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)

Seven immunocompetent, revaccinated patients with surgically incurable cutaneous melanoma underwent treatment of dermal and/or subcutaneous metastases with twice-weekly intratumoral injections of escalating doses (10(4)-2 x 10(7) plaque-forming units (PFU)/lesion; 10(4)-8 x 10(7) PFU/session) of a vaccinia/GM-CSF recombinant virus for 6 weeks. Patients with stable or responding disease were maintained on treatment until tumor resolution or progression. Systemic toxicity was infrequent, dose-related, and limited to mild flu-like symptoms that resolved within 24 hours. Local inflammation, at times with pustule formation, was consistently seen with doses of > or =10(7) PFU/lesion. Chronically treated lesions showed a dense infiltration, with CD4+ and CD8+ lymphocytes, histiocytes, and eosinophils. All seven patients developed an antivaccinia humoral immune response 14-21 days following revaccination. Despite the presence of these antivaccinia antibodies, the reporter gene was expressed, as judged by the development of anti-beta-galactosidase antibodies in all patients. Passenger cytokine gene function was evidenced by the presence of virally encoded GM-CSF mRNA at injection sites both early (weeks 1 and 5) and late (week 31) in the course of treatment. Eosinophilia at treatment sites indicated that physiologically significant levels of functional cytokine were generated. However, there were no changes in the total number of peripheral white blood cells or in the numbers or percentages of polymorphonuclear leukocytes, monocytes, or eosinophils. GM-CSF was not detected in the sera. The two patients with the largest tumor burdens failed to respond even at treatment sites. Three patients had mixed responses, with regression of treated and untreated dermal metastases and progression of disease elsewhere. One patient had a partial response, with regression of injected and uninjected regional dermal metastases. Residual melanoma was excised, rendering the patient disease free. One patient with only dermal metastases confined to the scalp achieved a complete remission. Sequential administration of escalating doses of a GM-CSF recombinant vaccinia virus is safe, effective at maintaining passenger gene function, and can induce tumor regression.
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PMID:Intratumoral recombinant GM-CSF-encoding virus as gene therapy in patients with cutaneous melanoma. 1050 51

Sialic acid is the receptor determinant for the human parainfluenza virus type 3 (HPF3) hemagglutinin-neuraminidase (HN) glycoprotein, the molecule responsible for binding of the virus to cell surfaces. In order for the fusion protein (F) of HPF3 to promote membrane fusion, HN must interact with its receptor. In addition to its role in receptor binding and fusion promotion, the HPF3 HN molecule contains receptor-destroying (sialidase) activity. The putative active sites are in the extracellular domain of this type II integral membrane protein. However, HN is not available in crystalline form; the exact locations of these sites, and the structural requirements for binding to the cellular receptor, which has not yet been isolated, are unknown. Nor have small molecular synthetic inhibitors of attachment or fusion that would provide insight into these processes been identified. The strategy in the present study was to develop an assay system that would provide a measure of a specific step in the viral cycle-functional interaction between viral glycoproteins and the cell during attachment and fusion-and serve to screen a variety of substances for inhibitory potential. The assay is based on our previous finding that CV-1 cells persistently infected (p.i.) with HPF3 do not fuse with one another but that the addition of uninfected CV-1 cells, supplying the critical sialic acid containing receptor molecules that bind HN, results in rapid fusion. In the present assay two HeLa cell types were used: we persistently infected HeLa-LTR-betagal cells, assessed their fusion with uninfected HeLa-tat cells, and then quantitated the beta-galactosidase (betagal) produced as a result of this fusion. The analog alpha-2-S-methyl-5-N-thioacetylneuraminic acid (alpha-Neu5thioAc2SMe) interfered with fusion, decreasing betagal production by 84% at 50 mM and by 24% at 25 mM. In beginning to extend our studies to different types of molecules, we tested an unsaturated derivative of sialic acid, 2,3-dehydro-2-deoxy-n-acetyl neuraminic acid (DANA), which is known to inhibit influenza neuraminidase by virtue of being a transition-state analog. We found that 10 mM DANA inhibited neuraminidase activity in HPF3 viral preparations. More significantly, this compound was active in our assay of HN-receptor interaction; 10 mM DANA completely blocked fusion and betagal production, and hemadsorption inhibition by DANA suggested that DANA blocks attachment. In plaque reduction assays performed with the compounds, the active analog alpha-Neu5thioAc2SMe reduced plaque formation by 50% at a 50 mM concentration; DANA caused a 90% inhibition in the plaque reduction assay at a concentration of 25 mM. Our results indicate that specific sialic acid analogs that mimic the cellular receptor determinant of HPF3 can block virus cell interaction and that an unsaturated n-acetyl-neuraminic acid derivative with affinity to the HN site responsible for neuraminidase activity also interferes with HN-receptor binding. Strategies suggested by these findings are now being pursued to obtain information regarding the relative locations of the active sites of HN and to further elucidate the relationship between the receptor-binding and receptor-destroying activities of HN during the viral life cycle. The quantitative assay that we describe is of immediate applicability to large-scale screening for potential inhibitors of HPF3 infection in vivo.
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PMID:The use of a quantitative fusion assay to evaluate HN-receptor interaction for human parainfluenza virus type 3. 1060 17

Gene transfer was performed using asialo-oroso-mucoid-polylysine (ASOR-PL) conjugates to allow targeted expression of the gene in cells of hepatic origin. In a gel-electrophoretic analysis, the ASOR-PL conjugate produced a complete DNA retardation effect at the optimal ratio of 222:1 (ASOR-PL conjugate/pCMV beta-gal plasmid). The gene-transfer efficiency of the ASOR-PL conjugate was evaluated in HepG2 cells that express asialoglycoprotein receptor and NIH 3T3 cells that do not. The expression was assayed by 5-bromo-4-chloroindol-3-yl beta-D-galactopyranoside ('X-Gal') staining and Chlorophenol Red beta-D-galactopyranoside. When an expression vector for the tumour-suppressor gene p53, pCMVp53, complexed to ASOR-PL conjugate, was transfected into HepG2 cells, the exogenously provided p53 gene was detected in the HepG2 cells by PCR. To improve the efficiency of DNA delivery and expression of the therapeutic proteins poloxamer 407, a fusogenic peptide, influenza-virus haemagglutinin HA2 and chloroquine were individually incorporated into the system. The expression level of beta-galactosidase in HepG2 cells was increased by about four times by the presence of poloxamer 407, whereas the fusogenic peptide HA2 and chloroquine had no effects. When HepG2 cells were transfected with pCMVp53 in the presence of poloxamer 407, the mRNA of transfected p53 could be detected by reverse transcriptase PCR. The current findings open the possibility that a receptor-mediated gene-delivery system for hepatic gene therapy using ASOR-PL conjugate in combination with poloxamer 407 may be developed in the future.
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PMID:Improvement of receptor-mediated gene delivery to HepG2 cells using an amphiphilic gelling agent. 1091 34

Intramuscular injection of plasmid DNA results in myofiber cell expression of proteins encoded by the DNA. The preferred vehicle for plasmid DNA injections has been saline (154 mM sodium chloride) or PBS (154 mM NaCl plus 10 mM sodium phosphate). Here, it is shown that injection of luciferase or beta-galactosidase encoding plasmid DNA in a 150 mM sodium phosphate vehicle into murine muscle resulted in a two- to seven-fold increase in transgene expression compared with DNA injected in saline or PBS. When the DNA encoded secreted alkaline phosphatase, preproinsulin or interferon, sodium phosphate vehicle increased their serum levels by two- to four-fold. When the DNA encoded mouse erythropoietin, sodium phosphate vehicle increased hematocrits by two-fold compared with DNA injected in saline. When the DNA encoded influenza nucleoprotein, sodium phosphate increased anti-nucleoprotein antibody titers by two-fold. The expression of luciferase from plasmid DNA instilled into lung was increased five-fold compared with that in vehicle without sodium phosphate. Incubation of plasmid DNA with muscle extract or serum showed that sodium phosphate protected the DNA from degradation. Thus, a change from sodium chloride to sodium phosphate vehicle can enhance the expression of plasmid DNA in a tissue, possibly by inhibiting DNA degradation. Gene Therapy (2000) 7, 1171-1182.
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PMID:Sodium phosphate enhances plasmid DNA expression in vivo. 1091 85

Replication-defective adenoviruses are arousing growing interest as both gene therapy and vaccine vectors. In a phase I clinical trial designed to evaluate the feasibility and tolerance of recombinant adenovirus (rAd)mediated gene transfer, we previously demonstrated that a single intratumoral injection of 10(9) PFU of rAd encoding the beta-galactosidase protein (Ad-beta-Gal) induced strong short-term (1-3 months) humoral, helper (Th1 type) and cytotoxic T cell responses specific for the transgene product in patients with advanced lung cancer. The purpose of the present study was to evaluate the persistence of long-lasting immunity to the transgene protein and in parallel, to assess patient immunocompetence revealed by responses to recall antigens (tetanus toxoid, purified protein derivative), viral pathogens (Epstein-Barr virus, influenza virus), and allogeneic antigens in mixed lymphocytic reactions. The beta-Gal-specific proliferative response declined rapidly in patients with progressive disease, as did responses to the other antigens. In contrast, a long-lasting proliferative response to beta-gal was maintained in an immunocompetent patient in complete remission 2 years after an injection of 108 PFU of Ad-beta-Gal. Anti-beta-Gal humoral (IgG and IgA) responses persisted notably, as did responses to TT and poliomyelytic antigens. While T cell effector cytotoxic responses specific for the viral peptides plummeted, the frequency of anti-beta-Gal CTL precursors remained particularly high, thus attesting to major immunization. Despite the impact of both advanced disease and chemotherapy on immunocompetence, we show the long-term persistence of immunity to the transgene protein vectorized by rAd.
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PMID:Longitudinal follow-up of cellular and humoral immunity induced by recombinant adenovirus-mediated gene therapy in cancer patients. 1098 63

This report characterizes Vaxfectin, a novel cationic and neutral lipid formulation which enhances antibody responses when complexed with an antigen-encoding plasmid DNA (pDNA). In mice, intramuscular injection of Vaxfectin formulated with pDNA encoding influenza nucleoprotein (NP) increased antibody titers up to 20-fold, to levels that could not be reached with pDNA alone. As little as 1 microg of pDNA formulated with Vaxfectin per muscle resulted in higher anti-NP titers than that obtained with 25 microg naked pDNA. The antibody titers in animals injected with Vaxfectin-pDNA remained higher than in the naked pDNA controls for at least 9 months. The enhancement in antibody titers was dependent on the Vaxfectin dose and was accomplished without diminishing the strong anti-NP cytolytic T cell response typical of pDNA-based vaccines. In rabbits, complexing pDNA with Vaxfectin enhanced antibody titers up to 50-fold with needle and syringe injections and also augmented humoral responses when combined with a needle-free injection device. Vaxfectin did not facilitate transfection and/or increase synthesis of beta-galactosidase reporter protein in muscle tissue. ELISPOT assays performed on bone marrow cells from vaccinated mice showed that Vaxfectin produced a three- to five-fold increase in the number of NP-specific plasma cells. Thus, Vaxfectin should be a useful adjuvant for enhancing pDNA-based vaccinations.
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PMID:Vaxfectin enhances the humoral immune response to plasmid DNA-encoded antigens. 1122 61

The association of HLA-B27 with certain forms of arthritis implies a role for MHC class I-restricted T cells in the arthritic process. Our aim was to study CD8(+) T cell responses towards specific antigens localized in joint tissue. Known determinants were introduced into chondrocytes of transgenic (TG) mice, under the control of the cis-regulatory sequences of the human type II collagen gene (COL2A1). Two Escherichia coli beta-galactosidase (beta-gal)-expressing lines were derived (CIIL73 and CIIL64) as well as two lines (CIINP) expressing influenza A virus nucleoprotein (NP). Expression of the antigens could be demonstrated in cartilaginous tissues. The TG lines showed variable degrees of responsiveness towards the transgene-introduced antigens; whilst 75% of CIIL73 mice had an impaired cytotoxic T lymphocyte (CTL) response towards beta-gal, the response in CIIL64 mice was essentially normal. However, both lines displayed normal proliferative and antibody responses to beta-gal. A reduced CTL response was seen to NP in the CIINP lines in approximately 65% of the animals. In spite of the persistence of T cell responses to the transgene antigens in these lines, induction of CTL responses alone has so far failed to induce clinical signs of arthritis. Interestingly, some animals expressing beta-gal were susceptible to arthritis following challenge with type II collagen alone, whilst their non-TG littermates and TG mice from other lines remained unaffected. As beta-gal is expressed by E. coli, a component of the normal gut flora, this suggests a possible role for gut-derived immune responses. We believe these lines could form the basis of a model for studying links between intestinal inflammation and arthritis.
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PMID:Chondrocyte antigen expression, immune response and susceptibility to arthritis. 1128 81

In this study, the potential of the bare skin as a non-invasive route for vaccination was examined. Following application of heat-labile enterotoxin (LT) of Escherichia coli onto bare skin of BALB/c mice, strong serum anti-LT antibody responses were observed, and mucosal immunoglobulin A (IgA) and IgG antibodies were measured in vagina washes. In addition, LT enhanced the serum and mucosal antibody and proliferative T-cell responses to the model protein antigen beta-galactosidase (beta-gal) when coadministered onto bare skin, highlighting its potential to exert an adjuvant effect. When a peptide representing a T-helper epitope (aa 307-319) from the haemagglutinin of influenza virus was applied onto bare skin with LT or cholera toxin (CT), it primed effectively peptide- and virus-specific T cells, as measured in vitro by the interleukin-2 (IL-2) secretion assay. LT was shown to be as immunogenic as CT. Binding activity to GM1 gangliosides was essential for effective induction of anti-CT serum and mucosal antibody responses. Finally, mice immunized onto bare skin with LT were protected against intraperitoneal challenge with a lethal dose of the homologous toxin. These findings give further support to a growing body of evidence on the potential of skin as a non-invasive route for vaccine delivery. This immunization strategy might be advantageous for vaccination programmes in Third World countries, because administration by this route is simple, painless and economical.
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PMID:Immunization onto bare skin with heat-labile enterotoxin of Escherichia coli enhances immune responses to coadministered protein and peptide antigens and protects mice against lethal toxin challenge. 1129 34

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

Tissue-specific gene transfer remains one of the main challenges to deliver genes into designated and/or disseminated cells. We have previously shown successful gene transfer with a nonviral gene delivery system based on the simple chemical conjugation of plasmid DNA with antibody. However, this approach was hampered by low efficiency due to the poor translocation rate of DNA to the nucleus. To improve this approach, we have modified our vector by introducing noncovalent binding between the antibody and DNA, allowing the possibility to introduce different important molecules. The noncovalent association was achieved with neutravidin and biotinylated components: (1) biotinylated antibodies; (2) a biotinylated hemagglutinin fusogenic peptide of influenza virus to favor endosomal escape; and (3) biotinylated histone H1 to compact, protect, and associate DNA to the complex. We report here that this delivery system can be internalized by tumor cells targeted by a specific monoclonal antibody, permits the protection of the transfected DNA, and allows its subsequent transfer into the nucleus after escape from the endosomal compartment. We also demonstrate that, in vitro, gene transfer with this vector showed much higher reporter activity in cells (15 vs. 0.5%) and a stronger production of murine interleukin 2 as compared with our previous vector. In vivo, a single intravenous injection of the vector containing an antibody directed to the G250 renal cell carcinoma-associated antigen led to beta-galactosidase expression in engrafted tumor bearing G250 but not in G250-negative tumor or in other tissues. Altogether, these results indicate that our antibody-based vector is suitable to promote gene delivery in vitro and in vivo in tumor cells.
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PMID:In vivo-targeted gene delivery using antibody-based nonviral vector. 1206 43


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