SummaryDisease activity in systemic lupus erythematosus is closely associated with the appearance of immunoglobulin (Ig)G antibody to native DNA in both humans and mice. Like normal antibody responses, the anti-DNA autoantibody first appears as IgM and then switches to IgG. Structural studies of IgG anti-DNA suggest that these antibodies are the products of clonally selected, specifically stimulated B cells. The origins of the IgM anti-DNA have been less clear. To determine whether the earlier appearing IgM anti-DNA antibody in autoimmune mice also derives from clonally selected, specifically stimulated B cells or B cells activated by nonselective, polyclonal stimuli, we have analyzed the molecular and serological characteristics of a large number of monoclonal IgM anti-DNA antibodies from autoimmune (NZB x NZW)F1 mice. We have also analyzed IgM and IgG anti-DNA hybridomas obtained from the same individual mice to determine how the later-appearing IgG autoantibody may be related to the earlier-appearing IgM autoantibody within an individual mouse. The results demonstrate that: (a) IgM anti-DNA, like IgG, has the characteristics of a specifically stimulated antibody; (b) IgM and IgG anti-DNA antibodies have similar variable region structures and within individual mice may be produced by B cells derived from the same clonal precursors; (c) recurrent germline and somatically derived VH and VL structures may influence the specificity of anti-DNA monoclonal antibody for denatured vs. native DNA; and (d) the results provide a structural explanation for the selective development of IgG antibody to native DNA as autoimmunity to DNA progresses in (NZB x NZW)F1 mice.

Results from our analyses of variable region gene usage among spontaneous anti-DNA antibodies in autoimmune mice have indicated that both the early IgM and later-appearing IgG autoantibodies to DNA are generated by clonally selected B cells. The recurrent usage of particular variable region genes among all the anti-DNA hybridomas analyzed and reported to date supports this hypothesis. The preferential expression of particular light and heavy chain variable region genes among selected populations of both IgM and IgG anti-DNA hybridomas likewise supports the hypothesis. Both IgM and IgG antibody-producing B cells are derived from the same clonal precursor population and may be derived from the same B cell clonal precursor within an individual mouse. The selective and recurrent expression of germline and somatically-derived structures that would be expected to promote protein binding to DNA within anti-DNA antibody variable regions, particularly arginines in both light and heavy chain complementarity-determining regions, indicates that DNA or DNA-containing complexes may be the antigen that stimulates anti-DNA antibody in autoimmune mice. The progressive increase in the specificity of spontaneous anti-DNA antibodies for native DNA as the autoimmune response matures from IgM to IgG likewise suggests that DNA may be the antigenic stimulus for spontaneous anti-DNA in autoimmune mice. A hypothetical, computer-generated model of anti-DNA antibody binding to DNA provides an interesting paradigm for the molecular basis of antibody specificity for DNA.

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Stem cell factor (SCF) gene expression is regulated by FSH in testicular Sertoli cells. Many functions of FSH are mediated through the second messenger cAMP. We show that cAMP activates transcription of the human SCF promoter in a Sertoli cell line. The human SCF promoter was cloned in cosmid vector pWE15, and its DNA sequence was determined for the promoter region extending 2.3 kilobase pairs upstream from the translation start site at +184 bp. The in vivo messenger RNA (mRNA) start site, by primer-extension studies, was located in exon 1 at +109 bp in human testis mRNA, and at +99 bp in mouse SF7 Sertoli cell line or GC1 germ cell line mRNA. To test which regions of the SCF promoter are necessary for regulation by cAMP, a series of 5'-end deletions of this region were cloned onto the luciferase reporter gene in plasmid pXP1. The SCF promoter region was fused to luciferase downstream (at +120) from its +109 mRNA start site, extending upstream a variable distance to BstXI (-162), BamHI (-313), Bgl2 (-853), or XbaI (-2185). The shortest of these fragments extending only to -162 bp, contains possible SP1 and AP-2 elements. When mouse Sertoli SF7 or human JEG.3 cell lines were transfected with these plasmids, all of the mutants were regulated by 8Br-cAMP or forskolin, as expected for the SCF gene, whereas FSH and TPA had no effect. In the shortest promoter deletion -162, luciferase expression from SF7 cells in serum-free media was at a moderate basal level, but it was induced in six h about 2-fold by 8Br-cAMP, and over 7-fold by forskolin (an adenylate cyclase activator) to high levels, similar to the SV40 positive control promoter. In SCF-luc plasmids extending to -853 or -2185, luciferase expression was still inducible by 8Br-cAMP and forskolin to high levels, but basal promoter activity was repressed to levels over 15-fold lower, in both the absence or presence of testosterone in the media for SF7 cells. The distal portion of the human SCF promoter (between -313 and -853, and also -853 and -2185) inhibits the basal level of transcription, while the proximal region (5' of -162) can mediate activation by cAMP.

Several problems remain before molecular biology-based techniques, such as PCR, are widely accepted for the detection of infectious agents. Among the most formidable of these problems are the inability of the tests to distinguish between viable and nonviable organisms. We approached this problem by using the fact that bacterial mRNA has an extremely short half-life, averaging only a few minutes. We reasoned that by targeting bacterial mRNA by a reverse transcriptase PCR (RT-PCR), a positive signal would indicate the presence of a recently viable organism. To test our hypothesis, we chose to target the mRNA coding for the ubiquitous 85B antigen of mycobacteria. After partially sequencing the gene coding for 85B, we developed primers that were specific for Mycobacterium tuberculosis. In a single-tube, nested, RT-PCR (STN RT-PCR), these primers detected fewer than 40 CFU in spiked sputum samples and as few as 12 CFU in clinical sputum specimens. The sensitivity of STN RT-PCR with smear-negative samples was as good as that of culture. The specificity was 100%. More importantly, when M. tuberculosis was cultured with and without 1 g of isoniazid per ml, this assay could distinguish between those cultures which contained the antibiotic and those which did not. Subcultures on Lowenstein-Jensen agar confirmed the viability assessments of the STN RT-PCR. Control experiments demonstrated that isoniazid did not inhibit the RT-PCR. In addition, when an IS6110-targeted, DNA PCR was used to examine the same samples, all samples through 13 days (the last sample) continued to be positive, irrespective of whether isoniazid was present, thereby demonstrating the superiority of an mRNA target in the detection of mycobacterial viability.