PubMed Health⌕ Search

Biomedical subjects

B Horowitz

Publications and source records attributed to B Horowitz.

At least 109 records · Page 6Linked to original sources
PubMed1993

Dihydropyridine-sensitive calcium channels expressed in canine colonic smooth muscle cells.

Experiments were performed to identify and characterize the types of calcium channels that regulate inward calcium current in canine colonic smooth muscle. Freshly dispersed smooth muscle cells from the circular layer of the canine proximal colon were used. Single-channel currents were measured with 80 mM Ba2+ as the charge carrier. Small-conductance (10 +/- 2 pS, EBa = 46 +/- 11 mV, n = 9) and large-conductance (21 +/- 1 pS, EBa = 52 +/- 3 mV, n = 19) single-channel currents were observed during depolarizing voltage steps positive to -30 mV. Both types of single-channel currents were inhibited by the addition of 10(-6) M nifedipine to the bath solution. The smaller current was infrequently observed and therefore was not further characterized. Open probability (P(o)) of the larger current amplitude was strongly dependent on voltage. Activation curves were well described by a Boltzmann function with half activation occurring at 4 mV, and a 5-mV increase in membrane potential resulted in an e-fold increase in P(o). BAY K 8644 (1 microM) shifted the activation curve to the left while nifedipine (1 microM) resulted in a right shift. Molecular analysis showed that only the C class of Ca2+ channel alpha 1-subunit is expressed in this tissue. Furthermore, only a single splice variant (rbc-II) was observed. The results suggest that a single class of dihydropyridine-sensitive calcium channels regulates inward calcium current in canine colonic smooth muscle cells.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1993

Characterization of the properties of canine colonic smooth muscle in culture.

We have developed and characterized an organ culture system that maintains the viability of colonic smooth muscles. Morphological, mechanical, electrical, and molecular properties of cultured canine colonic circular muscles were determined. Strips of circular muscle were cultured for up to 6 days. The smooth muscle phenotype was retained during culture; muscles contracted to agonists and responded to electrical field stimulation, suggesting that intrinsic nerves also survived in culture. Morphological analysis showed identifiable smooth muscle cells, enteric neurons, and interstitial cells, but some alterations in ultrastructure were also observed. Mechanical responses to acetylcholine suggested that the muscles developed supersensitivity during the culture period. The resting membrane potentials of cells near the submucosal surface of the circular muscle layer decreased from -82 mV on day 0 to -55 mV on day 3. Similar changes in the resting potential gradient occur when colonic muscles are treated with inhibitors of the Na(+)-K(+)-ATPase. Resting potentials of day 3 muscles remained constant in low external K+ (0.1 mM), suggesting little contribution of the pump to resting potential. Northern analysis of RNA from muscles cultured up to 6 days showed that the alpha 2-isoform of the pump decreased. The data suggest that organ-cultured strips of smooth muscle may provide a useful tool for evaluating electrical and mechanical events in conjunction with molecular analysis of functional components.

Acetylcholine↗
PubMed1993

Alternative splicing of CFTR Cl- channels in heart.

We have previously demonstrated that cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels are expressed in heart (Levesque et al., Circ. Res. 71: 1002-1007, 1992). However, the structural identity between this cardiac protein and CFTR in epithelial cells is unknown. We amplified cDNA from rabbit ventricle and cloned fragments corresponding to the 12 transmembrane spanning domains of the epithelial CFTR transcript. The deduced sequence from rabbit heart indicated deletion of a 30-amino acid segment in the first cytoplasmic loop of CFTR, which corresponds to known locations of intron-exon junctions bordering exon 5 in the CFTR gene, suggesting that CFTR is alternatively spliced in heart. Outside this region, the heart CFTR isoform displayed > 95% identity to human epithelial CFTR. Molecular analysis demonstrated CFTR expression only in cardiac tissues that exhibited a adenosine 3',5'-cyclic monophosphate-dependent Cl- conductance in native cells. The expression of a specific isoform of CFTR Cl- channels in mammalian heart may have functional and clinical significance.

Animals↗
PubMed1993

Viral safety of solvent-detergent treated blood products.

Laboratory research commencing in 1982 led to licensing in the United States in 1985 of a solvent/detergent (SD)-treated anti-haemophilic factor (AHF) concentrate. Licensing was based on (a) studies demonstrating the inactivation of several marker viruses [vesicular stomatitis virus (VSV), Sindbis virus, Sendai virus], human immunodeficiency virus (HIV), hepatitis B virus (HBV), and non-A, non-B hepatitis virus [NANBHV; now known to be principally hepatitis C virus (HCV)] added to AHF just before treatment, (b) the realization that the principal viruses of concern in a transfusion setting (e.g. HIV, HBV, NANBHV) were all lipid-enveloped, and (c) laboratory, preclinical and clinical evidence indicating that AHF and other proteins present in the preparation were unaffected. The applicability of the SD method to a wide range of products and preparations, high process recoveries, and a growing body of viral safety information linked with the failure of several other virus inactivation methods to eliminate hepatitis transmission fostered the adoption of SD technology by more than 50 organizations world-wide. SD mixtures are now used in the preparation of products as diverse as intermediate purity and monoclonal antibody purified AHF and other coagulation factor concentrates, fibrin glue, normal and hyperimmune IgG and IgM preparations including those derived from tissue culture, plasma for transfusion, and various diagnostic controls. Over four million doses of SD-treated products have been administered, and numerous laboratory and clinical studies designed to assess virus safety have been conducted. SD treatment has been shown to inactivate > or = 10(9.2) tissue culture infectious doses (TCID50) of VSV, > or = 10(8.8) TCID50 of Sindbis virus, > or = 10(6.0) TCID50 of Sendai virus, > or = 10(7.3) duck infectious doses of duck HBV, > or = 10(11.0) degrees TCID50 of HIV-1, > or = 10(6.0) TCID50 of HIV-2, > or = 10(6.0) chimpanzee infectious doses (CID50) of HBV, > or = 10(5.0) CID50 of HCV, > or = 10(6.0) TCID50 of cytomegalovirus, > or = 10(5.8) TCID50 of herpes simplex virus type 1, > or = 10(4.0) TCID50 of PI-1, > or = 10(6.0) TCID50 of murine leukemia virus (Mov-3), > or = 10(4.0) TCID50 of murine xenotropic virus, and > or = 10(2.0) TCID50 of Rauscher murine leukemia ecotropic virus. Moreover, in ten prospective clinical studies, 0/53, 0/427, and 0/455 patients susceptible to HBV, NANBHV (HCV), and HIV became infected on follow-up.(ABSTRACT TRUNCATED AT 400 WORDS)

Biological Products↗
PubMed1993

Hepatitis B viral clearance studies using duck virus model.

We have shown data to suggest that the in vivo duck hepatitis B virus system represents an excellent animal model system for the study of hepatitis B virus. Because of the similarity of DHBV to human HBV (including comparable results in virus inactivation studies), the high level of sensitivity of the DHBV assay, and the rapidity, ease, and relative low cost of obtaining results, we propose that the in vivo DHBV titration system be used as a model for human HBV in process validation studies. Data generated in such validation studies have, in fact, been submitted by a number of blood products manufacturers to the U.S. F.D.A. in support of IND applications.

Animals↗
PubMed1992

Solvent/detergent-treated plasma: a virus-inactivated substitute for fresh frozen plasma.

Fresh frozen plasma (FFP) is prepared in blood banks world-wide as a by-product of red blood cell concentrate preparation. Appropriate clinical use is for coagulation factor disorders where appropriate concentrates are unavailable and when multiple coagulation factor deficits occur such as in surgery. Viral safety depends on donor selection and screening; thus, there continues to be a small but defined risk of viral transmission comparable with that exhibited by whole blood. We have prepared a virus sterilized FFP (S/D-FFP) by treatment of FFP with 1% tri(n-butyl)phosphate (TNBP) and 1% Triton X-100 at 30 degrees C for 4 hours. Added reagents are removed by extraction with soybean oil and chromatography on insolubilized C18 resin. Treatment results in the rapid and complete inactivation of greater than or equal to 10(7.5) infectious doses (ID50) of vesicular stomatitis virus (VSV) and greater than or equal to 10(6.9) ID50 of sindbis virus (used as marker viruses), greater than or equal to 10(6.2) ID50 of human immunodeficiency virus (HIV), greater than or equal to 10(6) chimp infectious doses (CID50) of hepatitis B virus (HBV), and greater than or equal to 10(5) CID50 of hepatitis C virus (HCV). Immunization of rabbits with S/D-FFP and subsequent adsorption of elicited antibodies with untreated FFP confirmed the absence of neoimmungen formation. Coagulation factor content was comparable with that found in FFP. Based on these laboratory and animal studies, together with the extensive history of the successful use of S/D-treated coagulation factor concentrates, we conclude that replacement of FFP with S/D-FFP, prepared in a manufacturing facility, will result in improved virus safety and product uniformity with no loss of efficacy.

Animals↗
PubMed1992

High-temperature short-time heat inactivation of HIV and other viruses in human blood plasma.

An ultra-short-time heating system was used to process blood plasma spiked with various viruses (HIV, vesicular stomatitis virus, encephalomyocarditis virus). Virus reduction and recovery of plasma proteins were measured at various temperatures from 65 to 85 degrees C. Processing at 77 degrees C and 0.006 s resulted in a high level of virus kill, including greater than or equal to 4.4 log10 HIV, while maintaining protein structure and activity essentially intact.

Blood↗
PubMed1992

Importance of type I and type II mechanisms in the photodynamic inactivation of viruses in blood with aluminum phthalocyanine derivatives.

The relative importance of type I and type II mechanisms in the photodynamic treatment of red blood cell concentrations (RBCC) to inactivate viruses was studied using aluminum phthalocyanine tetrasulfonate (AlPcS4), visible light and quenching or enhancing agents of reactive forms of oxygen. Treatment of a human RBCC with 10-13 microM AlPcS4 and 25-26 mW/cm2 visible light resulted in the rapid and complete inactivation of added vesicular stomatitis virus (VSV). The addition of mannitol, glycerol, reduced glutathione (GSH), or superoxide dismutase (SOD), known quenching agents of type I mechanisms, had little to no effect on the rate of inactivation of VSV. Significant inhibition of VSV kill was observed on addition of tryptophan or sodium azide, known quenchers of type II mechanisms. Additionally, the rate of VSV kill was enhanced in the presence of D2O. Taken together, these results indicate a predominant role of singlet oxygen in the inactivation of VSV on photodynamic treatment of RBCC. The relative importance of type I and type II mechanisms on cellular toxicity was also evaluated. Little, if any hemoglobin release was observed on treatment of human or rabbit RBCC with 10 microM AlPcS4 and 44 J/cm2 of visible light in the presence or absence of the above mentioned quenchers. The effect of the addition of quenchers on the recovery and circulatory survival of treated, autologous rabbit RBCC, labeled with 51Cr, was also assessed.(ABSTRACT TRUNCATED AT 250 WORDS)

Aluminum↗
PubMed1992

Expression of cystic fibrosis transmembrane regulator Cl- channels in heart.

Cyclic AMP (cAMP)-dependent chloride channels modulate changes in resting membrane potential and action potential duration in response to autonomic stimulation in heart. A growing body of evidence suggests that there are marked similarities in the properties of the cAMP-dependent chloride channels in heart and cystic fibrosis transmembrane regulator (CFTR) chloride channels found in airway epithelia or in cells expressing the CFTR gene product. We isolated poly A+ mRNA from rabbit ventricle and converted it to cDNA for amplification using the polymerase chain reaction (PCR). A fragment corresponding to the nucleotide-binding domain 1 (NBD1) of the CFTR transcript was cloned. Comparison of the amino acid sequence of NBD1 of human CFTR with the deduced sequence of the rabbit heart PCR product indicated 98% identity. Northern blot analysis, using the heart amplification product as a cDNA probe, demonstrated expression of homologous transcripts in human atrium, guinea pig and rabbit ventricle, and dog pancreas. Xenopus oocytes injected with poly A+ mRNA extracted from rabbit and guinea pig ventricle or dog pancreas expressed robust time-independent chloride currents in response to an elevation of cAMP. We conclude that CFTR chloride channels are expressed in heart and are responsible for the observed cAMP-dependent chloride conductance.

Animals↗
PubMed1992

Inactivation of viruses in red cell and platelet concentrates with aluminum phthalocyanine (AIPc) sulfonates.

Aluminum phthalocyanine tetrasulfonates (AIPcS) are photoactive compounds with absorption maxima at 665-675 nm. The inactivation of viruses (vesicular stomatitis virus, VSV; human immunodeficiency virus, HIV) added to either whole blood or red blood cell concentrates (RBCC) and platelet concentrates (PC) on treatment with tetrasulfonated AIPc (AIPcS4) was evaluated. Treatment of RBCC with 10 microM AIPcS4 and 44 J/cm2 visible light resulted in the inactivation of greater than or equal to 10(5.5) infectious doses (TCID50) of cell-free VSV, greater than or equal to 10(5.6) TCID50 of cell-associated VSV, and greater than or equal to 10(4.7) TCID50 of cell-free sindbis virus. Both greater than or equal to 10(4.2) TCID50 of cell-free and greater than or equal to 10(3.6) TCID50 of cell-associated forms of HIV were also shown to be inactivated. Encephalomyocarditis virus, used as a model for nonenveloped viruses, was not inactivated. Equivalent virus kill with Photofrin II required a substantially higher concentration of dye and longer exposure to visible light. Following AIPcS4 treatment, red cell integrity was well maintained as judged by the low level (less than 2%) of hemoglobin release immediately following treatment and on subsequent storage, by measurements of erythrocyte osmotic fragility, and by the normal recovery and circulatory survival on infusion of treated, autologous red blood cells in baboons. Treatment of PC with 10 microM AIPcS4 and 44 J/cm2 visible light also resulted in effective virus kill (greater than or equal to 10(5.5) TCID50) of VSV; however, both the rate and extent of platelet aggregation in response to collagen addition declined by at least 50%. Based on these results, further characterization of AIPcS4-treated RBCC is justified.

Animals↗
PubMed1991

Sodium pump isozymes are differentially expressed in electrically dissimilar regions of colonic circular smooth muscle.

Molecular analyses of Na,K-ATPase abundance and alpha-subunit isoform distribution were performed to determine whether pump expression varies at different points through the thickness of the circular layer of colonic smooth muscle. The mRNA and polypeptides of Na,K-ATPase alpha 1 and beta subunits were twice as abundant in the submucosal region of the circular layer, which has previously been shown to generate large pump potentials. Sodium pump activity directly correlated with the relative abundance of the alpha 1 polypeptide. These data show that sodium pump expression varies in electrically dissimilar regions of the circular layer.

Animals↗
PubMed1991

Inactivation of viruses found with plasma proteins.

Plasma protein solutions such as albumin and intramuscular immune globulin have long histories of viral safety. Coagulation factor concentrates as traditionally manufactured frequently transmitted HBV, HCV, and HIV. Indeed, it is probable that every vial of concentrate contained infectious HCV. Modern coagulation factor concentrates have a greatly improved safety record arising, principally, from the implementation of virucidal procedures. It is interesting to note that the same methods that failed to substantially reduce NANBHV transmission in clinical studies are those that were found to inactivate less than 10(4) ID50 of HIV, HBV, and/or HCV in preclinical studies (Table 17-5). Implementation even of these methods nearly eliminated the transmission of HIV by coagulation factor concentrates. A summary of the results of the most successful procedures is given in Table 17-10. The results show 0/564 patients had evidence of HIV transmission, 6/151 patients had evidence of HBV transmission, and 2/301 patients had evidence of HCV transmission. As compared with those procedures described in Table 17-5, the greater kill of HIV, HBV, and NANBHV demonstrated preclinically, and the improved clinical results, are most notable. The data, examined in terms of units transfused, are presented in Figure 17-1. Since the average adult hemophiliac in the United States receives 80,000 units of clotting factor per year, the best of the concentrates show safety over the equivalent of at least 10-human-years of treatment. Are the best of today's coagulation factor concentrates safe from the transmission of HBV, NANBHV (including HCV), and HIV. Given the limited number of patients eligible for clinical studies, and the length, difficulty, and expense of such studies, the best answer comes from a knowledge of the initial virus load coupled with information regarding virus removal, serendipitous inactivation, and intentional sterilization. A recently completed analysis of these factors (Horowitz 1990) indicates that the best of the modern coagulation factor concentrates are likely to be as safe as albumin (Figure 17-2).

Blood↗
PubMed1991

Inactivation of viruses in blood with aluminum phthalocyanine derivatives.

The inactivation of viruses added to whole blood and a red cell concentrate with aluminum phthalocyanine and its sulfonated derivatives was studied. A cell-free form of vesicular stomatitis virus (VSV), used as a model, was completely inactivated (greater than 10(4) infectious units; TCID50) on treatment of whole blood with 10 microM (10 mumol/L) aluminum phthalocyanine chloride (AIPs) and visible light dosage of 88 to 176 J per cm2. At 44 J per cm2, complete VSV inactivation was achieved on raising the concentration of AIPc to 25 microM (25 mumol/L). Results at least as good were achieved on similar treatment of a red cell concentrate. Also inactivated were a cell-associated form of VSV and both cell-free and cell-associated forms of human immunodeficiency virus; encephalomyocarditis virus, used as a model for non-lipid-enveloped viruses, was not inactivated by this procedure. This inactivation of cell-free VSV suggests that a similar degree of inactivation could be achieved with a lower concentration of the sulfonated forms of aluminum phthalocyanine. Throughout the above studies, red cell integrity was well maintained, as judged by the absence of hemoglobin release (less than or equal to 2%) during the course of treatment or on subsequent storage. Red cell osmotic fragility was decreased on treatment of whole blood with AIPc. This study indicates that AIPc may be a promising method for the inactivation of viruses in cellular blood products.

Antiviral Agents↗
PubMed1991

Prevention of HIV infection by passive immunization with HIV immunoglobulin.

The use of a human immunodeficiency virus (HIV) immune globulin (HIVIG) in prevention of HIV infection in chimpanzees was investigated in the hope of ultimate application to interruption of vertical transmission. In previous experiments, no protection was observed when relatively high challenge doses were used. This study shows that HIVIG protected against a challenge dose (10 CID50) tenfold lower than that used previously. The protected animal remained free of HIV infection as determined by cocultivation and by polymerase chain reaction (PCR), and did not mount a primary immune response detectable by enzyme-linked immunosorbant assay (ELISA) and neutralization assays. These results imply that HIV vaccines should induce neutralizing antibody and may not need to induce cell-mediated immunity in order to be protective against exposure to HIV. They also provide an experimental basis for the conduct of clinical trials to evaluate prevention of maternal-infant transmission by HIVIG.

Animals↗
PubMed1991

Muscarinic receptors in canine colonic circular smooth muscle. I. Coexistence of M2 and M3 subtypes.

The parasympathetic neurotransmitter acetylcholine, acting postsynaptically at the smooth muscle muscarinic receptor, is a principle determinant of colonic motility. In order to elucidate the receptor signal-transduction events responsible for muscarinic receptor-induced contraction of colonic circular smooth muscle, we present here and in the accompanying work studies designed to characterize the muscarinic receptors present in colon and to determine their biochemical coupling. Muscarinic receptor subtypes in canine colonic circular smooth muscle were characterized using radioligand binding techniques. The nonselective muscarinic receptor antagonist radioligand [3H]quinuclidinyl benzilate ([3H]QNB) binds rapidly and reversibly to a single class of saturable sites in colon circular smooth muscle membranes, with an affinity (KD) for the antagonist radioligand of 79.8 +/- 12.6 pM and a density of 123.3 +/- 18.7 fmol/mg of protein. Experiments using membranes prepared from isolated cells purified from the circular smooth muscle layer of canine colon (KD = 102.4 +/- 13.5 pM) confirm the smooth muscle origin of the binding and yield a receptor density of 124,340 receptors/cell. The order of potencies of selective muscarinic receptor antagonists in competition with [3H]QNB for binding to colonic receptors is 4-diphenylacetoxy-N-methylpiperidine methobromide greater than methoctramine greater than AF-DX 116 greater than pirenzepine. Unlike other antagonists tested, pirenzepine competition of [3H]QNB binding is biphasic. The high and low affinities deduced from nonlinear fit of the binding data in colon correlate very well with affinities determined for pirenzepine in mixtures of both submandibular gland (M3) and atrium (M2), indicating the presence of two muscarinic receptor subtypes (82% M2, 18% M3) in colon circular smooth muscle. The muscarinic agonist carbachol binds to both high and low affinity sites in colon, and addition of guanine nucleotide (100 microM GTP gamma S) shifts the agonist competition curve to the right, without eliminating high affinity binding sites. Agonist competition studies with a known ratio of M2 and M3 receptors, obtained by mixing pure M2 and M3 populations, predict the result obtained in colon. cDNA probes specific for each of the muscarinic receptors m1 through m4 were hybridized to colon RNA in a Northern blot analysis. Only m2 and m3 probes hybridized to colon RNA, suggesting the presence of both M2 and M3 receptors. Our data demonstrate that the colon circular smooth muscle contains muscarinic receptors of both the M2 and M3 subtypes, which may be coupled to disparate signal transduction pathways important in the physiological actions of acetylcholine in this tissue.

Animals↗