Activation of the cardiac ATP-sensitive K+ channel by ER-001533, a newly synthesized vasorelaxant.

Effects of ER-001533 (ER), a newly synthesized vasorelaxant, on the membrane currents were examined in single ventricular cells of guinea pigs. The patch-clamp technique was used in the "whole-cell" and "inside-out" patch configurations. In the whole-cell clamp condition, ER induced a time-independent K(+)-dominant current, which was inhibited by glibenclamide (1-3 microM), suggesting that ER activated the cardiac ATP-sensitive K+ channel (KATP). To elucidate the mechanism of ER-mediated KATP channel activation, the drug was applied to the inside-out patches before and after channel "run-down." Since nucleotide diphosphates could induce the channel openings after complete run-down, effects of the drug on the nucleotide diphosphate-induced channel openings were also examined. Before run-down, ER activated the KATP channel only in the presence of ATP. ER shifted the relation between [ATP]i and the channel activity to the right in a concentration-dependent fashion without a significant alteration of the slope. After channel run-down, ER did not affect the channel openings either in the absence or in the presence of UDP. However, ER could relieve the ATP-gamma-S inhibition of the UDP-induced channel openings. Thus, we conclude that ER antagonizes the inhibitory effect of ATP on the KATP channel in a competitive manner, thereby enhancing the channel openings.     

Bronchoalveolar lavage cell counts as a predictor of short term outcome in pulmonary sarcoidosis.

Sixty seven patients with biopsy proven pulmonary sarcoidosis were prospectively studied to determine whether single point bronchoalveolar lavage cell counts were a useful indicator of functional outcome and whether repeated lavage helped in management. The mean follow up period was 25 (range 13-37) months. No patient was having corticosteroid treatment at the time of initial bronchoalveolar lavage. "High intensity alveolitis" (lymphocyte count greater than or equal to 28%) was present at the initial lavage in 42 patients. These patients showed a significant improvement in their pulmonary function and chest radiographs over the follow up period whereas patients with "low intensity alveolitis" did not. Of the 42 patients with high intensity alveolitis, 31 had chronic sarcoidosis (duration over two years, mean 80 months). These patients showed a significant improvement in FVC but not in TLCO. Corticosteroids resulted in greater functional and radiological improvement in the patients with high intensity alveolitis than in those with low intensity alveolitis. Repeat bronchoalveolar lavage in 34 patients, mean 8.4 months after the original lavage, showed a weak inverse relation between a reduced lymphocyte count and change in forced vital capacity and isotope uptake on a gallium scan. These correlations were too weak to make repeated cell counts useful in management. Our results suggest that high intensity alveolitis may be a favourable prognostic factor for lung function in pulmonary sarcoidosis, even in patients with chronic disease, but that repeat lavage adds little to the management of the individual patient.     

Inhibitability and enhanceability of basophil histamine release in asthmatic and normal subjects

Circulating human basophils contain histamine, a potent mediator of inflammation. Previous in vitro studies have shown that histamine 'releasability' in asthmatic subjects differs from normal subjects but have not evaluated possible differences in the immunopharmacological control of the release of this mediator which might account for these differences. The purpose of the present study was to examine the immunopharmacologic control of basophil histamine release in 14 asthmatics and 10 normal subjects who were characterized by pulmonary function tests, allergic status (skin tests and serum IgE levels) and nonspecific airways reactivity to methacholine and histamine. Basophils were stimulated with anti-IgE, and the inhibitory effects of the H2 agonist, dimaprit, and dibutyryl cyclic AMP (dbcAMP), as well as the enhancing properties of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and indomethacin on the modulation of histamine release, were investigated. Although no statistically significant differences were seen in the percent histamine release triggered by anti-IgE in these two groups, enhancement of histamine release by 5-HPETE was more consistent in the asthmatic subjects (10 of 10) than in control subjects (6 of 8). The percent increase in histamine release produced by 5-HPETE in asthmatic subjects averaged 3.9 +/- 1.3% using 0.03 micrograms anti-IgE/ml and 4.8 +/- 3.2% using 0.1 microgram anti-IgE/ml (p less than 0.002, Wilcoxon's signed rank test), and averaged 3.0 +/- 4.3 and 3.1 +/- 5.3%, respectively, in control subjects (p greater than 0.10).(ABSTRACT TRUNCATED AT 250 WORDS)     

Virus diversity and an outbreak of group C rotavirus among infants and children with diarrhea in Maizuru city, Japan during 2002-2003

A total of 236 fecal specimens collected from infants and children with gastroenteritis in Maizuru city, Japan from July 2002 to June 2003, were tested for the presence of rotaviruses, noroviruses, sapoviruses, astroviruses, and adenoviruses by RT-PCR, PAGE, RPHA, and latex agglutination methods. Among diarrheal viruses detected, group A rotavirus was the most prevalent (32.2%; 76 of 236) followed by norovirus GII (21.2%; 50 of 236), group C rotavirus (10.2%; 24 of 236), adenovirus (3.8%; 9 of 236), sapovirus (2.5%; 6 of 236), astrovirus (1.3%; 3 of 236), and norovirus GI (0.8%; 2 of 236), respectively. It is noteworthy that group C rotavirus infection was apparently confined only within the period of 5 months (December 2002 through April 2003). This pattern of infection implied that the outbreak of group C rotavirus in these patients, which was the first outbreak of gastroenteritis attributed to group C rotavirus in Maizuru city. Moreover, about half (12 of 24) of group C rotavirus infected cases were confined to infants and young children less than 3 years old. Another interesting feature of the study was the demonstration of the mixed infections with group C rotavirus and group A rotavirus, as well as group C rotavirus and norovirus GII in 20.8% (5 of 24) and 8.3% (2 of 24), respectively. This is the first report of gastroenteritis associated with the mixed infections with group C rotavirus and other viral enteropathogens such as norovirus. The results indicate that group C rotavirus could infect not only older children and adults but also infants and young children under 3 years old.     

Potent inhibition of SARS-associated coronavirus (SCOV) infection and replication by type I interferons (IFN-alpha/beta) but not by type II interferon (IFN-gamma).

We sought to investigate the anti-severe acute respiratory syndrome (SARS)-associated coronavirus (SCoV) activities of type I (alpha and beta) and type II (gamma) interferons (IFN) in vitro. Type I IFNs protected cells from cytopathic effects (CPE) induced by SCoV, and inhibited viral genomic RNA replication in FRhk-4 cells (measured by quantitative RT-PCR) in a dose-dependent manner. Intracellular viral RNA copies were reduced 50% by IFN-alpha at a concentration of 25 U/ml and by IFN-beta at a concentration of 14 U/ml. IFN-gamma had fewer effects on inhibition of viral infection and replication. The type I IFN receptor signaling pathway in host cells is mainly involved in the inhibition of SCoV infection and replication. Type I IFNs could be used as potential agents for anti-SARS treatment.     

Linkage of the MHC to familial multiple sclerosis suggests genetic heterogeneity. The Multiple Sclerosis Genetics Group

Multiple sclerosis (MS) is a demyelinating autoimmune disease of the central nervous system. While its etiology is not well understood, genetic factors are clearly involved. Until recently, most genetic studies in MS have been association studies using the case-control design testing specific candidate genes and studying only sporadic cases. The only consistently replicated finding has been an association with the HLA-DR2 allele within the major histocompatibility complex (MHC) on chromosome 6. Using the genetic linkage design, however, evidence for and against linkage of the MHC to MS has been found, fostering suggestions that sporadic and familial MS have different etiologies. Most recently, two of four genomic screens demonstrated linkage to the MHC, although specific allelic associations were not tested. Here, a dataset of 98 multiplex families was studied to test for an association to the HLA-DR2 allele in familial MS and to determine if genetic linkage to the MHC was due solely to such an association. Three highly polymorphic markers (HLA-DR, D6S273 and TNFbeta) in the MHC demonstrated strong genetic linkage (parametric lod scores of 4.60, 2.20 and 1.24, respectively) and a specific association with the HLA-DR2 allele was confirmed (TDT; P < 0.001). Stratifying the results by HLA-DR2 status showed that the linkage results were limited to families segregating HLA-DR2 alleles. These results demonstrate that genetic linkage to the MHC can be explained by the HLA-DR2 allelic association. They also indicate that sporadic and familial MS share a common genetic susceptibility. In addition, preliminary calculations suggest that the MHC explains between 17 and 62% of the genetic etiology of MS. This heterogeneity is also supported by the minority of families showing no linkage or association with loci within the MHC.      

Determination of optimal cryoprotectants and procedures for their addition and removal from human spermatozoa

The objective was to test the hypothesis that the optimal cryoprotective agent for cryopreservation of human spermatozoa would be a solute for which cells have the highest plasma membrane permeability, resulting in the least amount of volume excursion during its addition and removal. To test this hypothesis, theoretical simulations were performed using membrane permeability coefficients to predict optimal procedures for the addition and removal of a cryoprotectant. Simulations were performed using data from four different cryoprotectants: (i) glycerol, (ii) dimethyl sulphoxide, (iii) propylene glycol and (iv) ethylene glycol. Thermodynamic formulations were applied to determine approaches for the addition and removal of 1 M and 2 M final concentrations of cryoprotectant, allowing the spermatozoa to maintain a cell volume within their osmotic tolerance limits. Based on these data, ethylene glycol was predicted to be optimal for minimizing volume excursions among the solutes evaluated. These predictions were then experimentally tested using glycerol as the control cryoprotectant and ethylene glycol as the experimental cryoprotectant. The results indicate that there was a higher (P < 0.05) recovery of motile spermatozoa after cryopreservation when using 1 M ethylene glycol than with 1 M glycerol, supporting the hypothesis that use of the cryoprotectant for which the cell has the highest permeability will result in higher cell survival.      

Study of circulating immune complex size in systemic lupus erythematosus.

The molecular size of circulating immune complexes in patients with systemic lupus erythematosus was determined by the C1q solid-phase assay after the sera were fractionated by sucrose-gradient ultracentrifugation. Circulating immune complexes in patients with membranous glomerulonephritis were uniformly large, sedimenting exclusively above 19S, whereas the immune complexes in patients with cerebritis were small, at or just above 7S. In lupus patients with diffuse proliferative glomerulonephritis and patients without renal involvement, immune complexes of both large and small sizes were found. Of patients without renal involvement, more circulating immune complexes were associated with active disease (n = 22, prevalence = 82%, mean level = 24 standard deviations) than with inactive disease (n = 17, prevalence = 41%, mean level = 41%, mean level = 6 . 5 standard deviations). In patients with clinical evidence for renal involvement, circulating immune complexes were detected in all of five patients with membranous glomerulonephritis, in 88% of 17 patients with diffuse proliferative glomerulonephritis and in one of four patients with mesangial nephritis. Thus, in addition to the finding of an overall positive correlation between disease activity and circulating immune complex levels, circulating immune complexes of certain general molecular size ranges appear to be associated with different clinical manifestations of systemic lupus erythematosus.     

Use of Air-Liquid Two-Phase Flow in Hydrophobic Microfluidic Channels for Disposable Flow Cytometers

This paper describes a disposable flow cytometer that uses an air-liquid two-phase microfluidic system to produce a focused high-speed liquid sample stream of particles and cells. The susceptibility of thin liquid columns to instabilities may suggest that focusing of sample liquids with streams of air would be difficult. The design of channel geometry, control of flow rates, and use of appropriate surface chemistries on the channel walls, however, enabled the generation of thin (15–100 m) and partially bounded sample streams that were stable and suitable for rapid cell analysis. Using an inverted epi-fluorescence microscope with a photo-multiplier tube, we demonstrated that the system is capable of counting the number of beads and C₂C₁₂ myoblast cells. The effects of different flow rates and surface chemistries of the channel walls on the air-liquid two-phase flows were characterized using optical and confocal microscopy. Use of air instead of liquids as a sheath fluid eliminates the need for large sheath liquid reservoirs, and reduces the volume and weight requirements. The low manufacturing cost and high volumetric efficiency make the air-sheath flow cytometer attractive for use as a stand-alone device or as an integrated component of bio-artificial hybrid microsystems.     

Calcium ionophore-induced acrosome reaction correlates with fertilization rates in vitro in patients with teratozoospermic semen

The aim of this study was to determine the relationship between calcium ionophore A23187-induced acrosome reaction (AR) and sperm fertilizing ability. Semen samples remaining after preparation for standard IVF were studied in 109 patients who had sperm concentrations > or =20 x 10(6)/ml. Ionophore-induced AR was performed on motile spermatozoa selected by centrifugation on a Percoll gradient. Semen analysis was performed using standard methods. Patients with higher (>50%, n = 76) fertilization rates had significantly higher ionophore-induced AR than patients with lower (<50%, n = 33) fertilization rates (49 +/- 14 versus 38 +/- 21%, P < 0.05). When the data from all patients were analysed by logistic regression, only the percentage sperm motility in insemination medium and ionophore-induced AR were significantly related to fertilization rates. Similar results were also obtained when the data from a subgroup of patients with poor (<15% normal) sperm morphology were analysed. However, when patients with normal sperm morphology > or =15% were analysed separately, only sperm count and the percentage of spermatozoa with progressive motility in semen were significantly related to fertilization rates. In conclusion, ionophore- induced AR was significantly related to fertilization rates in vitro mainly in patients with teratozoospermic semen. Tests for ionophore- induced AR may provide additional information about sperm fertilizing ability but may not indicate specific defects of the physiological AR.