1997;40:936C44

1997;40:936C44. pp65422-439-immunized mice and patients with SLE with anti-pp65422-439 antibody. Epitope spreading from pp65428-437 to pp65430-439 was found in pp65422-439-immunized mice in which we generated monoclonal antibodies to pp65425-434 and pp65430-439. However, dsDNA positive reactivity was exclusively observed in stains with pp65430-439-reactive monoclonal antibody. Additionally, Levonorgestrel we observed the amelioration of autoimmunity following the elevation of IgM targeting pp65428-437. Conclusions Our data suggest that pp65428-437 may be an autoimmune or lupus-prone B cell epitope and may catalyze further epitope spreading for inducing autoantibodies in lupus-susceptible individuals. Electronic supplementary material The online version of this article (doi:10.1186/s13075-017-1268-2) contains supplementary material, which is available to authorized users. Keywords: Systemic lupus erythematosus, Human cytomegalovirus phosphoprotein 65, Glomerulonephritis, Anti-dsDNA antibody Background Systemic lupus erythematous (SLE) is a chronic autoimmune disease characterized by widespread loss of immune tolerance to self-antigens. Pathogen recognition and Levonorgestrel subsequent immune responses are potentially the important initiators of autoimmunity in genetically predisposed persons. Emerging evidence indicates that in patients with lupus, exposure to human cytomegalovirus (HCMV) or Epstein-Barr virus (EBV), often precedes the onset of tolerance break [1C3]. EBV is Levonorgestrel the most studied example for cross-reactive autoantibody-mediated autoimmunity. Cross-reactivity of anti-Epstein Barr virus antigen-1 (EBNA-1) antibody to Ro or spliceosomal proteins has been reported [4C6]. Anti-Sm antibody has been found to cross-react in EBNA-1-immunized animals, underlying the molecular mimicry between these antigens [7C10]. HCMV, a ubiquitous opportunistic pathogen, induces 60 kD/Ro expression on the surface of human keratinocytes [11]. Immunization of lupus-prone mice by HCMV recombinant glycoprotein B (gB) results in the production of significant autoantibody to the U1-70?kDa spliceosome protein [12]. Also, the significant Levonorgestrel correlation between antibody to HCMV and U1 small nuclear ribonucleoprotein (snRNP) in HCMV-infected patients with SLE implies that HCMV infection is associated with the development of SLE [13]. In addition, immunization of BALB/c mice with a surrogate octapeptide, DWEYSVWLSN, which induces anti-dsDNA antibody, suggests that the shared structural similarity of antigenic determinants among pathogens and self-proteins leads to autoantibody production [14]. The DNA-interacting amino acids of necrotic cells from post-infected hosts may contribute to induction of anti-dsDNA antibodies [15]. HCMV phosphoprotein 65 (pp65) is a viral scaffold protein and the most abundant constituent of the extracellular viral particle [16]. The pp65 is involved in modulating viral kinase activity and attenuating host antiviral responses [17, 18]. The pp65 protein is a target of both cellular and humoral immunity in healthy individuals, but dominant T cell epitope(s) leads to the robust cellular responses such as cytotoxic T lymphocyte response [19, 20]. Highly elevated anti-pp65 titers in patients with SLE and immunization of NZB/W?F1 mice by pp65 induces early onset of lupus-like symptoms, implying a potential role of pp65 in SLE [21]. The immunization of truncated pp65336-439-conjugated C3d has been shown to induce lupus-like autoantibodies and subsequent development of autoimmunity [22]. The current study aims to further identify the autoantibody-inducing B cell epitope(s) within pp65386-439 and the potential pathogenic immune response. Methods Characteristics of the study populations All patients were recruited from the clinics of Chang Gung Memorial Hospital, and rheumatology specialists confirmed that all patients fulfilled the 1982 and 1997 American College of Rheumatology (ACR) diagnostic criteria for SLE [23, 24] This study was approved by the Institutional Review Board of Chang Gung Medical Foundation. The study of methods was carried out in accordance with the relevant guidelines and informed consent was obtained from all subjects. Mice Normal female BALB/c mice, 3C5 weeks old, were purchased from the National Laboratory Animal Center (NLAC), Taiwan. Animals were housed in a pathogen-free facility with an independent ventilation cage system at the laboratory animal center of Chang Gung Memorial Hospital. All BALB/c mice were 8?weeks old at inoculation. Synthetic peptides For all synthetic peptides, the purity of the peptide was >95%, per the peptide manufacturer (GenScript, NJ, USA). The preparation of Levonorgestrel peptides followed the manufacturers instructions (20?g/l), with storage at -80?C prior to use. Six histidines and one cysteine were added at the C terminus of the peptide as a target or for crosslinking to a carrier protein via a disulfide bond. Plasmid construction The full-length pp65 sequence was amplified from pCMV6-pp65 (SKU Rabbit Polyclonal to 5-HT-6 VC101263, Origene, “type”:”entrez-nucleotide”,”attrs”:”text”:”FJ527563″,”term_id”:”219879600″,”term_text”:”FJ527563″FJ527563) using the.