Biljana Jovov

Molecular characterization of mucosal adherent bacteria and associations with colorectal adenomas.

The human large bowel is colonized by complex and diverse bacterial communities. However, the relationship between commensal bowel bacteria and adenomas (colorectal cancer precursors) is unclear. This study aimed to characterize adherent bacteria in normal colon and evaluate differences in community composition associated with colorectal adenomas. We evaluated adherent bacteria in normal colonic mucosa of 21 adenoma and 23 non-adenoma subjects enrolled in a cross sectional study. Terminal restriction fragment length polymorphism, clone sequencing and fluorescent in-situ hybridization analysis of the 16S rRNA genes were used to characterize adherent bacteria. A total of 335 clones were sequenced and processed for phylogenetic and taxonomic analysis. Differences in bacterial composition between cases and controls were evaluated by UniFrac and analysis of similarity matrix. Overall, Firmicutes (62%), Bacteroidetes (26%) and Proteobacteria (11%) were the most dominant phyla. The bacterial composition differed significantly between cases and controls (UniFrac P<0.001). We observed significantly higher abundance of Proteobacteria (p<0.05) and lower abundance of Bacteroidetes (p<0.05) in cases compared to controls. At the genus level, case subjects showed increased abundance of Dorea spp. (p<0.005), Faecalibacterium spp. (p<0.05), and lower proportions of Bacteroides spp. (p<0.03) and Coprococcus spp. (p< 0.05) than controls. Cases had higher bacterial diversity and richness than controls. These findings reveal that alterations in bacterial community composition associated with adenomas may contribute to the etiology of colorectal cancer. Extension of these findings could lead to strategies to manipulate the microbiota to prevent colorectal adenomas and cancer as well as to identify individuals at high risk.

Increased rectal microbial richness is associated with the presence of colorectal adenomas in humans.

Differences in the composition of the gut microbial community have been associated with diseases such as obesity, Crohn’s disease, ulcerative colitis and colorectal cancer (CRC). We used 454 titanium pyrosequencing of the V1–V2 region of the 16S rRNA gene to characterize adherent bacterial communities in mucosal biopsy samples from 33 subjects with adenomas and 38 subjects without adenomas (controls). Biopsy samples from subjects with adenomas had greater numbers of bacteria from 87 taxa than controls; only 5 taxa were more abundant in control samples. The magnitude of the differences in the distal gut microbiota between patients with adenomas and controls was more pronounced than that of any other clinical parameters including obesity, diet or family history of CRC. This suggests that sequence analysis of the microbiota could be used to identify patients at risk for developing adenomas.

Role of e-cadherin in the pathogenesis of gastroesophageal reflux disease

OBJECTIVES:An early event in the pathogenesis of gastroesophageal reflux disease (GERD) is an acid-induced increase in junctional (paracellular) permeability in esophageal epithelium (EE). The molecular events that account for this change are unknown. E-cadherin is a junctional protein important in barrier function in EE. Therefore, defects in barrier function in EE were sought in GERD as well as whether their presence correlated with abnormalities in e-cadherin.METHODS:Endoscopic biopsies of EE from GERD (n=20; male 10; female 10; mean age 50±10 years) and subjects with a healthy esophagus (controls; n=23; male 11; female 12; mean age 51±11 years) were evaluated in mini-Ussing chambers and by western blot and immunochemistry; and serum analyzed by enzyme-linked immunosorbent assay (ELISA). A role for e-cadherin was also assessed using a unique conditional knockout of e-cadherin in adult mouse esophagus.RESULTS:EE from GERD patients had lower electrical resistance and higher fluorescein flux than EE from controls; and the findings in GERD associated with cleavage of e-cadherin. Cleavage of e-cadherin in GERD was documented in EE by the presence of a 35-kDa, C-terminal fragment of the molecule on western blot and by an increase in soluble N-terminal fragments of the molecule in serum. Activation of the membrane metalloproteinase, A Disintegrin And Metalloproteinase (ADAM-10), was identified as a likely cause for cleavage of e-cadherin by western blot and immunostaining and a role for e-cadherin in the increased junctional permeability in EE from GERD supported by showing increased permeability after deletion of e-cadherin in mouse EE.CONCLUSIONS:The EE in GERD has increased junctional permeability and this is in association with proteolytic cleavage of e-cadherin. As loss of e-cadherin can, alone, account for the increase in junctional permeability, cleavage of e-cadherin likely represents a critical molecular event in the pathogenesis of GERD, and identification of cleaved fragments may, if confirmed in larger studies, be valuable as a biomarker of GERD.

The gastrointestinal microbiota and colorectal cancer

The human gut is home to a complex and diverse microbiota that contributes to the overall homeostasis of the host. Increasingly, the intestinal microbiota is recognized as an important player in human illness such as colorectal cancer (CRC), inflammatory bowel diseases, and obesity. CRC in itself is one of the major causes of cancer mortality in the Western world. The mechanisms by which bacteria contribute to CRC are complex and not fully understood, but increasing evidence suggests a link between the intestinal microbiota and CRC as well as diet and inflammation, which are believed to play a role in carcinogenesis. It is thought that the gut microbiota interact with dietary factors to promote chronic inflammation and CRC through direct influence on host cell physiology, cellular homeostasis, energy regulation, and/or metabolism of xenobiotics. This review provides an overview on the role of commensal gut microbiota in the development of human CRC and explores its association with diet and inflammation.

Differential Gene Expression between African American and European American Colorectal Cancer Patients

The incidence and mortality of colorectal cancer (CRC) is higher in African Americans (AAs) than other ethnic groups in the U. S., but reasons for the disparities are unknown. We performed gene expression profiling of sporadic CRCs from AAs vs. European Americans (EAs) to assess the contribution to CRC disparities. We evaluated the gene expression of 43 AA and 43 EA CRC tumors matched by stage and 40 matching normal colorectal tissues using the Agilent human whole genome 4x44K cDNA arrays. Gene and pathway analyses were performed using Significance Analysis of Microarrays (SAM), Ten-fold cross validation, and Ingenuity Pathway Analysis (IPA). SAM revealed that 95 genes were differentially expressed between AA and EA patients at a false discovery rate of ≤5%. Using IPA we determined that most prominent disease and pathway associations of differentially expressed genes were related to inflammation and immune response. Ten-fold cross validation demonstrated that following 10 genes can predict ethnicity with an accuracy of 94%: CRYBB2, PSPH, ADAL, VSIG10L, C17orf81, ANKRD36B, ZNF835, ARHGAP6, TRNT1 and WDR8. Expression of these 10 genes was validated by qRT-PCR in an independent test set of 28 patients (10 AA, 18 EA). Our results are the first to implicate differential gene expression in CRC racial disparities and indicate prominent difference in CRC inflammation between AA and EA patients. Differences in susceptibility to inflammation support the existence of distinct tumor microenvironments in these two patient populations.

Defective barrier function in neosquamous epithelium.

OBJECTIVES:Radiofrequency ablation (RFA) of Barretts esophagus (BE) is a common strategy for the prevention of esophageal adenocarcinoma (EAC). After RFA, the ablated esophagus heals on acid suppressive therapy, and is re-populated with a stratified squamous epithelium, referred to as “neosquamous epithelium (NSE).” Because the ability of the NSE to protect the underlying tissue from recurrent insult by reflux is unclear, we assessed the barrier function of NSE by comparing it to that of the native upper squamous epithelium (USE) in subjects having undergone RFA.METHODS:At varying intervals following RFA, the barrier function of NSE and USE were assessed in endoscopic biopsies by light and electron microscopy, and by measurement of electrical resistance (RT) and fluorescein flux in mini-Ussing chambers. Chamber results were further compared with results from control biopsies (healthy distal esophagus). A claudin expression profile in the tight junctions (TJs) of NSE and USE was determined using Quantitative reverse transcriptase PCR. Differential expression of claudin-4 between NSE and USE was assayed by immunoblots.RESULTS:USE was histologically normal whereas NSE showed dilated intercellular spaces and marked eosinophilia. NSE was also more permeable than USE and healthy controls, having lower mean RT and higher fluorescein fluxes. Abnormally low RT values for NSE were unrelated to the time period following RFA (or number of prior RFA sessions), being abnormal even 26 months after RFA. Abnormal permeability in NSE was associated with significantly lower values for claudin-4 and claudin-10 than in USE.CONCLUSIONS:NSE commonly exhibits defective barrier function. As this defect will make it vulnerable to injury, inflammation, and destruction by acidic and weakly acidic refluxates, it may in part explain incidences of recurrence of BE following ablation.

1060 Cleavage of E-Cadherin is More Specific Than Dilated Intercellular Spaces (DIS) Within Esophageal Epithelium (EE) for the Diagnosis of Gastroesophageal Reflux Disease (GERD)

INTRODUCTION: Maintenance of closure of the lower esophageal sphincter (LES) in resting state is central to the prevention of gastroesophageal reflux disease (GERD). There are many factors which contribute to the maintenance/failure of the “reflux barrier” of the LES. Here we present a detailed analysis of the anatomy and corresponding biomechanics of the gastroesophageal junction (GEJ) with the AIM of identifying structural differences, and consequent functional differences, in GEJ between normal subjects and GERD patients. METHODS: Three dimensional (3D) reconstructions of the GEJ from transverse magnetic resonance images (MRI) of 12 healthy volunteers and 12 reflux patients were studied for different breathing and meal phases. The orientation of the GEJ and the proximal stomach (angle φ in fig. 1), and the angle of His (θ in fig. 1) were evaluated with respect to an anatomical reference of the midpoint of the vertebrae. Biomechanical analysis of the GEJ was performed for both groups of subjects using the finite element (FE) methods to assess the mechanical consequences of the structural parameters of the GEJ. RESULTS: The angle of His increased with meal and inspiration in both normals and patients, however it always remained more acute (p=0.002) in volunteers (47.23±1.65°) than patients (54.62±1.66°). Interestingly, the orientation of GEJ and proximal stomach in patients was similar to volunteers during fasting, but became more postero-anterior during subsequent fed states. In addition the span of gastric contact with GEJ increased significantly with meal in volunteers (p=0.002) whereas patients showed no such change. FE simulations based on real geometry of the GEJ from 3D reconstruction showed higher intraluminal pressure in the GEJ with pressurization of the stomach when the angle of His was more acute. CONCLUSIONS: The difference in orientation of the GEJ and proximal stomach is possibly the reason for changes in angle of His between normals and patients. Structural and biomechanical differences in the GEJ between normals and patients indicate existence of a virtual “flap valve” in normals which might be dysfunctional in GERD patients.

Mucosal Adherent Bacteria, Inflammation and Colorectal Adenomas

G A A b st ra ct s 3 minority groups, with pancreatic cancer nationally has remained unchanged since 2002. Among all 5261 visits of minority patients to our medical center over the past 8 years, 1.3% (66) were in the Emergency Department, 11.1% (585) were in the inpatient setting, and 1.6% (84) were for surgery. These differences are statistically significant for the 3 sites of care (p<0.0001 using chi-square analysis) when compared withWhite/Non-Hispanic patients (0.2%, 7.3%, and 2.6%, respectively). CONCLUSION: A large percentage of the patients seen at our medical center for pancreatic cancer belong to a minority group but the percentage of these patients is declining, in contrast with the unchanged incidence observed nationally. Minority patients have significantly greater visits to the Emergency Department and inpatient services but significantly fewer surgical visits when compared with White/Non-Hispanic patients. This may reflect advanced stage of disease in the minority population, requiring inpatient care but precluding surgery. The causes of these trends are likely multifactorial but in need of further study in order to understand and improve the management and outcomes of minority patients with pancreas cancer.