The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Amano, K.
Right arrow Articles by Gershwin, M. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Amano, K.
Right arrow Articles by Gershwin, M. E.
The Journal of Immunology, 2004, 172: 6444-6452.
Copyright © 2004 by The American Association of Immunologists

Xenobiotic-Induced Loss of Tolerance in Rabbits to the Mitochondrial Autoantigen of Primary Biliary Cirrhosis Is Reversible1

Katsushi Amano2,*,§, Patrick S. C. Leung2,*, Qingchai Xu{ddagger}, Jan Marik{ddagger}, Chao Quan{dagger}, Mark J. Kurth{dagger}, Michael H. Nantz{dagger}, Aftab A. Ansari, Kit S. Lam{ddagger}, Mikio Zeniya§, Ross L. Coppel|| and M. Eric Gershwin3,*

* Division of Rheumatology, Allergy, and Clinical Immunology, and {dagger} Department of Chemistry, University of California, Davis, CA 95616; {ddagger} University of California Davis Cancer Center, Division of Hematology and Oncology, Department of Internal Medicine, University of California at Davis, Sacramento, CA 95817; § Division of Gastroenterology and Hepatology, Department of Internal Medicine, Jikei University School of Medicine, Tokyo, Japan; Department of Pathology, Emory University, Atlanta, GA 30322; and || Department of Microbiology, Monash University, Clayton, Victoria, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous work has demonstrated that immunization of rabbits with the xenobiotic 6-bromohexanoate coupled to BSA breaks tolerance and induces autoantibodies to mitochondria in rabbits. Such immunized rabbits develop high-titer Abs to pyruvate dehydrogenase complex (PDC)-E2, the major autoantigen of primary biliary cirrhosis. In efforts to map the fine specificity of these autoantibodies, rabbits were immunized biweekly with 6-bromohexanoate-BSA and screened for reactivity using a unique xenobiotic-peptide-agarose microarray platform with an emphasis on identifying potential structures that mimic the molecular image formed by the association of lipoic acid with the immunodominant PDC-E2 peptide. Essentially, a total of 23 xenobiotics and lipoic acid were coupled to the 12-mer peptide backbones, PDC, a mutant PDC, and albumin. As expected, we succeeded in breaking tolerance using this small organic molecule coupled to BSA. However, unlike multiple experimental methods of breaking tolerance, we report in this study that, following continued immunization, the rabbits recover tolerance. With repeated immunization, the response to the rPDC-E2 protein increased with a gradual reduction in autoantibodies against the lipoic acid-peptide, i.e., the primary tolerance-breaking autoantigen. Detailed analysis of this system may provide strategies on how to restore tolerance in patients with autoimmune disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
When autoimmune disease supervenes, there is an initial but poorly understood event in which mechanisms to preserve tolerance fail to function and an autoimmune response is induced. Several theories have been proposed to explain this phenomenon, including the sharing of epitopes between an immunogen, often called a molecular mimic, and self-molecules. A number of experimental models of autoimmunity have used this model and generally immunized in the presence of adjuvant (1, 2). In all cases, once tolerance is lost, the process continues inexorably with maintenance of the autoimmune response in the face of continuing immunization with the tolerance-breaking molecule.

Recently, we have shown that organic molecules known as xenobiotic agents can be used to induce an autoimmune response that mimics the one found in primary biliary cirrhosis (PBC)4 (3). PBC is a female dominant autoimmune liver disease, characterized by the destruction of intrahepatic bile ducts and the production of high-titer anti-mitochondrial Abs (AMA). The major mitochondrial autoantigens recognized by AMA are the E2 components of the 2-oxo-dehydrogenase pathway, particularly pyruvate dehydrogenase complexes (PDC-E2) (4). One of the hallmarks of these autoantigens is that they are each lipoylated in a region localized to the immunodominant peptide recognized by both humoral and cellular autoimmune responses by PBC patients (5, 6, 7, 8). To break tolerance, we first identified an organic mimic of the lipoic acid lysine region (3). Rabbits immunized with this mimic, 6-bromohexanoate, developed autoantibodies that reacted with mitochondrial Ags with many of the features of the Ab response found in PBC (9). These data demonstrated that xenobiotics could break tolerance to a self-Ag.

In efforts to extend our findings and to understand how xenobiotic immunization could break tolerance, we immunized rabbits with 6-bromohexanoate BSA and then used a small molecule conjugated peptide microarray to define the molecular requirements for the binding of the 6-bromohexanoate-BSA-immunized rabbit sera against a panel of lipoic acid mimic xenobiotics. Following immunization, tolerance was broken, but with continued immunization, reactivity to the primary tolerance-breaking epitope was lost, whereas reactivity to the rPDC-E2 protein increased.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Source of Abs

The production and specificity of murine anti-PDC mAbs, clones 2H4, C355.1, and 4C8, have been previously described (10). Anti-influenza hemagglutinin (HA) Abs were obtained from Roche Applied Science (Indianapolis, IN). Sera from the 6-bromohexanoate-BSA- and BSA-immunized rabbits were obtained as previously described (3).

Rabbit immunization

Female New Zealand White rabbits at 16 wk of age were immunized s.c. with 100 µg/animal 6-bromohexanoate-BSA (n = 10) or 100 µg/animal BSA alone (n = 8) incorporated in CFA and then boosted s.c. every 2 wk with the same dose of Ag in IFA. Sera were collected 8 wk after initial immunization and every 4 wk thereafter for 22 mo for analysis of AMA reactivity using the high-throughput xenobiotic-peptide-agarose assay described below. Animal protocols were approved by the Institutional Review Board of the University of California at Davis.

Peptide synthesis

Four peptides (influenza HA peptide YPYDVPDYA; PDC peptide DKATIGFEVQEE; mutant PDC peptide AKATIGFEVQEE; and the BSA peptide FKGLVLIAFSQY) were synthesized on Rink Amide MBHA Resin (GL Biochem, Shanghai, China) (11). Briefly, first, N{alpha}-F-moc-(N{beta}-Boc-amino-oxyacetyl)-L-diaminopropionic acid (F-moc-Dpr(Boc-Aoa)) (Novabiochem, Läufelfingen, Switzerland) (12) was attached to the solid support followed with the hydrophilic spacer (N-F-moc-2,2'-(ethylenedioxy)bis(ethylamine) monosuccinamide) (13) and the appropriate amino acid sequence. Amino acid coupling was conducted by a 3-fold molar excess of 9-fluorenylmethoxycarbonyl (F-moc) protected amino acid, 1-hydroxy-1H-benzotriazole/N,N-diisopropylcarbodiimide activation in N,N-dimethylformamide (DMF) until Kaiser test (14) was negative. The F-moc protecting group was removed by 20% piperidine in DMF (30 min). After removal of the F-moc group from the last residue (Asp), the N-terminal amino group was acylated with acetic anhydride and N,N-diisopropylethylamine. A mixture of trifluoroacetic acid, triisopropylsilane, and ddH2O (95:2.5:2.5 v/v/v) was applied to cleave compounds from the resin and remove the side chain protecting groups. Peptides were then purified by preparative C-18 reversed phase (Vydac, Hesperia, CA) HPLC to yield >95% purity.

Modification of agarose and conjugation to peptide

One gram of agarose (type XI: low gelling temperature; Sigma-Aldrich, St. Louis, MO) was melted in 50 ml of ddH2O. The agarose solution was added dropwise into 200 ml of stirred dichloromethane to form agarose beads. The beads or blocks were collected by filtration, washed with acetonitrile, crushed into smaller pieces (<5 mm) and lyophilized. The pretreated dry agarose (0.66 g, calculated to be 8.8 mmol of OH) was then dissolved in 50 ml of DMF with heating. A solution of levulinic acid (0.45 ml, 4.4 mmol), N,N-diisopropylcarbodiimide (0.34 ml, 2.2 mmol), and 4-(dimethylamino)pyridine (53 mg, 0.22 mmol) in 30 ml of DMF was added to the agarose (in DMF) solution. The mixture was stirred at room temperature overnight (>8 h). The solution was poured into 500 ml of diethyl ether. The resulting precipitates were filtered and washed with ether. Ten milliliters of this modified agarose solution (5 mg/ml) was added to 10 ml of the appropriate peptide solution (20 µM) in a 0.05 M NaAc/AcOH buffer (pH 4.5) containing 50% DMSO. The mixture was stirred for 5 h at 65–70°C. Ketones on modified agarose react selectively with amino-oxy groups on peptides to form oximes at slightly acidic pH (15, 16). The conjugation solution was subjected to dialysis and subsequently lyophilized. Loading of each peptide was calculated by a quantitative ninhydrin test at 570 nm and was determined to be as follows: PDC peptide, 95.5 µmol/g; mutant PDC peptide, 83.5 µmol/g; and albumin peptide, 81.5 µmol/g.

Synthesis of mimeotopes and coupling with peptide-agarose conjugate

Twenty-three xenobiotic compounds were synthesized and used in this study (9). Compound nos. 1–19 were synthesized as previously described (9). Compound nos. 20–23 were synthesized as follows: The desired carboxylic acid derivative (2.5 mmol) was placed in a clean, dry 10-ml round-bottom flask under nitrogen, and then N-hydroxysuccinimide (NHS) (345 mg, 3.0 mmol) and 1,2-dimethoxyethane (8 ml) were added to this flask. The solution was stirred at room temperature until dissolved, cooled to 0°C, and then dicyclohexylcarbodiimide (773 mg, 3.75 mmol) was added. The mixture was stirred at 0°C for 15 min, and the reaction continued at room temperature overnight. The white precipitate (dicyclohexylurea) was removed by filtration through Celite. The crude solid was obtained by removal of the solvent and recrystallized from isopropanol to give pure crystals of the NHS ester. Compound purity was verified by proton nuclear magnetic resonance and by the presence of only one spot on TLC. 1H Nuclear magnetic resonance analysis indicated four additional protons ({delta} 2.8–3.0) in each of the products and an indication of NHS incorporation. The 23 compounds, in addition to lipoic acid with NHS ester, were coupled to the lysine residue on the PDC-E2 peptide-agarose conjugate as follows. Briefly, 0.4 mg of the PDC-E2 peptide-agarose conjugate and 10 µmol of each of the NHS esters were mixed in 40 µl of DMSO. Mixtures were incubated at room temperature for 2 h. To ensure complete coupling, a quantitative ninhydrin test at 570 nm was performed. A schematic representation of the conjugation chemistry is shown in Fig. 1.



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 1. Synthesis of peptide agarose conjugate on microarray. Peptide synthesis was performed on resin. Dpr(Boc-Aoa) and a hydrophilic spacer were incorporated between resin and peptide. The hydroxyl group on agarose reacts with levulinic acid to form an ester; ketones on modified agarose bind to the amino-oxy groups of Aoa and form oximes. After this conjugation to agarose, xenobiotics (R) were added to the lysine of the peptide.

 
Preparation of microarray and analysis of immunoreactivity

Xenobiotic compounds-peptide-agarose mixture was diluted (0.1%) in 0.1 M Na2CO3/NaHCO3 buffer (pH 9.0) and transferred to 96-well plates. Thereafter, mixtures were spotted onto glass slides (Mercedes Medical, Sarasota, FL) using the Affymetrix 417 Microarrayer (Affymetrix, Santa Clara, CA). Each sample was spotted in triplicate, with a spot size of 150 µm in diameter. Spotted microarrays were stored at 4°C until use. Before use, microarrays were blocked with 3% nonfat dry milk in PBS buffer for 1 h at room temperature, and individual slides were thereafter incubated with diluted Ab samples (rabbit sera, 1/250; murine anti-PDC mAb, 1/1) in 1 ml of blocking buffer (3% nonfat dry milk in PBS with 0.05% Tween 20) (PBST) for 1 h at room temperature. After thorough washes with PBST, 1 ml of the Cy3-conjugated secondary Ab (1 µg/ml) (Zymed Laboratories, San Francisco, CA) in blocking buffer was added to each slide and incubated at room temperature for 30 min. Subsequently, slides were washed in PBST for 10 min and in water for 15 s. Arrays were then dried and scanned using the Affymetrix 428 Array Scanner. To validate peptide microarray sensitivity, four different concentrations (0.1, 0.03, 0.01, and 0.004%) of the control HA peptide were spotted. Serially diluted anti-HA mAbs (1000, 167, 28, and 5 ng/ml) were assayed individually. Data analysis was performed using the ImageQuant software (Molecular Dynamics, Sunnyvale, CA) (17). Mixtures of xenobiotics and agarose were also spotted and analyzed as controls. To derive net reactivity against xenobiotics coupled with peptide backbone, the mean intensity of reactivity of the experimental rabbit sera against the mixture of xenobiotics and agarose was subtracted from the mean intensity obtained on the corresponding peptide coupled with xenobiotics or lipoic acid. Statistical analysis was performed using JMP software (SAS Institute, Cary, NC). Paired t test was performed to compare differences of the signal intensity between pre- and postimmunized sera.

Comparison between ELISA and microarray assay

Rabbit sera (n = 5) at 1 mo postimmunization were serially diluted (1/250, 1/750, 1/2,250, 1/6,750, and 1/20,250), and IgG reactivity to small molecule-peptide-agarose conjugates was determined by both the ELISA and microarray assay. Briefly, ELISA plates were coated with 50 µl of each individual xenobiotic compounds-peptide-agarose mixture in DMSO (1 mg/ml) for 2 h at room temperature, and then Ags were removed, and plates were dried overnight at room temperature. Dried ELISA plates were thereafter blocked with 3% nonfat dry milk in PBS and incubated with serially diluted rabbit sera for 1 h at room temperature. After washing, the plates were incubated with HRP-conjugated mouse anti-rabbit IgG (Zymed Laboratories) Abs for 30 min at room temperature, washed, and incubated with ABTS containing hydrogen peroxide (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Likewise, IgG reactivity of rabbit sera at 0, 1, 6, 12, and 22 mo postimmunization against recombinant human PDC-E2 protein (3) and keyhole limpet hemocyanin (Sigma-Aldrich) was determined at 1/100 sera dilution by standard ELISA.

Inhibition assay

Rabbit sera (n = 5) at 1 mo postimmunization were absorbed in 3% milk in PBST at a final dilution of 1/250 with three different concentrations (50, 5, and 0.5 µg/ml) of xenobiotics (6-bromohexanoate or compound no. 9)-PDC-E2 peptide-agarose conjugate or lipoylated PDC-E2 peptide-agarose conjugate. After incubation at 4°C overnight, the mixture was centrifuged, and then the supernatant was saved. IgG reactivity of unabsorbed and absorbed sera against small molecule-peptide-agarose conjugates was determined by the microarray assay.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Detection of Ab against lipoic acid and xenobiotics

The unique xenobiotic-peptide-agarose microarray platform was used to screen for the fine specificity of the binding of anti-PDC-E2 Abs with an emphasis on identifying potential structures that mimic the molecular image formed by the association of lipoic acid with the immunodominant PDC-E2 peptide. Various peptide backbones, including PDC peptide, mutant PDC peptide, or albumin peptide, were each coupled with each of the 23 xenobiotics and lipoic acid. Reactivity of three different murine anti-PDC mAbs was also studied. The mAb 2H4 bound strongly to 6-bromohexanoate, compound nos. 8 and 10, and lipoic acid on the PDC peptide (Fig. 2A). Weak reactivity to compound nos. 3, 15, 16, 18, 21, and 22, and the nonlipoylated native PDC peptide was also detected. 2H4 did not react to xenobiotics conjugated to other peptides. Interestingly, clone 4C8 or C355.1, which are other murine mAbs against PDC-E2, did not react to any of these xenobiotics (data not shown). As noted, the normal murine IgG did not react to any of the xenobiotic conjugates (Fig. 2B), including lipoylated peptide or the peptide alone, demonstrating the specificity of the binding of the 2H4 Ab.



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 2. Detection of Ab against lipoic acid and xenobiotics. Twenty-three xenobiotics and lipoic acid coupled to either the 12-mer, PDC peptide, mutant PDC peptide, and/or control albumin peptide were spotted. Reactivity was determined using mAb against PDCE2 (2H4) (A) and control murine IgG (B).

 
Analysis of the 6-bromohexanoate-BSA- or BSA-immunized rabbit IgG reactivity against lipoic acid and xenobiotics

Sera IgG from rabbits immunized with either 6-bromohexanoate-BSA or BSA alone were analyzed for their reactivity against the same 23 xenobiotic compounds and lipoylated peptide by microarray (Fig. 3). It should be noted that no significant histological changes in liver or abnormal liver function was found in these rabbits. All 6-bromohexanoate-BSA immunized rabbits (n = 10) were positive for AMA. As expected, sera from the 6-bromohexanoate-BSA-immunized rabbits showed high IgG reactivity to 6-bromohexanoate on the PDC peptide (4.61 ± 0.73 x 106 pixel; p < 0.001) at 1 mo postimmunization. High reactivity to compound no. 8 (1.11 ± 0.34 x 106 pixel; p < 0.001) and to no. 10 (2.45 ± 0.44 x 106 pixel; p < 0.001) on the PDC peptide was also observed. 6-Bromohexanoate-BSA-immunized rabbits also showed weak but significant reactivity against lipoylated PDC peptide (0.7 ± 0.18 x 106 pixel; p < 0.002). Sera IgG reactivity against other compounds was weak and negligible (data not shown). Sera from the BSA-immunized rabbits (negative for AMA) did not react to any of the xenobiotics (data not shown). It was of interest to note the differences in the pattern of specificities being recognized as a function of kinetics. Thus, mean reactivity ± SE observed by analysis of the series of sequential sera from each of the rabbits against lipoylated peptides (Fig. 4A), nonlipoylated peptides (B), and compound no. 9-conjugated peptides (C) at 0, 1, 6, 12, and 22 mo postimmunization are presented. Sera from the rabbits immunized with 6-bromohexanoate-BSA showed increasing reactivity against the 6-bromohexanoate-conjugated PDC-E2 peptide following booster immunizations with the 6-bromohexanoate-BSA (at 6 mo, 17.4 ± 3.6 x 106 pixel; at 12 mo, 12.4 ± 1.7 x 106 pixel; at 22 mo, 20.9 ± 2.6 x 106 pixel). However, aliquots of the same sera appeared to show relatively lower but decreasing activity against the lipoylated PDC peptide following booster immunization with 6-bromohexanoate-BSA (Fig. 4A). Aliquots of the same rabbit sera did show some reactivity against the nonlipoylated PDC-E2 peptide (Fig. 4B) but negligible reactivity against lipoylated albumin peptide (A), nonlipoylated mutant PDC peptide and nonlipoylated albumin peptide (B), and compound no. 9-conjugated PDC, mutant PDC, and albumin peptides (C).



View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 3. Ab detection by microarray. PDC-E2 (A), mutant PDC-E2 (C), and albumin peptide (D) were coupled with 23 xenobiotic mimeotopes and lipoic acid, and then Ags were spotted on the on the glass slides with a spot size of 150 µm in diameter. Mixture of xenobiotic mimeotopes and agarose was also spotted (B). Individual arrays were incubated with rabbit sera (1/250) followed by secondary Ab (goat anti-rabbit IgG) conjugated to Cy3.

 


View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 4. Kinetics of IgG reactivity of sera from 6-bromohexanoate BSA-immunized rabbit against lipoic acid and xenobiotics. Sera from rabbits immunized with 6-bromohexanoate BSA (n = 10) at 0, 1, 6, 12, and 22 mo postimmunization were assayed for IgG reactivity using the small molecule-conjugated peptide microarray. The data shown are mean IgG reactivity (pixel number) ± SE of 6-bromohexanoate-BSA-immunized rabbit against lipoylated peptides (A), nonlipoylated native peptides (B), and peptides coupled with control compound no. 9 (C).

 
Inhibition assay

To characterize the antigenic specificity of 6-bromohexanoate-BSA-immunized rabbits, aliquots of these sera (n = 5) were absorbed with either xenobiotics (6-bromohexanoate or compound no. 9)-PDC-E2 peptide-agarose conjugate or lipoylated PDC-E2 peptide-agarose conjugate. Each aliquot was then analyzed for reactivity against 6-bromohexanoate conjugate PDC-E2 peptide and lipoylate PDC-E2 peptide on microarray. When sera were absorbed with 6-bromohexanoate-PDC-E2-agarose conjugates, the reactivity against 6-bromohexanoate conjugate PDC-E2 peptide and lipoylate PDC-E2 peptide was markedly decreased in a dose-dependent manner (Fig. 5). In contrast, when sera were absorbed with lipoylated PDC-E2-agarose conjugates, the reactivity against 6-bromohexanoate conjugate PDC-E2 peptide remained unchanged. Absorption with compound no. 9-PDC-E2-agarose conjugates did not affect the reactivity.



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 5. Inhibition assay. To characterize the antigenic specificity of 6-bromohexanoate-BSA-immunized rabbits, rabbit sera (n = 5) at 1 mo postimmunization were absorbed at a final dilution of 1/250 with three different concentrations (50, 5, and 0.5 µg/ml) of xenobiotics (6-bromohexanoate or compound no. 9)-PDC-E2 peptide-agarose conjugate or lipoylated PDC-E2 peptide-agarose conjugate. Each aliquot was then analyzed for IgG reactivity against 6-bromohexanoate conjugate PDC-E2 peptide (A) and lipoylate PDC-E2 peptide (B). *, p < 0.01; **, p < 0.001.

 
Rabbit sera IgG reactivity against recombinant human PDC-E2 protein

Sera IgG reactivity against human recombinant PDC-E2 protein was analyzed by ELISA (Fig. 6). The maximum reactivity was observed at 22 mo postimmunization (0.59 ± 0.11). Unlike reactivity to the lipoylated PDC-E2 peptide, sera IgG reactivity against rPDC-E2 protein was maintained over time.



View larger version (11K):
[in this window]
[in a new window]
  		FIGURE 6. Rabbit sera IgG reactivity against recombinant human PDC-E2 protein. IgG reactivity of rabbit sera at 0, 1, 6, 12, and 22 mo postimmunization against PDC-E2 protein and keyhole limpet hemocyanin (KLH) was determined at 1/100 sera dilution by standard ELISA.

 
Validation of peptide microarray assay

To validate the microarray assay, data obtained using the microarray and ELISA were compared. Rabbit sera (n = 5) at 1 mo postimmunization were serially diluted, and their IgG reactivity against 6-bromohexanoate, compound no. 9, lipoic acid on PDC-E2 peptide, and nonlipoylated native PDC-E2 peptide was determined by both ELISA and microarray assay (Fig. 7). Both results demonstrated a dilution-dependent response with sera at a dilution of 1/6750 or lower. To validate the sensitivity of the microarray, four different concentrations (0.1, 0.03, 0.01, and 0.004%) of HA peptide (YPYDVPDYA) were spotted. Individual arrays were incubated with murine monoclonal anti-HA Ab or normal murine IgG followed by secondary Ab (goat anti-murine IgG) conjugated to Cy3. Reactivity to HA peptide was dependent on anti-HA mAb concentration, and a dose-dependent response against the Ag was observed with Ab concentration of 5 ng/ml or higher in each case except for the lowest concentration of HA which required >50 ng/ml mAb (data not shown). Specificity of anti-HA binding was verified by the absence of the binding by normal murine Ig performed in parallel (data not shown).



View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 7. Comparison between ELISA and microarray assay. Rabbit sera (n = 5) at 1 mo postimmunization were serially diluted (1/250, 1/750, 1/2,250, 1/6,750, and 1/20,250), and IgG reactivity to small molecule-peptide-agarose conjugates was determined by both ELISA (B) and the microarray assay (A).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
AMA are the predominant autoantibody in patients with PBC. Several studies using oligopeptide or recombinant proteins have shown that the major AMA epitope is localized to a linear sequence within the inner lipoyl domain of PDC-E2 (5, 6). Although AMA recognize both lipoylated and nonlipoylated PDC-E2 (18), they are also reactive against the lipoyl domain of BCOADC-E2, OGDC-E2, and dihydrolipoamide dehydrogenase binding protein (E3BP) (19). In addition, the T cell epitope in patients with PBC also is localized to the same peptide location to the same inner lipoyl domain of PDC-E2 (7, 8). Furthermore, 82% of AMA-positive PBC patients are positive for anti-lipoic acid Ab (20). These findings suggest that both the peptides bearing the lipoyl domain and lipoic acid itself may play a significant but yet unknown role in the pathogenesis of PBC. Lipoic acid is a coenzyme essential to the activity of enzymes. Five lipoate-containing proteins are known in vertebrates: acyltransferase components of the pyruvate, 2-oxo-glutarate and branched chain 2-oxo-acid dehydrogenase complexes (E2p, E2k, and E2b, respectively), protein X of the PDC, and H-protein of the glycine cleavage system (21, 22, 23, 24). In each protein complex, lipoic acid is covalently bound to at least one subunit of these complexes via an amide linkage to the {epsilon}-amino group of a specific lysine residue. The lipoyl-lysine arm functions as a carrier of reaction intermediates and interacts with the active site of the components of the complexes (25, 26).

The underlying mechanism in breaking of immune self-tolerance, like all autoimmune diseases, is one of the most important issues in the etiopathology of PBC. One attractive theory is the concept of molecular mimicry. In fact, a number of previous reports supports the hypothesis that molecular mimicry plays a role in the pathogenesis of a number of autoimmune disease, including rheumatic fever, Reiter’s syndrome, myasthenia gravis, rheumatoid arthritis, insulin-dependent diabetes, ankylosing spondylitis, Guillain-Barré syndrome, and multiple sclerosis (27, 28, 29). In most cases, oligopeptides serve as a mimic epitope, but the possibility of small molecules/haptens as xenobiotics has not to date been entertained or studied.

Xenobiotics are foreign substances of synthetic, natural, or biological origin. They comprise industrial chemicals, drugs, and cytokines to which we may be exposed because of their presence in the environment or their therapeutic use. Many xenobiotics can cause organ- or tissue-specific autoimmune diseases. For example, {alpha}-methyldopa and high-dose penicillin are responsible for drug-induced autoimmune hemolytic anemia (30). Iodine supplementation induces reversible thyroid dysfunction and autoimmunity (31). In addition, many cases of membranous glomerulonephritis due to long-term contact with mercury compounds have been reported (32). Of particular interest to this study, sera of patients with halothane hepatitis contain a wide variety of autoantibodies against liver proteins, including PDC-E2 (33).

We hypothesized that the lipoyl domain of the immunodominant PDC-E2 peptide could be modified by a mimic xenobiotic, and such a modified mimic may alter and convert the self-protein into an immunogen and initiate AMA production. Our recent report (3) and data presented in this study provide support to our hypothesis. For example, cross-reactivity between lipoic acid and several xenobiotic compounds (6-bromohexanoate and compound nos. 8 and 10) was demonstrated by the pattern of reactivity seen with the mAb 2H4. This mAb was generated by immunizing mice with a recombinant trihybrid protein consisting of PDC-E2, BCOADC-E2, and OGDC-E2 (10). This result suggests that immunization with PDC-E2 may induce the production of Abs against lipoic acid and lipoic acid-like component besides PDC-E2 itself. Furthermore, rabbit sera immunized with 6-bromohexanoate-BSA also reacted to lipoic acid conjugated to the PDC peptide and a number of xenobiotics (6-bromohexanoate and compound nos. 8 and 10). Data from the inhibition study also support that 6-bromohexanoate-BSA-immunized rabbit sera were cross-reactive to lipoylated PDC-E2 peptide. Rabbits immunized with 6-bromohexanoate-BSA did not show concomitant histological or biochemical evidence of liver/bile duct damage during this study period. These results suggest that exposure and sensitization to xenobiotics results in autoantibodies to PDC-E2, but that additional factors are required to generate immunopathology. More interestingly, sera from rabbits immunized with 6-bromohexanoate-BSA showed the highest IgG reactivity against lipoylated PDC-E2 peptide at 1 mo postimmunization, and the reactivity decreased gradually by continuing immunization (Fig. 4A). In contrast, increasing reactivity against 6-bromohexanoate-conjugated PDC-E2 peptide and recombinant human PDC-E2 protein (Fig. 6) was observed. Thus, these data suggest that the 6-bromohexanoate-BSA serves as a mimotope for lipoylated peptide initially, and booster doses of 6-bromohexanoate-BSA lead to decreases in the mimic. However, IgG reactivity against other PDC-E2 epitopes was still maintained. This improvement in Ab specificity against 6-bromohexanoate may be explained by affinity maturation. The affinity maturation of Ab responses results from the accumulation of point mutations in the V region of Ig genes followed by Ag-driven selection of the B lymphocytes expressing high-affinity Abs (34). This process takes place in germinal centers where Ag-specific B cells differentiate into memory and/or plasma cells after switching of the H chain isotypes (35). The affinity maturation may also play a key role in several autoimmune responses (36, 37) including the production of AMA (38). In contrast, sustained IgG reactivity against rPDC-E2 protein may be explained by epitope spreading (39, 40). Although this is indeed the converse of what is described as "original antigenic sin" (41, 42), we note that regression of reactivity to a tolerance-breaking epitope during the emergence of epitope spreading has been described in experimental autoimmune encephalomyelitis (43, 44).

The development of microchip technology has greatly facilitated the understanding of the molecular basis of gene function and mRNA expression patterns using DNA microarrays. Recently, much effort has been directed in gaining information about the function of identified genes by protein chip technology (45, 46, 47, 48, 49, 50). The advantage of protein chips including a proteome microarray is that individual proteins can be directly screened for a variety of functional activities in high-throughput assays (51). Immunoassays on microarrays is an attractive approach in proteomics. Although considerable advances have been made on this subject (52, 53, 54), the use of protein microarrays in research and diagnostic settings is still limited. Several issues are important in developing peptide microrarrays. In most cases, the protein is immobilized on the slide via nonspecific covalent binding (50, 55). Site-specific binding is required on a peptide microarray to immobilize peptide with the correct orientation of C or N terminus.

Based on the principle of chemoselective attachment, a high-throughput peptide and small molecule microarray platform was developed to determine the binding specificities of cell adhesion (56). In addition, direct immobilization of peptides on slides does not necessarily result in high signal intensity, because direct immobilization on slides not only fails to provide sufficient space to react with an Ab, but also uses low concentration of the peptide Ag. This problem was solved using a hydrophilic spacer (57) and an agarose scaffold modified with levulinic acid (58). Agarose is a polysaccharide consisting of 1,3-linked {beta}-D-galactopyranose and 1,4-linked 3,6-anhydro-{alpha}-L-galactopyranose. This basic agarobiose repeat unit forms long chains with an average molecular mass of 120,000 Da, representing ~400 agarobiose units (59). The agarose modified with levulinic acid carries a ketone group to react with the synthesized peptide containing an amino-oxy group in an aqueous condition, and this approach allows relatively higher loading of amino acid (~2 mmol/g). Reaction between the ketone and the amino-oxy group is highly specific (15, 16); therefore, a peptide Ag may remain intact.

We demonstrated in this study the feasibility of this new technology in developing a peptide-small molecule microarray to assay for the reactivity of not only peptide-specific autoantibodies but also reactivity against Abs against a panel of haptens such as the xenobiotic compounds conjugated to peptide backbones. The peptide microarray technology we showed in this study may also be applied for fine epitope mapping. For example, the 4C8 is a mAb that recognizes the inner lipoyl domain (128–229) of PDC-E2, but it did not react to any xenobiotics. Our previous study (60) showed that the reactivity of the 2H4 clone requires both lipoic acid and the PDC-E2 inner lipoyl domain (128–229), whereas lipoic acid was not necessary for clone 4C8 or C355.1 binding. Although those three mAbs showed disease-specific apical staining pattern on bile duct (10, 60), the clones 4C8, C355.1, and 2H4 recognize distinctly different epitopes within the PDC-E2 inner lipoyl domain. Future studies using this peptide-small molecule microarray platform will be useful in defining the molecular requirement of chemical mimics involved in the breaking of tolerance in PBC.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants DK39588 and DK037003. Back

2 K.A. and P.S.C.L. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. M. Eric Gershwin, Division of Rheumatology, Allergy, and Clinical Immunology, University of California at Davis School of Medicine, TB 192, Davis, CA 95616. E-mail address: megershwin{at}ucdavis.edu Back

4 Abbreviations used in this paper: PBC, primary biliary cirrhosis; AMA, anti-mitochondrial Ab; PDC, pyruvate dehydrogenase complex; HA, hemagglutinin; F-moc, 9-fluorenylmethoxycarbonyl; DMF, N,N-dimethylformamide; NHS, N-hydroxysuccinimide. Back

Received for publication November 4, 2003. Accepted for publication March 1, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Swanborg, R. H.. 2001. Experimental autoimmune encephalomyelitis in the rat: lessons in T-cell immunology and autoreactivity. Immunol. Rev. 184:129.[Medline]
  2. Luross, J. A., N. A. Williams. 2001. The genetic and immunopathological processes underlying collagen-induced arthritis. Immunology 103:407.[Medline]
  3. Leung, P. S., C. Quan, O. Park, J. Van De Water, M. J. Kurth, M. H. Nantz, A. A. Ansari, R. L. Coppel, K. S. Lam, M. E. Gershwin. 2003. Immunization with a xenobiotic 6-bromohexanoate bovine serum albumin conjugate induces antimitochondrial antibodies. J. Immunol. 170:5326.[Abstract/Free Full Text]
  4. Nishio, A., E. B. Keeffe, M. E. Gershwin. 2001. Primary biliary cirrhosis: lessons learned from an organ-specific disease. Clin. Exp. Med. 1:165.[Medline]
  5. Surh, C. D., R. Coppel, M. E. Gershwin. 1990. Structural requirement for autoreactivity on human pyruvate dehydrogenase-E2, the major autoantigen of primary biliary cirrhosis: implication for a conformational autoepitope. J. Immunol. 144:3367.[Abstract]
  6. Surh, C. D., A. Ahmed-Ansari, M. E. Gershwin. 1990. Comparative epitope mapping of murine monoclonal and human autoantibodies to human PDH-E2, the major mitochondrial autoantigen of primary biliary cirrhosis. J. Immunol. 144:2647.[Abstract]
  7. Shimoda, S., J. Van de Water, A. Ansari, M. Nakamura, H. Ishibashi, R. L. Coppel, J. Lake, E. B. Keeffe, T. E. Roche, M. E. Gershwin. 1998. Identification and precursor frequency analysis of a common T cell epitope motif in mitochondrial autoantigens in primary biliary cirrhosis. J. Clin. Invest. 102:1831.[Medline]
  8. Kita, H., Z. X. Lian, J. Van de Water, X. S. He, S. Matsumura, M. Kaplan, V. Luketic, R. L. Coppel, A. A. Ansari, M. E. Gershwin. 2002. Identification of HLA-A2-restricted CD8+ cytotoxic T cell responses in primary biliary cirrhosis: T cell activation is augmented by immune complexes cross-presented by dendritic cells. J. Exp. Med. 195:113.[Abstract/Free Full Text]
  9. Long, S. A., C. Quan, J. Van de Water, M. H. Nantz, M. J. Kurth, D. Barsky, M. E. Colvin, K. S. Lam, R. L. Coppel, A. Ansari, M. E. Gershwin. 2001. Immunoreactivity of organic mimeotopes of the E2 component of pyruvate dehydrogenase: connecting xenobiotics with primary biliary cirrhosis. J. Immunol. 167:2956.[Abstract/Free Full Text]
  10. Migliaccio, C., A. Nishio, J. Van de Water, A. A. Ansari, P. S. Leung, Y. Nakanuma, R. L. Coppel, M. E. Gershwin. 1998. Monoclonal antibodies to mitochondrial E2 components define autoepitopes in primary biliary cirrhosis. J. Immunol. 161:5157.[Abstract/Free Full Text]
  11. Fields, G. B., R. L. Noble. 1990. Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids. Int. J. Pept. Protein Res. 35:161.[Medline]
  12. Wahl, F., M. Mutter. 1996. Analogs of oxytocin with an oxime bridge using chemoselectivity addressable building blocks. Tetrahedron Lett. 37:6861.
  13. Song, A., J. Zhang, C. B. Lebrilla, K. S. Lam. 2003. A novel and rapid encoding method based on mass spectrometry for "one-bead-one-compound" small molecule combinatorial libraries. J. Am. Chem. Soc. 125:6180.[Medline]
  14. Kaiser, E., R. L. Colescott, C. D. Bossinger, P. I. Cook. 1970. Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal. Biochem. 34:595.[Medline]
  15. Lemieux, G. A., C. R. Bertozzi. 1998. Chemoselective ligation reactions with proteins, oligosaccharides and cells. Trends Biotechnol. 16:506.[Medline]
  16. Shao, J., J. P. Tam. 1995. Unprotected peptides as building blocks for the synthesis of peptide dendrimers with oxime, hydrazone, and thiazolidine linkages. J. Am. Chem. Soc. 117:3893.
  17. Christ, S. A., R. N. Silbiger, M. Garg, S. E. Franson, G. P. Toth. 2000. Quality of assurance considerations for use of the Fluorlmager SI and fragmeNT analysis software. Electrophoresis 21:874.[Medline]
  18. Quinn, J., A. G. Diamond, J. M. Palmer, M. F. Bassendine, O. F. James, S. J. Yeaman. 1993. Lipoylated and unlipoylated domains of human PDC-E2 as autoantigens in primary biliary cirrhosis: significance of lipoate attachment. Hepatology 18:1384.[Medline]
  19. Leung, P. S., R. L. Coppel, A. Ansari, S. Munoz, M. E. Gershwin. 1997. Antimitochondrial antibodies in primary biliary cirrhosis. Semin. Liver Dis. 17:61.[Medline]
  20. Bruggraber, S. F. A., P. S. C. Leung, K. Amano, C. Quan, M. J. Kurth, M. H. Nantz, G. D. Benson, J. Van de Water, V. Luketic, T. E. Roche, et al 2003. Autoreactivity to lipoate and a conjugated form of lipoate in primary biliary cirrhosis. Gastroenterology 125:1705.[Medline]
  21. Fujiwara, K., K. Okamura-Ikeda, Y. Motokawa. 1986. Chicken liver H-protein, a component of the glycine cleavage system: amino acid sequence and identification of the N{epsilon}-lipoyllysine residue. J. Biol. Chem. 261:8836.[Abstract/Free Full Text]
  22. Fujiwara, K., K. Okamura-Ikeda, Y. Motokawa. 1992. Expression of mature bovine H-protein of the glycine cleavage system in Escherichia coli and in vitro lipoylation of the apoform. J. Biol. Chem. 267:20011.[Abstract/Free Full Text]
  23. Perham, R. N.. 1991. Domains, motifs, and linkers in 2-oxo acid dehydrogenase multienzyme complexes: a paradigm in the design of a multifunctional protein. Biochemistry 30:8501.[Medline]
  24. Reed, L. J., M. L. Hackert. 1990. Structure-function relationships in dihydrolipoamide acyltransferases. J. Biol. Chem. 265:8971.[Free Full Text]
  25. Yeaman, S. J.. 1989. The 2-oxo acid dehydrogenase complexes: recent advances. Biochem. J. 257:625.[Medline]
  26. Douce, R., J. Bourguignon, D. Macherel, M. Neuburger. 1994. The glycine decarboxylase system in higher plant mitochondria: structure, function and biogenesis. Biochem. Soc. Trans. 22:184.[Medline]
  27. Wucherpfennig, K. W., J. L. Strominger. 1995. Molecular mimicry in T cell-mediated autoimmunity: viral peptides activate human T cell clones specific for myelin basic protein. Cell 80:695.[Medline]
  28. Oomes, P. G., B. C. Jacobs, M. P. Hazenberg, J. R. Banffer, F. G. van der Meche. 1995. Anti-GM1 IgG antibodies and Campylobacter bacteria in Guillain-Barre syndrome: evidence of molecular mimicry. Ann. Neurol. 38:170.[Medline]
  29. Atkinson, M. A., M. A. Bowman, L. Campbell, B. L. Darrow, D. L. Kaufman, N. K. Maclaren. 1994. Cellular immunity to a determinant common to glutamate decarboxylase and coxsackie virus in insulin-dependent diabetes. J. Clin. Invest. 94:2125.
  30. Gehrs, B. C., R. C. Friedberg. 2002. Autoimmune hemolytic anemia. Am. J. Hematol. 69:258.[Medline]
  31. Kahaly, G. J., H. P. Dienes, J. Beyer, G. Hommel. 1998. Iodide induces thyroid autoimmunity in patients with endemic goitre: a randomised, double-blind, placebo-controlled trial. Eur. J. Endocrinol. 139:290.[Abstract]
  32. Aymaz, S., O. Gross, B. Krakamp, M. Ortmann, H. P. Dienes, M. Weber. 2001. Membranous nephropathy from exposure to mercury in the fluorescent-tube-recycling industry. Nephrol. Dial. Transplant. 16:2253.[Free Full Text]
  33. Gut, J., U. Christen, N. Frey, V. Koch, D. Stoffler. 1995. Molecular mimicry in halothane hepatitis: biochemical and structural characterization of lipoylated autoantigens. Toxicology 97:199.[Medline]
  34. Manser, T., K. M. Tumas-Brundage, L. P. Casson, A. M. Giusti, S. Hande, E. Notidis, K. A. Vora. 1998. The roles of antibody variable region hypermutation and selection in the development of the memory B-cell compartment. Immunol. Rev. 162:183.[Medline]
  35. Berek, C., A. Berger, M. Apel. 1991. Maturation of the immune response in germinal centers. Cell 67:1121.[Medline]
  36. Dammers, P. M., J. C. Bun, B. Bellon, F. G. Kroese, J. Aten, N. A. Bos. 2001. Immunoglobulin VH-gene usage of autoantibodies in mercuric chloride-induced membranous glomerulopathy in the rat. Immunology 103:199.[Medline]
  37. Owens, G. P., M. P. Burgoon, J. Anthony, B. K. Kleinschmidt-DeMasters, D. H. Gilden. 2001. The immunoglobulin G heavy chain repertoire in multiple sclerosis plaques is distinct from the heavy chain repertoire in peripheral blood lymphocytes. Clin. Immunol. 98:258.[Medline]
  38. Potter, K. N., R. K. Thomson, A. Hamblin, S. D. Richards, J. G. Lindsay, F. K. Stevenson. 2001. Immunogenetic analysis reveals that epitope shifting occurs during B-cell affinity maturation in primary biliary cirrhosis. J. Mol. Biol. 306:37.[Medline]
  39. Croxford, J. L., J. K. Olson, S. D. Miller. 2002. Epitope spreading and molecular mimicry as triggers of autoimmunity in the Theiler’s virus-induced demyelinating disease model of multiple sclerosis. Autoimmun. Rev. 1:251.[Medline]
  40. James, J. A., J. B. Harley. 1998. B-cell epitope spreading in autoimmunity. Immunol. Rev. 164:185.[Medline]
  41. Klenerman, P., R. M. Zinkernagel. 1998. Original antigenic sin impairs cytotoxic T lymphocyte responses to viruses bearing variant epitopes. Nature 394:482.[Medline]
  42. de St. Groth, F., R. G. Webster. 1966. Disquisitions of original antigenic sin. I. Evidence in man. J. Exp. Med. 124:331.[Abstract]
  43. Steinman, L.. 1999. Absence of "original antigenic sin" in autoimmunity provides an unforeseen platform for immune therapy. J. Exp. Med. 189:1021.[Free Full Text]
  44. Tuohy, V. K., M. Yu, L. Yin, J. A. Kawczak, R. P. Kinkel. 1999. Spontaneous regression of primary autoreactivity during chronic progression of experimental autoimmune encephalomyelitis and multiple sclerosis. J. Exp. Med. 189:1033.[Abstract/Free Full Text]
  45. Haab, B. B., M. J. Dunham, P. O. Brown. 2001. Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions. Genome Biol. 2:RESEARCH0004.[Medline]
  46. Joos, T. O., M. Schrenk, P. Hopfl, K. Kroger, U. Chowdhury, D. Stoll, D. Schorner, M. Durr, K. Herick, S. Rupp, et al 2000. A microarray enzyme-linked immunosorbent assay for autoimmune diagnostics. Electrophoresis 21:2641.[Medline]
  47. Robinson, W. H., C. DiGennaro, W. Hueber, B. B. Haab, M. Kamachi, E. J. Dean, S. Fournel, D. Fong, M. C. Genovese, H. E. de Vegvar, et al 2002. Autoantigen microarrays for multiplex characterization of autoantibody responses. Nat. Med. 8:295.[Medline]
  48. Robinson, W. H., P. Fontoura, B. J. Lee, H. E. de Vegvar, J. Tom, R. Pedotti, C. D. DiGennaro, D. J. Mitchell, D. Fong, P. P. Ho, et al 2003. Protein microarrays guide tolerizing DNA vaccine treatment of autoimmune encephalomyelitis. Nat. Biotechnol. 21:1033.[Medline]
  49. Zhu, H., M. Bilgin, R. Bangham, D. Hall, A. Casamayor, P. Bertone, N. Lan, R. Jansen, S. Bidlingmaier, T. Houfek, et al 2001. Global analysis of protein activities using proteome chips. Science 293:2101.[Abstract/Free Full Text]
  50. Zhu, H., J. F. Klemic, S. Chang, P. Bertone, A. Casamayor, K. G. Klemic, D. Smith, M. Gerstein, M. A. Reed, M. Snyder. 2000. Analysis of yeast protein kinases using protein chips. Nat. Genet. 26:283.[Medline]
  51. Wilson, D. S., S. Nock. 2003. Recent developments in protein microarray technology. Angew. Chem. Int. Ed. Engl. 42:494.[Medline]
  52. Miller, J. C., H. Zhou, J. Kwekel, R. Cavallo, J. Burke, E. B. Butler, B. S. Teh, B. B. Haab. 2003. Antibody microarray profiling of human prostate cancer sera: antibody screening and identification of potential biomarkers. Proteomics 3:56.[Medline]
  53. Pavlickova, P., A. Knappik, D. Kambhampati, F. Ortigao, H. Hug. 2003. Microarray of recombinant antibodies using a streptavidin sensor surface self-assembled onto a gold layer. BioTechniques 34:124.[Medline]
  54. Schweitzer, B., S. F. Kingsmore. 2002. Measuring proteins on microarrays. Curr. Opin. Biotechnol. 13:14.[Medline]
  55. MacBeath, G., S. L. Schreiber. 2000. Printing proteins as microarrays for high-throughput function determination. Science 289:1760.[Abstract/Free Full Text]
  56. Falsey, J. R., M. Renil, S. Park, S. Li, K. S. Lam. 2001. Peptide and small molecule microarray for high throughput cell adhesion and functional assays. Bioconjugate Chem. 12:346.[Medline]
  57. Zhao, Z. G., J. S. Im, K. S. Lam, D. F. Lake. 1999. Site-specific modification of a single-chain antibody using a novel glyoxylyl-based labeling reagent. Bioconjugate Chem. 10:424.[Medline]
  58. Xu, Q., S. Miyamoto, and K. S. Lam. A novel approach to chemical microarray using ketone-modified macromolecular scaffolds: application in micro cell-adhesion assay. Mol. Divers. In press.
  59. Rochas, C., M. Lahaye. 1989. Average molecular weight and molecular weight distribution of agarose and agarose-type polysaccharides. Carbohydrate Polymers 10:289.
  60. Migliaccio, C., J. Van de Water, A. A. Ansari, M. M. Kaplan, R. L. Coppel, K. S. Lam, R. K. Thompson, F. Stevenson, M. E. Gershwin. 2001. Heterogeneous response of antimitochondrial autoantibodies and bile duct apical staining monoclonal antibodies to pyruvate dehydrogenase complex E2: the molecule versus the mimic. Hepatology 33:792.[Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
A. Kawano, S. Shimoda, T. Kamihira, F. Ishikawa, H. Niiro, Y. Soejima, A. Taketomi, Y. Maehara, M. Nakamura, A. Komori, et al.
Peripheral Tolerance and the Qualitative Characteristics of Autoreactive T Cell Clones in Primary Biliary Cirrhosis
J. Immunol., September 1, 2007; 179(5): 3315 - 3324.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
P. S. C. Leung, O. Park, K. Tsuneyama, M. J. Kurth, K. S. Lam, A. A. Ansari, R. L. Coppel, and M. E. Gershwin
Induction of Primary Biliary Cirrhosis in Guinea Pigs following Chemical Xenobiotic Immunization
J. Immunol., August 15, 2007; 179(4): 2651 - 2657.
[Abstract] [Full Text] [PDF]

Home page
 NEJMHome page
M. M. Kaplan and M. E. Gershwin
Primary Biliary Cirrhosis
N. Engl. J. Med., September 22, 2005; 353(12): 1261 - 1273.
[Full Text] [PDF]

Home page
 J. Immunol.Home page
K. Amano, P. S. C. Leung, R. Rieger, C. Quan, X. Wang, J. Marik, Y. F. Suen, M. J. Kurth, M. H. Nantz, A. A. Ansari, et al.
Chemical Xenobiotics and Mitochondrial Autoantigens in Primary Biliary Cirrhosis: Identification of Antibodies against a Common Environmental, Cosmetic, and Food Additive, 2-Octynoic Acid
J. Immunol., May 1, 2005; 174(9): 5874 - 5883.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Amano, K.
Right arrow Articles by Gershwin, M. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Amano, K.
Right arrow Articles by Gershwin, M. E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS