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)

Adenovirus is endocytosed and efficiently destroyed by human and murine alveolar macrophages (AMs) and rapidly cleared from the lungs of wild-type but not GM-CSF(-/-) mice. We hypothesized that GM-CSF may regulate adenovirus clearance in AMs via the transcription factor PU.1 by redirecting virion trafficking from the nucleus to lysosomes. This hypothesis was tested in murine AM cell lines with altered GM-CSF and/or PU.1 expression including MH-S (GM-CSF(+/+)PU.1(Pos)), mAM (GM-CSF(-/-)/PU.1(Neg)), and mAM(PU.1+) (GM-CSF(-/-)/PU.1(Pos); PU.1-transduced mAM cells) and A549 (an epithelial-like cell line) using a human adenovirus expressing a beta-galactosidase reporter. In PU.1(Neg) mAM and A549 cells, adenovirus efficiently escaped from endosomes, translocated to the nucleus, and expressed the viral reporter in most cells. In marked contrast, in PU.1(Pos) mAM(PU.1+) and MH-S cells, adenovirus failed to escape from endosomes, colocalized exclusively with endosome/lysosome markers (Rab5, Rab7, and Lamp1), and rarely expressed the reporter. Retroviral expression of PU.1 in A549 cells blocked endosomal escape, nuclear translocation and reporter expression. Inhibition of endosome acidification also blocked escape, nuclear translocation, and reporter expression in PU.1(Neg) cells. The effect of PU.1 on viral trafficking and transduction could not be explained by an effect on endosome acidification or on differences in viral load. PU.1 reduced expression of integrin beta(5), a host factor important for endosomal escape of adenovirus, suggesting that PU.1 redirects adenoviral trafficking by modulating integrin signaling. These results demonstrate that PU.1 uncouples infection from internalization in AMs, providing a mechanism for AMs to avoid infection by adenovirus during clearance.
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PMID:PU.1 redirects adenovirus to lysosomes in alveolar macrophages, uncoupling internalization from infection. 1727 51

A lyophilization method was developed to locally release adenoviral vectors directly from biomaterials for in situ regenerative gene therapy. Adenovirus expressing a beta-galactosidase reporter gene (AdLacZ) was mixed with different excipient formulations and lyophilized on hydroxyapatite (HA) disks followed by fibroblasts culturing and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) staining, suggesting 1 M sucrose in phosphate-buffered saline had best viability. Adenovirus release studies showed that greater than 30% virus remained on the material surface up to 16 h. Lyophilized adenovirus could be precisely localized in defined patterns and the transduction efficiency was also improved. To determine if the lyophilization formulations could preserve viral bioactivity, the lyophilized AdLacZ was tested after being stored at varying temperatures. Bioactivity of adenovirus lyophilized on HA was maintained for greater than 6 months when stored at -80 degrees C. In vivo studies were performed using an adenovirus encoding BMP-2 (AdBMP-2). AdBMP-2 was lyophilized in gelatin sponges and placed into rat critical-size calvarial defects for 5 weeks. Micro-computed tomography (micro-CT) analysis demonstrated that free-form delivery of AdBMP-2 had only modest effects on bone formation. In contrast, AdBMP-2 lyophilized in gelatin sponges led to more than 80% regeneration of critical-size calvarial defects.
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PMID:Localized viral vector delivery to enhance in situ regenerative gene therapy. 1734 1

We tested whether cystic fibrosis (CF) airway epithelia have larger innate immune responses than non-CF or cystic fibrosis transmembrane conductance regulator (CFTR)-corrected cells, perhaps resulting from ER stress due to retention of DeltaF508CFTR in the endoplasmic reticulum (ER) and activation of cytosolic Ca(2+) (Ca(i)) and nuclear factor (NF)-kappaB signaling. Adenovirus infections of a human CF (DeltaF508/DeltaF508) nasal cell line (CF15) provided isogenic comparisons of wild-type (wt) CFTR and DeltaF508CFTR. In the absence of bacteria, there were no or only small differences among CF15, CF15-lacZ (beta-galactosidase-expressing), CF15-wtCFTR (wtCFTR-corrected), and CF15-DeltaF508CFTR (to test ER retention of DeltaF508CFTR) cells in NF-kappaB activity, interleukin (IL)-8 secretion, Ca(i) responses, and ER stress. Non-CF and CF primary cultures of human bronchial epithelial cells (HBE) secreted IL-8 equivalently. Upon infection with Pseudomonas aeruginosa (PA) or flagellin (key activator for airway epithelia), CF15, CF15-lacZ, CF15-wtCFTR, and CF15DeltaF508CFTR cells exhibited equal PA binding, NF-kappaB activity, and IL-8 secretion; cells also responded similarly to flagellin when both CFTR (forskolin) and Ca(i) signaling (ATP) were activated. CF and non-CF HBE responded similarly to flagellin + ATP. Thapsigargin (Tg, releases ER Ca(2+)) increased flagellin-stimulated NF-kappaB and ER stress similarly in all cells. We conclude that ER stress, Ca(i), and NF-kappaB signaling and IL-8 secretion were unaffected by wt- or DeltaF508CFTR in control and during exposure to PA, flagellin, flagellin + ATP, or flagellin + ATP + forskolin. Tg, but not wt- or DeltaF508CFTR, triggered ER stress. Previous measurements showing hyperinflammatory responses in CF airway epithelia may have resulted from cell-specific, rather than CFTR- or DeltaF508CFTR-specific effects.
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PMID:Effects of cystic fibrosis transmembrane conductance regulator and DeltaF508CFTR on inflammatory response, ER stress, and Ca2+ of airway epithelia. 1782 50

Targeted retrograde gene delivery into the injured spinal cord is less invasive for the damaged tissue. One of the advantages of this approach is the possible selection of target organs according to the level of spinal cord injury. We evaluated nine candidate target organs for retrograde delivery of an adenovirus vector carrying beta-galactosidase (AdV-LacZ) gene to cervical, thoracic and lumbar spinal cord segments. One week after vector injection into each muscle, we assessed the LacZ gene expression in the spinal cord by X-gal staining. The most appropriate target organs with high transduction efficacy were the sternomastoid and clavotrapezius muscles for cervical spinal cord, tibialis anterior and the gastrocnemius muscles for the lumbar spinal cord. Retrograde gene delivery to the thoracic spinal cord was inefficient probably due to the small number of anterior horn neurons in the region. Gene expression was mainly identified over the anatomical area of innervation and not into other body organs. Our results suggested that retrograde delivery of adenovirus genome to the cervical and lumbar spinal cord segments seems feasible by injection of an adenoviral vector into the appropriate target organ. Adenovirus vector is an efficient retrograde tracer since it can deliver the carried gene to a wide area of the spinal cord and not to other body organs.
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PMID:Target muscles for retrograde gene delivery to specific spinal cord segments. 1834 75

Gene therapy using adenoviral vectors in tissue regeneration is hindered by a short duration of transgene expression. It is hypothesized that a fibrin scaffold will enhance delivery of the adenovirus to a wound site, precluding the need for high repeated doses. It was aimed to analyze whether fibrin could deliver a low single dose of viral vector to a wound site, without compromising transfection efficiency. Fibrin scaffold containing adenovirus encoding beta-galactosidase, fibrin alone, adenovirus alone, and no treatment groups were applied to a rabbit ear ulcer model. beta-Galactosidase transgene expression was measured at 7 and 14 days. Transgene expression was enhanced in the fibrin containing adenovirus group at 7 days. By 14 days, there was low expression and no difference between groups. Stereological methods assessing wound healing aimed to determine whether the adenovirus capsid elicited an unfavorable inflammatory response and whether fibrin's beneficial properties were altered by addition of adenovirus. The fibrin adenovirus group showed a wound-healing response similar to fibrin alone, showing maximum cellularity and angiogenesis at 7 days. By 14 days, cellularity and angiogenesis subsided, and this effect was not inhibited by the presence of adenovirus. Adenovirus alone did not cause an unfavorable inflammatory response. It is concluded the fibrin aids in the delivery of a low-dose viral vector, thereby avoiding a chronic inflammatory response, and allowing superior transfection than viral vector alone. This has wide-ranging implications on the use of viral vectors in tissue engineering.
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PMID:Fibrin scaffold promotes adenoviral gene transfer and controlled vector delivery. 1846 22

Adenoviruses are powerful, widely utilized vectors for gene transfer. Limitations to their application, however, have not been well described. We used rat pituitary lactotrophs in primary culture as a model for studying how adenovirus vector infection modulates mitogen-induced proliferation and the activities of mitogen signaling pathways. Infection with adenovirus vectors expressing beta-galactosidase (betagal) raised basal proliferative levels and blocked fetal bovine serum (FBS)-induced proliferation of lactotrophs, but did not influence the changes in proliferation induced by forskolin, IGF-I, and bromocriptine. The betagal-expressing adenoviruses did not alter the inhibitory action of 17beta-estradiol (E(2)) in the presence of IGF-I; however, they blocked the stimulatory action of E(2) in the presence of dextran-coated charcoal-striped serum or forskolin. An adenovirus expressing no protein failed to block FBS-induced proliferation, but was effective in modulating basal proliferative levels and the stimulatory actions of E(2). The increased basal proliferative level and the blockade of FBS-induced proliferation were transient, and lost 5 days after infection while the blockade of the stimulatory action of E(2) in the presence of forskolin persisted. Adenovirus infection raised basal protein levels of the phosphorylated forms of cAMP response element-binding protein (pCREB) and ERK1/2 and increased the proportion of pCREB-immunoreactive lactotrophs. Adenoviruses also altered estrogen-induced responses in mRNA expression of several estrogen-responsive genes in a gene-specific manner. The results demonstrate that an adenovirus vector differentially interferes with lactotroph proliferation in response to various mitogens. Our results suggest that the effects of the adenovirus that are independent of the genes transferred must be considered when performing adenoviral gene transfer in the primary cultures of normal cells.
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PMID:Adenovirus vectors differentially modulate proliferation of pituitary lactotrophs in primary culture in a mitogen and infection time-dependent manner. 1857 72

Vein grafts are used to bypass coronary arterial stenosis, but many grafts thrombose soon after surgery. A model was developed in the pig to allow continuous measurement of blood flow and production of flow-restricting thrombi (cyclic flow reductions; CFRs). Saphenous vein lumen was exposed to adenovirus ex vivo, to over-express human tissue plasminogen activator (h-tPA), with beta-galactosidase adenovirus as a control. The vein segments were engrafted into carotid arteries and examined 0, 1 or 3 days later (4-7 animals/group). Untransduced grafts examined on the day of surgery developed repeated CFRs at both normal and restricted flow, but their frequency declined in grafts examined after 3 days. Adenovirus transduction was evident as beta-galactosidase or h-tPA expression 1 day after engraftment. Blood flow was increased 1.4-fold in h-tPA transduced grafts after 1 day [control 390 (280-510), h-tPA 550 (450-660) ml/min; p=0.02 (expressed as mean (95% confidence intervals)]. CFRs were less severe (p=0.002) in the h-tPA transduced grafts than beta-galactosidase-transduced grafts. CFRs were also less frequent in unstenosed undamaged h-tPA grafts [control 17 (6.1-29), h-tPA 7.6 (1.7-14) CFR/hr; p=0.02], but this difference was reduced after damage or stenosis. CFRs formed faster in h-tPA than in beta-galactosidase-transduced grafts [control 14 (11-17), h-tPA 23 (19-27) ml/min(2); p<0.001], and resolved twofold faster [control 25 (22-30), h-tPA 48 (39-60) ml/min(2); p<0.001]. Hence, in this model, local gene therapy with h-tPA increased graft blood flow and decreased measures of early graft thrombosis, namely quicker CFR resolution and decreased frequency and severity.
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PMID:Reduction of early vein graft thrombosis by tissue plasminogen activator gene transfer. 1957 59

Using an adenoviral system as a delivery mediator of therapeutic gene, we investigated the therapeutic effects of the use of combined MDR1 shRNA and human NIS (hNIS) radioiodine gene therapy in a mouse colon xenograft model. In vitro uptake of Tc-99m sestamibi was increased approximately two-fold in cells infected with an adenovirus vector that expressed MDR1 shRNA (Ad-shMDR1) and I-125 uptake was 25-fold higher in cells infected with an adenovirus vector that expressed human NIS (Ad-hNIS) as compared with control cells. As compared with doxorubicin or I-131 treatment alone, the combination of doxorubicin and I-131 resulted in enhanced cytotoxicity for both Ad-shMDR1- and Ad-hNIS-infected cells, but not for control cells. In vivo uptake of Tc-99m sestamibi and Tc-99m pertechnetate was twofold and 10-fold higher for Ad-shMDR1 and Ad-hNIS-infected tumors as compared with tumors infected with a control adenovirus construct that expressed beta-galactosidase (Ad-LacZ), respectively. In mice treated with either doxorubicin or I-131 alone, there was a slight delay in tumor growth as compared to mice treated with Ad-LacZ. However, combination therapy with doxorubicin and I-131 induced further significant inhibition of tumor growth as compared with mice treated with Ad-LacZ. We have shown successful therapeutic efficacy of combined MDR shRNA and hNIS radioiodine gene therapy using an adenoviral vector system in a mouse colon cancer model. Adenovirus-mediated cancer gene therapy using MDR1 shRNA and hNIS would be a useful tool for the treatment of cancer cells expressing multi-drug resistant genes.
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PMID:Enhanced anti-tumor effects of combined MDR1 RNA interference and human sodium/iodide symporter (NIS) radioiodine gene therapy using an adenoviral system in a colon cancer model. 2018 72

Adenovirus-mediated gene therapy shows remarkable promise as a new strategy for advanced pancreatic cancer, but satisfactory clinical results have not yet been obtained. To improve this gene therapy, we investigated the effects of gemcitabine (GEM) on transgene expression by adenoviral vectors and their biological effects. We used Ad-lacZ and adenoviral vector-expressing NK4 (Ad-NK4) as representative adenoviral vectors. These vectors express beta-galactosidase (beta-gal) and NK4 (which inhibits the invasion of cancer cells), respectively, under the control of the CMV promoter. Cells were infected with the individual adenoviruses and then treated with GEM. GEM increased beta-gal mRNA expression and beta-gal activity, and increased NK4 expression in both culture media and within infected cells, in dose-dependent manners. The increased expression of NK4 delivered by Ad-NK4 had biological effects by inhibiting the invasion of cancer cells. GEM also enhanced NK4 expression in SUIT-2 cells transfected with an NK4-expressing plasmid, suggesting that GEM enhanced CMV promoter activity. In in vivo experiments, NK4 expression within subcutaneously implanted tumors was increased in GEM-treated mice compared with control mice. These results suggest that adenovirus-mediated gene therapy with GEM may be a promising approach for treating pancreatic cancer, and that this combination therapy may decrease the risks of side effects.
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PMID:Gemcitabine synergistically enhances the effect of adenovirus gene therapy through activation of the CMV promoter in pancreatic cancer cells. 2039 79

EBV-associated nasopharyngeal cancer (NPC) occurs with high frequency in China and is a major cause of morbidity and mortality. To explore the potential use of adenovirus-mediated tumor suppressor p53 gene therapy In NPC, we first examined the in vitro effects of p53 introduced into the NPC cell lines RPMI 2650, Fadu and Detroit 562. p21(WAF1/CIP1) induction by chemotherapy was used as a functional assay which revealed that RPMI 2650 expresses wild-type p53 whereas Fadu and Detroit 562 encode mutant p53. Infection with p53-expressing adenovirus (Ad-p53) induced apoptosis and inhibited cell growth in all three NPC cell lines, regardless of the endogenous p53 status. Adenovirus infectivity was greatest in RPMI 2650 cells, with 100% of the cells expressing beta-galactosidase following Ad-LacZ infection using an MOI of 100, as compared to 20-30% infectivity with the other NPC lines. Using RPMI 2650 cells injected into nude mice, we developed an animal model for nasopharyngeal cancer. Established tumors (0.6-0.8 cm) were injected with 5x10(9) PFU Ad-LacZ, Ad-p53 or PBS in a 100 mu l volume. We found evidence for in vivo expression of beta-galactosidase or p53 and p21 up to two weeks following Ad-LacZ or Ad-p53 virus injection respectively. Objective regression of tumor size was observed at two weeks in 4/6 Ad-p53-treated tumors, but not in Ad-LacZ or PBS-treated tumors. The results provide an animal model for human nasopharyngeal cancer, and indicate a potential use of p53 in its therapy in vivo.
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PMID:Adenovirus-mediated p53 gene therapy in nasopharyngeal cancer. 2152 3


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