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Biomedical subjects

Barney S Graham

Publications and source records attributed to Barney S Graham.

45 records · Page 3Linked to original sources
PubMed2003

Role of plasma membrane lipid microdomains in respiratory syncytial virus filament formation.

The fusion protein (F) of respiratory syncytial virus (RSV) is the envelope glycoprotein responsible for the characteristic cytopathology of syncytium formation. RSV has been shown to bud from selective areas of the plasma membrane as pleomorphic virions, including both filamentous and round particles. With immunofluorescent microscopy, we demonstrated evidence of RSV filaments incorporating the fusion protein F and colocalizing with a lipid microdomain-specific fluorescent dye, 1,1-dihexadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate. Western blot analysis of Triton X-100 cold-extracted membrane fractions confirmed the presence of RSV proteins within the lipid microdomains. RSV proteins also colocalized with cellular proteins associated with lipid microdomains, caveolin-1, and CD44, as well as with RhoA, a small GTPase. ADP-ribosylation of RhoA by Clostridium botulinum exotoxin inactivated RhoA signaling and resulted in the absence of RSV-induced syncytia despite no significant change in viral titer. We demonstrated an overall decrease in both the number and length of the viral filaments and a shift in the localization of F to nonlipid microdomain regions of the membrane in the presence of C3 toxin. This suggests that the selective incorporation of RSV proteins into lipid microdomains during virus assembly may lead to critical interactions of F with cellular proteins, resulting in microvillus projections necessary for the formation of filamentous virus particles and syncytium formation. Thus, manipulation of membrane lipid microdomains may lead to alterations in the production of viral filaments and RSV pathogenesis and provide a new pharmacologic target for RSV therapy.

ADP Ribose Transferases↗
PubMed2002

Transgenic overexpression of interleukin (IL)-10 in the lung causes mucus metaplasia, tissue inflammation, and airway remodeling via IL-13-dependent and -independent pathways.

To address the complex chronic effector properties of interleukin (IL)-10, we generated transgenic mice in which IL-10 was overexpressed in the lung. In these mice, IL-10 inhibited endotoxin-induced tumor necrosis factor production and neutrophil accumulation. IL-10 also caused mucus metaplasia, B and T cell-rich inflammation, and subepithelial fibrosis and augmented the levels of mRNA encoding Gob-5, mucins, and IL-13. In mice bred to have null mutations of IL-13, IL-4R(alpha), or STAT-6, transgenic IL-10 did not induce mucus metaplasia but did induce inflammation and fibrosis. IL-10 was also a critical mucin regulator of virus-induced mucus metaplasia. Thus, IL-10, although inhibiting lipopolysaccharide-induced inflammation, also causes mucus metaplasia, tissue inflammation, and airway fibrosis. These responses are mediated by multiple mechanisms with mucus metaplasia being dependent on and the inflammation and fibrosis being independent of an IL-13/IL-4R(alpha)/STAT-6 activation pathway.

Animals↗
PubMed2002

Selective cyclooxygenase-1 and -2 inhibitors each increase allergic inflammation and airway hyperresponsiveness in mice.

Nonselective cyclooxygenase (COX) inhibition during allergic sensitization with ovalbumin in a murine model leads to an increase in the Type 2 cytokines interleukin-5 (IL-5) and IL-13; however, the effect of selective COX-1 and COX-2 inhibitors on these cytokines is unknown. We found that COX-1 protein was constitutively expressed in lung tissue. Expression of COX-1 protein did not increase with ovalbumin sensitization, but expression of COX-2 protein did. Ovalbumin-sensitized mice treated with either selective COX-1 inhibitor SC58560 (OVA-COX-1 inhibitor) or selective COX-2 inhibitor SC58236 (OVA-COX-2 inhibitor) had significantly greater airway hyperresponsiveness (p < 0.05) and higher levels of IL-13 (p < 0.05) in lung supernatants than did untreated mice that were ovalbumin sensitized (OVA). Lung mRNA levels for the chemokine receptors CCR1 through CCR5 (expressed on eosinophils, basophils, lymphocytes, and dendritic cells) were increased in the OVA-COX-2 inhibitor and OVA-indomethacin groups. We conclude that in the BALB/c mouse, COX inhibition with either a COX-1 or COX-2 inhibitor during allergen sensitization augments production of IL-13 and increases airway hyperresponsiveness.

Animals↗
PubMed2002

Illness severity, viral shedding, and antibody responses in infants hospitalized with bronchiolitis caused by respiratory syncytial virus.

The relationships between host factors, viral shedding, illness severity, and antibody response in respiratory syncytial virus (RSV)-induced bronchiolitis are poorly defined. These relationships were prospectively evaluated in 77 infants hospitalized with RSV bronchiolitis in multicenter, double-blind, placebo-controlled trials of RSV immunoglobulin therapy. Severity of illness was influenced by age and host risk factors but was not influenced by RSV neutralizing antibody titer or by the amount of virus in nasal secretions at enrollment. Virus recovery in nasal secretions was variable but was highest at enrollment. Viral shedding was not influenced by primary diagnosis, antibody titer, age, or duration of acute respiratory illness before enrollment. In intubated patients, the amounts of virus recovered in nasal secretions and endotracheal aspirates were highly correlated. A serum neutralizing antibody response was seen in 64% of subjects who received placebo. The response was not influenced by age, primary diagnosis, amount of virus recovered, or severity of illness but was suppressed by preexisting antibody.

Adolescent↗
PubMed2002

The role of IFN in respiratory syncytial virus pathogenesis.

Formalin-inactivated respiratory syncytial virus (RSV) vaccine preparations have been shown to cause enhanced disease in naive hosts following natural infection. In this study we demonstrate a similar pattern of enhanced disease severity following primary RSV infection of IFN-nonresponsive STAT1(-/-) mice. STAT1(-/-) mice showed markedly increased illness compared with wild-type BALB/c animals following RSV inoculation despite similar lung virus titers and rates of virus clearance. Histologically, STAT1(-/-) animals had eosinophilic and neutrophilic pulmonary infiltrates not present in wild-type or IFN-gamma(-/-)-infected mice. In cytokine analyses of infected lung tissue, IFN-gamma was induced in both STAT1(-/-) and wild-type mice, with preferential IL-4, IL-5, and IL-13 induction only in the STAT1(-/-) animals. Eotaxin was detected in the lungs of both wild-type and STAT1(-/-) mice following infection, with a 1.7-fold increase over wild-type in the STAT1(-/-) mice. Using a peptide epitope newly identified in the RSV fusion protein, we were able to demonstrate that wild-type memory CD4(+) T cells stimulated by this peptide produce primarily IFN-gamma, while STAT1(-/-)CD4(+) cells produce primarily IL-13. These findings suggest that STAT1 activation by both type I (alphabeta) and type II (gamma) IFNs plays an important role in establishing a protective, Th1 Ag-specific immune response to RSV infection.

Amino Acid Sequence↗
PubMed2002

NK T cells contribute to expansion of CD8(+) T cells and amplification of antiviral immune responses to respiratory syncytial virus.

CD1d-deficient mice have normal numbers of T lymphocytes and natural killer cells but lack Valpha14(+) natural killer T cells. Respiratory syncytial virus (RSV) immunopathogenesis was evaluated in 129xC57BL/6, C57BL/6, and BALB/c CD1d(-/-) mice. CD8(+) T lymphocytes were reduced in CD1d(-/-) mice of all strains, as shown by cell surface staining and major histocompatibility complex class I tetramer analysis, and resulted in strain-specific alterations in illness, viral clearance, and gamma interferon (IFN-gamma) production. Transient activation of NK T cells in CD1d(+/+) mice by alpha-GalCer resulted in reduced illness and delayed viral clearance. These data suggest that early IFN-gamma production and efficient induction of CD8(+)-T-cell responses during primary RSV infection require CD1d-dependent events. We also tested the ability of alpha-GalCer as an adjuvant to modulate the type 2 immune responses induced by RSV glycoprotein G or formalin-inactivated RSV immunization. However, immunized CD1-deficient or alpha-GalCer-treated wild-type mice did not exhibit diminished disease following RSV challenge. Rather, some disease parameters, including cytokine production, eosinophilia, and viral clearance, were increased. These findings indicate that CD1d-dependent NK T cells play a role in expansion of CD8(+) T cells and amplification of antiviral responses to RSV.

Animals↗
PubMed2002

Clinical trials of HIV vaccines.

Development of a preventive vaccine for HIV is the best hope of controlling the AIDS pandemic. Evidence from natural history studies and experiments in animal models indicates that immunity against HIV is possible, suggesting that vaccine development is feasible. These studies have shown that sufficient levels of neutralizing antibody against HIV can prevent infection, although the effect is type-specific. In contrast, HIV-specific cytotoxic T lymphocyte (CTL) activity has broad cross-reactivity, and although CTL activity alone cannot prevent HIV infection, it can control the level of viremia at a low level. Evaluation of candidate vaccines in human trials has focused on approaches that can safely elicit HIV-specific antibody and T cell responses. Current strategies have been unable to induce antibody with broad neutralizing activity against primary HIV isolates. However, recombinant poxvirus and DNA vaccines have elicited CTL responses that are broadly cross-reactive against primary HIV isolates from diverse clades. Future advances will require the discovery of new immunogens that can induce neutralizing antibody, as well as efficacy trial evaluation of regimens optimized for CTL induction.

AIDS Vaccines↗
PubMed2001

Construction and characterization of recombinant vaccinia viruses co-expressing a respiratory syncytial virus protein and a cytokine.

Recombinant vaccinia viruses are well-characterized tools that can be used to define novel approaches to vaccine formulation and delivery. While vector co-expression of immune mediators has enormous potential for optimizing the composition of vaccine-induced immune responses, the impact on antigen expression and vector antigenicity must also be considered. Co-expression of IL-4 increased vaccinia virus vector titres, while IFN-gamma co-expression reduced vaccinia virus replication in BALB/c mice and in C57BL/6 mice infected with some recombinant viruses. Protection against respiratory syncytial virus (RSV) challenge was similar in mice immunized with vaccinia virus expressing RSV G glycoprotein and IFN-gamma, even though the replication efficiency of the vector was diminished. These data demonstrate the ability of vector-expressed cytokine to influence the virulence of the vector and to direct the development of selected immune responses. This suggests that the co-expression of cytokines and other immunomodulators has the potential to improve the safety of vaccine vectors while improving the immunogenicity of vaccine antigens.

Animals↗