Regulation of fibrinolysis by platelet-released plasminogen activator inhibitor 1: light scattering and ultrastructural examination of lysis of a model platelet-fibrin thrombus

We have investigated the role of plasminogen activator inhibitor 1 (PAI- 1) in the regulation of fibrinolysis using a model thrombus composed of thrombin-stimulated platelets, fibrin(ogen), plasminogen, and recombinant tissue-type plasminogen activator. Laser light scattering kinetic measurements showed that clot lysis was significantly delayed both by thrombin-stimulated platelets and their cell-free releasate. This delay in lysis was almost fully reversed by the addition of a PAI- 1-specific monoclonal antibody that blocks the ability of PAI-1 to inhibit plasminogen activators. Lysis half-times exhibited a linear dependence on the concentration of PAI-1 antigen present, as determined by enzyme-linked immunosorbent assay (ELISA). Sodium dodecylsulfate- polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting confirmed the presence of PAI-1 antigen in the platelet releasates. Scanning electron micrographs of the model thrombus components sampled late in lysis showed considerable unproteolyzed fibrin still attached to platelets. Immunogold cytochemistry detected large amounts of PAI-1 antigen in the partially lysed platelet-fibrin thrombi. This PAI-1 appeared to be bound to the fibrin network rather than to the platelet surface itself. We conclude that the residual clots observed late in lysis represent platelet-associated fibrin to which platelet-released PAI-1 has bound, rendering it less susceptible to degradation.     

Basic and translational concepts of immune-mediated glomerular diseases

Genetically modified immune responses to infections and self-antigens initiate most forms of GN by generating pathogen- and danger-associated molecular patterns that stimulate Toll-like receptors and complement. These innate immune responses activate circulating monocytes and resident glomerular cells to release inflammatory mediators and initiate adaptive, antigen-specific immune responses that collectively damage glomerular structures. CD4 T cells are needed for B cell-driven antibody production that leads to immune complex formation in glomeruli, complement activation, and injury induced by both circulating inflammatory and resident glomerular effector cells. Th17 cells can also induce glomerular injury directly. In this review, information derived from studies in vitro, well characterized experimental models, and humans summarize and update likely pathogenic mechanisms involved in human diseases presenting as nephritis (postinfectious GN, IgA nephropathy, antiglomerular basement membrane and antineutrophil cytoplasmic antibody-mediated crescentic GN, lupus nephritis, type I membranoproliferative GN), and nephrotic syndrome (minimal change/FSGS, membranous nephropathy, and C3 glomerulopathies). Advances in understanding the immunopathogenesis of each of these entities offer many opportunities for future therapeutic interventions.     

Role of the terminal complement pathway in experimental membranous nephropathy in the rabbit.

Our recent observations of a complement-mediated, cell-independent mechanism of altered glomerular permeability in rat membranous nephropathy suggested a possible role for the terminal complement pathway in the mediation of proteinuria in certain forms of glomerular disease. To directly determine whether the membranolytic terminal complement components (C5b-C9) are involved in glomerular injury, we studied the development of proteinuria in normal and C6-deficient (C6D) rabbits, in both of which a membranous nephropathy-like lesion develops early in the course of immunization with cationized bovine serum albumin (cBSA) (pI 8.9-9.2). C6 hemolytic activity of C6D was 0.01% that of control rabbits. After 1 wk of daily intravenous injections of cBSA, proteinuria developed in 71% of controls (median 154, range 1-3,010 mg/24 h, n = 24), whereas none of C6D were proteinuric (median 6, range 2-12 mg/24 h, n = 12, P less than 0.01). After 1 wk of cBSA, both groups had qualitatively identical glomerular deposits of BSA, rabbit IgG, and C3 on immunofluorescence microscopy, predominantly subepithelial electron-dense deposits on electron microscopy, and minimal glomerular inflammatory cell infiltration of glomeruli. Glomeruli were isolated from individual animals after 1 wk of cBSA and deposits of rabbit IgG antibody were quantitated by a standardized in vitro assay using anti-rabbit IgG-125I. Rabbit IgG deposits were found to be similar in control (29.8 +/- 13.2, range 12.7-48.6 micrograms anti-IgG/2,000 glomeruli, n = 6) and C6D rabbits (32.6 +/- 13.8, range 16.8-48.8 micrograms anti-IgG/2,000 glomeruli, n = 5, P greater than 0.05). After 2 wk, coincident with a prominent influx of mononuclear cells and neutrophils, proteinuria developed in C6D rabbits. These results document, for the first time, a requirement for a terminal complement component in the development of immunologic glomerular injury. Since the only known action of C6 is in the assembly of the membrane attack complex, these observations suggest that the membranolytic properties of complement may contribute to glomerular damage.     

Experimental Membranous Glomerulonephritis in Rats: QUANTITATIVE STUDIES OF GLOMERULAR IMMUNE DEPOSIT FORMATION IN ISOLATED GLOMERULI AND WHOLE ANIMALS

Quantitation of immune deposit formation in glomeruli and correlation with immunohistologic and functional changes has been accomplished only in models of anti-glomerular basement membrane antibody-induced nephritis, or indirectly in immune complex disease by measuring radiolabeled antigen deposition. The kinetics of subepithelial immune deposit formation and the relationship between the quantity of antibody deposited and proteinuria are defined here for the first time in an established model of membranous immune complex nephritis (passive Heymann nephritis) induced by a single intravenous injection of ¹²⁵I-labeled sheep immunoglobulin (Ig)G antibody to rat tubular brush border antigen (Fx1A). Measurement of antibody deposition in glomeruli (GAb) isolated from rats injected with 10 mg of anti-Fx1A demonstrated a mean of 12 μg GAb in 4 h, which increased linearly to 48 μg in 5 d. GAb represented only 20 and 44% of total kidney antibody binding at these times. Proteinuria occurred only after 4-5 d of antibody deposition in rats with total kidney antibody binding exceeding ~200 μg/2 kidneys. Steroid treatment and vasoactive amine blockade did not significantly alter the quantity or localization of immune deposits. It was also demonstrated that isolated rat glomeruli specifically bound nephritogenic quantities of anti-Fx1A in vitro within hours. Analysis of the quantitative aspects of glomerular antibody deposition in vivo and glomerular antibody binding in vitro provides additional evidence that subepithelial immune deposits in passive Heymann nephritis may form in situ by reaction of free antibody with antigenic constitutents of the normal rat glomerulus. The observed kinetics of deposit formation differ markedly from those in anti-glomerular basement membrane disease and suggest a role for factors in addition to antigen-antibody interaction in determining this unique pattern of glomerular immune deposit formation.     

缩血管活性因子内皮素在缺血性心脏病中的作用及中医药干预效应

目的:总结内皮素在缺血性心脏病中的病理生理意义。方法:应用计算机检索维普、万方和中文生物医学期刊(光盘)数据库2000-01/2006-12相关缺血性心脏病及内皮素方面的文献,检索词“缺血性心脏病,内皮素”,限定文献语言种类为中文。同时计算机检索Medline数据库2000-01/2006-12相关缺血性心脏病及内皮素方面的文献,检索词“ischemic heart disease,endothelin”,限定文献语言种类为English。对资料进行初审,选取包括缺血性心脏病及内皮素方面的文献,开始查找全文。纳入标准:内皮素在缺血性心脏病中的作用及中医药的干预效应。排除标准:内皮素在其他疾病中的作用。结果:共检索到200余篇关于缺血性心脏病及内皮素方面的文献,最终纳入29篇符合标准的文献。内皮素是极强的缩血管活性因子,在缺血性心脏病中具有重要的病理生理意义,中医药从缺血性心脏病的发病机制及病理环节着手,进行干预,大量研究资料表明,中医药在保护内皮细胞功能、治疗缺血性心脏病方面疗效肯定,在目前寻找和研究新型有效的内皮素受体拮抗剂具有广阔的发展前景。结论:内皮素在心血管疾病中具有重要意义,但其治疗缺血性心脏病保护内皮功能的具体机制还有待进一步探讨。     

Responder analyses in randomised controlled trials for chronic low back pain: an overview of currently used methods

Purpose

To provide an overview and a critical appraisal of the use of responder analyses in published randomised controlled trials (RCTs) of interventions for chronic low back pain (LBP). The methodology used for the analyses, including the justification, as well as the implications of responder analyses on the conclusions was explored.

Methods

A convenience sample of four systematic reviews evaluating 162 RCTs of interventions for chronic LBP was used to identify individual trials. Randomised trials were screened by two reviewers and included if they performed and reported a responder analysis (i.e. the proportion of participants achieving a pre-defined level of improvement). The cutoff value for responders, the period of follow-up, and the outcome measure used were extracted. Information on how RCT authors justified the methodology of their responder analyses was also appraised.

Results

Twenty-eight articles (17 %) using 20 different definitions of responders were included in this appraisal. Justification for the definition of responders was absent in 80 % of the articles. Pain was the most frequently used domain for the definition of response (50 %), followed by back-specific function (30 %) and a combination of pain and function (20 %). A reduction in pain intensity ≥50 % was the most common threshold used to define responders (IQR 33–60 %).

Conclusions

Few RCTs of interventions for chronic LBP report responder analyses. Where responder analyses are used, the methods are inconsistent. When performing responder analyses authors are encouraged to follow the recommended guidelines, using empirically derived cutoffs, and present results alongside mean differences.     

Effectiveness of dexamethasone in preventing extubation failure in preterm infants at increased risk for airway edema.

We studied 50 preterm infants who had multiple or traumatic endotracheal intubations, or whose duration of endotracheal intubation was > or = to 14 days, and who were considered at high risk for airway edema. These infants were enrolled in a prospective, randomized, controlled clinical trial to assess whether prophylactic dexamethasone therapy would be effective in the prevention of postextubation stridor and respiratory distress. At study entry, both groups had similar weights, postnatal ages, methylxanthine use, ventilator settings, blood gas values, and pulmonary function test results (dynamic compliance, total respiratory resistance, tidal volume, peak-to-peak transpulmonary pressure, minute ventilation, and peak inspiratory and expiratory flow rates). Patients underwent blood gas studies, physical examinations, and pulmonary function testing at baseline (4 hours before extubation) and again 2 to 4 hours and 18 to 24 hours after extubation. Twenty-seven infants received dexamethasone, 0.25 mg/kg per dose, at baseline, and then every 8 hours for a total of three doses; 23 infants received saline solution at corresponding times. Eighteen to twenty-four hours after extubation, total pulmonary resistance increased by 225% from baseline in the control group compared with 33% in the dexamethasone group (p < 0.006), and the dexamethasone group had a greater tidal volume, a greater dynamic compliance, and a lower arterial carbon dioxide pressure. Of 23 control infants, 10 had postextubation stridor compared with 2 of 27 dexamethasone-treated patients (p < 0.006). Of the 23 control patients, 4 required reintubation compared with none of the treated group (p < 0.05). We conclude that the prophylactic use of corticosteroids for the prevention of postextubation stridor and respiratory distress is efficacious in low birth weight, high-risk preterm infants.     

Depletion of C6 prevents development of proteinuria in experimental membranous nephropathy in rats.

To study the possible role of the complement membrane attack complex, C5b-9, in an experimental rat model that is morphologically indistinguishable from membranous nephropathy in man (passive Heymann nephritis [PHN]), an antibody to rat C6 was used to deplete C6 levels to less than 5% of pretreatment values (C6D) during disease development. C3, C7, C8, and C9 levels were not different in C6D and control rats. After injection of nephritogenic quantities of 125I-anti-Fx1A antibody, the kinetics of disappearance of labeled IgG from the blood were identical in the complement deficient and sufficient groups, and glomerular deposition of 125I-antibody was the same in both groups at 5 days. Glomerular deposits of sheep IgG and C3 were also similar in C6D and controls, but glomerular deposits of C6 and C5b-9 neoantigens were markedly reduced or absent in C6 depleted rats. However, despite equivalent antibody deposits, proteinuria was abolished in C6D rats compared with normocomplementemic controls. Similar results were obtained when F(ab')2 anti-rat C6 IgG was used to deplete C6 during development of PHN. These results demonstrate that C6 is required for the development of the increased glomerular permeability that occurs in PHN, presumably because C6 is required for formation of C5b-9. We conclude that glomerular injury in the PHN model of membranous nephropathy in the rat is mediated by C5b-9.