Potential role of molecular mimicry between Helicobacter pylori lipopolysaccharide and host Lewis blood group antigens in autoimmunity.

Helicobacter pylori is involved in gastritis, gastric and duodenal ulcers, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma. Earlier studies already suggested a role for autoimmune phenomena in H. pylori-linked disease. We now report that lipopolysaccharides (LPS) of H. pylori express Lewis y, Lewis x, and H type I blood group structures similar to those commonly occurring in gastric mucosa. Immunization of mice and rabbits with H. pylori cells or purified LPS induced an anti-Lewis x or y or anti-H type I response, yielding antibodies that bound human and murine gastric glandular tissue, granulocytes, adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma cells. Experimental oral infections in mice or natural infection in humans yielded anti-Lewis antibodies also. The beta chain of gastric (H+,K+)-ATPase, the parietal cell proton pump involved in acid secretion, contained Lewis y epitopes; gastric mucin contained Lewis x and y antigenic determinants. Growth in mice of a hybridoma that secretes H. pylori-induced anti-Lewis y monoclonal antibodies resulted in histopathological evidence of gastritis, which indicates a direct pathogenic role for anti-Lewis antibodies. In conclusion, our observations demonstrate that molecular mimicry between H. pylori LPS and the host, based on Lewis antigens, and provide understanding of an autoimmune mechanism for H. pylori-associated type B gastritis.     

Low antigenicity of the polysaccharide region of Helicobacter pylori lipopolysaccharides derived from tumors of patients with gastric cancer.

We have examined the antibody response to Helicobacter pylori lipopolysaccharide (LPS) during natural infection in humans. The sera of over 70% of H. pylori-infected individuals were found to contain immunoglobulin G antibodies against the LPS fractions isolated from smooth strains of H. pylori but not against those derived from rough strains, as determined by enzyme-linked immunosorbent assay. These results taken together with the immunoblot data indicated that the polysaccharide region of H. pylori LPS is antigenic in humans. However, the antigenicity of the polysaccharide varied, depending on the strain. We found that smooth H. pylori strains isolated from the tumors of patients with gastric cancer showed significantly lower antigenicity than smooth strains derived from patients with chronic gastritis and gastric and duodenal ulcers. The results suggest that the levels of antigenicity of the polysaccharide region of H. pylori LPS in humans correlate with the nature of the gastroduodenal diseases and that they allow a particular distinction to be made between gastric cancer and other gastroduodenal diseases, especially chronic gastritis.     

Relationship between autoepitope and DNA-binding site on a histone H1 molecule.

Autoepitope and DNA-binding domain on a histone H1 molecule were compared using truncated histone H1 peptides as antigens. At least two epitopes (epitope A, N-terminal side; epitope B, C-terminal side) were found both of which were composed of approximately 20 amino acids. IgM from all 17 anti-histone H1-positive SLE sera reacted with epitope A. IgG from 12 sera reacted with epitope A and IgG from 4 sera reacted with epitope B. In one case, no IgG anti-histone H1 reactivities were found while IgM from the same patient reacted with epitope A. Epitope A had the ability to bind DNA. The reactivities against histone H1 of affinity-purified antiepitope A autoantibodies were inhibited by DNA. These data suggest that some anti-histone H1 antibodies are directed against a histone H1 DNA-binding site, raising the possibility that an idiotype/anti-idiotype network, at least in part, is involved in the generation of anti-histone H1 autoantibodies.     

Implication of antigenic conversion of Helicobacter pylori lipopolysaccharides that involve interaction with surfactant protein D

We propose two antigenic types of Helicobacter pylori lipopolysaccharides (LPS): highly antigenic epitope-carrying LPS (HA-LPS) and weakly antigenic epitope-carrying LPS (WA-LPS) based on human serum reactivity. Strains carrying WA-LPS are highly prevalent in isolates from gastric cancer patients. WA-LPS exhibits more potent biological activities compared to HA-LPS, namely, upregulation of Toll-like receptor 4 (TLR4) expression and induction of enhanced epithelial cell proliferation. The results of competitive binding assays using monosaccharides and methylglycosides, as well as binding assays using glycosidase-treated LPS, suggested that β-linked N-acetyl-D-glucosamine and β-linked D-galactose residues largely contributed to the highly antigenic epitope and the weakly antigenic epitope, respectively. WA-LPS exhibited greater binding activity to surfactant protein D (SP-D) in a Ca(2+)-dependent manner, and this interaction was inhibited by methyl-β-D-galactoside. The biological activities of WA-LPS were markedly enhanced by the addition of SP-D. Lines of evidence suggested that removal of β-N-acetyl-D-glucosamine residue, which comprises the highly antigenic epitope, results in exposure of the weakly antigenic epitope. The weakly antigenic epitope interacted preferentially with SP-D, and SP-D enhanced the biological activity of WA-LPS.     

幽门螺杆菌黏附素A有效T和B联合抗原表位噬菌体展示和抗原性测定

目的 分析并确定幽门螺杆菌黏附素A(HpaA)分子中有效T细胞和B细胞联合(T/B)抗原表位.方法 重组表达HpaA(rHpaA)并免疫家兔制备抗血清.采用生物信息学技术预测和分析HpaA分子中T细胞和B细胞表位,PCB扩增T/B联合表位肽片段并构建其噬菌体展示系统.采用PEG/NaCl沉淀法提纯展示了T/B联合表位的重组噬菌体PⅢ蛋白(rPⅢ).分别以商品化幽门螺杆菌全菌IgG抗体和rHpaA抗血清为一抗,采用Western blot和ELISA对rPⅢ蛋白中展示的T/B联合表位进行筛选和鉴定.采用MTT检测rHpaA免疫小鼠脾细胞在不同重组噬菌体蛋白刺激下的增殖情况.结果 HpaA分子中共有5个T/B联合表位:HpaA10、HpaA37、HpaA79、HpaA116和HpaA143.所有T/B联合表位均成功地展示于M13噬菌体PⅢ蛋白表面.Western blot、ELISA和淋巴细胞增殖试验结果 均显示,HpaA116是优势抗原表位,HpaA37和HpaA79为有效抗原表位,HpaA10和HpaA143为无效抗原表位.结论 本研究成功地构建了幽门螺杆菌HpaA的T/B联合表位肽噬菌体展示系统.HpaA37和HpaA79,尤其是HpaA116是HpaA有效T/B联合抗原表位.     

Fine specificity of autoantibodies to La/SSB: epitope mapping, and characterization

The B cell epitope mapping of La/SSB was performed using 20 mer synthetic peptides overlapping by eight amino acids covering the whole sequence of the protein. IgG, purified from sera of five patients with systemic lupus erythematosus (SLE) and four sera from patients with primary Sjögren's syndrome (pSS) were tested against the overlapping synthetic peptides. Peptides highly reactive with purified IgG were those spanning the regions 145–164, 289–308, 301–320 and 349–368 of the La protein. Determination of the minimum required length of the antigenic determinants disclosed the following epitopes:¹⁴⁷HKAFKGSI¹⁵⁴, ²⁹¹NGNLQLRNKEVT³⁰², ³⁰¹VTWEVLEGEVEKEALKKI³¹⁸ and ³⁴⁹GSGKGKVQFQGKKTKF³⁶⁴. Predicted features and molecular similarities of the defined epitopes were investigated using protein databases. The La epitope ¹⁴⁷HKAFKGSI¹⁵⁴ presented 83.3% similarity with the ¹³⁹HKGFKGVD¹⁴⁶ region of human myelin basic protein (MBP) and 72% similarity with the fragment YKNFKGTI of human DNA topoisomerase II. Peptides corresponding to these sequences cross-reacted with anti-La/SSB antibodies. Sixty-three sera with anti-La/SSB antibodies from patients with pSS or SLE, 35 sera without anti-La/SSB antibodies from patients with SS or SLE and 41 sera from age/sex-matched healthy blood donors were tested against biotinylated synthetic epitope analogues in order to determine their sensitivity and specificity for the detection of anti-La/SSB antibodies. Anti-La/SSB were detected with various frequencies ranging from 20% to epitope ¹⁴⁷HKAFKGSI¹⁵⁴ to 100% to epitope ³⁴⁹GSGKGKVQGKKTKF³⁶⁴. The overall sensitivity and specificity using all assays with the synthetic peptides were found to be 93.6% and 85.6%, respectively. In conclusion, antibodies to La/SSB constitute a heterogeneous population, directed against different linear B cell epitopes of the molecule. The epitope ¹⁴⁷HKAFKGSI¹⁵⁴ presents molecular similarity with fragments of two other autoantigens, i.e. human MBP and DNA topoisomerase II. Finally, synthetic epitope analogues exhibit high sensitivity and specificity for the detection of anti-La/SSB antibodies.     

Use of monoclonal antibodies to identify the distribution of A and M epitopes on smooth Brucella species.

The smooth types of Brucella species express two lipopolysaccharides (LPSs) (A and M) which are antigenically distinct. Their existence as cross-reactive antigenic complexes makes definitive serology difficult. Murine hybridomas were produced and selected for their ability to produce monoclonal antibodies to the specific A- or M-LPS epitopes. The specificity of the monoclonal antibodies was determined by microplate enzyme-linked immunoassay, binding inhibition assay, microplate agglutination, and dot blot assay. Monoclonal antibody 12AE6 was specific for an epitope on the A LPS of Brucella spp., which was also expressed on Yersinia enterocolitica O:9. A unique epitope of M LPS was detected by monoclonal antibody 33.1.5. The two monoclonal antibodies did not exhibit cross-reactions when assayed with whole Brucella cells or purified M- and A-LPS preparations. Cross-reactive serology with polyvalent sera can be attributed to the presence of common antigenic determinants on both molecules. The use of A- and M-LPS-specific monoclonal antibodies has the potential to replace the more laborious methods involved in the production of monospecific sera.     

The inflammatory response in CD1 mice shortly after infection with a CagA+/VacA+ Helicobacter pylori strain

To investigate the early events of Helicobacter pylori infection in a mouse model, CD1 mice were infected with a type I (CagA+/VacA+) H. pylori strain. Up to 4 weeks after infection the majority of gastric tissue biopsies were positive in culture. Immunohistochemical analysis showed that inflammatory changes started to occur after 3 weeks. Four weeks after infection a significant increase in T cells was observed in the cardia/corpus region of the stomachs of infected mice. These T cells were CD4+ and CD8+, and they were located in an area with increased expression of MHC class II antigens. In 50% of the infected mice also an increased number of mast cells was seen. Furthermore, aggregates of B and T cells were present in the submucosa. Characterization of cytokines by immunohistochemistry showed an increase in IL-5-secreting cells in the inflamed area of the infected stomach. No difference was observed between interferon-gamma (IFN-gamma)-, IL-4- and IL-10-secreting cells in control and infected mice. These results suggest that no polarized T-helper cell response was present at this early phase of infection. Infection with H. pylori also induced a serum response and especially IgG was increased after 4 weeks of infection. However, no particular increase in IgG1, IgG2a or IgG3 isotype was observed. Part of the serum antibodies was directed against lipopolysaccharide (LPS), but no evidence for anti-Lewis antibodies or antibodies against epitopes on the gastric mucosa was found.     

Detection of anti-VacA antibody responses in serum and gastric juice samples using type s1/m1 and s2/m2 Helicobacter pylori VacA antigens.

Several different families of vacuolating toxin (vacA) alleles are present in Helicobacter pylori, and they encode products with differing functional activities. H. pylori strains containing certain types of vacA alleles have been associated with an increased risk for peptic ulcer disease. In this study, we tested serum samples and gastric juice from 19 H. pylori-negative and 39 H. pylori-positive patients for enzyme-linked immunosorbent assay reactivity with two different types of VacA antigens (types s1/m1 and s2/m2), which were purified from H. pylori 60190 and 86-338, respectively. Both antigens were recognized better by serum immunoglobulin G (IgG) from H. pylori-positive persons than by serum IgG from H. pylori-negative persons (P < 0.01). The s1/m1 VacA antigen was better recognized by sera from patients carrying vacA type s1/m1 strains than by sera from patients carrying vacA type s2/m2 or s1/m2 strains (P < 0.01). Conversely, the s2/m2 VacA antigen was better recognized by sera from patients carrying type s2/m2 or s1/m2 strains (P = 0.03). Serum IgG anti-VacA antibodies were present more frequently in patients carrying type s1/m1 strains than in other H. pylori-positive patients (P = 0.0002). In addition, the highest levels of IgA anti-VacA antibodies were detected in the gastric juice of patients carrying type s1/m1 strains. These data indicate that different VacA isoforms have distinct antigenic properties and that multiple forms of VacA elicit antibody responses in H. pylori-positive humans.     

43-kilodalton glycoprotein from Paracoccidioides brasiliensis: immunochemical reactions with sera from patients with paracoccidioidomycosis, histoplasmosis, or Jorge Lobo''s disease.

Sera from patients with paracoccidioidomycosis (PCM), histoplasmosis (HP), or Jorge Lobo's disease (JL) were titrated against purified gp43 from Paracoccidioides brasiliensis by using both enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation (IPP) reactions with 125I-labeled antigens. In IPP, PCM sera and other sera could be distinguished on the basis of serum titers, whereas in ELISA, 53% of the HP sera and 29% of the JL sera reacted similarly to the PCM sera. To investigate the possible role of the carbohydrate epitopes in these reactions, we compared the reactivities of sera from several patients with native and deglycosylated gp43. Competition experiments were carried out with monosaccharides as inhibitors. The results suggest that greater than 85% of the reactions of the PCM sera with gp43 involved peptide epitopes. Cross-reactions with HP and JL sera in ELISA were predominantly attributed to periodate-sensitive carbohydrate epitopes containing galactosyl residues. HP and JL sera which reacted strongly with gp43 in ELISA were only weakly reactive or did not react in IPP with labeled antigens in solution. Moreover, ELISA reactions could be significantly inhibited either by monosaccharides or by periodate treatment. Apparently, carbohydrate epitopes in gp43 are more accessible to the antibodies when the molecule is bound to a plastic substrate than when it is in solution. Structural changes in the gp43 antigen arising by N deglycosylation abolish reactivity with PCM sera and support the existence of conformational peptide epitopes.     

Anti-IgG antibodies in rheumatic diseases cross-react with Streptococcus mutans SR antigen.

We have previously shown that SR protein, a S. mutans major cell wall protein, as well as the recombinant protein SR (rSR) share common epitopes with human IgG. Since this antigenic mimicry could play a role in the induction of anti IgG, we have examined, in k-ELISA, the presence of antibodies reacting with S. mutans SR proteins and S. mutans whole cells in sera from 36 patients with rheumatic diseases. The majority of the 36 sera showed a high reactivity with rSR when compared with control sera. Eight highly positive sera were further purified on rSR and human IgG sorbents and tested against both rSR and IgG in ELISA and Western blotting. The affinity-purified antibodies reacted strongly with rSR, IgG and IgG Fab fragments but failed to react with IgG Fc fragment. In Western blotting the addition of unlabelled IgG abolished the reactivity of affinity-purified biotinylated antibodies with all antigens, confirming the existence of a common epitope shared by rSR and human IgG heavy chain. We show the existence in rheumatic diseases of high titres of anti-human IgG antibodies cross-reactive with S. mutans SR proteins. Those antibodies are principally IgG and react with the Fd part of the Fab fragment. We can hypothesize from the above data that this antigenic mimicry existing between S. mutans SR-related antigens and human IgG could play a role in the synthesis of at least a part of the anti-IgG antibodies present in rheumatic diseases sera.     

Reactivities of Lewis antigen monoclonal antibodies with the lipopolysaccharides of Helicobacter pylori strains isolated from patients with gastroduodenal diseases in Japan.

We have examined the reactivity of monoclonal antibodies (MAbs) specific for Lewis antigens (Le(x), Le(y), Le(a), and Le(b)) with Helicobacter pylori lipopolysaccharides (LPS) by immunoblot analysis and enzyme-linked immunosorbent assay (ELISA). Sixty-eight strains of H. pylori were isolated from patients with chronic gastritis, gastric and duodenal ulcers, and gastric cancer in Japan. The cells were treated with proteinase K, and the resulting fractions were used as a source of LPS for the immunoassays. In the immunoblot analysis, 28 isolates (41%) and 29 isolates (42%) reacted with anti-Le(x) and anti-Le(y) MAbs, respectively, while 4 isolates (6%) and 7 isolates (10%) reacted with anti-Le(a) and anti-Le(b) MAbs. On the other hand, in ELISA, the number of isolates that reacted with anti-Le(x) MAbs fell significantly to 21 isolates (30%) but the number of isolates that reacted with the other anti-Lewis antigen MAbs remained relatively unchanged. These data show that the immunoblotting technique is more sensitive than the ELISA technique for the detection of immunocomplexes of anti-Le(x) MAbs and components of H. pylori LPS. Furthermore, human serum was found to react with the synthetic Lewis antigens regardless of the status of the individual's H. pylori infection. This means that humans may naturally possess antibodies against Lewis antigens in the absence of H. pylori infection.     

Identification of a B-cell antigenic epitope at the N-terminus of SARS-CoV M protein and characterization of monoclonal antibody against the protein

To identify the potential B-cell antigenic epitopes within the N-terminus of SARS-CoV (SARS-associated coronavirus, SARS-CoV) M protein and characterize monoclonal antibody (MAb) against the protein as well as its recognizing region, we expressed and purified a portion of SARS-CoV M protein (amino acid 1–43) in Escherichia coli (E. coli). By using Western blot and enzyme-linked immunosorbent assay (ELISA), we showed that the purified recombinant M protein could be recognized by four SARS-CoV-positive human sera even when those sera were 12,800-fold diluted. Furthermore, we characterized one representative IgG2 MAb, 3H9, which exhibited a strong immunoreaction to both recombinant M protein and native viral protein of SARS-CoV. We found a B-cell antigenic epitope located between amino acid 1–15 and defined the MAb recognizing region within amino acid 16–28 of M. These findings not only suggest that both recombinant M protein and its specific MAbs may be used as the diagnostic reagents for SARS, but also provide a potential target site for the design of an epitope-based vaccine against SARS. Chao Qian and Di Qin contributed equally to this work.     

Identification of antigenic epitopes in an alanine-rich repeating region of a surface protein antigen of Streptococcus mutants.

A surface protein antigen (PAc) of Streptococcus mutans with a molecular mass of 190 kDa is considered to play an important role in the initial attachment of this streptococcus to the tooth surface. Two internal repeating amino acid sequences are present in the PAc molecule. One repeating region located in the N-terminal region is rich in alanine (A-region), and the other, located in the central region, is rich in proline (P-region). To identify antigenic epitopes on the A-region of the PAc protein, 82 sequential overlapping synthetic decapeptides covering one of the repetitive units of the A-region were synthesized. In the epitope scanning analyses using murine antisera raised against recombinant PAc (rPAc), multiple antigenic epitopes were found in the repetitive unit of the A-region, and some of them reacted with antisera to rPAc from BALB/c, B10, B10.D2, and B10.BR mice. In particular, a peptide YEAALKQY (residues 366 to 373) was recognized by anti-rPAc sera from all four strains of mice. The reactivities of anti-rPAc sera in the epitope scanning were confirmed by using a purified synthetic peptide, NAKATYEAALKQYEADLAA (corresponding to residues 361 to 379). Furthermore, antisera against a surface protein antigen PAg (SpaA) of Streptococcus sobrinus from BALB/c mice reacted strongly to residues 330 to 337, 362 to 369, and 366 to 373 of the PAc protein by the epitope scanning analysis. An AKATYEAALKQY (residues 362 to 373 of the PAc protein)-like sequence, AKANYEAKLAQY, was found within the A-region of S. sobrinus PAg, suggesting that the amino acid sequences AKA-YEA and YEA-L-QY may be major cross-reactive epitopes of the S. mutans PAc protein and the S. sobrinus PAg protein.     

Anti-M9 antibodies in sera from patients with primary biliary cirrhosis recognize an epitope of glycogen phosphorylase.

Anti-M9 antibodies in sera from patients with primary biliary cirrhosis (PBC) were previously found to recognize two antigenic determinants at 98 and 59 kD, using a purified antigen fraction derived from rat liver mitochondria in the Western blot. Here we show that these antibodies are directed against an epitope of the enzyme glycogen phosphorylase. By Western blotting, a determinant at 98 kD was obtained testing anti-M9 positive sera against phosphorylase from skeletal muscle, and after plasmin treatment a degradation product appeared at 59 kD. Both determinants were identical to the M9-specific determinants 98 and 59 kD as shown by absorption studies. When these antibodies were eluted from the 98 and 59 kD determinants of the M9 antigen after immunoblotting, they again recognized the same epitopes on plasmin-treated phosphorylase. Furthermore, phosphorylase enzyme activity could be also demonstrated in the purified M9 fraction, and anti-M9-positive/anti-M2-negative but not anti-M9-negative/anti-M2-positive sera could be shown to stimulate phosphorylase activity. Testing sera from 1189 patients with different hepatic and non-hepatic disorders against M9 and phosphorylase from skeletal muscle by ELISA, 20% were positive with phosphorylase and only 2% with the M9 fraction. These data indicate that the commercially available phosphorylase from skeletal muscle cannot be recommended as M9 source. It may still contain non-PBC-specific epitopes which are probably recognized by naturally occurring antibodies directed against this highly conserved protein.     

Antibody response to gp E of tick-borne encephalitis virus: comparison between natural infection and vaccination breakdown

Human sera obtained after tick-borne encephalitis (TBE) without prior vaccination were compared with sera from patients after a vaccination breakdown. Most sera previously shown to have high titers of IgG and IgM against TBE virus as detected in the ELISA and hemagglutination inhibition (HI) tests also reacted in Western blot with TBE virus E protein which is involved in virus neutralization. The serum of a patient with a vaccination breakdown, however, reacted only very weakly with the E protein in the Western blot in spite of a high amount of antibodies detectable in ELISA. Using SDS-denaturated virus as an antigen in ELISA (imitating the blotting condition), this serum revealed a significant reduction in its reactivity with denatured virus compared to the control sera. This indicates that the patient had an insufficient immune response against certain denaturation resistant epitopes which might contribute to development of disease despite vaccination. The analysis of the immune response of human sera at the epitope level revealed a characteristic "fingerprint" for each serum reflecting the genetic control of the production of antibody populations against different antigenic determinants.     

Identification of immunodominant antigens from Helicobacter pylori and evaluation of their reactivities with sera from patients with different gastroduodenal pathologies

Colonization of the gastric mucosa by Helicobacter pylori is the major cause of gastroduodenal pathologies in humans. Studying the outcome of the humoral immune response directed against this gastric pathogen may contribute substantially to vaccine development and to the improvement of diagnostic techniques based on serology. By using two-dimensional gel electrophoresis, 29 proteins from H. pylori G27 were identified which strongly react with sera derived from H. pylori-infected patients suffering from different gastroduodenal pathologies. These antigens were characterized by mass spectrometry and proved to correspond to products of open reading frames predicted by the H. pylori genome sequence. The comparison of the antigenic patterns recognized by these sera revealed no association of specific H. pylori antigens with antibodies in patients with particular gastroduodenal pathologies.     

Fine specificity of the B-cell epitopes recognized in HIV-1 NEF by human sera.

We have previously used partially overlapping synthetic nonapeptides to characterize the human natural antibody response against HIV-1 negative regulatory factor (NEF), and identified nine 5 to 13 amino acid long regions that were recognized by sera of HIV-1-infected individuals. In this report we define the minimal size of these epitopes with the use of shorter, from 3 to 8 amino acid long partially overlapping peptides covering the complete sequence of the previously identified reacting regions and the N- and C-terminal flanking sequences. We also introduce a new method for the analysis of the reactivities obtained with peptides of different lengths. In six of the antigenic regions the epitopes were found to be noncontiguous and to consist of multiple, down to three amino acid long separate reactive stretches (epitope 1: WSK, VGW, TVRERMRR; epitope 3A: PLRPM, SHFLK; epitope 3B: SQRRQD, DLW; epitope 3C: IYHT, QGYFPDWQN; epitope 4: SLL, VSL; epitope 5: EVLEWRFDSR, VAR). Three epitopes were clearly linear (epitope 2: CAWLE; epitope 3D: LTFGWC; epitope 6: PEYF). Interestingly, five of the minimized B-cell epitopes (1, 3A, 3C, 3D, 5) recognized by human sera overlap totally or partly with the previously identified T-cell epitopes in HIV-1 NEF. Also, only three of the epitopes (3C, 3D, 5) were in a computer-based homology search shown to contain strictly NEF-specific sequences.     

Characterization of the B cell response of patients with anti-liver cytosol autoantibodies in type 2 autoimmune hepatitis

Anti-liver cytosol type 1 (LC1) autoantibody is detected in 30% of sera from patients with type 2 autoimmune hepatitis (AIH), and is the only circulating autoantibody in 10% of cases. Human formiminotransferase cyclodeaminase (FTCD) has been shown to be the specific liver antigen recognized by anti-LC1 autoantibodies. The aim of this study was to identify the dominant epitope on human FTCD and to analyze antigenic-site sequences for clues on the development of AIH. Recombinant proteins and peptides covering the entire cDNA of human FTCD were tested against anti-LC1 autoantibodies. Conformational epitopes were found throughout the protein but linear epitopes were found exclusively in the C-terminal 146 amino acids. Two groups of sera with different reactivities were found: 69%of the sera recognized two specific linear epitopes at positions 428-434 (NTPEEKD) and 440-447 (LQEGLRRA) of human FTCD; others reacted only with a discontinuous epitope between the amino acids at position 395 and 528. FTCD autoantibody production is thus a polyclonal-antigen-driven B cell response. Autoantibodies against conformational or discontinuous epitopes were found in all patients and two-thirds also recognized linear epitopes on human FTCD.     

Two chicken monoclonal antibodies specific for heterophil Hanganutziu-Deicher antigens.

Two chicken monoclonal antibodies (MAbs), HU/Ch2-7 and HU/Ch6-1, against heterophil Hanganutziu-Deicher (HD) antigens with N-glycolylneuraminic acid (NeuGc) at a terminal carbohydrate were established by cell fusions using chicken B cell lines lacking thymidine kinase and spleen cells from chickens immunized with II3NeuGc alpha-LacCer (HD3). The reactivities of these MAbs against several gangliosides including NeuGc-containing glycosphingolipids were examined by a thin-layer chromatography/immunostaining method. MAb HU/Ch2-7 (IgG) reacted strongly with HD3 and IV3NeuGc alpha-nLc4Cer (HD5) and weakly with VI3NeuGc alpha-nLc6Cer (HD7) and 4-O-acetyl-HD3. HU/Ch6-1 (IgG) reacted with HD3 and HD5, but did not react with the other HD antigens. The reactivities of these MAbs against HD antigen were greatly reduced by pre-treatment of the antigen with neuraminidase. These MAbs did not react with N-acetylneuraminic acid-containing gangliosides (GM1 and GM3). These results indicate that these two chicken MAbs are directed toward the antigenic epitope containing the NeuGc.