Human platelet glycoprotein V: its role in enhancing expression of the glycoprotein Ib receptor

Platelet adhesion to an injured blood vessel wall is a critical initiating step in hemostasis mediated by a four member receptor complex (glycoprotein Ib/V/IX) interacting with plasma von Willebrand factor (vWF). The function of the GPV subunit within this complex is presently undefined. To study the role of glycoprotein (GP) V within the GPIb receptor complex, we transfected the GPV subunit gene into a hematopoietic cell line that constitutively expresses the other three subunits (human erythroleukemia [HEL] cells). Using flow cytometry, we found transfected GPV was surface expressed in HEL cells; this, in turn, led to increased surface expression of the ligand-binding GPIb alpha and GPIX subunits. Radioligand binding assays showed that GPV- transfected HEL cells bound more vWF than their non- or mock- transfected counterparts. We employed confocal microscopy of GPV- transfected HEL cells to show that GPV colocalizes with GPIb alpha on the cell surface. These findings suggest that the GPV subunit plays a role within the GPIb receptor complex by enhancing Ib alpha surface expression.     

Pseudomonas fluorescens alters epithelial permeability and translocates across Caco-2/TC7 intestinal cells

Background

Pseudomonas fluorescens has long been considered as a psychrotrophic microorganism. Recently, we have shown that clinical strains of P. fluorescens (biovar 1) are able to adapt at a growth temperature of 37°C or above and induce a specific inflammatory response. Interestingly, a highly specific antigen of P. fluorescens, I2, is detected in the serum of patients with Crohn's disease but the possible role of this bacterium in the disease has not yet been explored. In the present study, we examined the ability of a psychrotrophic and a clinical strain of P. fluorescens to modulate the permeability of a Caco-2/TC7 intestinal epithelial model, reorganize the actin cytoskeleton, invade the target cells and translocate across the epithelium. The behaviour of these two strains was compared to that of the well known opportunistic pathogen P. aeruginosa PAO1.

Results

Both strains of P. fluorescens were found to decrease the transepithelial resistance (TER) of Caco-2/TC7 differentiated monolayers. This was associated with an increase in paracellular permeability and F-actin microfilaments rearrangements. Moreover, the invasion and translocation tests demonstrated that the two strains used in this study can invade and translocate across the differentiated Caco-2/TC7 cell monolayers.

Conclusions

The present work shows for the first time, that P. fluorescens is able to alter the intestinal epithelial barrier function by disorganizing the F-actin microfilament network. Moreover, we reveal that independently of their origins, the two P. fluorescens strains can translocate across differentiated Caco-2/TC7 cell monolayers by using the transcellular pathway. These findings could, at least in part, explain the presence of the P. fluorescens specific I2 antigen in the serum of patients with Crohn's disease.     

ORIGINAL ARTICLE: Association between 25‐hydroxyvitamin D,somatic muscle weakness and falls risk in end‐stage renal failure

Objective Suboptimal levels of 25‐hydroxyvitamin D (25OHD) are common in haemodialysis patients (Chronic Kidney disease‐5D: CKD‐5D) and may be associated with reduced muscle strength and increased falls risk. We tested the hypothesis that 25OHD levels may be independently associated with falls risk in CKD‐5D. Background Supplementation with calcium and cholecalciferol reduces hip and other nonvertebral fractures in elderly individuals, and this effect may in part be attributable to reduction in falls frequency. The relationship between 25OHD and falls risk has not been investigated in CKD‐5D. Design and Patients This is a cross‐sectional study of 25 CKD‐5D patients with predialysis 25OHD, 1,25‐dihydroxyvitamin D (1,25(OH)₂D) and intact parathyroid hormone (iPTH) measurement. Falls risk was assessed by quadriceps muscle strength, FallsScreen^© test (FST), Berg Balance Scale (BBS), timed ‘up and go’ (TUG) test, Modified Barthel Index (MBI) and Falls Efficacy Scale (FES). Results Mean age was 69·8 ± 12·1 years, and median time on dialysis was 3·1 years. Median 25OHD level was 55·3 nmol/l (range 20·8–125·8 nmol/l). Muscle strength was significantly positively correlated with 25OHD (P = 0·024) but not with 1,25(OH)₂D (P = 0·477) or PTH (P = 0·461). Statistically significant correlation between 25OHD levels and FST (P = 0·028) plus MBI (P = 0·0046) was noted. No significant correlation was detected between falls risk and 1,25(OH)₂D or PTH. Conclusions Suboptimal levels of 25OHD in CKD‐5D are associated with reduced quadriceps muscle strength and increased falls risk. 25OHD may be more important than the active renal metabolite 1,25(OH)₂D for muscle strength with implications for vitamin D choice and goals of supplementation. Further investigation is required to examine effectiveness of calciferol supplementation on the incidence of falls in CKD‐5D.     

Inactivation of Friend erythroleukemia virus and Friend virus- transformed cells by merocyanine 540-mediated photosensitization

The Friend virus complex was used as a model to study the effects of merocyanine 540 (MC 540)-mediated photosensitization on enveloped viruses. Simultaneous exposure to the lipophilic dye MC 540 and white light inactivated cell-free virus, cell-associated virus, and virus- transformed cells. When used under experimental conditions that are known to preserve most mature blood cells, at least some coagulation factors, and a significant portion of the pluripotent hematopoietic stem cell compartment, MC 540-mediated photosensitization reduced virus titers by greater than or equal to 4 log and the concentration of in vitro clonogenic erythroleukemia cells by greater than or equal to 5 log. Animals that received a single intravenous injection of photosensitized virus were resistant to a subsequent challenge with live virus. High sensitivity to MC 540-mediated photosensitization appears to be a property that is shared by other enveloped viruses. Thus, photosensitization mediated by MC 540 may be of benefit in the sterilization of blood products (in particular, cellular products), the production of vaccines, and selected areas of antiviral therapy.     

计算机辅助的EBRA-FCA法分析半髋表面置换治疗股骨头缺血性坏死假体生物力学变化

背景:对于中晚期(FicatⅢ,Ⅳ期)股骨头缺血性坏死且髋臼基本完好的患者,单纯股骨头表面置换是一种较好的治疗方法。股骨假体位置、下沉影响全髋关节置换术的治疗效果,但假体位置、下沉对半髋表面置换术疗效的影响尚需进一步研究。目的:观察钴铬合金半髋表面置换对中晚期(FicatⅢ,Ⅳ期)股骨头缺血性坏死且髋臼基本完好患者髋关节功能的影响,并分析疗效与股骨假体柄干角、下沉的关系,评估计算机辅助X射平片分析对假体失败的预测价值。设计:病例分析。单位:广西医科大学第一附属医院脊柱骨病科。对象:选择1997-06/2002-07广西医科大学第一附属医院脊柱骨病科收治的中晚期(FicatⅢ,Ⅳ期)股骨头缺血性坏死行半髋表面置换患者41例(48髋),患者手术时的年龄29～49岁,平均(37±9)岁,其中男30例,女11例。其中FicatⅢ期35髋,FicatⅣ期13髋,髋臼均相对正常。所有患者病变部位病理检查均为股骨头缺血性坏死;且均签署知情同意书。方法:①于术前和随访时采用UCLA(UniversityofCaliforniaLosAnge-les)髋关节功能评分标准对患者手术前后疼痛、步行、功能、活动进行评分,每一项记分为1～10分,10分为最佳。优,35～40分,好29～34分,一般22～28分,差<22分。术后评分为随访时评分平均值。②运用计算机辅助的EBRA-FCA法,在术后骨盆的X射线平片上测量股骨假体的柄干角、下沉,并对半髋表面置换术治疗股骨头缺血性坏死的疗效与股骨假体的柄干角、下沉的关系进行分析。③采用复诊的方式进行随访,于术后2个月开始第1次随访,每年随访1次,共随访8年。④两组样本间均数比较采用独立样本t检验(并做方差齐性检验)。在对临床结果的析因分析(posthocanalysis)基础上,比值比(oddsratio)分析用来确定假体早期失败的相对危险性。主要观察指标:①纳入患者半髋表面置换术前后UCLA评分比较。②FicatⅢ期和Ⅳ期患者术后疗效比较。③不同疗效患者随访期间股骨假体柄干角和下沉程度比较。结果:全部病例均获随访,其中8例随访3年,8例随访4年,8例随访5年,7例随访6年,6例随访7年,4例8年。①UCLA髋关节功能评分:疼痛、步行、功能、活动评分别由术前的(3.1±1.2),(4.4±1.7),(5.8±2.3),(5.5±2.7)分提高到术后的(9.1±2.5),(9.2±2.9),(9.1±3.4),(7.1±3.1)分,差异明显(P<0.01)。②疗效:FicatⅢ期35髋术后的满意率为89%与FicatⅣ期13髋术后的满意率相近(69%,P>0.05),其中疗效差8髋为失败组,其余40髋为成功组。③计算机辅助的X射线平片检查结果:失败组6个髋的股骨假体有明显下沉(其柄干角小于130°),2个髋的髋臼有破坏(1个柄干角为128°,1个柄干角为136°)。成功组平均柄干角明显大于失败组,分别为139°±6.2°,127°±5.3°,差异明显(P<0.01)。失败组中,6髋假体头中心和假体柄尖的下沉分别为(5.02±1.3)mm和(4.85±1.1)mm明显大于成功组[(1.48±0.2)和(1.04±0.2)mm,P<0.05];小于130°的柄干角其发生不良后果的机会增加了7.1倍。④股骨假体下沉时间:计算机辅助的X线片分析显示股骨假体下沉超过2mm的时间为(19.5±3.2)个月,明显早于临床出现症状的时间和X射线平片异常的时间[(35.6±4.2),(24.8±2.5)个月,P<0.01]。结论:①钴铬合金半髋表面置换是一种可供选择的向全髋关节置换术过渡的较好的治疗方法,可以恢复中晚期(FicatⅢ,Ⅳ期)股骨头缺血性坏死的且髋臼基本完好的患者的髋关节功能。②假体的松动下沉与假体的位置有关。③用来评估假体柄在骨盆平片上的下沉程度的计算机辅助的平片分析可以预测假体的失败。     

Macrophage tropism of feline leukemia virus (FeLV) of subgroup-C and increased production of tumor necrosis factor-alpha by FeLV-infected macrophages

Erythroid aplasia is induced in cats by feline leukemia virus (FeLV) of subgroup C but not by FeLV of subgroup A. In an investigation of the role of macrophages in FeLV-C-induced diseases, the concentrations of FeLV and tumor necrosis factor-alpha (TNF-alpha) were compared between feline peritoneal macrophages incubated with FeLV of subgroup A or C. FeLV of both subgroups infected macrophages, but expression of FeLV-C was 21-fold higher than FeLV-A in peritoneal macrophages (P = .004). The supernatants of FeLV-C-inoculated macrophage cultures contained significantly higher levels of TNF-alpha (70 +/- 14 U/mL) at 72 hours postincubation compared with FeLV-A-inoculated (38 +/- 8 U/mL) and uninoculated (31 +/- 8 U/mL) cultures. Moreover, a positive correlation was shown between cell-associated FeLV surface glycoprotein gp70 and TNF-alpha expression in FeLV-C-infected macrophages by immunofluorescence (r = .6; P = .001), measured with a computer- assisted, laser-based digital imaging system. The addition of TNF-alpha to a uniform population of FeLV-infected cells (feline embryonic fibroblasts) caused an enhancement of viral expression (P < .05). These results indicate that FeLV-C has tropism for macrophages, FeLV expression is positively correlated with TNF-alpha expression in macrophages, and TNF-alpha enhances FeLV replication in fibroblasts. We suggest that FeLV-C infection of macrophages and secretion of TNF-alpha may be important in hematopoietic suppression in FeLV-C-infected cats.     

Factor XII-independent activation of factor XI in plasma: effects of sulfatides on tissue factor-induced coagulation

Factor XI (FXI) may be activated in a purified system by thrombin and by autoactivation in the presence of negatively charged substances such as dextran sulfate or sulfatides. The current studies were performed to determine if these processes occur during the coagulation of plasma. FXII--deficient plasma was supplemented with 125I-FXI and clot formation was induced with tissue factor and/or sulfatides. Cleavage of FXI was studied by standard polyacrylamide gel electrophoresis and autoradiography. Activated FXI (FXIa) was detected after 20 minutes of incubation with sulfatides alone and this process was markedly accelerated by the addition of tissue factor (TF). The enhancing effect of TF was blocked by hirudin, which indicated thrombin involvement in FXI activation. The contribution of FXIa to FIX activation in this system was studied using a 3H-FIX activation peptide release assay. Sulfatides increased FIX activation about twofold in plasma induced to clot with TF but had no effect if the plasma was immunodepleted of FXI. FIX activation was also increased in plasma induced to clot with FXa if sulfatides were present. The enhanced generation of FIXa was dependent on FXI and was blocked by hirudin. Some activation was seen in the reactions with sulfatides and hirudin and is likely solely caused by FXI autoactivation. The data indicate that during the coagulation of plasma in the presence of sulfatides, FXI is activated by a mechanism that is thrombin dependent and does not require FXII.     

How to obtain excellent response rates when surveying physicians

This paper outlines ways to maximize response rates to surveysby summarizing the most relevant literature to date and demonstratinghow these techniques have resulted in consistently high ratesof return in family practice research. We describe the methodologyused in recent surveys of physicians conducted by the Centrefor Studies in Family Medicine through its Thames Valley FamilyPractice Research Unit, located in London, Ontario, Canada andfunded by the Ontario Ministry of Health and Long-Term Care.The identification and implementation of these techniques tomaximize response rates is critical, as primary health careresearchers often rely on information gathered through questionnairesto study physicians' practice profiles, experiences and attitudes.Four separate and distinct mailed surveys of physicians usinga modified Dillman approach were conducted from 2001 to 2004.The sampling strategies, topics, types of questions and responseformats of these surveys varied. The first survey did not useany incentives or recorded delivery/registered mail and receiveda response rate of 48%. In sharp contrast, the other three surveysobtained responses rates of 76%, 74%, 74%, respectively, achievedthrough the use of gift certificates and recorded delivery/registeredmail. Sending a survey by recorded delivery/registered mailtends to result in the survey package being given priority inthe physicians' incoming mail at the practice. Gift certificatespartially compensate physicians for time spent completing thesurvey and recognition of the time required is appreciated.The response rates achieved provide strong evidence to supportthe use of monetary incentives and recorded delivery/registeredmail (along with the Dillman approach) in survey research. Itis anticipated that this evidence will be used by other researchersto justify requests for funding to cover the costs associatedwith incentives and recorded delivery/registered mail. We recommendthe use of these strategies to maximize response rates and improvethe quality of this type of primary health care research. Keywords. Response rates, surveys, physicians.