Michelle Cheng

The antimicrobial peptide cathelicidin modulates Clostridium difficile-associated colitis and toxin A-mediated enteritis in mice

Background Clostridium difficile mediates intestinal inflammation by releasing toxin A (TxA), a potent enterotoxin. Cathelicidins (Camp as gene name, LL-37 peptide in humans and mCRAMP peptide in mice) are antibacterial peptides that also posses anti-inflammatory properties. Objectives To determine the role of cathelicidins in models of Clostridium difficile infection and TxA-mediated ileal inflammation and cultured human primary monocytes. Design Wild-type (WT) and mCRAMP-deficient (Camp−/−) mice were treated with an antibiotic mixture and infected orally with C difficile. Some mice were intracolonically given mCRAMP daily for 3 days. Ileal loops were also prepared in WT mice and treated with either saline or TxA and incubated for 4 h, while some TxA-treated loops were injected with mCRAMP. Results Intracolonic mCRAMP administration to C difficile-infected WT mice showed significantly reduced colonic histology damage, apoptosis, tissue myeloperoxidase (MPO) and tumour necrosis factor (TNF)α levels. Ileal mCRAMP treatment also significantly reduced histology damage, tissue apoptosis, MPO and TNFα levels in TxA-exposed ileal loops. WT and Camp−/− mice exhibited similar intestinal responses in both models, implying that C difficile/TxA-induced endogenous cathelicidin may be insufficient to modulate C difficile/TxA-mediated intestinal inflammation. Both LL-37 and mCRAMP also significantly reduced TxA-induced TNFα secretion via inhibition of NF-κB phosphorylation. Endogenous cathelicidin failed to control C difficile and/or toxin A-mediated inflammation and even intestinal cathelicidin expression was increased in humans and mice. Conclusion Exogenous cathelicidin modulates C difficile colitis by inhibiting TxA-associated intestinal inflammation. Cathelicidin administration may be a new anti-inflammatory treatment for C difficile toxin-associated disease.

Antifibrogenic Effects of the Antimicrobial Peptide Cathelicidin in Murine Colitis-Associated Fibrosis

Background & Aims Cathelicidin (LL-37 in human and mCRAMP in mice) represents a family of endogenous antimicrobial peptides with anti-inflammatory effects. LL-37 also suppresses collagen synthesis, an important fibrotic response, in dermal fibroblasts. Here, we determined whether exogenous cathelicidin administration modulates intestinal fibrosis in two animal models of intestinal inflammation and in human colonic fibroblasts. Methods C57BL/6J mice (n = 6 per group) were administered intracolonically with a trinitrobenzene sulphonic acid (TNBS) enema to induce chronic (6–7 weeks) colitis with fibrosis. We administered mCRAMP peptide (5 mg/kg every 3 day, week 5–7) or cathelicidin gene (Camp)-expressing lentivirus (107 infectious units week 4) intracolonically or intravenously, respectively. We then infected 129Sv/J mice with Salmonella typhimurium orally to induce cecal inflammation with fibrosis. Camp-expressing lentivirus (107 infectious units day 11) was administered intravenously. Results TNBS-induced chronic colitis was associated with increased colonic collagen (col1a2) mRNA expression. Intracolonic cathelicidin (mCRAMP peptide) administration or intravenous delivery of lentivirus-overexpressing cathelicidin gene significantly reduced colonic col1a2 mRNA expression in TNBS-exposed mice compared with vehicle administration. Salmonella infection also caused increased cecal inflammation associated with collagen (col1a2) mRNA expression that was prevented by intravenous delivery of Camp-expressing lentivirus. Exposure of human primary intestinal fibroblasts and human colonic CCD-18Co fibroblasts to transforming growth factor-β1 (TGF-β1) and/or insulin-like growth factor 1 induced collagen protein and mRNA expression, which was reduced by LL-37 (3–5 μM) through a MAP kinase-dependent mechanism. Conclusions Cathelicidin can reverse intestinal fibrosis by directly inhibiting collagen synthesis in colonic fibroblasts.

Cathelicidin suppresses colon cancer development by inhibition of cancer associated fibroblasts

Background Cathelicidin (LL-37 in humans and mCRAMP in mice) represents a family of endogenous antimicrobial and anti-inflammatory peptides. Cancer-associated fibroblasts can promote the proliferation of colon cancer cells and growth of colon cancer tumors. Methods We examined the role of cathelicidin in the development of colon cancer, using subcutaneous human HT-29 colon-cancer-cell-derived tumor model in nude mice and azoxymethane- and dextran sulfate-mediated colon cancer model in C57BL/6 mice. We also determined the indirect antitumoral mechanism of cathelicidin via the inhibition of epithelial–mesenchymal transition (EMT) of colon cancer cells and fibroblast-supported colon cancer cell proliferation. Results Intravenous administration of cathelicidin expressing adeno-associated virus significantly reduced the size of tumors, tumor-derived collagen expression, and tumor-derived fibroblast expression in HT-29-derived subcutaneous tumors in nude mice. Enema administration of the mouse cathelicidin peptide significantly reduced the size and number of colonic tumors in azoxymethane- and dextran sulfate-treated mice without inducing apoptosis in tumors and the adjacent normal colonic tissues. Cathelicidin inhibited the collagen expression and vimentin-positive fibroblast expression in colonic tumors. Cathelicidin did not directly affect HT-29 cell viability, but did significantly reduce tumor growth factor-β1-induced EMT of colon cancer cells. Media conditioned by the human colonic CCD-18Co fibroblasts promoted human colon cancer HT-29 cell proliferation. Cathelicidin pretreatment inhibited colon cancer cell proliferation mediated by media conditioned by human colonic CCD-18Co fibroblasts. Cathelicidin disrupted tubulin distribution in colonic fibroblasts. Disruption of tubulin in fibroblasts reduced fibroblast-supported colon cancer cell proliferation. Conclusion Cathelicidin effectively inhibits colon cancer development by interfering with EMT and fibroblast-supported colon cancer cell proliferation.

Fidaxomicin Inhibits Clostridium difficile Toxin A-Mediated Enteritis in the Mouse Ileum

ABSTRACT Clostridium difficile infection (CDI) is a common, debilitating infection with high morbidity and mortality. C. difficile causes diarrhea and intestinal inflammation by releasing two toxins, toxin A and toxin B. The macrolide antibiotic fidaxomicin was recently shown to be effective in treating CDI, and its beneficial effect was associated with fewer recurrent infections in CDI patients. Since other macrolides possess anti-inflammatory properties, we examined the possibility that fidaxomicin alters C. difficile toxin A-induced ileal inflammation in mice. The ileal loops of anesthetized mice were injected with fidaxomicin (5, 10, or 20 μM), and after 30 min, the loops were injected with purified C. difficile toxin A or phosphate-buffered saline alone. Four hours after toxin A administration, ileal tissues were processed for histological evaluation (epithelial cell damage, neutrophil infiltration, congestion, and edema) and cytokine measurements. C. difficile toxin A caused histologic damage, evidenced by increased mean histologic score and ileal interleukin-1β (IL-1β) protein and mRNA expression. Treatment with fidaxomicin (20 μM) or its primary metabolite, OP-1118 (120 μM), significantly inhibited toxin A-mediated histologic damage and reduced the mean histology score and ileal IL-1β protein and mRNA expression. Both fidaxomicin and OP-1118 reduced toxin A-induced cell rounding in human colonic CCD-18Co fibroblasts. Treatment of ileal loops with vancomycin (20 μM) and metronidazole (20 μM) did not alter toxin A-induced histologic damage and IL-1β protein expression. In addition to its well known antibacterial effects against C. difficile, fidaxomicin may possess anti-inflammatory activity directed against the intestinal effects of C. difficile toxins.

Circulating cathelicidin levels correlate with mucosal disease activity in ulcerative colitis, risk of intestinal stricture in Crohn’s disease, and clinical prognosis in inflammatory bowel disease

BackgroundCathelicidin (LL-37) is an antimicrobial peptide known to be associated with various autoimmune diseases. We attempt to determine if cathelicidin can accurately reflect IBD disease activity. We hypothesize that serum cathelicidin correlates with mucosal disease activity, stricture, and clinical prognosis of IBD patients.MethodsSerum samples were collected from two separate cohorts of patients at the University of California, Los Angeles. Cohort 1 consisted of 50 control, 23 UC, and 28 CD patients. Cohort 2 consisted of 20 control, 57 UC, and 67 CD patients. LL-37 levels were determined by ELISA. Data from both cohorts were combined for calculation of accuracies in indicating mucosal disease activity, relative risks of stricture, and odds ratios of predicting disease development.ResultsSerum cathelicidin levels were inversely correlated with Partial Mayo Scores of UC patients and Harvey-Bradshaw Indices of CD patients. Among IBD patients with moderate or severe initial disease activity, the patients with high initial LL-37 levels had significantly better recovery than the patients with low initial LL-37 levels after 6–18 months, suggesting that high LL-37 levels correlate with good prognosis. Co-evaluation of LL-37 and CRP levels was more accurate than CRP alone or LL-37 alone in the correlation with Mayo Endoscopic Score of UC patients. Low LL-37 levels indicated a significantly elevated risk of intestinal stricture in CD patients.ConclusionCo-evaluation of LL-37 and CRP can indicate mucosal disease activity in UC patients. LL-37 can predict future clinical activity in IBD patients and indicate risk of intestinal stricture in CD patients.

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

The Roles of Antimicrobial Peptides in the Regulation of Gastrointestinal Microbiota and Innate Immunity

Abstract Numerous antimicrobial peptides (AMPs) are expressed in the mucosa of the gastrointestinal tract where they are able to modulate innate immune responses and gut microbiota. Interestingly, the antimicrobial functions of AMPs are not necessarily the most important mechanism in the modulation of gastrointestinal diseases. Instead, AMPs may promote certain protective microbial species and modulate innate immune responses. The interactions of AMPs with innate immunity and gut microbiota reveal interesting drug targets. AMPs may serve as novel therapeutic approaches against gastrointestinal infection and inflammation, but the application of AMPs and their derivatives in treating gastrointestinal diseases remains at an early stage. AMPs may even directly modulate obesity and metabolic diseases. Additionally, AMPs may serve as biomarkers of gastrointestinal diseases, as expression of AMPs is often altered during the development of gastrointestinal diseases. This report summarizes the latest development of AMP-related gastrointestinal research with emphasis on innate immunity and gut microbiota.

Chronic Constipation and Its Complications An Interesting Finding to an Otherwise Commonplace Problem

Background. Fecalomas are hard dense masses separate from surrounding fecal material or bowel contents. This case report intends to provide a brief review of the literature and differential diagnosis for a pelvic mass in a pediatric patient. Case Presentation. The patient is a 5-year-old male presenting with worsening constipation and stool leakage over several months, found to have a rare calcified pelvic mass on abdominal X-ray consistent with a fecaloma. Conclusion. Fecalomas should be considered on the differential diagnosis of pediatric patients who present with chronic constipation and a calcified pelvic mass.

271 Cathelicidin Inhibits Colitis Associated Colon Cancer Development by Inhibition of Epithelial-Mesenchymal Transition and Cancer Associated Fibroblasts

Oxyntic atrophy and intestinal metaplasia are considered critical precursors for the development of gastric adenocarcinoma. Autoimmune atrophic gastritis (AAG) is characterized by immune-mediated destruction of gastric parietal cells, chronic inflammation, atrophic gastritis, and pernicious anemia. While AAG is associated with increased risk of carcinoids, the risk for adenocarcinoma in the absence of H. pylori infection is controversial. Here, we investigated the differences between AAG and chronic atrophic gastritis (CAG) with intestinal metaplasia (IM). Methods: Tissue from 6 patients with CAG were obtained at the time of resection for gastric adenocarcinomna and all patients showed IM. 20 AAG patients were diagnosed by upper endoscopy, oxyntic atrophy, and the presence of anti-parietal cell antibodies. 20/20 AAG patients were negative for H. pylori. 19/20 AAG patient showed marked elevations in both serum gastrin and serum chromogranin. In the body mucosa, we evaluated the presence of spasmolytic-polypeptide expressing metaplasia (SPEM) with TFF2 staining, IM with MUC2, macrophages with CD68, neutrophils with myeloperoxidase (MPO) and proliferative cells with Ki67. Results: 20/20 AAG patients showed profound parietal cell loss with SPEM and 15 demonstrated IM (75%). AAG patients had significantly fewer MPO positive neutrophils (6.4/1000 cells) compared to CAG patients (21.9/1000 cells) (p<0.01). The gastric biopsies from AAG patients also showed significantly fewer CD68 positive macrophages (10.1/1000 cells) compared to CAG patients (17.4/1000 cells, p= 0.01). Ki67 positive cells were markedly decreased in AAG patients (0.2/1000 cells) compared to CAG patients (14.5/1000 cells) (p<0.01). Conclusions: The metaplasia observed in AAG patients exhibited a low proliferative rate compared with the metaplasia samples from CAG patients. This lower proliferative rate was associated with smaller macrophage and neutrophil numbers in AAG patients compared to those in CAG patients. The lower proliferative rate in metaplasia and reduced macrophage and neutrophil infiltrates may explain in part the lower tendency of metaplasia in H. pylori-negative AAG patients to evolve into gastric adenocarcinoma compared with individuals with CAG.

Su1182 Intestinal Cathelicidin Levels Predict Prognosis of Ulcerative Colitis Patients

Introduction: Faecal Calprotectin (FC) is a cytosolic protein belonging to the S-100 family of calcium binding proteins found in neutrophils. It is excreted in the intestinal lumen in inflammatory conditions of the gut and can be used to distinguish irritable bowel syndrome (IBS) from other inflammatory bowel conditions such as colitis, diverticulosis, etc. Pointof-contact qualitative FC tests are now available and can be used in primary care to aid decision making for referrals to gastroenterologists for young patients presenting with chronic diarrhoea. Aims: To assess the feasibility and cost effectiveness of a primary care Pathway using a point-of-contact FC Test (Caldetect®) to aid decision making for referrals to gastroenterology in young patients presenting to their primary physicians with chronic diarrhoea. Methods: Primary Care data indicated that approximately 253 referrals are made annually to gastroenterologists from Primary Care to assess patients ,60years presenting with diarrhoea, costing approx. £119,000 for investigations and consultations. Using a Caldetect® (Preventis, GmbH) point of contact FC test, it was estimated that a saving of £89,000 could be achieved. A pathway for investigating chronic diarrhoea using Caldetect® was designed and implemented in the community (population 150,000) between September 2011-March 2012. (this will be presented). FC results were categorised using manufacturer cut-offs of ,15ug/ g, 15-60ug/g and .60ug/g. Patients with FC results of 15-60 and .60 were deemed to have an inflammatory process and referred to Gastroenterology Clinics. Cost analysis was carried out using the 2010-11 tariffs for the NHS. Results: 142 Caldetect® tests were carried out in Primary Care during this pilot phase. Of these, a negative result ( ,15ug/g) was present in 89, with 36 tests being .60ug/g. 3 tests were at the intermediate level and 14 tests could not be accurately reported. Negative results were managed in primary care as IBS. A monthly cost savings of £6100 was calculated taking consultation and endoscopy tariffs into account. Conclusion: This pilot study demonstrates the feasibility and cost effectiveness of a Pathway for decision making and a point-of-care faecal calprotectin test in rationalising referrals to Gastroenterologists for chronic diarrhoea.