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.

Pharmacology of the biological response modifier bropirimine (PNU-54461) on experimental autoimmune encephalomyelitis (EAE) in mice.

In murine severe experimental autoimmune encephalomyelitis (EAE), an animal model for the human disease multiple sclerosis (MS), we tested the efficacy of a 5-halo-6-phenyl pyrimidinone compound, bropirimine (PNU-54461). We observed that the compound is active in suppressing EAE when administered orally, a significant pharmacological advantage compared to some current therapies for the treatment of MS. Furthermore, bropirimine was most efficacious when dosing was begun 5-10 days after injection of myelin basic protein, the protein isolated from the central nervous system and used for inducing EAE in our model. This is a period of time following the initial immunological events leading to the disease, when large-scale leukocyte infiltration into the central nervous system begins. Following oral dosing, bropirimine peaked in the blood within 3 h and was cleared to undetectable concentrations within 16-18 h. Despite the pharmacokinetics in the blood, bropirimine was fully efficacious when dosed orally every two or three days. Surprisingly, bropirimine treatment did not result in a statistically significant decrease in leukocyte infiltration into the lower spinal cord, unless the compound was dosed daily at a high concentration. We also observed the concentration and time course of alpha-interferon in blood following oral dosing of bropirimine. The kinetics of interferon in the blood are similar to, but clearly distinguishable from, the pharmacokinetics of bropirimine in the blood. It is not clear whether or not the induction of interferon plays a key role in the efficacy of bropirimine. Nevertheless, the results using bropirimine in EAE suggest that the compound may be useful for the treatment of multiple sclerosis.

Pharmacokinetic properties, induction of interferon, and efficacy of selected 5-halo-6-phenyl pyrimidinones, bropirimine analogues, in a model of severe experimental autoimmune encephalomyelitis.

We showed previously that a 5-halo-6-phenyl-pyrimidinone, bropirimine (PNU-54461), inhibited progression of severe experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis. In the work presented here, we examined the activity of a group of chemically-related bropirimine analogues. First, the pharmacokinetic properties of the bropirimine analogues were examined in normal mice following oral dosing. After equal oral doses, both PNU-56169 and PNU-63693 were found in the blood of normal mice at equal or higher concentrations than bropirimine, but PNU-54462 and PNU-56359 were present in blood only at very low concentrations. Next, we examined the bropirimine analogues for activity in our model of severe EAE. At a dose of 400 mg/kg administered orally every second day PNU-56169 nearly completely blocked EAE progression, but was ineffective at 100 mg/kg. PNU-63693 was effective in EAE at concentrations of 200 mg/kg, 100 mg/kg, 50 mg/kg, and as low as 25 mg/kg. Histopathology was examined by observing leukocyte infiltration into the lower spinal cords of the mice. Treatment with 400 mg/kg of PNU-56169 and doses of 25, 50, 100, and 200 mg/kg of PNU-63693 significantly inhibited leukocyte infiltration into the lower spinal cord of treated mice in a dose-dependent manner. Orally administered PNU-56169 and PNU-63693 also stimulated significant concentrations of IFNalpha in the serum of treated mice, which may be related to the efficacy of the compounds in EAE. However, the correlation between IFNalpha in the blood and efficacy in treating EAE was not exact. Thus, PNU-56169 and PNU-63693 were delivered to the blood following oral dosing, induced significant concentrations of IFNalpha in the blood, and were equally or more potent than PNU-54461 in inhibiting clinical signs of EAE. The results suggest that 5-halo-6-phenyl-pyrimidinones are an interesting class of compounds to investigate for development in the treatment of multiple sclerosis.

Role of PECAM-1 (CD31) in neutrophil transmigration in murine models of liver and peritoneal inflammation.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is thought to be critical for transendothelial migration of leukocytes, including neutrophils. Because neutrophil-mediated liver injury during endotoxemia is dependent on transmigration, we investigated the role of PECAM-1 in the pathophysiology of endotoxin-induced liver injury. Male C3Heb/FeJ mice were treated with galactosamine (Gal) and endotoxin (ET) (700 mg/kg Gal/100 micrograms/kg ET), and liver sections were stained for PECAM-1 expression. Control livers showed the presence of PECAM-1 on endothelial cells of large vessels but not in sinusoids. Gal/ET treatment did not change the expression pattern of PECAM-1. Gal/ET-induced liver injury (area of necrosis: 38 +/- 3%) was not attenuated by treatment with 3 mg/kg of the antimurine PECAM-1 antibody 2H8. The antibody had no effect on sequestration and transmigration of neutrophils in sinusoids or the margination of neutrophils in large vessels. In contrast, 2H8 inhibited glycogen-induced neutrophil migration into the peritoneum by 74%; this effect correlated with PECAM-1 expression in the intestinal vasculature. Thus PECAM-1 is neither expressed nor inducible in hepatic sinusoids and is consequently not involved in neutrophil transmigration in the liver during endotoxemia. On the other hand, expression of PECAM-1 in mesenteric veins is critical for peritoneal neutrophil accumulation.

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.

Role of very late activation antigen-4 in the antigen-induced accumulation of eosinophils and lymphocytes in the lungs and airway lumen of sensitized brown Norway rats.

We used flow cytometry and treatment in vivo with a monoclonal antibody (mAb), TA-2, to the alpha 4 integrin to investigate the role of alpha 4 beta 1, CD49d/CD29 (VLA-4) in antigen-induced lung inflammation in Brown Norway (BN) rats. Ovalbumin (OVA) inhalation induced an accumulation of eosinophils and lymphocytes in the lungs and bronchoalveolar lavage (BAL) fluid of sensitized BN rats at 24 h after challenge. Phenotypic analyses demonstrated that the percentages of T cells expressing detectable alpha 4 and CD25 in the bronchial lumen after antigen challenge were dramatically increased compared with blood and lymph node T cells. The mean channel fluorescence values of alpha 4 expression were also increased on BAL T cells compared with blood or lymph node T cells. Treatment of OVA-sensitized rats in vivo with total cumulative doses of 0.75 to 6 mg/kg TA-2 mAb intraperitoneally produced dose-related increases in circulating TA-2 and a peripheral blood lymphocytosis, basophilia, and eosinophilia. Flow cytometric analysis of the peripheral blood T cells after in vivo TA-2 mAb administration showed decreases in detectable alpha 4 when these cells were examined ex vivo. Treatment with TA-2, but not an isotype-matched control mouse immunoglobulin G1 mAb, markedly inhibited the OVA-induced recruitment of lymphocytes and eosinophils into the airway lumen. Very few CD3+CD49d+ cells migrated into BAL fluid following anti-alpha 4 mAb treatment in vivo. Treatment with TA-2 also significantly attenuated OVA-induced inflammatory histopathology. We conclude that VLA-4 is a critically important adhesion molecule involved in antigen-specific lung inflammation in sensitized BN rats.

Molecular determinants of rotavirus virulence: localization of a potential virulence site in a murine rotavirus VP4.

The molecular basis of pathogenesis in vivo for a virulent mouse rotavirus (MRV) and a less virulent bovine rotavirus (BRV) were compared under in vitro and in vivo conditions. Obvious differences in the mobility of several genomic RNA segments were observed in one-dimensional gels. Under in vitro conditions, partial proteolytic peptide mapping identified differences between the two outer capsid proteins of these virus and no difference in inner capsid protein was observed. Since it has been observed by us and others that the gene coding for VP4 protein plays a significant role in determining virulence, the variability observed in the present study between the 84 k proteins (VP4) provided a basis for further investigations in order to locate a potential virulence determinant. A comparison of the carboxypeptidase digests of the MRV- and BRV-VP4 revealed an area of variability between amino acids 307 and 407, which may represent a site of virulence determinant. Under in vivo conditions the virulence of both parenteral BRV and MRV isolates and their corresponding reassortants (with replaced gene 4) were studied in murine and bovine hosts. Like their parents, BRV and MRV isolates, reassortants obtained by replacement of gene 4 in BRV with MRV gene 4 indicated that the dose of the virus isolate used and the clinical outcome in vivo was determined by gene segment 4. The implications of these findings to elucidate the molecular basis of pathogenesis of rotaviruses are discussed.

Characterization of two rotaviruses differing in their in vitro and in vivo virulence.

The proteins, genomic RNA and disassembly conditions and pathogenesis in vivo for a virulent mouse rotavirus (MRV) and a less virulent bovine rotavirus (BRV) were compared. An obvious difference in the mobility of several genomic RNA segments were observed in one-dimensional gels. Reassortants obtained by replacement of gene 4 in BRV with MRV gene 4 indicated that the dose of the virus used and the clinical outcome in vivo was determined by gene segment 4. Under in vitro conditions, a comparison of the inner capsid proteins by partial proteolytic peptide mapping did not reveal any difference between corresponding proteins. However, this technique did identify differences between the two corresponding outer capsid proteins of these viruses. These differences, in turn, may account for the increased stability of MRV, as compared to BRV, when subjected to calcium-chelating and chaotropic agents and may be one of the mechanisms involved in conferring virulence on the virus. The observed variability between the 84K proteins (VP4) provided a basis for further investigations in order to locate a potential virulence determinant, since it has been observed by us and others that the gene coding for this protein plays a role in determining virulence. A comparison of the carboxypeptidase digests of the MRV and BRV VP4 revealed an area of variability between amino acids 307 and 407, which may represent the site of a virulence determinant.

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.

Purification of fimbriae from Pasteurella haemolytica A-1.

Pasteurella haemolytica A-1 produces large, rigid fimbriae with a diameter of 12 nm and a denisty of 1.32. Their subunit molecular weight was 35,000 as determined by SDS-polyacrylamide gel electrophoresis and immunoblotting with monoclonal antibodies raised against native fimbriae. The isoelectric point of the purified fimbriae was 4.8. While Pasteurella haemolytica whole cells plus a crude shear fraction were capable of agglutinating bovine erythrocytes, purified fimbriae exhibited no such activity.

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.

The assembly of barrel cactus virus protein.

The coat protein of barrel cactus virus, a member of the potexvirus family, self-assembles into long tubular particles in the absence of the RNA. The particles have the same structure as the virus. The conditions under which they form suggest a new control mechanism not involving carboxyl-carboxylate interactions.