Congenital heart block (CHB) may develop in fetuses of women with anti-Ro/SSA autoantibodies, and carries substantial morbidity and mortality. The aim was to evaluate how information on CHB is ...imparted and identify areas of improvement.
A questionnaire was distributed to anti-Ro/SSA antibody-positive women who had either participated in a surveillance programme but whose expected child did not develop CHB (n = 100, denoted Doppler-Assessed Pregnancies (DAP) group) or given birth to a child with CHB (n = 88, denoted CHB-Affected Pregnancies (CAP) group).
The response rate was 83% (157/188). Most women received the information on CHB when they were already pregnant (DAP group 60%, CAP group 83%). However, a majority of them would have wanted the information before pregnancy (DAP group 52%, CAP group 56%), and most stated that it would not have influenced their decision to have a child (DAP group 77%, CAP group 58%). The ability to both understand the information and to perceive the information as sufficient were significantly higher when someone trained in paediatric cardiology gave the information.
Our findings indicate that information on CHB should be given to women before pregnancy. The data further highlight the importance of having specific knowledge for giving relevant and understandable, yet sufficient information.
Canine systemic lupus erythematosus (SLE) has a similar disease expression as human SLE, but the serological characterisation of the canine disease is as yet incomplete. In the present study, we ...examined the specificity of antinuclear antibodies (ANA) in indirect immunofluorescence (IIF) positive canine sera. Sixty-four canine IIF ANA positive sera were characterised using HeLa cell nuclear extract immunoblots and recombinant U1-70K ELISA. We compared these results with a previously shown concordance between indirect immunofluorescence and immunodiffusion in canine SLE serological diagnosis. One canine serum reacting with Sm proteins was observed, and five canine sera presented anti-RNP autoantibodies against the antigens 70K, A, C, and/or B/B′. The autoantigen most frequently recognised was a 43 kDa nuclear protein, previously described as hnRNP G. This prominent canine autoantigen was missing in the commercially available extract designed for immunodiffusion testing of human sera. Other prominent canine autoantigens were found not to be identical with the principal human ones, thus making present human test systems deficient for the use in canine systemic connective disease diagnosis. The development of antigenic extract designed for canine autoimmune autoantigens is necessary in order to make immunodiffusion a useful method in canine diagnosis. The anti-RNP positive canine sera were examined in more detail and we found that the human major antigenic region of the most prominent RNP antigen, the U1-70K protein, also is targeted by canine autoantibodies. Thus, the response against the RNP antigen seems to be conserved between man and dog.
The U1‐70K protein is specifically bound to stemloop I of the U1 small nuclear RNA contained in the U1 small nuclear ribonucleoprotein complex (U1 snRNP), which is involved in the splicing of ...pre‐mRNA. All components of the U1 snRNP complex, including the U1‐70K protein, are important autoantigens in patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD). Here we describe for the first time the selection and characterization of recombinant human anti‐U1‐70K single chain autoantibody fragments (anti‐hU1‐70K scFv) from autoimmune patient‐derived phage display antibody libraries. All scFv specifically recognize parts of the hU1‐70K protein and its apoptotic 40‐kDa cleavage product. In Western blotting assays a number of scFv preferentially recognize the 40‐kDa apoptotic cleavage fragment of the U1‐70K protein, suggesting a possible involvement of this apoptotic cleavage product in the autoimmune response of patients. The germ‐line gene usage of these recombinant autoantibodies was also determined. Using several U1‐70K deletion and point mutants of both human (h) and Drosophila melanogaster (Dm) origin, it was established that the U1‐70K epitope that is recognized by the anti‐hU1‐70K scFv is located within the RNA binding domain.
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.
Objective
Rituximab is a monoclonal antibody directed against the CD20 marker of B cells. Because of its ability to deplete B lymphocytes, it has been suggested that the drug could be of benefit in B ...cell–dependent diseases, including systemic lupus erythematosus (SLE). The purpose of this study was to investigate the histopathologic and clinical effects of combination treatment with rituximab and cyclophosphamide (CYC) in patients with CYC‐resistant proliferative lupus nephritis.
Methods
Seven female patients with proliferative lupus nephritis were treated with rituximab in combination with CYC. Renal biopsies were performed before treatment and during followup. SLE activity was evaluated by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) and the British Isles Lupus Assessment Group index. In 6 of the 7 patients, immunostaining of lymphocyte subpopulations in the renal tissue was performed before treatment and during followup.
Results
At 6 months of followup, significant clinical improvement was noted, with a reduction in SLEDAI scores (from a mean of 15 to 3), anti–double‐stranded DNA antibody levels (from a mean of 174 IU/ml to 56 IU/ml), and anti‐C1q antibody levels (from a mean of 35 units/ml to 22 units/ml). On repeat renal biopsy, improvement in the histopathologic class of nephritis occurred in a majority of patients, and a decrease in the renal activity index was noted (from 6 to 3). A reduction in the number of CD3, CD4, and CD20 cells in the renal interstitium was noted in 50% of the patients on repeat biopsy.
Conclusion
At 6 months of followup, all patients had responded both clinically and histopathologically to combination therapy. For patients with proliferative lupus nephritis who fail to respond to conventional immunosuppressive therapy including CYC, combined treatment with rituximab and CYC may constitute a new treatment option.
The small nuclear ribonucleoprotein 70K (snRNP 70K; U1‐70kDa) 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.
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.
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 melanogaster70K 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 antibodies 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.
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