A biomarker panel and psychological morbidity differentiates the irritable bowel syndrome from health and provides novel pathophysiological leads.

BACKGROUNDS: The development of a reliable biomarker for irritable bowel syndrome (IBS) remains one of the major aims of research in functional gastrointestinal disorders (FGIDs) and is complicated by the absence of a perfect reference standard. Previous efforts based on genetic and immune markers have showed promise, but have not been robust. AIM: To evaluate an extensive panel of gene expression and serology markers combined with psychological measures in differentiating IBS from health and between subtypes of IBS. METHODS: Of subjects eligible for analysis (N = 244), 168 met criteria for IBS (60 IBS-C, 57 IBS-D and 51 mixed), while 76 were free of any FGID. A total of 34 markers were selected based on pathways implicated in pathophysiology of IBS or whole human genome screening. Psychological measures were recorded that covered anxiety, depression and somatisation. Models differentiating disease and health were based on unconditional logistic regression and performance assessed through area under the receiver-operator characteristic curve (AUC), sensitivity and specificity. RESULTS: The performance of a combination of 34 markers was good in differentiating IBS from health (AUC = 0.81) and was improved considerably with the addition of four psychological markers (combined AUC = 0.93). Of the 34 markers considered, discrimination was derived largely from a small subset. Good discrimination was also obtained between IBS subtypes with the best being observed for IBS-C vs. IBS-D (AUC = 0.92); however, psychological variables provided almost no incremental discrimination subtypes over biological markers (combined AUC = 0.94). CONCLUSIONS: A combination of gene expression and serological markers in combination with psychological measures shows exciting progress towards a diagnostic test for IBS compared with healthy subjects, and to discriminate IBS-C from IBS-D.

Randomised clinical trial: alosetron improves quality of life and reduces restriction of daily activities in women with severe diarrhoea-predominant IBS.

BACKGROUND: Patients with irritable bowel syndrome with diarrhoea (IBS-D) experience restriction in daily activities and decreased health-related quality of life (QOL). AIM: To investigate effects of alosetron on patient-reported health-related QOL, satisfaction and productivity in women with severe IBS-D. METHODS: A total of 705 women (severe IBS-D, Rome II criteria) randomised to alosetron 0.5 mg QD, 1 mg QD, 1 mg BID, or placebo for 12 weeks were studied. IBSQOL, treatment satisfaction, daily activities, and lost workplace productivity (LWP) were evaluated at randomisation and Week 12. RESULTS: One or more doses of alosetron significantly improved all IBSQOL domains except for sexual function from baseline vs. placebo. The magnitude of IBSQOL changes was consistent with a clinically meaningful effect. Alosetron 0.5 mg QD and 1 mg BID significantly reduced IBS interference with social/leisure activities and LWP from baseline vs. placebo [social/leisure (mean ±S.E.) days lost: -6.7 ± 0.8, -7.0 ± 0.9, P < 0.01; LWP (mean ± S.E.) h lost: -11.0 ± 3.3, -21.1 ± 4.1, P < 0.05 respectively]. Significantly more patients treated with alosetron reported satisfaction vs. placebo. Improvements in IBSQOL, LWP, and treatment satisfaction significantly correlated with global improvement of IBS symptoms. The incidence of adverse events with alosetron was low with constipation being the most commonly reported event. A single case of ischaemic colitis occurred, in a patient receiving alosetron 0.5 mg QD. CONCLUSIONS: In women with severe IBS-D, alosetron treatment, including 0.5 mg QD, resulted in statistically significant and clinically relevant improvements in health-related QOL, restriction of daily activities and treatment satisfaction over placebo. IBS symptom improvement corresponded with positive changes in IBSQOL, LWP and treatment satisfaction.

Protein oxidation and 20S proteasome-dependent proteolysis in mammalian cells.

The generation of reactive oxygen species is an inevitable aspect of aerobic life. In addition to being exposed to free radicals in the environment, aerobic organisms must also deal with oxygen radicals generated as byproducts of a number of physiological mechanisms for example, by the mitochondrial and endoplasmic reticulum electron transport chains, and by cells of the immune system. Although most organisms are equipped with several lines of defense against oxidative stress, these defensive mechanisms are not 100% effective, and oxidatively modified forms of proteins accumulate during aging, and in many pathological conditions. Oxidatively modified proteins can form large aggregates due to covalent cross-linking or increased surface hydrophobicity. Unless repaired or removed from cells, these oxidized proteins are often toxic and can threaten cell viability. Mammalian cells exhibit only limited direct repair mechanisms, and oxidatively damaged proteins appear to undergo selective proteolysis, primarily by the major cytosolic proteinase, the proteasome. Interestingly, it appears that the 20S 'core' proteasome conducts the recognition and elimination of oxidized proteins in an ATP-independent and ubiquitin-independent pathway.

Age-related changes in protein oxidation and proteolysis in mammalian cells.

Reactive oxygen species generated as by-products of oxidative metabolism, or from environmental sources, frequently damage cellular macromolecules. Proteins are recognized as major targets of oxidative modification, and the accumulation of oxidized proteins is a characteristic feature of aging cells. An increase in the amount of oxidized proteins has been reported in many experimental aging models, as measured by the level of intracellular protein carbonyls or dityrosine, or by the accumulation of protein-containing pigments such as lipofuscin and ceroid bodies. In younger individuals, moderately oxidized soluble cell proteins appear to be selectively recognized and rapidly degraded by the proteasome. An age-related accumulation of oxidized proteins could, therefore, be a result of declining activity of the proteasome. Previous research to investigate the notion of an age-related decline in the content and/or activity of the proteasome has generated contradictory results. The latest evidence, including our own recent findings, indicates that proteasome activity does, indeed, decline during aging as the enzyme complex is progressively inhibited by oxidized and cross-linked protein aggregates. We propose that cellular aging involves both an increase in (mitochondrial) oxidant production and a progressive decline in proteasome activity. Eventually so much proteasome is inactivated that oxidized proteins begin to accumulate rapidly and contribute to cellular dysfunction and senescence.

Glutathiolation of the proteasome is enhanced by proteolytic inhibitors.

The proteasome inhibitors lactacystin, clastro lactacystin beta-lactone, or tri-leucine vinyl sulfone (NLVS), in the presence of [(35)S]cysteine/methionine, caused increased incorporation of (35)S into cellular proteins, even when protein synthesis was inhibited by cycloheximide. This effect was blocked by incubation with the glutathione synthesis inhibitor buthionine sulfoximine. Proteasome inhibitors also enhanced total glutathione levels, increased reduced/oxidized glutathione ratio (GSH/GSSG) and upregulated gamma-glutamylcysteine synthetase (rate-limiting in glutathione synthesis). Micromolar concentrations of GSH, GSSG, or cysteine stimulated the chymotrypsin-like activity of purified 20S proteasome, but millimolar GSH or GSSG was inhibitory. Interestingly, GSH did not affect 20S proteasome's trypsin-like activity. Enhanced proteasome glutathiolation was verified when purified preparations of the 20S core enzyme complex were incubated with [(35)S]GSH after pre-incubation with any of the inhibitors. NLVS, lactacystin or clastro lactacystin beta-lactone may promote structural modification of the 20S core proteasome, with increased exposure of cysteine residues, which are prone to S-thiolation. Three main conclusions can be drawn from the present work. First, proteasome inhibitors alter cellular glutathione metabolism. Second, proteasome glutathiolation is enhanced by inhibitors but still occurs in their absence, at physiological GSH and GSSG levels. Third, proteasome glutathiolation seems to be a previously unknown mechanism of proteasome regulation in vivo.

4-Hydroxynonenal-modified amyloid-beta peptide inhibits the proteasome: possible importance in Alzheimer's disease.

The amyloid beta-peptide (Abeta) is a 4-kDa species derived from the amyloid precursor protein, which accumulates in the brains of patients with Alzheimer's disease. Although we lack full understanding of the etiology and pathogenesis of selective neuron death, considerable data do imply roles for both the toxic Abeta and increased oxidative stress. Another significant observation is the accumulation of abnormal, ubiquitin-conjugated proteins in affected neurons, suggesting dysfunction of the proteasome proteolytic system in these cells. Recent reports have indicated that Abeta can bind and inhibit the proteasome, the major cytoslic protease for degrading damaged and ubiquitin-conjugated proteins. Earlier results from our laboratory showed that moderately oxidized proteins are preferentially recognized and degraded by the proteasome; however, severely oxidized proteins cannot be easily degraded and, instead, inhibit the proteasome. We hypothesized that oxidatively modified Abeta might have a stronger (or weaker) inhibitory effect on the proteasome than does native Abeta. We therefore also investigated the proteasome inhibitory action of Abeta1-40 (a peptide comprising the first 40 residues of Abeta) modified by the intracellular oxidant hydrogen peroxide, and by the lipid peroxidation product 4-hydroxynonenal (HNE). H2O2 modification of Abeta1-40 generates a progressively poorer inhibitor of the purified human 20S proteasome. In contrast, HNE modification of Abeta1-40 generates a progressively more selective and efficient inhibitor of the degradation of fluorogenic peptides and oxidized protein substrates by human 20S proteasome. This interaction may contribute to certain pathological manifestations of Alzheimer's disease.