An improved enzyme-linked immunosorbent assay for the detection of rotavirus in faeces of neonates

A direct enzyme-linked immunosorbent assay (EIA) for the detection of rotavirus in neonatal stools was developed. Rabbit antiserum against SA 11 rotavirus was incorporated as both coating and detector antibody, and rotavirus-negative rabbit serum was applied as a coating antibody control to eliminate false positive results. Pretreatment of stools with EDTA was found to increase both the sensitivity and specificity of the assay. This effect was greatest when 0.25 M EDTA (tetrasodium salt) was included in homogenized stool suspensions before the removal of solid debris by centrifugation. By electron microscopy, this EDTA pretreatment appeared to partly uncoat human rotavirus particles in faeces. Potentially suitable solid phase supports and horseradish peroxidase substrates were evaluated in the development of the assay. Screening of stool samples revealed that repeated freezing and thawing of stools eliminated positive EIA reactions. The SA 11 coating antibody compared favourably with a reference coating antiserum prepared against human faecal rotavirus strains. This EIA showed greater sensitivity for rotavirus detection than electron microscopy of stool concentrates prepared by ultracentrifugation, on testing 143 stools from 99 neonates and children. The assay has been applied successfully to detection of rotavirus in stools of neonates containing meconium, smaller amounts of viral antigen than in older children, and lacteal antirotaviral antibody. It is likely to be particularly useful for cross-infection studies in hospital wards and neonatal nurseries.     

Pharmacokinetics of amixin after repeated peroral administration to mice

Pharmacokinetics of amixin was studied after repeated administration (5 days) to animals. Perorally administered amixin is characterized by high bioavailability and is present in the circulation in high concentrations for a long time. The main pharmacokinetic parameters were estimated by the method of linear regression because of slow elimination of amixin from organs and tissues. Our results indicate that repeated treatment with amixin holds much promise for the prevention and therapy of chronic diseases (particularly hepatitides). __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 140, No. 12, pp. 661–663, December, 2005     

An improved technique for repeated bronchoalveolar lavage and lung mechanics measurements in individual rats

Lung function and bronchoalveolar lavage (BAL) fluid are commonly analyzed to assess the severity of lung disease in sacrificed animals. The input impedance of the respiratory system (Z(rs)) was measured and BAL fluid was collected in intubated, anesthetized, mechanically ventilated rats on three occasions 1 week apart. Measurements were performed in control animals (group C), while lung injury was induced in the other group (group LPS) by i.p. injection of lipopolysaccharide (LPS) before the second measurement. The airway resistance (R(aw)), tissue damping (G) and elastance (H) were determined from the Z(rs) spectra. The total cell counts (TC) from 0.3- to 0.4-ml BAL fluid were also determined. R(aw) exhibited no significant change in either group C (-6.7+/-3.6[S.E.]%) or LPS (-0.9+/-3.7%). Reproducible G and H values were obtained in group C (2.5+/-5.3%, -7.0+/-4.4%), while G and H increased in group LPS (18.4+/-6.5%, 14.9+/-13.8%, p<0.05). The changes in TC followed a similar pattern to those observed in G, with no change in group C (-7.9+/-30%), but with a marked increase in group LPS (580+/-456%, p<0.05). The method devised for repeated BAL measurements in another group of rats without intubation and muscle relaxant resulted in similar results in BAL profile. We conclude that longitudinal follow-up of the airway and tissue mechanics and inflammatory cells in the BAL fluid are feasible in rats. The current method allows an early detection of lung injury, even in a relatively mild form.     

An improved method for the isolation and culture of rat epidermal stem cells

The management of burns and injuries using novel treatment strategies involving epidermal stem cells (ESC) requires a better understanding of the biology of these cells, in particular, their isolation and the maintenance of their unique characteristics in culture. The purpose of this study was to describe an improved method for isolating putative ESC from fetal rat skin and to maintain them long term in culture. Single ESC suspensions were obtained from fetal rat skin by enzyme digestion containing 0.5% neutral protease. The target cells were harvested by rapid adherence on type IV collagen plates and were cultured in complex DMEM. After primary isolation, cells were continuously cultured in K-serum free medium. After reaching 70-80% confluence, the cells were digested with 0.25% trypsin at 37°C for 5-10 minutes, and passaged at a ratio of 1:2. The cultured ESC showed good growth, resulting in cell viability of over 98%. Four days later, clones containing 100-200 cells were detected, showing cobblestone-like characteristics. The rapidly adherent cells were positive for keratin 15, 19 and P63. Eighty three percent of cells expressed β1 integrin. The growth-curve showed that the rapidly adherent cells were in the exponential growth phase. The protocol described in this paper provides a simplified and effective method to isolate and maintain long-term culture of epidermal stem cells from fetal rat skin. This method should be valuable for isolating and studying ESC from various transgenic rat lines that are currently available.     

经鼻腔给予丙酸睾丸酮对老年大鼠骨骼肌的改善作用

目的:探讨经鼻腔给予丙酸睾丸酮对老年大鼠骨骼肌生理功能和形态结构的改善作用.方法:老年雄性大鼠经鼻腔给予丙酸睾丸酮12周,利用倾斜面实验和水平绳实验观察大鼠的行为变化;H-E染色显示大鼠肱二头肌的形态变化.结果:老年大鼠鼻腔给予丙酸睾丸酮对体质量无明显影响;老年大鼠连续2d的倾斜面下滑角度减小,50°角下滑次数增加,水平绳悬挂时间缩短,鼻腔给予丙酸睾丸酮后连续2d的倾斜面下滑角度增加,50°角下滑次数减少,水平绳悬挂时间延长;老年大鼠单位面积内肱二头肌肌细胞间隔的比例增多,鼻腔给予丙酸睾丸酮后单位面积内肱二头肌肌细胞间隔的比例减少.结论:鼻腔给予丙酸睾丸酮可以提高老年大鼠的平衡反应能力和肌张力,增加肱二头肌肌细胞面积,对老年大鼠肌细胞的生理功能及形态结构起到一定的保护作用,为鼻腔应用丙酸睾丸酮治疗老化过程引起的肌肉减少症提供实验依据.     

An improved technique for nasal inhalation challenge tests.

An improved technique for nasal inhalation challenge tests is described. It includes an improved delivery system utilizing a Maxi-Myst air compressor delivering a flow of room air controlled by an in-line, electroncially timed solenoid which precisely controls the duration of compressor activity. A No. 251 DeVilbiss atomizer will deliver 0.1 + 0.01 gm/spray when the flow rate is 11.5 L/min, the duration of atomizer activity is approximately 0.1 sec, and the amount of liquid in the atomizer insert is kept between 0.75 and 2.0 ml. Nasal aerosol challenge of 0.1 ml of isotonic phosphate-buffered saline per nostril produced less variability in nasal airway resistance (NAR) response than 0.2 ml, and the smaller volume proved satisfactory for 6 consecutive saline challenges at 15-min intervals. A new face mask, which did not impinge on the bridge of the nose or paranasal structures, yielded lower baseline values of nasal airway resistance and much less variability in these measurements. Techniques employed in objectively quantitating nasal responses to various exogenous substances are briefly but critically reviewed.     

大鼠原代小胶质细胞的分离和培养方法的改进

目的:探索改进大鼠原代小胶质细胞培养分离纯化方法,以获取数量多、功能活性稳定的小胶质细胞。方法:在Miriam Mecha的混合胶质细胞培养方法的基础上稍加改良,采用直立手摇法进行分离纯化小胶质细胞,细胞铺板后再采用差速贴壁法进一步得到更纯的小胶质细胞。使用台盼蓝法进行细胞计数;采用CD11b免疫荧光方法对纯化的小胶质细胞进行纯度鉴定;采用原代小胶质细胞吞碳粒实验对其功能活性进行检测。结果:改进的方法可以稳定地获取大约1×106个/培养瓶(75 cm2,10 ml)的小胶质细胞,纯度达到98%,并且其吞噬活性也非常强。结论:改进的原代小胶质细胞培养方法操作时间短,获得细胞数量多、稳定性好、纯度高、功能活性强,且可第二次收获细胞,对于离体条件下进行小胶质细胞的研究具有广泛的实用价值。     

A technique for improved blood sampling during sleep studies

Research protocols often require that blood samples be drawn during sleep. This study compares the efficacy of obtaining nocturnal blood samples using a standard heparinized intravenous setup versus the same intravenous setup used in conjunction with a small chemical heating pad. The chemical heating pad significantly improved the number of blood samples obtained and the maintenance of intravenous patency. The use of a chemical heating pad is an economical way to resolve the frustration of lost blood samples while maintaining a reasonable environment to monitor sleep.     

An improved autoradiographic technique for the detection of antibody-forming cells

An autoradiographic technique for the detection of antibody-forming cells has been developed for the assay of anti-DNP responses. The lymphoid cells suspension to be assayed was allowed to sediment on to a glass slide coated with DNP-conjugated gelatin to which the secreted antibody bound during subsequent incubation. The bound antibody and its Ig class was revealed by a second incubation using ¹²⁵I-anti-immunoglobulin reagents followed by autoradiography. Studies on the sensitivity and specificity of the method are presented and its advantages over other techniques described. The technique should be readily applicable to other haptens.     

An improved technique for monitoring the drinking behavior of mice

A set of calibrated lickometers provides continuous, quantitative monitoring of fluid consumption. It has been used in our laboratory at four levels of temporal resolution: 24 hr, 1 hr, 6 min, and for counting of individual licks. Convenient features are mounting of the licking tube-bottle assembly on the cage top (which permits the use of disposable plastic cages with litter) and automated collection of data with microcomputers.     

An improved plasma clot technique for initiating mammalian fibroblast cultures

Summary A modified plasma clot technique is described, which has been used with a high degree of success in initiating fibroblast cultures from tissues of human, cat, and dog origin. The technique offered real advantages in terms of production of healthy rapidly growing cultures, although human skin biopsies proved to take significantly longer to explant than cat or dog biopsies. There was no difference in explant time when cat biopsies were of skin or connective tissue origin. The skin from a series of insulin dependent (juvenile onset) diabetics explanted as rapidly as a series of normal biopsies.     

An improved method for producing an antiserum against T-lymphocytes of the mouse

C-reactive protein (CRP) is an acute-phase reactant the concentration of which increases significantly following tissue injury or inflammation. I report here the development of a rapid and sensitive solid-phase enzyme immunoassay (EIA) for determination of serum CRP. The inclusion of 4% (w/v) polyethylene glycol (PEG) in the diluent buffers made it possible to quantitate CRP within 4 h at room temperature. The minimum detectable amount was about 500 pg CRP/ml. Excellent correlation in determination of serum CRP was found between solid-phase EIA and nephelometric assay, radial immunodiffusion and the qualitative latex agglutination tests.     

Alprenolol fails to antagonize the metabolic changes following repeated thyroxine injections in the rat

Repeated injections of rat with 1-thyroxine (50 μg/kg daily for 5 five-day weeks) retarded the weight gain of the animals and increased the absolute and relative size of the heart, adrenals and interscapular brown adipose tissue. In the myocardium and thigh muscle, thyroxine treatment resulted in elevated activity of oxidative enzymes, succinate dehydrogenase, malate dehydrogenase and citrate synthase, while the activities of glycolytic enzymes remained unchanged. Glycogen content of the heart was decreased following thyroxine regime. In the brown fat, on the other hand, thyroxine injections resulted in a reduction of the activity of oxidative enzymes. This reduction can be accounted for by the decreased protein (enzyme) content of the tissue due to deposition of fat. Furthermore, thyroxine treatment delayed the body cooling of the rats swimming in water at 25^oC and enhanced hyperthermic response to injected noradrenaline. All these changes, which were not observable in rats treated with daily alprenolol (20 mg/kg) injections, were as pronounced in rats injected with alprenolol together with thyroxine as in rats injected with thyroxine only. It is concluded that beta blockers do not antagonize the metabolic changes due to hyperthyroidism.     

An experimental influenza subunit vaccine (iscom): induction of protective immunity to challenge infection in mice after intranasal or subcutaneous administration.

An experimental influenza virus (A/PR/8/34(H1N1] vaccine was tested and evaluated in mice. The mice were inoculated once or twice intranasally or subcutaneously with 1 or 10 micrograms of iscoms prior to challenge with high dose of live virus. It was demonstrated that two intranasal administrations were as efficient as two s.c. administrations, both routes inducing high levels of antibody and protection against challenge infection. With a one-dose regimen, the s.c. route induced a somewhat higher antibody response than the intranasal route; this might be explained by technical difficulties connected with an intranasal administration.     

Monoclonal antibodies specific for glial and neuronal antigens in the young rat cerebellum

Monoclonal antibodies from 15 different hybridomas derived from the fusion of mouse spleen cells with a myeloma cell line were selected. Mice were immunized with the particulate fraction from 10-13-day-old rat cerebella. Hybridoma secreting antibodies were screened simultaneously by both immunocytochemistry and binding assay. Each antibody reacts with specific cerebellar neuronal or glial cells and structures.     

An improved method for the determination of renin in rat kidneys

Summary An enzyme-kinetic method for the determination of renin activity in rat kidneys is described. Substrate (angiotensinogen) was prepared from plasma of rats nephrectomized 24 h previously. Under the existing conditions, the angiotensin formed during incubation was not inactivated by peptidases, and added angiotensin II-amide was completely recovered. The optimum pH for the reaction of renin extracted from rat kidneys with homologous plasma substrate was found to be 7.2 For the rate of angiotensin formation, aK _m of 2,400 ng angiotensin per ml was determined at pH 7.4 and 37°C. The renin activity in kidneys of normal adult rats corresponded to about 10 Goldblatt units of hog renin (NBC) under the same conditions of incubation.     

An immunochemical procedure to evaluate the degree of desialylation of alpha 1-acid glycoprotein in rat serum

When radial immunodiffusion (RID) and electroimmunodiffusion (EID) were used for the determination of rat alpha 1-acid glycoprotein (alpha 1-AGP) a significant discrepancy in the results was encountered depending on the degree of sialylation. When alpha 1-AGP was desialylated, the amounts estimated by EID were much lower than those actually present as assayed by the RID method. The relationship between the percentage of desialylation of alpha 1-AGP and the percentage of its underestimation by EID relative to RID was determined and a calibration curve was plotted to evaluate the degree of desialylation of rat alpha 1-AGP. When compared to other procedures (rat membrane inhibition assay and isoelectrofocusing), the proposed method was easier to perform and allowed the specific evaluation of the degree of undersialylation of the glycoprotein.     

An improved method for chronic catheterization of the rat spinal subarachnoid space

A method for chronic catheterization of the rat spinal subarachnoid space is described. The catheters are simple to make and the catheterization procedure circumvents many of the problems associated with earlier published technics.     

植入前遗传学诊断中卵裂球固定技术改进

目的改进植入前遗传学诊断卵裂球固定技术,减少卵裂球细胞丢失,获得更好的固定效率、更高的荧光原位杂交成功率,简化操作程序.方法体外授精治疗周期废弃胚胎的96个卵裂球细胞,分别用3种不同的固定技术进行固定,固定后使用X、Y着丝粒探针进行荧光原位杂交,比较其固定效率和杂交效率.结果改进方法固定率和杂交率均为100%.而两种传统技术的固定率分别为90%,83.3%;杂交率为80%,73.3%.结论这种新的固定卵裂球技术从根本上消除了卵裂球标本的丢失,使PGD结果更为可靠;同时简化了操作程序,值得推广.