[Laser microdissection and molecular typing of dysplastic cells from Pap smears: a new approach to early detection of cervical cancer]. / Lasermikrodissektion und molekulare Charakterisierung von dysplastischen Zellen aus Zervixabstrichen. Ein neues analytisches Konzept zur Frühdiagnose des Zervixkarzinoms.

The Papanicolaou smear (Pap) is a worldwide screening tool for early detection of cervical cancer and its precursor lesions. The transition from dysplasia to cancer is a highly complex genetic process and cannot be predicted based solely on cell morphology. Molecular characterization of precursor lesions could yield a better definition of lesions at high risk for progression. We developed an analytical concept comprising not only morphological characterization but also molecular analysis with multiple parameters of dysplastic cells from cervical smears. We isolated dysplastic cells from 52 fixed Pap-stained smears of various grades by laser microdissection and analyzed them for genetic lesions typical for cervical carcinoma. The loss of heterozygosity (LOH) as published for cervical carcinoma tissue was detected. Markers for early stages showed a LOH in 43% and 22%, and those for late stages in 26% of the cases. Combining morphological characterization with molecular analysis by multiple molecular markers could open up new opportunities for early detection of cervical carcinoma.

Molecular characterization of minimal residual cancer cells in patients with solid tumors.

The failure to reduce the mortality of patients with solid tumors is mainly a result of the early dissemination of cancer cells to secondary sites, which is usually missed by conventional diagnostic procedures used for tumor staging. PCR was shown to be superior to conventional techniques in detecting circulating tumor cells and micrometastases allowing the identification of one tumor cell in up to 10(7) normal cells in various sources such as blood, bone marrow, lymph nodes, urine or stool. The methods used are based on the detection of either genomic alterations in oncogenes and tumor suppressor genes or on the mRNA expression of tissue-specific and tumor-associated genes. The additional implementation of techniques for cancer cell purification had a significant impact on analytical sensitivity and specificity of MRCC detection. For patients with e.g. melanoma, breast, colorectal or prostate cancer it was demonstrated that the presence of disseminated cancer cells defines a subgroup of patients with reduced time to recurrence. The possibility to use easily accessible body fluids as a source for MRCC detection enables longitudinal observations of the disease. In this review we discuss the potential of molecular characterization of MRCC as a tool to improve prognostication, therapy selection and drug targeting as well as therapy monitoring.

Prognostic value of genomic alterations in minimal residual cancer cells purified from the blood of breast cancer patients.

The prognostic value of disseminated tumour cells derived from 353 breast cancer patients was evaluated. Disseminated tumour cells were purified from blood using a newly established method and nucleic acids were subsequently isolated. We investigated genomic imbalances (GI) such as mutation, amplification and loss of heterozygosity of 13 tumour suppressor genes and 2 proto-oncogenes using DNA from isolated minimal residual cancer cells. Significant correlations were found between genomic alterations of the DCC - and c-erbB-2 genes in disseminated breast cancer cells and actuarial relapse-free survival. Furthermore, increasing numbers of genomic imbalances measured in disseminated tumour cells were significantly associated with worse prognosis of recurrent disease. Logistic regression and Cox multivariate analysis led to the identification of genomic imbalances as an independent prognostic factor. Determination of disseminated tumour cells by genotyping of oncogenes and tumour suppressor genes seems not only to be a useful adjunct in follow up of carcinoma patients but provides also valuable additional individualized prognostic and predictive information in breast cancer patients beyond the TNM system.

Detection of mammaglobin expressing cells in blood of breast cancer patients.

Expression of human mammaglobin (hMAM) was published to be exclusively expressed in mammary tissue, in solid tumors, axillary lymph nodes and disseminated cancer cells in blood of breast cancer patients. A quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) test was applied to investigate hMAM expression in blood of breast cancer patients. Mammaglobin mRNA expression was found not only in breast cancer cell lines but also in cell lines of other cancer origin. In our patient cohort hMAM expression in 11/98 (11%) samples of breast cancer and 3/12 (25%) ovarian cancer patients could be detected. hMAM mRNA expression as a candidate marker for the detection of disseminated cancer cells in blood of breast cancer patients showed low sensitivity and reduced tissue specificity. A prognostic significance of hMAM expression could not be demonstrated.

Independent prognostication and therapy monitoring of breast cancer patients by DNA/RNA typing of minimal residual cancer cells.

Clinical relevance, purification techniques and molecular characterization of minimal residual cancer cells (MRCC) is a controversial topic in the literature. An analytical concept including a novel isolation procedure and a panel of tests for DNA and RNA typing of MRCCs is described and clinically evaluated in this paper. The purification procedure exploiting the physical characteristics of MRCCs shows superior performance leading to > 50% pure and viable tumor cells. Proof of the presence and purity of MRCCs in an isolated sample is given by multiparametric DNA typing (amplifications, mutations, losses of heterozygosity). On the basis of the proven presence of MRCCs tumor-relevant mRNAs can be adequately analyzed by normalized quantitative real-time RT-PCR. The molecular characterization of MRCCs isolated from blood of breast cancer patients could have a strong clinical impact on prognostication, drug targeting and therapy monitoring.

[Sensitivity studies on the demonstration of HIV-1 particles using RT HIV-1 PCR in pooled plasma before virus inactivation]. / Sensitivitätsstudien zum Nachweis von HIV-1-Partikeln mittels RT-HIV-1-PCR im Poolplasma vor Virusinaktivierung.

The goal of this study was the establishment of a reverse-transcription polymerase chain reaction (PCR) test and the evaluation of its sensitivity to detect an HIV-1 contamination in pooled plasma samples prior to the solvent-detergent (SD) inactivation procedure. Pooled plasma samples were spiked with known concentrations (1,000-0.1 TCID50/ml) of HIV-1, originated from tissue culture supernatants. Unspiked plasma samples were used as negative controls. After reverse transcription, a PCR in 4 different HIV-gene regions (gag, pol, env, ltr) followed by a hybridisation with specific probes was done. The achieved sensitivity in 3 tests (pol. gag, ltr) was 0.1 TCID50/ml and 1.0 TCID50/ml in the env-PCR system. It was shown in previous studies that by inactivation of pooled plasma with the SD technique a reduction of HIV-1 infectivity greater than 10(6) is achieved. The combination of PCR testing and the SD inactivation procedure makes it possible for the first time to define the maximum amount of contaminating HIV-1 in case of a negative PCR result. According to this procedure, the residual HIV-1 load of virus-inactivated plasma would not exceed 1 TCID50 per 1,000 litres at worst.

Pharmacokinetics of indoramin and its metabolite 6-hydroxyindoramin after single and multiple doses to cirrhotic liver patients.

Pharmacokinetic data obtained after intravenous and single and repeat chronic oral dosing of indoramin in nine patients with liver cirrhosis are described. Median plasma clearance is 11.2 ml/min/kg. Terminal disposition half-life is prolonged after intravenous as well as acute and chronic oral dosing (9.1 versus 10.7 versus 12.2 h). Median volume of distribution is 11.2 L/kg. Bioavailability is increased with a wide range of distribution from 12.2 to 75.4%. There is a slight tendency of accumulation during twice daily oral dosing that cannot be explained by the degree of prolongation of half-life. The kinetics of the main metabolite, 6-hydroxyindoramin, are substantially comparable to the kinetics of indoramin with a ratio of 6-hydroxyindoramin/indoramin calculated from the area under plasma concentration-time curve of 0.3, which is within the range of normal. All data suggest that the changed pharmacokinetics are due to altered liver perfusion as would be expected from a substance with a plasma clearance in the magnitude of liver perfusion in normal volunteers. It seems likely that, in patients with liver cirrhosis, similar alpha 1 blocking effects may be achieved with lower doses than in patients with normal liver function.

Antihypertensive therapy in the Federal Republic of Germany: clinical practice experience with indoramin (Wydora).

The clinical experience of 763 medical practitioners who treated 3,708 hypertensive patients (51% men) with indoramin, administered alone or in combination with diuretics and/or beta-adrenergic antagonists, is reported. All patients had baseline diastolic blood pressure (DBP) of 95 mm Hg or greater, even though most of the patients (62%) already were receiving optimum doses of diuretics (20%), beta-adrenergic antagonists (15%), or diuretics and beta-adrenergic antagonists (27%). After 6 to 10 weeks of indoramin therapy, the DBP of 70% of the patients was 90 mm Hg or lower; another 17% had treated DBP between 91 and 95 mm Hg. Response rates were similar among patients treated with indoramin alone and those who received concomitant antihypertensive treatment. Indoramin doses of 50 mg/day or less (dose range, 12.5 to 125 mg/day) were required in approximately 70% of the patients. Weight gain and reflex tachycardia were not observed. The most frequently reported side effects were drowsiness/tiredness, dizziness, and dry mouth. Only 6% of the patients discontinued indoramin treatment because of side effects. The results of this study indicate that indoramin, administered alone or in combination with diuretics and/or beta-adrenergic antagonists, is a safe and effective antihypertensive agent when used in relatively low doses in clinical practice.

Phospholipid-induced changes of gamma-aminobutyric acid transport in cortex grey matter in culture.

The function of membrane phospholipids (PL) in the regulation of gamma-aminobutyric acid (GABA) transport and GABA carrier binding has been investigated in organized cultures of rat cerebral cortex. The cellular lipid composition has been changed by growing the cells in a delipidated nutrient solution or by short-term exposure of the cells to PL emulsions. Introduction of PL into the cellular matrix was monitored by analysis of biologically active fluorescently labeled phosphatidylcholine (PC) or phosphatidylethanolamine (PE). Parinaroyl and dansyl derivatives were used. Conditions of maintenance as well as exogenously given PL affected the transport of GABA. Two transport systems were observed, one first-order system and one cooperative system. Saturated species of PC or PE reduced first-order GABA uptake with increase in chain length of the fatty acid residues. The effects of unsaturated PL were dependent upon the polar head. Unsaturated PC enhanced the capacity of the first-order transport of the amino acid. In comparison to cultures grown in lipid-free medium, introduction of diarachinoyl-PC into the cells increased the density of the first-order active transport sites by a factor of 8 and the affinity constant by a factor of 17. Diarachinoyl-PE reduced both kinetic parameters. GABA uptake via the cooperative system was enhanced by the unsaturated PE, not by PC. The role of endogenous PL and their asymmetric distribution was studied by application of phospholipase A2, C, and D. Stimulation of carrier activity was induced by hydrolysis of PL on the external leaflet. Inhibition occurred upon enzymatic degradation of external and cytoplasmic PL. Lipolysis also affected GABA receptor binding, suggesting that the effects observed represent the activity of both classes of binding sites, the carrier and the receptor. However the latter accounted for a small fraction of the binding. Transport of the amino acid was temperature sensitive. The temperature curve was shifted within two discontinuities, appearing in the Arrhenius plot as a function of membrane lipids. The results suggest a partitioning of the proteins between fluid and ordered lipid domains. Displacement of the protein may govern the rate constants and/or the effective protein concentration.

Lipid metabolism of developing central nervous tissues in organotypic cultures. III. Ganglionic control of glycerolipids and fatty acids in cortex grey matter.

Lipid changes in rat brain grey matter were observed in a coculture system of innervating and target explants. The de novo biosynthesis of individual glycerolipids and the metabolism of fatty acids were investigated. Innervating grey matter cultures exhibited a substantial increase in neutral glycerolipid formation. Only slight modifications were observed in the fatty acid fraction. Target cells responded to innervation by a marked increase in phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine formation. In addition, the biosynthesis of arachidonate and docosahexaenoate was drastically enhanced. It is shown that neuritic bridges connecting the explants, rather than soluble factors, account for the effects observed. Putative mechanisms involved in changes of lipid metabolism are discussed.

Effect of cocultivation of brain tissues on leucine and glycerol-3-phosphate incorporation.

Studies on the interaction between cortex grey matter (CC), diencephalon (DE), mesencephalon (ME) and pure glial cells free of neurons were undertaken in explant cultures. The culture system provided the formation of neuritic bridges between individual tissue pieces. Cocultivation of different brain tissues resulted in changes of leucine and glycerol-3-phosphate incorporation characteristic for individual brain areas. Glial cell (GC) did not respond to innervation by neurites of various sources. Glycerol-3-phosphate uptake rates were substantially altered in cocultures containing neurons and glia.

Lipid metabolism of developing central nervous tissues in organotypic cultures. I. Lipid distribution and fatty acid profiles of the medium for rat brain cortex in vitro.

The distribution and concentration of lipid components of different media used for organotypic cultures of rat brain cortex have been measured quantitatively. Five cholesterol ester species have been fractionated from chicken embryonic extract. Their fatty acid profiles have been determined by combined gas liquid chromatography-mass spectrometry. The depletion characteristics of medium cholesterol esters and some fatty acids during development of brain cortex were not symmetrical. Polyunsaturated fatty acids of the medium were lowered prior to the saturated and monoenoic fatty acids. The phenomenon of depletion may be correlated with enhanced esterase activity mediated by the tissue.

Lipid metabolism of developing central nervous tissues in organotypic cultures. II. A comparative study of medium and tissue fatty acids in developing rat cerebral cortex and cerebellum.

Organotypic cultures of rat cerebral cortex and cerebellum have been supplemented with different media. Fatty acid analysis in incubated media and in tissue explants revealed a characteristic differentiation of three periods during maturation: a posttraumatic repair phase, a synaptogenic period and a synaptic stage. Medium fatty acids are biphysically depleted from the nutrient. Both tissues differ in the depletion pattern of individual fatty acids. The development of tissue fatty acids is mainly observed during the synaptogenic period. Turnover studies of individual fatty acids indicate a tissue-specific and time-dependent characteristic.