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)

Measles virus (MV)-specific murine helper T cell clones (Thy-1.2+, CD4+, CD8-) were generated from mice immunized with MV-infected mouse brain homogenate by limiting dilution and in vitro stimulation of spleen cells with UV-inactivated MV Ag. The protein specificity of 7 out of 37 stable T cell clones, which displayed MHC-restricted MV Ag recognition, could be assessed by using purified MV proteins. Two fusion (F) protein-specific, two hemagglutinin-specific, and three nucleoprotein- or matrix protein-specific clones were shown to be established. The F protein-specific T cell clones together with a panel of previously generated F protein-specific T cell clones were characterized for their fine specificity by using beta-galactosidase fusion products, which contained different parts of the F protein. It was shown that at least two epitopes on the major part of the F protein (amino acid 2-513) can be recognized by mouse T cells. Functional characterization of three T cell clones showed that they were able to assist MV-specific B cells and bystander B cells for antibody production. Furthermore, they were shown to produce the lymphokines IL-2 and IFN-gamma. It was also shown that these T cell clones induced a MV-specific delayed type hypersensitivity response. These observations suggest that all of the T cell clones characterized belong to the TH1 helper subset.
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PMID:Measles virus-specific murine T cell clones: characterization of fine specificity and function. 252 70

Heterogeneity of human gamma interferon (IFN-gamma) induced by the combined treatment with OK-432 and Staphylococcal enterotoxin B (SEB) was demonstrated by chromatofocusing. Treatment of IFN-gamma with a mixture of neuraminidase, and beta-galactosidase eliminated the charge heterogeneity. Apparent molecular weight of IFN-gamma was decreased by enzyme treatment. These results suggest that the heterogeneity of IFN-gamma induced in our system was the result of the difference in the content of sialic acids.
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PMID:Effect of glycosidases on the properties of human interferon gamma. 642 24

Recombinant viruses with the lacZ gene placed under the control of the HSV-1 ICP4, TK and gD regulatory regions were constructed by recombination into the TK locus of HSV-1. Difficulty in isolating ICP4 and gD recombinant viruses with high level, regulated expression of beta-galactosidase was overcome by the use of HSV-1 translational initiation sequences of these genes in place of vector-derived sequences. beta-Galactosidase expression displayed the kinetics particular to each viral class. The maximal expression of beta-galactosidase from the recombinant viruses within a 22-h period (m.o.i. 5) (relative to the ICP4 virus) was gD(3) > gC(2) > ICP4(1) > TK(0.5). The ICP4 virus produces easily quantifiable levels of beta-galactosidase activity for multiplicities of infection from 5 x 10(-4) through 5 over 48 h postinfection. At multiplicities of infection between 2 and 5, ICP4-driven activity was measurable within 2 h postinfection from a monolayer of 3 x 10(4) Vero cells in microtiter wells. Mechanisms of inhibition of several antivirals were probed by using the regulated expression of beta-galactosidase from the ICP4 virus as a marker for viral growth. An experimental antiviral (E3925, IC50 1 microgram/ml) and a neutralizing gD MAb (DUP55306, IC50 0.6 microgram/ml) acted prior to immediate early synthesis, consistent with inhibition of viral entry or uncoating. IFN-gamma inhibited expression of immediate-early synthesis, while having no effect on viral entry. IC50 values for E3925 obtained using either the ICP4 or gD viruses at m.o.i. 0.005, were in good agreement with those obtained by standard plaque assays, but were determined in only 1 day, using a microtiter plate format. Thus, these reporter viruses are useful tools for defining the mechanisms of action of antiherpes agents, while quantitatively reproducing the results for IC50 determinations from standard plaque assays within 24 h in a microtiter plate format.
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PMID:Herpes simplex type 1:lacZ recombinant viruses. I. Characterization and application to defining the mechanisms of action of known antiherpes agents. 862 13

The immunogenic properties of a replication-defective herpes simplex virus HD-2, containing the Escherichia coli lacZ gene under control of the HSV ICP8 early gene promoter were studied in BALB/c mice. Experiments were designed to determine if the HD-2 virus preferentially stimulated either Th1- or Th2-associated immune responses to beta-galactosidase (beta gal). Sera from mice immunized i.p. or s.c. with virus HD-2, beta gal on aluminum phosphate adjuvant, or a control ICP8 deletion mutant, d301, were assayed for total and Ag-specific IgG1 and IgG2a Abs, beta gal-driven lymphocyte proliferation, and in vitro production of the cytokines IFN-gamma, IL-4, and IL-2. Viruses HD-2 and d301 preferentially stimulated the production of total serum IgG2a following two immunizations i.p. or a single immunization s.c., while only HD-2 virus stimulated in vivo production of beta gal-specific IgG2a serum Abs. In contrast, beta gal adsorbed on AIPO4 preferentially stimulated production of Ag-specific IgG1 serum Abs. The HD-2 virus also induced a potent cellular proliferative response to beta gal, which was still pronounced 5 wk after primary immunization. Cultured lymphocytes from HD-2-immunized mice produced IFN-gamma after 5 days in culture with soluble beta gal in an Ag- and dose-dependent fashion. These results demonstrate that replication-defective mutants of HSV can be used as vectors for eliciting Th1-associated immune responses to a heterologous Ag expressed from the viral genome.
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PMID:Th1-associated immune responses to beta-galactosidase expressed by a replication-defective herpes simplex virus. 875 44

Replication-deficient adenovirus (Ad) vectors are effective to specifically target the respiratory epithelium for either corrective gene therapy such as cystic fibrosis or for mucosal immunization. As a consequence of transducing the lower respiratory tract with an E1/E3 deleted Ad5 vector, host responses have been characterized by the duration of transgene expression and by the induction of CTL responses. However, limited emphasis has been devoted to understanding the contribution of CD4+ T cell responses to the Ad vector. Both CD4+ and CD8+ T cells migrate into the lung following sequential intratracheal Ad5 transgene instillations. Isolated CD3+ T lymphocytes from the lungs were predominantly of the Th2 type, and after cell sorting, the IL-4-producing T cells were largely CD4+, while IFN-gamma expression was associated with both CD4+ and CD8+ T cells. Ab responses to the Ad5 vector and to the expressed transgene beta-galactosidase (beta gal) revealed elevated bronchial and serum IgA and IgG Abs with low neutralization titers. Analysis of serum IgG subclass responses showed IgG1 and IgG2b with lower IgG2a Abs to Ad5 and IgG2a and IgG2b Ab responses to beta gal. Ad5-specifc CD4+ T cells produced both Th1 (IFN-gamma and IL-2)- and Th2 (IL-4, IL-5, IL-6)-type cytokines, while beta gal-specific CD4+ T cells secreted IFN-gamma and IL-6. This study provides direct evidence for the concomitant induction of Th2- with Th1-type responses in both the pulmonary systemic and mucosal immune compartments to the Ad5 vector as well as a Th1-dominant response to the transgene.
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PMID:Adenoviral gene delivery elicits distinct pulmonary-associated T helper cell responses to the vector and to its transgene. 921 83

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

The anti-CD40 ligand antibody MR-1, and macrophage-depleting liposomes were tested for their ability as transient immunosuppressive agents to: (1) prolong transgene expression; and (2) permit redosing after recombinant adenovirus infusion of mice. To test for effect on transgene duration, mice were infused with recombinant adenovirus coding for human factor IX (AdFIX), and plasma FIX levels monitored over time. Treatment with either agent significantly prolonged transgene expression. Persistence was accompanied by inhibition of anti-adenovirus (anti-Ad) IgG, and decreased IL-10 and IFN-gamma production from splenic lymphocytes re-exposed to virus particles in vitro. To test for effect on redosing, mice were given a primary infusion of recombinant adenovirus coding for bacterial beta-galactosidase (Ad beta gal), followed by secondary and tertiary infusions of AdFIX on days 24 and 63. Mice that had received MR-1 had low to undetectable anti-Ad on day 24, and efficient transduction occurred. Furthermore, FIX levels endured in these mice, with 40% retention of FIX on day 63, in contrast to rapid loss in naive controls. On day 63, the continuance of negligible anti-Ad levels correlated with successful tertiary transduction. These results suggest that both macrophage depletion and CD40 ligand blockade inhibit immune responses to recombinant adenovirus to slow decline of transgene expression, while only CD40 ligand blockade inhibits anti-Ad antibody generation sufficiently to allow redosing to the liver.
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PMID:Effects of macrophage depletion and anti-CD40 ligand on transgene expression and redosing with recombinant adenovirus. 961 66

Respiratory syncytial virus (RSV) infection is an important cause of lower respiratory tract illness, the severity of which may be partly due to cellular recruitment. RSV infection activates chemokine secretion from airway epithelial cells by largely unknown mechanisms. We investigated the regulation of RSV-induced activation of the chemokine RANTES in the bronchial epithelial cell line BEAS-2B and primary normal human tracheobronchial epithelial cultures. RANTES protein and mRNA were detected at 24 h and up until 72 h from cultures of BEAS-2B infected with replicating virus, but not with UV-inactivated RSV. RSV infection of BEAS-2B or normal human tracheobronchial epithelial cells stimulated NF-kappa B translocation to the nucleus and binding to the RANTES-specific kappa B-binding sequences within 2 h, with levels peaking at 24 h. Supershift assays indicated that binding was due to p50/p65 heterodimers. BEAS-2B cells were transfected with a replication-deficient adenoviral vector, expressing a mutated, nondegradable form of I kappa B alpha. I kappa B alpha overexpression specifically blocked NF-kappa B translocation and inhibited mRNA accumulation and secretion of RANTES induced by RSV or TNF-alpha plus IFN-gamma. Adenoviral transfection did not interfere with RSV replication or significantly induce apoptosis. Further, a control adenovirus, expressing the beta-galactosidase gene, did not alter cellular functions. Thus, NF-kappa B nuclear translocation is a critical step in RSV induction of RANTES secretion. Elucidating the mechanisms of cellular activation by RSV and targeting specific areas may lead to novel therapeutic approaches in the treatment of RSV.
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PMID:Respiratory syncytial virus-induced RANTES production from human bronchial epithelial cells is dependent on nuclear factor-kappa B nuclear binding and is inhibited by adenovirus-mediated expression of inhibitor of kappa B alpha. 967 Sep 82

Cells of a human glioblastoma line were stably transfected with a glial fibrillary acidic protein (GFAP) promoter sequence/lacZ reporter gene. Following this modification, they produced Escherichia coli beta-galactosidase constitutively in amounts that could be measured through their conversion of an added fluorophore into a product readily estimated by fluorimetry. Human interferons (IFN) selectively and in a dose-dependent manner reduce the formation of beta-galactosidase in this system. We have used it as the basis for a novel assay that is sensitive (4-40 pg/ml), precise, completed in 30 h, and applicable to both type I and type II human IFNs. Statistical analysis showed interassay relative standard deviations ranging from 5% to 11%, and most individual assays revealed potencies with limits of error within 85%-115%. Neither partially trypsin-digested IFN nor the other cytokines and mitogens we tested reacted in this system, except for tumor necrosis factor-alpha (TNF-alpha). The high selectivity was further shown by the loss of response to IFN in the presence of the appropriate specific anti-IFN or anti-IFN-gamma receptor antibodies.
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PMID:The beta-gal interferon assay: a new, precise and sensitive method. 971 60

Replication-deficient adenovirus vectors (Avs) have shown high-efficiency gene transfer in a variety of animal models, but demonstrated lower than expected efficiency in the intensely inflammatory milieu of the respiratory tract of individuals with cystic fibrosis (CF). Specific acquired immune responses directed at adenovirus capsid proteins are known to limit the duration of transgene expression and the effectiveness of vector readministration. In these models, however, nonspecific inflammation is also frequently noted to accompany specific immune responses. Because inflammation can occur early after Av administration, we hypothesized that inflammation may block Av-mediated gene transfer in the lung independent of specific immune responses. To evaluate this hypothesis, we measured pulmonary gene transfer and expression in the absence or presence of the potent antiinflammatory agent dexamethasone. To address and eliminate concerns over the potentially confounding effects of systemic, vector-specific acquired immune responses, evaluations were confined to a 3-day period following Av administration and were carried out, in parallel, in normal and immunodeficient (athymic) mice. Dexamethasone significantly reduced Av-associated inflammation in all animals as measured by a significant reduction of blinded, quantitative lung histopathology scores and by reduced proinflammatory cytokine release. Concomitant with reduced inflammation, gene transfer efficiency was significantly increased in both normal and immunodeficient animals as measured by transgene product activity (beta-galactosidase) in total lung homogenates 3 days after vector administration. This finding could not be explained by a direct effect of dexamethasone on transgene specific activity. To begin to understand the molecular mechanisms of Av-induced inflammatory responses, lung levels of the chemoattractive chemokines MIP-2, MIP-1alpha, and MCP-1 were quantified. All were elevated significantly in Av-exposed animals. Dexamethasone reduced levels of MCP-1 and MIP-1alpha, but not MIP-2, consistent with the observed pattern of inflammatory cell changes. Expression of several proinflammatory cytokines including TNF-alpha, IL-6, IL-1beta, and IFN-gamma were also elevated in Av-exposed animals and modulated by dexamethasone. These observations demonstrate that nonspecific inflammation is an important determinant of the efficiency of in vivo pulmonary gene transfer and expression independent of specific immune responses and may have important implications for human gene therapy for diseases of the lung.
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PMID:Nonspecific inflammation inhibits adenovirus-mediated pulmonary gene transfer and expression independent of specific acquired immune responses. 979 5


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