Detection of activated complement complex C5b-9 and complement receptor C5a in skin biopsies of patients with systemic sclerosis (scleroderma)

OBJECTIVE: Upregulated matrix synthesis is a hallmark of systemic sclerosis (SSc). There are indications that growth factors such as platelet derived growth factor (PDGF) are involved in proliferative pathways in SSc lesions. As activated complement releases PDGF from endothelial cells, we searched for activated complement and the complement receptor for C5a (C5aR) in skin biopsies of patients with SSc. METHODS: Snap frozen sections of 8 patients with early SSc and 5 patients with longterm SSc were examined. Using monoclonal antibodies against activated complement complex C5b-9 and the C5aR, skin biopsies derived from both clinically involved and non-involved skin were examined by APAAP immunohistochemistry. RESULTS: A pattern of activated complement C5b-9 and the CSaR could be detected in SSc microvasculature. Eleven of the 13 patients (7/8 patients with early SSc) showed positive staining for C5b-9. The CSaR was detected in 6 of the 8 patients with early SSc. In 3 patients with longterm disease, C5aR expression could also be detected in non-involved skin. CONCLUSION: Activated complement and complement receptors could be detected in early and late stages of SSc skin lesions. The presence of complement receptors in non-involved skin may indicate preclinical activation of pathways resulting in growth factor dependent matrix synthesis.     

Expression of interleukin-21 receptor in epidermis from patients with systemic sclerosis

OBJECTIVE: To examine the expression and regulation of interleukin-21 (IL-21) and IL-21 receptor (IL-21R) in patients with systemic sclerosis (SSc; scleroderma). METHODS: Skin biopsy specimens were obtained from 23 patients with SSc and 15 healthy controls. IL-21/IL-21R messenger RNA (mRNA) was quantified using real-time polymerase chain reaction (PCR). The expression pattern of IL-21/IL-21R was analyzed by in situ hybridization and Western blotting. Stimulation experiments were performed with cultured dermal fibroblasts from patients with SSc and healthy controls as well as with keratinocytes, using IL-1beta, platelet-derived growth factor BB, monocyte chemoattractant protein 1, transforming growth factor beta, and IL-21. The SCID-hu skin mouse model was used for in vivo experiments. RESULTS: IL-21R mRNA was detected in all biopsy specimens from patients with SSc and controls, with a 4.7-fold increase observed in SSc samples. In situ hybridization and immunohistochemical analysis showed an up-regulation of IL-21R in samples of epidermis from SSc patients, whereas no signal was detected in skin specimens from healthy controls. These results were confirmed in vitro, in that cultured keratinocytes expressed significant levels of IL-21R, whereas no signal was observed in fibroblasts. Interestingly, mRNA for IL-21 could not be detected by real-time PCR and in situ hybridization. Various concentrations of key cytokines in the pathogenesis of SSc did not stimulate the expression of IL-21R mRNA in cultured keratinocytes and dermal fibroblasts. In the SCID mouse transplantation model, the overexpression of IL-21R mRNA in SSc keratinocytes remained unchanged after transplantation. CONCLUSION: The up-regulation of IL-21R in keratinocytes indicates that, similar to fibroblasts and endothelial cells, the expression pattern is altered in SSc. Moreover, the expression of IL-21R appears to be independent of key cytokines that are operant in SSc.     

Changes in physical activity behaviour and physical function after bariatric surgery: a systematic review and meta‐analysis

Although physical activity performed after bariatric surgery is associated with enhanced weight loss outcomes, there is limited information on patients' physical activity behaviour in this context. This systematic review and meta‐analysis assessed pre‐operative to post‐operative changes in physical activity and physical function outcomes among obese adults undergoing bariatric surgery. A total of 50 studies met inclusion criteria with 26 papers reporting data for meta‐analysis. Increases in both objectively recorded and self‐reported physical activity at 12 months were demonstrated. Studies indicated that there was a shift towards a greater amount of active time, but of a lower intensity within the first 6 months of bariatric surgery, suggested by a reduction in moderate to vigorous physical activity but an increase in step count. A standardized mean difference (SMD) of 1.53 (95% CI: 1.02–2.04) based on nine studies indicated improved walking performance at 12 months. Similarly, analysis of five studies demonstrated increased musculoskeletal function at 3–6 months (SMD: 1.51; 95% CI: 0.60–2.42). No relationship was identified between changes in weight and walking performance post‐surgery. More studies assessing physical activity, physical function and weight loss would help understand the role of physical activity in optimizing post‐operative weight and functional outcomes.     

Use of a generally applicable tissue factor--dependent factor V assay to detect activated protein C-resistant factor Va in patients receiving warfarin and in patients with a lupus anticoagulant

The original activated partial thromboplastin time-based assay for activated protein C (APC)-resistant factor Va (FVa) requires carefully prepared fresh plasma and cannot be used in patients receiving warfarin or in patients with antiphospholipid antibodies. A new test is described here that circumvents these limitations and distinguishes without overlap heterozygotes for APC-resistant FVa from persons with normal FV. A diluted test plasma is incubated with an FV-deficient substrate plasma and tissue factor and then clotted with Ca2+ or Ca2+ plus APC. Test results are independent of the FV level or the dilution of the test plasma used. Of 39 controls, 37 gave normal results. Two controls (5%) gave results indicative of APC resistant FVa and on DNA analysis were found to be heterozygous for FV R506Q. Twenty of 21 randomly selected patients receiving warfarin gave normal results. In the single patient with abnormal results, heterozygous FV R506Q was confirmed by DNA analysis. Two of 15 patients with protein S deficiency and 5 of 29 patients with a lupus anticoagulant had abnormal results. APC resistance caused by FV R506Q was confirmed in the five of these seven patients available for DNA analysis. APC-resistant FVa was also detected in 10 of 21 (46%) stored plasma from unrelated patients with venous thrombosis and negative earlier evaluation for a lupus anticoagulant or a deficiency of protein C, protein S, or antithrombin, which confirms a high incidence of this defect among patients with venous thrombosis.     

Recombinant human thrombopoietin (Mpl ligand) enhances proliferation of erythroid progenitors

We have studied the effects of recombinant human thrombopoietin (TPO, Mpl ligand) on human hematopoiesis in vitro. TPO alone did not support erythroid burst formation but, in the presence of erythropoietin, it enhanced erythroid burst formation from CD34+ bone marrow and cord blood cells. The burst-promoting activity (BPA) was stronger under 5% serum than 30% serum conditions. The direct nature of BPA effects was documented by replating studies of early erythroid bursts. The BPA of TPO was less than that of interleukin-3 but was comparable with that of granulocyte/macrophage colony-stimulating factor and steel factor. The soluble form of Mpl receptor inhibited burst enhancing effects of TPO, suggesting that the BPA effects of TPO are mediated through the Mpl receptor. These results further delineate the physiologic roles of TPO and may aid in the determination of the clinical usages of TPO.     

Evidence of 5-lipoxygenase overexpression in the skin of patients with systemic sclerosis: a newly identified pathway to skin inflammation in systemic sclerosis

OBJECTIVE: Leukotrienes are a family of arachidonic acid derivatives with potent proinflammatory and profibrotic properties, and 5-lipoxygenase (5-LOX) catalyzes two key steps in the leukotriene biosynthetic pathway. Since inflammatory cell infiltrates and excessive fibrosis are hallmarks of systemic sclerosis (SSc) skin lesions, we undertook the present study to investigate the expression of 5-LOX in skin biopsy specimens from patients with SSc. METHODS: Expression of 5-LOX in skin sections from 10 SSc patients and 8 healthy controls was examined by in situ hybridization with specific riboprobes and by immunohistochemistry analysis with 5-LOX monoclonal antibodies. Synthesis of 5-LOX by cultured dermal fibroblasts from 7 patients with SSc and 4 controls was measured by fluorescence-activated cell sorter analysis. In addition, concentrations of leukotriene B4 (LTB4) and LTE4 in fibroblast supernatants after stimulation were determined using enzyme immunoassays. RESULTS: Expression of 5-LOX was found in all skin sections from SSc patients as well as from controls. However, the number and percentage of 5-LOX-positive cells were significantly higher in SSc skin sections compared with control sections. Expression of 5-LOX was seen in cells within perivascular inflammatory infiltrates as well as in fibroblasts throughout the skin. The experiments with cultured skin fibroblasts revealed that 5-LOX was constitutively expressed in these cells, which resulted in the production of leukotrienes after cell stimulation. Whereas no difference was found for LTE4, SSc fibroblasts produced significantly higher amounts of LTB4 after stimulation, compared with healthy control fibroblasts. CONCLUSION: The results of this study suggest that the 5-LOX pathway may be of significance in the pathogenesis of SSc and may represent a target for new treatment strategies.     

Treatment with imatinib prevents fibrosis in different preclinical models of systemic sclerosis and induces regression of established fibrosis

Objective

Imatinib is a small‐molecule tyrosine kinase inhibitor capable of selective, dual inhibition of the transforming growth factor β and platelet‐derived growth factor (PDGF) pathways. Imatinib has previously been shown to prevent the development of inflammation‐driven experimental fibrosis when treatment was initiated before administration of the profibrotic stimulus. The aim of this study was to confirm the efficacy of imatinib in a murine model of systemic sclerosis (SSc) that is less driven by inflammation and to investigate whether imatinib is also effective for the treatment of established fibrosis.

Methods

The tight skin 1 (TSK‐1) mouse model of SSc was used to evaluate the antifibrotic effects of imatinib in a genetic model of the later stages of SSc. In addition, the efficacy of imatinib for the treatment of preestablished fibrosis was analyzed in a modified model of bleomycin‐induced dermal fibrosis in which the application of bleomycin was prolonged and the onset of treatment was late.

Results

Treatment with imatinib reduced dermal and hypodermal thickening in TSK‐1 mice and prevented the differentiation of resting fibroblasts into myofibroblasts. In the model of preestablished dermal fibrosis, imatinib not only stopped further progression of fibrosis but also induced regression of preexisting dermal fibrosis, with a reduction in dermal thickness below pretreatment levels.

Conclusion

These results indicate that combined inhibition of the tyrosine kinase c‐Abl and PDGF receptor might be effective in the later, less inflammatory stages of SSc and for the treatment of established fibrosis. Thus, imatinib might be an interesting candidate for clinical trials in patients with longstanding disease and preexisting tissue fibrosis.

     

乙交酯-丙交酯共聚物与神经生长因子复合支架的细胞相容性及缓释作用

目的：体外实验观察生物可降解材料乙交酯-丙交酯共聚物与神经生长因子复合支架的细胞相容性及其缓释作用. 方法：实验于2005-05/09在首都医科大学附属北京市神经外科研究所损伤修复实验室完成.①实验方法：乙交酯-丙交酯共聚物采用专利技术（200510011350.9）制备,体外将许旺细胞和神经干细胞与乙交酯-丙交酯共聚物共培养.②实验评估：扫描电镜下观察许旺细胞和神经干细胞在乙交酯-丙交酯共聚物内生长状况;应用组织工程技术将神经生长因子整合入乙交酯-丙交酯共聚物内,0.02 g乙交酯-丙交酯共聚物（干质量）中含有1μg神经生长因子,ELISA法检测每天神经生长因子释放的质量浓度. 结果：①扫描电镜下乙交酯-丙交酯共聚物横断面可见其为网格状,纵断面可见其直孔道.②许旺细胞和神经干细胞在乙交酯-丙交酯共聚物支架上生长良好,与其紧密贴附.③培养后第4天神经生长因子释放的质量浓度达峰值,第7天趋于平稳,稳定释放的质量浓度为100μg/L. 结论：①乙交酯-丙交酯共聚物与许旺细胞和神经干细胞具有良好的生物相容性及细胞亲和性.②按神经生长因子（1μg）与乙交酯-丙交酯共聚物（干质量0.02 g）的比例合成的神经生长因子-乙交酯-丙交酯共聚物组织工程材料可缓慢稳定释放神经生长因子,达到其可以发挥生物学活性的质量浓度100μg/L.     

基于血氧水平依赖性的功能磁共振成像联合弥散张量成像技术纵向观察视觉通路病变中皮质功能和白质纤维结构的动态变化

目的：联合应用血氧依赖功能磁共振成像和弥散张量成像技术纵向观察视觉通路病变的皮质功能和白质纤维结构的动态变化,探讨病变后恢复期脑功能和结构重组的特点. 方法：病变组患者为2006-01/09解放军南京军区福州总医院收治,因视觉通路病变致单侧或双侧视觉障碍患者8例,采用美国GE公司Signa Excite HD 1.5T双梯度16通道磁共振成像系统,在病变早期和恢复期分别进行血氧依赖功能磁共振成像和弥散张量成像检查,对比治疗前后双眼刺激下脑激活区的体积以及视放射解剖结构、局部各向异性值,并与对照组12名正常被试比较. 结果：20例受试者均完成测试进入结果分析.①血氧依赖功能磁共振成像结果：正常被试采用黑白棋盘格刺激明显激活双侧枕叶距状回,平均激活皮质体素数为（9.12±2.47）万;病变组患者双眼刺激视皮质激活体积较对照组比较明显减少（P＜0.05）,治疗后复查,刺激的皮质激活像素数增多,激活区扩大.②弥散张量成像三维纤维束重建：对照组双侧视放射均能追踪至皮质下,12例被试视放射平均部分各项异性值为0.39±0.13.病变组中4例视觉通路病变患者视放射显示完整,与对照组间比较无明显差异,治疗前后纵向观察亦无显著变化;4例枕叶视中枢病变患者患侧视放射纤维部分中断、纤细,并可见移位,部分各项异性值均明显下降,治疗后视放射纤维束有一定程度恢复,部分各项异性值增加. 结论：血氧水平依赖功能磁共振成像联合弥散张量成像从皮质功能和纤维解剖方面诠释了病变的发展及康复过程,可帮助了解视觉通路病变后皮质功能区的重组.     

体外冲击波改善创伤性膝关节功能障碍

目的:比较体外冲击波与关节松动术两种治疗方法对创伤性膝关节功能障碍患者的疗效。方法:选择2004-01/2006-09佛山市第一人民医院收治的63例创伤性膝关节功能障碍患者,随机数字表法随机分为体外冲击波组28例和关节松动术组35例。①治疗方法:体外冲击波组采用骨科冲击波机(深圳慧康医疗器械有限公司)进行体外冲击波治疗,60次/min、强度6～11W/cm2,时间约25min,2次/周,共6次。关节松动术组选用澳式手法和CPM仪治疗,45min/次,次/周,共治疗18次,治疗部位均为患侧膝关节。②评估指标:同时采用测角器评估患者膝关节主动和被动运动时的6关节活动范围变化,并根据关节活动范围变化评估疗效;采用目测类比评分评定活动时疼痛强度;并观察不良事件和副反应。结果:57例患者进入结果分析。①关节活动范围变化:治疗3周后,体外冲击波组膝关节活动度平均增加73.8°,关节松动术组平均增加为35.6°,两者比较差异有非常显著性意义(χ2=26.645P<0.01)。②疼痛评分结果:治疗3周后,体外冲击波组疼痛减轻47.96%,关节松动术组疼痛减轻23.73%,两者比较差异有非常显著性意义(P<0.01)。③疗效:体外冲击波组总有效率100%,关节松动术组总有效率90%。④副反应:体外冲击波组治疗后无任何不良反应,关节松动术组治疗时和后膝关节局部有疼痛剧烈,有6例因此脱落。结论:体外冲击波组治疗创伤性膝关节功能障碍的疗效优于关节松动术组,且膝关节功能障碍改善明显、疗效持久,治疗时患者痛苦少。