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)

We have constructed a recombinant adeno-associated virus serotype 2 vector encoding human interleukin 10 (rAAVhIL10). IL-10 is a potent antiinflammatory/immune cytokine, which has received growing attention for its therapeutic potential. Human IL-10 (hIL-10) production was virus dose dependent after in vitro infection of HSG cells, a human submandibular gland cell line. The vector-derived hIL-10 produced was biologically active, as the medium from rAAVhIL10-infected HSG cells caused a dose-dependent blockade of IL-12 secretion from spleen cells of IL-10 knockout mice challenged with heat-killed Brucella abortus. Administration of rAAVhIL10 (10(10) genomes per gland) to both mouse submandibular glands led to hIL-10 secretion into the bloodstream (approximately 1-5 pg/ml), that is, in an endocrine manner, which was stable for approximately 2 months. Salivary gland administration of rAAVhIL10 under experimental conditions was more efficacious than intravenous administration (approximately 0.5-0.7 pg/ml). Also, hIL-10 was readily secreted in vitro from organ cultures of minced submandibular glands infected with rAAVhIL10, 6 or 8 weeks earlier. Consistent with these results, hIL-10 mRNA was detected by reverse transcription-polymerase chain reaction in submandibular glands of mice infected with rAAVhIL10 but not from control mice. At these doses, little to no hIL-10 was detected in mouse saliva. Using a rAAV serotype 2 vector encoding beta-galactosidase, we observed that the primary parenchymal target cells were ductal. These findings represent the first report of rAAV use to target exocrine glands for systemic secretion of a therapeutic protein, and support the notion that rAAV serotype 2 vectors may be useful in salivary glands for local (periglandular) and systemic gene-based protein therapeutics.
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PMID:Recombinant adeno-associated virus serotype 2 vectors mediate stable interleukin 10 secretion from salivary glands into the bloodstream. 1181 84

Duchenne muscular dystrophy (DMD) is an X-linked lethal disorder caused by a defect in the DMD gene, which encodes the cytoskeletal protein dystrophin. Utrophin is an autosomal homolog of the DMD gene product dystrophin, and augmented expression of endogenous utrophin is expected to provide an alternative therapeutic approach to DMD. We previously reported that an immune response against a beta-galactosidase-expressing adenovirus vector, AxCALacZ, resulted in an accumulation of endogenous utrophin on the extrasynaptic sarcolemma in dystrophin-deficient mdx mice. To determine which cytokine is involved in the regulation of utrophin expression, we directly injected several cytokines separately into neonatal mdx muscles and tested whether the expression of utrophin is increased on the sarcolemma. Importantly, among the cytokines tested, solely interleukin 6 (IL-6) successfully increased expression of utrophin. Moreover, the increase in utrophin mRNA was detected in recombinant IL-6-injected mdx muscles by quantitative real-time reverse transcriptase-polymerase chain reaction. Further, IL-6 expression was elevated in AxCALacZ-infected mdx muscle at an early stage, and anti-IL-6 receptor (IL-6R) antibody treatment blocked enhanced utrophin expression in AxCALacZ-infected mdx muscle. We should point out, however, that overexpression of utrophin due to recombinant IL-6 treatment lasted only 1 week. In addition, expression of utrophin was not evident in normal C57BL/10 neonatal muscles injected with IL-6. Taken together, these results suggest that IL-6 can induce overexpression of utrophin on the extrasynaptic sarcolemma but requires preexisting factors in neonatal mdx muscle to fully regulate utrophin expression.
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PMID:Interleukin 6 induces overexpression of the sarcolemmal utrophin in neonatal mdx skeletal muscle. 1187 29

Antitumor effects of combined transfer of P16 and cytokine genes were investigated in this study. The adenovirus harboring the P16 gene (AdP16) and murine granulocyte-macrophage colony-stimulating factor gene (AdGM-CSF) were utilized for the treatment of established tumors. The mice were inoculated subcutaneously with Renca cells and, 6 days later, received an intratumoral injection of AdP16 in the presence or absence of AdGM-CSF. The results demonstrated that tumor-bearing mice treated with AdP16 in combination with AdGM-CSF showed more potent inhibition of tumor growth and survived much longer than did mice treated with AdP16, AdGM-CSF, adenovirus expressing beta-galactosidase, or phosphate-buffered saline alone (P<.01). The tumor mass showed obvious necrosis and inflammatory cell infiltration, and more CD(4)(+) and CD(8)(+) T cells infiltrating the tumor after combined therapy. After combined therapy, the expression of MHC-1 (H-2K(d)) and Fas molecules on freshly isolated tumor cells increased greatly. The activity of specific cytotoxic T lymphocytes was also found to be induced more significantly after the combined therapy (P<.01). Our results demonstrated that combined therapy with P16 and GM-CSF genes can inhibit the growth of established tumors in mice significantly and induce antitumor immunity of the host efficiently.
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PMID:Adenovirus-mediated combined P16 gene and GM-CSF gene therapy for the treatment of established tumor and induction of antitumor immunity. 1222 22

Biomaterial surface morphology and chemistry influence cell responses mediated via signaling cascades that regulate a wide range of metabolic processes. These responses may range from changes in surface adhesion and remodeling of the extracellular matrix to activation of cytokine, cytoskeletal and other biochemical pathways regulating or modulating cellular morphology and function. The present study has focused on collagen Type I, a key extracellular matrix protein, and its potential impact on the process of cellular aging. This study was undertaken for several reasons. First, several investigators reported that growth of cells on a collagen matrix markedly enhanced the resistance of cells to stresses. Second, a large body of accumulated data strongly indicated a relationship between the potential to respond to stresses and cellular aging with the former strongly influencing the rate of the latter. Finally, it has been recently demonstrated that in aged cells one of the key aging-related processes previously considered irreversible, attenuation of the expression of a major stress response protein, Hsp70, can be reversed. This fact together with a probable regulatory role of the stress response potential in cellular aging suggested a possibility that the cellular aging process as a whole can be altered. Indeed, in the present study, growth on a denatured collagen matrices reversed in aged cells not only the attenuation of Hsp70 expression but also other aging-related processes, such as beta-galactosidase expression, increase in protein oxidation and changes in cell morphology. Moreover, it appeared to reduce the rate of aging in young cells. Understanding the nature of collagen matrix-mediated cellular rejuvenation might suggest approaches for interfering with organismic aging. Some immediate applications include cell rejuvenation for purposes of cloning and reduction of the rate of aging during expansion of stem cells for purposes of tissue engineering.
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PMID:Matrix-mediated cellular rejuvenation. 1239 64

Gene therapy may be an effective approach to thyroid carcinoma refractory to conventional treatment. A transcriptionally targeted retroviral vector for gene therapy of thyroid carcinomas was generated replacing the viral enhancer with the enhancer sequence of the human thyroglobulin (TG) gene, yielding a chimeric long-terminal repeat. The TG enhancer was used to drive the expression of either a reporter gene (beta-galactosidase) or two therapeutic genes, i.e. the prodrug-activating enzyme thymidine kinase of herpes simplex virus (HSV-TK) and human IL-2, separated by an internal ribosome entry site. The corresponding vector having an unmodified long-terminal repeat was used as control. The targeted vector allowed selective transgene expression and cell killing in differentiated thyroid tumor cells but not in anaplastic thyroid carcinoma cells and nonthyroid cells, as demonstrated by quantitative RT-PCR and cytotoxicity assays. Nude mice injected with tumor cells underwent near complete or complete regression of tumors transduced with the control vector after ganciclovir treatment. On the other hand, infection with the thyroid-specific vector led to regression only of TG-expressing tumors. In addition, tumors expressing human IL-2 showed significant growth retardation, compared with nontransduced tumors while exhibiting signs of necrosis and presence of an inflammatory infiltrate. However, HSV-TK/IL-2 plus ganciclovir was significantly more efficient than HSV-TK/IL-2 alone in eradicating tumor masses. Our results indicate that replacement of viral enhancer with TG enhancer confers selectivity of transgene expression in thyroid cells. Thus, the combined thyroid-specific expression of two therapeutic genes (cytokine and suicide genes), although a safe tumor-targeted treatment, would allow an increased anticancer effect.
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PMID:Transcriptionally targeted retroviral vector for combined suicide and immunomodulating gene therapy of thyroid cancer. 1241 7

The myofibroblast shares phenotypic features of both fibroblasts and smooth muscle cells. It plays a critical role in collagen deposition and wound healing and disappears by apoptosis when the wound is closed. Its abnormal persistence leads to hypertrophic scar formation and other fibrotic conditions. Myofibroblasts are present in the fibrotic plaque of the tunica albuginea (TA) of the penis in men with Peyronie's disease (PD), a localized fibrosis that is accompanied by a spontaneous induction of the inducible nitric oxide synthase (iNOS), also observed in the TGFbeta1-elicited, PD-like lesion in the rat model. iNOS expression counteracts fibrosis, by producing nitric oxide (NO) that reduces collagen deposition in part by neutralization of profibrotic reactive oxygen species. In this study we investigated whether fibroblast differentiation into myofibroblasts is enhanced in the human and rat PD-like plaque and in cultures of human tissue fibroblasts. We also examined whether NO reduces this cell differentiation and collagen synthesis. The myofibroblast content in the fibroblast population was measured by quantitative immunohistochemistry as the ratio between alpha-smooth muscle actin (ASMA; myofibroblast marker) and vimentin (general fibroblast marker) levels. We found that myofibroblast content was considerably increased in the human and TGFbeta1-induced rat plaques as compared to control TA. Inhibition of iNOS activity by chronic administration of L-iminoethyl-L-lysine to rats with TGFbeta1-induced TA lesion increased myofibroblast abundance and collagen I synthesis measured in plaque and TA homogenates from animals injected with a collagen I promoter construct driving the expression of beta-galactosidase. Fibroblast differentiation into myofibroblasts occurred with passage in the cell cultures from the human PD plaque, but was minimal in cultures from the TA. Induction of iNOS in PD and TA cultures with a cytokine cocktail and a NO donor, S-nitroso-N-acetyl penicillamine (SNAP), was detected by immunohistochemistry. Both treatments reduced the total number of cells and the number of ASMA positive cells, whereas only SNAP decreased collagen I immunostaining. These results support the hypotheses that myofibroblasts play a role in the development of the PD plaque and that the antifibrotic effects of NO may be mediated at least in part by the reduction of myofibroblast abundance and lead to a reduction in collagen I synthesis.
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PMID:Effect of nitric oxide on the differentiation of fibroblasts into myofibroblasts in the Peyronie's fibrotic plaque and in its rat model. 1244 75

Ethanol is known to cause both tolerance and sensitization to endotoxin (lipopolysaccharide). It is also known that ethanol modulates the expression and activity of several intracellular signaling molecules and transcription factors in monocytes and Kupffer cells, the resident hepatic macrophages. Expression of CD14, the endotoxin receptor, is up-regulated following chronic exposure to endotoxin and ethanol. Ethanol-induced oxidative stress is important in the regulation of transcription factor activation and cytokine production by Kupffer cells. Thus, it was hypothesized that acute ethanol increases CD14 expression through a mechanism dependent upon oxidant production. This hypothesis was tested by overexpression of superoxide dismutase via recombinant adenovirus. Mice were infected with adenovirus (3 x 10(9) plaque-forming units, intravenously) containing either Cu,Zn superoxide dismutase (Ad.SOD1) or beta-galactosidase (Ad.lacZ), which caused significant expression of Cu,Zn-SOD in hepatocytes and Kupffer cells. Three days post-infection, mice were given saline or ethanol (5 g/kg, intragastrically). A significant increase in CD14 mRNA was observed 3 h after ethanol, and this increase was almost completely blocked in mice overexpressing Cu,Zn-SOD. Additionally, overexpression of SOD also blunted ethanol-induced activation of redox-sensitive transcription factors NFkappaB and AP-1 and production of cytokines. However, only inhibition of AP-1 with dominant-negative TAK1 but not NFkappaB by dominant-negative IkappaBalpha significantly blunted ethanol-induced increases in CD14, suggesting that AP-1 is important for CD14 transcriptional regulation. It is also shown here that NADPH oxidase is important in the increase in CD14 due to ethanol. Moreover, these data suggest that acute ethanol causes sensitization to endotoxin through mechanisms dependent upon oxidative stress.
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PMID:Up-regulation of CD14 in liver caused by acute ethanol involves oxidant-dependent AP-1 pathway. 1248 56

Antigen-specific T cells demonstrate several potent effector functions during immune responses. Direct killing of infected cells is crucial for clearing viruses and other intracellular pathogens, but it has been difficult to measure the frequency of cytolytic cells. We have now developed a single-cell assay to measure the number of cytotoxic cells in a population, using a herpes simplex virus amplicon vector to express Escherichia coli beta-galactosidase in mouse or human target cells, and an Elispot to detect release of beta-galactosidase from killed target cells. This antigen-specific, perforin-dependent Lysispot assay has been combined with a cytokine Elispot in a two-color assay to confirm that cytotoxicity and interferon-gamma secretion are regulated independently. The simultaneous enumeration of cytokine-secreting and cytotoxic cells should be invaluable for ex vivo analysis of immune responses during infection and autoimmunity.
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PMID:Measuring the frequency of mouse and human cytotoxic T cells by the Lysispot assay: independent regulation of cytokine secretion and short-term killing. 1253 41

Previously, we reported on a novel cationic nanoparticle-based DNA vaccine delivery system. In the present studies, the effects of co-administration of two well-known adjuvants, cholera toxin (CT) and lipid A (LA), with plasmid DNA (pDNA)-coated nanoparticles were investigated. Balb/C mice (n=6) were immunized with either pDNA alone (cytomegalovirus-beta-galactosidase, 5 microg) or pDNA-coated nanoparticles with either 0 or 50 microg of LA on days 0, 7, and 14 subcutaneously (s.c.), or topically on shaved skin with either pDNA (5 microg) alone or pDNA-coated nanoparticles with 0, 10, or 100 microg of CT on days 0, 6, 21, and 35. Mice were sacrificed on day 28 or day 45. Serum IgG titer, in vitro cytokine release and cell proliferation of the isolated splenocytes were determined. By the topical route, immunization of mice with 'naked' pDNA together with 10 and 100 microg of CT significantly enhanced the antigen-specific serum IgG titer by four- and 20-fold, respectively, compared to immunization with pDNA alone. Moreover, co-administration of 100 microg CT with the pDNA-nanoparticles enhanced the IgG titer by more than 300-fold over immunization with 'naked' pDNA alone with no CT. In vitro interferon-gamma (IFN)-gamma release from splenocytes isolated from mice immunized with pDNA-coated nanoparticles with CT (100 microg) was increased by three-fold over immunization with pDNA-nanoparticles without CT. Similarly, in vitro IFN-gamma release from splenocytes isolated from mice immunized with 'naked' pDNA with CT (100 microg) was increased by two-fold over immunization with 'naked' pDNA without CT. Finally, pDNA-coated nanoparticles adjuvanted with 10 microg CT resulted in the strongest splenocyte proliferation. By the s.c. route, co-administration of LA (50 microg) with pDNA resulted in more than 16-fold enhancement in IgG titer over immunization with 'naked' pDNA alone. Immunization with pDNA-coated nanoparticles with LA (50 microg) led to 16-fold enhancement in specific serum IgG titer over immunization with pDNA-coated nanoparticles with no LA, and more than 250-fold enhancement over immunization with 'naked' pDNA alone with no LA. Moreover, in vitro IFN-gamma release and proliferation by splenocytes isolated from LA co-immunized mice was also significantly enhanced. In conclusion, CT (topical) and LA (s.c.) are potential adjuvants to further enhance immune responses using a novel cationic nanoparticle-based DNA vaccine delivery system.
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PMID:The effect of co-administration of adjuvants with a nanoparticle-based genetic vaccine delivery system on the resulting immune responses. 1255 99

Dendritic cells (DCs) are the most potent antigen-presenting cells and a prerequisite for the initiation of primary immune response. This study was performed to investigate the contribution of DCs to the initiation of Graves' hyperthyroidism, an organ-specific autoimmune disease in which the thyrotrophin receptor (TSHR) is the major autoantigen. DCs were prepared from bone marrow precursor cells of BALB/c mice by culturing with granulocyte macrophage-colony stimulating factor and interleukin-4. Subcutaneous injections of DCs infected with recombinant adenovirus expressing the TSHR (but not beta-galactosidase) in syngeneic female mice induced Graves'-like hyperthyroidism (8 and 35% of mice after two and three injections, respectively) characterized by stimulating TSHR antibodies, elevated serum thyroxine levels and diffuse hyperplasitc goiter. TSHR antibodies determined by ELISA were of both IgG1 (Th2-type) and IgG2a (Th1-type) subclasses, and splenocytes from immunized mice secreted interferon-gamma (a Th1 cytokine), not interleukin-4 (a Th2 cytokine), in response to TSHR antigen. Surprisingly, IFN-gamma secretion, and induction of antibodies and disease were almost completely suppressed by co-administration of alum/pertussis toxin, a Th2-dominant adjuvant, whereas polyriboinosinic polyribocytidylic acid, a Th1-inducer, enhanced splenocyte secretion of IFN-gamma without changing disease incidence. These observations demonstrate that DCs efficiently present the TSHR to naive T cells to induce TSHR antibodies and Graves'-like hyperthyroidism in mice. In addition, our results challenge the previous concept of Th2 dominance in Graves' hyperthyroidism and provide support for the role of Th1 immune response in disease pathogenesis.
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PMID:Dendritic cells infected with adenovirus expressing the thyrotrophin receptor induce Graves' hyperthyroidism in BALB/c mice. 1256 82


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