Validating biomarkers of treatable mechanisms in irritable bowel syndrome.

BACKGROUND: A valid biomarker is 'an indicator of normal biologic or pathogenic processes, or pharmacological responses to a therapeutic intervention'. There is no validated biomarker for irritable bowel syndrome (IBS). The aim of the study was to assess ability of three quantitative traits to identify treatable processes to discriminate between IBS-diarrhea (IBS-D) patients, IBS-constipation (IBS-C) patients and healthy volunteers (HV). METHODS: In 30 HV, 30 IBS-C patients and 64 IBS-D patients, we characterized bowel symptoms and quantitated pathophysiological mechanisms: bile acid (BA) synthesis (serum C4 and FGF19), fecal BA and fat, colonic transit (CT), and intestinal permeability (IP). We used multiple logistic regression and receiver-operating characteristic (ROCAUC ) to appraise three factors (fecal BA, CT, and IP) individually and in combination to identify discriminant targets for treatment in IBS. KEY RESULTS: There were significant associations between the three subgroups and symptoms reflecting bowel function and the quantitative traits. There were significant associations between fecal BA and CT at 48 h (r = 0.43; p < 0.001) and between fecal BA and IP (r = 0.23; p = 0.015). Individually, fecal BA and CT48 (but not IP) were significant independent predictors for distinguishing HV from IBS. In combination, they discriminated HV from IBS-D patients (ROCAUC 0.70), HV from IBS-C patients (ROCAUC 0.73), and IBS-C patients from IBS-D patients (ROCAUC 0.86). Colonic transit and fecal BA excretion together discriminate between healthy volunteers and IBS-C patients or IBS-D patients, or between the IBS subgroups with 75-90% specificity at 60% sensitivity. CONCLUSIONS & INFERENCES: Colonic transit and fecal BA individually and together constitute useful biomarkers to identify treatable mechanisms in IBS and to differentiate subgroups of IBS.

Understanding measurements of intestinal permeability in healthy humans with urine lactulose and mannitol excretion.

Our aim was to understand the information from differential two-sugar excretion (2-SE) in measuring intestinal permeability. In a crossover study in 12 healthy volunteers, we compared urinary excretion ratios of lactulose (L) to mannitol [(M) LMR] after ingestion in liquid formulation (LF) or in delayed-release, methacrylate-coated capsules (CAP). Both formulations were radiolabelled. Urine was collected every 2 h from 0 to 8 h, and from 8 to 24 h. Two hours after LF, gastric residual was 15.9 +/- 6.2% (SEM), and the percentage in colon was 49.6 +/- 7.8%; in 11/12 participants, liquid had entered colon within 2 h. Average CAP arrival time in colon was 5.16 +/- 0.46 h (mode 6 h). After LF, mannitol was extensively absorbed in the first 8 h; lactulose absorption was low throughout the 24 h. After the LF, the LMR (geometric mean, 95% CI per h) in the 0-2 h urine was [0.08 (0.05, 0.11)], which was lower than in 8-24 h urine [0.32 (0.16, 0.46); P < 0.05]. Urine LMRs at 8-24 h were similar after LF or CAP. We concluded that, after LF, sugar excretion in 0-2 h urine may reflect both SI and colon permeability. Colonic permeability is reflected by urine sugar excretion between 6 and 24 h. CAP delivery reduces mannitol excreted at 0-6 h, compared with LF. The 0-5 or 6 h 2-SE urine likely reflects both SI and colon permeability; the higher LMR in the 8-24 h urine relative to 0-2 h urine should be interpreted with caution and does not mean that colon is more permeable than SI.

Measurement of serum 7alpha-hydroxy-4-cholesten-3-one (or 7alphaC4), a surrogate test for bile acid malabsorption in health, ileal disease and irritable bowel syndrome using liquid chromatography-tandem mass spectrometry.

Bile acid malabsorption (BAM) is reported in up to 50% of patients with functional diarrhoea and irritable bowel syndrome with diarrhoea (IBS-D). Serum 7alpha-hydroxy-4-cholesten-3-one (7alphaHCO or 7alphaC4), an indirect measurement of hepatic bile acid synthesis, has been validated as a measurement of BAM relative to the (75)SeHCAT retention test. Our aim was to develop a serum 7alphaC4 assay, normal values, and compare results from healthy controls, patients with ileal Crohn's disease or resection, and patients with IBS-D or IBS with constipation (IBS-C). Stored serum samples were used from adult men and women in the following groups: 111 normal healthy controls, 15 IBS-D, 15 IBS-C, 24 with distal ileal Crohn's disease and 20 with distal ileal resection for Crohn's disease. We adapted a published high pressure liquid chromatography, tandem mass spectrometry (HPLC-MS/MS) assay. The HPLC-MS/MS assay showed good linearity in concentration range 0-200 ng mL(-1), sensitivity (lowest limit of detection 0.04 ng mL(-1)), and high analytical recovery (average 99%, range 93-107%). The 5th to 95th percentile for 111 normal healthy controls was 6-60.7 ng mL(-1). There were significant overall group differences (anovaon ranks, P < 0.001), with significantly higher values for terminal ileal disease or resection. There were significant differences between health and IBS (anova, P = 0.043) with higher mean values in IBS-D relative to controls (rank sum test, P = 0.027). We have established a sensitive non-isotopic assay based on HPLC-MS/MS, determined normal 7alphaC4 values, and identified increased 7alphaC4 in IBS-D and in distal ileal resection and disease. This assay has potential as a non-invasive test for BAM in IBS.

The long form of the leptin receptor (OB-Rb) is widely expressed in the human brain.

Leptin exerts important effects on the regulation of food intake and energy expenditure by acting in the brain. Leptin action is mediated by the interaction with a receptor that is alternatively spliced, resulting in at least five different isoforms. The long form (OB-Rb) has a long intracellular domain that is essential for intracellular signal transduction. The specific aim of this study was to further investigate the role that the brain may play in the pathogenesis of obesity in humans. We studied the expression of OB-R mRNA (both short or common and long isoforms) in the brains of obese, lean and diabetic subjects, by in situ hybridization, semiquantitative RT-PCR and Northern blots analysis. We used two alternative probes: one that recognizes all known splice variants (OB-Ra) and a second that recognizes only the long form, OB-Rb. Several brain regions, including hypothalamus, cerebellum, neocortex, entorrhinal cortex, amygdala, and rostral medulla, were evaluated. In situ hybridization studies revealed that both OB-Ra and OB-Rb mRNAs are widely distributed in the human brain. The specific hybridization signal with both probes was detected exclusively in the cytoplasm of the cell body, dendrites and proximal axons of neurons. Hypothalamic nuclei, Purkinje cells and dentate nuclei of the cerebellum, inferior olivary and cranial nerves nuclei in the medulla, amygdala and neurons from both neocortex and entorrhinal cortex demonstrated positive signals. The hybridization signal obtained in ependyma was lower than that in neurons and no specific hybridization was detected in glial cells. No significant differences were identified among the regions or among the three groups studied. These results match those previously obtained by us [Couce et al.: Neuroendocrinology 1997;66:145] in which the distribution of the OB-R protein in the human brain was first described. RT-PCR indicated that the OB-Rb was highly expressed in the hypothalamus and cerebellum. No significant differences of OB-Ra or OB-Rb mRNA expression were identified in lean or obese individuals in these two cerebral regions. The levels of OB-Rb were significantly higher in cerebellum compared to hypothalamus in lean and obese individuals. The original hypothesis that OB-Rb is present only in the hypothalamus needs to be reconsidered. This OB-Rb isoform seems to be widely expressed in the human brain with highest levels in the cerebellum. Obesity and hyperleptinemia appears not to be associated with down-regulation of the OB-Rb in the human brain.

Gene therapy for solid tumors.

Advances in molecular biology have proven that there is a genetic basis to the process of carcinogenesis that allows for the consideration of entirely new approaches to the treatment of cancer. The development of an ability to selectively destroy cancer cells through the manipulation of DNA may provide the opportunity to dramatically improve the quality of care and treatment of cancer patients by decreasing systemic toxicities and enhancing efficacy. These new therapies may occur through the restoration of genetic health, such as the insertion of normal tumor suppressor genes or via down-regulation of oncogene or growth factor receptor expression. Other possibilities include the targeting of genetic alterations in tumor cells that will enhance tumor immunogenicity or induce a specific sensitivity to a prodrug. In this chapter, we have reviewed the current status of gene therapy for solid tumors in the United States and evolving new approaches for this emerging clinical discipline.

Survival and toxicity of xenogeneic murine retroviral vector producer cells in liver.

Murine retroviral vector producer cells (VPC) can selectively transfer genes stably into proliferating cells. We injected LacZ gene producing VPC directly into normal rat liver. There was no measurable gene transfer into the surrounding hepatic parenchyma with X-GAL staining. Rejection of the xenogeneic murine VPC occurred 7-14 days after injection. Toxicity of this delivery method was evaluated with the herpes simplex-thymidine kinase (HS-tk) gene, which confers sensitivity to the antiherpes drug, ganciclovir (GCV). HS-tk VPC were injected and allowed to grow in normal liver for 7 days before GCV treatment. There was no clinical or histologic evidence of toxicity with GCV treatment. These findings suggest that the direct injection of murine VPC into xenogeneic human tumors may survive sufficiently long without immunosuppression to transfer genes to tumor cells in situ without attendant toxicity.