Anti-NPPB monoclonal antibody, clone MAAG5173

The cystic fibrosis transmembrane conductance regulator (CFTR) is present on the apical membrane of corneal endothelial cells. Increasing intracellular [cAMP] with forskolin stimulates an NPPB and glibenclamide-inhibitable apical Cl- and HCO3- permeability [Sun, X.C., Bonanno, J.A., 2002. Expression, localization, and functional evaluation of CFTR in bovine corneal endothelial cells. Am. J. Physiol. Cell Physiol. 282, C673-C683]. To definitively determine that the increased permeability is dependent on CFTR, we used an siRNA knockdown approach. Apical Cl- and HCO3- permeability and steady-state HCO3- flux were measured in the presence or absence of forskolin using cultured bovine corneal endothelial cells that were transfected with CFTR siRNA or a scrambled sequence control. CFTR protein expression was reduced by similar to 80% in CFTR siRNA treated cultures. Forskolin (10 mu M) increased apical chloride permeability by 7-fold, which was reduced to control level in siRNA treated cells. CFTR siRNA treatment had no effect on baseline apical chloride permeability. Apical HCO3- permeability was increased 2-fold by 10 mu M forskolin, which was reduced to control level in siRNA treated cultures. Similarly, there was no effect on baseline apical HCO3- permeability by knocking down CFTR expression. The steady-state apical-basolateral pH gradient (Delta pH) at 4 h in control cultures was increased similar to 2.5-fold by forskolin. In CFTR siRNA treated cells, the baseline Delta pH was similar to control, however forskolin did not have a significant effect. We conclude that forskolin induced increases in apical HCO3- permeability in bovine corneal endothelium requires CFTR. However, CFTR does not have a major role in determining baseline apical chloride or HCO3- permeability. (C) 2008 Elsevier Ltd. All rights reserved.

Background: Neuroprotection studies are generally unable to demonstrate efficacy in humans. Our specific hypothesis is that multiple pathophysiologic pathways, of variable importance, contribute to ischemic brain damage. As a corollary to this, we discuss the broad hypothesis that a multifaceted approach will improve the probability of efficacious neuroprotection. But to properly test this hypothesis the nature and importance of the multiple contributing pathways needs elucidation. Our aim is to demonstrate, using functional genomics, in human cardiac surgery procedures associated with cerebral ischemia, that the pathogenesis of perioperative human ischemic brain damage involves the function of multiple variably weighted proteins involving several pathways. We then use these data and literature to develop a proposal for rational design of human neuroprotection protocols. Methods: Ninety-four patients undergoing deep hypothermic circulatory arrest (DHCA) and/or aortic valve replacement surgery had brain damage biomarkers, S100 beta and neurofilament H (NFH), assessed at baseline, 1 and 24 h post-cardiopulmonary bypass (CPB) with analysis for association with 92 single nucleotide polymorphisms (SNPs) (selected by co-author WAK) related to important proteins involved in pathogenesis of cerebral ischemia. Results: At the nominal significance level of 0.05, changes in S100 beta and in NFH at 1 and 24 h post-CPB were associated with multiple SNPs involving several prospectively determined pathophysiologic pathways, but were not individually significant after multiple comparison adjustments. Variable weights for the several evaluated SNPs are apparent on regression analysis and, notably, are dissimilar related to the two biomarkers over time post CPB. Based on our step-wise regression model, at 1 h post-CPB, SOD2, SUMO4, and GP6 are related to relative change of NFH while TNF, CAPN10, NPPB, and SERPINE1 are related to the relative change of S100B. At 24 h post-CPB, ADRA2A, SELE, and BAX are related to the relative change of NFH while SLC4A7, HSPA1B, and FGA are related to S100B. Conclusions: In support of the proposed hypothesis, association SNP data suggest function of specific disparate proteins, as reflected by genetic variation, may be more important than others with variation at different post-insult times after human brain ischemia. Such information may support rational design of post-insult time-sensitive multifaceted neuroprotective therapies.