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A semiquantitative polymerase chain reaction (PCR) assay described in this study has been used lo analyse the VH1, VH3 and VH4 repertoire expressed by total IgM+ and IgG+ B cells from normal individuals and lupus patients. This approach consists of a combination of B cell selection, utilization of the anchored PCR technique to avoid technical bias in the amplification of different VH gene family cDNA templates, and screening of the amplified IgM or IgG cDNA rearrangements by family-specific oligonucleotide probes. In four lupus patients, VH family representation in IgM+ and IgG+ in vivo activated B cells, selected by anti-CD71 antibody, and in total CD19+ B cells were compared. In all patients VH4 gene family segments were preferentially underrepresented in IgM+ activated B cells. In IgG+ B cells the results suggest that VH4 expression is variable, depending on the phase of the disease. Polyclonal B cell activation, which is usually considered as being the first event in autoantibody production in SLE, cannot explain our results. The data evoke the possible involvement of a VH4-specific B cell superantigen in the onset or development of SLE. This hypothesis is also supported by the sequence conservation of the fourth β loop-a putative superantigen binding site-of functional VH4 gene segments which are preferentially used by anti-dsDNA lupus antibodies of established clones and hybridomas. [ABSTRACT FROM AUTHOR]

We have analysed the heavy and light chain variable region genes of two monoclonal antibodies, specific for the Sm antigen (RSP1; IgGκ) and for cardiolipin (RSP4; IgMλ), derived from a patient with active systemic lupus erythematosus (SLE). We have established that the variable region genes of the RSPI autoantibody are somatic mutants of two germ line genes from the VH4 and VκI gene families. RSP4 antibody uses gene segments closely related to a VH3 gene member and to a Vλ1 gene. The presence and distribution of the somatic mutations on both monoclonal autoantibodies are compatible with an antigen-driven immune process. These data suggest that in SLE a common antigenic stimulus may govern the autoantibody response against a wide spectrum of unrelated antigens, including native DNA, cardiolipin or Sm antigens, and provide further evidence that disease-associated autoantibodies are generated through antigen-selected somatic mutations. [ABSTRACT FROM AUTHOR]

We have evaluated the transduction profiles of an HIVbased lentiviral vector delivered regionally to ocular tissues in vivo. Following subretinal injection, a green fluorescent protein (GFP) reporter gene was efficiently and stably expressed in retinal pigment epithelial (RPE) cells. Limited transduction of adjacent photoreceptors occurred in newborn mice, but was inefficient in adult animals. Injection of the vector into the anterior chamber resulted in efficient and stable transduction of corneal endothelial cells. Efficient in vivo gene transfer into cells of the corneal endothelium and retinal pigment epithelium by lentiviral vectors may therefore offer a valuable approach to the treatment of disorders of the cornea and outer retina. [ABSTRACT FROM AUTHOR]