Stacy Weitsman

Overexpression of theca-cell messenger RNA in polycystic ovary syndrome does not correlate with polymorphisms in the cholesterol side-chain cleavage and 17α-hydroxylase/C17-20 lyase promoters

OBJECTIVE To determine whether overexpression of CYP17 or CYP11A messenger (m)RNA in theca cells from polycystic ovaries is related to polymorphic regions in the gene promoters that may increase transcription. DESIGN Case-control study. SETTING Research institute. PATIENT(S) Fifty-one women with PCOS and 280 regularly cycling controls underwent genotyping. Thecal cells were obtained from 23 women with PCOS and 51 controls. MAIN OUTCOME MEASURE(S) Ovarian tissue was obtained from women with PCOS undergoing wedge resection for treatment of their infertility and from controls undergoing ovariectomy for indications unrelated to the study. Expression of mRNA in theca cells was measured by using competitive reverse transcriptase polymerase chain reaction. Genotype analysis for polymorphisms in the CYP11A and CYP17 promoters was performed by using polymerase chain reaction. RESULT(S) Although expression of CYP11A and CYP17 mRNA was higher in women with PCOS, no significant dose effects of CYP11A or CYP17 alleles were observed with respect to serum testosterone; follicular fluid androstenedione, estradiol, and androstenedione-to-estradiol ratio; or CYP11A or CYP17 mRNA expression. CONCLUSION(S) Overexpression of CYP17 and CYP11A mRNA in theca cells from polycystic ovaries is explained by polymorphic differences in the gene promoters.

Development and Validation of a Biomarker for Diarrhea-Predominant Irritable Bowel Syndrome in Human Subjects

Diarrhea-predominant irritable bowel syndrome (IBS) is diagnosed through clinical criteria after excluding “organic” conditions, and can be precipitated by acute gastroenteritis. Cytolethal distending toxin B (CdtB) is produced by bacteria that cause acute gastroenteritis, and a post-infectious animal model demonstrates that host antibodies to CdtB cross-react with vinculin in the host gut, producing an IBS-like phenotype. Therefore, we assessed circulating anti-CdtB and anti-vinculin antibodies as biomarkers for D-IBS in human subjects. Subjects with D-IBS based on Rome criteria (n=2375) were recruited from a large-scale multicenter clinical trial for D-IBS (TARGET 3). Subjects with inflammatory bowel disease (IBD) (n=142), subjects with celiac disease (n=121), and healthy controls (n=43) were obtained for comparison. Subjects with IBD and celiac disease were recruited based on the presence of intestinal complaints and histologic confirmation of chronic inflammatory changes in the colon or small intestine. Subjects with celiac disease were also required to have an elevated tTG and biopsy. All subjects were aged between 18 and 65 years. Plasma levels of anti-CdtB and anti-vinculin antibodies were determined by ELISA, and compared between groups. Anti-CdtB titers were significantly higher in D-IBS subjects compared to IBD, healthy controls and celiac disease (P<0.001). Anti-vinculin titers were also significantly higher in IBS (P<0.001) compared to the other groups. The area-under-the-receiver operating curves (AUCs) were 0.81 and 0.62 for diagnosis of D-IBS against IBD for anti-CdtB and anti-vinculin, respectively. Both tests were less specific in differentiating IBS from celiac disease. Optimization demonstrated that for anti-CdtB (optical density≥2.80) the specificity, sensitivity and likelihood ratio were 91.6%, 43.7 and 5.2, respectively, and for anti-vinculin (OD≥1.68) were 83.8%, 32.6 and 2.0, respectively. These results confirm that anti-CdtB and anti-vinculin antibodies are elevated in D-IBS compared to non-IBS subjects. These biomarkers may be especially helpful in distinguishing D-IBS from IBD in the workup of chronic diarrhea.

Effect of insulin-like growth factor-I on cholesterol side-chain cleavage cytochrome P450 messenger ribonucleic acid expression in ovarian theca-interstitial cells stimulated to differentiate in vitro

Currently available evidence supports the hypothesis that insulin-like growth factor-I (IGF-I) may play a role in stimulating ovarian theca-interstitial cell (TIC) differentiation in preantral follicles. The purpose of the present studies was to examine the potential role of IGF-I in TIC differentiation by determining the effects of IGF-I on cholesterol side-chain cleavage cytochrome P450 (P450SCC) mRNA expression in TIC stimulated to differentiate in vitro. TIC were isolated from the ovaries of hypophysectomized immature rats by Percoll gradient centrifugation and cultured in the presence and absence of LH and IGF-I up to 6 days. At various times cytoplasmic RNA was extracted from the TIC and P450SCC mRNA was measured by specific assay using reverse transcription followed by the polymerase chain reaction. Increasing concentrations of LH (0-1 microgram/ml) stimulated a dose-related increase in P450SCC mRNA (ED50 = 36.2 +/- 5.5 ng/ml) which reached maximal levels at 100 ng/ml of LH. Addition of IGF-I (30 ng/ml) caused a small increase in P450SCC mRNA over TIC treated with LH alone but did not alter the ED50 for LH stimulation. IGF-I alone also stimulated an increase in P450SCC mRNA which reached approximately 3-fold over unstimulated levels at 100 ng/ml. In the presence of LH, IGF-I stimulated a dose-related increase in P450SCC mRNA (ED50 = 1.2 +/- 0.05 ng/ml). Time-course studies revealed that expression of P450SCC mRNA was greatest at 2 days in TIC treated with IGF-I alone, LH alone or LH plus IGF-I and then declined at 4 and 6 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular assessment of differences in the duodenal microbiome in subjects with irritable bowel syndrome.

Abstract Objective. Breath testing and duodenal culture studies suggest that a significant proportion of irritable bowel syndrome (IBS) patients have small intestinal bacterial overgrowth. In this study, we extended these data through 16S rDNA amplicon sequencing and quantitative PCR (qPCR) analyses of duodenal aspirates from a large cohort of IBS, non-IBS and control subjects. Materials and methods. Consecutive subjects presenting for esophagogastroduodenoscopy only and healthy controls were recruited. Exclusion criteria included recent antibiotic or probiotic use. Following extensive medical work-up, patients were evaluated for symptoms of IBS. DNAs were isolated from duodenal aspirates obtained during endoscopy. Microbial populations in a subset of IBS subjects and controls were compared by 16S profiling. Duodenal microbes were then quantitated in the entire cohort by qPCR and the results compared with quantitative live culture data. Results. A total of 258 subjects were recruited (21 healthy, 163 non-healthy non-IBS, and 74 IBS). 16S profiling in five IBS and five control subjects revealed significantly lower microbial diversity in the duodenum in IBS, with significant alterations in 12 genera (false discovery rate < 0.15), including overrepresentation of Escherichia/Shigella (p = 0.005) and Aeromonas (p = 0.051) and underrepresentation of Acinetobacter (p = 0.024), Citrobacter (p = 0.031) and Microvirgula (p = 0.036). qPCR in all 258 subjects confirmed greater levels of Escherichia coli in IBS and also revealed increases in Klebsiella spp, which correlated strongly with quantitative culture data. Conclusions. 16S rDNA sequencing confirms microbial overgrowth in the small bowel in IBS, with a concomitant reduction in diversity. qPCR supports alterations in specific microbial populations in IBS.

Intestinal Methanobrevibacter smithii but not total bacteria is related to diet-induced weight gain in rats.

It is increasingly understood that gastrointestinal (GI) methanogens, including Methanobrevibacter smithii, influence host metabolism.

Role of Cytolethal Distending Toxin in Altered Stool Form and Bowel Phenotypes in a Rat Model of Post-infectious Irritable Bowel Syndrome

Background/Aims Campylobacter jejuni infection is a leading cause of acute gastroenteritis, which is a trigger for post-infectious irritable bowel syndrome (PI-IBS). Cytolethal distending toxin (CDT) is expressed by enteric pathogens that cause PI-IBS. We used a rat model of PI-IBS to investigate the role of CDT in long-term altered stool form and bowel phenotypes. Methods Adult Sprague-Dawley rats were gavaged with wildtype C. jejuni (C+), a C. jejuni cdtB knockout (CDT-) or saline vehicle (controls). Four months after gavage, stool from 3 consecutive days was assessed for stool form and percent wet weight. Rectal tissue was analyzed for intraepithelial lymphocytes, and small intestinal tissue was stained with anti-c-kit for deep muscular plexus interstitial cells of Cajal (DMP-ICC). Results All 3 groups showed similar colonization and clearance parameters. Average 3-day stool dry weights were similar in all 3 groups, but day-to-day variability in stool form and stool dry weight were significantly different in the C+ group vs both controls (P < 0.01) and the CDT- roup (P < 0.01), but were not different in the CDT- vs controls. Similarly, rectal lymphocytes were significantly higher after C. jejuni (C+) infection vs both controls (P < 0.01) and CDT-exposed rats (P < 0.05). The counts in the latter 2 groups were not significantly different. Finally, c-kit staining revealed that DMP-ICC were reduced only in rats exposed to wildtype C. jejuni. Conclusions In this rat model of PI-IBS, CDT appears to play a role in the development of chronic altered bowel patterns, mild chronic rectal inflammation and reduction in DMP-ICC.

Effect of repeated Campylobacter jejuni infection on gut flora and mucosal defense in a rat model of post infectious functional and microbial bowel changes

Campylobacter jejuni infection is a leading cause of gastroenteritis and post infectious irritable bowel syndrome (PI‐IBS). Unanswered questions include the role of cytokines, effects on gut flora, and why IBS is not more prevalent in countries with higher gastroenteritis rates. Therefore, we determined the effects of early and repeat C. jejuni infections on post infectious phenotypes, gut flora, and cytokine levels in a rat model of functional bowel and microbial changes.

Metabolic effects of eradicating breath methane using antibiotics in prediabetic subjects with obesity

Methanogens colonizing the human gut produce methane and influence host metabolism. This study examined metabolic parameters in methane‐producing subjects before and after antibiotic treatment.

Mo2051 Lovastatin Improves Stool Form in Methanobrevibacter Smithii Colonized Rats With Constipation

30 adult, male Sprague-Dawley rats were placed on a high-fat diet (60.3% kcal from fat, Teklad high-fat diet TD.06414, Harlan Laboratories Inc, Madison, WI) for 7 weeks. The rats were assessed for increased M. smithii by qPCR before and after the diet, and then divided into 3 groups. Group 1 was given lovastatin in its lactone form, Group 2 was given lovastatin hydroxy acid (each 1.5 mg/rat), and Group 3 was gavaged with a placebo. Each group was gavaged daily for 10 days. Three day stool collections were performed to assess average stool wet weight and daily variability prior to commencing the highfat diet, after 7 weeks of high-fat diet, and the final days of the lovastatin gavage (still on high-fat diet). On day 10 of the gavage, rats were euthanized and DNA was extracted from contents of ligated bowel segments (duodenum, jejunum, ileum, cecum and left colon). qPCR was performed using primers for total luminal bacteria and M. smithii. RESULTS

311 Large Scale Validation of a Biomarker for Diarrhea-Predominant Irritable Bowel Syndrome

of these patients had fasting FGF19 measured. Alanine transaminase (ALT) and appearance of fatty liver on imaging (ultrasound, CT or MR) were retrospectively added to the database. Where multiple investigations had been performed, the test nearest to the date of the SeHCAT test was recorded. Patients with known chronic liver disease or alcohol abuse were excluded from the final analysis. Results: Of 578 SeHCAT values on the database, 303 (52%) were positive with a value 31IU/L (36% v 21%, p 31IU/L (21% v 7%, p 31IU/L (43% v 22%, p 31IU/L (23% v 7%, p 40 IU/L (40% vs 12%, p<0.05), OR 5.13 (95%CI 1.28-20.61, p<0.05). Conclusions: Primary bile acid diarrhea is associated with NAFLD and may share a common pathology in low FGF19. Both conditions may be presentations of the metabolic syndrome associated with low FGF19.