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Biomedical subjects

C A Guzman

Publications and source records attributed to C A Guzman.

At least 19 recordsLinked to original sources
PubMed2006

Enhanced protection against Streptococcus pyogenes infection by intranasal vaccination with a dual antigen component M protein/SfbI lipid core peptide vaccine formulation.

We investigated the efficacy of a synthetic Streptococcus pyogenes vaccine targeting two virulence factors using the Lipid Core Peptide (LCP) delivery system. BALB/c mice were immunised intranasally with LCPs containing peptides encompassing T-cell and B-cell epitopes of the conserved C-repeat region of the M protein (J8) or the fibronectin-binding repeats region (FNBR) of SfbI, or a combination formulation containing peptides representing both antigens. LCPs were co-administered with the TLR2/6 agonist MALP-2 as mucosal adjuvant. Humoral and cellular immune responses stimulated at systemic and mucosal levels were strongest in mice immunised with the dual antigen formulation. Mice were completely protected following a respiratory challenge with a lethal dose of a heterologous S. pyogenes strain, whereas there was 70% and 90% survival in mice immunised with LCP-J8 and LCP-FNBR, respectively. This is the first report demonstrating the elicitation of better protective immunity by a dual antigen component S. pyogenes vaccine.

Adhesins, Bacterial↗
PubMed2000

Wild-type HFE protein normalizes transferrin iron accumulation in macrophages from subjects with hereditary hemochromatosis.

Hereditary hemochromatosis (HC) is one of the most common single-gene hereditary diseases. A phenotypic hallmark of HC is low iron in reticuloendothelial cells in spite of body iron overload. Most patients with HC have the same mutation, a change of cysteine at position 282 to tyrosine (C282Y) in the HFE protein. The role of HFE in iron metabolism and the basis for the phenotypic abnormalities of HC are not understood. To clarify the role of HFE in the phenotypic expression of HC, we studied monocytes-macrophages from subjects carrying the C282Y mutation in the HFE protein and clinically expressing HC and transfected them with wild-type HFE by using an attenuated Salmonella typhimurium strain as a gene carrier. The Salmonella system allowed us to deliver genes of interest specifically to monocytes-macrophages with high transduction efficiency. The accumulation of (55)Fe delivered by (55)Fe-Tf was significantly lower in macrophages from patients with HC than from controls expressing wild-type HFE. Transfection of HC macrophages with the HFE gene resulted in a high level of expression of HFE protein at the cell surface. The accumulation of (55)Fe delivered by (55)Fe-Tf was raised by 40% to 60%, and this was reflected by an increase in the (55)Fe-ferritin pool within the HFE-transfected cells. These results suggest that the iron-deficient phenotype of HC macrophages is a direct effect of the HFE mutation, and they demonstrate a role for HFE in the accumulation of iron in these cells.

Cells, Cultured↗
PubMed2000

Co-operative binding of human fibronectin to Sfbl protein triggers streptococcal invasion into respiratory epithelial cells.

Streptococcal fibronectin binding protein I (SfbI) mediates adherence to and invasion of Streptococcus pyogenes into human epithelial cells. In this study, we analysed the binding activity of distinct domains of SfbI protein towards its ligand, the extracellular matrix component fibronectin, as well as the biological implication of the binding events during the infection process. By using purified recombinant SfbI derivatives as well as in vivo expressed SfbI domains on the surface of heterologous organism Streptococcus gordonii, we were able to dissociate the two major streptococcal target domains on the human fibronectin molecule. The SfbI repeat region exclusively bound to the 30 kDa N-terminal fragment of fibronectin, whereas the SfbI spacer region exclusively bound to the 45 kDa collagen-binding fragment of fibronectin. In the case of native surface-expressed SfbI protein, an induced fit mode of bacteria-fibronectin interaction was identified. We demonstrate that binding of the 30 kDa fibronectin fragment to the repeat region of SfbI protein co-operatively activates the adjacent SfbI spacer domain to bind the 45 kDa fibronectin fragment. The biological consequence arising from this novel mode of fibronectin targeting was analysed in eukaryotic cell invasion assays. The repeat region of SfbI protein is mediating adherence and constitutes a prerequisite for subsequent invasion, whereas the SfbI spacer domain efficiently triggers the invasion process of streptococci into the eukaryotic cell. Thus, we were able to dissect bacterial adhesion from invasion by manipulating one protein. SfbI protein therefore represents a highly evolved prokaryotic molecule that exploits the host factor fibronectin not only for extracellular targeting but also for its subsequent activation that leads to efficient cellular invasion.

Adhesins, Bacterial↗
PubMed1998

Gene transfer in dendritic cells, induced by oral DNA vaccination with Salmonella typhimurium, results in protective immunity against a murine fibrosarcoma.

A live attenuated AroA- auxotrophic mutant of Salmonella typhimurium (SL7207) has been used as carrier for the pCMVbeta vector that contains the beta-galactosidase (beta-gal) gene under the control of the immediate early promoter of Cytomegalovirus (CMV). We tested whether orally administered bacterial carrier could enter and deliver the transgene to antigen-presenting cells (APCs) through the natural enteric route of infection and whether beta-gal expression could generate a protective response against an aggressive murine fibrosarcoma transduced with the beta-gal gene (F1.A11) that behaves operationally as a tumor-associated antigen. After three courses, at 15-day intervals, mice developed both cell-mediated and systemic humoral responses to beta-gal. Mice vaccinated with the Salmonella harboring pCMVbeta, but not with plasmid-less carrier, showed resistance to a challenge with F1.A11 cells. These experiments suggest that Salmonella-based DNA immunization allows us to specifically target antigen expression in vivo to APCs. To prove that the transgene is actually expressed by APCs as a function of an eukaryotic promoter, the green fluorescent protein (GFP) was placed under the control of either the eukariotic CMV or a prokaryotic promoter. Using cytofluorometric analysis, GFP was detected only in splenocytes of mice receiving a Salmonella carrier harboring GFP under the CMV promoter. These results indicate that transgene expression occurs because of a Salmonella-mediated gene transfer to eukaryotic cells. Finally, approximately 19% of the splenocytes expressed GFP. Among them, F4/80(+) macrophages and CD11cbright dendritic cells (DCs) were scored as positive for GFP expression. Extensive work has been performed trying to optimize the way to transfect DCs, ex vivo, with genes coding for relevant antigens. We show here, for the first time, that DCs can be directly and specifically transduced in vivo such to induce DNA vaccination against tumors.

Administration, Oral↗
PubMed1997

Influence of different rol gene products on the chain length of Shigella dysenteriae type 1 lipopolysaccharide O antigen expressed by Shigella flexneri carrier strains.

Introduction of the rol genes of Shigella dysenteriae 1 and Escherichia coli K-12 into Shigella flexneri carrier strains expressing the heterologous S. dysenteriae type 1 lipopolysaccharide resulted in the formation of longer chains of S. dysenteriae 1 O antigen. In bacteria producing both homologous and heterologous O antigen, this resulted in a reduction of the masking of heterologous O antigen by homologous lipopolysaccharide and an increased immune response induced by intraperitoneal immunization of mice by recombinant bacteria. The rol genes of S. dysenteriae 1 and E. coli K-12 were sequenced, and their gene products were compared with the S. flexneri Rol protein. The primary sequence of S. flexneri Rol differs from both E. coli K-12 and S. dysenteriae 1 Rol proteins only at positions 267 and 270, which suggests that this region may be responsible for the difference in biological activities.

Amino Acid Sequence↗
PubMed1995

Interaction of Listeria monocytogenes with mouse dendritic cells.

In this study, the interaction of murine dendritic cells with Listeria monocytogenes was investigated. Dendritic cells are efficient antigen-presenting cells, play a key role in the immune response, and are capable of migrating over substantial distances between sites of infection and lymphoid tissues. L. monocytogenes EGD invaded dendritic cells, escaped from phagosomes into the cytoplasm, and there directed actin nucleation, polymerization, and polarization in a typical fashion, thereby achieving intracellular movement and cell-to-cell spread. The internalization process appears to be independent of the inl locus. Interestingly, an intact microtubular function was essential for efficient uptake, whereas in a previous report, microtubule disruption did not affect bacterial spread in Caco-2 cells. The results obtained also suggest that L. monocytogenes binds to glycosylated receptors of dendritic cells. Uptake of Listeria cells was mediated by a protein kinase-dependent transducing phosphorylation signal that induces the actin polymerization-dependent phagocytic process. To achieve efficient uptake, de novo protein synthesis of eukaryotic and prokaryotic cells is also required. Despite the killing of dendritic cells, wild-type bacteria were found to persist in small numbers in some cells for at least 24 h. When different isogenic mutants of the EGD strain were analyzed for their capability to interact with dendritic cells, it was observed that some virulence-attenuated mutants (i.e., prfA and delta hly) persisted in large numbers for even longer times. Invasion of dendritic cells by L. monocytogenes, which in turn could result in either cell death or persistent infection, might have an important role in the pathogenesis of listeriosis, leading to impaired immune responses with inefficient bacterial clearance and/or promoting bacterial spread.

Actin Cytoskeleton↗
PubMed1994

Invasion and intracellular survival of Bordetella bronchiseptica in mouse dendritic cells.

We have studied the interaction between the respiratory pathogen Bordetella bronchiseptica and murine spleen dendritic cells, important antigen-presenting cells that are found in the airway epithelium. Wild-type B. bronchiseptica 5376 attached very efficiently to dendritic cells, whereas the bvg mutant ATCC 10580, wild-type strain BB7865, and its spontaneous delta bvgS mutant BB7866 bound less efficiently. However, all tested B. bronchiseptica strains were able to invade dendritic cells and survive intracellularly for at least 72 h. These results suggest that bvg-independent or bvg-downregulated products are involved in the uptake and intracellular survival. Transmission electron microscopic analysis revealed that bacteria grew and replicated intracellularly and were present in typical phagosomes, which fused with lysosomes during the initial infection period. However, in later infection stages some bacteria seemed to escape into an unfused endocytic compartment, where individual bacteria were tightly surrounded by a membrane. The in vitro interaction of B. bronchiseptica with dendritic cells reported here may be relevant to natural infections caused by this organism that lead to chronicity or an altered immune response.

Animals↗
PubMed1994

Mechanisms involved in uptake of Bordetella bronchiseptica by mouse dendritic cells.

The invasion and intracellular survival of Bordetella bronchiseptica in mouse dendritic cells were investigated. The results obtained suggest that B. bronchiseptica binds specifically to glycosylated receptors present on the plasma membrane of dendritic cells, thereby inducing a signal that triggers an actin polymerization-dependent phagocytic process, probably via a protein kinase-dependent transducing phosphorylation signal. The energy required for the uptake process by host cells is provided mainly by the glycolytic pathway. An intact microtubule system and de novo protein synthesis in eukaryotic and prokaryotic cells are essential for efficient uptake and intracellular survival. The interaction of B. bronchiseptica with dendritic cells may be pertinent to natural infections that follow a chronic clinical course and predispose to secondary infections, and to the T-cell response involved in protective immunity following infections caused by Bordetella spp.

Actin Cytoskeleton↗
PubMed1993

Antibody responses in the serum and respiratory tract of mice following oral vaccination with liposomes coated with filamentous hemagglutinin and pertussis toxoid.

Mice were orally vaccinated with liposomes coated with filamentous hemagglutinin (FHA) and detoxified pertussis toxin (PT) of Bordetella pertussis. FHA- and PT-specific immunoglobulin G (IgG) was detected in serum, and both IgG and IgA were detected in lung washes following the immunization. Antibody responses in mice immunized with liposomes coated with FHA and PT were significantly higher than those in mice immunized with free FHA and PT, which demonstrated the adjuvanticity of the liposome carrier. The results indicate the potential usefulness of this approach for eliciting immune responses against FHA and PT (and perhaps other pertussis antigens) in humans and its possible utility in large-scale vaccination to protect against both B. pertussis infection and disease.

Adhesins, Bacterial↗