Model system for isolation of competent ovarian-carcinoma cells from fresh tumor-tissue by a magnetic separation technique (macs)

Flow cytometric analysis of tumor cells in carcinomas is hampered by the presence of a variety of different cells in the tumor tissue and the surrounding stroma. To obtain single competent tumor cells, we have established a model system which can be applied to separate living cells from fresh ovarian carcinoma tissue. Due to the lack of tumor-cell surface specific antibodies, we isolated tumor cells by a procedure called 'negative tumor cell selection'. For this purpose, fresh ovarian carcinoma tissue, immediately after surgery, was subjected to mechanical disintegration using an automated mincing device to obtain a single-cell suspension (approximately 10(7) cells/g). Collagenase D (0.005%) was added to prevent further aggregation. Cells other than tumor cells were then labeled with a set of monoclonal antibodies directed to cell surface antigens: CD3 (T-cells), CD14 (monocytes), CD15 (granulocytes), CD45R (T-/B-cells) and 5B5 (fibroblasts). Anti-isotype antibodies coupled to ferrit microbeads were then reacted with the cell suspension and those cells reacting with the microbeads retained on a steel wool matrix in a magnetic field (1). Tumor cells not reacting with the microbeads were recovered by a simple wash of the steel wool matrix. All incubation steps were at 4 degrees C. This procedure, which takes about 2 hours, enables fast and simple isolation of single, living competent tumor cells from fresh tumor tissue and also from ascitic or pleuritic effusions. In a model system with cultured ovarian carcinoma cells and human leukocytes, tumor cell purity was about 93% and about 97% when re-subjected to the same procedure (respective recovery rates 75% and 50%). The still unlabeled tumor cells can subsequently be analyzed by flow cytometry or by central laser scanning microscopy for the presence of various surface antigens including receptors for proteases or growth factors. Moreover, after detergent treatment and fixation, flow cytometric multiparameter analysis such as simultaneous labeling of intracellular and surface antigens as well as nuclear DNA staining for ploidy and S-phase determination becomes possible.     

Triple approach for diagnosis and grading of meningiomas: Histology,morphometry of Ki-67/feulgen stainings,and cytogenetics

Summary With regard to meningioma grading and the recently introduced atypical meningioma, we evaluated 160 cases retrospectively by conventional histology and image analysis. For that, the cell nuclei were stained with a Ki-67 (MIB1)/Feulgen-method on paraffin sections, thus enabling the assessment of both the Ki-67 proliferation index and nuclear morphometric features, such as tumour cell arrangement, nuclear pleomorphism, and cellularity.It could be demonstrated that the Ki-67 proliferation index is the most important criterion for distinguishing anaplastic meningiomas (WHO grade III) (mean Ki-67 index: 11%) from those of common type (WHO grade I) (mean Ki-67 index: 0.7%). The parameter for the relative volume weighted mean nuclear volume is another valuable morphometric feature. The atypical meningioma (WHO grade II) which should represent an intermediate category between common type and anaplastic meningiomas is characterized by a mean Ki-67 proliferation index of 2.1%. Common type meningiomas which comprise almost 50% of the cases of this series have a relapse rate of 9%. Atypical and anaplastic meningiomas recurred in 29% and 50%, respectively. Since the term atypical meningioma is confusing in the context of tumour grading, the term intermediate type meningioma is proposed.Furthermore, the results of cytogenetic analyses of 142 cases of this series were evaluated and compared with the meningioma grades. Thereby, 25 cases disclosed, independent of the typical loss of one chromosome 22, cytogenetic features assumed to be progression-associated, e.g., the gain or loss of different chromosomes and the deletion of the short arm of one chromosome 1 (hyperdiploidy, increased hypodiploidy, Ip-), when correlated to the histological and morphometric findings or the high relapse rate.For meningioma diagnosis and grading, a practical guideline is proposed based upon histology, morphometry (Ki-67), and cytogenetics.     

The effect of CO2 laser laparoscopy for treatment of pelvic endometriosis on serum concentrations of antiendometrial antibodies

This study uses antiendometrial antibodies as an indicator for follow-up of patients with endometriosis after surgical treatment. This is a prospective study of 32 patients with endometriosis. The patients were used as their own controls. Serum samples were obtained before surgery and 1 month, 3 months, and 6-9 months following surgery. All patients were treated with CO2 laser laparoscopy. Serum concentrations of antiendometrial antibodies decreased significantly at 1 month, 3 months, and 6-9 months following treatment in each group. There was no correlation between baseline concentration of antibodies and stage of the disease. This study confirms the value of using antiendometrial antibodies as an indicator for follow-up of endometriosis patients. Surgical treatment with CO2 laser laparoscopy is effective in lowering the antigenic effect of the disease in these patients. This beneficial effect is maintained for up to 9 months after treatment.     

Guillain-Barré syndrome as differential diagnosis of intervertebral disk-induced nerve root compression

INTRODUCTION: Diagnosis of Guillian-Barré Syndrome usually is not difficult, but diagnostic failure occurs for the variable initial presentation. Diagnosis is based on physical examination showing loss of motor strength in more than one limb and loss of deep tendon reflexes. Ventilatory assistance, pharmacologic maintenance of cardiovascular homeostasis, corticosteroids, IgG and plasma exchange are the dominant therapeutic measures. CASE: This article reports on a case of a 59-year old surgeon suffering from degenerative disc disease in the lumbar spine. The patient developed a severe course of the Guillian-Barré Syndrome with persisting motor weakness of the legs. CONCLUSION: If the primary problem at presentation is limb and back pain the pathology appears to be in the musculoskeletal rather than in neurological system. The awareness of this presentation of Guillian-Barré-Syndrome will eliminate delay in diagnosis.     

Household budget survey nutritional data in relation to mortality from coronary heart disease, colorectal cancer and female breast cancer in European countries. DAFNE I and II projects of the European Commission. Data Food Networking.

OBJECTIVE: We have undertaken a study to examine whether Household Budget Survey (HBS)-derived nutritional patterns are related to mortality from diseases with strong nutritional components, namely coronary heart disease, colorectal cancer and cancer of the female breast. DESIGN: Ecological correlation study. In the context of the Data Food Networking projects of the European Union, raw data from the national HBS of 10 European countries were provided. For each of the 10 participating countries, daily food availability per capita around 1990 was calculated. Individual foods were aggregated into 12 major food groups that were linked with the diseases under consideration. Mortality data were available from a World Health Organisation database. We have used a composite score to summarise the postulated influence of diet. SETTING: Ten European countries circa 1990. RESULTS: The correlation coefficients between the composite score and the age-adjusted mortality from each of the studied diseases were: + 0.51 (P approximately 0.14) for colorectal cancer; +0.72 (P approximately 0.02) for female breast cancer; and +0.60 (P approximately 0.07) for coronary heart disease, after adjustment for tobacco smoking. CONCLUSIONS: We conclude that dietary information from HBS is sufficiently reliable to reveal correlations with mortality rates from chronic diseases with fairly strong nutritional roots. HBS data could be used, with little cost, for monitoring secular trends in dietary patterns with a view to their health implications.     

Interspecies pharmacokinetic comparisons and allometric scaling of napsagatran, a low molecular weight thrombin inhibitor

The objective of this work was to assess the pharmacokinetics of napsagatran, a low molecular weight thrombin inhibitor, after intravenous administration in a variety of laboratory animals, and prospectively to help design the first pharmacokinetic studies in man. Napsagatran is actively excreted into the bile and urine of various species and pronounced species-differences in its pharmacokinetics are observed. It is, therefore, an interesting compound to use in tests of the limitations of presently available inter-species scaling methods. The present data suggest that allometric exponent values which are consistent with the values expected for physiological processes and small organic molecules are not necessarily associated with successful predictions in man when active transport processes are involved in the disposition of the compounds. For example, compared with the values observed in man, the clearance (CL), non-renal clearance (CL(nr)) and the volume of distribution at steady state (Vd(ss)) were over-predicted by 3-, 7- and 2-fold, respectively, by use of allometry. Of the species tested, the cynomolgus monkey seemed to be the most useful for predicting kinetics in man when the approach based on concentration-time transformations was used. Thus, for half-life (t(1/2)), CL and Vd(ss), the observed mean values of 1.7 h, 459 mL min(-1) and 24 L kg(-1) in man were very close to the values predicted from the cynomolgus monkey (1.7 h, 652 mL min(-1) and 22 L kg(-1), respectively). The results show that there are large inter-species differences for kidney and liver excretion of napsagatran. This is probably because of the involvement of active transport processes, which compromised the kinetic extrapolation from animal to man, although a more thorough investigation of the transporters involved in the disposition of napsagatran is necessary to enable better understanding of the species differences observed.     

Regulation of substantia nigra pars reticulata neuronal activity by excitatory amino acids

Midbrain non-dopaminergic neurons of the substantia nigra pars reticulata play an important role in the basal ganglia circuitry. The regulation of their electrical activity by excitatory amino acid (EAA) inputs was investigated using in vivo electrophysiological methods in chloral hydrate-anaesthetized rats. We first determined the subtypes of EAA receptors present on reticulata neurons, using microiontophoretic application of selective agonists: kainic acid (KA), (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), N-methyl-D-aspartic acid (NMDA), and trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD). Each agonist activated reticulata neurons and the apparent rank order of efficacy was: KA> or =AMPA=NMDA>trans-ACPD. Using pressure or iontophoretic microejections of ionotropic and metabotropic receptor antagonists, we then investigated EAA receptor subtypes involved in the spontaneous firing rate of reticulata neurons. Kynurenic acid and (+/-)-2-amino-5-phosphonopentanoic acid (AP-5) markedly decreased the spontaneous firing rate of reticulata neurons, while 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) was much less effective. The metabotropic receptor antagonist (R,S)-alpha-methyl-4-carboxyphenylglycine (MCPG) failed to affect the spontaneous electrical activity. In contrast to CNQX, microapplications of AP-5 sometimes produced total inhibition. This powerful effect may reflect the potential importance of NMDA receptors in regulating the activity of some reticulata neurons. These results indicate that both functional ionotropic (NMDA and non-NMDA) and metabotropic EAA receptors are present on non-dopaminergic substantia nigra pars reticulata neurons. Moreover, in the anaesthetized animal, the spontaneous firing rate of these neurons, mediated by EAA inputs, seems mainly due to the tonic activation of ionotropic, but not metabotropic, receptors.     

Multiple-dose pharmacokinetics, safety, and tolerability of bosentan, an endothelin receptor antagonist, in healthy male volunteers.

The multiple-dose pharmacokinetics, safety, and tolerability of oral bosentan, a selective endothelin receptor antagonist, were investigated in healthy male volunteers. In study A, an ascending-dose, double-blind, placebo-controlled trial, doses of 100, 200, 500, and 1000 mg bosentan or placebo were given once daily for 8 days as tablets (100 and 500 mg dose strength). In study B, a double-blind, placebo-controlled trial, 500 mg tablets of bosentan or placebo tablets were given once daily for 8 days with two additional single intravenous dose administrations of 250 mg bosentan 48 hours before the first and 24 hours after the last oral dose. The drug was very well tolerated. No effects on pulse rate, ECGs, or clinical laboratory tests were observed. Marginal effects on blood pressure were seen in subjects only when standing. The oral bioavailability of bosentan was 43% to 48%, with a small interindividual variability of 20%. Doses above 500 mg did not lead to significant further increases in plasma levels of bosentan. From the first to the last day of the oral treatment phase, plasma concentrations of bosentan decreased by 30% to 40% due to a 2-fold increase in plasma clearance. Absorption and plasma protein binding did not change. The 24-hour urinary excretion of 6 beta-hydroxycortisol was increased in parallel by approximately 1.7-fold, indicating induction of cytochrome P450 3A isozymes. The two metabolites of bosentan reached plasma concentrations well below those of bosentan and will most likely not contribute to the pharmacological activity.     

Contribution of patient and hospital characteristics to adverse patient incidents.

The 1974 medical malpractice "crisis" brought about extensive legislation and insurance regulation in the United States. Hospitals in many states are now required to support risk management programs that include investigation and systematic analyses of adverse patient incidents. However, no research supports the hypothesis that systematic analysis of adverse patient incidents can identify contributory factors. In this study, a simple prediction model was used to estimate relationships between adverse incidents and selected patient and environmental characteristics in a large hospital. While some of the incident-characteristic relationships were significant, none of the estimated equations yielded results that could be logically translated into policy recommendations for the hospital. These results point to the need for further research. The benefits that positive research results would have for patients, hospitals, an the bill-paying public are obvious. Additional negative results would suggest that many legislative bodies and regulatory agencies were presumptions in requiring hospitals to conduct analyses of incidents.