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M Pfreundschuh

Publications and source records attributed to M Pfreundschuh.

At least 91 records · Page 5Linked to original sources
PubMed1997

NPM/ALK fusion mRNA expression in Hodgkin and Reed-Sternberg cells is rare but does occur: results from single-cell cDNA analysis.

BACKGROUND: The translocation t(2;5)(p23;q35) leads to the fusion of the nucleophosmin gene (NPM) on chromosome 5q35 to the recently described receptor kinase ALK on 2p23. It is characteristic of a subgroup of CD30+ large-cell anaplastic non-Hodgkin's lymphoma (ALCL). Since some cases of Hodgkin's disease (HD) and ALCL share common features, a common pathogenesis has been proposed in a report of the expression of NPM/ALK fusion mRNA in 11/13 Hodgkin's lymphomas. PATIENTS AND METHODS: We approached this question by micro-manipulatory isolation of single Hodgkin and Reed-Sternberg (H-RS) cells and subsequent RT-PCR amplification of NPM/ALK fusion cDNA from these single cells. RESULTS: Specificity of cell selection was shown by the HD-specific pattern of EBV-gene expression in single H-RS cells. In 4 out of 7 cases, NPM/ALK fusion cDNA was detected in the RNA from whole lymph node tissue. In 2 out of 9 cases, NPM/ALK fusion sequences were amplified from single H-RS cells, albeit in a very low frequency (< 5%). CONCLUSIONS: These data indicate that NPM/ALK fusion transcripts do not play an early role in the pathogenesis of HD. Whether the rare expression of NPM/ALK is the result of clonal heterogeneity or an indication for clonal evolution and progression toward ALCL can only be answered by the repeated analysis of indicator cases during the course of the disease.

Adolescent↗
PubMed1996

The SSX-2 gene, which is involved in the t(X;18) translocation of synovial sarcomas, codes for the human tumor antigen HOM-MEL-40.

Using autologous serum for the serological analysis of recombinantly expressed clones (SEREX) from a cDNA derived from a human melanoma, several new melanoma antigens were identified that are immunogenic in the autologous host. Sequence analysis revealed that one of these antigens, HOM-MEL-40, was coded for by the SSX2 gene, which has recently been described to be involved in the t(X;18) translocation of human synovial sarcomas. Expression analysis performed by Northern blot and RT-PCR demonstrated the presence of HOM-MEL-40 transcripts in a significant proportion of human melanomas (50%), colon cancers (25 %), hepatocarcinomas (30%), and breast carcinoma (20%) but not in normal tissues except for testis. Sequence comparison with transcripts cloned from testis ruled out mutations in the melanoma-derived HOM-MEL-40. Antibodies against HOM-MEL-40 were found in 10 of 89 patients with melanoma, including 3 of 8 patients with HOM-MEL-40-positive tumors, but not in 41 apparently healthy controls. In view of the specific expression pattern and immunogenicity in cancer patients, HOM-MEL-40 holds promise as a target for immune interventions in a considerable population of patients with HOM-MEL-40-positive tumors.

Antigens, Neoplasm↗
PubMed1996

T cells from patients with Hodgkin's disease have a defective T-cell receptor zeta chain expression that is reversible by T-cell stimulation with CD3 and CD28.

To investigate the mechanisms underlying the deficiency of T lymphocytes from patients with Hodgkin's disease, we investigated the expression of the T-cell receptor (TCR) zeta chain in patients with Hodgkin's disease. By flow cytometry using an anti-zeta chain monoclonal antibody, peripheral blood T lymphocytes from patients with untreated Hodgkin's disease were shown to express decreased levels of the TCR zeta chain. After stimulation by combined CD3 and CD28 cross-linking, T cells from Hodgkin's disease patients upregulated zeta chain protein expression to normal values within 48 hours and achieved a cytolytic potential and levels of interleukin (IL)-2 secretion that were not different from T cells obtained from healthy controls. These results show that downregulation of the TCR zeta chain in Hodgkin's T lymphocytes is a reversible event. Costimulation of CD3 and CD28 is a novel approach for overcoming the T-cell deficiency in Hodgkin's disease and might be exploited clinically. As upregulation of the zeta chain can also be achieved using bispecific monoclonal antibodies (BI-MoAbs) with specificity for tumor antigens and CD3 and CD28, respectively, an immunotherapy with CD3/CD30 and CD28/CD30 Bi-MoAbs may overcome and should therefore, not be jeopardized by the inherent T-cell deficiency in patients with Hodgkin's disease.

Adolescent↗
PubMed1996

A novel cell cycle-dependent antinuclear antibody in a patient with a monoclonal gammopathy of unknown significance.

A patient with a monoclonal gammopathy, an erosive polyarthritis and a spastic paraparesis of both legs revealed a high titer of antinuclear antibodies with an immunofluorescence pattern hitherto unknown. A pleomorphic staining pattern of unsynchronized cells suggested a cell cycle-dependent autoantigen. Cell cycle-dependency of the antigen was confirmed by staining tissue sections, serum-starvation of tissue culture cells and mitogen-stimulation of peripheral blood lymphocytes. The antinuclear antibody does not belong to the paraprotein fraction of the serum immunoglobulins.

3T3 Cells↗
PubMed1996

Cure of disseminated xenografted human Hodgkin's tumors by bispecific monoclonal antibodies and human T cells: the role of human T-cell subsets in a preclinical model.

Cure of a single established human Hodgkin's tumor growing subcutaneously in severe combined immunodeficient (SCID) mice can be achieved with a complex protocol using two bispecific monoclonal antibodies (Bi-MoAb) directed against the Hodgkin's associated CD30 antigen and the T-cell triggering molecules CD3 and CD28, respectively, together with human T cells prestimulated in vitro with Bi-MoAbs in the presence of CD30+ cells. To adapt this model to the clinical situation, disseminated tumors were established in SCID mice by intravenous injection of 2 x 10(7) cells of the Hodgkin's derived cell line L540CY. Treatment of SCID mice bearing disseminated CD30+ Hodgkin's tumors with the combination of CD3/CD30 and CD28/CD30 Bi-MoAbs and naive (ie, not in vitro prestimulated) human T cells resulted in the cure of all appropriately treated animals. T lymphocytes obtained from patients with advanced stage untreated Hodgkin's disease were as effective as lymphocytes from healthy controls. Treatment was effective even when delayed until 2 weeks after tumor inoculation, and application of Bi-MoAbs into SCID mice with circulating human T cells was as effective as injecting the Bi-MoAbs before the lymphocytes. Treatment results with isolated CD4+ and CD8+ human T cells suggest that both subsets are necessary for the Bi-MoAb mediated cure of xenografted human tumors in vivo. The efficacy and practicability of this preclinical immunotherapy protocol support and form the basis for the clinical evaluation of this approach in patients with Hodgkin's disease resistant to standard therapy.

Animals↗
PubMed1996

[Integrative concept on the treatment of highly malignant non-Hodgkin lymphomas].

With CHOP, the standard protocol for the treatment of high-grade non-Hodgkin's lymphomas, about 40% of long term survival has been reported. A recent randomized comparison of CHOP vs. ProMACE-Cyta-BOM vs. m-BACOD vs. MACOP-B showed no advantage of these third generation protocols. The analysis of prognostic factors in several controlled trials and the results of the International Non-Hodgkin's Lymphoma Prognostic Factors Project identified stage, LDH, performance status and number of extranodal sites as the most important pretreatment factors. Already the pretreatment LDH-level (normal vs. enhanced) defines accurately cohorts of patients with low or high risk. Based on these results two cooperative trials were initiated: In trial A we are currently evaluating in a randomised fashion the concept of high dose chemotherapy (BEAM) with autologous stemcell rescue vs. a standard treatment for high risk patients < 60 years. In trial B low risk patients or high risk patients > 60 years are randomised to receive either CHOP or CHOEP at two or three weeks intervals.

Adult↗
PubMed1996

Demonstration of interleukin-1beta and interleukin-6 in cells of synovial fluids by flow cytometry.

Cytokine levels are increased in the synovial fluid of affected joints from patients with inflammatory joint diseases. The aim of our study was therefore to determine if and to what extent immmunologically defined subpopulations of mononuclear cells (MNC) in the synovial fluid are responsible for the increased levels of interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) in affected joints. Lipopolysaccharide (LPS) stimulated peripheral MNC were used as positive controls. While soluble IL-1beta (median 167 pg/ml) and IL-6 (median 508 pg/ml) levels were significantly elevated in the synovial fluids tested, IL-1beta and IL-6 were demonstrated by flow cytometry in only a small subpopulation (<=11%) of mononuclear synovial fluid cells in 7/13 patients. Our results suggest that elevated IL-1beta and IL-6 levels in the synovial fluid of inflammatory joints are derived mainly from cells in the synovial membrane and only to a minor extent from cells in the synovial fluid itself.

Arthritis, Reactive↗
PubMed1996

Analysis of bcl-2+ lymphocyte subpopulations in inflammatory synovial infiltrates by a double-immunostaining technique.

We used a double-immunostaining technique to analyze the distribution of bcl-2+ B and T lymphocytes within the synovial membranes (SM) of 13 patients with rheumatic diseases: 11 with rheumatoid arthritis (RA), 1 with ankylosing spondylitis (AS), and 1 with osteoarthritis (OA). A high proportion (up to 50%) of the lymphocytes belonged to the B cell subset. Most of both T and B lymphocytes were positive for the bcl-2 protein. In germinal centers B lymphocytes were also negative for bcl-2 protein expression, comparable to the situation in germinal centers of secondary lymphatic organs. We conclude that bcl-2- B lymphocytes are submitted to antigen selection in the inflamed SMs while bcl-2 protein expression provides survival signals for their persistence in the infiltrates. The expression of bcl-2 may be an important factor in protecting lymphocytes in SM from apoptosis by glucocorticoids, cytostatic drugs, and irradiation.

Adult↗
PubMed1996

Interleukin-12 increases bispecific-antibody-mediated natural killer cell cytotoxicity against human tumors.

The combination of CD16/CD30 bispecific monoclonal antibodies (bi-mAb) and unstimulated human resting natural killer (NK) cells can cure about 50% of mice with severe combined immunodeficiency (SCID) bearing subcutaneously growing established Hodgkin's lymphoma. As interleukin-2 (IL-2) and IL-12 have been shown to increase NK cell activity, we tested the capacity of these cytokines to increase bi-mAb-mediated NK cell cytotoxicity against two types of human tumors (Hodgkin's disease and colorectal carcinoma). Unstimulated NK cells needed a three- to five-times higher antibody concentration than cytokine-stimulated NK cells to exert similar levels of bi-mAb-mediated cytotoxicity. The augmented tumor cell lysis was achieved with IL-12 at considerably lower concentrations than with IL-2 and was associated with a significantly increased bi-mAb-mediated intracellular Ca2+ mobilization. The efficiency of IL-12 in this setting together with its low toxicity make it the ideal candidate for a combination therapy with NK-cell-activating bi-mAb in human tumors that are resistant to standard treatment.

Antibodies, Bispecific↗
PubMed1996

Evidence for a selected humoral immune response encoded by VH4 family genes in the synovial membrane of a patient with rheumatoid arthritis (RA).

The analysis of rearranged antibody-encoding genes from B cell foci in rheumatoid synovial tissue has characterized these cells as highly mutated memory B cells with a high proportion of members of the VH4 family. In order to characterize further the VH4 response in one patient, B cell-rich areas from different sections of synovial membrane (SM) were identified by CD20 staining, isolated by microdissection and pooled, in order to analyse highly enriched B cells without selection by in vitro culture procedures. From DNA of about 5 x 10(3) B cells rearranged VH genes were amplified by polymerase chain reaction (PCR) and cloned. Sequencing of 11 clones containing rearranged VH4 gene products revealed that seven were potentially functional, and all were mutated with 84-96% homology to known germ-line (gl) genes and VH4 gl genes amplified from the patient's genomic DNA. Analysis of the complementarity determining region (CDR) 3 revealed that two products represented members of one B cell clone which differed by five nucleotide changes. Three of the five mutations encoded amino acid replacements in CDRs indicating antigen-driven expansion of one specific clone. Additional analyses of 25 members of three B cell clones from isolated aggregates showing intraclonal diversity in one of three clones provided further evidence that antigen selection takes place in the SM. Overall, the pattern of mutations and the replacement to silent (R:S) ratios were diverse, with six products indicating antigen selection by their high R:S ratios in CDRs. Although DNA analysis does not allow a characterization of antibody specificities, we can conclude from our analysis does not allow a characterization of antibody specificities, we can conclude from our analysis of antibody-encoding genes that selection by antigen and expansion of specific clones occur in the SM against the background of polyclonal activation.

Aged↗
PubMed1996

Treatment of Hodgkin's disease with bispecific antibodies.

Bispecific monoclonal antibodies (Bi-MAbs) with dual specificity for tumor-associated antigens (TAA) and a triggering molecule of an immunologic effector cell, respectively, open the possibility to specifically target to and activate cytotoxic effector cells (macrophages, T-cells, NK cells) at the tumor site. Using appropriately designed Bi-MAbs and unstimulated human NK cells and T-cells, respectively, we were able to cure SCID mice xenografted with human Hodgkin's tumors. This approach was also effective in disseminated tumors and when treatment was delayed until three weeks after the inoculation of the tumor, thus establishing this approach as the most effective model of an immunomodulating therapy of human neoplasms. Early observations with an ongoing phase I/II study with CD16/CD30 Bi-MAb in patients with refractory Hodgkin's disease confirm the expected low toxicity. If these observations can be confirmed in larger clinical studies, effector cell activating Bi-MAbs could become an important weapon in the remaining fight for the conquest of Hodgkin's disease.

Animals↗
PubMed1996

Single cell PCR for the analysis of Hodgkin's disease: four years later.

BACKGROUND: Single cell-based studies represent a promising alternative to conventional molecular approaches in the study of Hodgkin's disease since the malignant Hodgkin and Reed-Sternberg cells (H & RS) represent only a small minority of the cellular infiltrate in affected nodes. METHODS: Single cell polymerase chain reaction (PCR) assays were developed for the analysis of specific genomic DNA sequences and the detection of gene expression. Single H & RS cells were isolated by micromanipulation from cytospin slides or fresh cell suspensions after staining with an anti-CD 30 MoAB. RESULTS: The status of oncogenes and immune receptor genes was examined by DNA-PCR. So far, no IgH or TCR gamma rearrangements were detected in H & RS cells of T- and B-antigen negative classical Hodgkin's cases but were detected in two cases of nodular paragranuloma. Global cDNA amplification was successfully performed from single H & RS cells, and specific gene transcripts were detected with a novel PCR method. CONCLUSION: Single cell PCR is a novel and promising method that will help to elucidate many of the open questions in the biology of Hodgkin's disease. In the case of contradictory results, collaborations between different groups utilizing similar approaches have to be performed.

DNA, Neoplasm↗