Estrogen up-regulates Bcl-2 and blocks tolerance induction of naive B cells.

Sex hormones are presumed to contribute to sexual dimorphism in the immune system. Estrogen, in particular, has been suggested to predispose women to systemic lupus erythematosus. We report here that estradiol (E(2)) can break B cell tolerance and induce a lupus-like phenotype in nonautoimmune mice transgenic for the heavy chain of a pathogenic anti-DNA antibody. E(2) treatment resulted in a rise in anti-DNA serum titers and in Ig deposition in renal glomeruli. ELISPOT analysis confirmed a significant increase in the number of high-affinity anti-DNA antibody-secreting B cells in the spleens of E(2)-treated mice. Hybridomas generated from E(2)-treated mice express high-affinity, unmutated anti-DNA antibodies, indicating that naive B cells that are normally deleted or anergized are rescued from tolerance induction. Finally, immunohistochemical studies revealed increased Bcl-2 expression in splenic B cells of E(2)-treated mice. These data demonstrate that estrogen interferes with tolerance induction of naive autoreactive B cells and that the presence of these B cells in the periphery is associated with up-regulation of Bcl-2.

Bcl-2 leads to expression of anti-DNA B cells but no nephritis: a model for a clinical subset.

Transgenic mice expressing anti-DNA antibodies have been extensively studied as a model for understanding B cell regulation in systemic lupus erythematosus (SLE). BALB/c mice transgenic for the R4A-gamma2b heavy chain of an anti-double-stranded DNA (dsDNA) antibody produce two populations of high-affinity anti-dsDNA B cells, one deleted, and the other anergized. We generated double-transgenic BALB / c mice expressing both the R4A-gamma2b heavy chain and the anti-apoptotic bcl-2 gene in the B cell compartment to study whether bcl-2 overexpression differentially affected anergic and deleted B cells. The double-transgenic mice (R4A/bcl-2) express elevated serum titers of both high- and low-affinity anti-dsDNA antibodies and display rescue of autoreactive B cells that are normally either deleted or anergized. Despite the presence of anti-dsDNA antibodies in their serum, R4A/bcl-2-transgenic mice do not develop nephritis, demonstrating that overexpression of bcl-2 is not by itself sufficient to allow disease progression. This phenotype resembles that of some SLE patients who have high titers of anti-DNA antibodies without nephritis.

Crossreactive B cells are present during a primary but not secondary response in BALB/c mice expressing a bcl-2 transgene.

While it is clear that multiple genetic factors lead to autoimmune diseases such as systemic lupus erythematosus (SLE), it appears that an environmental stimulus is also required to trigger the disease in susceptible individuals. We have previously demonstrated that B cells making crossreactive antibodies that bind to both phosphorylcholine (PC), a component of pneumococcal cell wall polysaccharide, and double stranded DNA (dsDNA) can be found in BALB/c mice immunized with PC coupled to a protein carrier. While these B cells are normally eliminated in vivo by apoptosis, they can be recovered ex vivo by fusion with a cell line overexpressing the anti-apoptotic gene bcl-2. This observation led us to ask whether in vivo expression of bcl-2 might abrogate immunologic tolerance during an ongoing immune response. In the present study, we have examined BALB/c mice that constitutively express a bcl-2 transgene in the B cell compartment. Bcl-2 transgenic BALB/c mice have an expanded B cell number, but display no evidence of anti-dsDNA antibodies in the serum even following immunization with PC coupled to a protein carrier. Crossreactive anti-DNA, anti-PC B cells can be recovered by hybridoma technology late in the primary response, but do not appear in the memory B cell compartment. Thus, in vivo expression of bcl-2 can rescue B cell autoreactivity in the primary immune response, but is not sufficient for activation of these B cells or for their maintenance in the memory compartment.

Characterization of anti-DNA B cells that escape negative selection.

One of the challenges in the study of autoimmunity is to understand which autoreactive cells are subject to regulation and what mechanisms of regulation are operative. In mice transgenic for the R4A-gamma2b heavy chain of an anti-double stranded (ds) DNA antibody, the gamma2b heavy chain can pair with the full spectrum of endogenous light chains to produce a multitude of antibodies, including anti-dsDNA antibodies of different affinities and fine specificities. We have previously demonstrated the existence of two populations of anti-DNA B cells in non-autoimmune hosts: a high-affinity population which is rendered anergic in vivo, and a second high-affinity population which is deleted. We have now identified a third population of dsDNA-binding B cells. These cells produce germ-line-encoded antibodies with an apparent affinity for dsDNA that is 1 to 4 logs lower than the apparent affinities of antibodies made by anergic or deleted B cells, and represent a non-tolerized population which escapes regulation. Based on its characterization, we can define a molecular threshold for tolerance induction, and can speculate on the fate of these B cells when they are recruited to an immune response and undergo somatic mutation to become high-affinity anti-DNA B cells.

Knowledge of diet and blood pressure among African Americans: use of focus groups for questionnaire development.

Though focus groups are widely used for development of interventions, little is known about their utility in questionnaire construction, particularly for health surveys in a south-eastern African-American population. In this study, focus groups aided in the development of questions, question sub-components, and response options identifying factors that may influence dietary behavior. Information was used for a survey of dietary knowledge, blood pressure knowledge, and measured blood pressure in a church-based, stratified random sample of middle-class African Americans in North Carolina. Each session, conducted in six churches, lasted 1-1 1/2 hours and had four to nine participants; thirty-four individuals participated. Recorded responses were reviewed and summarized by trained personnel. Results indicate that participants had a general understanding of hypertension, its risk factors, and modes of prevention. However, some misconceptions existed regarding blood pressure and sources of sodium. Television was the most common source of health information. Cost and Southern cultural traditions were deemed the major influences on dietary behavior. Many believed stress was strongly related to blood pressure. The focus group process generated useful information for developing questions about nutrition knowledge, blood pressure knowledge, and health attitudes and beliefs of the target population for the epidemiologic survey that followed.

Studies on the structure, regulation, and pathogenic potential of anti-dsDNA antibodies.

Studies of anti-double-stranded (anti-ds)DNA antibodies have provided insights into how and why these antibodies arise in systemic lupus erythematosus. In this review we discuss the experimental approaches that have been used by our laboratory to study these autoantibodies. Structure/function analyses including site-directed mutagenesis have helped characterize the molecular genetics of anti-dsDNA antibodies, and more recently peptide libraries have been used to define molecular motifs that these antibodies bind. Most of the pathogenic anti-dsDNA antibodies observed in lupus are somatically mutated. We demonstrated in vitro and in vivo that anti-bacterial antibodies can mutate to acquire specificity for dsDNA. Furthermore, using a fusion partner constitutively expressing bcl-2, NSO(bcl-2), we have shown the existence of anergic or preapoptotic B cells making antibodies that cross-react with both bacterial antigen and dsDNA. Whether defects in the regulation of these antibodies might contribute to serum expression of anti-dsDNA antibodies in some individuals remains unknown. A major emphasis of this review is the regulation of anti-dsDNA antibodies in a transgenic mouse model harboring the gene for the heavy chain of a pathogenic anti-dsDNA antibody. Nonautoimmune transgenic mice effectively regulate autoreactive B cells by anergy and deletion, while their autoimmune counterparts do not. The vast majority of anergic B cells expressing high-affinity transgenic anti-dsDNA antibody fail to display allelic exclusion of the heavy chain. We postulate that this may be one mechanism that allows them to escape deletion. Comparative studies on light chain usage in both the autoimmune and the nonautoimmune transgenic mouse strains have demonstrated that within the autoreactive B-cell population, there are subsets that are differentially regulated. Ultimately transgenic animals making pathogenic autoantibodies may provide us with a system for testing novel therapies for autoimmune disease.

Attenuation of human influenza A viruses.

The attenuation of two human influenza A viruses has been carried out, using the selection of inhibitor-resistant strains and multiple passages at low temperatures. A virus related to A2/Tokyo/3/67 was obtained in an inhibitor-resistant form. When this was compared with the inhibitor-sensitive strain in a volunteer trial it was relatively non-pathogenic. The second virus, A2/Hongkong/1/68, was subjected to much longer treatment, but nevertheless remained slightly sensitive to serum inhibitor. When given to volunteers it was less pathogenic than before but attenuation was incomplete. A2/Hongkong/1/68 was also modified by passage at low temperatures. Many of these passages are apparently necessary for full attenuation.All attenuated viruses were infective and antigenic.

Studies on parainfluenza type 2 and 4 viruses obtained from patients with common colds.

Four agents which were obtained from adults with common colds were cultivated and identified-one was influenza type B and the others were parainfluenza viruses of types 2 and 4. Their cultivation was assisted by the use of organ cultures of human embryo tracheal or nasal epithelium. They infected and caused typical common colds in volunteers.

Investigetion into the attenuation of influenza viruses by serial passage.

For vaccination live viruses are better than dead ones, but live influenza vaccines are difficult to prepare. One influenza A(2) and two influenza B viruses were passed in series in embryonated eggs. At several stages of their passage they were inoculated into volunteers, and their effects assessed by virus isolations, antibody rises, and clinical reactions. The A(2) virus and one of the influenza B viruses, both of which had grown readily in embryonated eggs on first isolation, continued to induce human infections and clinical reactions after 30 egg passes. The other influenza B virus acquired enhanced human pathogenicity after three passages from man to man. After adaptation to eggs in which it at first grew reluctantly, its human virulence was appreciably reduced. It underwent no further change during a total of 20 egg passes. There was little convincing evidence of an increased incidence of clinical reactions during the winter seasons, but the numbers of volunteers were too small to draw definite conclusions.

Production of common colds in human volunteers by influenza C virus.

Influenza C virus was intranasally administered to volunteers; most were infected and nine developed symptoms of common cold.Increasing titres of serum haemagglutination-inhibiting antibody and complement-fixing antibody were detected. Virus neutralizing activity in nasal secretion was not correlated with either resistance to infection or the occurrence of overt illness. Interferon was detected in the nasal secretions of some subjects.