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)

Investigation of the molecular basis of megakaryocyte (MK) and platelet biology has been limited by an inadequate source of genetically manipulable cells exhibiting physiologic MK and platelet functions. We hypothesized that ex vivo cultured MKs would exhibit agonist inducible glycoprotein (GP) IIb-IIIa activation characteristic of blood platelets and that these cultured MKs would be capable of transgene expression. Microscopic and flow cytometric analyses confirmed that human hematopoietic stem cells cultured in the presence of pegylated recombinant human MK growth and development factor (PEG-rHuMGDF) differentiated into morphologic and phenotypic MKs over 2 weeks. Cultured MKs expressed functional GPIIb-IIIa receptors as assessed by agonist inducible soluble fibrinogen and PAC1 binding. The specificity and kinetics of fibrinogen binding to MK GPIIb-IIIa receptors were similar to those described for blood platelets. The reversibility and internalization of ligands bound to MK GPIIb-IIIa also shared similarities with those observed in platelets. Cultured MKs were transduced with an adenoviral vector encoding green fluorescence protein (GFP) or beta-galactosidase (beta-gal). Efficiency of gene transfer increased with increasing multiplicities of infection and incubation time, with 45% of MKs expressing GFP 72 hours after viral infection. Transduced MKs remained capable of agonist induced GPIIb-IIIa activation. Thus, ex vivo cultured MKs (1) express agonist responsive GPIIb-IIIa receptors, (2) are capable of expressing transgenes, and (3) may prove useful for investigation of the molecular basis of MK differentiation and GPIIb-IIIa function.
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PMID:Ex vivo cultured megakaryocytes express functional glycoprotein IIb-IIIa receptors and are capable of adenovirus-mediated transgene expression. 1059 53

To investigate the effect of murine cytomegalovirus (MCMV) infection on the developing mouse brain in vitro, we developed an infection system using cerebral slice cultures. Using a micromanipulator, the cerebral slices from mouse embryos on day 18.5 of gestation were injected in the subventricular zone with recombinant MCMV in which the lacZ gene was inserted into a late gene, and were cultured for 7 days. Viral infection, detected by beta-galactosidase reaction, was developed at the injection sites of the slices. The virus-infected spots in the slices were enhanced by adding tumor necrosis factor-alpha to the medium and inhibited by adding phosphonoacetic acid or ganciclovir. Sections from paraffin-embedded slices were subjected to immunohistochemical analyses. Neuronal cells, labeled with 5-bromo-2-deoxyuridine 24 h before cutting the slices, migrated to the cerebral cortex in the slices. Virus-infected neuronal cells expressing only the early viral antigen migrated to the cortex, whereas glial cells expressing the immediate early and late antigens tended to remain at the injected sites. The neuronal migration of infected cells was not observed in the cerebral slices from 7-day-old mice and viral infection was not detected after injection in the cerebral slices from 14- and 21-day-old mice. These results from these cerebral slices may reflect the infectious dynamics in vivo, and this system may provide a useful model for analysis of disorders of brain development caused by CMV.
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PMID:Migration of virus-infected neuronal cells in cerebral slice cultures of developing mouse brains after in vitro infection with murine cytomegalovirus. 1060 34

The modified vaccinia virus Ankara (MVA) strain is a candidate vector for vaccination against pathogens and tumors, due to safety concerns and the proven ability of recombinants based on this vector to trigger protection against pathogens in animals. In this study we addressed the fate of the MVA vector in BALB/c mice after intraperitoneal inoculation in comparison with that of the replication-competent Western Reserve (WR) strain by measuring levels of expression of the reporter luciferase gene, the capability to infect target tissues from the site of inoculation, and the length of time of virus persistence. We evaluated the extent of humoral and cellular immune responses induced against the virus antigens and a recombinant product (beta-galactosidase). We found that MVA infects the same target tissues as the WR strain; surprisingly, within 6 h postinoculation the levels of expression of antigens were higher in tissues from MVA-infected mice than in tissues from mice infected with wild-type virus but at later times postinoculation were 2 to 4 log units higher in tissues from WR-infected mice. In spite of this, antibodies and cellular immune responses to viral vector antigens were considerably lower in MVA-inoculated mice than in WR virus-inoculated mice. In contrast, the cellular immune response to a foreign antigen expressed from MVA was similar to and even higher than that triggered by the recombinant WR virus. MVA elicited a Th1 type of immune response, and the main proinflammatory cytokines induced were interleukin-6 and tumor necrosis factor alpha. Our findings have defined the biological characteristics of MVA infection in tissues and the immune parameters activated in the course of virus infection. These results are of significance with respect to optimal use of MVA as a vaccine.
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PMID:Biology of attenuated modified vaccinia virus Ankara recombinant vector in mice: virus fate and activation of B- and T-cell immune responses in comparison with the Western Reserve strain and advantages as a vaccine. 1062 55

Recombinant adenoviruses have great potential as gene delivery systems because of their ability to infect a wide range of target cells. However, systemic delivery of viral vectors to tissues other than liver and spleen has been inefficient because of the rapid clearance of the circulating virus by the liver. In the present study we tested the hypothesis that a systemic administration of E1-deleted recombinant adenovirus vectors that bypasses the hepatic circulation will lead to enhanced expression of these vectors in extrahepatic tissues. The portal vein and hepatic artery in B6/129 F1 mice were clamped and an E1-deleted recombinant adenovirus carrying the beta-galactosidase gene (Ad.CBlacZ) was then administered through the retroorbital venous plexus. The clamp was released 30 min after viral injection with no major chronic ischemic consequences noted. High levels of LacZ expression were detected predominantly in the vessels and capillaries of the lung, intestinal wall, and renal glomeruli 7 days after viral infusion. The transgene expression persisted for at least 21 days. Intense LacZ staining was also observed in the liver, suggesting that liver infection occurred after the portal clamp was released. A retroorbital infusion of anti-adenovirus neutralizing antibodies 5 min before the release of the portal clamp significantly reduced postclamp viral infection to the liver, while LacZ expression in lung and intestine persisted after the antibody treatment. Taken together, these results suggest that liver bypass can significantly improve the transduction efficiency in the other target organs. This method could be used to develop animal models of human diseases that predominantly affect the vessels of the lung, intestine, and kidney.
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PMID:Liver bypass significantly increases the transduction efficiency of recombinant adenoviral vectors in the lung, intestine, and kidney. 1072 40

Treatment failure after radiation therapy of prostate cancer (PC) could be a significant problem. Our objective is to design genetic radiosensitizing strategies for the treatment of PC. Cells from individuals with the genetic disorder ataxia telangiectasia (AT) are hypersensitive to ionizing radiation. Therefore, we examined whether attenuation of the AT gene product, AT mutated (ATM), in PC cells could result in an increased intrinsic radiosensitivity. A p53-mutant PC cell line, PC-3 was infected with adenoviral vectors, expressing antisense ATM RNA to various domains of the ATM gene. Immunoblot analyses of cellular extracts from antisense ATM-transfected PC-3 cells showed attenuated expression of the ATM protein within 2 days of viral infection. Compared with cells infected with an adeno-beta-galactosidase vector, antisense ATM-transfected PC-3 cells showed aberrant control of S-phase cell-cycle checkpoints after exposure to ionizing radiation. Under these conditions, the intrinsic radiosensitivity of the PC-3 cells was enhanced. Consequently antisense ATM gene therapy could serve as a paradigm for strategies that target the cellular survival mechanisms of an irradiated tumor cell and may provide therapeutic benefit to patients undergoing radiation therapy for PC.
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PMID:Adenovirus-mediated antisense ATM gene transfer sensitizes prostate cancer cells to radiation. 1105 87

Epithelial intercellular adhesion is fundamental to the formation of the airway epithelial protective barrier. In this respect, cadherins are important because these adhesion molecules regulate formation and maintenance of epithelial intercellular junctions. To study the importance of airway epithelial integrity in determining susceptibility to virus infection, we used a replication-incompetent adenovirus, RAd35, and an E-cadherin specific function-blocking antibody, SHE78-7, to disrupt intercellular contacts in human bronchial epithelial cell line 16HBE14o- and primary bronchial epithelial cells. After exposure of 16HBE14o- cell cultures to SHE78-7, disruption of the transepithelial permeability barrier was indicated by a loss of transepithelial electrical resistance and an associated increase of mannitol, inulin, and dextran paracellular flux. Subsequent exposure of SHE78-7-treated cell cultures to RAd35 showed a remarkable increase in adenoviral infection as assessed by beta-galactosidase reporter gene expression. In cultures exposed to SHE78-7, disruption of E-cadherin function resulted in infection equivalent to that in control cultures using 16-fold lower viral titers. These studies show that manipulation of E-cadherin function provides a specific means of altering epithelial integrity that in turn determines resistance of airway epithelia to adenoviral infection.
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PMID:Loss of epithelial integrity resulting from E-cadherin dysfunction predisposes airway epithelial cells to adenoviral infection. 1106 39

A key impediment to the development of effective virus-mediated gene therapy for cancer is the low level of gene transfer that occurs after the administration of recombinant viral vectors. Improving in vivo infection and transduction efficiency is an important goal for gene therapy. The limited distribution of gene delivery is particularly problematic when large vectors such as recombinant adenoviruses and retroviruses are used to mediate transgene delivery to solid tumors. To facilitate the spread of virus, we have investigated the potential of administering proteases prior to the intratumoral inoculation of recombinant replication deficient adenovirus. For these studies, we chose proteases that are active against collagen and the other extracellular matrix proteins found in primary brain tumor tissue, but are not widely expressed in normal brain. Various concentrations of a mixture of collagenase/dispase or trypsin were inoculated into xenografts of human glioblastoma multiforme-derived brain tumor cell lines U87, U251, and SF767. Subsequently, recombinant adenovirus encoding the beta-galactosidase gene was administered and tumor tissue was examined for evidence of virus infection. Both collagenase/dispase and trypsin enhanced virus infection, indicating that protease pretreatment may be a useful strategy for enhancing virus-mediated gene transduction for many in vivo applications.
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PMID:Pretreatment with protease is a useful experimental strategy for enhancing adenovirus-mediated cancer gene therapy. 1108 79

To evaluate the efficiency of gene delivery in gene therapy strategies for malignant brain tumors, it is important to determine the distribution and magnitude of transgene expression in target tumor cells over time. Here, we assess the time- and vector dose-dependent kinetics of recombinant herpes simplex virus (HSV)-1 vector-mediated gene expression and vector replication in culture and in vivo by a recently developed radiotracer method for noninvasive imaging of gene expression (J. G. Tjuvajev et al., Cancer Res., 55: 6126-6132, 1995). The kinetics of viral infection of rat 9L gliosarcoma cells by the replication-conditional HSV-1 vector, hrR3, was studied by measuring the accumulation rate of 2-[14C]-fluoro-5-iodo-1-beta-D-arabinofuranosyl-uracil (FIAU), a selective substrate for viral thymidine kinase (TK). The level of viral TK activity in 9L cells was monitored by the radiotracer assay to assess various vector doses and infection times, allowing vector replication and spread. In parallel, viral yields and levels of Escherichia coli beta-galactosidase activity were assessed quantitatively. To study vector replication, spread and HSV-1-tk and lacZ gene coexpression in vivo, first- or second-generation recombinant HSV-1 vectors (hrR3 or MGH-1) were injected into s.c. growing rat 9L or human U87 deltaEGFR gliomas in nude rats at various times (8 h to 8 days) and at various vector doses [1 x 10(6) to 2 x 10(9) plaque-forming units (PFUs)] prior to imaging. For noninvasive assessment of HSV-1-tk gene expression (124I-labeled FIAU % dose/g), 0.15 mCi of 124I-labeled FIAU was injected i.v. 8 h after the last vector administration, and FIAU positron emission tomography (PET) was performed 48 h later. For the assessment of HSV-1-tk and lacZ gene coexpression, 0.2 mCi of 131I-labeled FIAU was injected i.v. 24 h after the last vector administration. Forty-eight h later, animals were killed, and tumors were dissected for quantitative autoradiographical and histochemical assessment of regional distribution of radioactivity (TK expression measured as 131I-labeled FIAU % dose/g) and coexpressed lacZ gene activity. The rates of FIAU accumulation (Ki) in hrR3-infected 9L cells in culture, which reflect the levels of HSV-1-tk gene expression, ranged between 0.12 and 3.4 ml/g/min. They increased in a vector dose- and infection time-dependent manner and correlated with the virus yield (PFUs/ml), where the PFUs:Ki ratios remained relatively constant over time. Moreover, a linear relationship was observed between lacZ gene expression and FIAU accumulation 5-40 h after infection of 9L cells with a multiplicity of infection of 1.5. At later times (> 52 h postinjection), high vector doses (multiplicity of infection, 1.5) led to a decrease of FIAU accumulation rates, viral yield, and cell pellet weights, indicating vector-mediated cell toxicity. Various levels of HSV-1-tk gene expression could be assessed by FIAU-PET after in vivo infection of s.c. tumors. The levels of FIAU accumulation were comparatively low (approximately ranging from 0.00013 to 0.003% injected dose/g) and were spatially localized; this may reflect viral-induced cytolysis of infected tumor cells and limited lateral spread of the virus. Image coregistration of tumor histology, HSV-1-tk related radioactivity (assessed by autoradiography), and lacZ gene expression (assessed by beta-galactosidase staining) demonstrated a characteristic pattern of gene expression around the injection sites. A rim of lacZ gene expression immediately adjacent to necrotic tumor areas was observed, and this zone was surrounded by a narrow band of HSV-1-tk-related radioactivity, primarily in viable-appearing tumor tissue. These results demonstrate that recombinant HSV-1 vector-mediated HSV-1-tk gene expression can be monitored noninvasively by PET, where the areas of FIAU-derived radioactivity identify the viable portion of infected tumor tissue that retains FIAU accumulation ability, and that the accumulation rate of FIAU in culture, Ki, reflects the number of HSV-1 viral particles in the infected tumor cell population [4.1 +/- 0.6 x 10(6) PFUs/Ki unit (PFUs divided by ml/min/g)]. Moreover, time-dependent and spatial relationships of HSV-1-tk and lacZ gene coexpression in culture and in vivo indicate the potential for indirect in vivo imaging of therapeutic gene expression in tumor tissue infected with any recombinant HSV-1 vector where a therapeutic gene is substituted for the lacZ gene.
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PMID:Positron emission tomography-based imaging of transgene expression mediated by replication-conditional, oncolytic herpes simplex virus type 1 mutant vectors in vivo. 1130 77

The loss of cochlear hair cells, or the loss of their capacity to transduce acoustic signals, is believed to be the underlying mechanism in many forms of hearing loss. To develop viral vectors that allow for the introduction of genes directly into the cochleae of adult animals, replication-deficient (E1(-), E3(-)) and replication-defective (E1(-), E3(-), pol(-)) adenovirus vectors were used to transduce the bacterial beta-galactosidase gene into the hair cells of the guinea pig cochlea in vivo. Distortion product otoacoustic emissions, which monitor the functional status of outer hair cells, were measured throughout the viral infection periods to identify hair cell ototoxicity. The results demonstrated that the use of the (E1(-), E3(-)) adenovirus vectors containing CMV-driven LacZ, compromised cochlear function when gradually introduced into scala tympani via an osmotic pump. However, when (E1(-), E3(-), pol(-)) adenoviral vectors containing CMV-driven LacZ were used to transduce cochlear hair cells, there was no loss of cochlear function over the frequency regions tested, and beta-galactosidase (beta-gal) was detected in over 80% of all hair cells. Development of a viral vector that infects cochlear hair cells without virus-induced ototoxic effects is crucial for gene replacement strategies to treat certain forms of inherited deafness and for otoprotective strategies to prevent hair cell losses to treat progressive hearing disorders. Moreover, in vivo (E1(-), E3(-), pol(-)) adenovirus mediated gene-transfer techniques applied to adult guinea pig cochleae may be useful in testing several hypotheses concerning what roles specific genes play in normal cochlear function.
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PMID:A modified adenovirus can transfect cochlear hair cells in vivo without compromising cochlear function. 1142 Jun 43

It has been shown that different types of pathogens induce different immune responses. Recovery from intracellular bacterial and viral infection is dependent on the secretion of Th1 cytokines, such as interferon-gamma (IFN-gamma), and on the generation of cytotoxic T cells. In contrast, responses to some parasitic invaders are of the Th2 type, characterized by secretion of interleukin-4 (IL-4). At present, it is not clear what directs this choice, and the most prevalent hypotheses are based on the dendritic cells (DC). In this work, we studied the immune responses generated in mice to a number of antigens, both replicating and nonreplicating, using bone marrow-derived DC as vehicles for immunization. We demonstrate that DC infected with influenza virus prime for a pure Th1 response in vivo devoid of IL-4 induction. This immune response correlates with the induction of DC maturation by the virus. In contrast, nonreplicating antigens, such as fetal bovine serum (FBS), beta-galactosidase, or inactivated influenza virus, do not mature the DC and prime for responses characterized by the secretion of large amounts of IL-4. These data support the hypothesis that myeloid DC are capable of eliciting both types of responses depending on the nature of the antigen.
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PMID:Myeloid dendritic cells stimulate both Th1 and Th2 immune responses depending on the nature of the antigen. 1157 70


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