Monoclonal antibody to human high-molecular-weight kininogen recognizes its prekallikrein binding site and inhibits its coagulant activity

We developed a mouse monoclonal antibody (MoAb 115-21) to human high- molecular-weight kininogen (HK) that recognizes its prekallikrein binding site (residues 565 through 595 of HK). The corresponding synthesized 31-amino acid peptide (peptide IV) was recently shown to retain native HK's prekallikrein binding property. The same peptide bound factor XI also, although less avidly. Our MoAb recognizes purified HK, peptide IV, and the light chain moiety of HK (where the peptide IV resides), as shown by enzyme-linked immunosorbent assay (ELISA) and Western blotting experiments. The apparent dissociation constant for the HK and MoAb 115-21 interaction was 2.2 nmol/L. It does not recognize low-molecular-weight kininogen (LK) with which HK shares its heavy chain moiety or any antigens in human plasma congenitally deficient in kininogens. The binding of MoAb 115-21 to purified light chain of HK was competitively inhibited by peptide IV. In addition, the antibody inhibits HK-dependent clotting activity of normal human plasma and dextran sulfate-mediated activation of prekallikrein in plasma and retards cleavage of HK in normal plasma after contact activation with dextran sulfate. Also, purified Fab fragments of MoAb 115-21 inhibited the HK-dependent coagulant activity and dextran sulfate-mediated prekallikrein activation in normal plasma. Since the kd for HK-MoAb 115- 21 interaction is ten times lower than that of HK-prekallikrein, our data suggest that binding of MoAb 115-21 to HK's peptide IV site increases the free prekallikrein concentration in plasma and thus results in the decreased efficiency of factor XIIa-mediated activation of prekallikrein. Decreased levels of kallikrein thus formed may be responsible for the inhibition of HK-dependent clotting activity and the decrease in rate and extent of HK cleavage in normal plasma on contact activation with dextran sulfate. MoAb 115-21 may thus prove very useful, especially with its high affinity for HK, in further delineation of the role of HK and prekallikrein in contact activation and kinin-related human pathology.     

经冠状动脉移植自体骨髓单个核细胞和间充质干细胞对急性心肌梗死模型心功能的改善

目的:为保护濒死心肌提供机会窗口,对比观察经冠脉移植自体骨髓单个核细胞或间充质干细胞后,实验性急性心肌梗死动物心功能变化及心肌组织核转录因子кB、心肌细胞凋亡情况。方法:实验于2005-03/2006-11在河北省人民医院实验中心完成。选用24只雄性冀中白猪,随机数字表法分为4组:正常对照组、模型组、单个核细胞组、间充质干细胞组,6只/组。①24只猪均以盐酸氯胺酮200mg臀部肌肉注射麻醉后,分别于各自右侧股骨抽取骨髓20mL,采用Fercoll法分离获得骨髓单个核细胞,加入胶体金溶液,培养12～16h待用。分离过程中取出含有骨髓单个核细胞成分的细胞层,常规培养传代,每3d换液1次,贴壁生长细胞即为骨髓间充质干细胞,加入胶体金溶液,培养24h待用。②除正常对照组外,其余各组均经导管球囊封闭第一对角支以远的前降支,复制猪急性心肌梗死模型。单个核细胞组、间充质干细胞组均于造模后立即开通前降支,分别经球囊注入预先分离的骨髓单个核细胞6×108个、间充质干细胞6×108个。模型组造模后于梗死1h开通前降支,经球囊注入磷酸盐缓冲液10mL。③各组分别于术前及术后4周经心脏超声检测心功能,取材行病理学检查、心肌组织核转录因子кB的免疫组织化学检测及心肌细胞凋亡检测。结果:24只雄性白猪均进入结果分析。①心功能变化:术前各组左心室收缩末内径、左心室舒张末内径、左心室射血分数、短轴缩短率基本相似。移植术后4周,正常对照组、单个核细胞组、间充质干细胞组左心室舒张末内径均明显低于模型组(F=4.68,P=0.01),左心室射血分数及短轴缩短率均明显高于模型组(F=5.14,P=0.01;F=3.32,P=0.04),各组左心室收缩末内径差异无显著性意义(F=1.64,P=0.21)。②心肌组织病理学改变:电镜下单个核细胞组、间充质干细胞组在梗死边缘区可见有胶体金颗粒的不成熟的心肌细胞,胞质中散在肌丝结构,肌丝排列紊乱不规则。③心肌组织核转录因子кB阳性率表达:与模型组比较,单个核细胞组、间充质干细胞组的梗死边缘区核转录因子кB阳性率明显降低(F=25.59,P=0.0001);正常心肌区核转录因子кB阳性率亦明显降低(F=18.20,P=0.0001)。④心肌细胞凋亡检测结果:与模型组比较,单个核细胞组、间充质干细胞组在心肌梗死区细胞凋亡率均明显降低(F=6.63,P=0.0027),梗死边缘区细胞凋亡率亦明显降低(F=36.07,P=0.0001)。正常心肌区单个核细胞组细胞凋亡率与模型组基本相似(F=9.69,P=0.004),但间充质干细胞组有所降低。⑤心功能与心肌细胞凋亡及心肌组织NF-кB的相关性:急性心肌梗死4周时,左心室射血分数与心肌细胞凋亡、心肌组织核转录因子кB均呈负相关(r=0.613,P=0.001;r=-0.437,P=0.033)。心肌细胞凋亡与心肌组织核转录因子кB呈正相关(r=0.672,P=0.002)。结论:经冠脉移植骨髓单个核细胞和间充质干细胞均可改善实验性急性心肌梗死动物的心功能,与梗死边缘区核转录因子кB表达降低及心肌细胞凋亡减少有关。骨髓单个核细胞移植的促血管增生作用优于间充质干细胞移植。     

自体骨髓间充质干细胞移植后心肌缝隙连接蛋白43表达与室性心动过速发生的关系

目的:缝隙连接蛋白43对维持心肌细胞的连接通讯功能、电信号传导和正常的节律性收缩起重要作用,其表达和分布的异常是多种室性心律失常的解剖学基础,建立小型猪急性心肌梗死模型.观察自体骨髓间充质干细胞移植后室性心动过速的发生及心肌缝隙连接蛋白43的表达方法:实验于2006-01/2007-01在河北省人民医院导管室完成。①材料:选取8～12月龄小型猪22头,由河北医科大学实验动物中心提供,体质量20～30 kg,随机数字表法分为细胞移植组12头、模型对照组10头.实验过程中对动物的处置符合动物伦理学际准②实验方法:无菌条件下抽取猪双侧股骨骨髓20 mL,percoll法 贴壁法分离培养骨髓间充质干细胞,待细胞生长达75%融合时用胰酶消化传代。将传至第2代细胞加入终浓度为10μmol/L的5-氮胞苷进行诱导,用胶体金标记12 h后继续培养20 d用于移植:两组小型猪均采用球囊堵闭法建立急性心肌梗死模型.心电图监测示相关至少2个导联ST段抬高大于0.2 mV、术后血肌钙蛋白和肌酸磷酸激酶同工酶升高超过正常的两倍为建模成功标准:细胞移植组于造模成功后经OTW球囊于第一对角支远端1 cm处再次阻断血流,注入经胶体金标记的10×10~7个骨髓间充质干细胞。③实验评估:于细胞移植后2 h及4周行电生理程序刺激.观察室性心动过速的发生情况。末次电生理检查后、采用免疫组化染色法检测心肌缝隙连接蛋白43的表达.计算其积分吸光度值。结果:①模型建立指标检测:与术前比较.造模后所有小型猪血肌钙蛋白含量和肌酸磷酸激酶同工酶活性均增高,峰值浓度分别为(21.3±3.6)μg/L和(178.3×41.4)IU/L,术中心电图ST段平均抬高(10.67±1.43)mm.证明急性心肌梗死模型成功建立。②骨髓间充质干细胞移植后室性心动过速的发生情况:与模型对照组诱发出室性心动过速的动物数量比较,术后2 h细胞移植组无明显变化(X~2=0.201,P=0.650),术后4周细胞移植组明显降低(X~2=4.455.P=0.035)。②骨髓间充质干细胞移植后梗死心肌缝隙连接蛋白43的表达:术后4周移植到梗死心肌的骨髓间充质干细胞与宿主心肌生长为一体,移植部位颜色变黑,苏木精-伊红染色示移植细胞的胞浆呈紫红色。细胞移植组心肌梗死区缝隙连接蛋白43积分吸光度值明显高于模型对照组(t=16,82.P=0.00),细胞移植组中未发生室性心动过速小型猪的梗死心肌缝隙连接蛋白43的表达明显高于发生室性心动过速小型猪(t=5.06,P=0.00)。结论:自体骨髓间充质干细胞移植可促进急性心肌梗死猪心肌缝隙连接蛋白43的表达,其表达程度可能与急性心肌梗死室性心动过速的发生有关。     

The Importance of Early Carotid Endarterectomy in Symptomatic Patients

INTRODUCTION

Early carotid endarterectomy (CEA) in symptomatic patients may prevent repeat cerebral events. This study investigates the relationship between waiting time for CEA and the incidence of repeat cerebral events prior to surgery in symptomatic patients.

PATIENTS AND METHODS

A prospective database of consecutive patients undergoing CEA between January 2002 and December 2006 was reviewed. Repeat event rates prior to surgery were calculated using Kaplan–Meier analysis and predictive factors identified using Cox regression analysis.

RESULTS

A total of 118 patients underwent CEA for non-disabling stroke, TIA and amaurosis fugax. Repeat cerebral events occurred in 34 of 118 (29%) patients at a median 51 days (range, 2–360 days) after the first event. The estimated risk of repeat events was 2% at 7 days and 9% at 1 month after first event (Kaplan–Meier survival analysis). Age (HR 1.059; 95% CI 1.014–1.106; P = 0.009] was identified as a predictor of repeat events. Patients underwent surgery at median 97 days (range, 7–621 days) after the first event. Eleven of 60 (18%) patients waiting ≤?97 days for surgery and 23 of 58 (40%) patients waiting >?97 days had repeat events. (P = 0.011, chi-squared test).

CONCLUSIONS

Delays in surgery should be reduced in order to minimise repeat cerebral events in patients with symptomatic carotid stenosis, particularly in the elderly population.     

Study of Disorders of Visual Acuity among Adolescent School Children in Pune

Background

Disorders of visual acuity are known to affect the learning abilities of school children. Various authors have reported varying prevalence in India and abroad.

Methods

A rapid, community based survey was conducted to assess the prevalence of disorders of visual acuity among adolescent school children in three schools in Pune Cantonment. A sample of 236 children studying in six sections were examined after random selection by a two stage sampling technique ensuring representation to each of the classes. Visual acuity was assessed for all the children available in the selected section on the date of survey. A questionnaire based study of the knowledge and attitude of the parents of the 50 affected children was also conducted.

Result

50 children (25 males and 25 females) were found to have errors of refraction, resulting in prevalence rate of 21.19% with confidence intervals of 15.97% to 26.40%.

Conclusion

The study highlights significantly high prevalence of uncorrected errors of visual acuity among adolescent school children as well as the need for instituting IEC activities aimed at improving the general lack of appreciation of the seriousness of the problem by the parents and consequent neglect of urgent corrective measures.Key Words: Visual defects, Adolescent school children     

Texas Children's Medication Algorithm Project: update from Texas Consensus Conference Panel on Medication Treatment of Childhood Major Depressive Disorder

OBJECTIVE: To revise and update consensus guidelines for medication treatment algorithms for childhood major depressive disorder based on new scientific evidence and expert clinical consensus when evidence is lacking. METHOD: A consensus conference was held January 13-14, 2005, that included academic clinicians and researchers, practicing clinicians, administrators, consumers, and families. The focus was to review, update, and incorporate the most current data to inform and recommend specific pharmacological approaches and clinical guidance for treatment of major depressive disorder in children and adolescents. RESULTS: Consensually agreed on medication algorithms for major depression (with and without psychosis) and comorbid attention-deficit disorders were updated. These revised algorithms also incorporated approaches to address issues of suicidality, aggression, and irritability. Stages 1, 2, and 3 of the algorithm consist of selective serotonin reuptake inhibitor and norepinephrine serotonin reuptake inhibitor medications whose use is supported by controlled, acute clinical trials and clinical experience. Recent studies provide support that selective serotonin reuptake inhibitors in addition to fluoxetine are still encouraged as first-line interventions. The need for additional assessments, precautions, and monitoring is emphasized, as well as continuation and maintenance treatment. CONCLUSIONS: Evidence and expert clinical consensus support the use of selected antidepressants in the treatment of depression in youths. The use of the recommended antidepressant medications requires appropriate monitoring of suicidality and potential adverse effects and consideration of other evidence-based treatment alternatives such as cognitive behavioral therapies.