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U Ebener

Publications and source records attributed to U Ebener.

At least 19 recordsLinked to original sources
PubMed2000

Quality assurance in RT-PCR-based BCR/ABL diagnostics--results of an interlaboratory test and a standardization approach.

Here we describe the results of an interlaboratory test for RT-PCR-based BCR/ABL analysis. The test was organized in two parts. The number of participating laboratories in the first and second part was 27 and 20, respectively. In the first part samples containing various concentrations of plasmids with the ela2, b2a2 or b3a2 BCR/ABL transcripts were analyzed by PCR. In the second part of the test, cell samples containing various concentrations of BCR/ABL-positive cells were analyzed by RT-PCR. Overall PCR sensitivity was sufficient in approximately 90% of the tests, but a significant number of false positive results were obtained. There were significant differences in sensitivity in the cell-based analysis between the various participants. The results are discussed, and proposals are made regarding the choice of primers, controls, conditions for RNA extraction and reverse transcription.

Base Sequence↗
PubMed1999

wt1 gene expression in childhood leukemias.

The expression of the Wilms' tumor gene (wt1) was detected in various tissues during embryonic development. Mutations in the wt1 gene probably play an important role in certain tumors, e.g. the Wilms' tumor. Furthermore the expression of wt1 gene was found in some human leukemias. In the present study we investigated the expression of wt1 gene in several types of childhood leukemia by reverse transcriptase-polymerase chain reaction. Bone marrow or peripheral blood of 61 pediatric patients (48 at initial diagnosis, 13 at first or second relapse) were analyzed. wt1 gene expression was detected in 35/48 patients (73%) with newly diagnosed leukemias and in 12/13 cases (92%) who had suffered from relapse. The expression levels were higher for AML than for ALL. The frequency of wt1 expression in different subtypes of acute leukemia was compared with results found in adult patients. Our results show that the frequency of wt1 gene expression in acute childhood leukemias is similar to previous data reported for adults.

Acute Disease↗
PubMed1997

Minimal residual disease in leukemia in children.

Many chromosomal translocations involved in leukemia have been defined at the molecular level in recent years. In addition to advancing the understanding of pathological mechanisms underlying the transformation process, the cloning and sequencing of the genes altered by the translocations have provided new tools for diagnosis and monitoring of patients. In particular, the polymerase chain reaction (PCR) method yields sensitive and accurate diagnostic and prognostic information. Minimal residual disease (MRD) is not clearly defined. In ALL we define MRD as fewer than 5% blast cells in the bone marrow by conventional cytology and proof of leukemic cells with more sensitive methods. The techniques for detecting MRD are imaging for detection of single leukemic cells in the blood, bone marrow, or other tissues by means of immunocytology or PCR/RT-PCR. Highly sensitive PCR, immunocytology, FACS analysis, or conventional cytology are important tools to use in the process of deciding on appropriate therapy. Detection limits at present are 10(-2) for cytology and FISH, up to 10(-4) for immunological procedures, and 10(-5) to 10(-6) for PCR. But multiple methods also imply the possibility of mistakes (e.g., PCR). The question must be raised what method should be decisive in assessing MRD for evaluating autologous peripheral blood stem cells (PBSC) or autologous bone marrow transplants? Prospective studies will have to answer the question whether MRD should be treated or not and whether purging of bone marrow or PBSC is useful or damaging. When applied, should a positive or a negative immunopurging or a chemotherapeutic purging be used? MRD refers to the organism of the patient as well as to the peripheral blood stem cells and autologous bone marrow that had been taken before myeloablative therapy and kept for retransfusion.

Bone Marrow Transplantation↗
PubMed1997

Direct molecular genetic diagnosis and heterozygote identification in X-linked Emery-Dreifuss muscular dystrophy by heteroduplex analysis.

X-linked Emery-Dreifuss muscular dystrophy (EMD) is a very rare, relatively benign muscle disorder. The disease is associated with potentially lethal cardiac arrhythmias in affected males and some heterozygous females. X-linked EMD can be genetically distinguished from phenotypically similar autosomal EMD. Heterogenic mutations are identified as the cause of X-linked EMD. We introduced heteroduplex analysis to follow the segregation of heterogenic emerin gene mutations in the families of six unrelated EMD patients. Heteroduplex analysis was proved to be a simple, fast and reliable tool for direct molecular genetic diagnosis of EMD in male patients and identification of heterozygotes even in families where affected males are not available as index cases.

DNA Mutational Analysis↗
PubMed1992

Increased HIV-1 production in chronically infected H9 cells grown in protein-free medium.

Human T cell line H9 was established in a protein-free 1:1 mixture of Ham's F-12 and IMDM. After 230 passages (3 years) in protein-free medium, the cells designated H9-PF were infected with HIV-1. The infectivity titers of HIV-1 in cell culture medium were monitored by determining the median tissue culture infectious doses (TCID50). Additionally, the production of viral antigen in cells was measured by an immunoenzymatical alkaline phosphatase anti-alkaline phosphatase (APAAP) method using a monoclonal antibody against HIV-1-p24 antigen. In acutely infected H9-PF and H9 cultures similar TCID50 values and percentage of cells positive for p24 antigen were found. In contrast, both TCID50 values and percentage of cells positive for p24 antigen were by far greater in chronically infected H9-PF than in H9 cultures.

Cell Line↗
PubMed1989

Uptake and release of iodine-labelled m-iodobenzylguanidine in a neuroblastoma cell culture system and its importance in neuroblastoma therapy.

A neuroblastoma cell line was established from bone marrow of a patient in stage IV of the disease and used as a model system in order to elaborate experimental data of importance in neuroblastoma therapy, such as cell-drug interactions, the mode of uptake and conditions for storage and release. m-Iodo benzylguanidine (MIBG) is rapidly taken up from culture medium, giving high concentrations of cell-bound radioactivity reaching a maximum level 4 h after the addition of the compound. A removal of the radiopharmacon from the culture medium causes a dramatic loss of cell-associated radioactivity, suggesting that neuroblastoma cells are not able to retain MIBG in a drug-free environment. Replacement of labelled by unlabelled MIBG prevents a similar release and maintains high levels of cellular radioactivity. Variations of cell culture conditions only result in minor changes of uptake rates, whereas a pretreatment with drugs used in neuroblastoma chemotherapy harms the cells extensively: even after a short-term exposure the cells lose the capacity for MIGB uptake and fail to recover within a long period of incubation in growth medium. The importance of our results is discussed, leading to the following suggestions: 1. The performance of radiotherapy with labelled MIBG is recommended prior to the chemotherapy protocols. 2. Therapeutically effective radioactivity in tumor tissues may be maintained by the additional infusion of unlabelled MIGB.

3-Iodobenzylguanidine↗
PubMed1989

Immunological detection and definition of minimal residual neuroblastoma disease in bone marrow samples obtained during or after therapy.

Immunological staining by the alkaline phosphatase/anti-alkaline phosphatase (APAAP) technique has been used to recognize low levels of neuroblastoma cells in bone marrow mononuclear cells. Immunological phenotyping with 11 well characterized monoclonal antibodies was performed on 16 children with neuroblastoma and BM involvement during or after therapy. Neuroblasts were detected in 11 of 16 patients (0.1-5%), whereas BM biopsies on six of these patients were classified as normal. Aspirates, stained conventionally, were positive for pathological cells in three patients only. The comparison of the phenotype of the neuroblastoma cells at the time of diagnosis to the phenotype of the residual cells within one patient revealed differences. The phenotype of residual disease in different patients on the other hand showed a unique pattern. The above mentioned results lead to the conclusion that the immunological procedure is particularly suitable for the analysis of minimal residual neuroblastoma since the technique allows very minor cell populations to be identified in BM samples.

Adolescent↗
PubMed1989

[Immunologic identification of undetectable neuroblastoma cells by current cytohistological studies of bone marrow samples].

Immuno-alkaline phosphatase staining (APAAP technique) has been used to identify neuroblastoma cells on bone marrow samples from 12 children at various stages of the disease. On 72 occasions immunological analyses were performed using a panel of monoclonal antibodies which selectively bind to cells of neuroectodermal origin. In 57 of these procedures, tumor cells were detected, whereas histological and cytological analyses revealed pathological cells in 45 and 37 cases respectively. Reactivity of minimal residual tumor cells--mainly with three MAbs (UJ13A, UJ167.11, A2B5)--points to the fact that these cells belong to a resistant neuroblastoma clone, which remains in bone marrow despite intense therapy. Our study demonstrates that immunological staining may identify and define a small number of neuroblastoma cells which are not yet detectable by traditional histological and cytological criteria.

Antibodies, Monoclonal↗
PubMed1988

[Initial results of genetic carrier diagnosis in risk pedigrees with hemophilia A and B in East Germany].

In 7 families at risk for hemophilia A 42 individuals were evaluated by the Taq I polymorphism of the extragenic probe St 14.1 and the Bcl I as well the Hind III polymorphism of the intragenic probe F8e16-19. 15 out of 20 females of the core families were identified as carriers, under them all of the obligate heterozygotes. 5 individuals were excluded as carriers. The heterozygosity for each of the RFLPs was found to be between 30% and 79%. Combining the single data in 96% heterozygosity was found under the individuals tested. A linkage disequilibrium was found between the Bcl I and Hind III polymorphismus of the probe F8e16-19. A family at risk for hemophilia B including 5 individuals was studied using the Taq I and Xmn I polymorphisms of the probe P1. In one of two females the carrier state could be excluded in the other one confirmed.

Genetic Carrier Screening↗
PubMed1981

[Implications of terminal deoxynucleotidyl transferase activity in various forms of childhood leukemia (author's transl)].

Terminal deoxynucleotidyl transferase (TdT) was examined in mononuclear peripheral blood cells (pB) and bone marrow (BM) specimens of 63 children with acute leukemia (AL). The enzyme activity in normal specimens (pB, BM) was below 0.2 U/10(8) cells; whereas, 49 of the 52 children with acute lymphoblastic leukemia (ALL) at diagnosis showed an activity in the range 1.2--60 U/10(8) cells. 2 of the remaining 3, devoid of TdT activity, were found to be B-cell leukemia. Patients with acute non-lymphoblastic leukemia (ANLL) were generally TdT-negative. Elevated level of TdT activity was detected in only one of 11 children with ANLL. In one patient with acute leukemia two distinct populations of cells with lymphoblastic (87%) and myeloblastic characteristics were evident. The clinical course and cell marker studies were consistent with the interpretation of a defect at the level of the common stem cell giving rise to a TdT-positive lymphoblastic cell population at diagnosis and, following the initial ALL-therapy (4 weeks), a predominant TdT-negative myeloblastic population. TdT as a marker for the modulation of chemotherapy was examined in the remission phase of the disease. Of the nine patients in the first 3 months of remission, one was found to have elevated level of TdT activity (2.5 U/10(8) cells). These data define the usefulness of TdT in the classification of acute leukemia.

Acute Disease↗
PubMed1979

Purification and biochemical characterization of a virus-specific reverse transcriptase from human osteosarcoma tissue.

A RNA-dependent DNA polymerase (RTase) was purified from human osteosarcoma tissue by successive column chromatography of the microsomal fraction on DEAE-cellulose (DE-23 and DE-52) and phosphocellulose. The purified enzyme has a molecular weight of about 68,000, a pH optimum of 8.1, a Mg2+ optimum of 0.8 mM, Mn2+ optimum of 1.0 mM and a KCl optimum of 60 mM. The enzyme transcribes (rA)n . (dT)12, (rC)n . (dG)12-18 and (2-O-methyl C)n . (dG)18, but is unable to transcribe (dA)n . (dT)10. The enzyme has no catalytic activity in the presence of oligodeoxynucleotide initiators alone, indicating the absence of terminal deoxynucleotidyl transferase. The purified enzyme is able to transcribe the heteropolymeric regions of a 70S RNA from R(Mu)LV. The presented data support the presence of a RNA-dependent DNA polymerase in human osteosarcoma tissue with biochemical properties, resembling those of C-type RNA tumor viruses.

Adolescent↗