人类免疫缺陷病毒感染细胞的Rer蛋白依赖性凋亡引导

目的:以人类免疫缺陷病毒(HIV)调节蛋白Rev与Rev反应区(RRE)的高亲和性建立引导HIV感染细胞凋亡的结构。方法:用分子克隆技术合成含RRE和肿瘤坏死因子受体-1(TNF-R1)的Rev依赖性凋亡引导质粒,流式细胞仪检测质粒表达。结果:新质粒pDM128-TNF-R1(pT128)HindIII内切有3.1、2.7、1.0和0.87kb片段;聚合酶链反应(PCR)法检测示TNF-R1的1360bp片段。DNA测序法确认其准确性。单纯Hup60TNF-R1在pDC302(pT60)转染Hela,TNF-R1表达可明显杀伤Hela(P<0.01),Rev存在时,pT128也能表达TNF-R1杀伤Hela(P<0.01),但不及pT60的作用(P<0.01);无Rev时,pT128不表达TNF-R1,不杀伤Hela(P>0.01)。单纯Rev不杀伤Hela(P>0.01)。pT128与pRev共转,TNF-R1表达较pT60慢(P<0.01),40h后才接近单纯pT60。结论:新质粒具有Rev依赖性表达作用,进而引导Rev表达细胞凋亡。     

Sustained effects of online genetics education: a randomized controlled trial on oncogenetics

Medical professionals are increasingly expected to deliver genetic services in daily patient care. However, genetics education is considered to be suboptimal and in urgent need of revision and innovation. We designed a Genetics e-learning Continuing Professional Development (CPD) module aimed at improving general practitioners'' (GPs'') knowledge about oncogenetics, and we conducted a randomized controlled trial to evaluate the outcomes at the first two levels of the Kirkpatrick framework (satisfaction, learning and behavior). Between September 2011 and March 2012, a parallel-group, pre- and post-retention (6-month follow-up) controlled group intervention trial was conducted, with repeated measurements using validated questionnaires. Eighty Dutch GP volunteers were randomly assigned to the intervention or the control group. Satisfaction with the module was high, with the three item''s scores in the range 4.1–4.3 (5-point scale) and a global score of 7.9 (10-point scale). Knowledge gains post test and at retention test were 0.055 (P<0.05) and 0.079 (P<0.01), respectively, with moderate effect sizes (0.27 and 0.31, respectively). The participants appreciated applicability in daily practice of knowledge aspects (item scores 3.3–3.8, five-point scale), but scores on self-reported identification of disease, referral to a specialist and knowledge about the possibilities/limitations of genetic testing were near neutral (2.7–2.8, five-point scale). The Genetics e-learning CPD module proved to be a feasible, satisfactory and clinically applicable method to improve oncogenetics knowledge. The educational effects can inform further development of online genetics modules aimed at improving physicians'' genetics knowledge and could potentially be relevant internationally and across a wider range of potential audiences.     

Receptor specificity of clinical Plasmodium falciparum isolates: nonadherence to cell-bound E-selectin and vascular cell adhesion molecule-1

The pathogenicity of Plasmodium falciparum is due largely to the parasite's unique ability to adhere to capillary and postcapillary venular endothelium during the second-half of the 48-hour life cycle. The resulting sequestration of infected erythrocytes (IRBC) in deep vascular beds leads to tissue hypoxia, metabolic disturbances, and organ dysfunction which characterize severe falciparum malaria. Several endothelial receptors of cytoadherence have been identified, but their clinical relevance remains controversial. In the present report, the receptor specificity of 60 clinical P falciparum isolates was determined using transfectants each expressing one of CD36, intercellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1 (VCAM-1). All isolates tested adhered to CD36 and ICAM-1, but the adherence to CD36 was at least 10-fold higher. Seven isolates adhered to E-selectin whereas none of 19 isolates adhered to VCAM-1. From a population standpoint, about 30% of IRBC in each isolate adhered to CD36, and 2% to 3% adhered to ICAM-1. The percentage adherent to E-selectin and VCAM-1 was negligible. IRBC selected on CD36 adhered almost exclusively to CD36 whereas 80% to 90% of IRBC selected on ICAM-1 could also adhere to CD36. Selected IRBC did not adhere to E-selectin or VCAM-1. These findings indicate that cytoadherence to multiple endothelial receptors is a rare occurrence with natural P falciparum isolates, but do not exclude a role for the adhesion molecules in promoting other IRBC-endothelial interactions such as rolling under flow conditions. Receptor specificity in vivo may be dictated by the ligand-receptor combination which provides the best survival potential for the parasite.     

Immunocytochemical localization of fibrinogen during thrombin-induced aggregation of washed human platelets

Because thrombin aggregates afibrinogenemic platelets and platelets from patients with the gray platelet syndrome and because antibodies to fibrinogen inhibit thrombin-induced aggregation only at low concentrations of thrombin, the role of fibrinogen in the formation of thrombin-induced aggregates was investigated further with human platelets washed and resuspended in Tyrode-albumin solution containing apyrase, either with or without added Ca2+ (2 mmol/L). Samples for immunocytochemical assessment of fibrinogen distribution were taken at several times (up to five minutes) after aggregation induced by 0.5 U/mL of thrombin. Glutaraldehyde-fixed samples were embedded in Lowicryl K4M, sectioned, incubated with goat antihuman fibrinogen, washed, reacted with gold-labeled antigoat IgG, and prepared for electron microscopy. By 10 seconds, small aggregates formed, and granules were centralized; alpha granules were heavily labeled with immunogold, but the platelet surface was not. As large aggregates formed, granule swelling or fusion occurred, and in some areas granule material seemed to be in contact with the exterior. In these experiments with no added fibrinogen, there were some clusters of gold particles on the platelet surfaces remote from sites of granule discharge, but there were large areas where platelets were in close contact with little or no fibrinogen detectable between them. No fibrin was visible up to five minutes after the addition of thrombin, which indicated that fibrinogen from the granules does not readily become available for fibrin formation in the ambient fluid. Similar results were obtained in media with and without added Ca2+. Thus at least some aggregation in response to thrombin can occur without the participation of released fibrinogen, and much of the granule fibrinogen appears to remain localized at sites where granules fuse with the plasma membrane or the open canalicular system. Incubation of unstirred samples with thrombin for ten minutes resulted in the formation of small aggregates, extensive gold label in regions connected to the exterior of the platelets, but very little gold labeling of the platelet membrane and no visible fibrin formation. When the platelets were aggregated in the presence of external fibrinogen, the morphological changes within the platelets were the same, but fibrinogen rapidly became associated with the entire platelet surface, and visible fibrin formed within 30 seconds in the medium containing 2 mmol/L Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparison of cytopathic changes in oral hairy leukoplakia with in situ hybridization for EBV DNA

OBJECTIVE: It has been observed that the cytopathic changes in hairy leukoplakia (HL) correlate with ultra-structural evidence of intra-keratinocyte herpes-type viral particles. In situ hybridization is considered to be the definitive confirmation of Epstein-Barr virus (EBV)-induced HL. This study evaluated the consistency of histopathological findings, which many believe to be diagnostic, with in situ hybridization for EBV-DNA in 60 patients with lesions clinically suggestive of HL.
MATERIALS AND METHODS: Hematoxylin and eosin (H&E)-stained sections were reviewed independently by three oral pathologists who did not know the hybridization results. The presence in keratinocytes of nuclear inclusions and/or homogenization, believed to be specific for EBV in these lesions, was used as an indicator for infection. Cytoplasmic changes were evaluated separately.
RESULTS: With in situ hybridization, 48 cases were positive and 12 were negative. When the two methods were compared, pathologist concurrence ranged from 83% to 92%. False negatives ranged from 6% to 19%, and false positives ranged from 8% to 25%. Cytoplasmic ballooning, homogenization, and perinuclear clearing were evident in all cases of hybridization-confirmed HL; however, these changes were also noted in 75% (9/12) of the cases with negative hybridization results. Most confirmed HL cases exhibited both nuclear homogenization and inclusions, although the former was more consistently seen.
CONCLUSION: Cytoplasmic changes did not agree well with EBV-DNA hybridization results, whereas nuclear changes demonstrated good, but not complete, agreement. In appropriate clinical settings, the finding of nuclear inclusions and/or homogenization may be of diagnostic value. However, because the potential for false positives and negatives is high, H&E cytopathology should not be used as a substitute for in situ hybridization in the definitive diagnosis of oral hairy leukoplakia.     

Decreased plasma folate concentration in young and elderly healthy subjects after a short-term supplementation with isotretinoin

Background In the last two decades, there has been an increasing use of isotretinoin (13‐cis‐retinoic acid or 13‐CRA) for treatment of severe, and recently mild and moderate, acne in Westernized populations. Recent human and animal studies emphasized alterations caused by 13‐CRA administration on folate‐dependent, one‐carbon metabolism. Folate deficiency and subsequent hyperhomocysteinemia increase the risk of degenerative diseases. Objectives We determine whether a short‐term supplementation with 13‐CRA alters folate status and homocysteinemia in young and elderly healthy human subjects. Methods Twenty young and 20 elderly (age mean, 26.1 and 65.4 years, respectively) healthy male volunteers were supplemented with ~0.5 mg/kg/day of 13‐CRA for 28 days. Fasting plasma concentrations of 13‐CRA, 5‐methyltetrahydrofolate (5‐mTHF) as the main circulating form of folate, and homocysteine (Hcy), as well as haematologic parameters and biochemical markers of liver and renal function, were measured at baseline and at the end of supplementation. Statistical analyses were carried out using two‐way anova and standard tests. Results In both groups, isotretinoin supplementation caused a dramatic increase in the circulating concentration of 13‐CRA and its derivatives. It also led to significant increases in serum triglyceride (P < 0.0001) and creatinine (P = 0.002) concentrations and γ‐glutamyltranspeptidase activity (P = 0.0001) and decrease in serum level of urea (P = 0.027). However, the latter four parameters remained within normal ranges. These changes were accompanied by a 17.7% and 13.5% decrease in the plasma level of 5‐mTHF (P = 0.001) in the young and elderly volunteers, respectively. Supplementation with 13‐CRA did not cause significant variations in their plasma Hcy concentration. However, the latter parameter seemed to respond differently in each group of age (P = 0.046). Conclusions Our data indicate that a 28‐day supplementation with isotretinoin alters the plasma folate in young and old healthy individuals. This stresses the necessity of studying the long‐term effects of retinoid therapy on folate status and homocysteinemia in acne patients, given that alteration in the latter parameters is known to increase the risk of degenerative diseases.