Use of precision-cut rat liver slices for studies of xenobiotic metabolism and toxicity: comparison of the Krumdieck and Brendel tissue slicers

1. In this study we have compared freshly cut and cultured precision-cut rat liver slices produced by the Krumdieck and Brendel-Vitron tissue slicers. 2. No significant differences were observed in levels of protein, potassium, total glutathione (i.e. GSH and GSSG), reduced glutathione (GSH) and cytochrome P450 and activities of 7-ethoxyresorufin O-deethylase and 7-benzoxyresorufin O-debenzylase in freshly cut rat liver slices produced by the two tissue slicers. However, levels of oxidized glutathione (GSSG) were significantly greater in liver slices produced with the Brendel-Vitron tissue slicer. 3. Precision-cut rat liver slices produced with both tissue slicers were cultured for 0 (i.e. a 1-h preincubation), 24 and 72 h in a dynamic organ culture system in an atmosphere of either 95% 02/5% CO2 or 95% air/5% CO2. 4. Apart from small differences in glutathione levels in 0 and 24 h cultured liver slices, no significant differences were observed in the parameters measured between liver slices prepared with both tissue slicers and cultured in both gas phases. 5. With liver slices produced by both tissue slicers 50 microM sodium arsenite produced a greater induction of heat shock protein 70 levels in slices cultured for 24 h in a high oxygen than in an air atmosphere. 6. These results suggest that both tissue slicers can readily produce precision-cut liver slices for studies of xenobiotic metabolism and toxicity. However, the data suggest that for any given application of precision-cut tissue slices it is desirable to establish optimal culture conditions     

Pulmonary tuberculosis in Harare, Zimbabwe: analysis by spoligotyping

BACKGROUND: Over the last 10 years there has been a fourfold increase in cases of tuberculosis in Harare, Zimbabwe. The use of molecular epidemiology to understand tuberculosis transmission in this epidemic has been hampered by the availability of suitable culture facilities. A study was therefore undertaken to explore the potential of spoligotyping, a polymerase chain reaction based technique that does not require tuberculosis culture. METHODS: Adults attending a chest clinic with clinical or radiological pulmonary tuberculosis and one smear positive sputum were enrolled over one month. Demographic, socioeconomic, and clinical data were gathered using a standardised questionnaire. Molecular fingerprinting of genomic DNA recovered from sputum was performed by spoligotyping. RESULTS: Sixty one subjects (median age 28 years (range 18-73); 61% men) were recruited and 57 provided adequate sputum samples. Recent rural-urban migration or immigration was not common; 40% of subjects lived in crowded living conditions. DNA suitable for spoligotyping was recovered from 28 patients and 20 different genotypes of Mycobacterium tuberculosis were identified. Fifteen patients were infected with an M tuberculosis strain shared by one or more individuals. Patients infected with a shared spoligotype were not closely linked geographically within Harare, but were more likely to live in overcrowded conditions (69% versus 23%; odds ratio 6.85 (95% CI 1.2 to 47), p = 0.026). Analysis of the patients' original rural family homes revealed two geographically related spoligotype clusters. CONCLUSIONS: Spoligotyping may yield valuable molecular typing information in populations where tuberculosis culture is not available. This novel technique requires further development and evaluation in larger epidemiological studies.     

Power Doppler ultrasound evaluation of the shear rate and shear stress dependences of red blood cell aggregation

The use of power Doppler ultrasound at 10 MHz is evaluated as a method to study the shear rate and the shear stress dependences of red blood cell aggregation. This evaluation was based on six in vitro experiments conducted in a 1.27-cm diameter tube under steady flow conditions. Porcine whole blood was circulated in the flow model at flow rates ranging between 125 to 1500 ml/min (mean shear rate across the tube ranging between 6 and 74 s-1). For each flow condition, the variation of the Doppler power across the tube and the velocity profile were measured by moving the Doppler sample volume across the tube diameter. For each radial position, the shear rate within the Doppler sample volume was also determined by considering the radial power pattern of the ultrasound beam. To estimate the shear stress within the Doppler sample volume, the apparent viscosity of blood samples withdrawn from the flow model was measured for each experiment. The variation of the Doppler power as a function of the shear rate within the sample volume showed a rapid reduction of the power between 1 and 5 s-1, a transition region between 5 and 10 s-1, and a very slow reduction beyond 10 s-1. Little variation of the Doppler power was measured for shear stress higher than 2 dyn/cm2. The maximum Doppler power for all flow rates was usually found near the center of the tube. Based on the ultrasonic scattering models, which predict that the Doppler power is related to the volume square of the scatterers, the method described in the present study showed a very high sensitivity to the presence of red blood cell aggregation for shear rates below 10 s-1.     

Design and synthesis of novel conformationally restricted HIV protease inhibitors

A set of HIV protease inhibitors represented by compound 2 has previously been described. Structural and conformational analysis of this compound suggested that conformational restriction of the P1/P2 portion of the molecule could lead to a novel set of potent protease inhibitors. Thus, probe compounds 3-7 were designed, synthesized, and found to be potent inhibitors of HIV protease.     

Development of the quality of life in epilepsy inventory

We developed an instrument to measure health-related quality of life (HRQOL) in epilepsy. A 99-item inventory was constructed from the RAND 36-Item Health Survey (generic core), with 9 additional generic items, 48 epilepsy-targeted items, and 6 other items concerning attitudes toward epilepsy and self-esteem. We administered the 99-item inventory to 304 adults with epilepsy at 25 epilepsy centers. Patients and patient-designated proxies completed the inventory and were retested 1-91 days later. A multitrait scaling analysis of these data led to retention of 86 items distributed in 17 multiitem scales (Cronbach's alpha ranged from 0.78 to 0.92). Factor analysis of the 17 multiitem scales yielded four underlying dimensions of health: an epilepsy-targeted dimension, a cognitive factor, mental health, and physical health. Construct validity was supported by significant patient-proxy correlations for all scales and correlations between neuropsychologic tests and self-reported emotional and cognitive function (all p values < 0.05). There were significant negative correlations between the four factor scores derived from the HRQOL scales and neurotoxicity, systemic toxicity, and health care utilization (except for the correlation between mental health factor and health care utilization; all p values < 0.05). Patients who were seizure-free in the preceding year reported better HRQOL for the overall score, three of the four factor scores, and 8 of the 17 scale scores than did patients with a high frequency of seizures. Relative validity analysis showed that the epilepsy-targeted factor and three of its four component scales were more sensitive to categorization of patients by severity of seizure frequency and type than scales tapping physical health, mental health, or cognitive function. These cross-sectional data support the reliability and validity of this measure of HRQOL in epilepsy. The addition of an epilepsy-targeted supplement to the generic core improved the sensitivity to severity of epilepsy. The 86 items included in the field testing were supplemented by three additional items to form the Quality of Life in Epilepsy (QOLIE-89) inventory.     

Rapidly progressive herpetic retinal necrosis: a blinding disease characteristic of advanced AIDS

Eleven patients with rapidly progressive herpetic retinal necrosis (RPHRN) complicating AIDS were investigated retrospectively to study the disease spectrum, systemic involvement, and therapy. The mean CD4 cell count was 24/microL. There was a characteristic disease pattern with rapid progression, 82% bilaterality, relative resistance to intravenous antiviral therapy, and 70% retinal detachment. Varicella-zoster virus was the probable cause in 10 patients (detected by polymerase chain reaction in two eyes investigated), and herpes simplex virus was the probable cause in one. Cutaneous zoster occurred previously in 73% but was not concurrent. Seventy-three percent had central nervous system disease, possibly virus-related. RPHRN may be a local herpetic recrudescence in an immune-privileged site with transneural spread. Only four of 20 affected eyes retained useful vision. Poor ocular bioavailability, retinal ischemia, acquired drug resistance, and strain pathogenicity may underlie treatment failure. Acyclovir therapy appears relatively ineffective. Combined intravenous and intravitreal therapy with foscarnet and ganciclovir may be the best current management. Research advances are needed urgently.