Automated assay of vitamin B-12 by the Abbott IMx analyzer.

A nonisotopic assay of vitamin B-12 in human serum or plasma is described, performed with the Abbott IMx analyzer. The sample is first treated at pH > 12.5 to release bound vitamin B-12 and to convert all forms to cyanocobalamin. Next, the analyte is bound, at lower pH, by vitamin B-12-specific binding protein, immobilized to a solid phase of polymeric microspheres. Detection involves monitoring the activity of the tracer enzyme (alkaline phosphatase) coupled to a derivative of cyanocobalamin. Total assay precision is 7.9% for vitamin B-12 at 200 ng/L, 6.6% at 400 ng/L, and 6.7% at 800 ng/L. Assay sensitivity, calculated as 2 SD from the zero calibrator, is 37 (+/- 9) ng/L. The dynamic range extends to 2000 ng/L. Analytical recovery of 300 and 600 ng/L additions of vitamin B-12 to sera with basal concentrations of 30-400 ng/L was 102.5%. Results of the assay correlated well with those of commercially available radioisotope assays. No interference was observed in specimens from patients with pernicious anemia, chronic or acute myelogenous leukemia, or renal failure. Cross-reactivity with cobinamide (1 g/L) was < 0.00003%. Vitamin B-12 measurements for blood specimens drawn into serum, EDTA, or heparinized plasma-collection tubes agreed within 3%.     

Coexistence of contractile and relaxant 5-hydroxytryptamine receptors coupled to distinct signaling pathways in intestinal muscle cells: convergence of the pathways on Ca2+ mobilization.

Muscle cells were dispersed separately from circular and longitudinal muscle layers of guinea pig intestine, and 5-hydroxytryptamine (5-HT) receptors were characterized in naive cells and in cells in which one receptor type was preserved by selective receptor protection. In naive cells from both regions, 5-HT caused contraction and stimulated increases in cytosolic free calcium concentration ([Ca2+]i) (3-fold; p < 0.01) and cAMP levels (40-60%; p < 0.01) that were inhibited, respectively, by the 5-HT2 antagonist ketanserin and the 5-HT1p antagonist N-acetyl-5-hydroxytryptophyl 5-hydroxytryptophan amide (5-HTP-DP). In circular muscle cells, where agonist-induced increase in [Ca2+]i is mediated by Ca2+ release from inositol (1,4,5)trisphosphate-sensitive stores, 5-HT caused an increase in inositol (1,4,5)trisphosphate levels that was inhibited by ketanserin. In cells maximally contracted with a non-5-HT agonist (cholecystokinin octapeptide), 5-HT caused relaxation when the contractile effect mediated by 5-HT2 receptors was blocked with ketanserin; relaxation and the concomitant increase in cAMP were inhibited by 5-HTP-DP. The singular contributions of the Ca2+ and cAMP signaling pathways were identified in cells where only one receptor type was preserved. In cells with only 5-HT2 receptors, 5-HT caused contraction and an increase in [Ca2+]i but not in cAMP levels; contraction and the increase in [Ca2+]i were inhibited by ketanserin. Conversely, in cells with only 5-HT1p receptors, 5-HT caused relaxation and an increase in cAMP levels but not in [Ca2+]i; relaxation and the increase in cAMP levels were inhibited by 5-HTP-DP. The two signaling pathways were functionally linked, converging to regulate the level of [Ca2+]i. Thus, the increase in [Ca2+]i was augmented 1) when cAMP production was inhibited by 5-HTP-DP in naive cells or 2) when cAMP production was suppressed in cells where 5-HT1p receptors were inactivated and only 5-HT2 receptors were preserved. The results imply that the increase in cAMP levels mediated by 5-HT1p receptors acted to attenuate the increase in [Ca2+]i mediated by 5-HT2 receptors. We conclude that the response to 5-HT in muscle cells is a compound effect involving activation of two receptor types coupled to distinct signaling pathways that converge on [Ca2+]i as the determinant of mechanical activity.     

Influence of α-tocopherol over the time course of experimental IgA nephropathy

 The present study investigated the pathogenesis and the time course of kidney injury in experimental IgA nephropathy. In order to determine an appropriate period in the course of experimental IgA nephropathy to study renal injury and repair, we examined proteinuria and IgA deposition in the renal mesangium after 4, 8, and 16 weeks of mucosal challenge by bovine gamma globulins (BGG) provided in the drinking water. The hallmark of IgA deposition in the mesangium was present after 4 weeks and 8 weeks of BGG inoculation, but by 16 weeks, the mesangial IgA deposition had resolved. In addition, we confirmed our previous report on the beneficial effects of α-tocopherol in reducing proteinuria in IgA nephropathy at 8 weeks, and extended this observation to investigate the effects of dietary supplementation of α-tocopherol at both 4 weeks and 16 weeks. Proteinuria resolved spontaneously at 16 weeks. There is oxidative stress, as suggested by the elevation in plasma and renal malondialdehyde content, and increased fibrogenic cytokine message, as suggested by elevated transforming growth factor β1 mRNA. These increases were clearly blunted by α-tocopherol at both 4 weeks and 8 weeks. Treatment with α-tocopherol was associated with a significant reduction in the severity of proteinuria. Thus, our data suggest that the period between 4 and 8 weeks of BGG vaccination could be relevant in designing an appropriate model to study the molecular biology of the pathogenesis of renal injury and the effects of treatment. The 16-week model may be useful in exploring gene expression involved with spontaneous resolution. Received: 17 February 1998 / Revised: 2 June 1998 / Accepted: 3 June 1998     

Prevalence and factors associated with anthropometric failure,vitamin A and iron deficiency among adolescents in a Nigerian urban community

BackgroundUnder nutrition is a problem of severe magnitude in low income countries like Nigeria. Adolescent school children might also be vulnerable. The dearth of data hinders planning of school health and nutrition programmes for school children.ObjectiveTo determine the prevalence of stunting, thinness; vitamin A and iron deficiencies among adolescent students in Nsukka urban, Nigeria and to determine factors that are associated with these nutritional problems.MethodsA total of 400 participants were randomly selected from 717 students aged 12 – 18 years in 3 randomly selected secondary schools. Questionnaires, anthropometric measurements, and blood analyses were the data collection methods employed.ResultsThe prevalence of stunting was 33.3% and thinness 31.0%. Neither overweight nor obesity was observed. While 64.0% were anaemic; 44.0% had vitamin A deficiency (VAD). A total of 48.0% had both anaemia and stunting, 42% had VAD + thinness; while 40% had anaemia + VAD. Household income was a predictor of vitamin A status. Children from medium/high income households had higher odds of having VAD than those from low income households (AOR=0.14; 95% CI=0.031, 0.607; P=0.009). Household income (AOR=0.12; 95% CI=0.021, 0.671; P=0.016), and age (AOR=0.09; 95% CI=0.014, 0.587; P=0.012) were independent determinants of height-for-age status.ConclusionAmong urban adolescent students in Nigeria, stunting, thinness, anaemia and VAD were problems of public health significance. Age and household monthly income played major roles.     

The fate of myofibroblasts during the development of fibrosis in Crohn's disease

Intestinal fibrosis is a devastating complication in patients with inflammatory bowel disease. Its characteristics include the loss of regular peristalsis and nutrition absorption, excessive deposition of extracellular matrix (ECM) components, thickness of intestinal lumen due to the formation of strictures and of scar tissue. As a major cell type involved in fibrogenesis, the myofibroblasts have already been shown to have a plastic and heterogeneous function in producing abundant collagen, fibronectin and connective tissue growth factor. The primary sources of ECM‐producing and vimentin‐positive myofibroblasts come from different precursor cells, including bone marrow‐derived mesenchymal cells, fibrocytes, pericytes, epithelial to mesenchymal transition and endothelial to mesenchymal transition. Recent immunological research findings suggest that numerous cytokines and chemokines made from macrophages, in addition to T cells and other myeloid cell types, are also important drivers of myofibroblast differentiation and hence of the activation of myofibroblast‐mediated transforming growth factor and collagen production. In this review we discuss the origins, roles and cell signaling of myofibroblasts during the development of fibrosis in different organs, particularly in Crohn's disease. Finally, we suggest that the epigenetic and immunological regulation of myofibroblast differentiation may provide a novel antifibrotic strategy in the near future.     

Growth hormone and growth hormone-related mRNA in uremic rats: effect of a growth hormone secretagogue

In experimental animals, the decreased growth during mild uremia is not accompanied by a loss in the capacity of the pituitary gland to secrete growth hormone (GH). With the development of orally administered GH "secretagogues" (GHS), it might be possible to stimulate growth during uremia without injections. This study was designed to determine the effects of the GHS, L-163,255. Uremia was induced by 5/6 nephrectomy (NX). GHS was given orally, 3 mg/kg, twice a week. Four groups of animals included: (1) sham-operated, (2) sham-operated, pair-fed, (3) uremic (NX), and (4) uremic, GHS-treated (NX+GHS). Blood sampling was conducted via intra-atrial catheters, and GH was quantitated. Pituitary GH mRNA was measured by Northern blot, and liver GH receptor and insulin-like growth factor-I mRNAs by RNAase protection. Untreated NX animals had a specific decrease in the "mass" of the GH pulses. A burst of GH was induced by GHS, but the pulsatile pattern of GH secretion over 6 h was not affected. An increase or a return to non-uremic levels of GH-related mRNAs occurred after GHS. Thus, GHS stimulated an acute burst of GH secretion and increased specific mRNAs encoding GH-related proteins in uremic animals.     

Gene expression after intrarenal injection of plasmid DNA in the rat

Effective gene therapy requires efficient delivery and expression of the necessary genetic information to the target tissue. We demonstrate here that plasmid DNA, injected as naked, uncomplexed DNA into the cortical region of rat kidney, or intravenously, is localized and expressed in the kidney. The plasmid pRSVZ contained the Rous sarcoma virus promoter and a reporter gene, the β-galactosidase gene, derived from bacteria. The β-galactosidase gene hydrolyzes the artificial substrate X-gal to produce an intense blue color in cells that have taken up and expressed the plasmid genes. We have used X-gal staining and Western blotting to study plasmid gene expression 1, 4, and 8 days and 6 months after intrarenal injection of 50 μg of plasmid DNA and at 1 and 4 days after intravenous injection. Expression was apparent in the kidneys and several other tissues 24 h after injection and persisted for at least 8 days; expressed proteins could still be detected in the injected kidney 6 months later. These observations were corroborated by use of a plasmid, pEGFP-Puro, harboring the cytomegalovirus promoter in conjunction with a different reporter gene, the green fluorescent protein (GFP). Histological localization and Western blotting analysis of GFP expression after intrarenal injection of pEGFP-Puro paralleled results obtained with the plasmid pRSVZ. Our findings support the suggestion that intrarenal or intravenous injection of naked plasmid DNA may be an effective means of delivering therapeutic genes to the kidney and several other tissues. Received: 21 September 1999 / Revised: 4 November 1999 / Accepted: 9 November 1999