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R Mertelsmann

Publications and source records attributed to R Mertelsmann.

At least 91 records · Page 5Linked to original sources
PubMed1996

In vitro differentiation of CD34+ hematopoietic progenitor cells toward distinct dendritic cell subsets of the birbeck granule and MIIC-positive Langerhans cell and the interdigitating dendritic cell type.

We have demonstrated recently that Birbeck granule-positive Langerhans cells (LC) can be derived from CD34+ peripheral blood progenitor cells in the presence of a seven-cytokine cocktail (CC7-7). Here, we show that the sequential use of early-acting hematopoietic growth factors, stem cell factor, interleukin (IL)-3, and IL-6, followed on day 8 by differentiation in the two-factor combination IL-4 plus granulocytemacrophage colony-stimulating factor (GM-CSF) (CC4GM) is more efficient and allows the cells to be arrested in the LC stage for more than 1 week while continuous maturation occurs in CC7-7. Maturation of LC to interdigitating dendritic cells (DC) could specifically be induced within 60 hours by addition of tumor necrosis factor-alpha (20 ng/mL) or lipopolysaccharide (100 ng/mL). Using LC that had been enriched to greater than 90% CD1a+ cells by an immunoaffinity column, we were able to define clear-cut differences between LC and DC that corroborate data of the respective cells derived from epithelial borders (LC) or from lymph nodes (LN) and spleen (DC). Thus, molecules and functions involved in antigen (AG) uptake and processing were highly expressed in LC, while those involved in AG presentation were at maximum in DC. LC were CD1a+2 DR+2, CD23+, CD36+, CD80-, CD86-, and CD25-, while DC were CD1a+/- DR+3, CD23-, CD36-, CD80+, CD86+2, and CD25+, CD40 and CD32 were moderately expressed and nearly unchanged on maturation, in contrast to monocyte-derived DC. Macropinocytosis of fluorescein isothiocyanate-dextran was dominant in LC, as were multilamellar major histocompatibility complex (MHC) class II compartments (MIICs), which were detected by electron microscopy. The functional dichotomy of these cell types was finally supported by testing the AG-presenting cell function for tetanus toxoid to primed autologous T-cell lines, which was optimal when cells were loaded with AG as LC and subsequently induced to become DC.

Antigen Presentation↗
PubMed1996

Interleukin-10 increases Bcl-2 expression and survival in primary human CD34+ hematopoietic progenitor cells.

Bcl-2 expression has been shown in hematopoietic progenitor cells. Through the use of Bcl-2 specific antisense oligonucleotides we herein report the biologic importance of Bcl-2 expression in primary human CD34+ hematopoietic progenitor cells committed to the myeloid lineage. In bone marrow or peripheral blood derived CD34+ cells Bcl-2 specific antisense decreased cell survival and inhibited the outgrowth of mixed myeloid colonies. A short-term overnight pretreatment of CD34+ cells with 25 mumol/L of Bcl-2 antisense in liquid culture completely ablated the growth of granulocyte-macrophage colony-forming cells (GM-CFC) in a subsequent 14 days methylcellulose colony assay. Control experiments using corresponding Bcl-2 sense or nonsense oligonucleotides did not significantly impair cell survival or growth of GM-colony-forming unit. Western blot analyses revealed the Bcl-2 antisense dependent inhibition of expression of the Bcl-2 protein in CD34+ progenitor cells. Furthermore, regulation of Bcl-2 expression by various cytokines including interleukin-10 (IL-10) was studied. IL-10's effects on the formation of mixed myeloid colonies were examined in the absence or presence of Bcl-2 specific antisense. In the absence of Bcl-2 antisense IL-10 significantly extended the colony forming potential of mixed myeloid colonies to 14 days. In the presence of Bcl-2 antisense rhIL-10 completely restored GM-CSF driven colony growth. Fluorescent microscopy, Western blot analysis, and reverse transcriptase-polymerase chain reaction revealed the IL-10 dependent increase in cellular expression of Bcl-2 protein and Bcl-2 mRNA transcripts in CD34+ cells. Thus these results show that Bcl-2 expression is necessary for the formation of GM-CSF-dependent colony growth in vitro and that rhIL-10 increases Bcl-2 expression and survival in primary human CD34+ hematopoietic progenitor cells that are committed to the myeloid lineage.

Antigens, CD34↗
PubMed1996

Constitutive activation of STAT proteins in primary lymphoid and myeloid leukemia cells and in Epstein-Barr virus (EBV)-related lymphoma cell lines.

Although various molecular mechanisms of STAT protein (signal transducers and activators of transcription) activation have been identified, little is known about the functional role of STAT-dependent transcriptional activation. Herein we report the constitutive nuclear localization, phosphorylation, and DNA-binding activity of STAT proteins in leukemia cells and lymphoma cell lines. With the use of oligonucleotide probes derived from the Fc gamma RI promoter, the beta-casein promoter and a STAT-binding element in the promoter of the Bci-2 gene constitutive activation of STAT proteins was detected in untreated acute T- and C/B-leukemia cells (3 of 5 and 12 of 19 patients, respectively). Supershift analyses using Stats 1-6 specific antisera showed the constitutive DNA binding activity of Stat5 in these cells. Confocal microscopy revealed the nuclear localization of Stat5 and Western blot analyses showed tyrosine phosphorylation of Stat5 in nuclear extracts of acute leukemia cells. In contrast, peripheral blood mononuclear cells did not display constitutive STAT-DNA interaction. Further studies were performed on freshly isolated acute myeloid leukemia cells as well as on cell line derived K562, lymphoblastoid cells (LCL), and Burkitt's lymphoma cells (BL). Fluorescence microscopy, gelshift, and supershift experiments showed the nuclear localization and constitutive DNA-binding activity of Stat5 in K562 cells. Stat1 and Stat3 were constitutively activated in freshly isolated AML cells (10 of 14 patients) and in Epstein Barr virus-positive or interleukin-10 expressing permanent LCL and BL cells. Thus, these data indicate a differential pattern of STAT protein activation in lymphoid or myeloid leukemia and in lymphoma cells.

Acute Disease↗
PubMed1996

Developmental regulation of myeloid gene expression and demethylation during ex vivo culture of peripheral blood progenitor cells.

Expression of tissue- and development-specific genes is coordinately regulated during maturation of hematopoietic precursor cells toward functional, end-stage peripheral blood (PB) cells. To study the expression and methylation of several myeloid-specific genes during in vitro differentiation of normal hematopoietic progenitor cells, we used a model of CD34+ selected PB progenitor cells (PBPCs). PBPCs from six patients with solid tumors were recruited by standard-dose chemotherapy and subsequent administration of recombinant granulocyte colony-stimulating factor (G-CSF). PBPCs were collected and CD34+ cells selected by immunoadsorption columns using a biotinylated anti-CD34 monoclonal antibody. Enriched cells contained between 78% and 90% (median, 84%) CD34+ cells as determined by fluorescence-activated cell sorting analysis. Cell preparations were cultured in the presence of interleukin-1 beta (IL-1 beta), IL-3, IL-6 and stem cell factor and with or without G-CSF for various time intervals up to 20 days. Genes for CD34 surface antigen, lysozyme (LZM) and myeloperoxidase (MPO) were examined by RNA and DNA analyses. A rapid and early downregulation of CD34 transcripts was observed, with concomitant, time-dependent upregulation of expression of both the LZM and MPO genes. These effects were enhanced in the presence of G-CSF. Analysis of the DNA methylation status at key sites within these genes showed a pattern of differentiation- and expression-associated demethylation of the LZM gene, which was also enhanced by G-CSF, and constitutive and unaltered demethylation at key regions of the CD34 and MPO genes. In conclusion, the genes for CD34, LZM, and MPO are regulated during in vitro culture of very immature PBPCs in the presence of stem cell factor, IL-1, IL-3, IL-6; their effects are enhanced by G-CSF.

Antigens, CD34↗
PubMed1996

Generation of antigen-presenting cells for soluble protein antigens ex vivo from peripheral blood CD34+ hematopoietic progenitor cells in cancer patients.

Peripheral blood CD34+ hematopoietic cells (PBPC) mediate hematopoietic reconstitution in cancer patients after autologous transplantation and can be expanded ex vivo in the presence of colony-stimulating factors. This study shows that functionally active antigen-presenting cells (APC) for soluble proteins are generated and expanded in these PBPC cultures. CD34+ cells were cultured ex vivo in medium containing stem cell factor, interleukin-1 beta (IL-1 beta), IL-3, IL-6, and erythropoietin (EPO). The cells from these cultures developed into very potent APC of tetanus toxoid and purified derivative of tuberculin for autologous T cells in vitro. The antigen-presenting capacity of these cell was maintained for at least 38 days of culture. These APC resembled immature cells of the myelomonocytic cell lineage by surface marker, immunocytochemistry and ultrastructural analysis. Such APC might be able to present antigens from certain tumors to the immune system.

Antigen-Presenting Cells↗
PubMed1996

Simultaneous detection of cytokine and immunophenotype at the single cell level by immunoenzymatic double staining.

The goal of this study was to establish a generally applicable immunoenzymatic method for the simultaneous detection of cytokine and immunophenotype at the single cell level. Evaluating various cell preparations and staining protocols, we found that permeabilization by saponin (0.1%) is very efficient, in combination with glutaraldehyde (0.04%) as fixative. Among various staining procedures, sequential immunoperoxidase labelling of the cytokine by use of diaminobenzidine, and detection of the immunophenotype by use of 4-chloronaphthol proved most discriminative. The typical localization of the cytokine reaction product ('Golgi staining') within the cell, and the 'ring-like' staining for the immunophenotype on the cell surface, allowed precise identification of double-labelled cells. Primary monoclonal antibodies from the same species could be used without loss of sensitivity and specificity for either or both antigens. This method thus provides the opportunity to study morphology, cytokine and immunophenotype simultaneously at the single cell level with standard equipment. Its application for the analysis of tissue samples is in progress, and may allow us to incorporate the cytokine-type as a new parameter in histopathological diagnostics.

Adult↗
PubMed1996

Recognition by human Vgamma9/Vdelta2 T cells of melanoma cells upon fusion with Daudi cells.

Daudi Burkitt's lymphoma cells, unlike other tumor cell lines, stimulate human T cells coexpressing the variable (V) region genes TCRG-V9 and V TCRD-V2 to proliferate and secrete lymphokines. Hybrids, derived by the fusion of Daudi cells with the human melanoma cell line MZ2-MEL 2.2, retain the morphology of melanoma cells. Unlike the parental melanoma cell line, these Daudi x MZ2-MEL 2.2 hybrids stimulate secretion of tumor necrosis factor (TNF) and granulocyte/macrophage colony stimulating factor (GM-CSF) by CD4-positive Vgamma9/Vdelta2 T-cell clones. Whereas the stimulator phenotype of Daudi cells behaves as a dominant trait in Daudi x melanoma hybrids, the expression of B-cell differentiation markers is suppressed. Thus, the gamma/delta T-cell ligand expressed by Daudi cells behaves as a dominant tumor antigen in Daudi x melanoma hybrids and is unrelated to the differentiated B-cell phenotype. Dominant expression of the Daudi ligand for human Vgamma9/Vdelta2 T cells in these hybrids may provide a basis for defining the stimulatory principle at the molecular level.

Burkitt Lymphoma↗
PubMed1996

Phase II trial of intravenous endotoxin in patients with colorectal and non-small cell lung cancer.

We report the immunological and clinical results of a phase II trial with intravenously administered highly purified endotoxin (Salmonella abortus equi) in patients with advanced cancer. 15 patients with non-small cell lung cancer and 27 with colorectal cancer were entered into the study. 37 evaluable patients received at least four injections of endotoxin (4 ng/kg body weight) and 1600 mg ibuprofen orally in 2-week intervals. Transient renal (WHO grade 0-1) and hepatic (WHO grade 0-4) toxicities occurred in several patients. Constitutional side-effects such as fever, chills and hypotension could not be prevented completely by pretreatment with ibuprofen. 3 patients in the colorectal cancer group demonstrated objective responses (1 complete remission (CR), 2 partial remission (PR)). The complete remission has been maintained for more than 3 years, while the partial remissions were stable for 7 and 8 months, respectively. Only marginal antitumour effects were seen in the lung cancer group. Tolerance of the macrophage system to the stimulatory effect of endotoxin, as measured by human necrosis factor alpha (TNF-alpha) release into serum, built up after the first administration and remained at a steady-state level after each subsequent injection. In constrast, rising CD4:CD8 ratio and release of tumour necrosis factor beta (TNF-beta) indicated the continuing activation of the lymphocyte system by repetitive injections of endotoxin.

Adult↗
PubMed1996

Umbilical cord blood: an alternative to the transplantation of bone marrow stem cells.

Recent in vitro analyses of human UCB have demonstrated the potential of UCB as a source for haematopoietic stem and progenitor cell harvest. Clinical data have further indicated that UCB can be given in vivo to fully and partially HLA-matched siblings or non-familial recipients for marrow reconstitution in genetic disorders as well as malignancies. In comparison to adult peripheral blood, UCB displayed decreased immune responses to alloantigens and was enriched in the numbers of CD34+ progenitor cells with high proliferative and long-term marrow reconstituting potential. Cord blood banks now store large transplantable resources of UCB that are analysed with respect to immunological parameters. Cryopreserved UCB cells may fill the gap in finding a stem-cell transplant for patients who lack a matched related or unrelated donor when a bone marrow transplant is needed.

Blood Preservation↗
PubMed1996

Minimal residual disease: detection, clinical relevance, and treatment strategies.

Improvement of established treatment strategies for cancer has resulted in increased survival times for patients with malignancies. However, success of surgery, chemotherapy, and radiotherapy is limited, as even combined and repeated therapy regimens and high-dose chemotherapy can only reduce tumor burden by several logarithmic steps and are not able to completely eradicate all neoplastic cells. If clinically complete remission is achieved--that is, if no sign of the tumor is detectable by standard diagnostic procedures, remaining minimal residual disease (MRD) can eventually give rise to clinically manifest relapse. More sensitive methods are, therefore, necessary to detect single tumor cells for exact staging, to assess the metastatic potential of an individual tumor, to evaluate the sensitivity to prior therapy, and to detect MRD-positive patients with remaining malignant cells who are at higher risk for relapse. Novel treatment approaches must be created to eradicate minimal residual disease after conventional therapy.

Humans↗
PubMed1996

Hematopoietic cell proliferation and differentiation.

The use of colony-stimulating factors to stimulate hematopoietic cell proliferation and differentiation in vivo is under still increasing investigation, particularly in the field of stem cell transplantation. This review discusses recently published results and tries to extrapolate lines of future development from the achievements reached to date.

Animals↗
PubMed1996

BCL-2/JH rearrangements in circulating B cells of healthy blood donors and patients with nonmalignant diseases.

PURPOSE: To answer the question whether t(14;18)-positive cells can be detected by polymerase chain reaction (PCR) in the peripheral blood of healthy blood donors and patients with nonmalignant diseases. PATIENTS AND METHODS: Peripheral-blood mononuclear cells (PBMNC) from healthy donors (n = 36) and patients with nonmalignant diseases (n = 21) were examined by two-step PCR for the detection of t(14;18)-positive cells with a breakpoint within the major breakpoint region (MBR). Approximate numbers of t(14;18)-positive cells were determined using limiting dilution assays, as well as the stochastic multiple-tube approach. RESULTS: We were able to detect t(14;18)-positive cells in PBMNC of approximately 50% of healthy donors and patients with nonmalignant diseases if DNA amounts up to 10 microg were tested. Compared with 17 t(14;18)-positive patients being in complete remission after radiotherapy for low-stage malignant follicular lymphoma, the majority of 26 healthy donors were found to have significantly lower numbers of t(14;18)-positive cells circulating in the peripheral blood. In the case of six healthy donors, more than one t(14;18) DNA fragment based on size and nucleotide sequence analysis was detected. In one healthy individual, four different t(14;18)-positive cell clones were found in nine samples found over 5 years. CONCLUSION: The occurrence of the t(14;18) translocation is not restricted to follicular lymphoma cells. In healthy donors, long-lived t(14;18)-positive cells can be detected by PCR if the sensitivity is high enough. Based on nucleotide sequence analysis, the t(14;18) DNA fragments detected in healthy donors cannot be distinguished from those found in follicular lymphomas.

Adult↗
PubMed1996

Biological and clinical advances in stem cell expansion.

One of the most exciting areas of hematological research is the ex vivo expansion of peripheral blood progenitor cells (PBPC). Several groups clearly demonstrated that the ex vivo expansion of hematopoietic progenitor cells is possible in the presence of various cytokine combination and/or different feeder layer models. This minireview summarizes recent developments in ex vivo expansion systems as well as first clinical applications.

Antigens, CD34↗
PubMed1996

HTLV-I-associated adult T cell leukemia/lymphoma in two patients from Bucharest, Romania.

Two middle-aged patients with T cell lymphoma, both natives of Bucharest, Romania, tested positive for HTLV-I antibodies. Malignant cells had the typical phenotype and morphology of adult T cell leukemia/lymphoma (ATL). Both cases presented with extranodal manifestation, hypercalcemia, early recurrence after initial responses to therapy, and subsequent resistance to conventional and intensified chemotherapy. Infection with HTLV-I was confirmed by PCR analyses of serial biopsies. Neither patient reported known risk factors for HTLV-I infection. This report points to the possibility that Romania may represent an endemic area for HTLV-I and should heighten the awareness towards HTLV-I infections in Romanian patients.

Adult↗