John Losurdo

A Compositional Look at the Human Gastrointestinal Microbiome and Immune Activation Parameters in HIV Infected Subjects

HIV progression is characterized by immune activation and microbial translocation. One factor that may be contributing to HIV progression could be a dysbiotic microbiome. We therefore hypothesized that the GI mucosal microbiome is altered in HIV patients and this alteration correlates with immune activation in HIV. 121 specimens were collected from 21 HIV positive and 22 control human subjects during colonoscopy. The composition of the lower gastrointestinal tract mucosal and luminal bacterial microbiome was characterized using 16S rDNA pyrosequencing and was correlated to clinical parameters as well as immune activation and circulating bacterial products in HIV patients on ART. The composition of the HIV microbiome was significantly different than that of controls; it was less diverse in the right colon and terminal ileum, and was characterized by loss of bacterial taxa that are typically considered commensals. In HIV samples, there was a gain of some pathogenic bacterial taxa. This is the first report characterizing the terminal ileal and colonic mucosal microbiome in HIV patients with next generation sequencing. Limitations include use of HIV-infected subjects on HAART therapy.

Endoscopy in eosinophilic esophagitis: “feline” esophagus and perforation risk

BACKGROUND & AIMS Idiopathic eosinophilic esophagitis is an underdiagnosed disease with typical endoscopic findings, which have not been well described. METHODS Charts and pathology reports at two tertiary care centers from June 1993 to April 2002 were reviewed to describe the endoscopic findings of this disease and to correlate them with clinical characteristics. Eight patients were identified as having eosinophilic esophagitis based on clinical symptoms and pathology reports. RESULTS Soft and subtle ring(s) in the esophagus were found in 7 of 8 patients. In 3 of 8 patients, the esophagus appeared rigid. Mucosal rents occurred with simple passage of the endoscope in 5 of 8 patients. One patient developed a perforation after simple passage of the endoscope. Endoscopic findings can be normal or very subtle in these patients, and the findings can easily be missed during endoscopy. Tearing of the esophagus can occur with simple passage of the endoscope or biopsy even in the absence of overt rings. A minimum of 8 weeks of medical therapy (proton pump inhibitor, histamine antagonists, immunosuppressants) should be undertaken before considering dilation because of the high risk involved with the procedure and the good response to medical therapy. CONCLUSIONS We recommend considering dilation only in patients with eosinophilic esophagitis who do not respond to medical therapy and have rings that appear to be obstructing the lumen.

Carbonylation and disassembly of the F-actin cytoskeleton in oxidant induced barrier dysfunction and its prevention by epidermal growth factor and transforming growth factor alpha in a human colonic cell line.

BACKGROUND Intestinal barrier dysfunction concomitant with high levels of reactive oxygen metabolites (ROM) in the inflamed mucosa have been observed in inflammatory bowel disease (IBD). The cytoskeletal network has been suggested to be involved in the regulation of barrier function. Growth factors (epidermal growth factor (EGF) and transforming growth factor α (TGF-α)) protect gastrointestinal barrier integrity against a variety of noxious agents. However, the underlying mechanisms of oxidant induced disruption and growth factor mediated protection remain elusive. AIMS To determine: (1) if oxidation and disassembly of actin (a key cytoskeletal component) plays a major role in ROM induced epithelial monolayer barrier dysfunction; and (2) if growth factor mediated protection involves prevention of theses alterations. METHODS Caco-2 monolayers were preincubated with EGF, TGF-α, or vehicle before incubation with ROM (H2O2 or HOCl). Effects on cell integrity, barrier function, and G- and F-actin (oxidation, disassembly, and assembly) were determined. RESULTS ROM dose dependently and significantly increased F- and G-actin oxidation (carbonylation), decreased the stable F-actin fraction (index of stability), and increased the monomeric G-actin fraction (index of disassembly). Concomitant with these changes were disruption of the actin cytoskeleton and loss of the monolayer barrier function. In contrast, growth factor pretreatment decreased actin oxidation and enhanced the stable F-actin, while in concert prevented actin disruption and restored normal barrier function of monolayers exposed to ROM. Cytochalasin-D, an inhibitor of actin assembly, not only caused actin disassembly and barrier dysfunction but also abolished the protective action of growth factors. Moreover, an actin stabilising agent, phalloidin, mimicked the protective actions of the growth factors. CONCLUSIONS Oxidation, disassembly, and instability of the actin cytoskeleton appears to play a key role in the mechanism of oxidant induced loss of intestinal barrier integrity. In contrast, organisation and stabilisation of actin through promotion of its assembly plays a critical role in the mechanism of growth factor mediated protection.

Successful and sustained treatment of chronic radiation proctitis with antioxidant vitamins E and C

OBJECTIVE:Chronic radiation proctitis, a common sequelae of pelvic radiation, is characterized by obliteration of the submucosal vasculature with subsequent ischemia and reperfusion injury. Oxidative stress is thought to be a major mechanism in radiation proctitis. Therefore, antioxidants (vitamins E and C) may be beneficial.METHODS:Twenty consecutive symptomatic outpatients with endoscopically documented radiation proctitis seen in a single gastroenterology clinic were given a combination of vitamin E (400 IU tid) and vitamin C (500 mg tid). Previous radiation therapy was given for prostatic (n = 10) or gynecological (n = 10) malignancies. These patients presented with one or more of the following symptoms: rectal bleeding, rectal pain, diarrhea, or fecal urgency. Using a questionnaire, these symptoms were rated by the patients in terms of their severity (grade 0–4) and frequency (grade 0–4) before and after treatment with vitamins E and C. A symptom index was calculated by the addition of the severity and frequency scores (8 = most symptomatic). The lifestyle impact of the symptoms was also assessed by questionnaire grading from 0 (no effect on daily activity) to 4 (afraid to leave home). Among these 20 patients, 10 patients who received vitamins E and C for 1 yr were assessed again to determine whether their initial responses were sustained.RESULTS:There was a significant (p < 0.05; Wilcoxon rank) improvement in the symptom index (before treatment vs after treatment with vitamins E and C) for bleeding (median score: 4 vs 0), diarrhea (median score: 5 vs 0), and urgency (median score: 6 vs 3). Patients with rectal pain did not improve significantly. Bleeding resolved in four of 11 patients, diarrhea resolved in eight of 16 patients, fecal urgency resolved in three of 16 patients, and rectal pain resolved in two of six patients. Lifestyle improved in 13 patients, including seven patients who reported a return to normal. Two of the patients with no improvement in their daily symptoms also had radiation ileitis. All 10 patients who underwent a second follow-up interview reported sustained improvement in their symptoms 1 yr later.CONCLUSION:A substantial number of patients with radiation proctitis seem to benefit from antioxidant therapy. A double-blind placebo-controlled trial is needed to confirm this open-labeled pilot study.

Inpatient capsule endoscopy leads to frequent incomplete small bowel examinations

AIM To examine the predictive factors of capsule endoscopy (CE) completion rate (CECR) including the effect of inpatient and outpatient status. METHODS We identified 355 consecutive patients who completed CE at Rush University Medical Center between March 2003 and October 2005. Subjects for CE had either nothing by mouth or clear liquids for the afternoon and evening of the day before the procedure. CE exams were reviewed by two physicians who were unaware of the study hypotheses. After retrospective analysis, 21 cases were excluded due to capsule malfunction, prior gastric surgery, endoscopic capsule placement or insufficient data. Of the remaining 334 exams [264 out-patient (OP), 70 in-patient (IP)], CE indications, findings, location of the patients [IP vs OP and intensive care unit (ICU) vs general medical floor (GMF)] and gastrointestinal transit times were analyzed. Statistical analysis was completed using SPSS version 17 (Chicago, IL). Chi-square, t test or fisher exact-tests were used as appropriate. Multivariate logistic regression analysis was used to identify variables associated with incomplete CE exams. RESULTS The mean age for the entire study population was 54.7 years. Sixty-one percent of the study population was female, and gender was not different between IPs vs OPs (P = 0.07). The overall incomplete CECR was 14% in our study. Overt obscure gastrointestinal bleeding (OGB) was significantly more common for the IP CE (P = 0.0001), while abdominal pain and assessment of IBD were more frequent indications for the OP CE exams (P = 0.002 and P = 0.01, respectively). Occult OGB was the most common indication and arteriovenous malformations were the most common finding both in the IPs and OPs. The capsule did not enter the small bowel (SB) in 6/70 IPs and 8/264 OPs (P = 0.04). The capsule never reached the cecum in 31.4% (22/70) of IP vs 9.5% (25/ 264) of OP examinations (P < 0.001). The mean gastric transit time (GTT) was delayed in IPs compared to OPs, 98.5 ± 139.5 min vs 60.4 ± 92.6 min (P = 0.008). Minimal SB transit time was significantly prolonged in the IP compared to the OP setting [IP = 275.1 ± 111.6 min vs OP = 244.0 ± 104.3 min (P = 0.037)]. CECR was also significantly higher in the subgroup of patients with OGB who had OP vs IP exams (95% vs 80% respectively, P = 0.001). The proportion of patients with incomplete exams was higher in the ICU (n = 7/13, 54%) as compared to the GMF (n = 15/57, 26%) (P = 0.05). There was only a single permanent SB retention case which was secondary to a previously unknown SB stricture, and the remaining incomplete SB exams were due to slow transit. Medications which affect gastrointestinal system motility were tested both individually and also in aggregate in univariate analysis in hospitalized patients (ICU and GMF) and were not predictive of incomplete capsule passage (P > 0.05). Patient location (IP vs OP) and GTT were independent predictors of incomplete CE exams (P < 0.001 and P = 0.008, respectively). CONCLUSION Incomplete CE is a multifactorial problem. Patient location and related factors such as severity of illness and sedentary status may contribute to incomplete exams.

The Effects of Bowel Preparation on Microbiota-Related Metrics Differ in Health and in Inflammatory Bowel Disease and for the Mucosal and Luminal Microbiota Compartments.

OBJECTIVES:Bowel preparations (BPs) taken before colonoscopy may introduce a confounding effect on the results of gastrointestinal microbiota studies. This study aimed to determine the effect of bowel preparation on the mucosa-associated and luminal colonic microbiota in healthy subjects (HC) and inflammatory bowel disease (IBD) patients.METHODS:Biopsy samples (n=36) and fecal samples (n=30) were collected from 10 HC and 8 IBD subjects pre- and post-BP. 16S rRNA gene was pyrosequenced using 454 Titanium protocols. We compared the differences between the pre- and post-BP samples (i.e., comparisons-across-bowel-prep); we examined the effect of BP on the expected separation of the mucosal vs. the luminal compartments (i.e., comparisons-across-compartments). Last, we compared the baseline differences between the HC vs. IBD groups (a secondary analysis), and examined whether the differences between the HC vs. IBD changed after BP.RESULTS:In comparisons-across-bowel-prep, the Shannon’s index (SI) decreased only in the biopsy samples of IBD subjects post-BP (P=0.025) and phylogenetic diversity-whole tree (PD-WT) metric decreased in biopsy samples of HC subjects post-BP (P=0.021). In secondary comparisons, the subtle differences between the fecal samples of the HC vs. IBD groups, in terms of evenness and the SI, were not apparent post-BP. In terms of β-diversity, in comparisons-across-bowel-prep, the proportion of shared operational taxonomic units (OTUs) in pre- and post-BP samples was low (~30%) and unweighted Unifrac distances between pre- and post-BP specimens ranged from 0.52 to 0.66. HC biopsies were affected more than IBD biopsies with BP (P=0.004). In comparisons-across-compartments, the proportion of shared OTUs between biopsy and fecal samples increased and Unifrac distances decreased post-BP in IBD subjects, reducing the differences between the mucosal and luminal compartments of the gut microbiota. Interindividual differences in Unifrac distances were preserved even with BP effects, although the effects were greater on weighted Unifrac distances. Bacteroidetes and its subtypes increased post-BP in both the luminal and mucosal compartments.CONCLUSIONS:Bowel preparations affect the composition and diversity of the fecal and luminal microbiota in the short term, introducing potential bias into experiments examining the gut microbiota. The magnitude of the effect of BP is not greater than that of interindividual variation. Both the luminal and mucosal compartments of the gut microbiota get affected, and samples from controls and IBD subjects may get affected differently. Studies of the colonic microbiota should take into account the direction and the magnitude of the change introduced by BP during the design stage of the experiments, and consider sample sizes so that potential bias is minimized.

HIV-associated mucosal gene expression: region-specific alterations

Objective:Despite the use of HAART to control HIV, systemic immune activation and inflammation persists with the consequence of developing serious non-AIDS events. The mechanisms that contribute to persistent systemic immune activation have not been well defined. The intestine is the major source of “sterile” inflammation and plays a critical role in immune function; thus, we sought to determine whether intestinal gene expression was altered in virally controlled HIV-infected individuals. Design and methods:Gene expression was compared in biopsy samples collected from HIV-uninfected and HIV-infected individuals from the ileum, right colon (ascending colon), and left colon (sigmoid). Affymetrix gene arrays were performed on tissues and pathway analyses were conducted. Gene expression was correlated with systemic markers of intestinal barrier dysfunction and inflammation and intestinal microbiota composition. Results:Genes involved in cellular immune response, cytokine signaling, pathogen-influenced signaling, humoral immune response, apoptosis, intracellular and second messenger signaling, cancer, organismal growth and development, and proliferation and development were upregulated in the intestine of HIV-infected individuals with differences observed in the ileum, right, and left colon. Gene expression in the ileum primarily correlated with systemic markers of inflammation (e.g., IL7R, IL2, and TLR2 with serum TNF) whereas expression in the colon correlated with the microbiota community (e.g., IFNG, IL1B, and CD3G with Bacteroides). Conclusion:These data demonstrate persistent, proinflammatory changes in the intestinal mucosa of virally suppressed HIV-infected individuals. These changes in intestinal gene expression may be the consequence of or contribute to barrier dysfunction and intestinal dysbiosis observed in HIV.

Hepatitis B vaccination in patients with inflammatory bowel disease

AIM To determine the prevalence for hepatitis B virus (HBV) and HBV screening and vaccination practices for inflammatory bowel disease (IBD). METHODS This study is a retrospective, cross-sectional observational study. A retrospective chart review was performed in 500 patients who have been consecutively treated for IBD between September 2008 and January 2013 at the Rush University Medical Center Gastroenterology section. The patients were identified through the electronic medical record with the criteria that they attended the gastroenterology clinic, and that they had a diagnosis of IBD at the time of visit discharge. Once identified, each record was analyzed to determine whether the subject had been infected with HBV in the past, already been vaccinated against HBV, or advised to get vaccinated and followed through with the recommended vaccination. RESULTS About 254 out of 500 patients (51%) had HBV screening ordered. Among those ordered to have screening tests, 86% followed through with HBV serology. Gastroenterology physicians had significantly different screening ratios from each other (P < 0.001). There were no significant differences in the ratios of HBV screening when IBD specialists were compared to other gastroenterology physicians (0.505 ± 0.023 vs 0.536 ± 0.066, P = 0.66). Of those 220 patients screened, 51% of IBD patients were found not to be immune against HBV. Approximately 50% of gastroenterology physicians recommended HBV vaccinations to their patients in whom serology was negative for antibodies against HBV. IBD specialists recommended vaccinations to a higher percentage of their patients compared to other gastroenterology physicians (0.168 ± 0.019 vs 0.038 ± 0.026, P = 0.015). Present and/or past HBV infection was found in 3.6% of the patients who had serology checked. There was no statistically significant difference in the prevalence of hepatitis B surface antigen (HBsAg) between our study and that reported in previous studies done in Spain (4/220 vs 14/2076 respectively, P = 0.070); and in France (4/220 vs 3/315 respectively, P = 0.159). But, the prevalence of anti-HBcAb in this study was less than that reported in the study in Spain (7/220 vs 155/2076 respectively, P = 0.006); and was not significantly different from that reported in the study in France (7/220 vs 8/315 respectively, P = 0.313). CONCLUSION The prevalence of HBsAg in our IBD patients was not higher than previously reported European studies. Most IBD patients are not routinely screened or vaccinated against HBV at a tertiary referral center in the United States.

Negative surveillance endoscopy occurs frequently in patients with short-segment non-dysplastic Barrett's esophagus.

Surveillance endoscopy of non-dysplastic Barretts esophagus (NDBE) that fails to detect intestinal metaplasia (IM), or negative surveillance, is known to occur in clinical practice, although the frequency and possible outcomes in a large cohort in clinical practice is not well described. The goals of this study were to define frequency in which negative surveillance occurs and endoscopic outcomes in a screening cohort of short segment NDBE. A retrospective cohort (n = 184) of patients newly diagnosed with short segment NDBE at an outpatient academic tertiary care center between 2003 and 2011 were reviewed. Only those with one or more surveillance endoscopies were included to define a frequency of negative surveillance. Included patients were further assessed if they had two or more surveillance endoscopies and were classified into groups as sampling error or negative IM on consecutive surveillances based on the results of their surveillance endoscopies. The frequency of a negative surveillance endoscopy in all short-segment NDBE patients was 19.66% (92 endoscopic exams were negative for IM of 468 total surveillance exams). A negative surveillance endoscopy occurred in 40.76% (n = 75) patients. Sampling error occurred in 44.12% and negative IM on consecutive surveillance endoscopies in 55.88% of those with ≥ 2 surveillance endoscopies and an initially negative surveillance exam. The frequency of negative IM on consecutive surveillances was 19.00% of all patients who had two surveillance endoscopies. When the index diagnostic Barretts esophagus segment length was < 1 cm, 32.14% (18/56) of all patients (with ≥ 2 surveillance endoscopies) had negative IM on consecutive surveillance endoscopies. Negative surveillance occurs frequently in short-segment NDBE. When an initial negative surveillance endoscopy occurs, it may be due to either a sampling error or lack of detectable IM on surveillance exam. When a <1 cm segment of NDBE is diagnosed, a significant proportion of patients may go on to have continuously undetected IM on consecutive surveillance endoscopic exams without intervention.

Effect of cytomegalovirus and Epstein–Barr virus replication on intestinal mucosal gene expression and microbiome composition of HIV-infected and uninfected individuals

Background: HIV-infection is associated with dramatic changes in the intestinal mucosa. The impact of other viral pathogens is unclear. Methods: One hundred and eight (108) biopsies from left and right colon (n = 79) and terminal ileum (n = 29) were collected from 19 HIV-infected and 22 HIV-uninfected participants. Levels of cytomegalovirus (CMV) and Epstein–Barr virus (EBV) DNA were measured by droplet digital PCR. Mucosal gene expression was measured via multiplex-assay. Microbiome analysis was performed using bacterial 16S-rDNA-pyrosequencing. The effect of CMV and EBV replication on the microbiome composition and mRNA-expression of selected cytokines (IL-6, IFN-&ggr;, IL-1&bgr;, CCL2, IL-8, and IFN-&bgr;1) was evaluated. Results: Overall, CMV and EBV were detected in at least one intestinal site in 60.5 and 78.9% of participants, respectively. HIV-infected individuals demonstrated less detectable CMV (P = 0.04); CMV was more frequently detected in terminal ileum than colon (P = 0.04). Detectable EBV was more frequent among HIV-infected (P = 0.05) without differences by intestinal site. The number of operational taxonomic units did not differ by CMV or EBV detection status. Among HIV-infected participants, higher CMV was only associated with lower relative abundance of Actinobacteria in the ileum (P = 0.03). Presence of CMV was associated with upregulated expression of all selected cytokines in the ileum (all P = 0.02) and higher expression of IL-8 and IFN-&bgr;1 in the colon (all P < 0.05) of HIV-uninfected participants, but not among HIV-infected. EBV had no effect on cytokine expression or microbiome composition whatsoever. Conclusion: These results illustrate a complex interplay among HIV-infection, intestinal CMV replication, and mucosal gut environment, and highlight a possible modulatory effect of CMV on the microbial and immune homeostasis.