A fast and simple in situ PCR method to detect human papilloma virus infection in archival paraffin-embedded tissues

We describe a simple and fast method for the detection and localization of low copy numbers of HPV DNA in formalin-fixed and paraffin-embedded archival tissues. We have developed a protocol for direct bl situ-PCR in order to demonstrate its convenience in rapid and reproducible assessment of HPV infection in unknown biopsies. The morphological aspect of the tissues has been maintained, despite the multiple steps of fixation, permeabilization and thermal cycling, and positivity has been detected only in virus target cells.     

Tumor-necrosis-factor and DNA topoisomerase-ii inhibitors in human ovarian-cancer - potential role in chemotherapy

Seven ovarian and one cervical human cancer cell lines were examined far their sensitivity or resistance to tumor necrosis factor, to three topoisomerase II inhibitors and to cisplatin. Only one line exhibited the multidrug-resistance phenotype and another one an 'atypical'-MDR phenotype. The combination of TNF and topoisomerase-II inhibitors produced enhanced cytotoxicity and overcame the MDR and the atypical resistance. No potentiation of cisplatin cytotoxicity was observed. These findings suggest that TNF enhances the activity of DNA topoisomerase II both in TNF resistant and sensitive cells.     

Genome scanning using endogenous LTR-like elements for rapid DNA-fingerprinting of breast-cancer and transformed human breast epithelial-cells

In the present work genomic DNAs from nine primary breast cancers and transformed human breast epithelial cell lines obtained by treatment of MCF 10F, a spontaneously immortalized human breast epithelial cell line, with benzo(a)pyrene (BP) or 7,12 dimethyl benz(a)anthracene (DMBA), were used for genomic scanning. The treatment of MCF 10F with BP gave rise to different clones designated BP1 and BP1E, the latter being a tumorigenic cell line. Treatment with DMBA gave rise to D3 and D3-1 clones. The clones D3-1 and BP1 have been transfected with the plasmid pH06T1 containing the mutated c-Ha-ras oncogene resulting in the D3-1Tras and BP1T-ras cell lines, that are highly tumorigenic in SCID mice. Genomic DNA are separately hybridized to two different probes representing different families of human endogenous retrovirus like sequences (RTLV-H and HERV-K LTR). The technique of genomic scanning allows the mapping of each tumor or cell line and comparison with its counterpart obtained from the adjacent normal tissue of the same patient or with the untreated MCF 10F cells. DNA changes such as deletions, amplifications and/or rearrangements were detected in 5 of the tumor pairs studied. We have identified genomic alterations that involved amplification of a 10 kb band in the transformed cell lines. The cell lines D3-1Tras and BP1T-ras show, in addition, the presence of a second band of 4.5 kb in size. A third band of 500 bp size was found in clones D3-1 and BP1E that have a more aggressive behavior in vitro than their precursors D3 and BP1 cells respectively. In conclusion the present report indicates that genomic scanning detects DNA aberrations in primary primary tumors and in human breast epithelial cells transformed with chemical carcinogens and/or oncogene transfection that are not present in their normal counterpart. These results further indicate that detection of endogenous retrovirus elements may help in genome mapping and can be a useful tool for detecting genomic changes in the preliminary screening of DNA extracted from primary breast cancer and transformed cells.     

Pharmacokinetics of thiopental after single and multiple intravenous doses in critical care patients

Thiopental was administered to neurosurgical patients for cerebral protection and its pharmacokinetic parameters were determined after a single bolus of 540, 1000 or 1500 mg (3 subjects) or after multiple doses of 250 mg (5 subjects) and 500 mg (2 subjects) every two hours for up to 7 days. The data were analysed by a two- or three- compartment model and linear kinetics. After a single IV bolus, the mean initial volume of distribution (V1) was 0.4811·kg^–1, and the steady-state volume of distribution (V_ss) was 2.16 1·kg^–1. The distribution (t_1/2) and elimination (t_1/2) half-lives were 0.590 and 5.89 h, respectively, and the mean residence time (MRT) was 7.44 h. The clearance was 5.41 ml·min^–1·kg^–1. With repeated injections, the pharmacokinetic parameters for each patient were estimated taking into account all administered doses and blood samples, which were taken whenever possible daily at steady state and after the last dose. The variability observed in the pharmacokinetic parameters of thiopental reflected by the coefficient of variation (CV%) was wide but was of similar magnitude within patients (CV_intra) as it was between patients (CV_inter). The steady-state trough plasma concentration (C_min obs) ranged from 4.8 to 30 mg·1^–1 (mean 16.0 mg·1^–1 and median 14.3 mg·1^–1). Peak concentrations (C_max obs) ranged from 8.35 to 45 mg·1^–1 (25.4 mg·1^–1, and median 23.3 mg·1^–1). The values of V1 and V_ss were similar to those obtained after a single dose. For V1, the mean was 0.333 1·kg^–1. The mean V_ss was 2.68 1·kg^–1, with a CV_intra of 12.6 to 56% and a CV_inter of 13.2%. A shorter distribution half-life t_1/2 was noted on multiple dosing; the mean value was 0.122 h. The elimination half-life t_1/2 and the mean residence time became longer due to a decrease in clearance. For t_1/2 the mean value was 16.3 h. The mean MRT was 21.9 h, CV_intra 9.19 to 48.5%, and the CV_inter 35.3%. The mean clearance was 2.16 ml·min^–1·kg^–1, CV_intra 7.28 to 25.5%, and the CV_inter 20.4%. This value is 50% lower than after a single dose.Identification of the kinetic parameters of thiopental allows simulation of the effects of doses on subsequent plasma levels and will permit a priori prediction of day to day adjustment of drug dosage.     

Plasma levels of antioxidant vitamins and cholesterol in a large population sample in Central-Northern Italy

Background: In a population-based multicenter case-control study of diet, life-style, and gastric cancer a large series of adults, aged 30–75 years (mean 58.9 yearss), were randomly sampled from the general population in 3 areas of Central-Northern Italy. Aim of the study: To evaluate the relationship between plasma levels of antioxidant vitamins and cholesterol, and sociodemographic characteristics, life-style factors, and dietary intake of selected nutrients in a sample of the Italian population. Methods: A fasting blood sample was available for 945 subjects (553 men, 392 women). The plasma concentrations of ascorbic acid, carotene, retinol, alpha-tocopherol, and cholesterol were determined by a centralized laboratory. All participants answered to a detailed questionnaire collecting information on sociodemographic and anthropometric characteristics, smoking, alcohol drinking, and dietary habits. Covariance analysis models, with post hoc Dunnett tests, including terms for age, sex, study center, and period of blood drawing, were used for selected multiple-way comparisons of mean values of plasma nutrients. Results: Mean plasma values of retinol were higher among men while women had higher levels of plasma carotene, ascorbic acid, alpha-tocopherol, and cholesterol. Plasma carotene levels showed an inverse association with body mass index, alcohol consumption, and smoking and a positive association with social class. Carotene concentrations were higher in plasma samples obtained in spring/summer, while ascorbic acid levels were higher in autumn/winter. Partial correlation coefficients between carotene and ascorbic acid (0.69 in men; 0.74 in women), between carotene and alpha-tocopherol (0.44; 0.37), and between alpha-tocopherol and ascorbic acid (0.45; 0.41). Plasma alpha-tocopherol and retinol correlated with plasma cholesterol. On the other hand, plasma carotene and ascorbic acid were correlated with their estimated dietary intakes, while the intakes of other nutrients, as expected, correlated rather poorly with the respective plasma concentrations. Conclusions: Socio-economic factors, life-style, and specific nutrient intake, in addition to gender, are related to nutrient plasma levels in Italian adults and may provide specific suggestions for the prevention of chronic diseases. Received: 8 December 1998, Accepted: 25 February 1999     

The ``LAP-LOOP'

The "LAP-LOOP," a device used to avoid the fourth trocar in the laparoscopic cholecystectomy, has been successfully applied by the author over more than 200 patients, even in the cases of acute cholecystitis.     

Sex Differences in Hemisphere Preference As Assessed by a Paper-and-Pencil Test

The present study examined sex differences in hemisphere preference (HP) assessed by the Preference Test (PT). This instrument is designed to measure the extent to which normal subjects rely on right-hemisphere or left-hemisphere cognition. Factor analysis on the total sample (N=1,057; 473 men and 584 women) revealed a clear two-factor structure (i.e., left-HP and right-HP), although separate analyses for men and women suggest that this structure is more straightforward in men than in women. The main differences between men and women have to do with PT items relevant to language abilities, where women tend to be more symmetrically distributed across the two factors. However, the frequency of right- and left-HP is similar in men and women and does not change for men when PT scores are recalculated after removal of unspecific items. Furthermore, once the items that assess verbal abilities were excluded, the corrected PT value for women showed higher right-HP. Our results provide some indications of a less pronounced lateralization of hemisphere-linked cognitive abilities in women.     

Interferon-gamma enhances TNF sensitivity in A172 human glioblastoma cell line

Although human glioblastoma cell line A172 presents tumor necrosis factor (TNF) high affinity receptors, it is resistant to TNF-mediated cytotoxicity. Preincubation of cells with 10 ng/ml interferon (IFN)-gamma for 6 h causes a great increase in TNF receptor numbers (about 477%). IFN-gamma alone is not cytotoxic in A172 cells. Cells preincubated with 10 ng/ml IFN-gamma for 6 h became more sensitive to the TNF cytotoxicity (about 400%). In A172 cells the enhanced TNF receptor expression may contribute to the enhancement of TNF cytotoxicity. This observation may have importance in designing new more effective therapies for human gliomas.     

High-dose chemotherapy and autologous bone marrow rescue for patients with refractory germ cell tumors. Early intervention is better tolerated.

Therapy with high-dose carboplatin plus etoposide-based chemotherapy plus autologous bone marrow rescue (AUBMR) was administered to 29 patients with advanced germ cell tumors (GCT) refractory to cisplatin-based chemotherapy. Two groups of patients with refractory disease were treated. Sixteen patients had been identified as "poor risk" at diagnosis and had an inappropriately slow decline of serum tumor markers after two cycles of induction cisplatin-based therapy (Group A). In addition, 13 patients were treated who had never had a complete response (CR) or had relapses after ifosfamide-based salvage chemotherapy (Group B). Patients in Group A were treated with high-dose carboplatin etoposide, and patients in Group B received high-dose carboplatin, etoposide, and cyclophosphamide. Fifteen of 29 (52%) patients had a CR (9, Group A; 6, Group B). The patients in Group A had fewer hematologic toxic effects, and the median number of days from day 0 to a granulocyte count greater than 0.5/microliters was 16 and to a platelet count of more than 50/microliters was 15, compared with 22 days and 23 days in Group B, respectively. There were fewer episodes of culture-positive sepsis in Group A (12%) compared with Group B (26%), and the only treatment-related death occurred in Group B. Therapy with high-dose carboplatin plus etoposide-based chemotherapy plus AUBMR is effective for patients with GCT refractory to regimens of cisplatin with or without ifosfamide. Early use of high-dose chemotherapy reduces hematologic toxic effects and allows patients to start treatment in a more predictable fashion after cytoreduction, rather than when the disease is progressing rapidly.     

Effects of ageing on the content in sulfur-containing amino acids in rat brain

Summary Concentrations of the sulfur-containing amino acids methionine, homocysteic acid, cysteic acid and taurine were measured in brain structures of young and old Wistar rats in an attempt to etablish a possible link between the increase in oxidative stress with ageing and changes in tissue levels of these amino acids. Contrary to data reported by others, in all brain structures of young and old rats homocysteic acid levels could not be quantified. Compared with young rats, in old animals taurine and methionine concentrations significantly decreased in striatum and cortex; decreased taurine levels were also found in nucleus accumbens and cerebellum and lower concentrations of methionine were found in midbrain, hippocampus and pons-medulla. Cysteic acid levels either did not change or significantly increased in cortex and hippocampus. These results are discussed taking into account the biosynthesis of sulfur-containing amino acids in rat brain and the decrease in glutathione in relation to oxidative stress with ageing.Changes in aspartic acid, glutamic acid, serine, glutamine, glycine and GABA concentrations with ageing were also determined in the same brain structures and were in good agreement with those previously reportedAbbreviations H ₂O₂ hydrogen peroxide - MAO monoamine oxidase - GSHP glutathione peroxidase - PAPS 3-phosphoadenosine-5-phosphosulfate - OPA O-phthaldialdehyde - HPLC high performance liquid chromatography - Asp aspartic acid - CA cysteic acid - CSA cysteine sulfinic acid - Cys cysteine - GABA -aminobutyric acid - Gln glutamine - Glu glutamic acid - Gly glycine - HCA homocysteic acid - Met methionine - Ser serine - Tau taurine