Characteristics of a highly labile human type 5 17beta-hydroxysteroid dehydrogenase

The overall disposition and hepatobiliary transport of BQ-123, an anionic cyclopentapeptide, and three analogs were examined in rats in vivo. Total body clearance (CLtotal) and biliary excretion clearance (CLbile, p) exhibited 4- to 8-fold differences between the compounds, with those for BQ-485 and compound A having the highest and lowest values, respectively. The CLbile, p values of BQ-485, BQ-123, and BQ-518 were almost equal to the CLtotal, suggesting that hepatobiliary transport is the major elimination pathway for these compounds. Hepatic uptake clearance (CLuptake, vivo) and biliary excretion clearance (CLbile, h/fT), which was defined for the hepatic unbound concentration, were separately determined to examine the hepatic uptake and excretion processes, respectively. Both the CLuptake, vivo and CLbile, h/fT of BQ-485 were higher than those of BQ-123, whereas the corresponding values for BQ-518 were similar to those for BQ-123. The CLuptake, vivo and CLbile, h/fT of compound A were, respectively, approximately two thirds and one half those of BQ-123, suggesting that the lower CLbile, p value is due to the low efficiency of both the uptake and excretion processes. The CLuptake, vivo of these four peptides in vivo was similar to the extrapolated values based on the carrier-mediated transport activity previously assessed in vitro in isolated rat hepatocytes. The primary active transport previously assessed in an in vitro study in canalicular membrane vesicles was also highest for BQ-485 and lowest for compound A, similar to CLbile, h/fT in vivo. Thus, the transporters on both the sinusoidal and canalicular membranes determine the efficiency of the peptide overall elimination from the circulation.     

Highly purified human immunodeficiency virus type 1 reveals a virtual absence of Vif in virions

The vif gene of human immunodeficiency virus type 1 (HIV-1) is essential for the productive infection of primary blood-derived lymphocytes, macrophages, and certain human T-cell lines. It has been shown that Vif is associated with HIV-1 virions purified by sucrose density-equilibrium gradient analysis. However, the specificity of Vif incorporation into virions has not been determined. Moreover, recent studies have demonstrated that standard HIV-1 particle preparations created with sucrose density-equilibrium gradients are contaminated with cell-derived microvesicles. Here we demonstrate, as previously reported, that Vif cosediments with HIV-1 particles in sucrose density-equilibrium gradient analysis. However, we also found that, when Vif was expressed in the absence of all other HIV-1-encoded gene products and then isolated by sucrose density-equilibrium gradient centrifugation from extracellular supernatants, its sedimentation pattern was largely unaltered, suggesting that Vif can be secreted from cells. Using a newly developed OptiPrep velocity gradient method, we were able to physically separate most of the extracellular Vif from the HIV-1 virions without disrupting the infectivity of the virus. By titrating serial dilutions of purified Vif and Gag against the viral peak fraction in the OptiPrep gradient, we demonstrate that <1.0 Vif molecule per virion was present. This study shows that Vif is not significantly present in HIV-1 virions, a finding which is consistent with the idea that Vif functions predominantly in the virus-producing cells during virus assembly. The OptiPrep velocity gradient technique described here could be an easy and rapid way to purify HIV and other enveloped viruses from microvesicles and/or cell debris.     

Stimulation of HCO3- secretion across cystic fibrosis pancreatic duct cells by extracellular ATP

Colonic endocrine cells from prediabetic and diabetic non-obese diabetic mice as well as of the sister strain, BALB/cJ, were investigated by immunocytochemistry and computer image analysis. In prediabetic mice, enteroglucagon-and serotonin-immunoreactive cells were significantly increased in number, whereas the cell secretory index of these two cell types was significantly reduced. No significant differences were found in numbers or cell secretory index of peptide YY (PYY)-immunoreactive cells. In diabetic mice, PYY-immunoreactive cells were significantly fewer, but there were no significant differences in the numbers of enteroglucagon-and serotonin-immunoreactive cells. Whereas the cell secretory index was reduced in serotonin-producing cells, no significant differences were found between diabetic and control mice regarding the cell secretory index of PYY- and enteroglucagon-immunoreactive cells. Nor was any statistically significant difference found between controls, prediabetic and diabetic non-obese diabetic mice, regarding the thickness of submucosa, of circular and longitudinal-muscle layers, or of the mucosal area/microm baseline. The present study showed that abnormalities in colonic endocrine cells do occur, in both prediabetic and diabetic mice, but they are different in nature and can be divided into primary and secondary to the diabetes onset. The present findings of abnormal colonic endocrine cells in non-obese diabetic mice, an animal model for human insulin-dependent diabetes mellitus, might help explain the gastrointestinal disorders observed in patients with diabetes. The study also showed that the change in the colonic endocrine cells is dynamic and started before the onset of the diabetic condition.     

Circulating, but not local lung, IL-5 is required for the development of antigen-induced airways eosinophilia

IL-5 is induced locally in the lung and systemically in the circulation during allergic airways eosinophilic inflammation both in humans and experimental animals. However, the precise role of local and systemic IL-5 in the development of allergic airways eosinophilia remains to be elucidated. In our current study, we demonstrate that compared with their IL-5(+/+) counterparts, IL-5(-/-) mice lacked an IL-5 response both in the lung and peripheral blood, yet they released similar amounts of IL-4, eotaxin, and MIP-1alpha in the lung after ovalbumin (OVA) sensitization and challenge. At cellular levels, these mice failed to develop peripheral blood and airways eosinophilia while the responses of lymphocytes, neutrophils, and macrophages remained similar to those in IL-5(+/+) mice. To dissect the relative role of local and systemic IL-5 in this model, we constructed a gene transfer vector expressing murine IL-5. Intramuscular IL-5 gene transfer to OVA-sensitized IL-5(-/-) mice led to raised levels of IL-5 compartmentalized to the circulation and completely reconstituted airways eosinophilia upon OVA challenge, which was associated with reconstitution of eosinophilia in the bone marrow and peripheral blood. Significant airways eosinophilia was observed for at least 7 d in these mice. In contrast, intranasal IL-5 gene transfer, when rendered to give rise to a significant but compartmentalized level of transgene protein IL-5 in the lung, was unable to reconstitute airways eosinophilia in OVA-sensitized IL-5(-/-) mice upon OVA-challenge, which was associated with a lack of eosinophilic responses in bone marrow and peripheral blood. Our findings thus provide unequivocal evidence that circulating but not local lung IL-5 is critically required for the development of allergic airways eosinophilia. These findings also provide the rationale for developing strategies to target circulating IL-5 and/or its receptors in bone marrow to effectively control asthmatic airways eosinophilia.     

Early and long-term results of 233 heart transplant patients in University Bordeaux in France: quality of life following heart transplantations

Between March 1986 and December 1993 we had 233 heart transplant patients who were 218 males and 15 females and had a mean age of 50.9 years (range, 2 to 65 years). We analyzed the actuarial survival for these patients and investigated the status of rehabilitation and return-to-work from the view point of quality of life after heart transplant. Actuarial survival (Kaplan-Meier) was 81.7% at 1 year, 76.3% at 3 years, and 72.2% at 5 years. In 57 dead patients 24 patients (42%) died in 1 month after heart transplant. In 176 living patients 165 patients (53%) returned to life. In 129 patients except 76 retired patients only 69 patients (53%) returned to work. In 60 patients, who didn't return to work, 38 patients (63%) were physically able to work.     

Survival of cells with bleomycin-induced chromosomal lesions in the cultured lymphocytes of lung cancer patients

OBJECTIVE: To investigate whether persistent T lymphocyte activation is a feature of steroid-resistant (SR) asthma and to study the inhibitory effects of dexamethasone and other immuno-inhibiting agents on PHA-induced proliferation of peripheral blood T lymphocytes from SR and steroid-sensitive (SS) asthmatics. MATERIAL AND METHODS: 15 SR and 15 SS asthmatics were studied. Serum levels of soluble interleukin-2 receptor (sIL-2R) were measured before and after prednisone therapy by an enzyme-linked immunosorbent assay. PHA-driven proliferative assay was performed to evaluate inhibition of T lymphocyte proliferation. RESULTS: Serum levels of sIL-2R were elevated in both patients with SR and SS asthma as compared with normal controls (P < 0.001). After a 7-day course of prednisone (20 mg/day), serum levels of sIL-2R decreased significantly in SS asthmatics (P < 0.001) but not in SR asthmatics (P > 0.1). Proliferation of T lymphocytes from the sensitive but not the resistant asthmatics was significantly (P < 0.002) inhibited by dexamethasone (10 mol/L), reflecting a shift of the dose-response curve. In contrast, oxymatrine and thymus-derived immunosuppressors inhibited proliferation of T lymphocytes to a similar degree between SR and SS asthmatics. CONCLUSIONS: The results suggest that persistent T lymphocyte activation due to a relative insensitivity of the cells to glucocorticoids is a feature of SR asthma. Immuno-inhibiting agents other than glucocorticoids may be of therapeutic benefit in patients with SR asthma.     

GABAA and GABAB receptors differentially regulate synaptic transmission in the auditory thalamo-amygdala pathway: an in vivo microiontophoretic study and a model

PURPOSE: Our purpose was to investigate the feasability of using sequential PCR and FISH analysis of single cells for preimplantation diagnosis. METHODS: Protocols for sequential PCR and FISH analysis of a single fibroblast (cell recycling) were optimized for six loci and the rates of allele specific dropout (ADO) were determined. RESULTS: Conditions that allow reliable genotyping of single cells in lysis buffer were not optimal for amplifying fibroblasts fixed to coverslips. After optimizing conditions, we observed a success rate of 85% for both analyses in sequential PCR-FISH experiments in single cells for the four loci studied. The individual success rates for each technique revealed a slightly higher rate for FISH (91-95%) than for PCR (85-87%) for single cells on coverslips. The presence of two hybridization signals in FISH experiments demonstrated that the failure to amplify both alleles from heterozygous cells on coverslips was due to true ADO, and not the loss of chromosomal material. The ADO rate observed on coverslips varied between 10 and 14%, which is significantly higher than that observed in solution, even after meticulous optimization. CONCLUSIONS: Sequential PCR and FISH analysis of single cells remains an attractive possibility. However, until the problem of the increased rate of ADO is resolved, cell recycling should be applied to clinical preimplantation genetic analysis.