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M Durm

Publications and source records attributed to M Durm.

15 recordsLinked to original sources
PubMed1998

Fast-FISH technique for rapid, simultaneous labeling of all human centromeres.

Fluorescence in situ hybridization (FISH) has become a powerful tool in chromosome analysis. This report describes the systematic optimization of the Fast-FISH technique for centromere labeling of human metaphase chromosomes for radiobiological dosimetry purposes. For the present study, the hybridization conditions and the efficiency of two commercially available alpha-satellite DNA probes were compared ("human chromosome 1 specific", Oncor, Gaithersburg, MD, vs. "all-human chromosomes specific", Boehringer-Mannheim, Germany). These probes were hybridized to human lymphocyte metaphase plates by using a hybridization buffer without formamide and without any other equivalent denaturing chemical agents. The results indicate the suitability of the method for automated image analysis on the basis of thresholding. The optimal conditions concerning hybridization time and temperature were determined by a systematic quantitative evaluation of the fluorescent labeling sites after the hybridization procedures. Under defined "low stringency" conditions, we found that the "human chromosome 1 specific" DNA probe labeled not only the centromere of the human chromosome 1 but also the other human centromeres in the same way as the "all-human chromosome specific" DNA probe. The optimized conditions to complete all centromere labeling were applied to the detection of dicentric chromosomes on irradiated human lymphocyte samples (gamma-rays of 60Co source, 0.5 Gy/min, for doses of 1, 3, and 4 Gy). The yield of dicentrics was determined after Fast-FISH and compared with results obtained after Giemsa staining. These results are very compatible and indicate that, because of its simplicity, this optimized Fast-FISH procedure would be useful for fast screening purposes in biological dosimetry after accidental overexposure.

Cells, Cultured↗
PubMed1998

Fast-painting of human metaphase spreads using a chromosome-specific, repeat-depleted DNA library probe.

For chromosome painting, in situ suppression of repetitive DNA sequences has been well established. Such standard protocols usually require large amounts of Cot-I DNA. Recently, it has become possible to deplete repetitive DNA sequences from library probes by magnetic purification and PCR-assisted affinity chromatography. These "repeat-depleted library probes" appear to be extremely useful for Fast-FISH, a technique that omits denaturing chemical agents such as formamide in the hybridization buffer, resulting in a substantial acceleration and simplification of the complete protocol. Shown here is the application of Fast-FISH to a repeat-depleted, directly fluorochrome-labeled library probe of the q-arm of chromosome 15 (Fast-Painting) for human lymphocyte metaphase spreads. Following painting without Cot-I DNA and without formamide, visual inspection revealed sufficient chromosome painting after a few hours of hybridization. The fluorescence signals of the labeling sites were analyzed after hybridization times of 1 and 2 h (in one case, 4 h) using digital fluorescence microscopy. The painting efficiency expressed in values of relative fluorescence signal ratios was quantitatively evaluated by image analysis using line-scan procedures and area-morphometry of mean luminance. Two preparation protocols (ethanol dehydration without and with RNase A treatment followed by pepsin digestion for four different exposure times) were compared. These results indicated that RNase A treatment and pepsin digestion are steps that can be omitted.

Adult↗
PubMed1997

Optimized Fast-FISH with alpha-satellite probes: acceleration by microwave activation.

It has been shown for several DNA probes that the recently introduced Fast-FISH (fluorescence in situ hybridization) technique is well suited for quantitative microscopy. For highly repetitive DNA probes the hybridization (renaturation) time and the number of subsequent washing steps were reduced considerably by omitting denaturing chemical agents (e.g., formamide). The appropriate hybridization temperature and time allow a clear discrimination between major and minor binding sites by quantitative fluorescence microscopy. The well-defined physical conditions for hybridization permit automatization of the procedure, e.g., by programmable thermal cycler. Here, we present optimized conditions for a commercially available X-specific alpha-satellite probe. Highly fluorescent major binding sites were obtained for 74 degrees C hybridization temperature and 60 min hybridization time. They were clearly discriminated from some low fluorescent minor binding sites on metaphase chromosomes as well as in interphase cell nuclei. On average, a total of 3.43 +/- 1.59 binding sites were measured in metaphase spreads, and 2.69 +/- 1.00 in interphase nuclei. Microwave activation for denaturation and hybridization was tested to accelerate the procedure. The slides with the target material and the hybridization buffer were placed in a standard microwave oven. After denaturation for 20 sec at 900 W, hybridization was performed for 4 min. at 90 W. The suitability of a microwave oven for Fast-FISH was confirmed by the application to a chromosome 1-specific alpha-satellite probe. In this case, denaturation was performed at 630 W for 60 sec and hybridization at 90 W for 5 min. In all cases, the results were analyzed quantitatively and compared to the results obtained by Fast-FISH. The major binding sites were clearly discriminated by their brightness.

Chromosomes↗
PubMed1996

Optimization of Fast-FISH for alpha-satellite DNA probes.

It has been shown for several highly repetitive DNA probes that the newly introduced Fast-FISH (fast-fluorescence in situ hybridization) technique is well suited for quantitative microscopy. The advantage of omitting denaturing chemical agents (e.g., formamide) in the hybridization buffer results in a short hybridization time and a considerable reduction of the number of subsequent washing steps. Choosing the appropriate hybridization temperature and time allows to clearly discriminate major and minor binding sites by quantitative fluorescence microscopy. To further optimize the procedure with reference to reproducibility, a fully programmable thermal-cycler was applied for thermal de- and renaturation. Here, the optimized renaturation conditions for two commercially available alpha-satellite probes (specific for chromosomes 1 and X) are described. For the Boehringer chromosome-1-specific DNA probe, two highly fluorescent binding sites were obtained for 72 degrees C hybridization temperature and 60 min hybridization time. For the Oncor chromosome-X-specific DNA probe, the optimal conditions were found at 74 degrees C and 60 min hybridization time. In both cases the major binding sites were clearly discriminated from only a few weakly fluorescent minor binding sites on metaphase spreads as well as in interphase cell nuclei.

DNA Probes↗
PubMed1994

Rapid fluorescence in situ hybridization with repetitive DNA probes: quantification by digital image analysis.

Fluorescence in situ hybridization (FISH) has become an important tool not only in cytogenetic research but also in routine clinical chromosome diagnostics. Here, results of a quantification of fluorescence signals after in situ hybridization with repetitive DNA probes are reported using a non-enzymatic hybridization technique working with a buffer system not containing any formamide or equivalent chemical denaturing agents. Following simultaneous denaturation of both cells and DNA probes, the renaturation time was reduced to less than 30 min. For one of the DNA probes reasonable FISH-signals were even achieved after about 30 s renaturation time. In addition, the number of washing steps was reduced drastically. As a model system, two repetitive DNA probes (pUC 1.77, D15Z1) were hybridized to human metaphase spreads and interphase nuclei obtained from peripheral blood lymphocytes. The probes were labelled with digoxigenin and detected by FITC-anti-digoxigenin. The hybridization time was reduced step by step and the resulting fluorescence signals were examined systematically. For comparison the pUC 1.77 probe was also hybridized according to a FISH protocol containing 50% formamide. By renaturation for 2 h and overnight two FISH signals per nucleus were obtained. Using shorter renaturation times, no detectable FISH signals were observed. Quantification of the FISH signals was performed using a fluorescence microscope equipped with a cooled colour charge coupled device (CCD) camera. Image analysis was made interactively using a commercially available software package running on a PC (80486). For the pUC 1.77 probe the major binding sites (presumptive chromosomes 1) were clearly distinguished from the minor binding sites by means of the integrated fluorescence intensity. For the two (pUC 1.77) or four (D15Z1) brightest spots on the metaphase spreads and in the interphase nuclei hybridized without formamide, integrated fluorescence intensity distributions were measured for different renaturation times (0.5, 15, 30 min). The intra-nuclear variation in the intensity of the two brightest in situ hybridization spots appeared to be slightly higher (CV between 16 and 32%) than the corresponding variation in the metaphase spreads (CV between 10 and 19%). For the D15Z1 probe FISH signals were detected after hybridization without formamide and 15 min and 30 min renaturation. Always four bright spots were visible and tentatively assigned on the metaphase spreads (presumptive chromosome 15 and 9). The intensity variation of each pair of homologues in a metaphase spread showed a CV of 14 or 15%, respectively, for the presumptive chromosome 15, and 8 or 9%, respectively, for the presumptive chromosome 9.

Analog-Digital Conversion↗
PubMed1994

A rapid FISH technique for quantitative microscopy.

Results of quantitative microscopy for fluorescence in situ hybridization (FISH) signals with repetitive DNA probes (pUC 1.77 and D15Z1) are reported. A nonenzymatic hybridization technique was applied using fluorescein-12-dUTP labeled DNA probes and a buffer system not containing any formamide or equivalent chemical denaturing agents. Following thermal denaturation, the renaturation time was reduced to less than 30 min. The number of wash steps was reduced to one. For the pUC 1.77 probe, the major binding sites (chromosome 1) were distinguished from the minor binding sites by means of fluorescence intensity and spot size. The intensity variation of the two brightest FISH spots (major binding sites) in the same metaphase was 19% for 15 min renaturation time and 16% for 30 min renaturation time. For the D15Z1 probe, generally four bright spots were visible and tentatively assigned according to chromosome length and centromere position (chromosomes 15 and 9). The intensity variation of each two homologues in the same metaphase spread showed a coefficient of variation of 47% (15 min) and 22% (30 min) for chromosome 15, and 19% (15 min) and 15% (30 min) for chromosome 9. The results indicate that the applied technique can considerably accelerate the FISH procedure and is suited for quantitative microscopy.

DNA Probes↗
PubMed1978

Characterization of a suppressor-cell leukemia. Evidence for the requirement of an interaction of two T cells in the development of human suppressor effector cells.

To characterize the suppressor activity of neoplastic T cells from a child with acute lymphoblastic leukemia and hypogammaglobulinemia, we applied an in vitro assay that determines the capacity of pokeweed-mitogen-stimulated lymphocytes to mature into immunoglobulin-secreting cells. The geometric mean synthesis by peripheral blood lymphocytes from 12 normal persons was 3200 ng for IgM, 2447 ng for IgG and 1825 for IgA (2 X 10(6) cells per 12 days in culture). The patient's leukemic cells produced no detectable immunoglobulin and depressed the immunoglobulin production of normal lymphocytes by 85 to 100 per cent in co-culture experiments. However, suppression was observed only when co-operating normal T cells were present. Prior irradiation of either the leukemic T cells or the co-operating normal T cells nullified the suppressor effect. Therefore, an interaction between at least two different T-cell subsets may be required for the generation of suppressor effector T cells in man.

Agammaglobulinemia↗
PubMed1976

The role of suppressor cells in the pathogenesis of common variable hypogammaglobulinemia and the immunodeficiency associated with myeloma.

The role of suppressor cells in the pathogenesis of immunodeficiency was analyzed using a technique that permits study of the differentiation of B lymphocytes into immunoglobulin-synthesizing plasma cells. Lymphocytes from normals synthesized 4,910 ng of IgM, 1,270 ng of IgA, and 1,625 ng of IgG per 2 X 10(6) cells when cultured for 7 days in the presence of pokeweed mitogen. In contrast the lymphocytes from patients with common variable hypogammaglobulinemia did not synthesize significant quantities of immunoglobulin. When lymphocytes from 9 of 13 patients with common variable hypogammaglobulinemia studied were cocultured with normal lymphocytes, the synthesis of immunoglobulin by the normal lymphocytes was depressed by 75-100%. A comparable suppression of immunoglobulin synthesis by normal lymphocytes was observed when they were cocultured with T cells from hypogammaglobulinemic patients. These studies suggest that in some patients the disease common variable hypogammaglobulinemia may not be due to an intrinsic defect of B cells alone but may be cuased or perpetuated by an abnormality of regulatory T cells that act to suppress B-cell maturation and antibody production. Peripheral blood lymphocytes from myeloma patients also had a drastically reduced capacity to produce polyclonal immunoglobulins. Three of 6 myeloma patients tested had circulating mononuclear cells that suppressed immunoglobulin production by cocultured normal lymphocytes. Purified T cells from myeloma patients did not mediate this suppressor effect. These observations suggest that one mechanism for the humoral immune deficiency observed in myeloma patients is a block of polyclonal B-cell maturation by suppressor cells.

Agammaglobulinemia↗
PubMed1976

Defect in IgA secretion and in IgA specific suppressor cells in patients with selective IgA deficiency.

The nature of the defect in patients with selective IgA deficiency was investigated using a technique established to study terminal differentiation of B lymphocytes into immunoglobulin synthesizing and secreting cells. The peripheral blood lymphocytes from normal individuals had geometric mean synthetic rates of 4910 ng for IgM, 1625 ng for IgG and 1270 ng for IgA per 2 x 10(6) cells in culture for 7 days in the presence of pokeweed mitogen. The cultured lymphocytes from each of the 14 patients with selective IgA deficiency studied synthesized normal quantities of IgG and IgM but secreted less than 100 ng of IgA into the media. However, 11 of the 14 patients studied synthesized IgA by the 7th day in PWM stimulated cultures as assessed by staining for cytoplasmic IgA using fluorescein-labeled anti-IgA antisera. Synthesis and secretion of IgA by normal cells was not suppressed when they were co-cultured with lymphocytes from these patients that synthesize but do not secrete IgA. Three of the 14 patients did not have lymphocytes with IgA demonstrable in their cytoplasm following culture. When the lymphocytes from these 3 patients were co-cultured with normal lymphocytes and pokeweed mitogen the synthesis of IgA by the normal cells was depressed by 80 to 100%. Synthesis of IgG and IgM was not depressed. These studies suggest that lymphocytes cultured with pokeweed mitogen from the majority of patients with selective IgA deficiency can synthesize IgA but have a defect in IgA secretion. A smaller group of the patients do not synthesize IgA and have IgA specific suppressor cells that prevent B cells from maturing into IgA synthesizing and secreting cells.

Adolescent↗
PubMed1975

Impaired synthesis of polyclonal (non-paraprotein) immunoglobulins by circulating lymphocytes from patients with multiple myeloma Role of suppressor cells.

Since patients with myeloma have serious abnormalities of humoral immunity, we applied an in vitro assay to determine the capacity of B lymphocytes to mature into immunoglobulin-secreting cells. In peripheral blood lymphocytes from 22 normal persons, geometric mean immunoglobulin synthesis was 4910 ng for IgM, 1270 ng for IgA and 1625 ng for IgG. The synthesis rates of peripheral blood lymphocytes of 22 patients with myeloma were 458 ng for IgM, 321 ng for IgA and 218 ng for IgG. Circulating mononuclear cells from three of six patients tested suppressed polyclonal immunoglobulin synthesis by cocultured normal lymphocytes. Suppressive activity was not mediated by purified T cells alone. Removal of phagocytic mononuclear cells from lymphocyte populations of one patient nullified suppressive activity. Removal of phagocytic mononuclear cells from lymphocyte populations of a second patient led to a nearly 10-fold increase in polyclonal immunoglobulin synthesis. Therefore, host suppressor cells may play a part in the decreased capacity of B lymphocytes to secret immunoglobulin in certain patients with myeloma.

Adolescent↗
PubMed1975

Suppressor T cells in the pathogenesis of hypogammaglobulinemia associated with a thymoma.

The nature of the immunological defect in patients with hypogammaglobulinemia associated with a thymoma was investigated using a technique established to study the differentiation of lymphocytes into immunoglobulin synthesizing and secreting cells. Exhaustively washed peripheral blood lymphocytes were cultured for 7 days in RPMI-1640 medium supplemented with fetal calf serum in the presence of the lectin, pokeweed mitogen. The IgG, IgA, and IgM synthesized and secreted into the medium were measured by competitive double antibody radio-immunoassays. Twenty-two normal individuals synthesized 1625 ng of IgG, 1270 ng of IgA, and 4910 ng of IgM per 2 million lymphocytes in culture. In contrast, the three patients with hypogammaglobulinemia and a thymoma synthesized less than 100 ng of each class of immunoglobulin. When lymphocytes from 2 of the 3 patients studied were cocultured with normal lymphocytes and pokeweed mitogen, the synthesis of immunoglobulin by normal lymphocytes was depressed by a factor of 66 to 97%. Co-cultue of purified T cells from the hypogammaglobulinemic patients with normal lymphocytes resulted in an 87% suppression of immunoglobulin synthesis by the normal cells. However, no suppression of immunoglobulin synthesis was observed when preparations of B cells and macrophages depleted of T cells from the hypogammaglobulinemic patients were co-cultured with normal lymphocytes. In addition, in control studies no such suppression of immunoglobulin synthesis was seen when normal cells were co-cultured with lymphocytes from unrelated normals, patients with isolated IgA deficiency, patients with chronic lymphocytic leukemia or patients with the Sezary syndrome, a T cell leukemia nor were they inhibited when incubated with T cells from unrelated normals. These observations suggest that in some patients the hypogammaglobulinemia associated with a thymoma may be caused or perpetuated by an abnormality of regulatory T cells which suppress the maturation of lymphocytes into antibody producing cells.

Adolescent↗
PubMedDate not supplied

Optimization of fast-fluorescence in situ hybridization with repetitive alpha-satellite probes.

A rapid FISH (fluorescence in situ hybridization) technique (Fast-FISH) for quantitative microscopy has been recently introduced. For highly repetitive DNA probes the hybridization (renaturation) time and the number of necessary washing steps were reduced considerably by omitting formamide or equivalent denaturing chemical agents. Due to low stringency conditions major and minor binding sites of the probes used showed visible FISH signals well suited for quantitative image-microscopy. The discrimination of minor and major binding sites was possible by automated image-processing. Here, a further, quantitative optimization of the Fast-FISH technique is described that allows to clearly discriminate major and minor binding sites of alpha-satellite probes by an easy image classification parameter. With respect to the optimization it was necessary to verify two sensitive parameters (hybridization time and temperature) of the given rapid FISH protocol. As examples the systematic optimization for the two probes D12Z2 (major binding site on the centromere of chromosome 12) and D8Z2 (major binding site on the centromere of chromosome 8) are shown. The optimal hybridization conditions concerning rapidness and quality of chromosome morphology were obtained using a hybridization temperature of 70 degrees C and a hybridization time of 60 min. For these conditions major and minor binding sites were clearly discriminated by the intensity maximum Smax of the corresponding FISH-spots.

Binding Sites↗
PubMedDate not supplied

Painting of human chromosome 8 in fifteen minutes.

The technique of chromosome-in-situ suppression (CISS)-hybridization (chromosome painting) has now been well established. However, all standard protocols so far require long renaturation times (typically 12 hours and more). Here, we describe a new, extremely fast protocol for chromosome painting using a commercially available, directly fluorescence labelled probe for chromosome 8. The hybridization conditions used omit separate preannealing procedures and denaturing chemical agents. The renaturation time required for chromosome painting was reduced to 15 minutes. In addition, most washing steps were eliminated. As a consequence, the entire painting procedure was feasible in less than half an hour.

Chromosomes, Human, Pair 8↗
PubMedDate not supplied

Non-enzymatic, low temperature fluorescence in situ hybridization of human chromosomes with a repetitive alpha-satellite probe.

In all DNA-DNA in situ hybridization (ISH) procedures described so far in the literature, the production of single-stranded target DNA sequences plays a decisive role. This can be achieved either by enzymatic treatment at physiological temperatures or by the separation of double-stranded DNA sequences. Denaturation by heat and chemical agents (e.g. formamide) is regarded as a prerequisite for the non-enzymatic ISH process. However, additional mechanisms of a non-enzymatic ISH procedure are conceivable which do not require high temperature treatment combined with formamide. Here, we report on a non-enzymatic, non-formamide, low temperature, fluorescence, in situ hybridization (FISH) procedure which allowed a microscopic visualization and quantitative fluorescence analysis of the binding sites of a repetitive DNA probe. Following only probe denaturation at 94 degrees C, hybridization was performed at 52 degrees C for 30 min, i.e. at nearly physiological temperatures. Moreover, increasing the hybridization time to 3 hours indicated that hybridization sites became also visible at 37 degrees C. Since the protocols are based on recently described Fast FISH developments, the technique will be called Low Temperature Fast-FISH (LTFF).

Cell Nucleus↗