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Biomedical subjects

C Streffer

Publications and source records attributed to C Streffer.

At least 37 records · Page 2Linked to original sources
PubMed1999

Automated comet assay analysis.

BACKGROUND: Recently the "comet assay" or "single-cell gel electrophoresis assay" has been established as a sensitive method for the detection of DNA damage and repair. Most of the software now available to quantify various parameters for DNA damage requires the interaction of a human observer. In this report, we describe an automated analysis system that is based on self-developed software and hardware and needs minimal human interaction. METHODS: The image analysis is divided into two parts: 1) automatic cell recognition and comet classification and 2) quantification of desired comet parameters. Image preprocessing, segmentation, and feature classification were developed with algorithms based on mathematical morphology. To enhance evaluation speed, we have introduced parallel processing of data under the Windows NT operating system (Microsoft Corporation, Redmond, WA). Use of an analogue real-time autofocus unit (Böcker et al.: Phys Med Biol 1997;42:1981-1992) allows for faster analysis. RESULTS: Our recognition software shows a sensitivity of 95.2% and a specificity of 92.7% when tested on test samples from routine work with DNA damage by low-dose radiation (0-2 Gy). The parallel hardware and software concept enables us to analyze 100 comets on one slide in less than 15 min. CONCLUSIONS: A comparison of measurements made on the same samples by manual and automated analysis systems revealed that there are no significant differences. The slope of the dose-response curves and the repair kinetics are very similar and demonstrate that automatic comet assay analysis is possible.

Algorithms↗
PubMed1999

Radiation and/or hyperthermia sensitivity of human melanoma cells grown for several days in media with reduced pH.

BACKGROUND: There are a number of reports in the literature dealing with the influence of reduced extracellular pH on cellular radiation and/or heat sensitivity. The focus of these earlier studies has been on the effects of a short-term incubation under acidic conditions, whereas much less attention has been given to chronic acidosis which would seem to be more relevant with respect to the tumor micromilieu. As far as mechanisms are concerned, many authors have taken the view that the intracellular rather than the extracellular pH is decisive at least for heat sensitivity, but only rodent cells have been studied so far and again the effects of chronically low pH in the incubation medium have been largely neglected. We therefore studied human melanoma cells after incubation for up to 6 days in media with different pH between 6.5 and 7.3. MATERIAL AND METHODS: MeWo cells were used throughout. Cells were incubated for 0, 3 or 6 days in media with pH 6.5, 6.7, 6.9, 7.1 or 7.3. The sensitivity against 250 kV X-rays and hyperthermia at 43 degrees C were determined in the colony-forming assay. The intracellular pH was measured flow cytometrically using 5(and 6)-carboxyfluorescein. Calibration curves were established with cells incubated in different buffers containing nigericin to equilibrate intra- and extracellular pH. RESULTS: Cell growth was optimal with pH 7.3 and 7.1 in the medium, somewhat reduced at pH 6.9, and largely inhibited at pH 6.7 and 6.5. Radiation and/or hyperthermia sensitivities were noticeably increased after several days of incubation at reduced pH; the surviving fraction after 4 Gy and 1 h at 43 degrees C either alone or in combination being a factor of 2 to 4 lower at pH 6.5 than at 7.3. These changes in sensitivity could not be correlated with changes in the intracellular pH. Cells seemed to be capable of regulating this parameter very well; the flow cytometric measurements revealed that the intracellular pH was 7.2 +/- 0.2 irrespective of the extracellular pH in the range considered here. CONCLUSIONS: In contrast to the almost generally accepted hypothesis that intracellular pH is decisive for the heat sensitivity, the human melanoma cells studied here became sensitive after a few days of incubation under acidic conditions without changes in the intracellular pH. Other factors seem to be influencing the cellular response to radiation and/or heat under chronically low pH.

Cell Division↗
PubMed1999

Optimization and standardization of the "comet assay" for analyzing the repair of DNA damage in cells.

BACKGROUND: The "comet assay" has become an interesting and a very useful tool for the analysis of the induction and amount of DNA damage in single cells thus offering the opportunity to measure the effectiveness of DNA repair. On the basis of the Ostling and Johanson protocol we have developed a modified method with increased sensitivity and high reproducibility. MATERIAL AND METHODS: Human tumor cells or isolated human peripheral blood lymphocytes were analyzed in the experiments. The amount of DNA damage and the effectiveness of DNA repair was measured after X-irradiation using the "comet assay" technique. RESULTS: In this presentation the influences of different methodological factors like agarose concentration, buffer pH, electrophoresis time, electric field strength on the applicability of the "comet assay" are described in detail and optimum conditions for "comet assay" experiments have been evaluated. Additionally the authors will show a comparison of different fluorescent DNA dyes pointing out their advantages or disadvantages for "comet" analysis. The usefulness of this technique and its capabilities are exemplified by showing DNA repair kinetics of human lymphocytes of different healthy or radiosensitive donors after in-vitro irradiation with 2 Gy X-rays. CONCLUSIONS: This paper presents data on the optimization and standardization of the original "comet assay" leading to an extremely fast and practicable protocol in the field of single cell gel electrophoresis. After irradiation with 0.1 Gy an increase in the amount of DNA damage can be measured with high statistical significance and the DNA repair capacity of individual cells after X-ray doses of 2 Gy can be analyzed with high reproducibility. The results comparing DNA repair capacities of different donors point out that the "comet assay" may have the potential for the estimation of individual radiosensitivity.

Adult↗
PubMed1999

In vitro studies on the cellular uptake of melanoma imaging aminoalkyl-iodobenzamide derivatives (ABA).

The cellular uptake of 11 radioiodinated aminoalkyl-iodobenzamides (ABA) was studied using cultivated murine melanoma cells (B16/C3). All derivatives showed a high uptake (up to about 80%) of radioactivity in melanotic melanoma cells; hence, accumulation of all compounds radioiodinated in the ortho position was reduced by approximately 30%. Using the compound para-[131I]iododiethyl-aminoethylbenzamide (p-131I-ABA-2-2) a close correlation of the cellular melanin content with the tracer uptake (R2 = 0.95) was verified. The presence of extracellular melanin, however, had no effect on the cellular tracer uptake. Because the accumulation was independent of the specific activity of p-131I-ABA-2-2, a significant contribution to the uptake process by binding to receptor sites could be excluded.

Benzamides↗
PubMed1998

Micronuclei-biological indicator for retrospective dosimetry after exposure to ionizing radiation.

Micronuclei can be measured through a conventional method after staining with Giemsa or fluorescence dyes for DNA. However, a technique with cell proliferation control should be preferred. This is done by incubation with cytochalasin B and counting the micronuclei in binucleated cells. Satisfactory dose relationships are observed after irradiation of human lymphocytes in vitro. The RBE for fast neutrons is around three. An automatic analysis is possible by image analysis. The dose range in which significant increases can be observed is 0.3 to 5 Gy X-rays. The assay becomes more sensitive when the micronuclei are determined only in B-lymphocytes. Another possibility exists by determination of the number of micronuclei with centromeres. For this purpose the hybridization with pancentromeric DNA probes and fluorescence labelling is of advantage. By this technique a radiation dose of 0.1 Gy X-rays can be detected. It is apparently also possible under these conditions to detect radiation exposures which have taken place decades before the measurements.

Cell Division↗
PubMed1998

The sensitivity of the in vitro cytokinesis-blocked micronucleus assay in lymphocytes for different and combined radiation qualities.

PURPOSE: The dose-response relationship and the relative biological effectiveness (RBE) for the induction of micronuclei in lymphocytes was analyzed after irradiation in vitro with a 6-MeV neutron beam that was followed by 240-kV X-rays. The dose range of the combined exposure comprised 1 to 3 Gy. For reference, the dose-effect relationships found after X-ray (0.5 to 5 Gy)- and neutron (0.5 to 4 Gy) exposure applied separately are presented. The possibility of an interaction between the 2 radiation qualities is investigated by the method of isobole calculation termed "envelope of additivity". METHODS: Micronuclei were analyzed in PHA-stimulated, cytokinesis-blocked human lymphocytes. RESULTS: The dose-response relationships for the micronucleus frequencies induced by the neutron irradiation, as well as by the mixed exposure, were linear. A saturation effect was indicated after neutron doses higher than 3 Gy. After low LET exposure the dose-response curves were describable by a linear-quadratic model. For neutron-induced micronucleus frequencies, RBE-values of 2 to 3 and for the combined exposure RBE values of 1.5 to 2 were calculated for a range of effect of 0.5 to 1.5 micronuclei/binucleated lymphocyte. No indication was found for an interaction between the damage induced by X-rays and that produced by neutrons under our experimental conditions. CONCLUSIONS: These studies demonstrate a clear dependence of micronucleus induction on radiation quality and emphasize the usefulness of the micronucleus assay in biological dosimetry, also in cases in which high LET radiation or a mixed beam is involved as the radiation source.

Adult↗
PubMed1998

A locus for radiation-induced gastroschisis on mouse Chromosome 7.

Gastroschisis (abdominal wall defects) occurs with a high frequency in the mouse inbred strain HLG compared with C57BL/6J mice. The risk of gastroschisis increases significantly after exposure to irradiation with X-rays during preimplantation development and follows a recessive mode of inheritance for the HLG susceptibility alleles. We have used a backcross strategy and genome-wide microsatellite typing to chromosomally map this trait. A suggestive linkage for a locus responsible for radiation-induced gastroschisis (Rigs1) was found in a region of mouse Chromosome 7.

Abdominal Muscles↗
PubMed1998

Comparison of radiation-induced aberration frequencies in chromosomes 1 and 2 of two human donors.

PURPOSE: The purpose of this study was to analyse donor and time dependent variations in the frequencies of radiation-induced aberrations in chromosomes 1 and 2. MATERIALS AND METHODS: Human lymphocytes from two donors were irradiated with 1 and 2 Gy of X-rays. Chromosomal aberrations were scored in chromosomes 1 and 2 painted with different fluorochromes and in Giemsa stained cells. Two time displaced experiments were performed with lymphocytes of each donor. RESULTS: In cells of both donors chromosome 1 was generally more frequently involved in translocations than chromosome 2. This result was not always reproducible. Chromosome 2 showed a higher frequency of acentric fragments, especially following a dose of 1 Gy. Again interexperimental variations were observed. No differences between the two chromosomes were seen with regard to other aberration types. Both chromosomes showed less dicentrics and more acentric fragments than proportional to their DNA content. CONCLUSIONS: Chromosomes 1 and 2 show a different sensitivity to ionizing radiation. The difference is dependent on the aberration type and is not always reproducible. This variability could contribute to the difficulty in reaching a consensus regarding the radiosensitivity of individual chromosomes.

Chromosome Aberrations↗
PubMed1998

Radiosensitivity of normal fibroblasts from breast cancer patients assessed by the micronucleus and colony assays.

PURPOSE: To investigate whether radiation-induced micronucleus formation as expressed by the cytochalasin-blocked Mn-assay correlates with cellular radiosensitivity measured by a colony assay in primary fibroblast cultures from cancer patients. MATERIALS AND METHODS: Studies were made on skin fibroblasts from 36 breast cancer patients. The micronucleus assay was performed using treatment with cytochalasin-B to create binucleate cells. Response was scored in terms of the percentage of binucleate cells with micronuclei, also as the number of micronuclei per binucleate cell. The data were related to previously published results of cell survival measurements on these cell lines. RESULTS: Neither endpoint for micronucleus formation showed a correlation with radiosensitivity by the colony assay. The fraction of fibroblasts that reach mitosis without micronuclei also failed to correlate with cell survival. CONCLUSIONS: Among these primary fibroblast cell lines radiation-induced micronucleus formation was not associated with radiosensitivity as measured by a colony assay.

Adult↗
PubMed1998

Evaluation of boron neutron capture effects in cell culture using sulforhodamine-B assay and a colony assay.

PURPOSE: The purpose of this study was to find an in vitro method for determining the cytotoxicity of boronated drugs as well as their potential suitability for neutron capture therapy. MATERIALS AND METHODS: The survival of human melanoma cells has been determined by a colony assay and the sulforhodamine-B assay after X-irradiation and irradiation with fast d(14) + Be-neutrons using the boronated compound borocaptate sodium (BSH). The cytotoxic effects of BSH have been studied using both methods. RESULTS: Under well-defined experimental conditions, and after a sufficient amount of time for the expression of radiation damage, the results of the sulforhodamine-B assay are qualitatively comparable with the results of the colony assay. CONCLUSION: The sulforhodamine-B assay is suitable for the screening of compounds for potential use in neutron capture therapy because it is a fast and efficient method that is reproducible and technically advantageous.

Boron Neutron Capture Therapy↗
PubMed1997

Polymorphisms in the p53 gene in thyroid tumours and blood samples of children from areas in Belarus.

We present changes in the p53 gene in a group of 70 thyroid tumours and 40 blood samples obtained from children from Belarus. Three thyroid tumours show a polymorphism in exon 6 (codon 213) and 5 tumours show a polymorphism in intron 6, 37 bp upstream to the 5'-end of exon 7. Only one patient has a mutation in exon 7 (codon 258) resulting in an amino acid substitution in the protein p53. The distribution of polymorphisms in the 40 blood samples was as follows: three patients had a polymorphism in exon 6 and two persons had a polymorphism in intron 6. One polymorphism in intron 6 was also found in the group of 30 healthy children from Belarus. The fact that the differences in the sequence in p53 found in the tumours was also seen in the blood of these patients demonstrates that they are polymorphisms not induced by radiation exposure. It is difficult to conclude, if the polymorphisms found by us could be associated with the predisposition to radiation-induced cancer.

Adolescent↗
PubMed1997

NIR reflection measurements of hemoglobin and cytochrome aa3 in healthy tissue and tumors. Correlations to oxygen consumption: preclinical and clinical data.

OBJECTIVES: A new near infrared reflectance spectroscopy based technology (MULTISCAN OS 10/30) for non-invasive measurements of tissue oxygenation allows detection of absolute tissue hemoglobin concentration and saturation values in real time. METHODS: MULTISCAN OS 10/30 scans a tissue sector of defined geometry at 400 to 1200 nm wavelength with 0.3 nm intervals at a scan rate of up to 400 Hz in reflection mode. The newly developed algorithms are based on the entropy pattern of photons and allow the detection of absolute values of total hemoglobin (tHb), deoxy- (Hb) and oxy-hemoglobin (HbO2) in mg/ml tissue as well as tissue oxygen (saturation = tiSO2) in the range of 0 to 100% in real time. Cytochrome aa3 (Cyt) can be monitored simultaneously. RESULTS: Physiological Stimulation. Clamping experiments at the finger of volunteers, in the intestine of a pig, in the kidney of mice and rats showed a very fast, sensitive and tissue specific reaction. Changes in breathing and/or anesthesia conditions were immediately followed by corresponding changes of tissue oxygenation (brain, finger, skin). Simultaneous measurements of oxygen (HbO2), and cytochrome showed a strong correlation between both parameters. For validation purposes parallel polarographic measurements (pO2 measurements) were performed in mice. Furthermore angiographic data of patients as well as NMR/I data were compared with the NIR spectroscopy findings. Tumor Measurements. Patients with tumors of different origins showed significantly different oxygen values. The lowest level was found in glioblastoma (< 10% sat, < 1.0 mg/ml tHb), whereas renal carcinoma showed tremendously increased values (> 90% sat, > 5.0 mg/ml tHb). In contrast the surrounding healthy kidney tissue was significantly less oxygenated and perfused compared to the tumors. Oxygen Consumption Measurements. Oxygen consumption was measured in 16 patients after ligation of the blood supply (A. and V. renalis) to the kidney affected by tumor. All tumors showed a significantly lower consumption rate compared to the healthy tissue. These findings were controlled by animal experiments of human renal carcinomas on nude mice. The same results were obtained under these experimental conditions. CONCLUSION: MULTISCAN OS 10/30 is a new and useful tool for in vivo characterization of oxygen and cytochrome in healthy and tumor tissues. In many clinically relevant situations oxygen measurements can be helpful and support the clinical routine diagnostics.

Adult↗
PubMed1997

Detection of vital germ cell tumor cells in short-term cell cultures of primary tumors and of retroperitoneal metastasis--clinical implications.

By establishing short-term cell cultures derived from retroperitoneal metastasis after neoadjuvant chemotherapy, our aim was to improve the diagnosis and prognosis in patients with advanced testicular germ cell tumors. The histological evaluation of surgically removed metastatic tissue by retroperitoneal lymphadenectomy (RLA) is extremely complicated after previous chemotherapy, but knowledge of persistence of vital tumor cells in residual lesions is of great prognostic value and therapeutic consequence in patients with testicular germ cell tumors. We therefore investigated whether vital tumor tissue could be detected in short-term cell cultures derived from such metastatic lesions by measuring the concentration of the tumor markers beta human chorionic gonadotropin (beta HCG) and alpha-1 fetoprotein (AFP) in cell culture supernatants. We initially demonstrated the specificity of the determination in cell cultures of human transitional-cell carcinoma cell lines, human foreskin fibroblasts and normal testicular tissue. In a group of 20 patients with untreated primary testicular germ cell tumors, detection of beta HCG and AFP was increased about threefold in cell culture supernatants in comparison to the serum concentration. Finally, we prepared primary cell cultures from surgically removed retroperitoneal metastasis of 12 patients with testicular germ cell tumors after chemotherapy. The serum concentrations of beta HCG and AFP of all patients were at normal values when RLA was performed. However, pathologically increased concentrations of beta HCG (3/3) and AFP (2/3) in cell culture supernatants were found in 3 of 12 cell cultures. Interestingly, these three patients with a pathological increase in beta HCG and AFP as determined in the supernatant of the short-term cell cultures had tumor progression within a mean follow-up of 3 +/- 1 months (P < 0.01), whereas 9 of 12 patients who had no pathological increase in beta HCG and AFP as determined in the supernatant of the short-term cell culture were in complete remission (CR) after a mean follow-up of 40 +/- 11.6 months.

Adult↗
PubMed1997

[Genetic predisposition and radiation sensitivity of normal tissue].

BACKGROUND: After radiotherapy there are always some patients who develop strong acute and late reactions in normal tissues. In these patients frequently a genetic predisposition is observed. There are found DNA-repair deficiencies and changes in the regulation of the cell cycle which are responsible for the increased radiosensitivity with enhanced cell killing. METHODS: The micronucleus test and the comet assay appear to be appropriate tests in order to measure this increased radiosensitivity. Both tests are characterized by being relatively quick and simple and can be performed with small cell numbers. It is possible to study blood lymphocytes and fibroblasts with these tests. RESULTS: Both tests can predict the radiosensitivity of normal tissues especially if they are applied in combination. CONCLUSIONS: Epidemiological studies with patients after radiotherapy show evidence that the increased radiosensitivity also causes an enhanced induction of secondary tumors by ionizing radiation. This is supported by corresponding animal models.

Ataxia Telangiectasia↗
PubMed1997

Image analysis of comet assay measurements.

In the last decade the 'comet assay' or 'single cell gel electrophoresis assay' has been established as a sensitive method for the detection of DNA damage and the measurement of its recovery. The results published in the literature have often been obtained with different methods for comet structure measurement. In most cases these data are not comparable with each other. Even when using similar systems for the analysis, it is difficult to obtain matching data. This presentation will describe some technical aspects of our measurement equipment and evaluation software. It focuses on necessary experimental conditions to minimize errors in obtaining such data. The software developed here allows the rapid analysis of the microscopic samples (< 2 s per image). The image analysis was designed with respect to the morphological shapes of comet cells, which were investigated with a confocal laser microscope. The system is built with standard components which are commercially available. As a measure of the amount of DNA damage the ratio of fluorescence intensity was used inside the comet tail and the fluorescence intensity of the comet head. Other parameters such as DNA content, comet area, head radius, tail length and tail moment are also determined. The reproducibility of the system has been evaluated in several experiments over a period of 5 years.

Ataxia Telangiectasia↗