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:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Daily systemic administration of hematopoietic growth factors can be associated with dose-limiting systemic side effects. To overcome this, we have investigated hematopoietic cytokine gene transfer to the marrow cavity of dogs by direct intramarrow injection of adenoviral vectors. In marrow culture, replication-deficient (E1-deleted) adenoviral vectors were able to transduce marrow stromal cells, demonstrating 30-fold greater expression than from other marrow cell types. High-level (ng/ml) cytokine production from transduced stromal cells persisted for 14 days in culture. Because adenovectors could efficiently transduce marrow stromal cells in culture, we investigated if stromal cells could also be transduced in vivo following direct intramarrow vector injection. Adenovectors with genes for interleukin 6 (IL-6) and Lac Z (beta-galactosidase) were injected directly into the marrow cavity of dogs resulting in protein expression localized to within the treated marrow. To evaluate this approach further in dogs, we constructed a vector expressing biologically active canine granulocyte-macrophage colony stimulating factor (GM-CSF). 293 cells infected with ADGM-CSF demonstrated prevalent GM-CSF mRNA by Northern blot and 135 +/- 30 ng/ml of protein as measured by enzyme-linked immunosorbent assay (ELISA). In vitro bioactivity of protein expressed was confirmed by canine GM colony-forming assay (CFU-GM). In vivo high-level protein production was noted in supernatants of marrow aspirates 72 hr following direct intramarrow administration of ADGM-CSF (baseline mean +/- SEM, 27 +/- 22 ng/ml, 72-hr sample 921 +/- 461 ng/ml). A localized myeloid expansion of marrow and significant peripheral leukocytosis (neutrophilia) were noted in all ADGM-CSF-treated dogs. Peripheral blood changes lasted for up to 3 weeks in dogs following single intramarrow injection. Thus, adenoviral cytokine expression from the marrow of a single large bone (ilium) led to compartmentalized expression of growth factor and an increase of hematopoiesis sufficient to cause peripheral blood changes in a large animal model.
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PMID:Intramarrow cytokine gene transfer by adenoviral vectors in dogs. 909 6

In order to realize a novel vaccination treatment for malignant gliomas using tumor cells genetically modified to express certain cytokines, it is essential to achieve an efficient gene transduction into primary cultured cells. We investigated the feasibility of preparing a glioma vaccine through retrovirus-mediated gene transduction. Glioma cells were cultured primarily from surgically resected tumor tissues of six patients. We obtained more than 1000-fold proliferation of cultures within eight weeks in all six cases. In vitro infection with a recombinant retrovirus GKlacZ carrying an Escherichia coli beta-galactosidase marker gene resulted in over 65% gene transfer to the primary cultured glioma cells. Further enrichment (approximately 90%) of transduced cells was possible by employing repeated infections or using vectors with neo' marker gene. Two cytokine genes, granulocyte-macrophage colony-stimulating factor and interleukin-4, were introduced into glioma cells by sequential transduction with two single-expression GK vectors. The transduced glioma cells produced high levels of both cytokines. We also evaluated simultaneous introduction of two genes with double-expression GK vectors containing internal ribosomal entry site (IRES) or internal promoter (PGK). Although the cells transduced with double-expression vectors secreted both cytokines, the level of the gene product following IRES or PGK was 10 times lower than that of the upstream gene product. The transduced cells retained cytokine secretion in vitro for 14 days after 100 Gy irradiation. Our data indicate the feasibility of retrovirus-mediated preparation of gene-modified tumor vaccines from clinical glioma materials, which could be useful for potentiating antitumor immunity in glioma patients.
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PMID:Efficient retrovirus-mediated cytokine-gene transduction of primary-cultured human glioma cells for tumor vaccination therapy. 914 Jan 15

Like most other normal cells, human endothelial cells possess a limited replicative life span, and, after multiple passages in vitro, develop an arrest in cell division referred to as replicative senescence. For many cell types senescence can be delayed by oncogenes or tumor suppressor genes or prevented altogether by malignant transformation; however, once developed, senescence has been regarded as irreversible. We now report that a cytokine, vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), significantly delays senescence in human dermal microvascular endothelial cells (HDMEC). Typically, VPF/VEGF-treated HDMEC could be cultured for at least 15-20 more population doublings (PD) than control cells. Protection from senescence was reversible in that subsequent withdrawal of VPF/VEGF returned cells to the senescent phenotype. Expression of several cell cycle-related genes (p21, p16 and p27) was significantly reduced in VPF/VEGF-treated cells but p53 expression was not significantly altered. Of particular importance, VPF/VEGF was able to rescue senescent HDMEC, restoring them to proliferation, to a more normal morphology, and to reduced expression of a senescence marker, neutral beta-galactosidase. Taken together, VPF/VEGF delayed the onset of senescence and also reversed senescence in microvascular endothelial cells without inducing cell transformation.
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PMID:Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) delays and induces escape from senescence in human dermal microvascular endothelial cells. 916 Aug 82

Endogenously released or exogenously administered glucocorticosteroids are relevant hormones for controlling inflammation. Only 11beta-hydroxy glucocorticosteroids, but not 11-keto glucocorticosteroids, activate glucocorticoid receptors. Since we found that glomerular mesangial cells (GMC) express 11beta-hydroxysteroid dehydrogenase 1 (11beta-OHSD1), which interconverts 11-keto glucocorticosteroids into 11beta-hydroxy glucocorticosteroids (cortisone/cortisol shuttle), we explored whether 11beta-OHSD1 determines the antiinflammatory effect of glucocorticosteroids. GMC exposed to interleukin (IL)-1beta or tumor necrosis factor alpha (TNF-alpha) release group II phospholipase A2 (PLA2), a key enzyme producing inflammatory mediators. 11beta-hydroxy glucocorticosteroids inhibited cytokine-induced transcription and release of PLA2 through a glucocorticoid receptor-dependent mechanism. This inhibition was enhanced by inhibiting 11beta-OHSD1. Interestingly, 11-keto glucocorticosteroids decreased cytokine-induced PLA2 release as well, a finding abrogated by inhibiting 11beta-OHSD1. Stimulating GMC with IL-1beta or TNF-alpha increased expression and reductase activity of 11beta-OHSD1. Similarly, this IL-1beta- and TNF-alpha-induced formation of active 11beta-hydroxy glucocorticosteroids from inert 11-keto glucocorticosteroids by the 11beta-OHSD1 was shown in the Kiki cell line that expresses the stably transfected bacterial beta-galactosidase gene under the control of a glucocorticosteroids response element. Thus, we conclude that 11beta-OHSD1 controls access of 11beta-hydroxy glucocorticosteroids and 11-keto glucocorticosteroids to glucocorticoid receptors and thus determines the anti-inflammatory effect of glucocorticosteroids. IL-1beta and TNF-alpha upregulate specifically the reductase activity of 11beta-OHSD1 and counterbalance by that mechanism their own proinflammatory effect.
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PMID:Tumor necrosis factor alpha and interleukin 1beta enhance the cortisone/cortisol shuttle. 922 48

Lymphotoxin-beta is a newly recognized member of the tumor necrosis factor ligand family. Recent studies have suggested a role for this cytokine in delayed-type hypersensitivity responses. To determine whether lymphotoxin-beta contributes to the development of contact sensitivity, we utilized an inhibitor protein that can effectively block binding of lymphotoxin-beta to its receptor. An adenoviral vector was created that encodes for a lymphotoxin-beta inhibitor protein consisting of the extracellular domain of the lymphotoxin-beta receptor fused to IgG heavy chain. Intravenous injection of the recombinant virus into BALB/c mice yielded plasma levels of inhibitor protein > 500 micrograms that persisted for 1 week. Mice treated in this manner were compared with control animals injected with adenovirus encoding beta-galactosidase, with respect to their ability to mount contact sensitivity responses to epicutaneously applied dinitro-fluorobenzene. Mice transduced with the lymphotoxin-beta inhibitor prior to the induction of contact sensitivity showed significantly suppressed ear swelling responses. By contrast, mice treated with the lymphotoxin-beta inhibitor prior to the elicitation of contact sensitivity showed no change in ear swelling responses in comparison to controls. These findings indicate that lymphotoxin-beta plays an important role in the afferent phase of the contact sensitivity response.
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PMID:Adenovirus-mediated blockade of lymphotoxin-beta inhibits the induction of contact sensitivity in mice. 929 89

Although adenoviral vectors are attractive for gene transfer, their effectiveness is limited by host antiviral immune responses. In this study, we determined if host antiallograft and antiviral immunity could be diminished with an adenoviral vector encoding the immunosuppressive cytokine viral interleukin-10 (vIL-10). AdSV40vIL-10, a vIL-10-expressing adenoviral vector with an SV40 promoter, induced significant prolongation of murine cardiac allograft survival to 32.2 +/- 1.7 days compared to 14.2 +/- 1.0 days for controls (p < 0.01). This effect was specific for vIL-10 encoding vector and could be inhibited by anti-vIL-10 monoclonal antibody (mAb). In vivo administration of adenovirus facilitated the generation of adenovirus-specific cytotoxic T lymphocytes (CTL), whereas treatment with AdSV40vIL-10 prevented CTL priming and generation of virus-specific immunity. AdSV40vIL-10 also induced extended expression of a beta-galactosidase reporter from a co-injected LacZ-encoding adenoviral vector. These results demonstrate that adenovirus-mediated gene transfer and expression of vIL-10 prolong allograft survival and inhibit the immune response to adenoviral antigens, thereby improving the persistence of the vector and extending transgene expression. The efficacy of adenoviral vectors can be improved by incorporating immunosuppressive genes into the vector.
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PMID:Adenovirus-mediated gene transfer of viral interleukin-10 inhibits the immune response to both alloantigen and adenoviral antigen. 929 31

Retrovirus-mediated gene transfer is commonly used in gene therapy protocols and has the potential to provide long-term expression of the transgene. Although expression of a retrovirus-delivered transgene is satisfactory in cultured cells, it has been difficult to achieve consistent and high-level expression in vivo. In this investigation, we explored the possibility of modulating transgene expression by host-derived cytokines. Normal human keratinocytes and dermal fibroblasts were transduced with recombinant retroviruses expressing a reporter gene (lacZ). Treatment of transduced cells with a proinflammatory cytokine, gamma interferon (IFN-gamma), significantly reduced lacZ expression to less than 25% of that of nontreated cells. The inhibition was concentration dependent (peak at 5 ng/ml) and time dependent (maximal at 16 h for transcript and 24 h for protein); expression remained repressed in the continued presence of IFN-gamma but returned to normal levels 24 h after IFN-gamma withdrawal. The decrease in beta-galactosidase activity appeared to result from decrease in steady-state lacZ mRNA levels. Inhibitors of transcription and translation blocked IFN-gamma-induced repression, suggesting involvement of newly synthesized protein intermediates. Similar results were obtained by treatment of transduced cells with IFN-alpha but not with other proinflammatory cytokines, including tumor necrosis factor alpha, interleukin-2 (IL-1), IL-4, and granulocyte colony-stimulating factor. Although the level of lacZ mRNA was reduced by >70% following IFN treatment, the rate of lacZ transcription was not significantly different from that for nontreated cells. These results suggest that IFN-mediated regulation of transgene expression is at a posttranscriptional level. Interestingly, IFN-gamma also suppressed transgene expression driven by a cellular promoter (involucrin) inserted in an internal position in the retroviral vector. The presence of the overlapping 3' untranslated regions in transcripts initiated from the internal promoter and the long terminal repeat is suggestive of a posttranscriptional regulation, likely at the level of RNA stabilization. These results provide direct evidence for modulatory effects of IFNs on retrovirus-mediated transgene expression and suggest that gene therapy results may be altered by host inflammatory responses.
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PMID:Repression of retrovirus-mediated transgene expression by interferons: implications for gene therapy. 937 74

We evaluated the efficiency of adenovirus-mediated gene transfer into normal and malignant human hematopoietic cells. An E-1 and E-3 deleted, replication-defective recombinant Ad.RSV beta gal vector was used and the transduction efficiency was studied at a multiplicity of infection of 13 p.f.u. per cell. Approximately 40-50% of normal monocytes were transduced, whereas purified normal resting T cells and B cells were resistant to infection. We showed that 50-80% of primary chronic myeloid leukemia cells (CML, n = 12) were efficiently transduced in contrast to CML, successful transduction of resting primary chronic B lymphocytic leukemia cells required appropriate preactivation of targeted cells. A novel protocol for the efficient transduction of adenovirus into B-CLL cells was presented. We showed that anti-CD40 mAb or CD40 ligand acts in synergy with rhIL-4 to enable the transduction of approximately 50-75% of B-CLL cells (B-CLL, n = 6). Expression of beta-galactosidase in transduced CML cells and B-CLL cells was detected for at least 15 days after transduction. The present studies underline the utility of adenovirus vectors for the construction of cytokine gene-modified tumor vaccines for the treatment of hematopoietic malignancies such as CML and B-CLL.
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PMID:Efficient adenovirus-mediated gene transduction of normal and leukemic hematopoietic cells. 941 16

TNF-alpha is one of the major proinflammatory cytokines involved in the pathogenesis of chronic inflammatory joint disease, in human rheumatoid arthritis as well as in murine models of this disease. It was previously described that a highly destructive chronic spontaneous inflammatory arthritis develops in mice expressing a human TNF-alpha transgene modified with the 3' untranslated region of beta-globin. The present study investigates in this mouse model the effects of the anti-inflammatory cytokines IL-4, IL-10 and IL-13 administered in vivo on proinflammatory cytokine expression. Groups of TNF-alpha-transgenic mice were engrafted with xenogeneic transfected Chinese hamster ovary (CHO) fibroblasts secreting murine IL-4, IL-10 or IL-13. In vivo treatments consisted of 3 or 4 weekly engraftments, starting when the mice were 4weeks old. Control groups of transgenic mice were engrafted with beta-galactosidase gene-transfected CHO cells or injected with medium. A significant decreased expression of TNF-alpha transgene, endogenous mouse TNF-alpha and IL-1 mRNA was observed in splenocytes of mice treated for 3 or 4 weeks with CHO/IL-4 and CHO/IL-13, and, to a lesser extent, with CHO/IL-10, compared with controls. Finally, attenuation of histological scores of arthritides was statistically significant only in the group of CHO/IL-4-treated mice after 3weeks of treatment (P<0.05), and was not significant in any other group. These results show that IL-4, IL-10 or IL-13, administered by gene therapy, can decrease the mRNA steady state levels of both endogenous and transgenic cytokines in human TNF-alpha transgenic mice. In addition, IL-4 can slightly attenuate the development of arthritides in this model.
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PMID:Modulation of proinflammatory cytokine production in tumour necrosis factor-alpha (TNF-alpha)-transgenic mice by treatment with cells engineered to secrete IL-4, IL-10 or IL-13. 948 9

The hepatic stellate cell (HSC), following a fibrogenic stimulus, is transformed from a quiescent to an activated cell. Cytokines induce NFkappaB activity in activated but not in quiescent HSCs with subsequent expression of NFkappaB-responsive genes, such as intercellular adhesion molecule (ICAM)-1 and interleukin (IL)-6. We investigated the effect of proteasome inhibitors and an IkappaB super-repressor on the cytokine mediated activation of NFkappaB, ICAM-1, and IL-6 in activated HSCs. Culture-activated HSCs were stimulated with IL-1beta or tumor necrosis factor alpha (TNFalpha) in the presence or absence of proteasome inhibitors, ALLN or MG-132, or after infection with an adenovirus expressing the IkappaB super-repressor (Ad5IkappaB) or beta-galactosidase (Ad5LacZ) as a control. NFkappaB activity was evaluated by immunofluorescence and by electrophoretic mobility shift assay. The steady state level of cytoplasmic IkappaB protein was measured by Western Blot. ICAM-1 and IL-6 expression was measured by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbant assay. Proteasome inhibitors, which block the degradation of IkappaB, and the Ad5IkappaB, which provides an exogenous nondegradable IkappaB, block the stimulation of NFkappaB activity by TNFalpha and IL-1beta in activated HSCs. These reagents block the subsequent nuclear translocation of p65 NFkappaB and induction of ICAM-1 and IL-6 by cytokines. The specificities of the proteasome inhibitors and the IkappaB super-repressor are demonstrated by their failure to block c-Jun N-terminal kinase induction by cytokines. Cytokine-induced stimulation of NFkappaB, ICAM-1, and IL-6 is blocked by proteasome inhibitors and Ad5IkappaB in activated HSCs. Inhibition of IkappaBalpha degradation is a potential target for anti-inflammatory therapy in the liver and might influence the activation process of HSCs following fibrotic stimuli.
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PMID:Inhibition of NFkappaB in activated rat hepatic stellate cells by proteasome inhibitors and an IkappaB super-repressor. 958 6


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