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Biomedical subjects

D Niethammer

Publications and source records attributed to D Niethammer.

At least 127 records · Page 7Linked to original sources
PubMed1994

High mdr1- and mrp-, but low topoisomerase II alpha-gene expression in B-cell chronic lymphocytic leukaemias.

Fifteen samples from 11 patients suffering from chronic lymphocytic leukaemia (CLL; 5 untreated, 6 chemotherapeutically treated) were analysed for their individual gene expression of the multidrug resistance (MDR) associated genes encoding mdr1/P-glycoprotein, mrp, and topoisomerase II alpha/beta-isoenzymes by a complementary DNA polymerase chain reaction (cDNA-PCR) approach. The expression of glyceraldehyde-3-phosphate dehydrogenase (gapdh) served as standard. Thereby, we generally found high mdr1- and mrp-, but low topoisomerase II alpha-mRNA levels. While mdr1 levels of the CLL samples were mostly found to be in the range of values measured in the T-lymphoblastoid, P-glycoprotein MDR cell line CCRF VCR 100, mrp levels were usually found to be 2-4-fold higher compared therewith. This might represent a multifactorial MDR in CLL. In contrast to the low or even absent topoisomerase II alpha gene expression, however, the expression of the topoisomerase II beta gene was generally high in the CLL lymphocytes exceeding the value observed in the cell line CCRF VCR 100 up to 5-fold. mdr1 gene expression correlated significantly with mrp gene expression in samples from patients having received chemotherapy (rs = 0.5833, P < 0.05, n = 10). In two patients the follow-up analysis revealed combined increases in mdr1- and mrp-gene expression levels in the course of the disease.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Determination of 2-5A synthetase and 2-5A phosphodiesterase in neuroblastoma cells by analytical capillary isotachophoresis: effects of cytokines and comparison with radioenzymatic methods.

2-5A Synthetase and 2-5A phosphodiesterase were determined by analytical capillary isotachophoresis in comparison to radioenzymatic methods. By means of isotachophoretic analysis, a frequently used radioenzymatic 2-5A synthetase assay was optimized and the results of both assays were compared. Using the isotachophoretic assay the influence of interferon-related cytokines (tumor necrosis factor-alpha and interleukin-2) on 2-5A synthetase induction in neuroblastoma cells was estimated. In contrast to mononuclear blood cells, the tumor necrosis factor induced 2-5A synthetase in these cells. 2-5A Phosphodiesterase was determined using an isotachophoretic assay and a radioenzymatic method. Degradation of A2'p5'A2'p5'A (trimeric form of 2-5A core) was measured by isotachophoresis whereas degradation of a mixture of phosphorus-32 labeled 2-5A cores was registered by radioenzymatic assay. Activity of 2-5A phosphodiesterase was only insignificantly enhanced by interferon in mononuclear blood and neuroblastoma cells. In contrast to the radioenzymatic assays, an accurate determination of 2-5A synthetase as well as of 2-5A phosphodiesterase is possible using the isotachophoretic method because the reactions are followed by measuring the substrates ATP and A2'p5'A2'p5'A, respectively.

2',5'-Oligoadenylate Synthetase↗
PubMed1994

Rapid method for purification of CD56+ natural killer cells with preferential enrichment of the CD56bright+ subset.

A rapid method for the purification of CD56+ natural killer (NK) cells with the preferential enrichment of the CD56bright+ subset is described. The method is based on the adsorption of CD56+ cells indirectly stained with a biotinylated antibody on an avidin-coated column. The adsorbed CD56+ cells can then be squeezed out mechanically without damaging the viability or the function of the cells. Starting from peripheral mononuclear cells from adult donors, the recovery of the CD56+ cells was 11.9 +/- 9.0% (n = 8), the purity 93.0 +/- 4.9% (n = 10), and the enrichment factor 7.5 +/- 1.7 (n = 8). Further phenotypic classification of the CD56+ cells into CD56dim+ and CD56bright+ cells showed a preferential enrichment of the CD56bright+ phenotype with a recovery of 40.5 +/- 19.6% (n = 8), a purity of 30.3 +/- 13.2% (n = 8), and an enrichment factor of 29.8 +/- 7.2 (n = 8). In conclusion, the described method allows the rapid purification of CD56+ NK cells with the preferential enrichment of the CD56bright+ subset.

Adult↗
PubMed1994

Ascorbic-acid-mediated iron release from cellular ferritin and its relation to the formation of DNA strand breaks in neuroblastoma cells.

Ascorbic acid at pharmacologically attainable concentrations effectively inhibited the growth of the catecholamine-positive neuroblastoma cell line SK-N-SH; it inhibited LS cells to a smaller extent and catecholamine-negative SK-N-LO cell growth least effectively. In all three cell lines high concentrations of H2O2 were found. Since ascorbic acid was shown to release iron from ferritin in vitro and to keep it in the reduced state, we suggested that it acted as a pro-oxidant in ferritin-rich neuroblastoma cells in the presence of H2O2 and Fe2+ (Fenton reaction), implying iron release from cellular ferritin. We show here that iron could be mobilized from cellular ferritin by 1 mM ascorbic acid in iron-59-preloaded SK-N-SH and LS cells, but not in SK-N-LO cells. In agreement with these results, DNA strand break formation by ascorbate was only observed in SK-N-SH and LS cells. In SK-N-LO cells, DNA strand breaks could be induced by a combination of 1 mM ascorbic acid and 100 microM H2O2. Since cell-damaging effects caused by chemotherapy further facilitate iron release from ferritin, we conclude that ascorbate could be a powerful enhancer of some cytostatic drugs in neuroblastoma therapy.

Ascorbic Acid↗
PubMed1994

Drug-induced changes in the expression of MDR-associated genes: investigations on cultured cell lines and chemotherapeutically treated leukemias.

The induced expression of multiple drug resistance (MDR)-associated genes as a direct response of tumor cells to antineoplastic drugs could be an important factor influencing the success of cancer chemotherapy. We investigated the effects of such compounds on mdr1/P-glycoprotein (P-gp) gene expression and drug sensitivities in the T-lymphoblastoid human cell line CCRF-CEM and MDR sublines. Thereby, we observed that actinomycin D or adriamycin administered at sublethal concentrations induced increases of mdr1 mRNA levels and resistance within 72 h. Furthermore, on leukemia cell samples collected before and after chemotherapy we checked by a complementary DNA polymerase chain reaction (cDNA-PCR) approach for similar alterations in the relative expression levels of the MDR-associated genes (a) mdr1/P-gp (b) mrp (MDR related protein), and (c) the topoisomerase II isoforms alpha and beta. We found a concomitant increase in mdr1 and mrp gene expression combined with a decreased expression of topoisomerase II alpha in the course of the second relapse of an acute lymphoblastic leukemia (ALL). This points to the emergence of at least three different MDR mechanisms in this type of leukemia unresponsive to chemotherapy. A chronic myeloid leukemia (CML) in blast crisis, however, showed combined increases in mdr1 (about 20-fold) and mrp (about four fold) gene expression after intense but unsuccessful chemotherapy over a 6-month period. Our results indicate the occurrence of induced resistance in vitro and in vivo and suggest a contribution of the newly identified ATP-binding cassette (ABC) transporter MRP in MDR.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Chemotactic activity of substances derived from antibody-loaded tumor cells on granulocytes.

Chemotactic activity of granulocytes attracted by tumor cells loaded either with anti-ganglioside monoclonal antibodies (mAb) or with antibody-glucose oxidase conjugates (mAb-GO) was investigated. The melanoma cell line SK-Mel-28 which expresses the ganglioside GD3 at high density as well as the neuroectodermal cell line SK-N-LO which expresses GD2 were used for the experiments. In the presence of 50% human AB-serum, antibody-loaded tumor cells induced chemotactic activity on granulocytes, probably due to the generation of C3a/C5a which could be detected in serum incubated with anti-GD3 loaded SK-Mel-28 cells. Both compounds could also be detected in vivo in the plasma of patients suffering from neuroblastoma during therapy with anti-GD2 antibodies. In another set of experiments mAb-GO conjugates generating high amounts of H2O2 in the presence of glucose were bound to these tumor cells. A significant lipid peroxidation could be observed in the simultaneous presence of iron and ascorbate. The lipid peroxidation products were measured as thiobarbituric acid-reactive substances (TBARS) and were also shown to induce chemotactic effects on granulocytes.

ABO Blood-Group System↗
PubMed1994

Activation of human T cells by the superantigen Staphylococcus enterotoxin B: analysis on a cellular level.

Superantigens interact with and activate a sizeable fraction of T cells characterized by expression of specific V beta gene segments of their antigen receptor. The massive activation of T cells in an organism is considered responsible for clinical symptoms associated with superantigen-producing bacteria. Here we studied the in vitro activation of human T cells by the superantigen Staphylococcus Enterotoxin B on a cell by cell basis. Superantigen-reactive T cells were stained with a V beta 12-specific monoclonal antibody and analyzed in a cytofluorograph. Blast formation of SEB-reactive T cells occurs within 12 h and reaches a plateau after 24 h. Double-staining of V beta 12+ T cells with antibodies against different T cell activation or adhesion surface molecules revealed a time-dependent differential upregulation for CD2, CD11 = LFA-1, CD25, CD28, CD69, and HLA-DR. The expression of CD3, CD4 and CD5 was not influenced by the superantigen. The rapid phenotypic changes of superantigen reactive T cells in terms of marker expression and cell size could provide early tools in diagnosing diseases caused by superantigens.

Antigens, CD↗
PubMed1994

[Technical aspects of autologous bone marrow transplantation].

Autologous bone marrow transplantation has become an established procedure in the treatment of malignancies. Especially in patients with hematological malignant diseases, who have no compatible allogeneic bone marrow donor or in patients with solid tumors, in whom the threshold of irreversible bone marrow toxicity is reached by the intensified chemotherapy, this procedure allows the use of a myeloablative therapy. As stem cell sources, bone marrow (BM), peripheral blood-derived stem cells (PBSC), and umbilical cord blood can be used. The risk of contaminating malignant cells in BM or PBSC can be reduced by an in vitro purging of the stem cells. In principle, two different methods exist: one is the negative depletion of contaminating tumor cells and the other is the positive selection of non-malignant hematopoietic pluripotent stem cells. Which of the purging principles will be most effective can only be answered through clinical studies.

Bone Marrow Purging↗
PubMed1994

[Autologous bone marrow transplantation].

Autologous bone marrow transplantation (ABMT) is a treatment modality increasingly used to treat a growing number of malignancies both in children and in adults. The theoretical rationale is the transfer of dose-action relationship from pharmacological theory to clinical practice. The basic principles, the important indications and the problems of this particular approach are discussed. Normally bone marrow from the iliac crest is used as stem cell source, but the possibilities of the approach become broader by using peripheral blood stem cells (PBSC). This treatment modality and its particular use in neuroblastoma is described demonstrating also the results.

Adult↗
PubMed1993

Isotachophoresis of nucleic acid constituents.

Determination methods for purine and pyrimidine bases, nucleosides, nucleotides and related compounds using analytical capillary isotachophoresis are reviewed. First, the isotachophoretic characterization of these compounds, as well as methods for sample preparation prior to analysis, and the different ways of detecting unknown substances in complex biological systems are described. Then applications of isotachophoretic analysis in medical diagnosis and biomedical research are reviewed. In particular, the analysis of purines, pyrimidines, nucleosides and related compounds in blood and serum for the diagnosis and treatment of inherited diseases and cancer is described. Selected applications of nucleotide analysis in biomedical research using different tissue extracts are also reviewed, and some examples of nucleotide-dependent enzymic reactions, which were performed by means of analytical isotachophoresis, are presented.

Animals↗
PubMed1993

Synovial sarcoma of childhood and adolescence. Report of the German CWS-81 study.

BACKGROUND: Synovial sarcoma is the third most common pediatric soft tissue tumor. It requires an aggressive approach to achieve a cure. However, optimal treatment modalities adapted to disease extension and histologic variants have not been determined because there is little information about prospectively treated patients. METHODS: A multicenter trial for soft tissue sarcomas (Protocol CWS 81) was conducted in West Germany between 1981-1985, and 31 patients with synovial sarcoma were registered. Treatment included multiagent chemotherapy and irradiation after initial tumor excision or biopsy. The male-female ratio in this group was 1:1.6 with a median age of 14 years (range, 1-19 years). The median follow-up time after diagnosis was 101 months (range, 77-131 months). RESULTS: The overall event-free survival (EFS) for patients with synovial sarcoma was 74.2% at 5 years. Group I-II tumors had a significantly better prognosis than those in Group III-IV (EFS at 5 years 84.4% and 58.3%, respectively; P = 0.024). Small tumors (< 5 cm) responded better than larger tumors (> or = 5 cm; EFS, 93% versus 58%; P = 0.029). Synovial sarcoma involved the extremities in 28 patients who had a better outcome compared with those with extremity rhabdomyosarcoma in this study (EFS for Group I-IV was 82% versus 24%, P = 0.001). CONCLUSIONS: The results appeared superior to previous experience using radical surgery alone and suggested that after initial, nonmutilating surgery, adjuvant chemotherapy, and irradiation contributed to the improved long-term survival.

Adolescent↗
PubMed1993

Determination of ascorbic acid by isotachophoresis with regard to its potential in neuroblastoma therapy.

Analytical capillary isotachophoresis was used to determine ascorbic acid (AA) in different matrices (cell-free system, neuroblastoma cell extracts and urine). The system for purging bone marrow of neuroblastoma cells, including 6-hydroxydopamine (6-OHDA) and AA, was analysed with regard to the interaction of AA with 6-OHDA and its autoxidation product, hydrogen peroxide. Furthermore, analyses concerning the uptake of AA into neuroblastoma cells as well as its excretion in urine after uptake of large amounts were carried out.

Ascorbic Acid↗
PubMed1993

Mdr1/P-glycoprotein expression in natural killer (NK) cells enriched from peripheral or umbilical cord blood.

The sensitivity to antineoplastic agents of subpopulations of haematopoietic cells during cancer chemotherapy is an open question. The performance of natural killer (NK) cells, possibly assisting the elimination of tumour cells under drug treatment might be of particular interest. We examined the expression of the transmembrane multidrug transporter mdr1/P-glycoprotein in NK-cells (CD56+) enriched from the peripheral blood or the umbilical cord blood from healthy donors by indirect immunocytofluorescence using the monoclonal P-glycoprotein antibody C219 and a polymerase chain reaction (PCR) approach with amplimers specific for the human mdr1 cDNA. As the antibody C219 apparently cross-reacts with the human mdr3 gene product whose functions are as yet unclear we also checked expression of this gene by PCR using mdr3 specific amplimers. Distinct, but rather inhomogeneous mdr1/P-glycoprotein expression was found in NK-cells enriched from the peripheral blood. NK-cells enriched from the umbilical cord blood showed quite strong mdr1 expression levels throughout, exceeding the values found in the moderately multidrug-resistant cell line CCRF VCR 100 which is permanently cultivated in the presence of 100 ng/ml vincristine. Mdr1/P-glycoprotein expression was mirrored by lowered sensitivities of the cultivated NK-cells towards actinomycin D or adriamycin. The drug sensitivity could be modulated by treatment of the cells with the immunosuppressive drug cyclosporin A. Expression of the mdr3 gene was low or absent in all NK-cell samples examined so far.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Expression of an early myelopoietic antigen (CD33) on a subset of human umbilical cord blood-derived natural killer cells.

A new subset of natural killer (NK) cells was identified in human umbilical cord blood. This subset of CD56+/CD3- NK cells co-expressed the CD33 antigen, which is present on early hematopoietic progenitors confined to the myeloid lineage. The percentage of the CD56+/CD33+ cells among the CD56+/CD3- NK cells was 7.9 +/- 6.6% (n = 27) with a range of 1.4-25.5% and a considerable individual variability. Additionally, the majority of freshly isolated CD56+/CD33+ cells co-expressed the CD2 and CD7 antigen, a minor proportion co-expressed the CD8 antigen but essentially all of the cells stained negative for CD16 and CD57. Morphological analysis of the CD56+/CD33+ cells showed the features of large agranular lymphocytes. From some of the samples, the CD56+/CD33+ NK cells were cultivated and expanded in vitro by incubation of the cells with interleukin 2 (IL-2) for up to 50 days. Morphological analysis of the cultured CD56+/CD33+ cells showed the features of large granular lymphocytes (LGL). The IL-2-expanded CD56+/CD33+ NK cells showed only a low cytolytic activity against K562 target cells, whereas most of the NK activity of the expanded cells was contributed by the CD56+/CD33- NK cells.

Antigens, CD↗
PubMed1993

Differential expression of T cell receptor variable beta genes on CD4+ and CD8+ T cells: influence by sex linked genes?

We examined the expression of seven V alpha or V beta T cell receptor (TCR) segments on human CD4+ and CD8+ T cells. Confirming previously published results, we found a preferential expression of four V segment gene products on CD4+ T cells. One of these markers (V beta 6.7) was constantly expressed on more CD4+ T cells than CD8+ T cells. None of the analyzed blood samples showed a complete deletion of T cells expressing a particular V beta gene segment. In addition, our data provide the first evidence that genes on sex chromosomes may influence the formation of the human T cell repertoire. The ratio of CD4+/CD8+ T cells expressing V beta 12 gene products was always > or = 1 in female donors, whereas approximately 30% male donors exhibited more CD8+V beta 12+ T cells than CD4+V beta 12+ T cells.

Adult↗