Ami Desai‐Mehta

We investigated the role of the costimulatory molecules, CD40 and its ligand CD40L, in the pathogenesis of human SLE. In comparison to normal subjects or patients in remission, PBMC from active lupus patients had a 21-fold increase in the frequency of CD40L-expressing, CD4+T cells. However, the expression of CD40L induced in either lupus or normal T cells by mitogenic stimulation could be down-regulated equally well by CD40 molecules on autologous B cells. Active lupus patients also had a 22-fold increase in percentage of CD8+ T cells expressing CD40L, consistent with their unusual helper activity in SLE. Surprisingly, patients with active lupus had a 20.5-fold increase in B cells that spontaneously expressed high levels of CD40L, as strongly as their T cells. Although lupus patients in remission had low levels of CD40L+ cells in the range of normal subjects, mitogen-induced upregulation of CD40L expression in the T and B cells was markedly greater than normal, suggesting an intrinsic defect. A mAb to CD40L blocked significantly the ability of lymphocytes from lupus patients with active and established disease to produce the pathogenic variety of antinuclear autoantibodies in vitro, bolstering the possibility of anti-CD40L immunotherapy for lupus. Future studies on the hyperexpression of CD40L could elucidate a regulatory defect in the pathogenic T and B cells of lupus.

The inherited chromosomal instability disorder Nijmegen breakage syndrome (NBS) results from truncating mutations in the NBS1 gene, which encodes the protein nibrin. Nibrin is part of a nuclear multiprotein complex that also contains the DNA repair proteins Mre11 and Rad50. Upon irradiation, this complex redistributes within the nucleus, forming distinct foci that have been implicated as sites of DNA repair. In NBS cells, nibrin is absent and Mre11 and Rad50 are cytoplasmic. In this study, the interacting domains on nibrin and Mre11 were mapped using the yeast two-hybrid system and expression of epitope-tagged constructs in NBS fibroblasts. Deletion of the carboxy-terminal 101 amino acids of nibrin eliminated its ability to interact with Mre11 and to complement the radiation sensitivity of NBS cells. However, this truncated form of nibrin could localize to the nucleus and form radiation-inducible foci. Expression of a carboxy-terminal 354-amino-acid fragment of nibrin was sufficient to direct the nuclear localization of nibrin, as well as that of Mre11 and Rad50. Despite providing some partial complementation of the radiation-sensitive phenotype, the nibrin-Mre11-Rad50 complexes in these cells were unable to form foci. These results indicate that nibrin directs not only the nuclear localization of the nibrin-Mre11-Rad50 complexes but also radiation-induced focus formation. However, direct interaction between nibrin and Mre11 is required for normal cellular survival postirradiation. Distinct domains of nibrin are required for each of these functions, focus formation, nuclear localization, and Mre11 interaction.

The production of potentially pathogenic anti-DNA autoantibodies in SLE is driven by special, autoimmune T helper (Th) cells. Herein, we sequenced the T cell receptor (TCR) alpha and beta chain genes expressed by 42 autoimmune Th lines from lupus patients that were mostly CD4+ and represented the strongest inducers of such autoantibodies. These autoimmune TCRs displayed a recurrent motif of highly charged residues in their CDR3 loops that were contributed by N-nucleotide additions and also positioned there by the recombination process. Furthermore, Th lines from four of the five patients showed a marked increase in the usage of the V alpha 8 gene family. Several independent Th lines expressed identical TCR alpha and/or beta chain sequences indicating again antigenic selection. 10 of these Th lines could be tested further for antigenic specificity. 4 of the 10 pathogenic anti-DNA autoantibody-inducing Th lines responded to the non-histone chromosomal protein HMG and two responded to nucleosomal histone proteins; all presented by HLA-DR molecules. Another Th line responded to purified DNA more than nucleosomes. Thus, these autoimmune Th cells of lupus patients respond to charged epitopes in various DNA-binding nucleoproteins that are probably processed and presented by the anti-DNA B cells they selectively help.

BACKGROUND: Nonviral gene transfer vectors have the potential to deliver much larger DNA constructs than current viral vectors but suffer from a low transfection efficiency. The LID vector, composed of Lipofectin (L), an integrin-targeting peptide (I) and DNA (D), is a highly efficient synthetic vector, both in vitro and in vivo, which may allow the transfer of genomic loci for gene therapy. METHODS: Transfection efficiencies were quantitated using the green fluorescent protein (GFP) reporter. Expression of a large genomic locus (NBS1 [Nijmegen breakage syndrome], encoding nibrin) was assessed by immunofluorescence. RESULTS: We report a systematic study of the parameters influencing delivery of BAC-based plasmids ranging in size from 12 to 242 kb using the LID vector. We showed 60% of cells were transfected with the smaller plasmids while plasmids up to 242 kb were consistently delivered to over 10% of cells. The number of transfected cells was related to number of plasmids in the transfection complex independent of plasmid size. Atomic force microscopy showed that LID particle size increased with plasmid size consistent with one plasmid molecule per particle. When LID vectors were used to deliver the NBS1 gene as a 143 kb construct to primary NBS cells, at least 57% of cells expressing GFP also expressed functional nibrin. CONCLUSIONS: We show that LID vectors represent a promising tool for the transfer of complete genomic loci.