Tolerant B lymphocytes acquire resistance to Fas-mediated apoptosis after treatment with interleukin 4 but not after treatment with specific antigen unless a surface immunoglobulin threshold is exceeded

Susceptibility to Fas-mediated apoptosis in nontolerant B cells is regulated in a receptor-specific fashion. To explore the regulation of Fas killing in tolerant, autoreactive B cells, mice doubly transgenic for hen egg lysozyme (HEL)-specific B cell receptors and soluble HEL were examined. Engagement of CD40 led to enhanced Fas expression and acquisition of sensitivity to Fas-mediated apoptosis in tolerant B cells, similar to that observed in nontolerant, receptor transgenic B cells. Engagement of surface immunoglobulin by specific (HEL) antigen failed to induce Fas resistance in tolerant B cells, in contrast to its effect on nontolerant B cells; however, cross-linking of biotinylated HEL with streptavidin induced similar levels of Fas resistance in tolerant and nontolerant B cells, which approximated the degree of Fas resistance produced by anti-Ig. Unlike surface Ig (sIg) engagement, physiological engagement of IL-4 receptors produced similar levels of Fas resistance in tolerant and nontolerant B cells. Thus, tolerant B cells differ from nontolerant B cells in the diminished capacity of surface immunoglobulin engagement to produce Fas resistance; however, tolerant B cells can be induced to become resistant to Fas-mediated apoptosis by IL-4 or by higher order cross-linking of sIg receptors.     

Cyclophosphamide and 4-Hydroxycyclophosphamide/aldophosphamide kinetics in patients receiving high-dose cyclophosphamide chemotherapy

Using a recently developed gas chromatography and mass spectrometry method to determine whole-blood cyclophosphamide (CP) and 4-hydroxycyclophosphamide/aldophosphamide (4-HO-CP/AP) concentrations, we investigated their pharmacokinetics in women receiving CP therapy. Patients (n = 18) received one or two courses of CP: (a) a 90-min i.v. infusion (4 g/m2) followed by a 96-h i.v. infusion (6 g/m2) in combination with high-dose thiotepa; or (b) a 96-h i.v. infusion (6 g/m2) in combination with high-dose thiotepa. Whole-blood exposures to CP [area under the whole blood concentration versus time curve (AUCCP)] and 4-HO-CP/AP (AUC4HOCP) between courses 1 and 2 were compared after normalization to dose (g/m2). A nonproportional increase was observed for the AUCCP between the first course [1112 micrometer. h/g/m2 +/- 14% coefficient of variation (CV)] and the second course (1579 micrometer . h/g/m2 +/- 28% CV) (P < 0.001). In contrast, the AUC4HOCP (27 micrometer . h/g/m2 +/- 25% CV) determined for the first course was 29% higher than the AUC4HOCP (21 micrometer . h/g/m2 +/- 26% CV) for the second course (P < 0.01). The interpatient whole-blood exposures to both CP and 4-HO-CP/AP were remarkably consistent in this patient population with percent CVs ranging from 14 to 28%. Because thiotepa (800 mg/m2) was administered simultaneously with CP during the second course of treatment, possible inhibition of CP metabolism by thiotepa was investigated using human liver microsomes in vitro. IC50 values determined for inhibition of CP metabolism in three individual liver donors ranged from 1.0 to 40 micrometer. However, the clinical relevance of this observation has not been established.     

A long-range pseudoknot is required for activity of the Neurospora VS ribozyme

Four small RNA self-cleaving domains, the hammerhead, hairpin, hepatitis delta virus and Neurospora VS ribozymes, have been identified previously in naturally occurring RNAs. The secondary structures of these ribozymes are reasonably well understood, but little is known about long-range interactions that form the catalytically active tertiary conformations. Our previous work, which identified several secondary structure elements of the VS ribozyme, also showed that many additional bases were protected by magnesium-dependent interactions, implying that several tertiary contacts remained to be identified. Here we have used site-directed mutagenesis and chemical modification to characterize the first long-range interaction identified in VS RNA. This interaction contains a 3 bp pseudoknot helix that is required for tertiary folding and self-cleavage activity of the VS ribozyme.     

Prospective, randomized trial of prolonged intracoronary urokinase infusion for chronic total occlusions in native coronary arteries

OBJECTIVES: The purpose of this study was to determine the safety and efficacy of three dosing regimens of intracoronary urokinase for facilitated angioplasty of chronic total native coronary artery occlusions. BACKGROUND: Percutaneous transluminal coronary angioplasty of chronically occluded (>3 months) native coronary arteries is associated with low initial success secondary to an inability to pass the guide wire beyond the occlusion. METHODS: Patients were enrolled if a chronic total occlusion >3 months old could not be crossed with standard angioplasty equipment. Of the 101 patients enrolled, 41 had successful guide wire passage and were excluded from urokinase treatment. The remaining 60 patients were randomized to receive one of three intracoronary dosing regimens of urokinase over 8 h (group A = 0.8 million U; group B = 1.6 million U; group C = 3.2 million U), and angioplasty was again attempted after completion of the urokinase infusion in 58 patients. RESULTS: Coronary angioplasty was successful in 32 patients (53%) (group A 52%, group B 50%, group C 59%, p = 0.86). This study had a 90% power to detect at least a 50% difference between dosing groups at alpha 0.05. Bleeding complications requiring blood transfusion did not differ significantly among the dosing groups (A 0%, B 15%, C 6%, p = 0.14), although major bleeding episodes were less common in group A (p < 0.05). There were no major procedural or in-hospital complications. Angiographic follow-up in 69% of the patients with successful angioplasty revealed target vessel patency in 91% but an angiographic restenosis rate of 59%. CONCLUSIONS: A prolonged supraselective intracoronary infusion of urokinase can be safely administered and may facilitate angioplasty of chronic total occlusions. Lower doses of urokinase are equally effective and result in fewer bleeding complications than do higher dosage regimens. Vessel patency is frequently maintained, but restenosis remains a problem.     

Expansion of transplanted hepatocytes during liver regeneration

A method for volume selective proton spectroscopy is presented based on a multiecho sequence with short refocusing interval tcp. It is demonstrated, that by appropriate choice of tcp on the order of 4-6 ms, signals from overlapping multiplets like the glutamine and glutamate (Glu/Gln) resonances in spectra of the human brain are considerably increased compared with a conventional PRESS volume selection scheme. Thus proton spectra from J-coupled multiplet signals can be acquired with TE on the order of 20-30 ms avoiding the baseline problems arising at shorter echo times due to broad resonances. This allows to selectively acquire spectra from substances with longer T2 without the confounding effects from J-coupling occurring in conventional volume selection techniques.     

Endothelial cell VE-cadherin functions as a receptor for the beta15-42 sequence of fibrin

The contact of fibrin with the apical surface of human umbilical vein endothelial cells (HUVEC) can induce capillary tube formation via the interaction of fibrin beta15-42 with a putative cell receptor (Chalupowicz, D. G., Chowdhury, Z. A., Bach, T. L., Barsigian, C., and Martinez, J. (1995) J. Cell Biol. 130, 207-215). To characterize this interaction, we studied the binding of the thrombin-cleaved N-terminal disulfide knot of fibrin (NDSK II), a dimeric fragment with exposed beta15-42, to HUVEC in three separate assay systems. Time-course binding of 125I-NDSK II to HUVEC monolayers or suspensions revealed that binding was specific at 50-60%, as determined by the addition of unlabeled NDSK II. Specific binding of 125I-NDSK II to HUVEC was 70% reversible by dilution or by competition, and was found to be divalent cation-independent. Binding plateaued after 10 min at a saturation of 15-20 nM. Scatchard analysis using the LIGAND computer program defined a single population of receptors with a KD of 7.7 +/- 1.6 nM and approximately 21,000 +/- 7000 binding sites/cell. N-terminal disulfide knot derivatives in which beta15-42 was absent (NDSK 325) or unexposed (NDSK, NDSK I) did not show specific binding. Specific binding of 125I-NDSK II could not be inhibited by RGDS or by antibodies to the alphavbeta3 or beta1 integrins, PECAM-1, ICAM-1, or N-cadherin. In contrast, a synthetic beta15-42/ovalbumin conjugate inhibited total 125I-NDSK II binding by 47 +/- 19% (corresponding to 95% of specific 125I-NDSK II bound) and a monoclonal antibody to vascular endothelial cadherin (VE-cadherin) inhibited binding by 35 +/- 8% (corresponding to 70% of specific 125I-NDSK II bound). Another assay was based on the capture of cadherins from HUVEC lysates by a polyclonal pan-cadherin antibody immobilized on plastic dishes. Binding of NDSK II to the captured cadherins was 89 +/- 5% specific, while specific binding of NDSK 325 and NDSK was negligible. An immortalized line of human adipose-derived microvascular endothelial cells, which express N-cadherin but not VE-cadherin, demonstrated no specific binding of NDSK II by the capture assay. These data define a novel interaction of fibrin with VE-cadherin, which is mediated by the fibrin N-terminal beta15-42 sequence, and may contribute to the mechanism through which fibrin induces angiogenesis.     

The sleep of abstinent pure primary alcoholic patients: natural course and relationship to relapse

Sleep in male pure primary alcoholic inpatients was examined at a mean of 16 days (n = 29), 19 weeks (n = 29), 14 months (n = 9), and 27 months (n = 4) of abstinence. Results were as follows: (1) the sleep of abstinent alcoholic patients is short, fragmented, and shallow early in abstinence; (2) a patient's sleep improves slowly over at least the first year of abstinence; however, (3) some facets of a patient's sleep remain abnormal even after 27 months of abstinence; (4) insomnia and sleep fragmentation after approximately 5 months of abstinence may be related to relapse by 14 months. The mechanism underlying the relationship between sleep and withdrawal in alcoholic patients is not well understood, and the issue of treating sleep problems as an adjunct to prevention of relapse warrants further investigation.     

Renal medullary rim sign in 2 adult quarter horses

This report describes a renal ultrasonographic abnormality (medullary rim sign), which was identified in 2 separate cases of spontaneously occurring disease associated with chronic and acute overdosage of phenylbutazone therapy. In horses, medullary rim sign has only been documented in neonatal foals experimentally administered large doses of phenylbutazone.     

Osteonecrosis of the knee following arthroscopic laser meniscectomy

An unusual case of osteonecrosis of the knee following an arthroscopic laser meniscectomy is presented. The unusual presentation of the osteonecrosis and the chronology suggest that the osteonecrosis of the knee resulted from damage to the articular cartilage and subchondral bone at the time of the arthroscopic laser meniscectomy.     

1,25-Dihydroxyvitamin D induces lipoprotein lipase expression in 3T3-L1 cells in association with adipocyte differentiation

1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is known to modulate the development of bone and other mesenchymal cell types. Since osteoblasts and adipocytes are thought to arise in bone marrow from a common progenitor, this work examined the effects of 1,25-(OH)2D3 on adipocyte development, and in particular on the expression of lipoprotein lipase (LPL), which is an early marker for the differentiated adipocyte. 3T3-L1 preadipocytes were cultured in the presence of 1,25-(OH)2D3 (10(-9) to 10(-7) M) for up to 7 days. LPL activity was measured in the medium and cell extracts, and LPL messenger RNA levels were measured by Northern blotting. When compared to control cells, 10(-7) M 1,25-(OH)2D3 increased medium LPL activity by 2- to 3-fold and cellular LPL by 1.5-fold. Significant increases in medium and cellular LPL were observed at 10(-9) M and were maximal at 10(-7) M. Along with the increase in LPL activity, there was an increase in LPL messenger RNA by 2-fold at 5 days, and by 5-fold at 7 days. In addition to an increase in LPL, 1,25-(OH)2D3 increased expression of aP2, an adipocyte-specific marker associated with differentiation. After the addition of 1,25-(OH)2D3, there was a decrease in 3T3-L1 cell number, which is consistent with differentiation, and a decrease in vitamin D receptors. Finally, these cells developed a different morphology. 1,25-(OH)2D3-treated cells assumed a rounded appearance, although without detachment from the dish and without the degree of lipid accumulation usually associated with the addition of insulin, isbutylmethylxanthine, and dexamethasone. It is concluded that 1,25-(OH)2D3 induced LPL expression in 3T3-L1 cells through an induction of differentiation-dependent mechanism(s). These findings suggest an important role for 1,25-(OH)2D3 in normal adipocyte differentiation.