Communication of BRCA1 and BRCA2 genetic test results to health care providers following genetic testing at a tertiary care center.

Individuals at high risk for hereditary cancers often receive genetic counseling and testing at tertiary care centers; however, they may receive care for long-term management of their cancer risk in community settings. Communication of genetic test results to health care providers outside of tertiary care settings can facilitate the long-term management of high risk individuals. This study assessed women's communication of BRCA1/BRCA2 genetic test results to health care providers outside of tertiary care settings (termed "outside" health care providers, or OHCPs) and women's perceptions regarding communication of results. Women (n = 312) who underwent BRCA1/BRCA2 genetic counseling and testing completed a questionnaire assessing whether or not they shared test results with OHCPs and perceptions regarding the communication of test results to OHCPs. Most (72%) shared genetic test results with OHCPs. Women with no personal history of cancer were more likely to have shared results compared to women with a personal history of cancer. Mutation status did not significantly predict sharing of genetic information. Most reported positive perceptions regarding the disclosure of genetic test results to OHCPs. The majority did not report any concerns about potential insurance discrimination (88%) and indicated that OHCPs were able to appropriately address their questions (81%). Although most women shared their genetic test results with OHCPs, those with a personal history of cancer may need further encouragement to share this information. Tertiary care centers should facilitate outreach and education with OHCPs in order to assure appropriate long-term cancer risk management for high risk populations.

Involvement of intercellular adhesion molecule-1 in the antigen-induced infiltration of eosinophils and lymphocytes into the airways in a murine model of pulmonary inflammation.

We investigated the effects of in vivo intraperitoneal treatment with the rat monoclonal antibody (mAb), YN1.7.4 (YN1) against intercellular adhesion molecule-1 (ICAM-1) on the ovalbumin (OA)-inhalation-induced infiltration of leukocytes into the airways of OA-sensitized mice. YN1 (100 to 400 microg) given over a period of 72 h dose-dependently reduced the influx of lymphocytes and eosinophils into the bronchial lumen by > 60% and > or = 70%, respectively, when compared with saline or purified rat IgG-treated controls. Alveolar macrophages (AM) in the bronchoalveolar lavage fluid (BALF) were also decreased by > 50%. Lung tissue inflammation as determined by histopathologic examination was reduced. The number of neutrophils in the blood of OA-sensitized mice 3 days after challenge was significantly increased by treatment with YN1. However, at 24 h and 72 h after OA-challenge, the numbers of eosinophils and mononuclear cells in the bone marrow were reduced by YN1 treatment. Additionally, at 72 h after OA-challenge, the numbers of bone-marrow neutrophils were depressed. BALF levels of interleukin-5 (IL-5) and of IgA were lower for YN1-treated mice than for controls. With increasing doses of YN1, the levels of anti-ICAM-1 mAb in the plasma were proportionally increased. To correlate these results with YN1 treatment, blood and BALF T cells and BALF eosinophils were examined with flow cytometry. Blood T cells from YN1-treated mice were unable to bind phycoerythrin (PE)-labeled anti-ICAM- mAb ex vivo. These results implied that ICAM-1 on these cells was bound (occupied) by YN1 administered in vivo. Dose-related decreases were observed in the percentage and mean channel fluorescence (MCF) values of ICAM-1+ BALF T cells and eosinophils. The percentages of CD11a+ or CD49d+ eosinophils were also suppressed. Our data suggest that ICAM-1 is an important molecule involved in the recruitment of leukocytes into the airways of sensitized mice after pulmonary challenge.

Biochemical and immunological characterization of a novel peptide carrier system using rotavirus VP6 particles.

A system which allows for the efficient attachment of synthetic peptides to spherical virus-like particles assembled from the VP6 rotavirus nucleocapsid protein is described. This attachment was shown to be mediated by peptide-protein interactions and did not require additional chemicals for conjugation. The resulting large macromolecular structure was highly immunogenic for both the VP6 protein and the coupled peptides. The antibody response to peptides bound to VP6 particles was of higher titre and longer duration than that induced by other carriers. In addition, the response to VP6-coupled peptides was not affected by prior exposure to rotavirus and exhibited a range of immunoglobulin subclasses in the absence of an adjuvant. These data demonstrate that assembled VP6 spherical particles are useful carriers for low doses of synthetic peptides.

In vitro assembly of the outer capsid of bovine rotavirus is calcium-dependent.

The nucleocapsid protein (VP6) and outer capsid glycoprotein (VP7) of bovine rotavirus (BRV) assemble in vitro, in 0.01 M Tris-HCl, pH 8.0, 50 mM CaCl2, into smooth particles resembling double-shelled BRV. That the two proteins interact is demonstrated by the immunoprecipitation of both by antibody directed against either VP6 or VP7. The calcium-dependence, particle morphology, and immunoreactivity in ELISA suggest that VP7 is presented authentically on the outer capsid. The implications for rotavirus morphogenesis are discussed.

The structure of tubes of bovine rotavirus nucleocapsid protein (VP6) assembled in vitro.

The structure of tubes of reassembled nucleocapsid protein (VP6) from bovine rotavirus (BRV) was determined using optical diffraction of electron micrographs. The tubes consist of a five-start helix of hexagons, with 38 hexagons per helix in a true repeat of three turns. The morphological subunits comprising the hexagons are probably elongated trimers. The structure of naturally occurring tubes (D. Chasey and J. Labram, 1983, J. Gen. Virol. 64, 863-872) was also examined and shown to be similar but not identical to that of tubes assembled in vitro. Considerations of the assembly process are discussed.

In vitro assembly of bovine rotavirus nucleocapsid protein.

The nucleocapsid protein (VP6) of bovine rotavirus was purified from in vitro-derived single shelled particles by CaCl2 or LiCl treatment. The protein exhibits polymorphism. Specifically, hexamers and small hexagonal lattices were present in many of the samples. Tubular particles formed between pH 5.0 and 9.0 were moderately stable to changes in temperature and ionic strength and were shown to be composed of nucleocapsid protein. Their formation is fully reversible. Spherical particles resembling single-shelled virus formed at pH 4.0. A novel structure in the form of sheets composed of a small-hole lattice formed in samples shifted from pH 6.0 to 4.0. The results demonstrate the importance of the nucleocapsid protein and of protein-protein interactions for rotavirus assembly.

Biochemical evidence for the oligomeric arrangement of bovine rotavirus nucleocapsid protein and its possible significance in the immunogenicity of this protein.

The nucleocapsid protein of bovine rotavirus was shown to exist in trimeric units in both the virus particle and in infected cells, with the subunits linked by non-covalent interactions. These trimeric units complex further by disulphide bridges into larger units which may represent the hexameric structures observed by electron microscopy. Visualization of various nucleocapsid protein complexes was also achieved on polyacrylamide gels by treating virus preparations with urea at 37 degrees C or boiling in the presence and absence of 2-mercaptoethanol. Since virus particles devoid of nucleic acid were also broken down into trimeric subunits by such treatments, assembly of virus particles appears not to require an RNA-protein interaction. Four nucleocapsid-specific monoclonal antibodies with low neutralizing ability reacted with the monomeric (45,000 mol. wt., 45K), dimeric (90K), trimeric (135K) and trimeric pair (270K) subunits, indicating that a site responsible for neutralization is probably exposed after assembly of these subunits. Analysis of radiolabelled virus revealed that a high proportion (80%) of infectious particles could be immunoprecipitated by these monoclonal antibodies, suggesting that the virus particles are either partially double-shelled or have the nucleocapsid exposed on the surface. The monoclonal antibodies also cross-reacted with the nucleocapsid proteins of simian (SA11), pig (OSU), bovine (NCDV and UK) and human (Wa and ST4) rotaviruses in an immunoblot ELISA reaction. Since these six viruses belong to two different subgroups, it is likely that the antibodies did not recognize the subgroup-specific site, but a shared exposed antigenic determinant. Due to the hexameric configuration of the nucleocapsid in virus particles the neutralizing epitope may be repeatedly presented and, therefore, may contribute to the immunogenicity of this protein.