A cystic fibrosis pancreatic adenocarcinoma cell line.

We established a pancreatic adenocarcinoma cell line (CFPAC-1) from a patient with cystic fibrosis (CF) and assessed some of its properties. The cells show epithelial morphology and express cytokeratin and oncofetal antigens characteristic of pancreatic duct cells. Basal and stimulated levels of cAMP and cAMP-dependent protein kinase and the biophysical properties of single Cl- channels in CFPAC-1 are similar to those of airway and sweat gland primary cultures and Cl(-)-secreting epithelial cell lines. Anion transport and single Cl- channel activity was stimulated by Ca2+ ionophores but not by forskolin, cAMP analogs, or phosphodiesterase inhibitors. The cells express the CF gene and manifest the most common CF mutation, deletion of three nucleotides resulting in a phenylalanine-508 deletion. These properties have been stable through greater than 80 passages (24 months), suggesting that CFPAC-1 can serve as a continuous cell line that displays the CF defect.     

A randomised controlled trial of extended anticoagulation treatment versus standard treatment for the prevention of recurrent venous thromboembolism (VTE) and post-thrombotic syndrome in patients being treated for a first episode of unprovoked VTE (the ExACT study)

Venous thromboembolism (VTE) is prevalent and impactful, with a risk of death, morbidity and recurrence. Post-thrombotic syndrome (PTS) is a common consequence and associated with impaired quality of life (QoL). The ExACT study was a non-blinded, prospective, multicentred randomised controlled trial comparing extended versus limited duration anticoagulation following a first unprovoked VTE (proximal deep vein thrombosis or pulmonary embolism). Adults were eligible if they had completed ≥3 months anticoagulation (remaining anticoagulated). The primary outcome was time to first recurrent VTE from randomisation. The secondary outcomes included PTS severity, bleeding, QoL and D-dimers. Two-hundred and eighty-one patients were recruited, randomised and followed up for 24 months (mean age 63, male:female 2:1). There was a significant reduction in recurrent VTE for patients receiving extended anticoagulation [2·75 vs. 13·54 events/100 patient years, adjusted hazard ratio (aHR) 0·20 (95% confidence interval (CI): 0·09 to 0·46, P < 0·001)] with a non-significant increase in major bleeding [3·54 vs. 1·18 events/100 patient years, aHR 2·99 (95% CI: 0·81–11·05, P = 0·10)]. Outcomes of PTS and QoL were no different between groups. D-dimer results (on anticoagulation) did not predict VTE recurrence. In conclusion, extended anticoagulation reduced VTE recurrence but did not reduce PTS or improve QoL and was associated with a non-significant increase in bleeding. Results also suggest very limited clinical utility of D-dimer testing on anticoagulated patients.     

富血小板血浆对人骨髓间充质干细胞成骨分化的抑制效应

目的:一些理论质疑富血小板血浆对骨前体细胞成骨分化的作用,本实验拟验证富血小板血浆对体外培养的人骨髓间充质干细胞成骨分化的抑制效应。方法:实验于2005-05/11在南方医科大学组织工程试验室(省级)完成。①实验方法:抽取6名健康志愿者髂前上棘骨髓5mL进行体外细胞培养扩增,静脉血10mL以二次离心法制得富血小板血浆。诱导骨髓间充质干细胞时富血小板血浆与骨髓间充质干细胞均来自同一个体。②碱性磷酸酶染色:取第4代骨髓间充质干细胞,分为两组:富血小板血浆组加入富血小板血浆使终浓度为100g/L,单纯血清培养组仅加入等量胎牛血清。培养后第7天进行碱性磷酸酶染色,阳性细胞为胞质中呈现黑色颗粒或块状沉淀。③矿化结节染色:取第4代骨髓间充质干细胞,分组同上。培养后第19天以0.1%茜素红-TrisHcl(pH8.3)37℃下放置30min,矿盐沉积染色阳性为红色。④Cbfa1基因表达:取第4代骨髓间充质干细胞,分组同上。培养后第3,7,12,16天RT-PCR法检测骨髓间充质干细胞Cbfa1基因的表达。⑤形态学观察:实验过程中使用相差显微镜观察各组细胞生长情况及形态学变化。结果:①骨髓间充质干细胞碱性磷酸酶染色结果:培养后第7天,富血小板血浆组碱性磷酸酶阳性细胞数量较单纯血清培养组明显减少,且阳性细胞内灰黑色颗粒也明显减少,为弱阳性。②骨髓间充质干细胞矿化结节染色结果:培养后第19天,单纯血清培养组可见细胞表面有较多的矿盐沉积,但未形成明显的矿化结节。富血小板血浆组细胞表面只有稀少的矿盐沉积。③骨髓间充质干细胞cbfa1mRNA的表达:培养后第3,7,12,16天,随着培养时间的延长单纯血清培养组与富血小板血浆组cbfa1基因表达量均逐渐增高,同一时间点两组间cbfa1基因的表达基本相似。④骨髓间充质干细胞形态学变化:富血小板血浆组骨髓间充质干细胞增殖旺盛,细胞达到单层汇合的时间较单纯血清培养组明显缩短。单纯血清培养组细胞在完全汇合后开始出现聚合现象(14～16d),但趋向性不明显,未完全形成团簇;富血小板血浆组细胞在完全汇合后未出现聚合现象,细胞密集生长。培养初期两组细胞以梭形为主,多角形细胞较少,培养至14～16d单纯血清培养组多角形细胞较富血小板血浆组增多。结论:富血小板血浆可抑制人骨髓间充质干细胞碱性磷酸酶的分泌与矿盐沉积,对人骨髓间充质干细胞成骨分化的直接效应是抑制其分化。     

Development of urease conjugated enzyme-linked immunosorbent assays (ELISA) for the detection of IgM and IgG antibodies against Mycoplasma pneumoniae in human sera

Urease conjugated enzyme linked immunosorbent assays (ELISA) were developed for the detection of human IgM and IgG antibodies against Mycoplasma pneumoniae. Results obtained by ELISA were compared with complement fixation test (CFT); which showed that of the 214 serum specimens tested, 80 were found to have antibody against M. pneumoniae. ELISA revealed that 70 of these specimens were IgG antibody, and 27 of them also contain IgM antibody. CFT failed to detect the presence of antibody against M. pneumoniae in five serum specimens tested. However, by using ELISA, three of them were found to have IgG and IgM antibodies. and the other two sera have IgG antibody only. Four out of the five specimens tested were the first serum specimens collected from patients with clinical and serological evidence of M. pneumoniae infection. In addition, 28 serum specimens, including 10 sera containing IgM rheumatoid factors and sera known to contain IgM antibody to other infectious organisms, were also tested for IgM antibody against M. pneumoniae by ELISA. None of these specimens showed a nonspecific reaction. ELISA had a sensitivity of 87.5% and a specificity of 96.3% when compared with CFT. Thus, ELISA developed in our laboratory is a specific test, and the results indicated that IgM ELISA might be used as a rapid diagnosis for M. pneumoniae infection.     

Greater Epoetin alfa Responsiveness Is Associated With Improved Survival in Hemodialysis Patients

Background and objectives: Among hemodialysis patients, achieved hemoglobin is associated with Epoetin alfa dose and erythropoietin responsiveness. A prospective erythropoietin responsiveness measure was developed and its association with mortality evaluated.Design, setting, participants, & measurements: Data from 321 participants were used and randomized to the hematocrit normalization arm of the Normal Hematocrit Cardiac Trial. Subjects were to receive a 50% Epoetin alfa dose increase at randomization. The prospective erythropoietin responsiveness measure was defined as the ratio of weekly hematocrit change (over the 3 wk after randomization) per Epoetin alfa dose increase (1000 IU/wk) corresponding to the mandated 50% dose increase at randomization. The distribution of responsiveness was divided into quartiles. Over a 1-yr follow-up, Cox proportional hazard modeling evaluated associations between this responsiveness measure and mortality.Results: Erythropoietin responsiveness values ranged from −2.1% to 2.4% per week per 1000 IU. Although subjects were similar across response quartiles, mortality ranged between 14% and 34% among subjects in the highest and lowest response quartiles (P = 0.0004), respectively. After adjusting for baseline prognostic indicators, highest versus lowest responsiveness was associated with a hazard ratio of 0.41 (95% confidence interval, 0.20 to 0.87).Conclusion: Lower erythropoietin responsiveness is a strong, independent predictor of mortality risk and should be considered when evaluating associations between clinical outcomes and potential prognostic indicators, such as Epoetin alfa dose and achieved hemoglobin values.More than 90% of end-stage renal disease patients require exogenous erythropoietin or transfusion to achieve and maintain target hemoglobin values (1,2) because of decreased endogenous erythropoietin production. The ability to achieve and maintain target hemoglobin levels is complicated by a variety of mediating factors that impact responsiveness to erythropoietin, including comorbidities, inflammation, and malnutrition. These factors are independently associated with poor clinical outcomes (3–9).The impact of erythropoietin responsiveness on mortality is not well understood. Although higher hemoglobin levels have been associated with reduction in mortality in observational studies (10,11), evidence from randomized clinical trials of hemodialysis patients does not suggest a mortality benefit (12). Paradoxically, in the Normal Hematocrit Cardiac Trial (13), the largest randomized trial conducted to date in hemodialysis patients, survival rates were higher among those achieving higher hematocrit values, but targeting a higher hematocrit was associated with a 1.3-fold increased risk of mortality or nonfatal myocardial infarction (95% confidence interval [CI], 0.9 to 1.9). This suggests that unknown/unmeasured patient characteristics associated with the ability to achieve greater hemoglobin values may confound analyses assessing mortality risks among dialysis patients.Achieved hemoglobin level is associated with both the Epoetin alfa doses administered and patient responsiveness to erythropoietin. Greater survival among patients with higher hemoglobin values may be partly due to greater erythropoietin responsiveness (14) in addition to a direct result of anemia correction. Likewise, lower survival among those with lower achieved hemoglobin values may be partly the result of lower relative erythropoietin responsiveness. Patients who require higher Epoetin alfa doses to achieve a given hemoglobin level, that is, who are less responsive to erythropoietin, may experience poorer outcomes at any achieved hemoglobin value (15).In this study, data from the hematocrit normalization arm of the Normal Hematocrit Cardiac Trial (13) were used to develop a prospective measure of erythropoietin responsiveness, which was then evaluated in relation to mortality.     

Myectomy versus anterior transposition for inferior oblique overaction

BACKGROUND: Inferior oblique overaction can be either secondary (as a sequela of ipsilateral superior oblique palsy) or primary (commonly associated with horizontal strabismus). Superior oblique underaction often coexists with both primary and secondary inferior oblique overaction. This retrospective case series compares the efficacy of inferior oblique myectomy versus anterior transposition in improving inferior oblique overaction and superior oblique underaction in eyes with either primary or secondary inferior oblique overaction. METHODS: One hundred twenty eyes of 81 patients were included in this retrospective case series, of which 20 had anterior transposition of the inferior oblique and 100 eyes underwent myectomy. Inferior oblique myectomy was compared with inferior oblique anterior transposition in improving inferior oblique overaction and superior oblique underaction in each diagnostic subgroup. Postoperative outcome was qualitatively and quantitatively assessed. Fisher's exact test was used to compare the outcomes. The quantitative improvement of function in terms of inferior oblique overaction and superior oblique underaction was analyzed by regression analysis. RESULTS: When postoperative inferior oblique overaction was considered, there was no statistically significant difference between myectomy and anterior transposition in both primary and secondary inferior oblique overaction. Myectomy was superior to anterior transposition in improving superior oblique underaction in both primary inferior oblique overaction (OR = 0.14; 95% CI, 0.015-1.45; p = 0.056) and secondary inferior oblique overaction (OR = 0; 95% CI, 0-0.027; p < 0.001). The quantitative improvement of function showed a significant difference between procedures for superior oblique underaction (t-test; p = 0.005; 95% CI, 0.25-1.3) but not inferior oblique overaction (t-test; p = 0.8; 95% CI, -0.67-0.54). CONCLUSIONS: This study demonstrates both inferior oblique myectomy and inferior oblique anterior transposition to be effective in correcting primary and secondary inferior oblique overaction. Myectomy is more effective in improving superior oblique underaction associated with both primary and secondary inferior oblique overaction. On this basis, we feel that inferior oblique myectomy has some advantage over anterior transposition in treating combined inferior oblique overaction and superior oblique underaction and can be considered the procedure of choice.