Faculty Bibliography

BACKGROUND: The pathogenesis of inflammatory bowel disease (IBD) may be caused by abnormal interactions between the immune system and microbiome. Recent studies using 16S ribosomal sequencing have shown that IBD is associated with dysbiosis of the microbiota. Inflammation may alter nutrient availability to adherent mucosal bacteria and impact their metabolic function. Microbial metabolites may also regulate intestinal CD4+ T cell homeostasis. We investigated the relationship between inflammation and microbial function by inferred metagenomics of the mucosal microbiota from colonic pinch biopsies of IBD patients to characterize differences in microbial metabolic pathways between inflamed and non-inflamed biopsy sites. METHODS: Institutional review board approval was obtained before involving patients in the study. Paired pinch biopsy samples of known inflammation states were analyzed from UC (23), CD (21) and controls (24) by 16S ribosomal sequencing and inferred metagenomics with comparison to pathology results and flow cytometry data. The V4 region of the 16S rRNA gene was amplified and sequenced on a MiSeq sequencer. Sequences were assigned to operational taxonomic units (OTUs) and classified taxonomically according to the Ribosomal Database Project (RDP) for use in taxonomic analysis. PICRUSt was then used with the Greengenes OTU database to generate metagenomic data, and derive relative Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway abundance information. Leukocytes were isolated from paired biopsy samples and activated with PMA/Ionomycin for intracellular cytokine (IL-22, IL-17, IFNg, IL-4 and TNFa) as well as nuclear antigen (Foxp3) analysis by multi-color flow cytometry on a BD LSRII. Active inflammation was defined by neutrophil infiltration into the epithelium, in the setting of epithelial cell damage. RESULTS: Carriage of metabolic pathways in the mucosal microbiota was relatively stable among IBD patients despite large variations in individual bacterial community structures (!

Electronic laboratory notebooks (ELNs) offer significant advantages over traditional paper laboratory notebooks (PLNs), yet most research labs today continue to use paper documentation. While biopharmaceutical companies represent the largest portion of ELN users, government and academic labs trail far behind in their usage. Our lab, a translational science laboratory at New York University School of Medicine (NYUSoM), wanted to determine if an ELN could effectively replace PLNs in an academic research setting. Over 6 months, we used the program Evernote to record all routine experimental information. We also surveyed students working in research laboratories at NYUSoM on the relative advantages and limitations of ELNs and PLNs and discovered that electronic and paper notebook users alike reported the inability to freehand into a notebook as a limitation when using electronic methods. Using Evernote, we found that the numerous advantages of ELNs greatly outweighed the inability to freehand directly into a notebook. We also used imported snapshots and drawing program add-ons to obviate the need for freehanding. Thus, we found that using Evernote as an ELN not only effectively replaces PLNs in an academic research setting but also provides users with a wealth of other advantages over traditional paper notebooks.

Diet influences host metabolism and intestinal microbiota; however, detailed understanding of this tripartite interaction is limited. To determine whether the nonfermentable fiber hydroxypropyl methylcellulose (HPMC) could alter the intestinal microbiota and whether such changes correlated with metabolic improvements, C57B/L6 mice were normalized to a high-fat diet (HFD), then either maintained on HFD (control), or switched to HFD supplemented with 10% HPMC, or a low-fat diet (LFD). Compared to control treatment, both LFD and HPMC reduced weight gain (11.8 and 5.7 g, respectively), plasma cholesterol (23.1 and 19.6%), and liver triglycerides (73.1 and 44.6%), and, as revealed by 454-pyrosequencing of the microbial 16S rRNA gene, decreased microbial alpha-diversity and differentially altered intestinal microbiota. Both LFD and HPMC increased intestinal Erysipelotrichaceae (7.3- and 12.4-fold) and decreased Lachnospiraceae (2.0- and 2.7-fold), while only HPMC increased Peptostreptococcaceae (3.4-fold) and decreased Ruminococcaceae (2.7-fold). Specific microorganisms were directly linked with weight change and metabolic parameters in HPMC and HFD mice, but not in LFD mice, indicating that the intestinal microbiota may play differing roles during the two dietary modulations. This work indicates that HPMC is a potential prebiotic fiber that influences intestinal microbiota and improves host metabolism.

Our aim was to examine the humoral immune response against Streptococcus gallolyticus subspecies gallolyticus antigens in individuals subjected to a routine colonoscopy in which colon adenomatous polyps were present or not. Serum samples from 133 individuals with adenomatous polyps and serum samples from 53 individuals with a normal colonoscopy were included. Western blot was performed in all subjects using a whole cell antigen from S. gallolyticus ATCC 9809, and rabbit antisera against the whole cell bacteria was prepared as a control. By analyzing the immune profile of the rabbit-immunized sera by Western-blot, at least 22 proteins were identified as immunogenic in S. gallolyticus. When we evaluated sera from human subjects, two proteins of approximately 30 and 22 kDa were most prominent. Based on this 2-protein band pattern, Western-blot profiles from human subjects were compared. The detection of a protein band of 22 kDa was associated with the presence of adenomatous polyps in colon [odds ratios (OR) 7.98, 95% confidence intervals (CI): 3.54-17.93], p < 0.001. When the presence of the 30 kDa protein alone or both the 22 and 30 kDa proteins were analyzed, the OR increased to 22.37 (95% CI: 3.77-131.64), p < 0.001. The specificity was 84.9 for the presence of the 22 kDa protein, and 98.1 for the presence of the 30 kDa protein alone or both 22 and 30 kDa bands. Serum from individuals with adenomatous polyps recognized two proteins from S. gallolyticus. This result confirmed the possible association of S. gallolyticus with adenomatous polyps in the colon.

OBJECTIVES/SPECIFIC AIMS: Th e potential role of microbes in the pathogenesis of colorectal cancer remain unclear. Recent studies suggest that Fusobacterium species are associated with CRC but did not provide evidence of causation. Th e objective of this study was to determine the relationship between Fusobacterium and adenomas found on screening colonoscopies. METHODS/STUDY POPULATION: Patients were enrolled at the New York Harbor VA Hospital Screening Colonoscopy Clinic. Stool specimens were obtained from patients prior to undergoing bowel preparation. Study subjects were matched by age, gender, and ethnicity to normal controls (n = 5/group). High-grade adenoma was defined as size >1 cm or villous features on histology while low-grade was defined as fewer than three adenomas <1 cm. DNA was extracted from stool and quantitative PCR performed using degenerate primers targeting the 16S rDNA and Fusobacterium species. Fusobacterium gene copies were normalized to total 16S DNA. Comparisons amongst the groups were performed using the Mann-Whitney U test. RESULTS/ANTICIPATED RESULTS: Th ere were no significant differences in total 16S gene copies amongst the control, low-grade adenoma, and high-grade adenoma groups. Fusobacterium gene copies were significantly lower in patients with high-grade than with low-grade adenomas (p < 0.05). Fusobacterium gene copies were lower in high-grade adenomas compared to controls but the difference was not statistically significant. DISCUSSION/SIGNIFICANCE OF IMPACT: Our data indicates that there is no positive association between Fusobacteria and low- or high-grade premalignant colonic adenomas. Fusobacteria appear to be decreased in high-grade adenomas. Th ese findings suggest that Fusobacteria may not play a causative role in the development of colorectal cancer from adenomas but may preferentially colonize the mucosal surface of colorectal cancers

Interest in the role of the microbiome in human health has burgeoned over the past decade with the advent of new technologies for interrogating complex microbial communities. The large-scale dynamics of the microbiome can be described by many of the tools and observations used in the study of population ecology. Deciphering the metagenome and its aggregate genetic information can also be used to understand the functional properties of the microbial community. Both the microbiome and metagenome probably have important functions in health and disease; their exploration is a frontier in human genetics.

There is increasing evidence that dysregulation of CD4(+) T cell populations leads to intestinal inflammation, but the regional distribution of these populations throughout the intestinal tract in healthy individuals remains unclear. Here, we show that T(H)17, T(H)22 and T(Reg) cells are enriched in the healthy human cecum compared to the terminal ileum and sigmoid colon, whereas T(H)1 and T(H)2 cells do not significantly vary by location. Transcriptional profiling analysis of paired pinch biopsies from different regions of the intestine identified significant differences in the metabolic state of the terminal ileum, cecum, and sigmoid colon. An increased proportion of T(H)17 cells was positively associated with expression of resistin (RETN) and negatively associated with expression of trefoil factor 1 (TFF1). These results suggest that CD4(+) T helper cells that are important in maintaining mucosal barrier function may be enriched in the cecum as a result of metabolic differences of the surrounding microenvironment.

Colorectal cancer is a major cause of cancer-related morbidity and mortality in the United States and many other regions of the world. Our understanding of the pathogenesis of colorectal cancer, from the precursor adenomatous polyp to adenocarcinoma, has evolved rapidly. Colorectal carcinogenesis is a sequential process characterized by the accumulation of multiple genetic and molecular alterations in colonic epithelial cells. However, the development of colorectal cancer involves more then just a genetic predisposition. External or environmental factors presumably play a significant role, and inflammatory bowel diseases, obesity, alcohol consumption, and a diet high in fat and low in fiber have all been implicated as risk factors for the development of either colonic adenomas or carcinomas. We are becoming increasingly aware of microbes as causes of malignancies. This article reviews the various microbes that have been associated with the development of colorectal carcinomas