一氧化氮外源性供体L-arginine对破骨细胞骨吸收功能的影响

目的:一氧化氮在维持机体多个系统的生理功能中起重要作用,许多慢性疾病可造成一氧化氮产生减少,此时一氧化氮供体是一种必要的补充。观察一氧化氮外源性供体L-arginine对体外培养破骨细胞增殖及骨吸收功能的影响。方法:实验于2005-06/2006-05在四川大学华西医院生物治疗国家重点实验室干细胞与组织工程研究室完成。选择出生1d的清洁级SD大鼠乳鼠,采用骨髓诱导法体外培养破骨细胞,培养液内分别加入0.3,0.6,1.0g/L不同浓度的L-arginine,并以等体积三蒸水作为对照。培养7d后,以抗酒石酸酸性磷酸酶染色观察破骨细胞数目、形态,MIAS-2000图像分析仪检测骨片上骨吸收陷窝的数目和面积,并用扫描电镜观察不同浓度L-arginine对骨吸收陷窝的影响。结果:①破骨细胞的一般形态:破骨细胞较其他细胞大,形态不规则,呈油煎蛋形、长条形、腊肠形或漏斗形等,细胞内可见几个至几十个核不等。抗酒石酸酸性磷酸酶染色酶活性部分酒红色,颗粒状。②抗酒石酸酸性磷酸酶阳性细胞数:各组破骨细胞数目随着L-arginine浓度增加而减少(P<0.05)。③骨吸收陷窝的面积和数目:骨片培养7d,吸收陷窝计数的结果显示,0.3g/L以上浓度L-arginine对破骨细胞吸收功能均有明显抑制作用,并呈剂量相关性。0.3g/LL-arginine组陷窝面积为对照组的91%(P<0.05),0.6g/LL-arginine组为对照组的80%(P<0.05),1.0g/LL-arginine组为对照组的69%(P<0.01)。结论:采用骨髓诱导法培养的破骨细胞数量多、纯度高,且具有明显的骨吸收功能。L-arginine抑制破骨细胞增殖,抑制破骨细胞骨吸收功能,并呈剂量相关性。     

Molecular remedy of complex I defects: Rotenone-insensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae mitochondria restores the NADH oxidase activity of complex I-deficient mammalian cells

The NDI1 gene encoding rotenone-insensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae mitochondria was cotransfected into the complex I-deficient Chinese hamster CCL16-B2 cells. Stable NDI1-transfected cells were obtained by screening with antibiotic G418. The NDI1 gene was shown to be expressed in the transfected cells. The expressed Ndi1 enzyme was recognized to be localized to mitochondria by immunoblotting and confocal immunofluorescence microscopic analyses. Using digitonin-permeabilized cells, it was shown that the transfected cells, but not nontransfected control cells, exhibited the electron transfer activities with glutamate/malate as the respiratory substrate. The activities were inhibited by flavone, antimycin A, and KCN but not by rotenone. Added NADH did not serve as the substrate, suggesting that the expressed Ndi1 enzyme was located on the matrix side of the inner mitochondrial membranes. Furthermore, although nontransfected cells could not survive in a medium low in glucose (0.6 mM), which is a substrate of glycolysis, the NDI1-transfected cells were able to grow in the absence of added glucose. When glycolysis is slow, either at low glucose concentrations or in the presence of galactose, respiration is required for cells to survive. The mutant cells do not survive at low glucose or in galactose, but they can be rescued by Ndi1. These results indicated that the S. cerevisiae Ndi1 was expressed functionally in CCL16-B2 cells and catalyzed electron transfer from NADH in the matrix to ubiquinone-10 in the inner mitochondrial membranes. It is concluded that the NDI1 gene provides a potentially useful tool for gene therapy of mitochondrial diseases caused by complex I deficiency.     

Long-Term Outcomes and Dynamics of Mutants Associated with Lamivudine-Adefovir Rescue Therapy in Patients with Lamivudine-Resistant Chronic Hepatitis B

Background/Aims

To investigate the association between the baseline profiles and dynamics of hepatitis B virus (HBV) DNA polymerase gene mutations and the long-term virological response of lamivudine (LAM)-adefovir (ADV) combination therapy in patients with LAM-resistant chronic hepatitis B.

Methods

Seventy-five patients who received LAM-ADV combination therapy for more than 12 months were analyzed. Restriction fragment mass polymorphism assays were used to detect and monitor the dynamics of LAM- and ADV-resistant mutations.

Results

The median duration of LAM-ADV combination therapy was 26 months (range, 12 to 58 months). The baseline mutation profiles, rtM204I (p=0.992), rtM204I/V (p=0.177), and rtL180M (p=0.051), were not correlated with the cumulative virological response, and the baseline HBV DNA level (p=0.032) was the only independent predictive factor for cumulative virological response. Tests for LAM- and ADV-resistant mutations were performed in 12 suboptimal responders in weeks 48 and 96. The population of rtM204 mutants persisted or increased in 8 of 12 patients, and rtA181T mutants newly emerged as a minor population in four patients until 96 weeks. Nevertheless, the viral loads progressively decreased during rescue therapy, and these dynamics did not correlate with virological response.

Conclusions

The baseline profile and dynamics of LAM-resistant mutations during LAM-ADV combination therapy are not associated with a virological response.     

Air pollution exposure estimation using dispersion modelling and continuous monitoring data in a prospective birth cohort study in the Netherlands

Background

High temperature and humidity conditions are associated with short-term elevations in the mortality rate in many United States cities. Previous research has quantified this relationship in an aggregate manner over large metropolitan areas, but within these areas the response may differ based on local-scale variability in climate, population characteristics, and socio-economic factors.

Methods

We compared the mortality response for 48 Zip Code Tabulation Areas (ZCTAs) comprising Philadelphia County, PA to determine if certain areas are associated with elevated risk during high heat stress conditions. A randomization test was used to identify mortality exceedances for various apparent temperature thresholds at both the city and local scale. We then sought to identify the environmental, demographic, and social factors associated with high-risk areas via principal components regression.

Results

Citywide mortality increases by 9.3% on days following those with apparent temperatures over 34°C observed at 7:00 p.m. local time. During these conditions, elevated mortality rates were found for 10 of the 48 ZCTAs concentrated in the west-central portion of the County. Factors related to high heat mortality risk included proximity to locally high surface temperatures, low socioeconomic status, high density residential zoning, and age.

Conclusions

Within the larger Philadelphia metropolitan area, there exists statistically significant fine-scale spatial variability in the mortality response to high apparent temperatures. Future heat warning systems and mitigation and intervention measures could target these high risk areas to reduce the burden of extreme weather on summertime morbidity and mortality.     

Bell's phenomenon during screening examination for retinopathy of prematurity

Purpose

Bell''s phenomenon (BP), which may disturb screening examinations for retinopathy of prematurity (ROP), is known to present infrequently in premature babies. Stress associated with the examinations can influence expression of BP. The authors of the present study evaluated BP during examinations for ROP.

Methods

The present study included 102 eyes of 51 premature babies. Expression of BP was assessed at 3 steps of the examination in the following order: after insertion of a speculum, after illumination of an indirect ophthalmoscope and after scleral depression. The relationship between the expression of BP and the gestational age at the examination was analyzed in each step of the examination.

Results

The frequency of BP after the speculum insertion and the illumination was 77% to 92% in infants 32 weeks of age or younger, and decreased significantly to 16% to 57% in infants 42 weeks of age or older (p < 0.005). BP after the scleral depression had no significant association with the gestational age. Frequency of BP increased significantly as the steps of the examination proceeded (p < 0.01).

Conclusions

BP was frequent in premature infants during ROP examination in spite of neurological immaturity. The examiner should take BP into consideration, which frequently occurs in younger infants.     

Wire-guided intraductal US in the assessment of bile duct strictures with Mirizzi syndrome-like features at ERCP

BACKGROUND: Mirizzi syndrome refers to common hepatic duct obstruction caused by extrinsic compression that is usually from a stone impacted in the cystic duct. The utility of transpapillary intraductal US for assessment of biliary strictures with radiographic features of Mirizzi syndrome was studied retrospectively. METHODS: Intraductal US was performed in 16 patients with a common hepatic duct stricture caused by extrinsic compression and nonvisualization of the gallbladder by endoscopic retrograde cholangiography. An over-the-wire catheter US probe (20 MHz) was inserted into the bile duct. Intraductal US findings were compared with the final diagnoses at surgery. RESULTS: Intraductal US was successful in all patients. The extraluminal cause of the common hepatic duct stricture was detected in 14 patients (87.5%). In 9 patients, intraductal US detected an impacted stone outside the common hepatic duct. Intraductal US demonstrated extraluminal lesions without evidence of a stone in 5 patients, including a mass in 4 and asymmetrical, irregular thickening of the bile duct wall in 1 patient. In the remaining 2 patients, intraductal US demonstrated only a distended gallbladder. CONCLUSIONS: Transpapillary wire-guided intraductal US is useful for assessing biliary strictures with features that suggest Mirizzi syndrome and optimizes management of patients with these findings.     

Endoscopic treatment of retained bile-duct stones by using a balloon catheter for electrohydraulic lithotripsy without cholangioscopy

BACKGROUND: Electrohydraulic lithotripsy is a highly effective method for fragmenting biliary stones, but direct visual control is required. The efficacy and the safety of electrohydraulic lithotripsy without cholangioscopy by using a balloon catheter were evaluated in patients with bile-duct stones that could not be extracted by using standard techniques. METHODS: Nineteen patients with extrahepatic bile-duct stones that could not be extracted by using conventional endoscopic methods, e.g., mechanical lithotripsy, were selected to undergo electrohydraulic lithotripsy without peroral cholangioscopy. An electrohydraulic lithotripsy probe with a 3.0 F radio-opaque tip was inserted through a balloon catheter. Electrohydraulic lithotripsy was performed under fluoroscopy until the fragmented stone could be captured in a large basket for mechanical lithotripsy. Endoscopic removal of the fragments was attempted during the electrohydraulic lithotripsy session. RESULTS: Stones were successfully fragmented in 17 of 19 patients. In 16 patients (84.2%), the bile duct was cleared of all stones. A mean of 1.8 endoscopic sessions was required for complete removal. Additional mechanical lithotripsy was performed in 9 (56.2%) of the 16 patients. Minor complications were noted in 4 patients (2 hemobilia, 1 pancreatitis, 1 cholangitis). There was no 30-day mortality. CONCLUSIONS: For a selected group of patients with bile-duct stones not extractable by using standard techniques, fluoroscopically controlled electrohydraulic lithotripsy with a balloon catheter seems to be an effective method of fragmentation.     

Defective adhesion of blood platelets to vascular microfibrils in the Bernard-Soulier syndrome

Bernard-Soulier syndrome (BSS) platelets, which lack the membrane glycoprotein complex Ib-IX, do not adhere to subendothelium. The adhesion of platelets from two patients with BSS to subendothelial microfibrils (MFs) and type I collagen was compared in an in vitro assay adapted to patients with low platelet count. With both patients, platelet adhesion to MFs was strongly defective, whereas the adhesion to collagen was normal. The involvement of GPIb in the MFs-induced platelet adhesion was confirmed by the inhibitory effect of a MoAb (AN51) to the von Willebrand (vWF) factor binding domain of GPIb. The adhesion of platelets to MFs thus requires GPIb-IX and an axis MFs-vWF- GPIb-IX seems therefore to be prevalent in the reactivity of platelets with subendothelium.