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)

In vivo electroporation dramatically enhances plasmid vaccine efficacy. This enhancement can be attributed to increased plasmid delivery and, possibly, to some undefined adjuvant properties. Previous reports have demonstrated CD8(+) T cell priming by plasmid vaccines is strongly dependent upon CD4(+) T cell help. Indeed, the efficacy of a plasmid vaccine expressing Escherichia coli beta-galactosidase was severely attenuated in MHC class II-deficient (C2D) mice. To determine whether electroporation could compensate for the absence of CD4(+) T cell help, C2D mice were immunized by a single administration of plasmid in combination with electroporation using two conditions which differed only by the duration of the pulse (20 or 50 msec). Both conditions elicited robust cellular and humoral responses in wild-type mice, as measured by IFN-gamma ELISPOT, anti-beta-galactosidase ELISA, and protection from virus challenge. In C2D mice, the cellular response produced by the vaccine combined with the 50-msec pulse, as measured by ELISPOT, was identical to the response in wild-type mice. The 20-msec pulse elicited a milder response that was approximately one-fifth that of the response elicited by the 50-msec pulse. By contrast, the 20-msec conditions provided comparable protection in both wild-type and C2D recipients whereas the protection elicited by the 50-msec conditions in C2D mice was weaker than in wild-type mice. Further investigation is required to understand the discordance between the ELISPOT results and outcome of virus challenge in the C2D mice. Nonetheless, using this technique to prime CD8(+) T cells using plasmid vaccines may prove extremely useful when immunizing hosts with limiting CD4(+) T cell function, such as AIDS patients.
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PMID:Electroporation enables plasmid vaccines to elicit CD8+ T cell responses in the absence of CD4+ T cells. 1450 Jun 31

Although several observations show local T cell recognition of retinal Ag, there has been no direct demonstration that the APC were retinal derived, rather than recruited. In this study, CD45(+) cells isolated from immunologically quiescent murine retina were tested in vitro for functional evidence of Ag presentation to naive and Ag-experienced CD4 T cells specific for beta-galactosidase. Because CD45(+) cells from brain have been reported to be efficient APC, they were included for comparison. Measures of activation included changes in CD4, CD25, CD44, CD45RB, CD62L, CD69, caspase-3 activation, CFSE dilution, size, number of cells recovered, and cytokine production. Retinal CD45(+) cells gave no evidence of Ag-dependent TCR ligation in naive T cells, unlike splenic APC and CD45(+) cells from brain, which supported potent responses. Instead, addition of retinal CD45(+) cells to cocultures of naive 3E9 T cells plus splenic APC reduced the yield of activated T cells and cytokine production by limiting T cell activation at early time points. Ag-experienced T cells responded weakly to Ag presented by retinal CD45(+) cells. Activating the retinal cells with IFN-gamma, anti-CD40, or LPS incrementally increased their APC activity. Addition of neutralizing Abs to TGF-beta did not reveal suppressed retinal APC activity. Because retina lacks tissue equivalents of meninges and choroid plexus, rich sources of dendritic cells in brain, cells from retina may better represent the APC activity of fresh, adult CNS parenchymal and perivascular cells. The activity of the retinal CD45(+) cells appears to be directed to limiting T cell responses.
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PMID:The antigen-presenting activity of fresh, adult parenchymal microglia and perivascular cells from retina. 1515 73

Dendritic cells (DCs) loaded with antigens can effectively stimulate host immune responses to syngeneic tumors, but there is considerable controversy as to which forms of antigen-loading are most immunogenic. Here, the authors compared immunotherapeutic reactivities of DCs loaded with a variety of antigen preparations. Because DC maturation stages affect their capacities of antigen processing and presentation, two DC populations were used for the current analysis: in vivo Flt-3 ligand-induced mature DCs and in vitro bone marrow-derived DCs, which were less mature. To facilitate a direct comparison, the LacZ gene-transduced B16 melanoma model system was used, where beta-galactosidase served as the surrogate tumor-rejection antigen. DC loading strategies included pulsing with the beta-galactosidase protein, H-2K restricted peptide, tumor cell lysate, and irradiated tumor cells and fusion of DCs with tumor cells. Our results demonstrated that electrofusion of DCs and tumor cells generated a therapeutic vaccine far superior to other methods of DC loading. For the treatment of 3-day established pulmonary tumor nodules, a single intranodal vaccination plus IL-12 resulted in a significant reduction of metastatic nodules, while other DC preparations were only marginally effective. Immunotherapy mediated by the fusion cells was tumor antigen-specific. Consistent with their therapeutic activity, fusion hybrids were the most potent stimulators to induce specific IFN-gamma secretion from immune T cells. Furthermore, fusion cells also stimulated a small amount of IL-10 production from immune T cells. However, this IL-10 secretion was also induced by other DC preparations and did not correlate with in vivo therapeutic reactivity.
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PMID:Comparative analysis of antigen loading strategies of dendritic cells for tumor immunotherapy. 1523 87

The presence of unmethylated CpG motifs in bacterial plasmids is thought to provide necessary immunoadjuvant signals to DNA vaccination. We took advantage of CpG-unresponsive toll-like receptor 9 (TLR9) knock-out mice to study whether this pathway was required to generate immune responses to DNA vaccination. We compared two vectors, one encoding the surface glycoprotein C of pseudorabies virus shown to protect target animals against challenge, and the other encoding the cytoplasmic enzyme beta-galactosidase. In the absence of TLR9, bone marrow-derived dendritic cells lost their ability to secrete IL-12 and type I IFN in response not only to CpG as expected but also to the plasmids used for vaccination. In contrast, DNA vaccination experiments showed that TLR9-deficient mice were able to mount Th1-biased antigen-specific antibody and IFN-gamma responses, albeit at lower levels than normal mice. Thus, TLR9 signaling is not needed for eliciting T- and B-cell responses to DNA encoded antigens. However, TLR9 signaling tended to enhance plasmid-adjuvant effects on antigen-specific immune responses.
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PMID:TLR9 pathway is involved in adjuvant effects of plasmid DNA-based vaccines. 1565 68

Pseudomonas aeruginosa is an important opportunistic pathogen that can cause chronic and often life-threatening infections of the respiratory tract, particularly in individuals with cystic fibrosis (CF). Because infections with P. aeruginosa remain the major cause of the high morbidity and mortality of CF, a vaccine against P. aeruginosa would be very useful for preventing this disorder. The outer membrane protein F (OprF) of P. aeruginosa is a promising vaccine candidate and various B cell epitopes within OprF have been identified. Given that adenovirus (Ad) vectors have strong immunogenic potential and can function as adjuvants for genetic vaccines, the present study evaluates the immunogenic and protective properties of a novel replication-deficient Ad vector in which the Ad hexon protein was modified to include a 14-amino acid epitope of P. aeruginosa OprF (Epi8) in loop 1 of the hypervariable region 5 of the hexon (AdZ.Epi8). Immunization of C57BL/6 mice with AdZ.Epi8 resulted in detectable serum anti-P. aeruginosa and anti-OprF humoral responses. These responses were haplotype dependent, with higher serum anti-OprF titers in CBA mice than in BALB/c or C57BL/6 mice. AdZ.Epi8 induced Epi8-specific IFN-gamma-positive CD4 and CD8 T cell responses and resulted in protection against a lethal pulmonary challenge with agar-encapsulated P. aeruginosa. Importantly, repeated administration of AdZ.Epi8 resulted in boosting of the anti-OprF humoral and anti-Epi8 cellular response, whereas no boosting effect was present in the response against the transgene beta-galactosidase. These observations suggest that Ad vectors expressing pathogen epitopes in their capsid will protect against an extracellular pathogen and will allow boosting of the epitope-specific humoral response with repeated administration, a strategy that should prove useful in developing Ad vectors as vaccines where humoral immunity will be protective.
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PMID:Protection against P. aeruginosa with an adenovirus vector containing an OprF epitope in the capsid. 1584 Dec 17

We formulated adenovirus (AdV) vectors with cationic steroid liposomes containing dexamethasone-spermine (DS)/dioleoylphosphatidylethanolamine (DOPE) in an effort to overcome the lack of apically expressed AdV vector receptors on airway epithelial cells and to reduce the inflammation associated with AdV vector exposure. An AdV vector (1 to 2.5 x 10(11) genome copies) expressing human placental alkaline phosphatase or beta-galactosidase (LacZ) was delivered alone or complexed with DS/DOPE, DC-Chol/DOPE, or dexamethasone to C57Bl/6 mice via intranasal instillation. Formulation of the AdV vector with DS/DOPE and DC-Chol/DOPE resulted in transgene expression targeted only to the airway epithelial cells with minimal expression in alveolar cells, while AdV alone caused high alveolar transduction. The DS/DOPE and dexamethasone formulations greatly reduced cellular infiltrates compared to AdV vector alone, while formulation with DC-Chol/DOPE did not. IFN-gamma was significantly elevated at day 7 in mice receiving only the AdV vector compared to the AdV vector formulated with DS/DOPE, DC-Chol/DOPE, or dexamethasone. Lipid formulation of adeno-associated virus vector expressing LacZ also produced airway epithelial targeting, similar to the AdV vector. Viral vectors can be formulated with DS/DOPE to improve targeting to the airway epithelium in vivo and to attenuate vector-induced inflammation through the pharmacological activity of DS.
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PMID:Targeting viral-mediated transduction to the lung airway epithelium with the anti-inflammatory cationic lipid dexamethasone-spermine. 1609 13

The avidity of Ag-specific CTL is a critical determinant for clearing viral infection and eliminating tumor. Although previous studies have demonstrated that vaccines using enhanced costimulation will enhance the level and avidity of Ag-specific T cells from naive mice, there are conflicting data about the effects of vaccines using enhanced costimulation (vector or dendritic cell based) on the survival of memory T cells. In this study we have first extended previous observations that primary vaccination with a recombinant vaccinia virus (rV-) expressing a model Ag (LacZ) and a triad of T cell costimulatory molecules (B7-1, ICAM-1, and LFA-3 (designated TRICOM)) enhances the level and avidity of T cells from naive vaccinated C57BL/6 (Thy1.2) mice. Adoptive transfer of Thy1.1 memory CD8(+) T cells into naive Thy1.2 C57BL/6 mice was followed by booster vaccinations with a recombinant fowlpox (rF-)-expressing LacZ (rF-LacZ) or booster vaccinations with rF-LacZ/TRICOM. Analysis of levels of beta-galactosidase tetramer-positive T cells and functional assays (IFN-gamma expression and lytic activity) determined that booster vaccinations with rF-LacZ/TRICOM were superior to booster vaccinations with rF-LacZ in terms of both maintenance and enhanced avidity of memory CD8(+) T cells. Antitumor experiments using a self-Ag (carcinoembryonic Ag (CEA) vaccines in CEA transgenic mice bearing CEA-expressing tumors) also demonstrated that the use of booster vaccinations with vaccines bearing enhanced costimulatory capacity had superior antitumor effects. These studies thus have implications in the design of more effective vaccine strategies.
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PMID:Vaccines with enhanced costimulation maintain high avidity memory CTL. 1614 17

The feasibility of using avipox virus as a vector for gene delivery to islet tissue (adult islets and fetal proislets) was examined using a recombinant fowlpox virus (FPV) engineered to express the reporter gene LacZ (FPV-LacZ). The efficiency of in vitro transduction was dose-dependent and influenced by the donor species and maturation status of the islet tissue. Reporter gene expression in FPV-LacZ-transduced islet grafts was transient (3-7 days) in immunoincompetent nude mice and was not prolonged by in vivo treatment with anti-IFN-gamma mAb. In contrast, FPV-LacZ-transduced NIT-1 cells (a mouse islet beta cell line) expressed the LacZ gene beyond 18 days in vitro. Silencing of transgene expression therefore appeared to occur in vivo and was T cell- and IFN-gamma-independent. Isografts of FPV-LacZ-transduced islets in immunocompetent mice underwent immunological destruction by 7 days, suggesting that either FPV proteins or the reporter protein beta-galactosidase induced an adaptive immune response. Co-delivery of the rat bioactive immunoregulatory cytokine gene TGF-beta to islets using FPV-TGF-beta led to enhanced expression of TGF-beta mRNA in isografts but no long-term protection. Nevertheless, compared to control islet isografts at 5 days, FPV-transduced islets remained embedded in the clotted blood used to facilitate implantation. This phenomenon was TGF-beta transgene-independent, correlated with lack of cellular infiltration, and suggested that the FPV vector transformed the blood clot into a temporary immunological barrier.
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PMID:Recombinant fowlpox virus for in vitro gene delivery to pancreatic islet tissue. 1626 13

HIV-1 p17 is a viral cytokine that acts on preactivated, but not on resting, human T cells promoting proliferation, proinflammatory cytokines release and HIV-1 replication, after binding to a cellular receptor (p17R). Here, we demonstrate that p17Rs are expressed on activated murine T cells, which respond to p17 stimulation similarly to their human counterpart. We developed a mouse model of abortive HSV-1 infection to induce T cell activation in vivo. Preactivated cells expressed p17Rs and were highly susceptible to p17 stimulation, which triggered proinflammatory cytokines release and promoted CD4+ T cell survival and expansion. Coculture of in vivo activated splenocytes with macrophages in the presence of p17 further increased their ability to produce IFN-gamma. The presence of macrophages and activated T cells at mucosal sites prompted us to investigate the immunomodulatory activities of p17 in vivo. Intranasal coadministration of p17 with beta-galactosidase (beta-gal) resulted in improved beta-gal specific cellular and humoral immune responses at systemic and mucosal levels. It is well established that HIV-1 replication is driven in an autocrine/paracrine manner by endogenously produced proinflammatory cytokines. Our results highlight the role of p17 in sustaining cellular activation and inflammation, thereby promoting a permissive microenvironment for HIV-1 replication. In addition, p17 is a promising candidate antigen, exhibiting immunomodulatory/adjuvant properties, that need to be exploited in the development of HIV/AIDS vaccines.
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PMID:HIV-1 matrix protein p17 modulates in vivo preactivated murine T-cell response and enhances the induction of systemic and mucosal immunity against intranasally co-administered antigens. 1681 60

Gene gun immunization has been associated with the induction of a heterologous type of immune response characterized by a T(H)1-like immune reaction on the cellular level, i.e. generation of IFN-gamma secreting CD8(+) T-cells, yet a T(H)2 biased serology as indicated by high IgG1:IgG2a ratios and induction of IgE. Nevertheless, gene gun immunization using the model molecule beta-galactosidase has been argued to prevent IgE induction and to promote T(H)1 cells with respect to allergy DNA immunization. In our current study, we evaluated the potential of gene gun immunization to prevent type I allergic reactions comparing beta-galactosidase with two clinically relevant allergens, and further investigated the effect of gene gun immunization on relevant lung parameters. BALB/c mice were immunized with plasmids encoding the birch pollen allergen Bet v 1, the grass pollen allergen Phl p 5, or the model molecule beta-galactosidase, either by gene gun or intradermal injection followed by sensitization and intranasal provocation with the respective allergen. IgG1 and IgG2a antibody titers were determined by ELISA. IgE levels were evaluated in a rat basophil release assay. The severity of eosinophilia was determined in bronchoalveolar lavages, and the overall infiltrate was analyzed by histology on lung paraffin sections. Gene gun immunization induced a T(H)2-biased immune reaction, which did not prevent from production of IgE after subsequent sensitization. This T(H)2 effect was influenced by the nature of the antigen, with a more pronounced T(H)2-bias for the allergens Bet v 1 and Phl p 5 compared to beta-galactosidase. Gene gun immunization with all three antigens promoted eosinophil influx into the lung and did not alleviate lung pathology after intranasal provocation. In contrast to needle injection of plasmid DNA, which triggers a clearly T(H)1-biased and allergy-preventing immune response, gene gun application fails to induce anti-allergic reactions with all tested antigens and is therefore contraindicated for allergen-specific immunotherapy.
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PMID:Gene gun immunization with clinically relevant allergens aggravates allergen induced pathology and is contraindicated for allergen immunotherapy. 1707 Sep 9


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