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.     

Identification of a glutathione peroxidase inhibitor that reverses resistance to anticancer drugs in human B-cell lymphoma cell lines

Cancer cells isolated from two patients with malignant non-Hodgkin B-cell lymphomas that became resistant to chemotherapy during clinical treatment were made ?fourfold resistant in culture to anticancer drugs, that is cisplatin, etoposide, methotrexate and bortezomib. Because most resistant lines showed significantly increased expression of the anti-oxidative enzyme glutathione peroxidase 1 (GPx1), GPx1 was investigated as a target for inhibitor development. Virtual screening of a library of diverse structures by docking them to the active site of the X-ray crystal structure of bovine GPx1 uncovered compounds that might block the enzyme. An enzyme assay confirmed an acylhydrazone heterocycle (3) with GPx inhibitory activity. Combinations of 3 with the anticancer drugs listed above led to reversal of resistance in the lymphoma cell lines.     

Glutamate induces oxidative stress and apoptosis in cerebral vascular endothelial cells: contributions of HO-1 and HO-2 to cytoprotection

In cerebral circulation, epileptic seizures associated with excessive release of the excitatory neurotransmitter glutamate cause endothelial injury. Heme oxygenase (HO), which metabolizes heme to a vasodilator, carbon monoxide (CO), and antioxidants, biliverdin/bilirubin, is highly expressed in cerebral microvessels as a constitutive isoform, HO-2, whereas the inducible form, HO-1, is not detectable. Using cerebral vascular endothelial cells from newborn pigs and HO-2-knockout mice, we addressed the hypotheses that 1) glutamate induces oxidative stress-related endothelial death by apoptosis, and 2) HO-1 and HO-2 are protective against glutamate cytotoxicity. In cerebral endothelial cells, glutamate (0.1–2.0 mM) increased formation of reactive oxygen species, including superoxide radicals, and induced major keystone events of apoptosis, such as NF-B nuclear translocation, caspase-3 activation, DNA fragmentation, and cell detachment. Glutamate-induced apoptosis was greatly exacerbated in HO-2 gene-deleted murine cerebrovascular endothelial cells and in porcine cells with pharmacologically inhibited HO-2 activity. Glutamate toxicity was prevented by superoxide dismutase, suggesting apoptotic changes are oxidative stress related. When HO-1 was pharmacologically upregulated by cobalt protoporphyrin, apoptotic effects of glutamate in cerebral endothelial cells were completely prevented. Glutamate-induced reactive oxygen species production and apoptosis were blocked by a CO-releasing compound, CORM-A1 (50 µM), and by bilirubin (1 µM), consistent with the antioxidant and cytoprotective roles of the end products of HO activity. We conclude that both HO-1 and HO-2 have anti-apoptotic effects against oxidative stress-related glutamate toxicity in cerebral vascular endothelium. Although HO-1, when induced, provides powerful protection, HO-2 is an essential endogenous anti-apoptotic factor against glutamate toxicity in the cerebral vascular endothelium. endothelium; carbon monoxide; bilirubin; injury; reactive oxygen species; heme oxygenase     

The role of integrin glycosylation in galectin-8-mediated trabecular meshwork cell adhesion and spreading

Primary open angle glaucoma (POAG) is a major blindness-causingdisease, characterized by elevated intraocular pressure dueto an insufficient outflow of aqueous humor. The trabecularmeshwork (TM) lining the aqueous outflow pathway modulates theaqueous outflow facility. TM cell adhesion, cell–matrixinteractions, and factors that influence Rho signaling in TMcells are thought to play a pivotal role in the regulation ofaqueous outflow. In a recent study, we demonstrated that galectin-8(Gal8) modulates the adhesion and cytoskeletal arrangement ofTM cells and that it does so through binding to β₁ integrinsand inducing Rho signaling. The current study is aimed at thecharacterization of the mechanism by which Gal8 mediates TMcell adhesion and spreading. We demonstrate here that TM cellsadhere to and spread on Gal8-coated wells but not on galectin-1(Gal1)- or galectin-3 (Gal3)-coated wells. The adhesion of TMcells to Gal8-coated wells was abolished by a competing sugar,β-lactose, but not by a noncompeting sugar, sucrose. Also,a trisaccharide, NeuAc2-3Galβ1-4GlcNAc, which binds specificallyto the N-CRD of Gal8, inhibited the spreading of TM cells toGal8-coated wells. In contrast, NeuAc2-6Galβ1-4GlcNAc whichlacks affinity for Gal8 had no effect. Affinity chromatographyof cell extracts on a Gal8-affinity column and binding experimentswith plant lectins, Maakia Amurensis and Sambucus Nigra, revealedthat ₃β₁, ₅β₁, and _vβ₁ integrins are major counterreceptorsof Gal8 in TM cells and that TM cell β₁ integrins carrypredominantly 2-3-sialylated glycans, which are high-affinityligands for Gal8 but not for Gal1 or Gal3. These data lead usto propose that Gal8 modulates TM cell adhesion and spreading,at least in part, by interacting with 2-3-sialylated glycanson β₁ integrins.     

Galectin-3 functions as an opsonin and enhances the macrophage clearance of apoptotic neutrophils

Galectin-3, a β-galactoside binding, endogenous lectin,takes part in various inflammatory events and is produced insubstantial amounts at inflammatory foci. We investigated whetherextracellular galectin-3 could participate in the phagocyticclearance of apoptotic neutrophils by macrophages, a processof crucial importance for termination of acute inflammation.Using human leukocytes, we show that exogenously added galectin-3increased the uptake of apoptotic neutrophils by monocyte-derivedmacrophages (MDM). Both the proportion of MDM that engulfedapoptotic prey and the number of apoptotic neutrophils thateach MDM engulfed were enhanced in the presence of galectin-3.The effect was lactose-inhibitable and required galectin-3 affinityfor N-acetyllactosamine, a saccharide typically found on cellsurface glycoproteins, since a mutant lacking this activitywas without effect. The enhanced uptake relied on the presenceof galectin-3 during the cellular interaction and was paralleledby lectin binding to apoptotic cells as well as MDM in a lactose-dependentmanner. These findings suggest that galectin-3 functions asa bridging molecule between phagocyte and apoptotic prey, actingas an opsonin. The process of clearance, whereby apoptotic neutrophilsare removed by macrophages, is crucial for the resolution ofacute inflammation and our data imply that the increased levelsof galectin-3 often found at inflammatory sites could potentlyaffect this process.     

Cyclooxygenase products stimulate carbon monoxide production by piglet cerebral microvessels

Products of arachidonic acid (AA) metabolism by cyclooxygenase (Cox) are important in regulation of neonatal cerebral circulation. The brain and cerebral microvessels also express heme oxygenase (HO) that metabolizes heme to carbon monoxide (CO), biliverdin, and iron. The purpose of this study in newborn pig cerebral microvessels was to address the hypothesis that Cox products affect HO activity and HO products affect Cox activity. AA (2.0-20 microM) increased prostaglandin E2 (PGE2) measured by radioimmunoassay (RIA) and also CO measured by gas chromatography/mass spectrometry (GC/MS). Further, 10(-4) M indomethacin, which inhibited Cox, reduced both AA and heme-induced CO production. Conversely, neither exogenous 2 x 10(-6) M heme, which markedly increased CO production, nor the inhibitor of HO, chromium mesoporphyrin, altered PGE2 synthesis. Because AA metabolism by Cox generates both prostanoids and superoxides, we determined the effects of the predominant prostanoid and superoxide on CO production. Although PGE2 caused a small increase in CO production, xanthine oxidase plus hypoxanthine, which produces superoxide, strongly stimulated the production of CO by cerebral microvessels. This increase was mildly attenuated by catalase. These data suggest that Cox-catalyzed AA metabolites, most likely superoxide and/or a subsequent reactive oxygen species, increase cerebrovascular CO production. This increase seems to be caused, at least in part, by the elevation of HO-2 catalytic activity. Conversely, Cox activity is not affected by HO-catalyzed heme metabolites. These data suggest that some cerebrovascular functions attributable to Cox activity could be mediated by CO.     

Phylogenetic analysis of the vertebrate galectin family

Galectins form a family of structurally related carbohydrate binding proteins (lectins) that have been identified in a large variety of metazoan phyla. They are involved in many biological processes such as morphogenesis, control of cell death, immunological response, and cancer. To elucidate the evolutionary history of galectins and galectin-like proteins in chordates, we have exploited three independent lines of evidence: (i) location of galectin encoding genes (LGALS) in the human genome; (ii) exon-intron organization of galectin encoding genes; and (iii) sequence comparison of carbohydrate recognition domains (CRDs) of chordate galectins. Our results suggest that a duplication of a mono-CRD galectin gene gave rise to an original bi-CRD galectin gene, before or early in chordate evolution. The N-terminal and C-terminal CRDs of this original galectin subsequently diverged into two different subtypes, defined by exon-intron structure (F4-CRD and F3-CRD). We show that all vertebrate mono-CRD galectins known to date belong to either the F3- or F4- subtype. A sequence of duplication and divergence events of the different galectins in chordates is proposed.     

Ionic mechanisms mediating the myogenic response in newborn porcine cerebral arteries

Mechanisms that underlie autoregulation in the newborn vasculature are unclear. Here we tested the hypothesis that in newborn porcine cerebral arteries intravascular pressure elevates wall tension, leading to an increase in intracellular calcium concentration ([Ca2+]i) and a constriction that is opposed by pressure-induced K+ channel activation. Incremental step (20 mmHg) elevations in intravascular pressure between 10 and 90 mmHg induced an immediate transient elevation in arterial wall [Ca2+]i and a short-lived constriction that was followed by a smaller steady-state [Ca2+]i elevation and sustained constriction. Pressures between 10 and 90 mmHg increased steady-state arterial wall [Ca2+]i between approximately 142 and 299 nM and myogenic (defined as passive-active) tension between 25 and 437 dyn/cm. The relationship between pressure and myogenic tension was strongly Ca2+ dependent until forced dilation. At low pressure, 60 mM K+ induced a steady-state elevation in arterial wall [Ca2+]i and a constriction. Nimodipine, a voltage-dependent Ca2+ channel blocker, and removal of extracellular Ca2+ similarly dilated arteries at low or high pressures. 4-Aminopyridine, a voltage-dependent K+ (Kv) channel blocker, induced significantly larger constrictions at high pressure, when compared with those at low pressure. Although selective Ca2+-activated K+ (KCa) channel blockers and intracellular Ca2+ release inhibitors induced only small constrictions at low and high pressures, a low concentration of caffeine (1 microM), a ryanodine-sensitive Ca2+ release (RyR) channel activator, increased KCa channel activity and induced dilation. These data suggest that in newborn cerebral arteries, intravascular pressure elevates wall tension, leading to voltage-dependent Ca2+ channel activation, an increase in wall [Ca2+]i and Ca2+-dependent constriction. In addition, pressure strongly activates Kv channels that opposes constriction but only weakly activates KCa channels.     

Sphingomyelinase activates GLUT4 translocation via a cholesterol-dependent mechanism

A basis for the insulin mimetic effect of sphingomyelinase on glucose transporter isoform GLUT4 translocation remains unclear. Because sphingomyelin serves as a major determinant of plasma membrane cholesterol and a relationship between plasma membrane cholesterol and GLUT4 levels has recently become apparent, we assessed whether GLUT4 translocation induced by sphingomyelinase resulted from changes in membrane cholesterol content. Exposure of 3T3-L1 adipocytes to sphingomyelinase resulted in a time-dependent loss of sphingomyelin from the plasma membrane and a concomitant time-dependent accumulation of plasma membrane GLUT4. Degradation products of sphingomyelin did not mimic this stimulatory action. Plasma membrane cholesterol amount was diminished in cells exposed to sphingomyelinase. Restoration of membrane cholesterol blocked the stimulatory effect of sphingomyelinase. Increasing concentrations of methyl--cyclodextrin, which resulted in a dose-dependent reversible decrease in membrane cholesterol, led to a dose-dependent reversible increase in GLUT4 incorporation into the plasma membrane. Although increased plasma membrane GLUT4 content by cholesterol extraction with concentrations of methyl--cyclodextrin above 5 mM most likely reflected decreased GLUT4 endocytosis, translocation stimulated by sphingomyelinase or concentrations of methyl--cyclodextrin below 2.5 mM occurred without any visible changes in the endocytic retrieval of GLUT4. Furthermore, moderate loss of cholesterol induced by sphingomyelinase or low concentrations of methyl--cyclodextrin did not alter membrane integrity or increase the abundance of other plasma membrane proteins such as the GLUT1 glucose transporter or the transferrin receptor. Regulation of GLUT4 translocation by moderate cholesterol loss did not involve known insulin-signaling proteins. These data reveal that sphingomyelinase enhances GLUT4 exocytosis via a novel cholesterol-dependent mechanism. vesicular trafficking; signal transduction; sphingolipids