Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.21.1 (DNase)
7,655 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

DNase/collagenase treatments are widely used to obtain single-cell suspensions of tumour cells and tumour-infiltrating T lymphocytes (TIL) from solid tumours. Since the functional integrity of such cells has been questioned, we have studied whether treatments with commonly used preparations of these enzymes could affect the expression of lymphocyte surface molecules and lymphocyte proliferative responsiveness. With peripheral-blood-derived T cells as a model, flow-cytometric analysis revealed strongly reduced expression of distinct CD molecules for each enzyme, notably CD2, CD4, CD8 and CD44 for DNase, and CD4, CD14, CD16, and CD56 for collagenase. The effects were found to be due to protease contaminations present in all but the purest enzyme preparations tested. Addition of serum or trypsin inhibitor abolished the effects. Since serum-free media are widely used to expand tumour-infiltrating T cells for clinical therapeutic use, data from early phenotypic analyses can be strongly misleading. Even after an 18-h rest period following the enzyme treatments, re-expression of the affected membrane markers was still far from complete. On the other hand, despite strongly reduced expression of CD2 molecules on the lymphocyte membrane, anti-CD2-induced proliferation was not affected, showing the redundancy of this signal molecule. Since other important T cell activation molecules (TCR, CD3, CD28) were not affected by enzymatic treatment, the use of expensive, highly purified collagenase/DNase preparations does not seem to be mandatory in clinical studies with expanded TIL.
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PMID:Reduced expression of distinct T-cell CD molecules by collagenase/DNase treatment. 816 20

In order to understand further the effects of Newcastle-disease-virus(NDV)-modified tumour vaccines we investigated the feasibility of isolating lymphocytes from the site of injection of patients undergoing postoperative active specific immunization (ASI) with autologous NDV-modified tumour cells. Delayed-type-hypersensitivity(DTH)-like reactions from five cancer patients were surgically removed, minced and the tissue particles were digested with collagenase and DNase. Lymphoid cells recovered were expanded in a highly efficient limiting-dilution analysis system optimized for T cell growth [Moretta et al. (1983) J Exp Med 157: 743] and lymphocyte microcultures (clonal probability > 0.8) could be grown for up to 1 year. Analysis of the microcultures for phenotype and function showed that the majority were positive for CD4 (92%) and TCR alpha beta (96%). Concanavalin-A-induced production of interleukin-2 (IL-2), IL-6, interferon gamma and tumour necrosis factor alpha was detected in more than 70% of the microcultures. Lectin-dependent cytotoxicity was only very rarely observed. The general characteristics of the microcultures obtained support the notion of a DTH-like reaction taking place at the site of tumour cell challenge. The possibility of in vitro expansion and cultivation of T lymphocytes from ASI vaccination sites should help to elucidate further the role of these cells in active specific immunization against autologous tumour cells.
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PMID:In vitro expansion and analysis of T lymphocyte microcultures obtained from the vaccination sites of cancer patients undergoing active specific immunization with autologous Newcastle-disease-virus-modified tumour cells. 834 63

Genes encoding the accessory molecules CD8 and CD4 are activated early in thymocyte development, generating CD4+8+ double positive intermediates, which give rise to two functionally distinct mature T cell subsets that express either CD4 or CD8. The mechanisms that govern the activation or suppression of the CD8 gene are likely to be central to the T cell development program. To identify the key regulatory factors, we have initiated an analysis of the transcriptional regulation of the murine CD8 alpha gene. We have identified three CD8+ cell-specific DNAase I hypersensitive sites (HSS) located upstream of the murine CD8 alpha gene. In vitro mobility shift analysis of the -4.0-kb HSS region has revealed multiple binding sites for the T cell-restricted transcription factor GATA-3. In vitro translated murine GATA-3 binds specifically to both CD8 GATA sites, and coexpression of this factor in transient transfection assays transactivates a reporter construct containing these sequences. These results provide the first evidence for the role of a T cell-restricted factor in the regulation of either CD8 or CD4 genes.
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PMID:Functional GATA-3 binding sites within murine CD8 alpha upstream regulatory sequences. 835 61

Using transgenic mice, we have identified a human CD4 silencer contained within a 484-bp fragment in the first intron of the CD4 gene. Further experiments have mapped a lineage-specific silencing activity to a region of 190 bp. This region contains two protein-binding sites detected by deoxyribonuclease I footprinting analyses. Tested in transient transfection assays, these two DNA elements showed significant silencing activity restricted to the CD8 phenotype. In CD4 cells, either no clear effect (FP I) or strong enhancing activity (FP II) was observed by transient transfection assays. Despite the lineage-specific activity of these two elements, electrophoretic mobility shift assays (EMSA) showed similar levels of protein binding to the silencer element FP I in CD4 and CD8 T cells. Base substitutions in the FP I fragment abolished the silencing activity in transfected CD8 cells as well as the protein binding in EMSA, suggesting an important role of this protein-DNA interaction in CD4 gene regulation.
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PMID:Identification and characterization of a human CD4 silencer. 861 22

Identification of expanded clones engaged in immune and autoimmune responses is still imperfect, since they are often diluted by irrelevant cells expressing diverse specificities. To efficiently characterize T cell receptors expressed by clonally expanded lymphocytes in rheumatoid arthritis (RA) and other inflammatory conditions, we developed an assay system, termed sequence enrichment nuclease assay (SENA). Key elements of SENA are the efficiency of heat-denatured DNA strand reassociation, which increases exponentially with concentration, and the elimination of unhybridized sequences by single-strand-specific DNase. T cell clonal expansions were identified primarily in synovial fluids, but also in peripheral blood of RA patients. Synovial fluids had more prominent expansions in the CD8 than the CD4 subset, whereas clonal expansions in the CD4 subset predominated among peripheral blood lymphocytes. Dominant clones exhibited diverse sequences with no clear conservation of junctional motifs, although the same amino acid sequence was identified in two patients. In most instances, dominant clones in the blood were discordant to those in the corresponding synovial fluid, suggesting local stimulation or preferential sequestration of T cells displaying particular specifities.
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PMID:Identification of clonally expanded T cells in rheumatoid arthritis using a sequence enrichment nuclease assay. 863 47

The prevalence, quantity, temporal pattern, and clinical and immunologic correlates of shedding of Kaposi's sarcoma (KS)-associated herpesvirus (KSHV; or human herpesvirus [HHV]-8) DNA in saliva were studied. KSHV DNA was detected in saliva from 18 (75%) of 24 human immunodeficiency virus (HIV)-positive patients with KS and from 1 of 1 HIV-negative patient with KS, 3 (15%) of 20 HIV-positive patients without KS, and none of 24 controls. KSHV DNA levels ranged from 10(2.4) to 10(6) copies/mL and were lower than levels for Epstein-Barr virus but comparable to those for HHV-6. Detection of KSHV DNA in saliva was not associated with oral KS or decreased peripheral blood CD4 cell counts. KSHV DNA was not detected in semen. Resistance of KSHV DNA from saliva to DNase treatment was consistent with the presence of virions. These data suggest that KSHV can replicate in the oropharynx and that salivary contact could contribute to KSHV transmission.
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PMID:Frequent detection of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) DNA in saliva of human immunodeficiency virus-infected men: clinical and immunologic correlates. 920 54

The coreceptors CD4 and CD8 play a crucial role during thymocyte development and T cell effector function, and their expression is developmentally regulated. To determine the underlying molecular mechanisms of CD8 gene regulation we cloned the murine CD8 gene locus from genomic libraries and analyzed this region for deoxyribonuclease (DNase I) hypersensitive sites (HSS). Here we report, using transgenic mice, deletion analysis of one of the identified clusters of DNase I hypersensitivity, consisting of three DNase I-HSS and located in the intergenic region between the CD8alpha and CD8beta genes. Our data show that at least two of the DNase I-HSS constituting this cluster are individually sufficient to direct CD8alpha or heterologous transgene expression to the mature CD8 single-positive T cell subset and that this expression coincides temporally with the appearance of positively selected T cells.
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PMID:A region in the CD8 gene locus that directs expression to the mature CD8 T cell subset in transgenic mice. 935 73

CD4 and CD8 are crucial for the development and function of T cells. An intergenic deoxyribonuclease I hypersensitive site region (cluster CIII) directs expression in mature CD8 T cells only. Here, we show that two further independent regions from the CD8 gene locus in conjunction with cluster CIII restore transgene expression in appropriate immature thymocytes. Deletion of two of the intergenic cluster CIII DNaseI-HSS in homozygous mutant mice affects expression of CD8alphaalpha homodimers on intraepithelial T cells (IEL), particularly on the gammadeltaTCR+ subset. Surprisingly, none of the thymocyte or peripheral alphabetaTCR T cell subsets are affected by this mutation, indicating hierarchical activation of these elements within the different T cell subsets.
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PMID:Hierarchical interactions of control elements determine CD8alpha gene expression in subsets of thymocytes and peripheral T cells. 980 36

The GATA-3 transcription factor is required for development of the T-cell lineage and Th2 cytokine gene expression in CD4 T-cells. We have mapped the DNase-I-hypersensitive (HS) regions of the human GATA-3 gene in T-cells and non-T-cells and studied their transcriptional activities. HS I-III, located 5' from the transcriptional initiation site, were found in hematopoietic and non-hematopoietic cells, whereas HS IV-VII, located 3' from the transcriptional start site, were exclusively observed in T-cells. Among these hypersensitive sites, two transcriptional control elements were found, one in the first intron of the GATA-3 gene and the other between 8.3 and 5.9 kilobases 5' from the GATA-3 transcriptional initiation site. The first intron acted as a strong transcriptional activator in a position-dependent manner and with no cell-type specificity. The upstream regulatory element could confer T-cell specificity to the GATA-3 promoter activity, and analysis of this region revealed a 707-base pair silencer that drastically inhibited GATA-3 promoter activity in non-T-cells. Two CAGGTG E-boxes, located at the 5'- and 3'-ends of the silencer, were necessary for this silencer activity. The 3'-CAGGTG E-box could bind USF proteins, the ubiquitous repressor ZEB, or the basic helix-loop-helix proteins E2A and HEB, and we showed that a competition between ZEB and E2A/HEB proteins is involved in the silencer activity.
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PMID:T-cell expression of the human GATA-3 gene is regulated by a non-lineage-specific silencer. 1003 51

TCR engagement of immature CD4(+)CD8(+) thymocytes induces clonal maturation (positive selection) as well as clonal deletion (negative selection) in the thymus. However, the cell death execution events of thymocytes during the negative selection process remain obscure. Using a cell-free system, we identified two different DNase activities in the cytosol of in vivo anti-TCR-stimulated murine thymocytes: one that induced chromosomal DNA fragmentation, which was inhibited by an inhibitor of caspase-activated DNase, and another that induced plasmid DNA degradation, which was not inhibited by an inhibitor of caspase-activated DNase. We purified the protein to homogeneity that induced plasmid DNA degradation from the cytosol of anti-CD3-stimulated thymocytes and found that it is identical with cyclophilin B (Cyp B), which was reported to locate in endoplasmic reticulum. Ab against Cyp B specifically inhibited the DNA degradation activity in the cytosol of anti-CD3-stimulated thymocytes. Furthermore, recombinant Cyp B induced DNA degradation of naked nuclei, but did not induce internucleosomal DNA fragmentation. Finally, we demonstrated that TCR engagement of a murine T cell line (EL4) with anti-CD3/CD28 resulted in the release of Cyp B from the microsome fraction to the cytosol/nuclear fraction. Our data strongly suggest that both active caspase-activated DNase and Cyp B may participate in the induction of chromosomal DNA degradation during cell death execution of TCR-stimulated thymocytes.
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PMID:Possible involvement of cyclophilin B and caspase-activated deoxyribonuclease in the induction of chromosomal DNA degradation in TCR-stimulated thymocytes. 1103 62


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