A new microcellular cytotoxicity test based on calcein AM release

We present a microtest for cell-mediated immunity, based on the use of the Tarasaki tray and calcein AM vital dye. The number of target cells needed has been reduced to 500 per test with a corresponding tenfold reduction in the number of effector cells needed. Results were read at the rate of 1 second per test using a fluorimeter attached to a microscope. Each reaction was also confirmed visually with the use of ethidium bromide as a counterstain for dead cells. The calcein AM dye used to stain the living cells was shown to have a low spontaneous leakage rate—less than 15% in 4 hours at 37°C. Dilutions of targets stained by calcein AM had a linear relationship with measured fluorescence values. NK cells, LAKs, and CTLs were readily detectable by this microtest. Quantitation of killing and kinetic analysis was readily performed with this test system. A significant positive correlation to ⁵¹Cr-release results was found. We conclude that the microtest should find wide application in studies of cell-mediated immunity.     

Evaluation of the Microbact-24E bacterial identification system.

The Microbact 24E (MB24E) system is a commercial microsystem for the identification of common clinical isolates of Enterobacteriaceae and non-fermenting Gram negative bacilli, and consists of dehydrated substrates distributed in the wells of microtitre trays. This system was compared with the API20E for the identification of 386 bacterial isolates, which included 284 clinical and 102 environmental organisms. There was 97% and 91% agreement for the identification of clinical isolates of Enterobacteriaceae and other Gram negative bacilli, respectively. The identification of environmental isolates by both systems was less satisfactory. The API20E has a more extensive database than the MB24E and is thus more reliable for the identification of rare or unusual organisms, but the MB24E is cheaper, is easy and convenient to use, and is suitable for a routine microbiology laboratory.     

Application of Serum-Free Culture Medium for Preparation of A-NK Cells

To compare the differences between proliferation and cytotoxicity of adherent natural killer （A-NK） cells cultured with serum-free medium AIMV and standard serum-containing medium in vitro, and also observe the assisting effect of IL-12 on the activation and the morphology character of IL-2-treated A-NK cells, cellular proliferation was evaluated by MTT method in vitro. The morphology of the target cells killed by A-NK cells was observed through electroscope. All of the A-NK cells cultured in serum-free medium AIMV could rapidly proliferate and keep high cytotoxicity compared with that in standard serum-containing medium. A-NK cells activated by both moderate-dose IL-2 and IL-12 were superior to the high-dose IL-2-treated A-NK cells. These data indicated that serum-free medium AIMV could replace standard serum-containing medium for culturing A-NK cells, and moderate-dose IL-2 and IL-12 could reduce side effects caused by high-dose IL-2. The study provided a new experimental basis for experimental and clinical preparation of A-NK cells. Cellular ＆ Molecular Immunology.     

Preparation of Aminated Macroporous Polyvinyl Alcohol Resins and Evaluation for Bilirubin Adsorption

In the present study we prepared macroporous polyvinyl alcohol beads. A series of bilirubin adsorbents were generated by immobilization of eight amine agents to the beads as ligands. The adsorption of bilirubin was evaluated by in vitro static and dynamic adsorption tests. The results show that these adsorbents have excellent adsorption efficiency and capacity. Among the eight ligands, trimethylamine (TMA), triethylamine (TEA) and 1,6- hexanediamine(HDA) showed the highest adsorption capacity. The adsorption equilibrium can be achieved in half an hour, and the adsorption percentage of bilirubin was up to 80%. Static electricity and hydrophobic interaction played the main role in bilirubin adsorption, and the adsorption was found to match the monolayer model. The excellent adsorption of these adsorbents indicates their potential in clinical treatment.     

Pulmonary pathological features in coronavirus associated severe acute respiratory syndrome (SARS)

BACKGROUND: Severe acute respiratory syndrome (SARS) became a worldwide outbreak with a mortality of 9.2%. This new human emergent infectious disease is dominated by severe lower respiratory illness and is aetiologically linked to a new coronavirus (SARS-CoV). METHODS: Pulmonary pathology and clinical correlates were investigated in seven patients who died of SARS in whom there was a strong epidemiological link. Investigations include a review of clinical features, morphological assessment, histochemical and immunohistochemical stainings, ultrastructural study, and virological investigations in postmortem tissue. RESULTS: Positive viral culture for coronavirus was detected in most premortem nasopharyngeal aspirate specimens (five of six) and postmortem lung tissues (two of seven). Viral particles, consistent with coronavirus, could be detected in lung pneumocytes in most of the patients. These features suggested that pneumocytes are probably the primary target of infection. The pathological features were dominated by diffuse alveolar damage, with the presence of multinucleated pneumocytes. Fibrogranulation tissue proliferation in small airways and airspaces (bronchiolitis obliterans organising pneumonia-like lesions) in subpleural locations was also seen in some patients. CONCLUSIONS: Viable SARS-CoV could be isolated from postmortem tissues. Postmortem examination allows tissue to be sampled for virological investigations and ultrastructural examination, and when coupled with the appropriate lung morphological changes, is valuable to confirm the diagnosis of SARS-CoV, particularly in clinically unapparent or suspicious but unconfirmed cases.     

Recurrent and novel mutations of GCDH gene in Chinese glutaric acidemia type I families

Glutaric acidemia type I is caused by mutations of the glutaryl-CoA dehydrogenase (GCDH) gene resulting in loss of GCDH enzyme activity. Patients present with progressive dystonia and lesions in basal ganglia. Dietary treatment, when instituted from the early neonatal period, markedly reduces dystonia and morbidity. Early diagnosis and prenatal diagnosis will be facilitated by knowledge of locally prevalent GCDH mutations. Several common GCDH mutations have been found in different ethnic groups. GCDH mutations were studied in 5 Chinese glutaric acidemia type I families. We detected two novel recurrent mutations (A219T and IVS10-2A>C) which were found in two unrelated families. An asymptomatic carrier of IVS10-2A>C was also found on screening of 120 individuals. Other mutations were identified, including two other novel (R386G & IVS3+1G>A) and two known mutations (G178R & R355H). Fibroblasts from patients carrying the novel mutations were confirmed to be deficient for GCDH activity. This is the first report of GCDH mutations describing recurrent mutations in Chinese patients. The carrier rate of IVS10-2A>C may be particularly high in Chinese.     

重复电刺激前肢神经引起成年大鼠运动皮层的可塑性改变

为了了解成年大鼠运动皮层的功能可塑性，利用皮层内微刺激方法测定ＭＩ代表区并观察重复电刺激前肢神经对ＭＩ代表区的影响。实验组大鼠（９例）持续１．２－２小时的前肢神经电刺激导致前肢运动区与面部触须运动区边界向ＶＩ方向，移动２６３．３±９０．９μｍ并同时伴有运动阈值的改变；ＦＬ内ＭＴ降低５．０±１３．３μＡ，而在ＶＩ内ＭＴ升高９．６±１１．６μＡ对照组大鼠间隔１．５－２小时的两次测定结果，ＦＬ－ＶＩ边界     

34例鹿角形肾结石手术取石的疗效观察

目的探讨肾下极肾盂肾盏联合切开术治疗复杂性鹿角形肾结石的疗效。方法回顾性分析采用肾下极肾盂肾盏联合切开术式治疗复杂性鹿角形肾结石34例临床资料。手术方法:分离肾窦内肾盂后,用1-0肠线在肾后唇中下1/3连接处紧贴肾盂肾盏表面缝合肾实质2针,中间尖刀切开肾实质,然后弧形切开肾盂及肾下盏,用神经剥离子将结石与肾盂肾盏粘膜粘连分离后,取石钳夹住结石轻轻撬出。盏颈狭窄者必要时先用气压弹道碎石器将其在分支处击断。4-0肠线缝合肾盂及肾下盏,再用2-0肠线间断全层缝合肾实质切口数针,包膜层用4-0肠线缝合。结果34例均一次性取净结石。术中平均出血量约90ml。术后无大出血病例。结论肾下极背侧肾实质与肾盂联合切开取石术具有术中出血少,无须阻断肾蒂,肾功能受损轻,便于一次取尽结石等优点。适合于肾窦内肾盂特别是肾下盏盏颈相对狭窄的复杂性鹿角形肾结石的治疗。