Treatment of refractory SLE with rituximab plus cyclophosphamide: clinical effects, serological changes, and predictors of response.

OBJECTIVE: To evaluate efficacy, serological responses, and predictors of response in patients with severe and refractory systemic lupus erythematosus (SLE) treated with rituximab plus cyclophosphamide. METHODS: 16 patients entered a treatment protocol using rituximab plus cyclophosphamide. Disease activity was assessed by the SLE disease activity index (SLEDAI) and by the British Isles Lupus Assessment Group (BILAG) index. RESULTS: At six months follow up, mean SLEDAI values decreased significantly from (mean (SD)) 12.1 (2.2) to 4.7 (1.1). Clinical improvement (50% reduction in SLEDAI) occurred in all but three patients. All but one patient responded according to BILAG. Remission defined as SLEDAI <3 was achieved in nine of 16 patients. Isotype analysis of anti-dsDNA antibodies revealed preferential decreases of IgG and IgA, but not IgM. Higher absolute numbers of CD19+ cells at baseline were correlated with shorter depletion time (r = -0.6). CONCLUSIONS: The majority of patients improved following rituximab plus cyclophosphamide. The differential downregulation of anti-DNA of the IgG and IgA but not the IgM isotypes supports the hypothesis that cells producing pathogenic autoantibodies are preferentially targeted by the treatment. The fact that greater absolute numbers of CD19+ cells at baseline predict a less impressive clinical and serological response suggests that more flexible dosing could be advantageous.

Autoepitope-mapping of the U1-70K protein with human-Drosophila chimeric proteins.

The 70K protein is the major autoantigen for anti-RNP autoantibodies directed against the U1 small nuclear ribonucleoprotein complex particle. The U1-70K protein has been epitope-mapped by various groups, and a major antigenic region of about 70 amino acids has been found which overlaps with the RNA binding motif. Attempts to map the major antigenic region further with smaller cloned fragments or with peptides have been hampered by total loss of, or strongly reduced, antigenicity. Thus the major antigenic region is composed of conformational epitopes and a detailed analysis of particular epitopes has not been possible. In the present work, we examine the antigenicity of chimeric proteins assembled from the highly conserved Drosophila melanogaster 70K proteins grafted with human 70K segments. With this approach, the effects on antigenicity of exchanging particular segments can be assayed with the overall structure of the major antigenic domain kept relatively constant. Our results, supported by depletion experiments, show that residues 99-128 from the human protein are essential for recognition by both human and canine anti-RNP autoantibodies. These residues have to be presented in a manner that allows correct conformational interaction between the different protein domains.

Human anti-RNP sera contain both human-specific and cross-reactive anti-70K autoantibodies.

The U1 snRNP (small nuclear ribonucleoprotein complex) associated 70K protein is the main autoantigen for the anti-RNP autoantibodies which are directed against the U1 snRNP particle. The major antigenic region of the 70K protein has by various laboratories been mapped to an RNA binding domain required for the 70K-U1 snRNA interaction. We have used recombinant proteins comprising this region from the human and the Drosophila melanogaster 70K proteins to examine the species specificity of the human anti-70K autoantibodies found in 42 patient sera. Most, but not all, anti-70K positive sera in this cross-sectional sample contained both human 70K specific anti-bodies and Drosophila 70K reactive antibodies. Results of a longitudinal follow-up of 14 patients indicated that the cross-reactive anti-70K antibodies developed secondarily to the establishment of a species-specific anti-70K reaction. In a fraction of the patient sera this broadening of the response never occurred. Taken together, the data in this study support the hypothesis that the endogenous human 70K protein is the immunogen driving the production of anti-70K autoantibodies.

Deficient brain snRNP70K in patients with Down syndrome.

The small nuclear ribonucleoprotein 70K (snRNP 70K; U1-70 kDa) is an integral part of the spliceosome, a large RNA-protein complex catalyzing the removal of introns from nuclear pre-mRNA. snRNP is one of the best-studied essential subunits of snRNPs, is highly conserved and its inactivation was shown to result in complete inhibition of splicing. Applying subtractive hybridization, we found a sequence with 100% identity to snRNP absent in fetal Down syndrome (DS) brain. This observation made us determine snRNP-mRNA steady-state levels and protein levels in brains of adult patients with DS. snRNP-mRNA and protein levels of five individual brain regions of DS and controls each, were determined by blotting techniques. snRNP-mRNA steady state levels were significantly decreased in DS brain. Performing Western blots with monoclonal and human antibodies, snRNP protein levels were decreased in several regions of DS brain, although one monoclonal antibody did not reveal different snRNP-immunoreactivity. Although decreased snRNP-protein could be explained by decreased mRNA-steady state levels, another underlying mechanism might be suggested: snRNP is one of the death substrates rapidly cleaved during apoptosis by interleukin-1-beta-converting enzyme-like (ICE) proteases, which was well-documented by several groups. As apoptosis is unrequivocally taking place in DS brain leading to permanent cell loses, decreased snRNP-protein levels may therefore reflect decreased synthesis and increased apoptosis-related proteolytic cleavage.