Autoantibodies to the Nuclear Sp100 Protein in Primary Biliary Cirrhosis and Associated Diseases: Epitope Specificity and Immunoglobulin Class Distribution

Sp100, a protein with a dot-like intranuclear localization in immunofluorescence microscopy, is a major target for patient autoantibodies in primary biliary cirrhosis (PBC) and occasionally in rheumatic disorders. The human Sp100 cDNA has recently been cloned, and the deduced amino acid sequence was found to contain sequence similarities with an MHC class I domain and several transacting regulatory proteins, including HIV-1 nef proteins. In this study, recombinant Sp100 fusion proteins were used to differentiate the immunoglobulin isotypes and to map the epitopes involved in the anti-Sp100 autoimmune response. PBC patients developed IgG as well as IgM and/or IgA class anti-Sp100 autoantibodies whereas most patients with rheumatic diseases developed IgG class autoantibodies only. For epitope mapping, truncated versions of the Sp100 protein were probed for immunoreactivity in ELISA and immunoblotting. With 55 sera, 17 different reaction patterns were obtained, and at least three non-overlapping major autoantigenic domains were recognized by the majority of sera. One domain, which contains the sequence similarity with HIV nef proteins, was recognized by all anti-Sp100 sera and harbours multiple, in part discontinuous, epitopes. These data demonstrate a heterogeneous and patient-specific anti-Sp100 autoimmune response which is antigen-driven and, at least in terms of isotype composition, different in PBC and non-PBC patients.     

原发性胆汁性肝硬化患者抗线粒体抗体亚型检测的临床意义

为探讨原发性胆汁性肝硬化(PBC)患者血清抗线粒体抗体(AMA)亚型抗M2、M4、M9抗体的临床意义,采用间接免疫荧光法检测96例PBC患者血清AMA水平,采用酶免疫斑点法检测AMA-M2、M4、M9抗体水平,应用全自动生化分析仪检测ALT等生化指标。结果表明96例PBC患者中,荧光法AMA阳性率为84.4%,抗M2抗体为81.3%;斑点法抗M2抗体阳性率为72.9%,抗M4抗体为44.8%,抗M9抗体为18.8%,抗M2和M4抗体同时阳性阳性率为43.8%,抗M2和M9抗体同时阳性阳性率为16.7%,抗M2、M4和M9抗体同时阳性阳性率为13.5%,抗M4抗体阳性的PBC患者ALT、AST和IgM水平明显高于抗M4抗体阴性(P<0.05),抗M9抗体阳性的PBC患者ALT、AST和IgG水平明显低于抗M9抗体阴性(P<0.05)。AMA-M2抗体的检测对PBC患者有诊断意义,抗M4抗体和抗M9抗体的检测对于PBC患者的病情判断有意义。     

Primary Biliary Cirrhosis (PBC): Characterization of a Monoclonal Antibody (PBC-MoAb) having Specificity Identical with Disease-Associated Auto-antibodies

We have raised a monoclonal antibody (PBC-MoAb) directed against mitochondria which resembles patent anti-mitochondrial autoantibodies (AMA) (M2 type) in several respects. The reaction pattern of PBC-MoAb was characterized by western blot experiments, immunoaffinity purification and enzyme inhibition studies. PBC-MoAb reacts specifically with an epitope on the E2 subunit of pyruvate dehydrogenase (dihydrolipoamide acyltransferase) which is essential for enzymatic activity. This was shown as follows: (1) PBC-MoAb, like PBC-AMA, completely inhibited PDH enzyme activity and reacted weakly with OGDH; (2) PBC-MoAb bound strongly to the E2 subunit in western blots, with weaker binding to a doublet of about 56 kDa; and (3) in immunosorbent experiments, PBC-MoAb absorbed most (greater than 95%) of the AMA reactive material found in solubilized mitochondria. The present data together with earlier findings that the majority of PBC patient autoantibodies bind to epitopes defined by the PBC-MoAb, makes this antibody a valuable tool for characterizing the major PBC-associated epitope on PDH-E2 and localizing this epitope in liver tissue.     

Mitochondrial Antibodies in Primary Biliary Cirrhosis: II. The Complement Fixing Antigen as a Component of Mitochondrial Inner Membranes

The autoantibodies found in the sera of patients with primary biliary cirrhosis have been shown to react with a component of the mitochondrial inner membranes. Outer membranes were inactive. The purity of the inner and outer membrane fractions obtained by 2 different methods was assessed by electron microscopy and marker enzyme tests. Using negative-staining it was possible to visualize antibody binding to mitochondrial membranes. At high resolution it could be seen that the 90° particles on the cristal membranes were not involved in the reaction with antibody, but it was not possible to establish in the present studies the precise antigenic site upon the mitochondrial inner membranes.     

The Immunophysiology and Apoptosis of Biliary Epithelial Cells: Primary Biliary Cirrhosis and Primary Sclerosing Cholangitis

Biliary epithelial cells (BECs) provide the first line of defense against lumenal microbes in the biliary system. BECs express a variety of pathogen recognition receptors and can activate several intracellular signaling cascades to initiate antimicrobial defenses, including production of several anti-microbial peptides, cytokines, chemokines, and adhesion molecules. BECs also secrete immunoglobulin A and interact with other cells through expression and release of adhesion molecules and immune mediators. Recently, several reports suggest a correlation between apoptosis and autoimmunity through ineffective clearance of self-antigens. Primary biliary cirrhosis (PBC) is a slowly progressive, autoimmune cholestatic liver disease characterized by highly specific antimitochondrial antibodies (AMAs) and the specific immune-mediated destruction of BECs. We have demonstrated that the AMA self-antigen, namely the E2 subunit of the pyruvate dehydrogenase complex, is detectable in its antigenically reactive form within apoptotic blebs from human intrahepatic biliary epithelial cells and activates innate immune responses. Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by inflammation and the presence of concentric fibrosis of intrahepatic and/or extrahepatic bile ducts, eventually leading to cirrhosis. However, apoptosis does not appear to play a central role in PSC. Despite both diseases involving immune-mediated injury to bile ducts, apoptosis occurs more commonly overall in PBC where it likely plays a unique role.     

Mitochondrial Antibodies in Primary Biliary Cirrhosis: V. Ultrastructural Localization of the Antigen to the Inner Mitochondrial Membrane Using a Direct Peroxidase Conjugate

The IgG fraction of a primary biliary cirrhosis serum with high titre of mitochondrial antibodies was conjugated with peroxidase and applied to 20 μm cryostat sections of various rat tissues (stomach, kidney and liver). Results at the ultrastructural level confirm the localization of the antigen to the inner mitochondrial membrane. It was found that fixation with 1% formaldehyde in 0·1 mol/l phosphate, pH 7·4, containing 0·25 mol/l sucrose for 40 minutes at 4° achieved reasonably good preservation of morphology without undue loss of antigen. Staining variations among mitochondria of different cell types are discussed with regard to antigenic distribution.     

Differentiation of Two Bovine Lentiviruses by a Monoclonal Antibody on the Basis of Epitope Specificity

Bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV) are bovine lentiviruses that are closely related genetically. A recombinant fusion protein containing the capsid protein of BIV expressed in Escherichia coli was used to immunize mice and produce monoclonal antibodies. Six hybridomas specific for BIV capsid protein were identified, and one antibody, designated 10H1, was characterized further. Competitive binding assays were performed to analyze the topography of antigenic determinants by enzyme-linked immunosorbent assay and demonstrated the existence of at least three distinct antigenic determinants on capsid protein. The monoclonal antibody reacted specifically with both BIV capsid and the recombinant fusion protein in Western immunoblot analyses. However, it did not react with the recombinant capsid fusion protein of JDV, indicating that BIV contains at least one unique epitope in the capsid protein that is absent in JDV. Further mapping of the epitope by chemical cleavage analysis identified that the epitope is located at the 6.4-kDa N terminus of the 29-kDa capsid protein. This monoclonal antibody assay will be valuable for distinguishing the two closely related lentiviruses by Western blotting.     

Oral Tolerisation to Pyruvate Dehydrogenase Complex as a Potential Therapy for Primary Biliary Cirrhosis

Primary biliary cirrhosis (PBC) is characterised by immune-mediated damage to the intra-hepatic biliary epithelial cells (BEC). Immuno-modulatory/suppressive therapy represents, therefore, a logical approach to treatment in this disease. Conventional immuno-suppressive and immuno-modulatory agents suffer from the breadth of their action and/or excessive side effects. Autoreactive responses to pyruvate dehydrogenase complex (PDC) have been extensively characterised in PBC, and implicated in target cell damage. The aim of the current study was to study the potential efficacy of an antigen specific approach (oral tolerisation with autoantigen) to modulation of anti-PDC immune responses characteristic of PBC, utilising a mouse model of PDC immuno-reactivity. Groups of SJL/J mice were orally dosed with PDC alone, dosed with carrier only (saline) but systemically sensitised with PDC in adjuvant, or orally dosed with PDC at high (5 mg) or low (0.01 mg) dose and systemically sensitised with PDC. Oral dosing with PDC in isolation had no adverse effects on the animals and did not prime anti-PDC responses at doses below 1 mg. Pre-dosing with PDC at both high and low doses was effective at skewing the phenotype of the T-cell response to PDC induced by subsequent sensitisation away from the disease associated Th-1 phenotype (IL-2 and IFN- &#110 secreting) and towards a theoretically protective Th-2 phenotype (IL-4 secreting) in a majority of, but not all, animals. No augmentation of Th-1 response was seen in any animal. Although the effects on liver histology remain to established oral tolerisation with PDC holds promise as a novel, antigen specific approach to therapy in PBC.     

Characterization of the Common Immunodominant Antigenic Determinant in the Lipopolysaceharide of Bacteroides fragilis by a Monoclonal Antibody

The target determinant of a monoclonal antibody (MoAb) to Bacteroides fragilis lipopolysaccharide (LPS) was characterized by inhibition enzyme immunoassay (EIA), immunoblotting (IB), immunofluorescence technique (IF) and electron immunocytochemical (EIC) technique. The MoAb has been shown to react positively with 96% of B. fragilis isolates. LPS preparations from 14 different B. fragilis strains were tested by EIA and IB. Two LPS preparations did not react in any of the tests. In both preparations the D-galactose was either lacking or present in low amount compared with the other LPSs. In addition, inhibition experiments with synthetic disaccharides confirmed that the target determinant is composed of beta-1,6-linked galactose disaccharide. EIC showed that the target of the LPS-MoAb is located on the surface of the outer membrane. These results show that the galactose chain present in LPS isolated from most B. fragilis strains contains the immunodominant antigenic determinant.     

Characterization of the Immune Response to an Epitope on Mycobacterium leprae Antigen 7 Defined by a Monoclonal Antibody

A mouse monoclonal antibody (038D-C6) was shown by crossed immunoelectrophoresis and radioimmunoassay to react with an epitope on the Mycobacterium leprae antigen 7. This epitope was highly crossreactive with BCG/M. tuberculosis and of a non-arabinogalactan-arabinomannan nature. A solid-phase radioimmunoassay (SPRIA) was applied, based on competitive inhibition by human sera of antigen binding by this anti-M. leprae monoclonal antibody. Inhibitory activity determined by this assay decreased markedly upon treatment in both lepromatous and tuberculoid leprosy patients. A correlation was found between the bacterial load of the patient and the inhibitory activity measured in the SPRIA assay. Serum-inhibitory activity could therefore perhaps be used as a follow-up test for patients on treatment or as a screening method to detect infective cases. A dot enzyme-linked immunosorbent assay based, like the SPRIA assay, on competitive inhibition by human sera, was explored as an inexpensive and technically simple alternative also applicable under field conditions.     

Case ReportInduction of T-cell Tolerance in a Patient with Idiopathic Thrombocytopenic Purpura by Single Injection of Humanized Monoclonal Antibody to CD40 Ligand

Normal B cells can be induced to express immune costimulatory molecules by activated T cells, and activated CD4 T cells can express CD40 ligand, a molecule that can engage CD40 on the B-cell surface. CD40–CD40 ligand interaction plays an important role in the pathology of certain autoimmune diseases. We report a patient with chronic idiopathic thrombocyopenic purpura (ITP) who was effectively treated with a single injection of humanized monoclonal antibody to CD40 ligand (E6040). The patient was refractory to steroid therapy, and had baseline platelet counts below 30×10⁹/l during the 3-month period before antibody treatment. The patient's platelet counts have increased to more than 100×10⁹/l long-term after E6040 administration. Platelet-associated IgG was decreased with thrombocytosis. Compared with the initial period of E6040 administration, the number of anti-GPIIb/IIIa antibody-producing B cells decreased, and proliferative response of autoreactive T cells to GPIIb/IIIa was also improved. A single injection of humanized monoclonal antibody to CD40 ligand may induce T-cell tolerance in patients with ITP.     

Production, Characterization, and Epitope Mapping of a Monoclonal Antibody against Aspartic Proteinase of Candida albicans

A monoclonal antibody (MAb; MAb CAP1) that was reactive with extracellular aspartic proteinase of Candida albicans (CAP) was produced. The MAb showed strong sensitivity and reactivity to CAP but not to the aspartic proteinases of Candida parapsilosis, Candida tropicalis, and Aspergillus fumigatus or to human cathepsin D or porcine pepsin. The epitope of the CAP recognized by the MAb was the proteinaseous part of CAP and the putative epitope of the MAb was located in the Asp⁷⁷ to Gly¹⁰³ sequence. This antibody could be useful for the characterization of CAP and would be a valuable probe for the detection of CAP antigen in the sera of patients with invasive candidiasis.     

Acetylcholine Receptor Antibodies in Primary Biliary Cirrhosis: Characterization of Antigen and Idiotypic Specificity

Auto-antibodies against the acetylcholine receptor of skeletal muscle are considered to be the cause of the neuromuscular dysfunction in myasthenia gravis. However, such auto-antibodies also occur in disease states not accompanied by neuromuscular symptoms. Patients with primary biliary cirrhosis have a high prevalence of different auto-antibodies, including antibodies against the acetylcholine receptor. In primary biliary cirrhosis, these anti-receptor antibodies are predominantly of IgM isotype. The IgM antibodies show a broader reactivity with receptors from other species than antibodies from myasthenic patients. Immunoglobulins from patients with primary biliary cirrhosis bear the same receptor antibody-associated idiotypes, but the repertoire is quantitatively different from that found in myasthenia gravis patients. The IgM receptor antibody activity in a serum from a patient with primary biliary cirrhosis could be inhibited by cardiolipin, poly[dT], poly[I], and ssDNA, whereas this antibody activity in serum from a patient with myasthenia gravis was slightly reduced only by cardiolipin. Generally, IgM antibodies from patients with primary biliary cirrhosis had a broader reactivity with polynucleotides and phospholipids than IgG antibodies and antibodies from patients with myasthenia gravis. These results indicate a difference in the fine specificity between acetylcholine receptor antibodies in primary biliary cirrhosis and in myasthenia gravis.     

Production and Characterization of a Novel Monoclonal Antibody Inhibitory for Murine Natural Killer Cell Activity

Natural killer (NK) cells are considered to play an important role in tumor surveillance. The killing of tumor target cells by NK cells is the result of a complex series of sequential binding, signal processing and lytic events. However, the mechanism which NK cells use to recognize tumor targets is poorly understood. To further study the cell-surface molecules involved in tumor recognition, we immunized rats against cloned murine T cells with NK activity (DBA/2.1) and generated rat-mouse hybridomas which were screened for the ability to block lytic activity of DBA/2.1 effector cells. Culture supernatants from one IgM-producing hybridoma, designated S1C4, were found to consistently inhibit DBA/2.1-mediated lysis of YAC-1 target cells. Endogenous splenic NK activity was also diminished in the presence of S1C4 monoclonal antibody (mAb) while alloantigen-specific cytotoxic T lymphocyte (CTL) activity was not affected. S1C4 mAb appears to react with effector cell-surface structures involved in the recognition/adhesion phase of NK activity since pretreatment of effector cells with mAb S1C4 inhibits their ability to bind to YAC-1 target cells. ELISA studies revealed that the S1C4 antigen is expressed by a range of lymphoid cell lines, as well as by DBA/2.1 cells and fresh splenic NK cells. S1C4 mAb were shown to react with 22, 24, 30, and 46 kiloDalton (kDa) DBA/2.1 cell membrane components on immunoblots performed under reducing conditions. These structures do not correspond to any known recognition/adhesion molecules, suggesting that mAb S1C4 defines novel cell membrane components involved in NK cell function.     

Characterization by Monoclonal Antibody of a Highly Conserved Antigenic Determinant Expressed on Human Platelet Membranes and Intermediate Filament Type III

The murine monoclonal antibody (MoAb) CB21, raised after immunization with sonicated extracts of human platelets, has been shown to react with a line-restricted surface molecule and also a cytoplasmic structure displaying no restriction in terms of lineage and species. The surface structure recognized by the CB21 MoAb is exclusively expressed on the surface membrane of human platelets, being undetectable on other cells or lines so far tested. After permeabilization, the majority of the cells and lines tested with the CB21 MoAb displayed strong cytoplasmic reactivity with a constant typical filamentous distribution. Biochemical and morphological analyses showed that the cytoplasmic counterpart recognized by the CB21 MoAb is the intermediate filament type III.     

A Murine Monoclonal Antibody (928) Recognizing a New Epitope Formed with a Combination of and Gene Products

ABSTRACT: A murine monoclonal antibody (mAb), 928, that recognizes a cell surface antigen (928 Ag) on a human Epstein-Barr virus-transformed fetal liver-derived lymphoid progenitor cell line (FL4.4) was generated. The 928 mAb reacted with only FL4.4; it did not react with any other 57 cell lines tested. Two color flowcytometry analysis of peripheral blood mononuclear cells (PBMC) revealed that the 928 mAb reacted with B cell and monocyte fractions from only two individuals out of 63 unrelated donors. Biochemical analyses showed that the 928 Ag composes of two molecules (33 and 34 K_d) and forms a SDS-resistant, noncovalently linked dimer conformation, the feature being similar to that of peptide-bound MHC class II molecules. Treatment of FL4.4 cells with the 928 mAb significantly facilitated homotypic cell aggregation. In addition, treatment of PBMC of the 928 Ag⁺ donor with recombinant IL-4 augmented the expression of the 928 Ag on CD64⁺ monocytes. Typing of and alleles of the 928 Ag expressing and nonexpressing cells revealed that the 928 Ag is expressed only on PBMC of and positive donors. Finally, anti-DP antibody precleared 928 Ag from the cell lysate. These results demonstrate that the 928 mAb recognizes a polymorphic determinant of - gene products. The possibility that amino acids in the groove of the peptide-binding site of HLA-DP molecules are critical for the 928 epitope is discussed.     

Clonotypic Dominance and Variable Gene Elements of Pathogenic Anti-DNA Autoantibodies from a Single Patient with Lupus

This study explores the usage and diversity of the variable gene elements expressed by human lupus antibodies to DNA bearing the 0–81 idiotype, a marker of pathogenic anti-DNA autoantibodies. Rather than studying DNA-specific clonotypes from different patients, a panel of idiotype positive anti-DNA autoantibody-secreting clones from a single individual were analysed. By cloning and nucleotide-sequeneing the heavy-chain variable gene segments, evidence was found for dominance of clonotypic patterns. Also noted was a high rate of diversification among the variable (V_H), diversity (D_h) and junctional (J_H) gene segments utilized, with a pattern of mutations indicative of antigenic selection. These features suggest that the clones secreting the lupus pathogenic autoantibodies have been selected over multiple generations through an affinity-maturation process that is reminiscent of antigen-driven immune responses.     

Association of Single Nucleotide Polymorphisms of the Interleukin-10 Promoter Gene and Susceptibility to Primary Biliary Cirrhosis: Immunogenetic Differences in Italian and Japanese Patients

Several lines of data suggest that genetic factors play an important role in the onset and/or progression of primary biliary cirrhosis (PBC). Since PBC is an autoimmune disease, it is reasoned to assume that genes encoding cytokines may confer susceptibility to disease. Amongst these factors, interleukin-10 (IL-10) has received significant attention. The promoter region of IL-10 gene has three single nucleotide polymorphisms (SNPs) at positions &#109 1082, &#109 819 and &#109 592. To elucidate the association of the three SNPs of IL-10 promoter region with susceptibility of PBC in two different genetic populations, 159 unrelated patients with PBC (94 Italian and 65 Japanese) and 143 local controls (72 Italian and 71 Japanese) were enrolled. SNPs were determined using allele-specific PCR/RFLP. In Italian PBC patients, the frequency of homozygosity for G/G at position &#109 1082 was significantly higher than that of local controls (p <0.041, OR=2.44, 95% C.I.; 1.02-5.86). The frequencies of haplotype GCC in PBC patients, possibly linked to higher IL-10 production, were also significant higher than local controls (p <0.033). However, in Japanese population, there were no significant differences in the three SNPs and haplotypes between PBC patients and controls. Excessive production of IL-10 may play an important role in some populations in modulating the onset of PBC. Further, immunogenetic studies of PBC should take into account ethnic and geographic variations; this makes such studies in heterogeneous population, like the USA, more difficult.     

Alloresponses to HLA-DP Detected in the Primary MLR: Correlation with a Single Amino Acid Difference

The one-way mixed lymphocyte reaction (MLR-1) response was measured in both directions in 50 HLA-A, B, DR and DQ identical pairs and the role of DP studied in MLR stimulation. DR, DQ and DP typing was performed at the allele level by the polymerase chain reaction-sequence specific oligotyping (PCR-SSO) technique. The group consisted of 19 potential bone marrow transplant recipients and 34 matched unrelated donors. When more than one matched donor was available for a patient, donor/donor MLR-1 was also studied. DP identity was observed in 3 out of 50 pairs (6%), however due to homozygosity no incompatibility was present in the stimulating cells in 21 out of 100 cases (21%). There was a significant difference in the range of relative responses (RR) between zero DPB1 mismatches and one (p = 0.002) and two (p = 0.02) DPB1 mismatches: 52.4% of cases in the zero DPB1 mismatch group had RR < 1.0% compared with 31.6% and 27.3% in the one and two DPB1 mismatches. Stimulation by DPB1*0201 and 0301 gave the highest RR (12.9 ± 22.5 and 17.5 ± 17.0, respectively) while stimulation with DPB1*0401 and 0402 resulted in low levels of T cell response (1.3 ± 8.2 and 0.6 ± 11.5, respectively). When the responses were restricted to DPB1*0401 homozygotes to standardise for responder type similar results were obtained (DPB1*0201 v DPB1*0402 p = 0.008). The protein products of the DPB1*0201 and 0402 alleles differ by a single amino acid at position 69 (DPB1*0402—Lysine, DPB1*0201—glutamic acid). A further analysis was performed therefore scoring responders and stimulators as glutamic acid positive (E+) or negative (E−). There was a highly significant increase in the response to E+ stimulators compared with E− stimulators (p = 0.004). There was also a significant difference in the distribution of relative responses between the E+ stimulator group and the subgroups of E− responders/E− stimulators (p = 0.012) and E+ responders/E− stimulators (p = 0.009). However the amino acid difference at position 69 does not explain all responses due to DP in the MLR-1 as evidenced by the strong responses observed in cases where DPB1*0301 (lysine pos.) was the only difference on the stimulator cells. The results indicate that not all DP incompatibilities elicit a measurable T cell MLR response, but where a response does occur residue 69 in the first domain of DP appears to be pivotal. These results may have implications with respect to GVHD in bone marrow transplantation.