Determination of a buspirone metabolite in plasma samples

A high-performance liquid chromatographic (HPLC) method is described for the assay of the active metabolite [1-(2-pyrimidinyl)piperazinel of buspirone, an anxiolytic agent, in rat plasma.

The method is based on the use of ion-pair HPLC coupled to a liquid—solid extraction scheme. Samples of rat plasma (2 ml) with internal standard (1-phenylpiperazine), adjusted to pH 10.5 with borate buffer, were loaded on to a preactivated C-18 cartridge. The metabolite and the internal standard were eluted with 5 ml of methanol and injected on to a reversed-phase 10-μm Spherisorb ODS-2 column. The column was eluted with a mobile phase of 0.005 M sodium lauryl sulphate in citrate buffer (pH 3.6)-acetonitrile (65:35, v/v) at 2 ml min⁻¹. Detection was carried out at 248 nm. The recovery of the metabolite was 55%. The method was applied to the determination of the metabolite in rat plasma after oral dosing (25 mg kg⁻¹) of the parent compound.     

Action of cocaine and chronic sympathetic denervation on vagal escape

1. The effect of cocaine has been studied on vagal escape and on the tachycardia due to vagal stimulation in the atropinized dog. All the dogs were submitted to acute cervical section of the spinal cord and acute or chronic sympathetic denervation.2. Cocaine, 5 mg/kg or 40 mug/kg/min, I.V., induces a significant enhancement of the ventricular escape. The effects of a continuous infusion of cocaine are more reproducible than those of a single injection of the drug.3. Cocaine, 40 mug/kg/min, I.V., potentiates the tachycardia due to vagal stimulation in the atropinized dog.4. Chronic thoracic sympathectomy markedly retards the recovery of the ventricular rate from the inhibitory action of the vagus. Under this condition, the infusion of cocaine does not significantly enhance the ventricular escape.5. These findings suggest that an adrenergic mechanism located at the sympathetic nerves supplying the heart is substantially involved in the phenomenon of vagal escape.     

Lethal encephalitis in myeloid differentiation factor 88-deficient mice infected with herpes simplex virus 1

Herpes simplex virus 1 (HSV-1), a large DNA virus from the Herpesviridae family, is the major cause of sporadic lethal encephalitis and blindness in humans. Recent studies have shown the importance of Toll-like receptors (TLRs) in the immune response to HSV-1 infection. Myeloid differentiation factor 88 (MyD88) is a critical adaptor protein that is downstream to mediated TLR activation and is essential for the production of inflammatory cytokines. Here, we studied the relationship between MyD88 and HSV-1 using a purified HSV-1 isolated from a natural oral recurrent human infection. We observed the activation of TLR-2 by HSV-1 in vitro using Chinese hamster ovary cells stably transfected with a reporter gene. Interestingly, we found that only peritoneal macrophages from MyD88-/- mice, but not macrophages from TRL2-/- or from wild-type mice, were unable to produce tumor necrosis factor-alpha in response to HSV-1 exposure. Additionally, although TLR2-/- mice showed no enhanced susceptibility to intranasal infection with HSV-1, MyD88-/- mice were highly susceptible to infection and displayed viral migration to the brain, severe neuropathological signs of encephalitis, and 100% mortality by day 10 after infection. Together, our results suggest that innate resistance to HSV-1 is mediated by MyD88 and may rely on activation of multiple TLRs.     

Abnormal B cell function in haemophiliacs and their relationship with factor concentrates administration.

The present study was performed to evaluate B cell function in haemophiliacs. Spontaneous and pokeweed mitogen (PWM)-induced immunoglobulin (Ig) production was determined by ELISA in the supernatants of cultured peripheral blood lymphocytes (PBL) from 14 haemophiliacs and 17 normal donors. Spontaneous IgM, IgA and IgG production was three times higher in patients than normal controls, while PWM-induced IgM, IgA and IgG production was markedly reduced in patients compared to normal donors (P less than 0.025). Allogeneic co-cultures of haemophiliacs and normal B plus T cell fractions revealed that these results are due to a defect of the patients' T cell depleted fraction. These abnormalities were not found in three patients who had received no clotting factor concentrates for at least 1 year prior to the study. Additionally, the annual amount of clotting factor concentrates received by treated patients correlates well with the enhancement of spontaneous Ig production (r = +0.688, P less than 0.02), the decrease of PWM-induced Ig secretion (r = -0.655, P less than 0.02), and the elevation of serum IgG levels (r = +0.610, P less than 0.05). These findings suggest that the administration of clotting factor concentrates play an important role in the altered B cell function in haemophiliacs.     

Androgen receptor independent cardiovascular action of the antiandrogen flutamide

We have previously shown that flutamide (specific antagonist of the androgen receptor) has antihypertensive effects. In the present study we examined the mechanisms of flutamide action in the vasculature. The vascular effects of flutamide were assayed in aortae isolated from male or female Sprague-Dawley rats and from rats or mice lacking a functional androgen receptor ( tfm, testicular feminization mutation). The effect of flutamide on coronary flow was tested in isolated hearts. In addition, male hypertensive rats with tfm mutation were treated with flutamide, and blood pressure was monitored. Flutamide induced a relaxation of rat aortae from all the strains used (maximum relaxation at 10 microM: 51.3+/-5.2% of phenylephrine contraction) and increased the coronary flow. The aortic relaxation to flutamide was abolished by endothelium removal, or by inhibition of nitric oxide synthase, guanylyl cyclase, and tyrosine kinase but not by calmodulin inhibition. Flutamide treatment attenuated the development of hypertension in mouse renin transgenic rats with the tfm mutation. Flutamide produces direct vasodilation by inducing release of NO from the endothelium and causes subsequent activation of soluble guanylyl cyclase in an active androgen receptor independent manner. This response may contribute to the observed antihypertensive actions of flutamide.     

Haplotype analysis of the BRCA2 9254delATCAT recurrent mutation in breast/ovarian cancer families from Spain

A frame-shift 9254del5 mutation was independently identified in 12 families, eleven of them with Spanish ancestors, in a BRCA2 screening performed in 841 breast and/or ovarian cancer families and in 339 women with breast cancer diagnosed before the age of 40 at different centers in France and Spain. We sought to analyze in detail the haplotype and founder effects of the 9254del5 and to estimate the time of origin of the mutation. Eight polymorphic microsatellite markers and two BRCA2 polymorphisms were used for the haplotype analyses. The markers were located flanking the BRCA2 gene spanning a region of 6.1 cM. Our results suggest that these families shared a common ancestry with BRCA2 9254del5, which is a founder mutation originating in the Northeast Spanish, with an estimated age of 92 (95% CI 56-141) generations.     

Laboratory methods for early detection of human immunodeficiency virus type 1 in newborns and infants.

Cumulative data on serological testing of newborns and infants have shown that (i) maternal and newborn anti-HIV-1 IgG titers are high at delivery, which may explain the persistence of antibody in the infants of seropositive mothers; (ii) in some situations, serial HIV-1 antibody testing may identify infected infants; and (iii) detection of anti-HIV-1 IgA or IgM is specific for infection but the sensitivity of this assay may be compromised in certain situations, such as when infected infants are hypogammaglobulinemic or when the rise and fall of HIV-1-specific IgM synthesis following acute infection has been completed before delivery of the infant. Cumulative data on PCR, viral culture, and tests for antigen in newborns and infants have shown that (i) among all age groups, viral culture is probably the most specific test available for detection of HIV-1, as PCR and the p24 antigen test may (though rarely) give false-positive results; (ii) the sensitivity of these tests increases in the order of antigen, culture, and PCR, with relatively insensitive results in the first 3 months of life for all of these tests; (iii) the sensitivity of all of these tests improves and approximates 90 to 100% when infants over 6 months of age are tested; and (iv) data regarding the sensitivity, specificity, and usefulness of these virological assays in infants under 3 months of age are very scant and inconclusive.     

Evaluation of E-Test for determination of antimicrobial MICs for Pseudomonas aeruginosa isolates from cystic fibrosis patients.

We determined the E-Test and National Committee for Clinical Laboratory Standards standardized agar dilution MICs of ceftazidime, ciprofloxacin, piperacillin, and tobramycin for Pseudomonas aeruginosa during tests of 100 rough and mucoid P. aeruginosa isolates from cystic fibrosis patients. The levels of agreement (+/- 1 log2 dilution) between quantitative E-Test and agar dilution MIC results were 80, 97, 73, and 89% for ceftazidime, ciprofloxacin, piperacillin, and tobramycin, respectively. Comparison of the results after converting the MIC data to qualitative categories (susceptible, intermediate, and resistant) yielded levels of agreement of 84, 96, 88, and 93% for the same agents, respectively. Of the 39 qualitative discrepancies, 36 were minor and 3 were very major. We conclude that use of the E-Test is easier and more practical than use of the agar dilution method for most laboratories and that the E-Test furnishes results which are at least as accurate as those obtained by the agar dilution method. However, the higher cost of the E-Test method would likely discourage most laboratories from selecting it over disk diffusion for routine antimicrobial susceptibility testing of P. aeruginosa isolates from cystic fibrosis patients.     

Shape,F-actin,and surface morphology changes during chemotactic peptide-induced polarity in human neutrophils

Background: The exposure of human neutrophils to uniform concentrations of chemoattractants, such as N-formyl peptides, induces morphological cell polarization. In this study we report the temporal sequence of changes in cell shape, F-actin, and cell surface morphology during cellular polarization induced by N-formylmethionyl-leucyl-phenyl-alanine (fMLP) in human neutrophils in suspension. Methods: Neutrophil shape changes induced by 10^?8 M fMLP were observed with DIC microscopy. Size and Cellular granularity were analyzed by flow cytometry measuring their forward and side scattered light. To visualize F-actin distribution, neutrophils were labeled with the fluorescence probe FITC-phalloidin, and were examined with fluorescence and confocal laser scanning microscopy. Cell surface morphology was assessed with scanning electron microscopy (SEM). Results: The stimulation of round-smooth neutrophils with nanomolar concentrations (10^?8 M) of fMLP in suspension induced a temporal sequence of morphological changes during cell polarization, characterized by 1) increase in size as determined by forward angle scattered light, 2) rapid redistribution of F-actin from a diffuse cytoplasmic localization to the cell periphery, and 3) rapid reorganization of cell surface morphological features, with accumulation of plasma membrane in the front of polar cells. Four cell shapes were identified with SEM after stimulation of round-smooth neutrophils: round-ridged, round-ruffled, nonpolar ruffled, and polar cells. These cell shapes were correlated with a cortical localization, focal aggregates, and multipolar distribution of F-actin. In polar neutrophils, F-actin became concentrated in the front of the cell. Conclusions: These findings show the relation between reorganization of the microfilamentous cytoskeleton and modifications in cell shape and surface features during cell polarization induced after fMLP activation in neutrophils. This approach offers a powerful tool for further analysis of receptor distribution in polarized, motile neutrophils. © 1995 Wiley-Liss, Inc.