPOT analysis can provide novel insights into the role of CMV infection in IBD. Additionally, our data have implications for the fields of transplantation, HIV, cancer, and autoimmune diseases, in all of which patient care critically depends on sensitive and reliable detection of a reactivation of CMV infection.

Nicotinamide treatment ameliorates the course of experimental colitis mediated by enhanced neutrophil-specific antibacterial clearance

SCOPE In previous studies, we could show that the B vitamin nicotinamide (NAM) enhanced antimicrobial activity of neutrophils. Here, we assessed the effects of NAM in two models of experimental colitis. METHODS AND RESULTS Colitis was induced in C57BL/6 mice either by oral infection with Citrobacter rodentium or by DSS (dextran sodium sulphate) administration, and animals were systemically treated with NAM. Ex vivo bacterial clearance was assessed in murine and human whole blood, as well as isolated human neutrophils. In C. rodentium-induced colitis, NAM treatment resulted in markedly decreased systemic bacterial invasion, histological damage and increased fecal clearance of C. rodentium by up to 600-fold. In contrast, NAM had no effect when administered to neutrophil-depleted mice. Ex vivo stimulation of isolated human neutrophils, as well as murine and human whole blood with NAM led to increased clearance of C. rodentium and enhanced expression of antimicrobial peptides in neutrophils. Moreover, NAM treatment significantly ameliorated the course of DSS colitis, as assessed by body weight, histological damage and myeloperoxidase activity. CONCLUSION Pharmacological application of NAM mediates beneficial effects in bacterial and chemically induced colitis. Future studies are needed to explore the clinical potential of NAM in the context of intestinal bacterial infections and human inflammatory bowel disease (IBD).

Isolation and eradication of a clinical isolate of Helicobacter pylori resistant to five antimicrobials in Germany

Sir, Colonization/infection with Helicobacter pylori is contracted during childhood, persists lifelong and causes chronic gastritis, which may be complicated by peptic ulcer disease, gastric cancer or mucosa-associated lymphoid tissue (‘MALT’) lymphoma. Failed H. pylori eradication therapy or antimicrobial therapy due to unrelated infections are the main risk factors for the development of resistance. In Germany, primary resistance amounts to 29% for metronidazole, 6% for clarithromycin, 15% for fluoroquinolones, 4% for double resistance (clarithromycin and metronidazole) and 1% for triple resistance (clarithromycin, metronidazole and fluoroquinolones) (E. Glocker, M. Kist and N. Wüppenhorst, unpublished data). After the first eradication therapy, resistance rates rise to 50% for metronidazole, 58% for clarithromycin, 30% for double resistance and 8% for triple resistance. Resistance to rifampicin/rifabutin (1.4%) and tetracycline is rare; amoxicillin resistance has not been described in Germany, so far. – 4 H. pylori resistant to three or more antimicrobials (multiresistant) have been described before, e.g. in Germany and Bulgaria. We describe the isolation and successful eradication of H. pylori showing resistance to clarithromycin, metronidazole, fluoroquinolones and rifampicin/rifabutin, and reduced susceptibility to tetracycline. H. pylori was identified twice from a middle-aged male patient with functional dyspepsia. Gastroduodenoscopy revealed a moderate antral gastritis; peptic ulcer disease and any other pathologies were ruled out. Relevant co-morbidities were asthma bronchiale and obstructive sleep apnoea. The patient received two courses of standard first-line H. pylori treatment, consisting of a proton pump inhibitor (PPI; standard dose twice daily), amoxicillin (1 g twice daily) and clarithromycin (250 mg twice daily) for 7 days (French triple), followed by one course of PPI (standard dose twice daily), clarithromycin (500 mg twice daily) and metronidazole (400 mg twice daily) for 7 days (Italian triple), and, afterwards, a rescue therapy with PPI (standard dose twice daily), amoxicillin (1 g twice daily) and rifabutin (150 mg twice daily) for 10 days. Following each eradication therapy, the patient reported significant but transient improvement of symptoms. However, H. pylori was still present, as proven by histopathology, rapid urease test and [C]urea breath test. Due to several respiratory tract infections, the patient had been treated in the past with moxifloxacin, clindamycin and azithromycin. There was no information about treatment with tetracycline during the last 12 months. Gastric tissue samples (antrum and corpus) were sent to the Institute of Medical Microbiology and Hygiene (Freiburg, Germany) for microbiological examination. Grown bacteria were identified as H. pylori and antimicrobial susceptibility testing (Etest) was performed as described previously. The following breakpoints were used: metronidazole, 8 mg/L; clarithromycin, 1 mg/L; levofloxacin, 1 mg/L; amoxicillin, 2 mg/L; tetracycline, 1 mg/L; and rifampicin, 4 mg/L. The strain showed resistance to metronidazole (MIC ≥256 mg/L), clarithromycin (MIC 16 mg/L), levofloxacin (MIC ≥32 mg/L) and rifampicin (MIC ≥32 mg/L), but was susceptible to amoxicillin (MIC 0.047 mg/L); the MIC of tetracycline was slightly higher than usually observed (0.75 mg/L). Based on these results, the patient received PPI (40 mg of omeprazole three times daily) and amoxicillin (1 g three times daily) for 14 days, which resulted in clinical improvement. Six months later, the patient presented again with dyspepsia and a positive [C]urea breath test. Re-gastroduodenoscopy followed by susceptibility testing of H. pylori revealed susceptibility to amoxicillin (MIC 0.032 mg/L), but resistance to metronidazole (MIC ≥256 mg/L), clarithromycin (MIC 24 mg/ L), levofloxacin (MIC ≥32 mg/L), rifampicin (MIC ≥32 mg/L) and tetracycline (MIC 1.5 mg/L). Genotyping resistance-associated genes showed mutations in the 23S rRNA (A2147G) and gyrA (D91G) genes, confirming phenotypic resistance to clarithromycin and levofloxacin. Phenotypic resistance to rifampicin/rifabutin was confirmed by detection of a D530V mutation in the rpoB gene in the strain isolated first and a D530N mutation in the latter strain. A possible explanation for these apparently inconsistent findings may be a mixed infection with different strains or clones. A single base pair A926G mutation in the 16S rRNA genes (rrnA/B) was found in both isolates, which was shown to be associated with resistance or reduced susceptibility to tetracycline. Whether this point mutation leads to treatment failures or Research letters

Isolated jejunal stricture formation two years after corrosive ingestion: Case report and review of literature

The ingestion of caustic agents is mostly accidental, especially in children. In adults, intentional, mainly suicidal cases of corrosive ingestion are frequently reported, which are more often fatal. Most corrosive ingestion is of cleaning substances, which mainly contain alkali substances. Since declaration rules for harmful substances have been restricted, the incidental ingestion of caustic substances has become rarer in the last years. However, the American Association of Poison Control Centres reported 216 228 poisonous human exposures to cleaning substances in 2007, which were mainly accidental.