Effects of verapamil on the release of different neurotransmitters

The effect of verapamil on resting and depolarization-induced monoamine release was investigated in rat hippocampal synaptosomes prelabeled with [³H]-5-hydroxytryptamine (HT) or [³H]-norepinephrine (NE) and rat striatal synaptosomes prelabeled with [³H]-dopamine (DA). Verapamil (50 μM) completely abolishes high K⁺-induced [³H]-NE release, but paradoxically facilitates high K⁺-induced [³H]-5-HT and [³H]-DA release. All these high K⁺-evoked responses were Ca²⁺ dependent. Verapamil does not modify [³H]-NE baseline release, but increases dose dependently [³H]-5-HT and [³H]-DA baseline release. Verapamil (10 μM, for 5 min) increases endogenous DA release (70%) and endogenous 5-HT release (40%) independently on the presence of external Ca²⁺. The total amount of these monoamines (released plus retained by the preparation) and their metabolites (DOPAC and 5-HIAA) was similar in control and verapamil-treated synaptosomes. Verapamil displaces [³H]-spiroperidol specific binding (K_i of 2.4 × 10^?6M) and [³H]-SCH-23390 specific binding (K_i of 9 × 10^?6M) from striatal synaptosomal membranes, and [³H]-5-HT specific binding (K_i of 3 × 10^?5M) from hippocampal synaptosomal membranes. It is concluded that in addition to the Ca²⁺ antagonistic properties of verapamil on the Ca²⁺-dependent, depolarization-induced release of some neurotransmitters [gamma aminobutyric acid (GABA and NE)], another mechanism probably mediated by presynaptic receptors underlies the effects of verapamil on DA and 5-HT release from discrete brain regions. © 1995 Wiley-Liss, Inc.     

Antinuclear antibodies in scleroderma,mixed connective tissue disease and “primary” Raynaud's phenomenon

Summary The diversity of antibodies in patients with scleroderma, mixed connective tissue disease or primary Raynaud's phenomenon could be used as a laboratory aid in the clinical diagnosis. In serum samples of 75 patients we screened for antinuclear antibodies (HEp 2 cells), anti DNA, soluble nucleoprotein and extractable nuclear antigens (Sm, rRNP, Ul-nRNP, SSA/Ro, SSB/La and Scl-70). Distinctive antinuclear antibodies pattern was identified in each group of patients. This immunologic profile is valuable for clinical diagnosis and the preferential association of certain autoantibodies with some diseases and not with others, suggest an antigen-driven stimulus for its production.     

Computer-assisted assignment of functional domains in the nonstructural polyprotein of hepatitis E virus: delineation of an additional group of positive-strand RNA plant and animal viruses.

Computer-assisted comparison of the nonstructural polyprotein of hepatitis E virus (HEV) with proteins of other positive-strand RNA viruses allowed the identification of the following putative functional domains: (i) RNA-dependent RNA polymerase, (ii) RNA helicase, (iii) methyltransferase, (iv) a domain of unknown function ("X" domain) flanking the papain-like protease domains in the polyproteins of animal positive-strand RNA viruses, and (v) papain-like cysteine protease domain distantly related to the putative papain-like protease of rubella virus (RubV). Comparative analysis of the polymerase and helicase sequences of positive-strand RNA viruses belonging to the so-called "alpha-like" supergroup revealed grouping between HEV, RubV, and beet necrotic yellow vein virus (BNYVV), a plant furovirus. Two additional domains have been identified: one showed significant conservation between HEV, RubV, and BNYVV, and the other showed conservation specifically between HEV and RubV. The large nonstructural proteins of HEV, RubV, and BNYVV retained similar domain organization, with the exceptions of relocation of the putative protease domain in HEV as compared to RubV and the absence of the protease and X domains in BNYVV. These observations show that HEV, RubV, and BNYVV encompass partially conserved arrays of distinctive putative functional domains, suggesting that these viruses constitute a distinct monophyletic group within the alpha-like supergroup of positive-strand RNA viruses.     

Osteogenic differentiation of human bone marrow stromal cells on partially demineralized bone scaffolds in vitro

Tissue engineering has been used to enhance the utility of biomaterials for clinical bone repair by the incorporation of an osteogenic cell source into a scaffold followed by the in vitro promotion of osteogenic differentiation before host implantation. In this study, three-dimensional, partially demineralized bone scaffolds were investigated for their ability to support osteogenic differentiation of human bone marrow stromal cells (BMSCs) in vitro. Dynamic cell seeding resulted in homogeneous cell attachment and infiltration within the matrix and produced significantly higher seeding efficiencies when compared with a conventional static seeding method. Dynamically seeded scaffolds were cultured for 7 and 14 days in the presence of dexamethasone and evaluated on biochemical, molecular, and morphological levels for osteogenic differentiation. Significant elevation in alkaline phosphatase activity was observed versus controls over the 14-day culture, with a transient peak indicative of early mineralization on day 7. On the basis of RT-PCR, dexamethasone-treated samples showed elevations in alkaline phosphatase and osteocalcin expression levels at 7 and 14 days over nontreated controls, while bone sialoprotein was produced only in the presence of dexamethasone at 14 days. Scanning electron microscopy evaluation of dexamethasone-treated samples at 14 days revealed primarily cuboidal cells indicative of mature osteoblasts, in contrast to nontreated controls displaying a majority of cells with a fibroblastic cell morphology. These results demonstrate that partially demineralized bone can be successfully used with human BMSCs to support osteogenic differentiation in vitro. This osseous biomaterial may offer new potential benefits as a tool for clinical bone replacement.     

Pediatric solid-organ transplant recipients carry chronic loads of Epstein-Barr virus exclusively in the immunoglobulin D-negative B-cell compartment

Solid-organ transplant recipients are at risk for development of lymphoproliferative diseases. The purpose of this study was to examine the distribution of Epstein-Barr virus (EBV) load in the peripheral blood of pediatric transplant recipients who had become chronic viral load carriers (>8 copies/10(5) lymphocytes for >2 months). A total of 19 patients with viral loads ranging from 20 to 5,000 viral genome copies/10(5) lymphocytes were studied. Ten patients had no previous diagnosis of posttransplant lymphoproliferative disease (PT-LPD), while nine had recovered from a diagnosed case of PT-LPD. No portion of the peripheral blood viral load was detected in the cell-free plasma fraction. Viral DNA was found in a population of cells characterized as CD19(hi) and immunoglobulin D negative, a phenotype that is consistent with the virus being carried exclusively in the memory B-cell compartment of the peripheral blood. There was no difference in the compartmentalization based upon either the level of the viral load or the past diagnosis of an episode of PT-LPD. These results have implications for the design of tests to detect EBV infection and for the interpretation and use of positive EBV PCR assays in the management of transplant recipients.     

Functional analysis of CD8 lymphocytes in long-term surviving patients after bone marrow transplantation

The recovery of T-cell populations after bone marrow transplantation (BMT) is characterized by a persistent expansion of CD8 lymphocytes. Previously, we have shown that beyond 1 year posttransplantation the CD8 lymphocytes consist, to a large extent, of CD8⁺ HNK1⁺ cells that suppress, like normal CD8 lymphocytes, immunoglobulin productionin vitro. We have further investigated the functional capabilities of CD8 lymphocytes, mostly HNK1⁺ (from 50 to 77%), in seven long-term BMT patients. As normal, patient CD8 lymphocytes do not suppress (1) phytohemagglutinin (PHA)-induced interleukin 2 (IL2) receptor expression and IL2 responsiveness by normal T cells or (2) the mixed lymphocyte reaction of donor cells. Also as normal, patient CD8 lymphocytes can be activated into potent cytotoxic effectors. Therefore, under the present experimental conditions, the increase in the absolute number of CD8 lymphocytes in the long-term BMT patients is characterized by an expansion of the CD8⁺ HNK1⁺-cell subpopulation and a normal suppressor/cytotoxic potential on a per-CD8⁺ cell basis.     

Performance of hydroxyapatite bone repair scaffolds created via three-dimensional fabrication techniques

The current study analyzes the in vivo performance of porous sintered hydroxyapatite (HA) bone repair scaffolds fabricated using the TheriForm solid freeform fabrication process. Porous HA scaffolds with engineered macroscopic channels had a significantly higher percentage of new bone area compared with porous HA scaffolds without channels in a rabbit calvarial defect model at an 8-week time point. An unexpected finding was the unusually large amount of new bone within the base material structure, which contained pores less than 20 microm in size. Compared with composite scaffolds of 80% polylactic-co-glycolic acid and 20% beta-tricalcium phosphate with the same macroscopic architecture as evaluated in a previous study, the porous HA scaffolds with channels had a significantly higher percentage of new bone area. Therefore, the current study indicates that scaffold geometry, as determined by the fabrication process, can enhance the ability of a ceramic material to accelerate healing of calvarial defects.     

Intrathoracic pressure fluctuations move blood during CPR: Comparison of hemodynamic data with predictions from a mathematical model

Whether blood flow during cardiopulmonary resuscitation (CPR) results from intrathoracic pressure fluctuations or direct cardiac compression remains controversial. We developed a mathematical model that predicts that blood flow due to intrathoracic pressure fluctuations should be insensitive to compression rate over a wide range but dependent on the applied force and compression duration. If direct compression of the heart plays a major role, however, the model predicts that flow should be dependent on compression rate and force, but above a threshold, insensitive to compression duration. These differences in hemodynamics produced by changes in rate and duration form a basis for determining whether blood flow during CPR results from intrathoracic pressure fluctuations or from direct cardiac compression. The model was validated for direct cardiac compression by studying the hemodynamics of cyclic cardiac deformation following thoracotomy in four anesthetized, 21–32-kg dogs. As predicted by the model, there was no change in myocardial or cerebral perfusion pressures when the duration of compression was increased from 15% to 45% of the cycle at a constant rate of 60/min. There was, however, a significant increase in perfusion pressures when rate was increased from 60 to 150/min at a constant duration of 45%. The model was validated for intrathoracic pressure changes by studying the hemodynamics produced by a thoracic vest (vest CPR) in eight dogs. The vest contained a bladder that was inflated and deflated. Vest CPR changed intrathoracic pressure without direct cardiac compression, since sternal displacement was <0.8 cm. As predicted by the model and opposite to direct cardiac compression, there was no change in perfusion pressures when the rate was increased from 60 to 150/min at a constant duration of 45% of the cycle. Manual CPR was then studied in eight dogs. There was no surgical manipulation of the chest. Myocardial and cerebral blood flows were determined with radioactive microspheres and behaved as predicted from the model of intrathoracic pressure, not direct cardiac compression. At nearly constant peak sternal force (378–426 N), flow was significantly increased when the duration of compression was increased from short (13%–19% of the cycle) to long (40%–47%), at a rate of 60/min. Flow was unchanged, however, for an increase in rate from 60 to 150/min at constant compression duration. In addition, myocardial and cerebral flow correlated with their respective perfusion pressures. Thus vital organ perfusion pressures and flow for manual external chest compression are dependent on the duration of compression, but not on rates of compression of 60 and 150/min. These data are of course similar to those produced by vest CPR, where intrathoracic pressure is manipulated without sternal displacement, and to those predicted for movement of blood by intrathoracic pressure changes. These data are, however, opposite to those produced by cardiac deformation and to those predicted for movement blood by direct cardiac compression. We conclude that intrathoracic pressure fluctuations generate blood flow during manual CPR.     

HLA alleles and haplotypes among the Lakota Sioux: report of the ASHI minority workshops, part III

Human leukocyte antigen (HLA) class I and II alleles were defined for 302 Lakota Sioux American Indians as part of the American Society for Histocompatibility and Immunogenetics coordinated studies on minority populations. The study group was comprised of adult volunteers from the Cheyenne River and Ogala Sioux tribes residing, respectively, on the Cheyenne River and Pine Ridge Reservations in South Dakota. Of the participants, 263 (87%) claimed full American Indian ancestry through both maternal and paternal grandparents. The study group included 25 nuclear families that were informative for genotyping. HLA phenotypes from 202 adults with no other known first-degree relative included in the study were used for calculation of allele and haplotype frequencies by maximum likelihood estimation. HLA-A, -B, and -Cw alleles were found to be in Hardy Weinberg equilibrium. Deviation from equilibrium was observed for DRB1 alleles (p=0.01), but could be attributed to the sample size and the occurrence of some genotypes with low expected frequencies. Polymorphism among the Sioux was limited with four to seven alleles comprising >80% of those observed at each locus. Several alleles were found at high frequency (0.05-0.30) among the Sioux that are also prevalent in other Native Americans and Alaska Natives, including: A*2402, *3101, and *0206; B*3501,*3901, *5101, and *2705; Cw*0702, *0404, and *03041; DRB1*0407, *0404, *1402, and *16021; and DQB1*0301, *0302, and *0402. DRB1*0811, which has been only previously described in Navajo and Tlingit Indians, was found to occur at a frequency of 0.119 among the Sioux. Two new alleles were defined among the Sioux: Cw*0204 and DRB1*040703, which were found in two and four individuals, respectively. In the haplotype analyses, significant linkage disequilibrium (p<0.00001) was seen in all pairwise comparisons of loci and numerous two and three locus haplotypes were found to have strong, positive linkage disequilibrium values. The two most common extended haplotypes among the Sioux, determined by maximum likelihood estimation and genotyping were: A*31012, B*3501, Cw*0404, DRB1*0407; and A*24021, B*3501, Cw*0404, DRB1*0404.