A new microperfusion system for the cultivation of tumor-cells invitro - approach to integrate pharmacokinetic parameters in screening assays for cytostatic drugs

By a newly introduced microperfusion system absorption and elimination rates can be simulated in vitro. This article describes the optimization of culture conditions (medium composition, membrane filters, pumping rates, and stirring speeds) of tumor cell lines (L1210, KB) maintained in suspension in an ultrafiltration-flat chamber. Viability and colony-forming ability are measured. Our results indicate that tumor cells can be cultured under serum-free conditions over a five hour incubation period with only minimal decrease in colony-forming ability. Survival of cells is independent from the pumping rate in the tested range, but is dependent of the stirring speed. Each cell line requires its own stirring speed. Ultrafiltration membranes with minimal nonspecific adsorption properties proved to be the best in terms of cell adsorption and toxicity to retain cells in the chamber. This system might improve the tumor cell colony assay for cytostatic drug screening.     

A semiautomatic microassay to measure human tumor clonogenic growth-invitro

A semi-automatic micromethod was developed using 96-well microtiter plates in combination with a video monitoring system to assay human tumor clonogenic growth in vitro. The conditions to follow the growth of K562 human myeloid colonies were optimized and validated: About 250 cells in a 40 mul agar mixture are to be seeded per well. The colony number is proportional to the number of seeded cells. The cloning efficiency is about 75% and correlates well with the established glass capillary method. The system precision of the automatic colony counter, expressed by the coefficient of variation, is less than 2%. The mean of the intra- and inter assay coefficient of variation is low (8.5% and 7%, respectively). The microassay was applied to measure the lymphokine-activated killer (LAK)-activity of peripheral blood mononuclear cells (PBMC) from healthy donors against K562 target cells and to examine the effects of various thymic hormone preparations on the cytotoxicity of generated LAK cells. Thus, the new microassay provides a useful tool for measuring clonogenic tumor cell growth and its modulation by different agents on many samples in a short time.     

Systematic toxicological analysis procedures for acidic drugs and/or metabolites relevant to clinical and forensic toxicology and/or doping control

This paper reviews systematic toxicological analysis (STA) procedures for acidic drugs and/or metabolites relevant to clinical and forensic toxicology or doping control using gas chromatography, gas chromatography-mass spectrometry, liquid chromatography, thin-layer chromatography and capillary electrophoresis. Papers from 1992 to 1998 have been taken into consideration. Screening procedures in biosamples (whole blood, plasma, serum, urine, vitreous humor, brain, liver or hair) of humans or animals (horse, or rat) are included for the following drug classes: angiotensin-converting enzyme (ACE) inhibitors and angiotensin II (AT-II) blockers, anticoagulants of the 4-hydroxy coumarin type, barbiturates, dihydropyridine calcium channel blockers (calcium antagonists), diuretics, hypoglycemic sulfonylureas and non-steroidal anti-inflammatory drugs (NSAIDs). Methods for confirmation of preliminary results obtained by screening procedures using immunoassay or chromatographic techniques are also included. Furthermore, procedures for the simultaneous detection of several drug classes are reviewed. The toxicological question to be answered and the consequences for the choice of an adequate method, the sample preparation and the chromatography itself are discussed. The basic information about the biosample assayed, work-up, separation column, mobile phase or separation buffer, detection mode and validation data of each procedure is summarized in 16 tables. They are arranged according to the drug class and the analytical method. Examples of typical applications are presented. Finally, STA procedures are reviewed and described allowing simultaneous screening for different (acidic) drug classes.     

Gabapentin leads to remission of somatoform pain disorder with major depression

Gabapentin, a novel antiepileptic drug, is effective in the treatment of partial seizures with and without secondary generalization. Evidence suggests that it may have mood-stabilizing and possibly antidepressant properties in bipolar depression. We report on a 48-year-old woman who had recurrent major depressive disorder. Following inguinal herniorrhaphy, she developed severe stabbing pain in the lower abdomen and inguinal area that progressed to constant pain in her whole body. She was depressive, hopeless, and had given up her social activities. A diagnosis of major depressive disorder and somatoform pain disorder was made. Antidepressants and carbamazepine were ineffective, and she had attempted suicide. Gabapentin resulted in remission of both the pain and the depressive mood at a dose of 1.800 mg/day.     

Transsexual development after committing an offense

Connections between transsexualism and delinquency have been considered in only a few publications to date. The authors report on a forensic psychiatric case with a transsexual development after committing an offence based on sexual deviations. Transsexualism in this context seems to represent an attempt for the symbolic solution of a conflict of identity. This should be interpreted in the context of psychotherapy and not be misunderstood as a symptom proving the diagnosis of manifest transsexualism by orientation on pure phenomenological criteria.     

Growth of human tumor cells in macroporous microcarriers results in p53-independent, decreased cisplatin sensitivity relative to monolayers

Multicellular contact has been shown to influence the in vitro sensitivity of cells to drug treatment. We investigated the use of macroporous gelatin microcarriers, CultiSpher-G, as a convenient laboratory system for the molecular analysis of this "contact effect". We determined that human A549 cells can be grown in CultiSphers with growth and cell cycle parameters similar to those of monolayers. In addition, cells in CultiSphers express less p27/kip1, an indicator of cell cycle arrest, than equivalent cells in monolayers. When treated with drugs, A549 cells grown in CultiSphers or monolayers accumulate equivalent amounts of platinum-DNA adducts and similar amounts of doxorubicin. Moreover, A549 and KB-3-1 cells in CultiSphers have significantly decreased sensitivity to cis-platinum(II)diammine dichloride (cisplatin), 4-hydroperoxycyclophosphamide, doxorubicin, and paclitaxel (taxol) compared with cells in monolayers when assayed by clonogenic survival. Cisplatin treatment in monolayers or CultiSphers did not result in apoptotic cell death. In contrast, paclitaxel caused a significant amount of sub-G1 DNA, an indicator of apoptosis, which was diminished when cells were grown in CultiSphers compared with monolayers. When grown in CultiSphers, cells with abrogated p53 function (A549/16E6 and NCI-H1299) were less sensitive to cisplatin than the corresponding monolayer cells, indicating that the decrease in sensitivity is p53 independent. Taken together, the data suggest that CultiSpher-G microcarriers are a useful in vitro system to examine the effects of three-dimensional cell contact on drug sensitivity of human tumor cells.     

Intensity dependence of auditory evoked potentials (AEPs) as biological marker for cerebral serotonin levels: effects of tryptophan depletion in healthy subjects

Rationale: The intensity dependence of the auditory evoked potentials (AEP) has been suggested to be a specific biological marker of central serotonergic activity. Objective: While previous studies used circumstantial evidence to support this hypothesis, we manipulated (decreased) cerebral levels of serotonin directly by using tryptophan depletion. Methods: Twelve healthy young subjects were investigated using placebo and two different amino acid mixtures in a double blind cross over design on three different occasions. AEPs recorded during tryptophan depletion were analyzed by dipole analysis and regional sources using methods published in the literature. Results: For none of the mixtures a significant effect of tryptophan depletion was found. There was a trend towards reduced intensity dependency after tryptophan depletion, especially in the right hemisphere. This reduction correlated with the amount of reduced tryptophan in plasma. Conclusions: The results indicate, in contrast to earlier indirect studies, that the intensity dependence of AEPs is not a specific marker of central serotonergic activity. Received: 8 March 1999 / Final version: 25 May 1999     

Cytogenetic diagnosis of cancer: abnormalities of chromosomes and polyploid levels in the bone marrow of patients with small cell anaplastic carcinoma of the lung.

The chromosomes of metastatic cells and polyploid levels in the bone marrow of 26 patients with small cell anaplastic carcinoma were studied by direct bone marrow preparation and trypsin-Giemsa banding. Eighteen of these patients had received no tumor therapy and 8 had had chemotherapy and/or radiation therapy; 18 patients, including 5 who had received therapy, had karyotypic abnormalities with or without elevation of the polyploid level. Modal numbers and chromosome abnormalities were highly variable in treated and untreated patients. Modes ranged from hypodiploid to polyploid, but polyploid modes were the most frequently observed abnormal modes. Polyploid modes were not seen, however, in post-therapy patients with the exception of one who had received radiation therapy to the mediastinum for only 4 days prior to withdrawal of the specimen for chromosome analysis. Ten patients had elevated polyploid levels that ranged from 4.24 to 44.8% and always occurred in conjunction with karyotypic abnormalities. Both aneusomy (abnormal number) of normal chromosomes and structural aberrations (markers) occurred frequently. Some markers were consistent within an individual, but other variable aberrations were also typically present. Very few markers were common to 2 or more patients. The no. 1 chromosome participated in marker formation in 14 of the 18 patients with karyotypic abnormalities. Of the 26 patients, 5 were negative for metastasis to the marrow by pathologic examination but positive by cytogenetic diagnosis, whereas none were positive by pathologic examination and negative by cytogenetic diagnosis; this demonstrated that cytogenetics may be used as a rapid adjunct diagnostic procedure for the detection of metastasis in the marrow.