Unusual manifestations of B-cell disorders. Case 2: chronic lymphocytic leukemia presenting with cutaneous lesions.
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Aged↗
Biomedical subjects
C M Wendtner
Publications and source records attributed to C M Wendtner.
Recombinant adeno-associated virus for the generation of autologous, gene-modified tumor vaccines: evidence for a high transduction efficiency into primary epithelial cancer cells.
To explore the potential of recombinant vectors based on recombinant adeno-associated virus (rAAV) for cancer vaccination, we investigated the transduction efficiency of rAAV into cancer cells ex vivo. Infection of human epithelial cancer cell lines with rAAV carrying reporter genes encoding beta-galactosidase (rAAV/LacZ) or luciferase (rAAV/Luc) resulted in high levels of reporter gene expression (>90% positive cells). In marked contrast, rAAV poorly transduced all murine tumor cell lines, as well as human hematopoietic cell lines. Either irradiation or adenovirus infection of tumor cells prior to rAAV infection induced a 10- to 100-fold increase of reporter gene expression. To determine the transduction efficiency of rAAV into primary cancer cells, freshly isolated, irradiated tumor cells from malignant melanoma and ovarian carcinoma patients were infected with rAAV/Luc, resulting in up to 6.9-fold higher levels of gene expression than in a HeLa tumor cell line. Time course experiments with freshly isolated tumor cells infected with rAAV/Luc showed maximal levels of luciferase expression between days 3 and 9 posttransduction. Simultaneous infection of primary tumor cells with up to three rAAV vectors containing genes encoding the immunostimulatory proteins B7-2 (CD86), p35 subunit of IL-12, and p40 subunit of IL-12 resulted in high expression of B7-2 in more than 90% of the tumor cells and in the secretion of high levels of IL-12. Taken together, our results demonstrate that rAAV efficiently transduces freshly isolated human, epithelial tumor cells and might therefore be a potent tool to produce improved, gene-modified cancer vaccines.
Antigens, CD↗
Gene transfer of the costimulatory molecules B7-1 and B7-2 into human multiple myeloma cells by recombinant adeno-associated virus enhances the cytolytic T cell response.
Gene transfer of the costimulatory molecules B7-1 and B7-2 induces a potent antitumor immune response in a variety of tumor models. B cell neoplasms including multiple myeloma (MM) often show little or no expression of B7 antigens; they are therefore a potential target for this approach. To increase the expression of human B7 genes in MM cells, both genes and the neomycin phosphotransferase gene were packaged into recombinant adeno-associated virus vectors (rAAV). The resulting recombinant viruses rAAV/B7-1, rAAV/B7-2 and rAAV/Neo were used to transduce the MM cell lines LP-1 and RPMI 8226. This allowed the transduction of up to 80% of LP-1 cells 4 days after infection with purified rAAV particles. The response of human allogeneic T cells to rAAV/B7-1 and rAAV/B7-2 transduced, gamma-irradiated LP-1 cells was assessed by [3H]thymidine incorporation, by RT-PCR-based detection of immunostimulatory cytokine transcripts and by ELISA quantification of cytokines in the supernatant. Stimulation of T cells with rAAV/B7-1 or rAAV/B7-2 transduced LP-1 cells resulted in an up to 10-fold increase of T cell proliferation when compared with LP-1 cells transduced with rAAV/Neo. Similar results were obtained with RPMI 8226 cells. Both rAAV/B7-1 and rAAV/B7-2 transduced LP-1 cells stimulated the T cell secretion of IL-2 and IFN-gamma. Furthermore, [51Cr] release assays showed that rAAV/B7-1 or rAAV/B7-2 transduced LP-1 cells induced a cytolytic T cell (CTL) response, in contrast to LP-1 cells transduced with rAAV/Neo. In all assays, the effects of rAAV/B7-1 and rAAV/B7-2 were similar. Taken together, the results show that rAAV-mediated transfer of B7 genes into MM cell lines is able to enhance the antitumor T cell response and to elicit a cytolytic T cell response.
Antigens, CD↗
Recombinant adeno-associated virus (rAAV) vectors for somatic gene therapy: recent advances and potential clinical applications.
Adeno-associated virus (AAV) is a single-stranded DNA dependovirus of the family of Parvoviridae that has promising features as a vector for somatic gene therapy. Different recombinant (r) AAV vectors have been generated that seem to have some advantages compared with other vector systems, such as the transduction of terminally differentiated and non-dividing cells, the lack of any apparent pathogenicity, low immunogenicity, relatively high stability of transgene expression, and the potential of targeted integration. Recent improvements in rAAV packaging should allow the generation of sufficient quantities of rAAV for clinical trials. Preclinical studies with rAAV are currently being performed for the treatment of a variety of inherited monogenic defects, such as beta-thalassemia, sickle cell anemia. Fanconi anemia, chronic granulomatous disease, Gaucher disease, metachromatic leukodystrophy and cystic fibrosis, and of acquired diseases, such as HIV infection and non-Hodgkin lymphoma. The diversity of these studies indicates that rAAV might have a broad range of clinical applications. A first clinical trial with rAAV vectors has been started for cystic fibrosis. While several important issues, including safety, tissue tropism and methods to achieve site-specific integration, need further clarification, rAAV seems to have a sufficient number of advantages to be seriously considered as a future gene therapy vector.
Anemia↗
CD30 ligand signal transduction involves activation of a tyrosine kinase and of mitogen-activated protein kinase in a Hodgkin's lymphoma cell line.
CD30 is a transmembrane receptor of the nerve growth factor/tumor necrosis factor receptor superfamily. Its expression associated with Hodgkin's lymphoma and a subset of non-Hodgkin's lymphoma. Recently, its ligand (CD30L) has been cloned. CD30L enhances the proliferation of peripheral T cells and the Hodgkin's cell line HDLM-2 but seems to exert antiproliferative effects on large cell anaplastic lymphoma cell lines. Since tyrosine kinases are critical regulators of cell growth, we investigated whether CD30L induced changes in cellular tyrosine phosphorylation in CD30-positive lymphoma cell lines. Stimulation with CD30L or with an agonistic mAb against CD30, M44, induced a rapid, transient, and concentration-dependent tyrosine phosphorylation of a cytosolic protein of M(r) 42,000 (p42) in the Hodgkin's lymphomas cell line HDLM-2 but not in other CD30-positive lymphomas. In HDLM-2 cells, the phrobol ester phorbol 12-myristate 13-acetate also stimulated tyrosine phosphorylation of p42, and this effect was enhanced by M44. In marked contrast, agents stimulating the protein kinase A pathway, like forskolin or dibutyryl cAMP, did not affect tyrosine phosphorylation of P42. By immunoprecipitation with mAbs against mitogen-activated protein kinase (MAPK; p42ERKII), a M(r) 42,000 protein was identified which comigrated with p42 on SDS gels and which was phosphorylated on tyrosine residues in response to stimulation of CD30. Immune complex kinase assays showed that M44 mAb induced the activation of MAPK (p42ERKII) and the phosphorylation of a MAPK substrate, myelin basic protein. Taken together, the results suggest that CD30L induces the tyrosine phosphorylation and activation of the MAPK p42ERKII isoform in HDLM-2 cells. These findings may have implications for the understanding of the pathogenesis of Hodgkin's disease.
CD30 Ligand↗
High-efficiency transfer of the T cell co-stimulatory molecule B7-2 to lymphoid cells using high-titer recombinant adeno-associated virus vectors.
Adeno-associated virus (AAV) is a single-stranded DNA virus that can either integrate or replicate in host cells. Production of recombinant viral particles (rAAV) requires expression of the viral structural genes and the viral inverted terminal repeats in cis. By using an SV40 replicon to amplify the structural genes, the yield of recombinant viral particles was increased 60-fold over a nonreplicating helper plasmid. The rAAV particles produced by this system have similar physical properties to wild-type particles, including buoyant density, size, and morphology. This novel rAAV packaging system was used to produce rAAV particles that contain the gene for the T cell co-stimulatory protein B7-2. Transduction of the human nonadherent lymphoid cell line LP-1 with these particles significantly increased the percentage of cells expressing B7-2 from 6.8% to 78.0%. Expression of B7-2 in the human lymphoid cell line RPMI-8226 was also substantially increased. Targeting of tumor cells grown in suspension was hampered by low-efficiency transduction using other viral or nonviral vector systems. Our new packaging system for recombinant AAV should allow generation of sufficient quantities of B7-2 containing particles to develop tumor vaccines for non-Hodgkin's lymphoma.
Animals↗
Hormone and growth factor subunits: a novel perception of cell growth regulation.
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Animals↗
Prediction of homologous binding sites on RB and p107 common for viral oncoproteins and cellular ligands.
Hydropathic anticomplementarity of amino acids specifies that peptides translated from complementary DNA strands may acquire amphiphilic conformations and bind to each other. This concept has been coined 'Molecular Recognition Theory' (MRT) or 'complementary peptide theory'. Inactivation of retinoblastoma protein (RB), a tumor suppressor gene product, has been shown to be involved in the pathogenesis of many tumors and to be due to either mutation of the RB gene, hyperphosphorylation or complex formation with viral oncoproteins. The viral oncoproteins share a common RB binding motif with cellular ligands. The exact site on RB associating with this common RB binding motif of viral oncoproteins and cellular ligands has not been identified yet. This study is the first to predict putative binding sites on RB and p107, a cellular protein with RB sequence homology, respectively, by using the hydropathic complementarity approach. These sites are residues 649-654 of RB and 657-662 of p107. Moreover, this paper proposes a structure for a potential antineoplastic agent based on the amino acid sequence of the predicted RB binding site. The data presented herein should have important implications both for the understanding of cancer pathophysiology and for the drug design of antineoplastic compounds.
Amino Acid Sequence↗
Proposed interaction between insulin and retinoblastoma protein.
Retinoblastoma protein (RB) is a tumor suppressor gene product involved in embryogenesis and cell cycle progression. One of the major mechanisms leading to RB dysfunction is complex formation with viral oncoproteins using the common RB binding motif Leu X Cys X Glu (LXCXE) which has also been identified in cellular ligands, e.g., RBP-1 and RBP-2. p107, a cellular protein with RB sequence homology, has been shown to bind to the same viral oncoproteins associating with RB and is therefore thought to contribute to cell cycle regulation. It has recently been suggested that insulin stimulates gene transcription through direct association with an, as yet, unidentified intracellular transcription factor. Due to the central roles of RB and p107 in coupling external growth signals with the progression of the cell cycle clock, we have hypothesized that these two proteins might be candidates for mediating the effects of insulin on DNA. We report here the identification of a region in the B-chain of human insulin that has the sequence LXCXE. Based on this finding we predict that the insulin B-chain may interact with RB and/or p107. Since we have also identified sequences hydropathically related to LXCXE in insulin-like growth factor I (IGF-I) and II (IGF-II), but not in relaxin, nerve growth factor, epidermal growth factor, glucagon or beta-endorphin, we further propose that both IGF-I and -II may assemble with RB and/or p107, too. Moreover, binding sites on RB and p107 identical with those suggested for viral oncoproteins and cellular ligands are predicted for insulin/IGF-I/IGF-II by using the hydropathic complementarity approach.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Single-cell detection of ets-1 transcripts in human neuroectodermal cells.
The genes of the ets family are thought to code for a novel class of transcriptional factors. These proteins have a specific DNA-binding domain different from the basic domain of both the helix-loop-helix and leucine zipper families of DNA-binding proteins. The ets-1 gene product has been shown to bind to the enhancer region of the human T-cell receptor alpha gene during thymocyte ontogeny. This finding explains the high expression of ets-1 observed in T cells and the correlation between ets-1 expression and the expression of the T-cell receptor gene during fetal development. The ets-1 gene is also possibly biologically active in neural cells. By using RNA in situ hybridization analysis, we demonstrate the presence of ets-1 transcripts in cells of peripheral embryonal neuroectodermal tumors, specifically neuroepithelioma and neuroblastoma. In addition, the gene is found transcribed in Ewing's sarcoma, postulated to be ontogenetically related to tumors derived from the neural crest.
Gene Expression↗
MR-imaging changes of musculoskeletal soft-tissue sarcomas associated with neoadjuvant chemotherapy and hyperthermia.
PURPOSE: To evaluate early changes in musculoskeletal soft-tissue sarcomas under neoadjuvant chemotherapy combined with regional hyperthermia (RHT). PATIENTS AND METHODS: Nineteen consecutive patients with high-grade soft-tissue sarcomas of the musculoskeletal system were treated with neoadjuvant chemotherapy combined with RHT. Patients were imaged, using a high field MR-scanner, before onset of therapy, immediately after one and after four cycles of therapy. The images were evaluated for volume reduction and development of tumour necrosis. In addition, side effects such as surrounding soft-tissue oedema, bleeding and muscle or bone marrow necrosis were analysed. RESULTS: Tumour volume reduction was significant after the completion of neoadjuvant therapy (mean 49%, range 5-91%; (p < 0.001). Extent of tumour necrosis was also significantly different before (mean 22%) and after therapy (mean 58%, p < 0.001). Three patients showed strong tumour necrosis already after one cycle of treatment. Tumour volume reduction was not associated with the extent of pre-existing necrosis or necrosis development. The extent of tumour volume before start of therapy did not affect volume reduction or necrosis induction after therapy. Reduction of tumour oedema was significant after therapy (p < 0.001). No side effects were observed during thermochemotherapy. CONCLUSION: Neoadjuvant chemotherapy combined with RHT resulted in significant tumour volume reduction and induction of tumour necrosis, which can be detected early and monitored closely with MRI.
Adult↗
Pyoderma gangrenosum associated with the secondary antiphospholipid syndrome.
A 64-year-old woman with an 11-year history of systemic lupus erythematosus and amputation of her left lower leg as a consequence of arterial embolism, presented with two large, non-healing ulcers on her right shank. Pyoderma gangrenosum associated with secondary antiphospholipid syndrome was diagnosed based upon the typical clinical features and increased antibodies to cardiolipin. Although an aggressive therapy with corticosteroids and cyclosporine was started, her condition continued to worsen. She finally died as a result of sepsis. We discuss the difficulties in diagnosis and therapy of SLE combined with the antiphospholipid syndrome and pyoderma gangrenosum.
Adrenal Cortex Hormones↗