Effects of Adjuvants for Human Use in Systemic Lupus Erythematosus (Sle)-Prone (Nzb/Nzw) F1 Mice

The safety of four different adjuvants was assessed in lupus-prone New Zealand black/New Zealand white (BW)F1 mice. Four groups of mice were injected intraperitoneally with incomplete Freund's adjuvant (IFA), complete Freund's adjuvant (CFA), squalene (SQU) or aluminium hydroxide (ALU). An additional group received plain phosphate-buffered saline (PBS) (UNT group). Mice were primed at week 9 and boosted every other week up to week 15. Proteinuria became detectable at weeks 17 (IFA group), 24 (CFA group), 28 (SQU and ALU groups) and 32 (UNT group). Different mean values were obtained among the groups from weeks 17 to 21 [week 17: one-way analysis of variance (anova) P = 0·016; weeks 18 and 19: P = 0·048; weeks 20 and 21: P = 0·013] being higher in the IFA group than the others [Tukey's honestly significant difference (HSD) post-test P < 0·05]. No differences in anti-DNA antibody levels were observed among groups. Anti-RNP/Sm antibody developed at week 19 in only one CFA-treated mouse. Mean mouse weight at week 18 was lower in the ALU group than the IFA (Tukey's HSD post-test P = 0·04), CFA (P = 0·01) and SQU (P < 0·0001) groups, while the mean weight in the SQU group was higher than in the IFA (P = 0·009), CFA (P = 0·013) and UNT (P = 0·005) groups. The ALU group weight decreased by almost half between weeks 29 and 31, indicating some toxic effect of ALU in the late post-immunization period. Thus, SQU was the least toxic adjuvant as it did not (i) accelerate proteinuria onset compared to IFA; (ii) induce toxicity compared to ALU or (iii) elicit anti-RNP/Sm autoantibody, as occurred in the CFA group

Extra-articular manifestations of rheumatoid arthritis: an update

Rheumatoid arthritis (RA) is an immune-mediated disease involving chronic low-grade inflammation that may progressively lead to joint destruction, deformity, disability and even death. Despite its predominant osteoarticular and periarticular manifestations, RA is a systemic disease often associated with cutaneous and organ-specific extra-articular manifestations (EAM). Despite the fact that EAM have been studied in numerous RA cohorts, there is no uniformity in their definition or classification. This paper reviews current knowledge about EAM in terms of frequency, clinical aspects and current therapeutic approaches. In an initial attempt at a classification, we separated EAM from RA co-morbidities and from general, constitutional manifestations of systemic inflammation. Moreover, we distinguished EAM into cutaneous and visceral forms, both severe and not severe. In aggregated data from 12 large RA cohorts, patients with EAM, especially the severe forms, were found to have greater co-morbidity and mortality than patients without EAM. Understanding the complexity of EAM and their management remains a challenge for clinicians, especially since the effectiveness of drug therapy on EAM has not been systematically evaluated in randomized clinical trials. Keywords: Rheumatoid arthritis; Extra-articular manifestation; Co-morbidities; Classification; Incidence/rate; Vasculitides; Amyloidosis; Felty's syndrom

Autoantibodies Recognizing the Amino Terminal 1-17 Segment of CENP-A Display Unique Specificities in Systemic Sclerosis

Centromere-associated protein A (CENP-A), a common autoimmune target in a subset of systemic sclerosis patients, appears to have no role to explain why its corresponding auto-antibodies are more frequently found in the limited than the diffuse form of systemic sclerosis. Therefore, we investigated the fine specificity of anti-CENP-A antibodies as a first step to understanding their role in systemic sclerosis pathology. We focused on the amino-terminal portion of CENP-A spanning amino acids 1 to 17 (Ap(1-17)), which represents, along with Ap(17-30), an immunodominant epitope of the protein. Peptide Ap(1-17) was used to purify antibodies from 8 patients with systemic sclerosis. Anti-Ap(1-17) antibodies specifically reacted with human CENP-A but did not cross-react with CENP-B or Ap(17-30). Panning of a phage display peptide library with anti-Ap(1-17) antibodies from 2 patients identified two novel, partially overlapping motifs, and as the result of the alignment of specific phage clone insert sequences. Anti-Ap(1-17) IgG from the 8 patients had different reactivities to isolated phage clone insert sequences. Scanning the Swiss-Prot database revealed a large number of different types of proteins containing the two Ap(1-17) antigenic motifs. These data show that anti-CENP-A(1-17) antibodies are generated independently from anti-CENP-B antibodies and display great heterogeneity in their specificity by recognizing different motifs within that peptide sequence. This finding, along with the widespread interspecies and human tissue distribution of the two motifs, suggests that the number of motif-expressing proteins which can be the potential target of these antibodies is markedly higher than that estimated from the peptide-based epitope spreading model

Autoantibodies Recognizing the Amino Terminal 1-17 Segment of CENP-A Display Unique Specificities in Systemic Sclerosis.

Centromere-associated protein A (CENP-A), a common autoimmune target in a subset of systemic sclerosis patients, appears to have no role to explain why its corresponding auto-antibodies are more frequently found in the limited than the diffuse form of systemic sclerosis. Therefore, we investigated the fine specificity of anti-CENP-A antibodies as a first step to understanding their role in systemic sclerosis pathology. We focused on the amino-terminal portion of CENP-A spanning amino acids 1 to 17 (Ap(1-17)), which represents, along with Ap(17-30), an immunodominant epitope of the protein. Peptide Ap(1-17) was used to purify antibodies from 8 patients with systemic sclerosis. Anti-Ap(1-17) antibodies specifically reacted with human CENP-A but did not cross-react with CENP-B or Ap(17-30). Panning of a phage display peptide library with anti-Ap(1-17) antibodies from 2 patients identified two novel, partially overlapping motifs, and as the result of the alignment of specific phage clone insert sequences. Anti-Ap(1-17) IgG from the 8 patients had different reactivities to isolated phage clone insert sequences. Scanning the Swiss-Prot database revealed a large number of different types of proteins containing the two Ap(1-17) antigenic motifs. These data show that anti-CENP-A(1-17) antibodies are generated independently from anti-CENP-B antibodies and display great heterogeneity in their specificity by recognizing different motifs within that peptide sequence. This finding, along with the widespread interspecies and human tissue distribution of the two motifs, suggests that the number of motif-expressing proteins which can be the potential target of these antibodies is markedly higher than that estimated from the peptide-based epitope spreading model

Clinical correlates of a subset of anti-CENP-A antibodies cross-reacting with FOXE3p53-62 in systemic sclerosis.

INTRODUCTION: In a subset of patients with limited cutaneous (lc) systemic sclerosis (SSc), anti-CENP-A antibodies (Ab) cross-react with a peptide (FOXE3p53-62) that presents striking homology with one of the two immunodominant epitopes of CENP-A (Ap17-30). We searched for clinical correlates of anti-FOXE3p53-62 Ab by measuring their levels along with those of Ab to Ap17-30 and to the second immunodominant epitope of CENP-A, namely Ap1-17. METHODS: Serum samples were obtained from 121 patients with SSc, 46 patients with systemic lupus erythematosus (SLE) and 25 healthy blood donors (HBD). The reactivity of serum IgG to Ap1-17, Ap17-30 and FOXE3p53-62 was measured by ELISA. The corresponding anti-peptide Ab were affinity-purified from pooled SSc sera and used to establish standard curves for quantifying these Ab in patients and HBD. Receiver operating characteristics (ROC) analysis, comparing SSc patients who were positive for anti-CENP Ab (ACA+) to those who were negative, was used to find cut-off points for dichotomizing the anti-peptide Ab levels into positive and negative. Clinical records were reviewed to extract demographic data and information about organ involvement and disease activity. RESULTS: Of 121 SSc sera, 75 were ACA+; 88.0% of these samples reacted with Ap1-17, 82.6% with Ap17-30 and 53.3% with FOXE3p53-62. Among the 46 ACA- SSc sera, 2.2% reacted with Ap1-17, 4.3% with Ap17-30 and 11% with FOXE3p53-62. The levels of these Ab were low in ACA-, SLE and HBD groups and not significantly different among them. When ACA+ SSc patients were divided into subgroups positive or negative for anti-FOXE3p53-62 Ab, the only variables that were significantly different between groups were the levels of anti-Ap17-30 Ab and disease activity index (DAI). There was a significant association between negativity for anti-FOXE3p53-62 Ab and active disease defined as either DAI ≥3 (Fisher exact test, P = 0.045) or less restrictive DAI≥2.5 (P = 0.009). CONCLUSIONS: ACA+-Anti-FOXE3p53-62+Ab identifies a subgroup of patients with lcSSc who are less likely to develop active disease. In lc SSc patients at presentation, anti-FOXE3p53-62+ can be a marker with prognostic significance