Tara C. Rubinas

Modulation of the Intestinal Microbiota Alters Colitis-Associated Colorectal Cancer Susceptibility

It is well established that the intestinal microbiota plays a key role in the pathogenesis of Crohns disease (CD) and ulcerative colitis (UC) collectively referred to as inflammatory bowel disease (IBD). Epidemiological studies have provided strong evidence that IBD patients bear increased risk for the development of colorectal cancer (CRC). However, the impact of the microbiota on the development of colitis-associated cancer (CAC) remains largely unknown. In this study, we established a new model of CAC using azoxymethane (AOM)-exposed, conventionalized-Il10−/− mice and have explored the contribution of the host intestinal microbiota and MyD88 signaling to the development of CAC. We show that 8/13 (62%) of AOM-Il10−/− mice developed colon tumors compared to only 3/15 (20%) of AOM- wild-type (WT) mice. Conventionalized AOM-Il10−/− mice developed spontaneous colitis and colorectal carcinomas while AOM-WT mice were colitis-free and developed only rare adenomas. Importantly, tumor multiplicity directly correlated with the presence of colitis. Il10−/− mice mono-associated with the mildly colitogenic bacterium Bacteroides vulgatus displayed significantly reduced colitis and colorectal tumor multiplicity compared to Il10−/− mice. Germ-free AOM-treated Il10−/− mice showed normal colon histology and were devoid of tumors. Il10−/−; Myd88−/− mice treated with AOM displayed reduced expression of Il12p40 and Tnfα mRNA and showed no signs of tumor development. We present the first direct demonstration that manipulation of the intestinal microbiota alters the development of CAC. The TLR/MyD88 pathway is essential for microbiota-induced development of CAC. Unlike findings obtained using the AOM/DSS model, we demonstrate that the severity of chronic colitis directly correlates to colorectal tumor development and that bacterial-induced inflammation drives progression from adenoma to invasive carcinoma.

Clinical, endoscopic, and histologic findings distinguish eosinophilic esophagitis from gastroesophageal reflux disease.

BACKGROUND & AIMS Features of eosinophilic esophagitis (EoE) and gastroesophageal reflux disease (GERD) overlap; because they cannot be differentiated on the basis of eosinophil counts alone, it can be a challenge to distinguish these disorders. We aimed to characterize the clinical, endoscopic, and histologic features of EoE and GERD and to identify factors that might be used to differentiate them. METHODS We performed a retrospective case-control study on data collected from 2000 to 2007. Cases were patients of any age with EoE, as defined by recent consensus guidelines; controls were patients of any age with GERD. Clinical and endoscopic data were collected, and all esophageal biopsy specimens were reassessed by gastrointestinal pathologists. Cases and controls were compared, unconditional logistic regression was performed to develop a model to predict EoE, and receiver operator characteristic curves were constructed. RESULTS Data from 151 patients with EoE and 226 with GERD were analyzed. Compared with GERD, features that independently predicted EoE included younger age; symptoms of dysphagia; documented food allergies; observations of esophageal rings, linear furrows, white plaques, or exudates by upper endoscopy; an absence of a hiatal hernia, observed by upper endoscopy; a higher maximum eosinophil count; and the presence of eosinophil degranulation observed in biopsy specimens. The area under the curve for this model was 0.934. CONCLUSIONS We identified a set of readily available and routinely measured variables that differentiate EoE from GERD. Use of this type of analysis with patients suspected to have EoE might lead to more accurate diagnoses.

Esophageal dilation in eosinophilic esophagitis: safety and predictors of clinical response and complications

BACKGROUND Esophageal strictures resulting from eosinophilic esophagitis present management challenges, and high rates of rents and perforation have been reported. OBJECTIVE To assess the safety of esophageal dilation in eosinophilic esophagitis and to characterize predictors of both clinical response and complications of the procedure. DESIGN Retrospective study of the University of North Carolina eosinophilic esophagitis database. SETTING Tertiary care referral center. PATIENTS Cases of eosinophilic esophagitis were defined as per consensus guidelines. INTERVENTION Dilation with either Savary or through-the-scope balloon techniques. MAIN OUTCOME MEASUREMENTS Complications (deep mucosal rents, contained or free perforation, and chest pain requiring medical attention or hospitalization) and the global clinical symptom response. RESULTS Of 130 eosinophilic esophagitis cases identified, 70 dilations (12 Savary, 58 balloon) were performed in 36 patients. Esophageal size improved from 12 to 16 mm (P < .001), with an overall symptom response rate of 83%. The only predictor of clinical response was final dilation diameter. There were 5 complications (7%): 2 deep mucosal rents and 3 episodes of chest pain. There were no perforations. There was one hospitalization for chest pain. All complications occurred in patients being treated with topical steroids, who underwent balloon dilation. Complications were associated with younger age (23 vs 42; P = .02) and more dilations (4 vs 1.7; P = .009). LIMITATIONS Single center, retrospective study. CONCLUSIONS Esophageal dilation can be performed in eosinophilic esophagitis with low rates of tears, chest pain, and hospitalization. No perforations were found in our database. The effectiveness of dilation was best when a larger esophageal caliber was achieved, but patients undergoing more procedures was associated with complications.

KRAS/BRAF mutation status and ERK1/2 activation as biomarkers for MEK1/2 inhibitor therapy in colorectal cancer

Phase II clinical trials of mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors are ongoing and ERK1/2 activation is frequently used as a biomarker. In light of the mutational activation of BRAF and KRAS in colorectal cancer, inhibitors of the Raf-MEK-ERK mitogen-activated protein kinase are anticipated to be promising. Previous studies in pancreatic cancer have found little correlation between BRAF/KRAS mutation status and ERK1/2 activation, suggesting that identifying biomarkers of MEK inhibitor response may be more challenging than previously thought. The purpose of this study was to evaluate the effectiveness of MEK inhibitor therapy for colorectal cancer and BRAF/KRAS mutation status and ERK1/2 activation as biomarkers for MEK inhibitor therapy. First, we found that MEK inhibitor treatment impaired the anchorage-independent growth of nearly all KRAS/BRAF mutant, but not wild-type, colorectal cancer cells. There was a correlation between BRAF, but not KRAS, mutation status and ERK1/2 activation. Second, neither elevated ERK1/2 activation nor reduction of ERK1/2 activity correlated with MEK inhibition of anchorage-independent growth. Finally, we validated our cell line observations and found that ERK1/2 activation correlated with BRAF, but not KRAS, mutation status in 190 patient colorectal cancer tissues. Surprisingly, we also found that ERK activation was elevated in normal colonic epithelium, suggesting that normal cell toxicity may be a complication for colorectal cancer treatment. Our results suggest that although MEK inhibitors show promise in colorectal cancer, KRAS/BRAF mutation status, but not ERK activation as previously thought, may be useful biomarkers for MEK inhibitor sensitivity. [Mol Cancer Ther 2009;8(4):834–43]

Liver allograft antibody‐mediated rejection with demonstration of sinusoidal C4d staining and circulating donor‐specific antibodies

The importance of antibody‐mediated rejection (AMR) in ABO‐compatible liver transplantation is controversial. Here we report a prospective series of liver recipients with a preoperative positive crossmatch. To establish the diagnosis of AMR in liver recipients, the criteria described for kidney allografts were adopted. In approximately 10% of 197 liver transplants, we observed a positive T and B cell flow crossmatch before transplantation. Fifteen of 19 patients converted to negative crossmatches early after transplantation and displayed normal liver function while they were on routine immunosuppression. Four patients maintained positive crossmatches. Three of the 4 met the criteria for AMR and showed evidence of graft dysfunction, the presence of donor‐specific antibodies (DSAs), morphological tissue destruction with positive C4d linear staining on the graft sinusoidal endothelium, and improved function with attempts to eliminate DSAs. A persistently positive crossmatch after liver transplantation may lead to early, severe AMR and liver failure. C4d staining in the liver sinusoidal endothelium should alert one to the possibility of AMR. In our experience, patients with a positive crossmatch should have it repeated at 2 weeks and, if it is positive, again at 3 to 5 weeks. Recipients with an unknown preoperative crossmatch who develop early cholestasis of unclear etiology should be crossmatched or tested for the presence of DSAs to evaluate for AMR. Liver Transpl, 2011.

Tryptase staining of mast cells may differentiate eosinophilic esophagitis from gastroesophageal reflux disease

OBJECTIVES:Mast cells may contribute to the pathogenesis of eosinophilic esophagitis (EoE), but their role in diagnosis is unknown. Our aim was to determine whether tryptase staining of esophageal mast cells differentiates EoE from gastroesophageal reflux disease (GERD) and has utility for diagnosis of EoE.METHODS:We performed a case–control study comparing patients with EoE, defined by consensus guidelines, to GERD patients with eosinophils on esophageal biopsy. Immunohistochemistry was performed with mast cell tryptase. The density (mast cells/mm2) and intensity (0–4 scale) of mast cell staining was compared between groups after masking the diagnosis. Receiver operating characteristic (ROC) curves were constructed, and the area under the curve (AUC) was calculated to assess mast cell staining as both a stand-alone diagnostic test and an adjunctive assay with eosinophil counts.RESULTS:Fifty-four EoE (mean age 24 years; 69% male; mean 146 eosinophils per high-power field (eos/hpf)) and 55 GERD (mean age 34 years; 60% male; mean 20 eos/hpf) patients were analyzed. The maximum epithelial tryptase density was higher in EoE than in GERD (162±87 mast cells/mm2 vs. 67±54; P<0.001). Mast cells were diffusely distributed throughout the biopsy in more EoE than GERD patients (41 vs. 7%; P<0.001). Tryptase density and eosinophil count were only weakly correlated (R2=0.09; P=0.002). The AUC was 0.84 for tryptase staining alone, and 0.96 for the combination of mast cells and eosinophils.CONCLUSIONS:Patients with EoE have higher levels of tryptase-positive mast cells compared with GERD patients, improving the diagnostic value of biopsies beyond eosinophil counts alone. Mast cell tryptase may have utility as a diagnostic assay for EoE.

Cutting Edge: IFN-γ Is a Negative Regulator of IL-23 in Murine Macrophages and Experimental Colitis

IL-23 regulation is a central event in the pathogenesis of the inflammatory bowel diseases. We demonstrate that IFN-γ has anti-inflammatory properties in the initiation phase of IL-23–mediated experimental colitis. IFN-γ attenuates LPS-mediated IL-23 expression in murine macrophages. Mechanistically, IFN-γ inhibits Il23a promoter activation through altering NF-κB binding and histone modification. Moreover, intestinal inflammation is inhibited by IFN-γ signaling through attenuation of Il23a gene expression. In germ-free wild-type mice colonized with enteric microbiota, inhibition of colonic Il23a temporally correlates with induction of IFN-γ. IFN-γR1/IL-10 double-deficient mice demonstrate markedly increased colonic inflammation and IL23a expression compared with those of IL-10−/− mice. Colonic CD11b+ cells are the primary source of IL-23 and a target for IFN-γ. This study describes an impor-tant anti-inflammatory role for IFN-γ through inhibition of IL-23. Converging genetic and functional findings suggest that IL-23 and IFN-γ are important pathogenic molecules in human inflammatory bowel disease.

Evaluation of a Non-invasive Method to Detect Cytomegalovirus (CMV)-DNA in Stool Samples of Patients with Inflammatory Bowel Disease (IBD): A Pilot Study

BackgroundA severe flare of colitis in patients with IBD treated with immunosuppressive therapy may be complicated by an underlying CMV infection. The aim of this pilot study was to investigate the diagnostic efficacy of quantitative polymerase chain reaction (PCR) to detect CMV DNA in stool samples of IBD patients.MethodsTwenty-one patients with a severe flare of IBD, incompletely responding or refractory to either steroids or immunosuppressive agents, were included in the study. Nineteen patients completed the study according to the protocol undergoing an endoscopy with biopsies and collection of stool samples. Biopsy and stool samples were qualitatively and quantitatively analyzed for CMV DNA using real-time PCR.ResultsThirty-two percent (6/19) of the patients had detectable CMV DNA in colonic biopsies and in five (83%) of those patients CMV DNA was detected in the stool. Thirteen patients had negative findings for CMV DNA in biopsy and stool samples. The sensitivity, specificity, and accuracy of the PCR-based stool test for detection of CMV DNA compared to PCR-based detection of CMV in mucosal biopsies were 83, 93, and 90%, respectively.ConclusionsThe pilot study suggests a high accuracy of this non-invasive testing method to detect CMV DNA in stool samples as compared to mucosal biopsies. This approach may offer a non-endoscopic testing modality for underlying CMV infection in patients with a severe flare of IBD, which could also be applied more broadly to determine the prevalence of CMV infections in patients with IBD.

Oral Budesonide for the Therapy of Post-liver Transplant De Novo Inflammatory Bowel Disease: A Case Series and Systematic Review of the Literature

Background: The therapy for posttransplant IBD is clinically challenging. Patients receiving liver transplants are immunosuppressed to prevent rejection, but via an unknown mechanism develop de novo IBD in spite of receiving some of the same medications used for therapy in traditional IBD. In the published literature most of the patients who developed de novo IBD were treated with traditional corticosteroids. Exposure to systemic corticosteroids increases risks of infection, diabetes mellitus, and osteoporosis among other complications. Budesonide, a luminally active steroid with low systemic absorption, is an established therapeutic agent for IBD that should receive special considerations as first‐line therapy in this patient population. Methods: We describe 3 cases of de novo IBD after liver transplantation. None of these patients had a history of IBD prior to their transplant. All 3 were treated with oral budesonide in lieu of systemic corticosteroids. Additionally, a Medline MeSH search was performed using the terms “inflammatory bowel disease” and “liver transplant” as part of a systematic review of the literature. Results: All 3 cases of de novo post transplant IBD went into clinical remission with oral budesonide. The Medline search ultimately revealed 19 case reports, case series or retrospective reviews on de novo post liver transplant IBD. Most reports focused on the diagnosis and risk factors and did not have an emphasis on therapy. Conclusions: Given the track record for budesonide in traditional IBD, and its documented efficacy and systemic steroid‐sparing benefit, in our opinion this drug should be considered first‐line therapy for de novo posttransplant IBD.

Multifocal gastrointestinal stromal tumor (GIST) of the stomach in an 11-year-old girl

A previously healthy 11-year-old girl presented with an 8-month history of anemia and left upper quadrant abdominal pain. US examination demonstrated a 9-cm cystic mass with a fluid-fluid level in the left upper quadrant with unclear organ of origin. Abdominal MR imaging demonstrated a complex cystic mass, likely arising from the stomach. Additional T2 hyperintense submucosal lesions were identified in the gastric wall. Surgical excision confirmed the diagnosis of multifocal gastric gastrointestinal stromal tumor (GIST). MR imaging was helpful in suggesting a gastric origin of the primary mass and in demonstrating multifocal disease within the stomach.