用自旋捕捉技术检测争光霉素A_6产生的活泼自由基

本文用自旋捕捉技术与ESR相结合的方法,研究了争光霉素A_6-Fe~(2+)复合物产生的活泼自由基。结果发现用NOS自旋捕捉剂可检测到该体系所产生的超氧阴离子自由基。在水溶剂中用PBN自旋捕捉到了羟基自由基。根据PBN-OH自旋加合物在水溶剂和甲醇溶剂中的超精细分裂常数,进一步确证了羟基自由基的生成。     

Does muscle inflammation influence recovery of muscle strength and function in patients undergoing total hip replacement?

BackgroundGenetic markers of muscle inflammation (eg, tumour necrosis factor α [TNFα] and interleukin [IL] 6 are downregulated following repeated transient increases after bouts of exercise. Total hip replacement (THR) typically resolves preoperative pain, although strength deficits of 10–21% persist in the affected hip at 1 year postoperatively. This study assessed whether mRNA expression of TNFα and IL6 in the vastus lateralis (VL) of the operated leg was related to changes in the strength of the operated leg quadriceps in patients following THR.MethodsTen patients were recruited prospectively after ethical approval. Distal VL (5 cm proximal to lateral suprapatellar pouch) biopsy samples were obtained intraoperatively and at 6 weeks postoperatively, with maximal voluntary contraction of the operated leg quadriceps (MVCOLQ) in Newtons (N), assessed preoperatively and at 6 weeks postoperatively. RT-PCR was used to assess mRNA expression in the biopsy samples and associations evaluated with Spearman's correlation coefficient.FindingsMean mRNA relative quotient (RQ) for comparison of 6 week intraoperative VL samples was 6·23 [SD 12·85] for TNFα and 17·10 [47·46] for IL6. Preoperatively, mean MVCOLQ was 188·90 N [76·84] and at 6 weeks it was 217·00 N [53·91]. There was no significant relation between TNFα or IL-6 RQ and absolute MVCOLQ at 6 weeks (r=0·115 [p=0·376] and ?0·491 [p=0·075], respectively). No statistically significant relation existed between TNFα mRNA RQ and the improvement in MVCOLQ at 6 weeks (r=–0·498, p=0·071) nor with IL6 and the same measure (r=0·091, p=0·401).InterpretationThere is a trend to correlation that exists for improvement in MVCOLQ with a reduction in TNFα mRNA expression, as well as between absolute MVCOLQ and reduction in IL-6 mRNA expression at 6 weeks postoperatively. Improvement in muscle strength may be mediated by reduced muscle inflammation and the associated reduction in pain in patients with severe osteoarthritis.FundingWales Deanery and Betsi Cadwaladr University Health Board Small Grants Scheme.     

Platelets inhibit the induction of nitric oxide synthesis by interleukin-1 beta in vascular smooth muscle cells

We have investigated the role of platelets in regulating the hemostatic and vasomotor properties of vascular smooth muscle. Experiments were performed to examine the effect of the releasate from activated platelets on the production of nitric oxide from interleukin-1 beta (IL- 1 beta)-treated cultured rat aortic smooth muscle cells. Treatment of vascular smooth muscle cells with IL-1 beta resulted in significant accumulation of nitrite in the culture media and in marked elevation of intracellular cyclic guanosine monophosphate (GMP) levels. The releasate from collagen-aggregated platelets blocked the IL-1 beta- mediated production of nitrite and the accumulation of cyclic GMP in smooth muscle cells in a platelet number-dependent manner. In functional assays, the perfusates from columns containing IL-1 beta- treated smooth muscle cells relaxed detector blood vessels without endothelium and the addition of IL-1 beta-treated smooth muscle cells to suspensions of platelets inhibited their thrombin-induced aggregation. The simultaneous treatment of smooth muscle cells with IL- 1 beta and the platelet releasate abolished both the vasorelaxing activities of the perfusates and the inhibition of platelet aggregation. Platelet releasates treated with a neutralizing antibody to platelet-derived growth factor (PDGF) failed to block IL-1 beta- induced nitric oxide production by the smooth muscle cells, as measured by both biochemical and functional assays. The platelet releasate from a patient with gray platelet syndrome likewise failed to block IL-1 beta-induced nitrite release by smooth muscle cells. These results demonstrate that platelets downregulate the production of nitric oxide by IL-1 beta-treated vascular smooth muscle cells through the release of PDGF. This effect may represent a novel mechanism by which platelets regulate vasomotor tone and thrombus formation at sites of vascular injury.     

Immunoglobulin G from patients with heparin-induced thrombocytopenia binds to a complex of heparin and platelet factor 4

Heparin-induced thrombocytopenia (HIT) is an important complication of heparin therapy. Although there is general agreement that platelet activation in vitro by the HIT IgG is mediated by the platelet Fc receptor, the interaction among the antibody, heparin, and platelet membrane components is uncertain and debated. In this report, we describe studies designed to address these interactions. We found, as others have noted, that a variety of other sulfated polysaccharides could substitute for heparin in the reaction. Using polysaccharides selected for both size and charge, we found that reactivity depended on two independent factors: a certain minimum degree of sulfation per saccharide unit and a certain minimum size. Hence, highly sulfated but small (< 1,000 daltons) polysaccharides were not reactive nor were large but poorly sulfated polysaccharides. The ability of HIT IgG to recognize heparin by itself was tested by Ouchterlony gel diffusion, ammonium sulfate and polyethylene glycol precipitation, and equilibrium dialysis. No technique demonstrated reactivity. However, when platelet releasate was added to heparin and HIT IgG, a 50-fold increase in binding of radio-labeled heparin to HIT IgG was observed. The releasate was then depleted of proteins capable of binding to heparin by immunoaffinity chromatography. Only platelet factor 4-immunodepleted releasate lost its reactivity with HIT IgG and heparin. Finally, to determine whether the reaction occurred on the surface of platelets or in the fluid phase, washed platelets were incubated with HIT IgG or heparin and after a wash step, heparin or HIT IgG was added, respectively. Reactivity was only noted when platelets were preincubated with heparin. Consistent with these observations was the demonstration of the presence of PF4 on platelets using flow cytometry. These studies indicate that heparin and other large, highly sulfated polysaccharides bind to PF4 to form a reactive antigen on the platelet surface. HIT IgG then binds to this complex with activation of platelets through the platelet Fc receptors.     

In vivo expression of the B7 costimulatory molecule by subsets of antigen-presenting cells and the malignant cells of Hodgkin's disease

The B-lymphocyte/accessory-cell activation antigen B7 (BB1) has been shown in vitro to stimulate T-lymphocyte proliferation and cytokine production via CD28 present on the latter cells. In this study, benign lymphoid tissues, lymphomas, and extralymphoid inflammatory sites were examined immunohistochemically using anti-B7 and other relevant monoclonal antibodies. B7 was expressed by benign transformed germinal center B cells, as it was by B cells of follicular lymphomas. B7 was also expressed by a subpopulation (a mean of 31% to 65%) of macrophages and dendritic cells in a variety of lymphoid tissues. It was present in abundance on all macrophages constituting sarcoid granulomas in lymph nodes. In extralymphoid inflammation, 17% to 35% of macrophages expressed B7 only weakly. Cases of Hodgkin's disease showed expression of B7 by the majority of Reed-Sternberg cells or malignant mononuclear variants, a phenomenon that potentially contributes to the lymphocytic accumulation that is a feature of this condition. CD28+ T cells were seen in all areas where T cells were present. B7+ and CD28+ cells colocalized in, for example, lymphoid follicles, lymph node paracortex, sarcoid granulomas, and Hodgkin's disease tissue, indicating a potential for cellular interaction via these molecules at these sites.