Antibody reactivity against thyroid peroxidase and myeloperoxidase in autoimmune thyroiditis and systemic vasculitis.

Potential cross-reactivity between thyroid peroxidase (TPO) and myeloperoxidase (MPO) molecules was evaluated by analysing the binding of 199 TPO antibody- and MPO antibody-positive sera to TPO and MPO molecules. Sera from six patients with autoimmune thyroiditis (AITD) and four patients with systemic vasculitis (SV) with different TPO-MPO antibody findings were then chosen for further analyses. All six patients with AITD had TPO antibodies in enzyme immunoassay (EIA) and four of them had simultaneously MPO antibodies in EIA. In AITD patients antibody binding to TPO could not be inhibited by adding native MPO to the serum diluent, suggesting that the possible cross-reactive epitopes were exposed in the denaturated MPO molecule. Similarly, the MPO ab reactivity of patients with systemic vasculitis could not be inhibited by native TPO. To study whether TPO and MPO antibodies recognize linear epitopes, the binding of antibodies to synthetic TPO and MPO peptides was analysed. Several TPO and MPO peptides were reactive, including peptides reacting with both TPO and MPO antibody-positive sera. One of the most cross-reactive peptides contained AA 586-601 in TPO, showing also particularly high AA homology (88%) with MPO (AA 594-609). The results suggest that TPO and MPO molecules contain cross-reactive epitopes that are exposed in denaturated molecules and may thus cause false positive antibody findings in solid phase EIA assays.

Definition of three minimal T helper cell epitopes of rubella virus E1 glycoprotein.

To characterize T cell-recognized epitopes on rubella virus (RV) E1 glycoprotein, IL-2-dependent RV-specific T cell lines were established from 14 rubella-seropositive healthy donors. The responses of these lines were studied by using a panel of 94 partially overlapping synthetic peptides of 15 amino acids (aa) length covering the known nucleotide sequence of RVE1 glycoprotein. Two to seven peptide-defined epitopes were recognized by the T cell lines, but a large interindividual variation was found. T cell reactivity was most often localized to the regions between aa 276 and 290, aa 381 and 395 and aa 410 and 420. Analysis of overlapping, truncated peptides revealed three minimal T helper cell epitopes VIGSQARK, KFVTAALLN and RVIDPAAQ in aa positions 280-287, 385-393 and 412-419, respectively.

Antibody response to B cell epitopes of Chlamydia trachomatis 60-kDa heat-shock protein and corresponding mycobacterial and human peptides in infants with chlamydial pneumonitis.

To study antibody response to the hypersensitivity protein B of Chlamydia trachomatis, also known as the 60-kDa heat-shock protein (hsp60), epitope scanning was done over the entire protein. Human sera with antibodies to C. trachomatis identified 5 major antigenic regions (peptides 2, 5, 9, 17, and 21) and several minor regions (peptides 34-37, 39, 50, and 59-62). Clear-cut IgG antibody responses to chlamydial peptide 2 (YNEEARKKIQKGVKT) and a corresponding mycobacterial peptide (YDEEARRGLERGLNA) were found in 8 of 16 infants with chlamydial pneumonitis and in 1 of 18 controls. Peptide 50 (RLAKLSGGVAVIRVG) showed an 80% identity with its human counterpart (RLAKLSDGVAVLKVG), which was derived from human mitochondrial protein P1, but specific antipeptide antibody responses were found in 3 of 16 cases only. In summary, both IgG antibody response to C. trachomatis hsp60 and occasional autoantibody formation in infants with chlamydial pneumonitis were found.

Antibody reactivity to an Epstein-Barr virus BERF4-encoded epitope occurring also in Asp-57 region of HLA-DQ8 beta chain. Childhood Diabetes in Finland Study Group.

A five amino acids-long sequence (GPPAA) in the region of the 57th amino acid of HLA-DQ8 beta chain, which seems to be important in defining the risk for type 1 diabetes, occurs also in the BERF4-encoded EBNA3C protein of Epstein-Barr virus (EBV) in six successive repeats. The antigenicity of this region was analysed using synthetic peptides containing different modifications of the GPPAA sequence. Two of the seven individuals who had acute EBV infection produced antibodies against an EBV-derived peptide (GPPAAGPPAAGPPAA) paralleling the EBNA2 antibodies. These two cases also contracted type 1 diabetes immediately after the infection. High antibody levels against this peptide were found in a total of 12% of EBV+ individuals, and in most cases antibodies remained at high levels for several years. Human sera as well as affinity-purified antibodies specific for the GPPAAGPPAAGPPAA peptide reacted also with shorter peptide analogues (GPPAAGPPAA and GPPAA), as well as with peptides containing the surrounding motifs from DQ8 beta chains. However, none of these antibodies bound to denatured DQ8 beta chains in immunoblotting. The charge of the 57th amino acid modulated the antigenicity of this epitope, as peptides from Asp-57-negative DQ molecules were reactive, while peptides from Asp-57-positive DQ molecules were not. The responsiveness was seen in both HLA-DQ8-positive and -negative subjects as well as in type 1 diabetic individuals. The results suggest that some individuals who carry the GPPAA sequence in their HLA-DQ molecule recognize this epitope in EBV. This phenomenon may have potential importance in EBV-induced immune abnormalities, although cross-reactivity against DQ molecules could not be demonstrated in the present study.

Antibodies to E1b protein-derived peptides of enteric adenovirus type 40 are associated with celiac disease and dermatitis herpetiformis.

Sera from 41 children with untreated celiac disease (CD), and 16 children with dermatitis herpetiformis (DH), and 57 matched controls were studied for serum antibodies to synthetic peptides derived from an early E1b protein of adenovirus type 12 and type 40 (Ad12, Ad40) and A-gliadin. In addition to peptides which share homology with A-gliadin, "nonhomologous" peptides derived from Ad12 and Ad40 E1b proteins were used as antigens in ELISA. Patients with CD and DH had significantly higher IgG antibody levels than those of controls to the nonhomologous Ad40 E1b peptide. Concomitant presence of antipeptide IgA antibodies to A-gliadin and Ad12 E1b or Ad40 E1b peptides (which share no homology with A-gliadin) increased the risk of CD/DH suggesting that both adenovirus infections and gluten exposure contribute to the development of CD/DH.

Evaluation of serum antibody response to a newly identified B-cell epitope in the minor nucleocapsid protein L2 of human papillomavirus type 16.

The aim of this work was to identify B-cell epitopes in the minor nucleocapsid (L2) protein of human papillomavirus (HPV) type 16 and characterization of allied antibody response. Serum samples of 513 individuals (323 women with various degrees of cervical atypia, 150 men and 40 small children) were available for the study. Synthetic peptides overlapping the L2 protein of HPV 16 twice were applied in ELISA for epitope scanning and antibody determination. An HPV 16 L2 derived dodecamer SGYIPANTTIPF (amino acids 391-402) proved to be the major B-cell epitope. Both IgA antipeptide antibody positivity (range 7-28%) and mean IgA antibody levels (range 13.2 EIU to 42.4 EIU, P < 0.05) increased with the degree of cervical atypia, whereas antipeptide IgG antibodies showed an opposite trend. During a 2-years follow-up significantly (P < 0.0005) decreasing IgA antibody levels to the SGYIPANTTIPF peptide were associated with regression of koilocytotic atypia. Analysis of anti-peptide IgA antibodies of 118 women with known HPV type revealed that a majority of positives had HPV 16/18 DNA. It was concluded that antibody response to the newly discovered peptide was partially type- and disease-specific. Our results also suggest an impairment of the IgG but not IgA class antibody response to HPV 16 in patients with persistent cervical HPV infection.

Defective HLA class II expression in monocytes of type 1 diabetic patients. The Childhood Diabetes in Finland Study Group.

Activation of T-helper cells is modulated by the intensity of HLA class II expression on antigen-presenting cells. We evaluated whether any abnormalities could be found in the expression of HLA-DR and -DQ molecules on monocytes in type 1 diabetic subjects. DR and DQ molecules were induced by human recombinant interferon-gamma on cultured peripheral blood monocytes obtained from children with type 1 diabetes (N = 28), their siblings (N = 18) and unrelated healthy controls (N = 21). The response in DQ induction varied considerably between different individuals, but the average responsiveness was significantly lower in patients compared to siblings and unrelated controls. In addition to the diabetic subjects deficient DQ induction was also observed in three siblings. One of them had high levels of islet cell antibodies and presented with diabetes 6 months later, and another had active rheumatoid arthritis. The response in DR induction was also slightly lower in patients than in siblings, but did not differ from that in unrelated controls. The results suggest abnormalities in the regulation of HLA class II expression in type 1 diabetic subjects possibly reflecting the ongoing autoimmune process.

Common peptide epitope in mumps virus nucleocapsid protein and MHC class II-associated invariant chain.

The present study describes a 7 amino acid-long sequence (YQQQGRL) which is identical in HLA-associated invariant chain and mumps virus nucleocapsid protein and is additionally followed by one conservative amino acid pair. As such a long amino acid homology is extremely rare in two evolutionarily unrelated proteins the possibility that it could induce immunological cross-reactivity was evaluated. Several antigenicity indices suggested high antigen potential within this region. Synthetic peptides containing this sequence were reactive with 31% of monoclonal antibodies specific for mumps virus nucleocapsid protein in ELISA. High antibody levels against this epitope were found in 7% of mumps-seropositive human sera and antibody levels clearly increased after natural mumps infections and mumps vaccinations. Rabbit antibodies raised against a synthetic invariant chain peptide AYF-LYQQQGRLDKL-C reacted with corresponding nucleocapsid peptide RFAKYQQQGRLEAR-C and antibodies against the nucleocapsid peptide reacted with the invariant chain peptide. Rabbit antibodies against the invariant chain peptide also reacted with nucleocapsid molecules in formaldehyde-fixed mumps virus-infected cells, and antibodies against the nucleocapsid peptide reacted with invariant chains expressed in methanol-fixed cells. One monoclonal antibody specific for the nucleocapsid molecule also reacted with cells expressing invariant chains. In immunoprecipitation rabbit antibodies against the invariant chain peptide bound to invariant chains while antibodies against the nucleocapsid peptide did not. The results suggest that there is antigenic similarity in mumps virus nucleocapsid molecule and HLA-associated invariant chain which may cause immunological cross-reactivity between these molecules.

Cross-reactivity between antibodies to thyroid microsomal antigens and myeloperoxidase.

Antibodies against thyroid microsomal antigen (thyroid peroxidase, TMA/TPO) and myeloperoxidase (MPO) were measured from 115 patients with vasculitic disorders and 144 patients with suspected thyroid disorders. Nineteen patients, three with vasculitis and 16 with thyroid disorders, were shown to have both TPO and MPO antibodies, suggesting cross-reactivity of these antibodies. Their cross-reactivity was further strengthened by studying the capacity of antibodies to tolerate dilution in enzyme immunoassay and reactivity with synthetic TPO/MPO peptides.

Determination of antibodies to non-structural and structural adenovirus antigens by ELISA.

We evaluated possibilities to analyze serum antibodies to non-structural (peptides derived from adenovirus E1b protein) and structural (hexon) adenovirus antigens by ELISA. Synthetic dodecapeptides covering a putative A-gliadin cross-reactive antigenic determinant of the E1b protein were used. The aminoterminus of the peptides appeared to be important for antibody binding but the exact sequence of a possible common B-cell epitope within the peptides remained open. Coupling of the peptides to a carrier protein was essential for ELISA analyses of serum antipeptide antibodies. IgA antibodies to both adenovirus derived E1b peptides and hexon antigen could be detected already two weeks after the onset of an acute adenovirus infection, while antipeptide IgG antibodies were seen in a restricted number of patients only.

Mumps virus infects beta cells in human fetal islet cell cultures upregulating the expression of HLA class I molecules.

The ability of mumps virus to infect pancreatic Beta cells and cause alterations in their HLA expression was evaluated in cultured human fetal islet cell clusters. Mumps virus could be isolated during the whole culture period (6-8 days) and 60% of cells, including Beta cells, contained viral nucleocapsid protein at the end of the culturing. A minor decrease in insulin secretion was observed in some of the infected cultures. The infection was invariably associated with an increase in the expression of HLA class I molecules. This enhancement was mediated by soluble factors secreted by infected cells. The infection could not induce the expression of HLA-DR molecules. However, external interferon-gamma was able to cause a clear rise in DR-expression which was observed only on non-Beta-cells. Rubella and coxsackie B4 viruses were also able to enhance the expression of class I molecules while herpes simplex virus type 2 was not. The results suggest that certain viruses are able to infect Beta cells and cause alterations in their immunological appearance. Increased HLA class I expression in infected islets may exaggerate the autoimmune process in pre-diabetic individuals by increasing the activity of autoreactive cytotoxic T cells.

Demonstration of evolutionary differences between conserved antigenic epitopes in the minor nucleocapsid protein of human papillomavirus types 6b, 16 and 18.

We studied human papillomavirus (HPV) minor nucleocapsid protein (L2) by epitope scanning. Conserved antigenic epitopes identified by rabbit antiserum to bovine papillomavirus (BPV) were revealed in HPV-6b (amino acids, aa, 196-205); HPV-16 (aa:s 376-85) and HPV-18 (aa:s 221-230). L2 proteins. The first two epitopes were situated in hydrophilic regions of the proteins. Aligning the aa-sequences that corresponded to the epitopes with the total L2 sequences of BPV and HPV1a revealed consensus motifs between BPV, HPV1a and the reactive HPV type. In the non-reactive types amino acid alterations were noted. Mismatch between HPV1a sequences and the corresponding HPV-6b and HPV-16, HPV-6b and HPV-18, and HPV-16 and HPV-18 sequences suggests that the alterations may have evolved to facilitate immune surveillance of the genital HPV types.