Chemical and physical characterization of a novel poly(carbonate urea) urethane surface with protein crosslinker sites

A major complication which occurs with implantable polyurethane biomaterials is bioincompatibility between blood and the biomaterial surface. Development of a novel biodurable polyurethane surface to which biological agents, such as growth factors or anticoagulants could be covalently bound, would be beneficial. The purpose of this study was to synthesize a novel poly(carbonate urea) urethane polymer with carboxylic acid groups which would serve as "anchor" sites for protein attachment. Physical characteristics such as tensile strength, initial modulus, ultimate elongation, tear strength, water/alcohol uptake and water vapor permeation were then evaluated and compared to other biomedical-grade polyurethanes. Covalent linkage of the blood protein albumin to this novel surface was then examined. A biodurable polycarbonate-based polyurethane containing carboxylic acid groups (cPU) was synthesized using a two step procedure incorporating the chain extender 2,2-bis(hydroxymethyl)-propionic acid (DHMPA). Tensile strength of this cPU film was 2.7 and 2.6 fold greater than both a polycarbonate-based polyurethane synthesized with a 1,4-butanediol chain extender (bdPU) and Mitrathane (Mit) controls, respectively. The cPU polymer also possessed 7.8 and 31 fold greater structural rigidity upon evaluation of initial modulus as compared to the bdPU and Mit, respectively. Ultimate elongation for the bdPU films was slightly higher than the cPU and Mit films, which had comparable elongation properties. The force required to tear the bdPU film was 1.9 and 32 fold greater than the cPU and Mit films, respectively. Alcohol solution uptake by all of the polyurethane segments increased with increasing alcohol concentrations, with the cPU having the greatest uptake. Water uptake was minimal for all the polyurethanes examined and was not affected by altering pH. Water vapor permeation was lowest for the cPU films as compared to both bdPU and Mit. Swelling the cPU in 50% ethanol prior to evaluation slightly increased water vapor permeation through the films. Covalent linkage of the radiolabelled blood protein albumin (125I-BSA) to the cPU segments incubated with the heterobifunctional crosslinker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was greatest in the higher percent of ethanol as compared to controls. These results serve as foundation for developing a novel poly(carbonate urea) urethane with physical characteristics comparable to other medical-grade polyurethanes while having protein binding capabilities.     

Bone matrix proteins: isolation and characterization of a novel cell-binding keratan sulfate proteoglycan (osteoadherin) from bovine bone

The distribution and function of the neurotransmitter adrenaline in the primate brain are poorly understood. Biochemical studies have shown the presence of adrenaline or its biosynthetic enzyme, phenylethanolamine-N-methyltransferase, in the rat and human thalamus. However, the distribution of the adrenergic fibres in the thalamus has only been demonstrated in rats. We study the adrenergic innervation of the macaque monkey thalamus using immunohistochemistry against phenyletanolamine-N-methyltransferase. The distribution of phenyletanolamine-N-methyltransferase-immunoreactive fibres is markedly heterogeneous and principally restricted to those nuclei, or their portions, that are located in or close to the midline, with the highest density being found in the paraventricular, parafascicular and mediodorsal nuclei. The paraventricular nucleus is densely innervated by adrenergic axons throughout, while the densest innervation of the parafascicular nucleus is located in its medial part and the strongest mediodorsal nuclear immunolabelling is found in its most posterior and medial region. Moderate or low concentrations of phenyletanolamine-N-methyltransferase-immunopositive fibres are present in the paratenial nucleus, and all parts of the central nucleus, nucleus reuniens, central medial nucleus, centromedian nucleus, medial geniculate body and medial pulvinar nucleus, while only scattered immunoreactive axons are found in other thalamic nuclei. The morphology of the phenyletanolamine-N-methyltransferase-immunoreactive axons is quite diverse, as they have different diameters and most are endowed with diversely-shaped varicosities. These findings are the first morphological evidence for the presence of adrenergic innervation in the primate thalamus and reveal that this innervation is highly selective, heterogeneous and more widely distributed in primates than in rats. The thalamic nuclei innervated by adrenaline are connected to widespread limbic and associative cortical areas as well as to subcortical structures, in particular the neostriatum and amygdala. We hypothesize that thalamic adrenaline may be implicated in emotional, social and attentional mechanisms through its facilitation of co-ordinated action by these brain regions.     

Modulation of lipoprotein(a) atherogenicity by high density lipoprotein cholesterol levels in middle-aged men with symptomatic coronary artery disease and normal to moderately elevated serum cholesterol. Regression Growth Evaluation Statin Study (REGRESS) Study Group

OBJECTIVES: This study sought to examine whether lipoprotein(a) levels predict coronary artery lumen changes in patients with symptomatic coronary artery disease (CAD) and normal to moderate hypercholesterolemia. BACKGROUND: Recent conflicting reports have confirmed or refuted the association of lipoprotein(a) with clinical events or angiographically verified disease progression. METHODS: The association between serum lipoprotein(a) and changes in coronary artery lumen was studied in 704 men entered into the Regression Growth Evaluation Statin Study (REGRESS), a double-blind, placebo-controlled, quantitative angiographic study that assessed the effect of 2 years of pravastatin treatment. The primary end points were changes in average mean segment diameter (MSD) and average minimal obstruction diameter (MOD). Pravastatin- and placebo-treated patients were classified as having progressing, regressing or stable CAD, and median lipoprotein(a) concentrations were compared. Bivariate and multivariate regression analyses were performed in the overall patient group and in high risk subgroups. RESULTS: Pravastatin treatment did not affect serum apolipoprotein(a) levels. Median in-trial (sampled at 24 months) apolipoprotein(a) levels for regressing, stable and progressing CAD were, respectively, 130, 162 and 251 U/liter in placebo-treated patients and 143, 224 and 306 U/liter in pravastatin-treated patients. Predictors of MSD and MOD changes were baseline MSD and MOD, in-trial apolipoprotein(a), in-trial high density lipoprotein (HDL) cholesterol and baseline use of long-acting nitrates. The multivariate models predicted 14% of MSD changes and 12% of MOD changes; apolipoprotein(a) predicted only 2.6% and 4.8%, respectively. However, in patients with in-trial HDL cholesterol levels <0.7 mmol/liter, apolipoprotein(a) predicted up to 37% of the arteriographic changes. CONCLUSIONS: Serum lipoprotein(a) levels predict coronary artery lumen changes in normal to moderately hypercholesterolemic white men with CAD; its atherogenicity is marked in the presence of concomitant hypoalphalipoproteinemia.     

Further characterization of the thrombasthenia-related idiotype OG. Antiidiotype defines a novel epitope(s) shared by fibrinogen B beta chain, vitronectin, and von Willebrand factor and required for binding to beta 3

A patient (OG) with Glanzmann thrombasthenia became refractory to platelet transfusion after the production of an immunoglobulin G (IgG) isoantibody (Ab1) specific for the integrin subunit beta 3. To determine the frequency at which the OG idiotype is found in the general population and in immune-mediated disease states, we developed a rabbit polyclonal antibody (Ab2) specific for affinity-purified OG anti-beta 3 Fab. The binding of Ab2 to Ab1 is inhibited by purified alpha IIb beta 3. Ab2 als binds to IgG specific for alpha IIb beta 3 obtained from one nonrelated Glanzmann thrombasthenia patient ES who has developed isoantibodies of similar specificity. On the other hand, Ab2 does not recognize alpha IIb beta 3-specific antibodies produced by two Glanzmann thrombasthenia patients, AF and LUC, who have developed isoantibodies with specificities distinct from that of the OG isoantibody. Moreover, Ab2 does not recognize alpha IIb beta 3-specific antibodies developed by three representative patients with (autoimmune) thrombocytopenic purpura or six representative patients with alloimmune thrombocytopenias, nor does it bind to IgG from any of 13 nonimmunized individuals. We have found that Ab2 also binds to selected protein ligands of alpha IIb beta 3 namely, fibrinogen, vitronectin, and von Willebrand factor, but not to other protein ligands or control proteins, such a fibronectin, type I collagen, and albumin. The epitope(s) recognized by Ab2 on each adhesive protein are either very similar or identical since each protein can inhibit the binding of Ab2 to any of the other proteins. The epitope on fibrinogen recognized by Ab2 resides in the B beta chain, and is likely contained within the first 42 amino acids from the NH2 terminus. Since OG IgG inhibits fibrinogen binding to alpha IIb beta 3, the specificity of the OG idiotype defines a novel binding motif for the integrin alpha IIb beta 3 that is shared by fibrinogen, vitronectin, and von Willebrand factor, but distinct from previously described RGD-containing sites on the fibrinogen, A alpha chain or the fibrinogen gamma chain COOH-terminal decapeptide site. Our findings reported here represent an excellent example of molecular mimicry in which an antigen-selected, IgG inhibitor of alpha IIb beta 3 function shares a novel recognition sequence common to three physiologic protein ligands of that receptor.