人白内障球结膜微血管的电镜观察

目的 :观察人球结膜微血管的超微结构 ,认识白内障时球结膜微血管的电镜改变 ,为微循环和眼科研究工作提供参考。方法 :观察对象为 5例老年性白内障、1例糖尿病性白内障 ,术前一般状态良好。在白内障手术时取球结膜标本。电镜常规固定、包埋、定位、超薄切片、用电镜观察。结果 :球结膜毛细血管多由 2～ 3个内皮细胞围绕而成。核多为长椭圆形 ,核周缘有轻度凸凹。近腔侧胞浆细胞器较多 ,微丝和微管十分发达。基底膜、周细胞与一般毛细血管相似。 6例白内障球结膜毛细血管的内皮细胞、基底膜、周细胞、管腔都有不同程度的变化。其电镜改变可以分为三类 :( 1)代偿反应性增强改变 :微丝、微管十分发达、核周深凹、胞体突入管腔内、内皮细胞突起穿越基底膜、白细胞穿壁 ;( 2 )可恢复性退变 :线粒体退变、核质周集、胞浆出现空泡、基底膜分层、弥散、小量出血 ;( 3)严重不可恢复性改变 :核质固缩、腔内红细胞融合、血红蛋白溢出弥散、管壁断裂、腔内充满退变、裂解结构 ( 2例 ) ,并通过断裂处出至基底膜外。结论 :白内障病人球结膜微血管的超微结构有程度不等的改变     

主要常见病甲襞微循环改变重复性的研究

在目前不能直接观测病人内部器官微循环改变的条件下，应用模糊聚类的理论和方法，对主要常见病有意义的的甲襞微循环改变进行聚类，同时参考国内外相关文献的结果，提出了主要常见病有意义的甲襞微循环改变群。在改变群中任意提出3、4、5、6、7、8项改变指标，形成改变组合，一种组合重复出现二次以上的重复率，随组合改变指标数的增多而减少（70．36％～4．36％）。将改变群分成重要指标和参考指标二类，从每类中提出三项形成组合，重复率仅为0．34％。说明组合方式的影响十分巨大。经过反复探索、大量实践，有可能找出对疾病的符合率较高、漏诊率较低、误诊率较小的最佳组合方式。     

Isolation and characterization of human and rabbit sperm tail fibrous sheath

Using mechanical and chemical dissection methods, fibrous sheath was isolated both from normal ejaculated human spermatozoa and from rabbit cauda epididymal spermatozoa. The same techniques did not produce a pure preparation of fibrous sheath from ejaculated rabbit spermatozoa, suggesting that further cross-linking and stabilization of sperm structures occurs in response to components of the seminal plasma. The isolation procedures were monitored by phase contrast microscopy and the purity of the fibrous sheath was verified by electron microscopy. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of isolated human fibrous sheath revealed at least 14 protein bands of which the most intensely stained were of molecular weight 84, 72, 66.2, 57, 32 and 28.5 kDa. The rabbit fibrous sheath revealed at least 10 protein bands, of which the most intensely stained were 35.2, 32.7 and 28.5 kDa. The amino acid composition of the purified fibrous sheath from human and rabbit spermatozoa was similar, being high in aspartic acid and/or asparagine and glutamic acid and/or glutamine, serine, alanine, leucine, lysine and glycine, but low in histidine, tyrosine and isoleucine. This composition is similar to that reported for the rat and suggests that mammalian sperm tail fibrous sheaths are composed of similar types of proteins, although there are apparent differences in protein components between species.      

Reduced expression of tissue inhibitor of metalloproteinase (TIMP)-2 in gestational trophoblastic diseases

To elucidate the involvement of type IV collagenases [matrix metalloproteinase (MMP)-2 and MMP-9] and their tissue inhibitors (TIMP-1 and TIMP-2) in the development of gestational trophoblastic disease (GTD), we quantified their levels in hydatidiform mole and choriocarcinoma tissues using specific enzyme-linked immunosorbent assays, and the results were compared with those from normal first trimester placenta. Levels of pro-MMP-2 were increased in hydatidiform mole, and they were further elevated in choriocarcinoma. Levels of pro-MMP-9 in choriocarcinoma and those of TIMP-1 in both hydatidiform mole and choriocarcinoma were also increased. In contrast, TIMP-2 levels were markedly decreased in both hydatidiform mole and choriocarcinoma. Similar results were obtained by the tissue culture of first trimester placenta and hydatidiform mole. Gelatin zymography indicated that the levels of both pro- and activated forms of MMP-2 and MMP-9 were higher in hydatidiform mole and choriocarcinoma. The decreased expression of TIMP-2 in hydatidiform mole and choriocarcinoma was confirmed by Western blot, Northern blot and immunohistochemistry, with the decrease being more pronounced in choriocarcinoma. Taken together, the present study shows that both TIMP-2 mRNA and protein levels are markedly decreased in GTD and the imbalance of MMP-TIMP production, shifted toward greater MMP activity, may be involved in the pathogenesis of GTD.     

House dust mite allergen avoidance and self-management in allergic patients with asthma: randomised controlled trial

BACKGROUND: The efficacy of bed covers that are impermeable to house dust mites has been disputed. AIM: The aim of the present study was to investigate whether the combination of 'house dust mite impermeable' covers and a self-management plan, based on peak flow values and symptoms, leads to reduced use of inhaled corticosteroids (ICS) than self-management alone. DESIGN OF STUDY: Prospective, randomised, double blind, placebo-controlled trial. SETTING: Primary care in a south-eastern region of the Netherlands. METHOD: Asthma patients aged between 16 and 60 years with a house dust mite allergy requiring ICS were randomised to intervention and placebo groups. They were trained to use a self-management plan based on peak flow and symptoms. After a 3-month training period, the intervention commenced using house dust mite impermeable and placebo bed covers. The follow-up period was 2 years. Primary outcome was the use of ICS; secondary outcomes were peak expiratory flow parameters, asthma control, and symptoms. RESULTS: One hundred and twenty-six patients started the intervention with house dust mite impermeable or placebo bed covers. After 1 and 2 years, significant differences in allergen exposure were found between the intervention and control groups (P<0.001). No significant difference between the intervention and control groups was found in the dose of ICS (P = 0.08), morning peak flow (P = 0.52), peak flow variability (P = 0.36), dyspnoea (P = 0.46), wheezing (P = 0.77), or coughing (P = 0.41). There was no difference in asthma control between the intervention and control groups. CONCLUSION: House dust mite impermeable bed covers combined with self-management do not lead to reduced use of ICS compared with self-management alone.     

Formaldehyde in cryoprotectant propanediol and effect on mouse zygotes

Cryopreservation of human zygotes and embryos has been routinely performed by in-vitro fertilization clinics for many years. Karran and Legge (1996) first reported that formaldehyde (FA) present in the cryoprotective solutions can have a deleterious effect on mouse oocytes. FA is a cytotoxic, carcinogenic and mutagenic chemical. The effect of FA on mouse zygotes was investigated. In addition, the concentrations of FA in propanediol (PROH) obtained from various sources were determined. Pooled 1-cell embryos were dispensed into droplets of modified Ham's F10 or human tubal fluid containing various concentrations of FA. Since bovine serum albumin (BSA) may minimize toxicity additional trials were done as above in the absence of BSA. FA concentration in the standard 1.5 M PROH, from different sources in water, was measured in the same assay using a standard curve of 0-100 microM FA. FA in a complex medium had a significant deleterious effect on embryo development and hatching but only at 1 mM concentration (P < 0.000001; see Tables I-III). There was no significant effect of FA at 100 microM. However, in a simple medium even 50 microM FA decreased embryo hatching. FA was present in 1.5 M PROH from different sources (range 1.0-35.3 microM concentration). It appears that FA concentrations do not increase with storage because FA concentrations were low even after opening and storage for 3 years on the shelf. This suggests that FA is a contaminant during the manufacturing process and may vary from manufacturer to manufacturer and batch to batch. Until further studies are done to confirm the lack of toxicity to embryos during cryopreservation (with or without FA scavengers) it may be prudent to screen all batches of cryoprotectants for FA as part of quality control.      

High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.      

Oocyte morphology predicts outcome of intracytoplasmic sperm injection

To examine the influence of cytoplasmic morphology on the success rate of intracytoplasmic sperm injection (ICSI), the morphology of 837 metaphase II oocytes was assessed after cumulus stripping. The main abnormalities detected were excessive granularity, cytoplasmic inclusions such as vacuoles, smooth endoplasmic reticulum clustering and refractile bodies. Microinjection was performed in 538 oocytes with normal cytoplasm, 142 out of 161 with excessive granularity and 112 out of 138 with cytoplasmic inclusions. Very poor oocytes were not injected. No difference was found in fertilization rate. The embryos achieved cleaved normally and a similar number of good quality embryos among the three groups was noted. The outcome of transfer of embryos derived solely from normal oocytes (group A: 72 patients, 183 embryos) was compared with those from oocytes with cytoplasmic abnormalities (group B: 34 patients, 85 embryos). In group A, 17 clinical pregnancies (24% per patient, implantation rate 10%) were established. In group B, only one clinical pregnancy (3% per patient, implantation rate 1%) was established, from the transfer of embryos derived from oocytes with homogeneous granularity of the cytoplasm. No pregnancy resulted following the transfer of embryos from eggs with cytoplasmic inclusions. The difference was statistically significant. The outcome of ICSI is dependent on the quality of the oocytes retrieved. Normal fertilization and early embryo development were achieved in oocytes with abnormal cytoplasm morphology, but the resulting embryos failed to demonstrate the same implantation potential as those derived from oocytes with normal cytoplasm.