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Antibodies to single-stranded and double-stranded DNA in antinuclear antibody-positive type 1-autoimmune hepatitis
Antibodies to Single-Stranded and Double-Stranded DNA in Antinuclear Antibody-Positive Type 1–Autoimmune Hepatitis ALBERT J. CZAJA,1 SYED AHMED MORSHED,2 SALINA PARVEEN,2
To determine the significance of antibodies to singlestranded (anti-ssDNA) and double-stranded DNA (antidsDNA) in antinuclear antibody (ANA)-positive type 1 autoimmune hepatitis, sera from 53 patients were tested by enzyme immunosorbent assay (ELISA) and indirect immunofluorescence using the Crithidia luciliae substrate. AntidsDNA were detected in 18 patients (34%) by ELISA and 12 patients (23%) by the Crithidia-based assay. Twenty patients with anti-dsDNA by either assay (38%) had higher serum levels of immunoglobulin G (3971 { 270 mg/dL vs. 3201 { 247 mg/dL, P Å .05) than seronegative patients. They also had human leukocyte antigen (HLA) DR4 more commonly than other patients (83% vs. 41%, P Å .006) and normal subjects (83% vs. 30%, P Å .00007). In contrast to patients seropositive by the Crithidia-based assay, those seropositive by ELISA failed corticosteroid therapy more commonly (24% vs. 3%, P Å .04). Anti-ssDNA were found in 45 patients (85%) and they did not distinguish patients with different clinical features or outcomes. We conclude that anti-dsDNA are common in ANA-positive type 1 autoimmune hepatitis. HLA DR4 is associated with their production, and seropositivity by ELISA characterizes patients who have a poorer immediate response to corticosteroid treatment. Anti-ssDNA are common but they do not have important clinical implications. (HEPATOLOGY 1997;26:567-572.) Antibodies to double-stranded DNA (anti-dsDNA) have a high specificity for the diagnosis of systemic lupus erythematosus (SLE) and they correlate closely with disease activity.1-7 Nonrheumatic diseases, including autoimmune hepatitis, may also have anti-dsDNA and the specificity of these antibodies for SLE has been challenged.8-10 In our earlier experience using an enzyme-linked immunosorbent assay (ELISA) and highly purified dsDNA, anti-
Abbreviations: Anti-dsDNA, antibodies to double-stranded DNA; SLE, systemic lupus erythematosus; ELISA, enzyme-linked immunosorbent assay; ssDNA, singlestranded DNA; ANA, antinuclear antibodies; anti-ssDNA, antibodies to single-stranded DNA; SMA, smooth muscle antibody; PBS, phosphate buffered saline; IU, international unit; HLA, human leukocyte antigens. From the 1Division of Gastroenterology and Internal Medicine, Mayo Clinic and Mayo Foundation, Rochester, MN; and the 2Third Department of Internal Medicine, Kagawa Medical University, Kagawa, Japan. Received November 26, 1996; accepted April 9, 1997. Presented in part at the meeting of the American Association for the Study of Liver Diseases, May 11, 1997, Washington, D.C. Address reprint requests to: Albert J. Czaja, M.D., Mayo Clinic, 200 First St. S.W., Rochester, MN 55905. Fax: (507) 284-0538. Copyright q 1997 by the American Association for the Study of Liver Diseases. 0270-9139/97/2603-0007$3.00/0
AND
MIKIO NISHIOKA2
dsDNA were shown in 64% of patients with autoimmune hepatitis, 46% with cryptogenic hepatitis, and 43% with chronic hepatitis B.8 Tsuchiya and colleagues, using an ELISA based on a similar substrate, found anti-dsDNA in 48% of patients with autoimmune hepatitis and 17% of patients with primary biliary cirrhosis.9 Testing of the same sera by an indirect immunofluorescence assay based on the kinetoplast of Crithidia luciliae, a pure source of dsDNA, disclosed similar results.9 A smaller study from Australia, using an immunofluorescence assay based on Crithidia luciliae, also showed a significant serologic overlap with SLE.10 Methodological differences can affect the detection of antidsDNA and the assessment of its clinical significance. Contamination of the dsDNA substrate with single-stranded DNA (ssDNA) may result in false positive results in some instances.5 Differences in antibody avidity and class may also influence detection. ELISAs detect antibodies of low avidity that may predominate in diseases other than SLE whereas the Crithidia assay may detect only high avidity antibodies of greater pathogenic importance.11 In this report, we determine the concordance of tests for anti-dsDNA by an ELISA and a Crithidia-based assay in patients with well-characterized antinuclear antibody (ANA)positive type 1 autoimmune hepatitis. We correlate seropositivity by each assay to clinical features and treatment outcome to determine the clinical pertinence of the findings and we evaluate the association between antibody expression and the known genetic risk factors for type 1 autoimmune hepatitis. We also determine the frequency of antibodies to ssDNA (anti-ssDNA) to evaluate further the clinical relevance of anti-dsDNA. PATIENTS AND METHODS Study Population. Fifty-three patients were selected for further study from the 303 patients entered in our chronic hepatitis treatment program. Patients were selected because: 1) each satisfied international criteria for autoimmune hepatitis;12 2) each had ANA in serum that justified their designation as ANA-positive type 1 autoimmune hepatitis;13 3) each had been screened and seronegative for serological markers of hepatitis B and C virus infection by second generation assays; 4) none satisfied the criteria of the American Rheumatism Association for the diagnosis of SLE;14 and 5) each had stored frozen serum samples available in their untreated state. The clinical, laboratory, and histological findings of the study population at presentation are shown in Table 1. Our study was part of a program project approved by the Institutional Review Board of the Mayo Clinic. Clinical Assessments. All patients underwent a complete physical examination by one investigator (A.J.C.) before therapy. Concomitant extrahepatic disorders of an immunological nature were sought in accordance with previously published guidelines.15 Twenty-four
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TABLE 1. Findings at Presentation of 53 Patients With Type 1 Autoimmune Hepatitis Clinical Features
Findings
Age (yr) Women:Men Duration of illness (mo) AST (NR:°31 U/L) Bilirubin (NR:°1.1 mg/dL) g-Globulin (NR:0.7-1.6 g/dL) Immunoglobulin G (NR:700-1,500 mg/dL) SMA¢1:40 ANA¢1:40 DR3//DR40 DR4//DR30 DR3 and DR4/ A1-B8-DR3/ Periportal hepatitis Bridging necrosis Multilobular necrosis Active cirrhosis
51 { 2 38:15 18 { 4 513 { 53 4 { 0.7 3.6 { 0.1 3490 { 191 47 (89) 53 (100) 13/45 (29) 19/45 (42) 7/45 (16) 15/45 (33) 25 (47) 7 (13) 5 (10) 16 (30)
NOTE. Numbers in parentheses are percentages. Abbreviation: AST, serum aspartate aminotransferase level; NR, normal.
of 53 patients (45%) had concurrent nonhepatic immunological diseases, including 8 patients with autoimmune thyroiditis, 3 patients with Graves’ disease, 4 patients with ulcerative colitis, 2 patients with synovitis, 5 patients with either rheumatoid arthritis, systemic sclerosis, Coomb’s positive hemolytic anemia, leukocytoclastic vasculitis, or asthma, and 2 patients with multiple diseases. Treatment Regimens. Fifty-one patients (96%) received prednisone alone (14 patients) or in combination with azathioprine (37 patients) in accordance with previously published protocols.16 Two patients had been randomized to investigational regimens and they did not receive conventional treatment. These patients were excluded from analyses of treatment outcome. Complete evaluations were performed every 6 months during and immediately after therapy and then at annual intervals if the clinical condition was stable. Treatment was continued until satisfaction of the previously reported treatment end points of remission, treatment failure, incomplete response, and drug toxicity.16 Clinical and laboratory manifestations of recrudescent inflammatory activity after cessation of therapy connoted relapse and it justified reinstitution of the original treatment regimen.16 Liver Tissue Examination. Liver tissue was obtained by needle biopsy in all patients at the time of presentation. Additional assessments were made as indicated to document histological remission of the disease or to clarify the clinical status. Specimens were interpreted under code and the diagnosis of cirrhosis required fibrosis and the presence of a complete regenerative nodule.17 Thirty-four of 51 patients who received corticosteroid therapy did not have cirrhosis at presentation and each also underwent subsequent liver biopsy examination. These patients were used to assess the frequency of progression to cirrhosis during or after therapy. Virological Assessments. All patients were tested for antibodies to hepatitis C virus by a second generation ELISA (Ortho Diagnostic Systems, Inc., Raritan, NJ) and in each instance, seronegativity was documented. Hepatitis B surface antigen was also sought in each patient by ELISA (Abbott Laboratories, North Chicago, IL) and its absence was confirmed in each instance. Determinations of ANA, SMA, and Antibodies to Liver/Kidney Microsome Type 1. All patients were screened at presentation for the presence
of smooth muscle antibody (SMA) and ANA by indirect immunofluorescence on murine kidney and stomach sections as described
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previously.18,19 A serum titer of 1:40 or higher was considered positive. ANAs were present in all patients in titers ranging from 1:40 to 1:10,240 (median ANA titer, 1:320). SMAs were present in 47 patients (89%) and in each instance, ANA were present concurrently. Serum titers of SMA ranged from 1:40 to 1:10,240 (median SMA titer, 1:320). Fifty-seven patients (89%) were also assessed for antibodies to liver/kidney microsome type 1 by indirect immunofluorescence on combined mouse kidney/stomach sections.20 A serum titer of 1:10 or higher was considered positive. None of the tested patients was seropositive and their designation as type 1 autoimmune hepatitis was thereby strengthened.13 Determinations of Anti-dsDNA and Anti-ssDNA. Stored frozen (0407 C) serum samples were each assessed for anti-dsDNA using ELISA, anti-dsDNA by indirect immunofluorescence using the Crithidia luciliae substrate, and anti-ssDNA using ELISA in one laboratory (Morshed and colleagues). Anti-dsDNA were determined by indirect immunofluorescence using the kinetoplast of Crithidia luciliae as the dsDNA source (AFT System II, Behring-Hoechst Japan, Ltd., Tokyo, Japan). Sera were diluted at 1:10 in phosphate buffered saline (PBS) and incubated with the Crithidia luciliae substrate in accordance with the instructions of the manufacturer. After 15 minutes, the slides were washed and then incubated with fluorescein-conjugated goat anti-human immunoglobulin G. The slides were then washed again, mounted, and viewed at 201 to 401 magnification on a Leitz immunofluorescence microscope (Ernst Leitz Canada Ltd., Midland, Ontario, Canada). The anti-dsDNA were identified by distinctive staining patterns of the kinetoplast. Positive and negative control slides were included in each determination. Seropositivity for anti-dsDNA connoted a serum titer of at least 1:10. Enzyme immunoassays were performed using the following commercial kits, MESACUP DNA-II, ssDNA, and dsDNA (Medical Biological Laboratories, Ltd., Nagoya, Japan). dsDNA and ssDNA were purified from a bacteriophage l vector grown in Escherichia coli according to a standard protocol.21 Ten mg/mL of each DNA was placed in PBS (100 mL/well) and used for coating the ELISA plate. After aspiration of the wells, the plate was blocked by PBS and 1% bovine serum albumin (100 mL/well) for 1 hour at room temperature. Serum samples were diluted 1:100 with diluting buffer (PBS and 1% bovine serum albumin). One hundred mL of diluted sample were added in duplicate to wells of the microtiter plates (Nunc, Maxisorp, Sweden) coated with either ssDNA or dsDNA. Plates were incubated at room temperature for 1 hour. Samples were then removed and washed three times with a washing buffer consisting of PBS and 0.1% Tween 20 in an automated washing machine (Micro Plate Washer, Model 1550, Bio-Rad Laboratories, Hercules, CA). Following incubation with 100 mL/well of peroxidase-conjugated monoclonal anti-human immunoglobulin G (g-chain, Fab fraction) for 1 hour, the plates were washed three times under the same conditions as before. The plates were incubated with a tetramethyl benzedene staining solution (100 mL/well) for 30 minutes at room temperature and the reaction was terminated by adding 100 mL/ well of a Stop solution (2N H2SO4 ). The plates were then read at a 450 nm wavelength with an ELISA microplate reader (MTP-120, Corona Electric Company, Ltd., Ibaraki, Japan). A standard curve was made for each ELISA plate, using standard (World Health Organization) reference samples of known concentrations, and results were reported in International Units (IU). The cut-off value was 40 IU/mL for anti-ssDNA or anti-dsDNA. This value was higher than the mean { 3 SD found in sera from 30 healthy subjects. All healthy control subjects were seronegative by three consecutive assays on three ELISA plates using duplicate wells. The serum levels of each control subject were always õ20 IU/mL (range, 5-20 IU/mL) in each assay. Previous studies in 49 patients with various nonhepatic autoimmune disorders, including systemic lupus erythematosus (10 patients), rheumatoid arthritis (19 patients), and Sjo¨gren’s syndrome (20 patients), indicated con-
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cordance of the ELISA and Crithidia results in 94%. Monoclonal antibodies to either ssDNA or dsDNA were used in a separate experiment to ensure the absence of kit contamination. HLA Determinations. All patients were caucasoid and each underwent typing for the class I (A and B locus) antigens. Forty-five patients (85%) were evaluated for the class II (DR locus) antigens. A standard microlymphocytotoxicity technique was used for class I and class II typing.15 Multiple locally derived and commercially available sera were used to define each specificity as described elsewhere.15 Restriction fragment length polymorphism was also used to assess the class II antigens in patients.22 Eighty healthy caucasoid adults participating in the volunteer blood donor program of the Mayo Clinic constituted the normal population. Each individual had indicated the absence of significant illness by a standard blood donation questionnaire. Typing for class I antigens was performed in 79 of 80 normal individuals (99%) by the standard microlymphocytotoxicity technique. Typing for class II antigens was accomplished in all normal subjects by both the standard microlymphocytotoxicity technique and restriction fragment length polymorphism. Statistical Analyses. Dichotomous variables were compared using the Fisher Exact test. The unpaired t test was used to evaluate the significance of differences in means for continuous variables. The Mann Whitney test was used to compare nonparametric variables in independent samples. Human leukocyte antigen (HLA) DR3, DR4, and A1-B8-DR3 are known risk factors for type 1 autoimmune hepatitis23 and only the frequencies of these antigens were analyzed in our study population. Because the variables for comparison had been formulated a priori and then assessed systematically in our study group, an unadjusted P-value of .05 was used to determine statistical significance. Data are presented as the mean { SE of the mean in tables and text. RESULTS Frequencies of Anti-dsDNA and Anti-ssDNA. Anti-dsDNA were detected in 18 patients (34%) by ELISA and 12 patients (23%) by indirect immunofluorescence using the Crithidia substrate. Both assays were positive for anti-dsDNA in 10 patients (19%). Twenty patients (38%) had anti-dsDNA by ELISA or the Crithidia-based assay. Only 2 individuals (4%) were seropositive by the Crithidia-based assay alone whereas 8 individuals (15%) had anti-dsDNA by the ELISA assay alone. Patients with anti-dsDNA by ELISA were seropositive for anti-dsDNA by the Crithidia-based assay more commonly than patients seronegative by ELISA (52% vs. 6%, P Å .0001) (Table 2). Similarly, patients with anti-dsDNA by the Crithidia-based assay were seropositive for anti-dsDNA by ELISA more frequently than patients seronegative by the Crithidiabased assay (83% vs. 20%, P Å .0001) (Table 2). Using the Crithidia-based assay as the gold standard, the sensitivity of the ELISA for anti-dsDNA was 83% and its specificity was 80%. Using the ELISA as the gold standard, the sensitivity of the Crithidia-based assay for anti-dsDNA was 55% and its specificity was 94%. Antibodies to ssDNA were found in 45 patients (85%). In 25 patients (47%), these antibodies were an isolated finding. In 10 patients (19%), they were accompanied by anti-dsDNA as determined by both assays. Antibodies to ssDNA were present only with anti-dsDNA by ELISA in 8 patients (15%) and they were present only with anti-dsDNA by the Crithidiabased assay in 2 patients (4%). Individuals seropositive for anti-ssDNA had anti-dsDNA by ELISA (40% vs. 0%, P Å .04) or by the Crithidia-based assay (27% vs. 0%, P Å .2) more frequently than seronegative counterparts (Table 2). Only the occurrence of anti-dsDNA by ELISA, however, was statis-
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tically greater in patients with anti-ssDNA than in patients without anti-ssDNA (Table 2). Seronegativity for antibodies to ssDNA and dsDNA by all assays was shown in 8 patients (15%). Associations Between Clinical Features and Antibodies to DNA. The 20 patients with anti-dsDNA by either the ELISA
or Crithidia-based assay had higher serum levels of immunoglobulin G (3,971 { 270 mg/dL vs. 3201 { 247 mg/dL, P Å .05) than seronegative patients and they also had HLA DR4 more commonly than other patients (83% vs. 41%, P Å .006) and normal subjects (83% vs. 30%, P Å .00007). Patients seropositive for anti-dsDNA by either assay were also less frequently HLA DR3-positive/HLA DR4-negative than patients seronegative for anti-dsDNA (6% vs. 44%, P Å .006). Both groups were otherwise indistinguishable by clinical criteria. The 18 patients with anti-dsDNA by ELISA were indistinguishable from seronegative patients by clinical or laboratory findings at presentation (Table 2). HLA DR4, however, occurred more commonly in these patients than in seronegative counterparts (81% vs. 45%, P Å .03) and in normal subjects (81% vs. 30%, P Å .0004) (Table 2). ELISA-positive patients were HLA DR3-positive/HLA DR4-negative less frequently than ELISA-negative patients (6% vs. 41%, P Å .02) (Table 2) but the frequencies of this phenotype in patients and normal subjects were not statistically significant (6% vs. 14%, P Å .7). The 12 patients who had anti-dsDNA by the Crithidiabased assay had higher serum concentrations of g-globulin and immunoglobulin G at presentation than seronegative counterparts (Table 2). They were also more commonly HLA DR4-positive than seronegative patients (92% vs. 54%, P Å .007) and normal subjects (92% vs. 30%, P Å .00007) (Table 2). Heterozygosity for HLA DR3-DR4 was common compared with seronegative counterparts (42% vs. 6%, P Å .01) and normal subjects (42% vs. 3%, P Å .0003) (Table 2) whereas the HLA DR3-positive/HLA DR4-negative phenotype was rare compared with other patients (0% vs. 39%, P Å .01) and normal subjects (0% vs. 14%, P Å .3) (Table 2). Patients with anti-ssDNA had no distinctive laboratory features or HLA phenotype (Table 2). Associations Between Treatment Outcomes and Anti-dsDNA.
Treatment outcomes were assessed in the 51 patients who had received conventional therapies. The 20 patients with anti-dsDNA by either assay entered remission as commonly (13 of 19 patients vs. 29 of 32 patients, P Å .06) and experienced treatment failure as often (4 of 19 patients vs. 1 of 32 patients, P Å .06) as patients seronegative by either assay. The frequencies of death from hepatic failure (0 of 9 patients vs. 1 of 32 patients, P ú .9) and liver transplantation (0 of 19 patients vs. 2 of 32 patients, P Å .5) were also similar in each group. Importantly, the 17 patients with anti-dsDNA by ELISA failed corticosteroid treatment more often (24% vs. 3%, P Å .04) than ELISA-negative patients (Table 3). A similar distinction was not found between the patients seropositive or seronegative by the Crithidia-based assay (Table 3). The serum concentrations of anti-dsDNA by ELISA did not characterize patients with different treatment outcomes. Indeed, the mean serum levels of antibody were similar between patients who failed therapy and those who entered remission (56 { 14 IU/mL vs. 73 { 19 IU/mL, P Å .8).
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HEPATOLOGY September 1997 TABLE 2. Associations Between Clinical Features and Antibodies to dsDNA and ssDNA Anti-dsDNA
Clinical Features
Age (yr) Female:Male Immune disease AST (NR:°31 U/L) Bilirubin (NR°1.1 mg/dL) g-Globulin (NR:0.7-1.6 g/dL) IgG (NR:700-1,500 mg/dL) Anti-dsDNA by ELISA Anti-dsDNA by Crithidia HLA DR3 HLA DR3//DR40 HLA DR4 HLA DR4//DR30 HLA DR3-DR4 A1-B8-DR3
Anti-ssDNA
ELISA/ (n Å 18)
ELISA0 (n Å 35)
Crithidia/ (n Å 12)
Crithidia0 (n Å 41)
ELISA/ (n Å 45)
ELISA0 (n Å 8)
50 { 3 13:5 8 (44) 650 { 110 4.1 { 1.2 3.8 { 0.2 3964 { 290
51 { 3 25:10 16 (46) 443 { 54 4.0 { 0.9 3.4 { 0.2 3252 { 239
55 { 3 9:3 4 (33) 622 { 135 5.8 { 1.9 4.2 { 0.2\ 4454 { 333‡ 10 (83)*
49 { 3 29:12 20 (49) 481 { 56 3.5 { 0.7 3.4 { 0.2\ 3236 { 207‡ 8 (20)*
10 (56)* 5/16 (31) 1/16 (6)\ 13/16 (81)Ø 9/16 (56) 4/16 (25) 3/16 (19)
2 (6)* 15/29 (52) 12/29 (41)\ 13/29 (45)Ø 10/29 (34) 3/29 (10) 12/29 (41)
5/12 0/12 11/12 6/12 5/12 3/12
15/33 13/33 18/33 13/33 2/33 12/33
51 { 2 32:13 21 (47) 551 { 57 4.5 { 0.8 3.6 { 0.1 3618 { 205 18 (40)# 12 (27) 16/37 (43) 9/37 (24) 23/37 (62) 16/37 (43) 7/37 (19) 12/37 (32)
48 { 7 6:2 3 (38) 297 { 120 1.5 { 0.3 3.2 { 0.3 2589 { 373 0 (0)# 0 (0) 4/8 (50) 4/8 (50) 3/8 (38) 3/8 (38) 0/8 (0) 3/8 (38)
(42) (0)§ (92)† (40) (42)§ (25)
(45) (39)§ (54)† (39) (6)§ (36)
NOTE. Significantly different from each other at level of *P Å .0001, †P Å .007, ‡P Å .008, §P Å .01, \P Å .02, ØP Å .03, and #P Å .04. Numbers in parentheses are percentages. Abbreviation: NR, normal.
Long-term outcomes, including development of cirrhosis, death from hepatic failure, and need for liver transplantation, were similar in patients with and without anti-dsDNA by ELISA (Table 3). Follow-up, however, was significantly longer in the ELISA-negative patients and a comparable longterm outcome in the ELISA-positive patients could not be ensured (Table 3). Patients with anti-dsDNA by ELISA who were seronegative by the Crithidia-based assay also had a poor immediate treatment outcome. Three of seven patients deteriorated on corticosteroid treatment compared with only 2 of 44 other patients (43% vs. 4%, P Å .01). Long-term outcomes were similar between the 7 patients with anti-dsDNA by ELISA but not by the Crithidia-based assay and the 44 others but follow-up was shorter in the former group (mean follow-up, 70 { 18 mo vs. 108 { 11 mo, P Å .2). Associations Between Treatment Outcomes and Anti-ssDNA. Antibodies to ssDNA did not characterize patients with different treatment outcomes (Table 3). These antibodies were present in 25 of 33 patients seronegative for anti-dsDNA by either
assay (76%). Thirty-two of 33 patients received corticosteroid therapy and patients with anti-ssDNA as their only marker entered remission (22 of 24 patients vs. 7 of 8 patients, P ú .9) and failed corticosteroid therapy (0 of 24 patients vs. 1 of 8 patients, P Å .2) as commonly as others. Additionally, they were not distinguished from seronegative patients by HLA DR3, DR4, DR3-DR4, or A1-B8-DR3. DISCUSSION
Our findings reconfirm that anti-dsDNA by ELISA or by the indirect immunofluorescence assay based on Crithidia luciliae occur commonly in patients with ANA-positive type 1 autoimmune hepatitis and that these serologic markers are not restricted to individuals with SLE.8-10 Seropositivity for anti-dsDNA by the Crithidia-based assay is less frequent than by ELISA (23% vs. 34%) but the former assay may have greater specificity for these antibodies because Crithidia luciliae is a pure source of dsDNA.3,7 Indeed, using the Crithidiabased assay as the gold standard, the ELISA assay had only an 80% specificity for anti-dsDNA. Alternatively, the Crithidia-
TABLE 3. Associations Between Treatment Outcomes and Antibodies to dsDNA and ssDNA Anti-dsDNA
Anti-ssDNA
Treatment Outcome
ELISA/ (n Å 17)
ELISA0 (n Å 34)
Crithidia/ (n Å 12)
Crithidia0 (n Å 39)
ELISA/ (n Å 43)
ELISA0 (n Å 8)
Remission Relapse Treatment failure Developed cirrhosis Death Hepatic death Liver transplant Hepatic death or transplant Treatment duration (mo) Follow-up (mo)
12 (71) 10/12 (83) 4 (24)‡ 4/12 (33) 1 (6) 0 (0) 0 (0) 0 (0) 13 { 2 58 { 11*
30 (88) 23/30 (77) 1 (3)‡ 7/21 (33) 4 (12) 1 (3) 2 (6) 3 (9) 21 { 3 124 { 12*
9 (75) 7/9 (78) 1 (8) 2/8 (25) 1 (8) 0 (0) 0 (0) 0 (0) 13 { 2 58 { 16†
33 (85) 26/33 (79) 4 (10) 9/25 (36) 4 (10) 1 (3) 2 (5) 3 (8) 20 { 3 116 { 11†
35 (81) 27/35 (77) 4 (9) 9/30 (30) 5 (12) 1 (2) 2 (5) 3 (7) 18 { 3 102 { 11
7 (88) 6/7 (86) 1 (12) 2/3 (67) 0 (0) 0 (0) 0 (0) 0 (0) 18 { 3 104 { 16
NOTE. Significantly different from each other at levels of *P Å .0009, †P Å .01, and ‡P Å .04. Numbers in parentheses are percentages.
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based assay had a 94% specificity for anti-dsDNA, using ELISA as the gold standard. Because the Crithidia-based assay binds only the most avid antibodies,5,11 its specificity may be enhanced at the expense of sensitivity. In our study, the sensitivity of the Crithidia-based assay was low compared with ELISA (55% vs. 83%) and it may thereby underestimate the frequency and importance of low avidity antibodies.5,11 Our study suggests this latter possibility because the presence of anti-dsDNA by ELISA had a greater prognostic significance than those detected by the Crithidia-based assay (Table 3). Patients with anti-dsDNA by ELISA had a significantly higher frequency of treatment failure than ELISA-negative patients whereas similar differences in treatment outcome were not observed if seropositivity was defined by the Crithidia-based assay (Table 3). These observations suggest that anti-dsDNA by ELISA have a greater prognostic significance than those detected by the Crithidia-based assay, either as surrogate markers of increased disease severity or as pathogenic factors. Pathogenic anti-dsDNA have been described in SLE and these antibodies are capable of binding to cell surfaces and migrating to nuclei where they can interfere with essential nuclear functions.6,24-26 Such interactions in autoimmune hepatitis may facilitate hepatocyte damage and enhance the clinical consequences of the disease. Antibodies detected by the Crithidia-based assay may be highly specific for anti-dsDNA but less sensitive to the pertinent antibody species that affects nuclear function. The high frequency of HLA DR4 in our patients with antidsDNA by either the ELISA or Crithidia-based assays (Table 2) suggests that there is a genetic basis for their production. This genetic association implies that anti-dsDNA reflect a particular immunopathic process rather than a random event because of liver cell destruction and release of nuclear antigens.18,19,27 Future studies must compare the binding characteristics of the antibodies in autoimmune hepatitis with those in SLE. Our findings extend the earlier observations of Tsuchiya and colleagues who also ascribed a prognostic significance to the presence of anti-dsDNA (9). Our studies indicate for the first time that anti-dsDNA detected by ELISA identify patients with ANA-positive type 1 autoimmune hepatitis who respond less well to corticosteroids. We also indicate that the expression of anti-dsDNA in these patients is associated with HLA DR4 and that the detection of clinically pertinent antibodies is assay-dependent. Unfortunately, the durations of follow-up in our study groups were not comparable and we could not assess the long-term outcomes of the seropositive patients. Eighty-five percent of our ANA-positive patients were seropositive for anti-ssDNA and 47% had anti-ssDNA in the absence of anti-dsDNA. Seropositivity for anti-ssDNA, however, was not associated with distinctive clinical or laboratory features or the DR3 and DR4 antigens (Table 2). Furthermore, patients with anti-ssDNA had responses to corticosteroid therapy that were comparable with seronegative patients (Table 3). These findings indicated that anti-ssDNA lacked clinical and prognostic value and they strengthened the hypothesis that anti-dsDNA by ELISA had distinctive implications. In summary, anti-dsDNA are commonly found in patients with ANA-positive type 1 autoimmune hepatitis by ELISA or by indirect immunofluorescence using the Crithidia luciliae
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substrate and their expression is associated with HLA DR4. Patients with anti-dsDNA by ELISA have a poorer immediate response to corticosteroid therapy than seronegative patients and immunoreactivity by this assay may identify individuals who require closer monitoring and treatment adjustments. Antibodies to dsDNA detected by the Crithidia-based assay lack prognostic significance possibly because the assay lacks sensitivity or it does not detect collateral immunoreactivities associated with liver cell destruction. Antibodies to ssDNA are present in most patients and they do not have clinical relevance. Acknowledgment: The authors ackowledge Linda Grande for her secretarial assistance. REFERENCES 1. Tan EM, Schur PH, Carr RI, Kunkel HG. Deoxyribonucleic acid (DNA) and antibodies to DNA in the serum of patients with systemic lupus erythematosus. J Clin Invest 1966;45:1732-1740. 2. Koffler D, Carr R, Agnello V, Thoburn R, Kunkel HG. Antibodies to polynucleotides in human sera: antigenic specificity and relation to disease. J Exp Med 1971;134:294-312. 3. Sontheimer RD, Gilliam JN. An immunofluorescence assay for doublestranded DNA antibodies using the Crithidia luciliae kinetoplast as a double-stranded DNA substrate. J Clin Lab Med 1978;91:550-558. 4. Stollar BD. Antibodies to DNA. CRC Crit Rev Biochem 1986;20:1-36. 5. Sontheimer RD, McCauliffe DP, Zappi E, Targoff I. Antinuclear antibodies: clinical correlations and biologic significance. In: Callen JP, Dahl MV, Golitz LE, Greenway HT Jr, Schachner LA, eds. Advances in Dermatology. St. Louis: Mosby Year Book, Inc., 1992:3-53. 6. Reichlin M, Hahn B, Koren E. Characterization of anti-dsDNA antibodies: cross-reaction with SnRNP polypeptides and cell-bonding abilities. The Immunologist 1995;3:84-88. 7. Gurian LE, Rogoff TM, Ware AJ, Jordan RE, Combes B, Gilliam JN. The immunologic diagnosis of chronic active ‘‘autoimmune’’ hepatitis: distinction from systemic lupus erythematosus. HEPATOLOGY 1985;5: 397-402. 8. Wood JR, Czaja AJ, Beaver SJ, Hall S, Ginsburg WW, Kaufman DK, Markowitz H. Frequency and significance of antibody to doublestranded DNA in chronic active hepatitis. HEPATOLOGY 1986;6:976980. 9. Tsuchiya K, Kiyosawa K, Imai H, Sodeyama T, Furuta S. Detection of anti-double and anti-single stranded DNA antibodies in chronic liver disease: significance of anti-double stranded DNA antibody in autoimmune hepatitis. J Gastroentol 1994;29:152-158. 10. Leggett BA, Collins RJ, Cooksley WGE, Prentice RL, Powell LW. Evaluation of the Crithidia assay to distinguish between auto-immune chronic active hepatitis and systemic lupus erythematosus. J Gastroenterol Hepatol 1987;2:205-211. 11. Werle E, Blazek M, Fiehn W. The clinical significance of measuring different anti-dsDNA antibodies by using the Farr assay, an enzyme immunoassay and a Crithidia luciliae immunofluorescence test. Lupus 1992;1:369-377. 12. Johnson PJ, McFarlane IG, Alvarez F, Bianchi FB, Bianchi L, Burroughs A, Chapman RW, et al. Meeting Report. International Autoimmune Hepatitis Group. HEPATOLOGY 1993;18:998-1005. 13. Czaja AJ, Manns MP. The validity and importance of subtypes of autoimmune hepatitis: a point of view. Am J Gastroenterol 1995;90:12061211. 14. Hall S, Czaja AJ, Kaufman DK, Markowitz H, Ginsburg WW. How lupoid is lupoid hepatitis? J Rheumatol 1986;13:95-98. 15. Czaja AJ, Carpenter HA, Santrach PJ, Moore SB. Genetic predispositions for the immunological features of chronic active hepatitis. HEPATOLOGY 1993;18:816-822. 16. Czaja AJ. Diagnosis and therapy of autoimmune liver disease. Med Clin N Amer 1996;80:973-994. 17. Czaja AJ, Carpenter HA. Sensitivity, specificity and predictability of biopsy interpretations in chronic hepatitis. Gastroenterology 1993;105: 1824-1832. 18. Czaja AJ, Nishioka M, Morshed SA, Hachiya T. Patterns of nuclear immunofluorescence and reactivities to recombinant nuclear antigens in autoimmune hepatitis. Gastroenterology 1994;107:200-207.
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19. Czaja AJ, Cassani F, Cataleta M, Valentini P, Bianchi FB. Frequency and significance of antibodies to actin in type 1 autoimmune hepatitis. HEPATOLOGY 1996;24:1068-1073. 20. Czaja AJ, Manns MP, Homburger HA. Frequency and significance of antibodies to liver/kidney microsome type 1 in adults with chronic active hepatitis. Gastroenterology 1992;103:1290-1295. 21. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning. A laboratory manual. New York: Cold Spring Harbor Laboratory Press, 1989. 22. Bidwell JL, Bidwell EA, Savage DA, Middleton D, Klouda PT, Bradley BA. A DNA-RFLP typing system that positively identifies serologically well-defined and ill-defined HLA-DR and DQ alleles, including Drw10. Transplantation 1988;45:640-646. 23. Czaja AJ, Strettel MDJ, Thomson LJ, Santrach PJ, Moore SB, Donaldson PT, Williams R. Associations between alleles of the major histocompati-
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26. 27.
bility complex and type 1 autoimmune hepatitis. HEPATOLOGY 1997; 25:317-323. Okudaira K, Yoshizawa H, Williams RC Jr. Monoclonal murine antiDNA antibody interacts with living mononuclear cells. Arthritis Rheum 1987;30:669-678. Vlahakos D, Foster MH, Ucci AA, Barrett KJ, Datta SK, Madaio MP. Murine monoclonal anti-DNA antibodies penetrate cells, bind to nuclei, and induce glomerular proliferation and proteinuria in vivo. J Am Soc Nephrol 1992;2:1345-1354. Ohnishi K, Ebling FM, Mitchell B, Singh RR, Hahn BH, Tsao BP. Comparison of pathogenic and non-pathogenic murine antibodies to DNA: antigen binding and structural characteristics. Int Immunol 1994;6:817-830. Czaja AJ. Autoantibodies. Bailliere’s Clinical Gastroenterology. 1995;9: 723-744.
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To determine the significance of antibodies to singlestranded (anti-ssDNA) and double-stranded DNA (antidsDNA) in antinuclear antibody (ANA)-positive type 1 autoimmune hepatitis, sera from 53 patients were tested by enzyme immunosorbent assay (ELISA) and indirect immunofluorescence using the Crithidia luciliae substrate. AntidsDNA were detected in 18 patients (34%) by ELISA and 12 patients (23%) by the Crithidia-based assay. Twenty patients with anti-dsDNA by either assay (38%) had higher serum levels of immunoglobulin G (3971 { 270 mg/dL vs. 3201 { 247 mg/dL, P Å .05) than seronegative patients. They also had human leukocyte antigen (HLA) DR4 more commonly than other patients (83% vs. 41%, P Å .006) and normal subjects (83% vs. 30%, P Å .00007). In contrast to patients seropositive by the Crithidia-based assay, those seropositive by ELISA failed corticosteroid therapy more commonly (24% vs. 3%, P Å .04). Anti-ssDNA were found in 45 patients (85%) and they did not distinguish patients with different clinical features or outcomes. We conclude that anti-dsDNA are common in ANA-positive type 1 autoimmune hepatitis. HLA DR4 is associated with their production, and seropositivity by ELISA characterizes patients who have a poorer immediate response to corticosteroid treatment. Anti-ssDNA are common but they do not have important clinical implications. (HEPATOLOGY 1997;26:567-572.) Antibodies to double-stranded DNA (anti-dsDNA) have a high specificity for the diagnosis of systemic lupus erythematosus (SLE) and they correlate closely with disease activity.1-7 Nonrheumatic diseases, including autoimmune hepatitis, may also have anti-dsDNA and the specificity of these antibodies for SLE has been challenged.8-10 In our earlier experience using an enzyme-linked immunosorbent assay (ELISA) and highly purified dsDNA, anti-
Abbreviations: Anti-dsDNA, antibodies to double-stranded DNA; SLE, systemic lupus erythematosus; ELISA, enzyme-linked immunosorbent assay; ssDNA, singlestranded DNA; ANA, antinuclear antibodies; anti-ssDNA, antibodies to single-stranded DNA; SMA, smooth muscle antibody; PBS, phosphate buffered saline; IU, international unit; HLA, human leukocyte antigens. From the 1Division of Gastroenterology and Internal Medicine, Mayo Clinic and Mayo Foundation, Rochester, MN; and the 2Third Department of Internal Medicine, Kagawa Medical University, Kagawa, Japan. Received November 26, 1996; accepted April 9, 1997. Presented in part at the meeting of the American Association for the Study of Liver Diseases, May 11, 1997, Washington, D.C. Address reprint requests to: Albert J. Czaja, M.D., Mayo Clinic, 200 First St. S.W., Rochester, MN 55905. Fax: (507) 284-0538. Copyright q 1997 by the American Association for the Study of Liver Diseases. 0270-9139/97/2603-0007$3.00/0
AND
MIKIO NISHIOKA2
dsDNA were shown in 64% of patients with autoimmune hepatitis, 46% with cryptogenic hepatitis, and 43% with chronic hepatitis B.8 Tsuchiya and colleagues, using an ELISA based on a similar substrate, found anti-dsDNA in 48% of patients with autoimmune hepatitis and 17% of patients with primary biliary cirrhosis.9 Testing of the same sera by an indirect immunofluorescence assay based on the kinetoplast of Crithidia luciliae, a pure source of dsDNA, disclosed similar results.9 A smaller study from Australia, using an immunofluorescence assay based on Crithidia luciliae, also showed a significant serologic overlap with SLE.10 Methodological differences can affect the detection of antidsDNA and the assessment of its clinical significance. Contamination of the dsDNA substrate with single-stranded DNA (ssDNA) may result in false positive results in some instances.5 Differences in antibody avidity and class may also influence detection. ELISAs detect antibodies of low avidity that may predominate in diseases other than SLE whereas the Crithidia assay may detect only high avidity antibodies of greater pathogenic importance.11 In this report, we determine the concordance of tests for anti-dsDNA by an ELISA and a Crithidia-based assay in patients with well-characterized antinuclear antibody (ANA)positive type 1 autoimmune hepatitis. We correlate seropositivity by each assay to clinical features and treatment outcome to determine the clinical pertinence of the findings and we evaluate the association between antibody expression and the known genetic risk factors for type 1 autoimmune hepatitis. We also determine the frequency of antibodies to ssDNA (anti-ssDNA) to evaluate further the clinical relevance of anti-dsDNA. PATIENTS AND METHODS Study Population. Fifty-three patients were selected for further study from the 303 patients entered in our chronic hepatitis treatment program. Patients were selected because: 1) each satisfied international criteria for autoimmune hepatitis;12 2) each had ANA in serum that justified their designation as ANA-positive type 1 autoimmune hepatitis;13 3) each had been screened and seronegative for serological markers of hepatitis B and C virus infection by second generation assays; 4) none satisfied the criteria of the American Rheumatism Association for the diagnosis of SLE;14 and 5) each had stored frozen serum samples available in their untreated state. The clinical, laboratory, and histological findings of the study population at presentation are shown in Table 1. Our study was part of a program project approved by the Institutional Review Board of the Mayo Clinic. Clinical Assessments. All patients underwent a complete physical examination by one investigator (A.J.C.) before therapy. Concomitant extrahepatic disorders of an immunological nature were sought in accordance with previously published guidelines.15 Twenty-four
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TABLE 1. Findings at Presentation of 53 Patients With Type 1 Autoimmune Hepatitis Clinical Features
Findings
Age (yr) Women:Men Duration of illness (mo) AST (NR:°31 U/L) Bilirubin (NR:°1.1 mg/dL) g-Globulin (NR:0.7-1.6 g/dL) Immunoglobulin G (NR:700-1,500 mg/dL) SMA¢1:40 ANA¢1:40 DR3//DR40 DR4//DR30 DR3 and DR4/ A1-B8-DR3/ Periportal hepatitis Bridging necrosis Multilobular necrosis Active cirrhosis
51 { 2 38:15 18 { 4 513 { 53 4 { 0.7 3.6 { 0.1 3490 { 191 47 (89) 53 (100) 13/45 (29) 19/45 (42) 7/45 (16) 15/45 (33) 25 (47) 7 (13) 5 (10) 16 (30)
NOTE. Numbers in parentheses are percentages. Abbreviation: AST, serum aspartate aminotransferase level; NR, normal.
of 53 patients (45%) had concurrent nonhepatic immunological diseases, including 8 patients with autoimmune thyroiditis, 3 patients with Graves’ disease, 4 patients with ulcerative colitis, 2 patients with synovitis, 5 patients with either rheumatoid arthritis, systemic sclerosis, Coomb’s positive hemolytic anemia, leukocytoclastic vasculitis, or asthma, and 2 patients with multiple diseases. Treatment Regimens. Fifty-one patients (96%) received prednisone alone (14 patients) or in combination with azathioprine (37 patients) in accordance with previously published protocols.16 Two patients had been randomized to investigational regimens and they did not receive conventional treatment. These patients were excluded from analyses of treatment outcome. Complete evaluations were performed every 6 months during and immediately after therapy and then at annual intervals if the clinical condition was stable. Treatment was continued until satisfaction of the previously reported treatment end points of remission, treatment failure, incomplete response, and drug toxicity.16 Clinical and laboratory manifestations of recrudescent inflammatory activity after cessation of therapy connoted relapse and it justified reinstitution of the original treatment regimen.16 Liver Tissue Examination. Liver tissue was obtained by needle biopsy in all patients at the time of presentation. Additional assessments were made as indicated to document histological remission of the disease or to clarify the clinical status. Specimens were interpreted under code and the diagnosis of cirrhosis required fibrosis and the presence of a complete regenerative nodule.17 Thirty-four of 51 patients who received corticosteroid therapy did not have cirrhosis at presentation and each also underwent subsequent liver biopsy examination. These patients were used to assess the frequency of progression to cirrhosis during or after therapy. Virological Assessments. All patients were tested for antibodies to hepatitis C virus by a second generation ELISA (Ortho Diagnostic Systems, Inc., Raritan, NJ) and in each instance, seronegativity was documented. Hepatitis B surface antigen was also sought in each patient by ELISA (Abbott Laboratories, North Chicago, IL) and its absence was confirmed in each instance. Determinations of ANA, SMA, and Antibodies to Liver/Kidney Microsome Type 1. All patients were screened at presentation for the presence
of smooth muscle antibody (SMA) and ANA by indirect immunofluorescence on murine kidney and stomach sections as described
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previously.18,19 A serum titer of 1:40 or higher was considered positive. ANAs were present in all patients in titers ranging from 1:40 to 1:10,240 (median ANA titer, 1:320). SMAs were present in 47 patients (89%) and in each instance, ANA were present concurrently. Serum titers of SMA ranged from 1:40 to 1:10,240 (median SMA titer, 1:320). Fifty-seven patients (89%) were also assessed for antibodies to liver/kidney microsome type 1 by indirect immunofluorescence on combined mouse kidney/stomach sections.20 A serum titer of 1:10 or higher was considered positive. None of the tested patients was seropositive and their designation as type 1 autoimmune hepatitis was thereby strengthened.13 Determinations of Anti-dsDNA and Anti-ssDNA. Stored frozen (0407 C) serum samples were each assessed for anti-dsDNA using ELISA, anti-dsDNA by indirect immunofluorescence using the Crithidia luciliae substrate, and anti-ssDNA using ELISA in one laboratory (Morshed and colleagues). Anti-dsDNA were determined by indirect immunofluorescence using the kinetoplast of Crithidia luciliae as the dsDNA source (AFT System II, Behring-Hoechst Japan, Ltd., Tokyo, Japan). Sera were diluted at 1:10 in phosphate buffered saline (PBS) and incubated with the Crithidia luciliae substrate in accordance with the instructions of the manufacturer. After 15 minutes, the slides were washed and then incubated with fluorescein-conjugated goat anti-human immunoglobulin G. The slides were then washed again, mounted, and viewed at 201 to 401 magnification on a Leitz immunofluorescence microscope (Ernst Leitz Canada Ltd., Midland, Ontario, Canada). The anti-dsDNA were identified by distinctive staining patterns of the kinetoplast. Positive and negative control slides were included in each determination. Seropositivity for anti-dsDNA connoted a serum titer of at least 1:10. Enzyme immunoassays were performed using the following commercial kits, MESACUP DNA-II, ssDNA, and dsDNA (Medical Biological Laboratories, Ltd., Nagoya, Japan). dsDNA and ssDNA were purified from a bacteriophage l vector grown in Escherichia coli according to a standard protocol.21 Ten mg/mL of each DNA was placed in PBS (100 mL/well) and used for coating the ELISA plate. After aspiration of the wells, the plate was blocked by PBS and 1% bovine serum albumin (100 mL/well) for 1 hour at room temperature. Serum samples were diluted 1:100 with diluting buffer (PBS and 1% bovine serum albumin). One hundred mL of diluted sample were added in duplicate to wells of the microtiter plates (Nunc, Maxisorp, Sweden) coated with either ssDNA or dsDNA. Plates were incubated at room temperature for 1 hour. Samples were then removed and washed three times with a washing buffer consisting of PBS and 0.1% Tween 20 in an automated washing machine (Micro Plate Washer, Model 1550, Bio-Rad Laboratories, Hercules, CA). Following incubation with 100 mL/well of peroxidase-conjugated monoclonal anti-human immunoglobulin G (g-chain, Fab fraction) for 1 hour, the plates were washed three times under the same conditions as before. The plates were incubated with a tetramethyl benzedene staining solution (100 mL/well) for 30 minutes at room temperature and the reaction was terminated by adding 100 mL/ well of a Stop solution (2N H2SO4 ). The plates were then read at a 450 nm wavelength with an ELISA microplate reader (MTP-120, Corona Electric Company, Ltd., Ibaraki, Japan). A standard curve was made for each ELISA plate, using standard (World Health Organization) reference samples of known concentrations, and results were reported in International Units (IU). The cut-off value was 40 IU/mL for anti-ssDNA or anti-dsDNA. This value was higher than the mean { 3 SD found in sera from 30 healthy subjects. All healthy control subjects were seronegative by three consecutive assays on three ELISA plates using duplicate wells. The serum levels of each control subject were always õ20 IU/mL (range, 5-20 IU/mL) in each assay. Previous studies in 49 patients with various nonhepatic autoimmune disorders, including systemic lupus erythematosus (10 patients), rheumatoid arthritis (19 patients), and Sjo¨gren’s syndrome (20 patients), indicated con-
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cordance of the ELISA and Crithidia results in 94%. Monoclonal antibodies to either ssDNA or dsDNA were used in a separate experiment to ensure the absence of kit contamination. HLA Determinations. All patients were caucasoid and each underwent typing for the class I (A and B locus) antigens. Forty-five patients (85%) were evaluated for the class II (DR locus) antigens. A standard microlymphocytotoxicity technique was used for class I and class II typing.15 Multiple locally derived and commercially available sera were used to define each specificity as described elsewhere.15 Restriction fragment length polymorphism was also used to assess the class II antigens in patients.22 Eighty healthy caucasoid adults participating in the volunteer blood donor program of the Mayo Clinic constituted the normal population. Each individual had indicated the absence of significant illness by a standard blood donation questionnaire. Typing for class I antigens was performed in 79 of 80 normal individuals (99%) by the standard microlymphocytotoxicity technique. Typing for class II antigens was accomplished in all normal subjects by both the standard microlymphocytotoxicity technique and restriction fragment length polymorphism. Statistical Analyses. Dichotomous variables were compared using the Fisher Exact test. The unpaired t test was used to evaluate the significance of differences in means for continuous variables. The Mann Whitney test was used to compare nonparametric variables in independent samples. Human leukocyte antigen (HLA) DR3, DR4, and A1-B8-DR3 are known risk factors for type 1 autoimmune hepatitis23 and only the frequencies of these antigens were analyzed in our study population. Because the variables for comparison had been formulated a priori and then assessed systematically in our study group, an unadjusted P-value of .05 was used to determine statistical significance. Data are presented as the mean { SE of the mean in tables and text. RESULTS Frequencies of Anti-dsDNA and Anti-ssDNA. Anti-dsDNA were detected in 18 patients (34%) by ELISA and 12 patients (23%) by indirect immunofluorescence using the Crithidia substrate. Both assays were positive for anti-dsDNA in 10 patients (19%). Twenty patients (38%) had anti-dsDNA by ELISA or the Crithidia-based assay. Only 2 individuals (4%) were seropositive by the Crithidia-based assay alone whereas 8 individuals (15%) had anti-dsDNA by the ELISA assay alone. Patients with anti-dsDNA by ELISA were seropositive for anti-dsDNA by the Crithidia-based assay more commonly than patients seronegative by ELISA (52% vs. 6%, P Å .0001) (Table 2). Similarly, patients with anti-dsDNA by the Crithidia-based assay were seropositive for anti-dsDNA by ELISA more frequently than patients seronegative by the Crithidiabased assay (83% vs. 20%, P Å .0001) (Table 2). Using the Crithidia-based assay as the gold standard, the sensitivity of the ELISA for anti-dsDNA was 83% and its specificity was 80%. Using the ELISA as the gold standard, the sensitivity of the Crithidia-based assay for anti-dsDNA was 55% and its specificity was 94%. Antibodies to ssDNA were found in 45 patients (85%). In 25 patients (47%), these antibodies were an isolated finding. In 10 patients (19%), they were accompanied by anti-dsDNA as determined by both assays. Antibodies to ssDNA were present only with anti-dsDNA by ELISA in 8 patients (15%) and they were present only with anti-dsDNA by the Crithidiabased assay in 2 patients (4%). Individuals seropositive for anti-ssDNA had anti-dsDNA by ELISA (40% vs. 0%, P Å .04) or by the Crithidia-based assay (27% vs. 0%, P Å .2) more frequently than seronegative counterparts (Table 2). Only the occurrence of anti-dsDNA by ELISA, however, was statis-
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tically greater in patients with anti-ssDNA than in patients without anti-ssDNA (Table 2). Seronegativity for antibodies to ssDNA and dsDNA by all assays was shown in 8 patients (15%). Associations Between Clinical Features and Antibodies to DNA. The 20 patients with anti-dsDNA by either the ELISA
or Crithidia-based assay had higher serum levels of immunoglobulin G (3,971 { 270 mg/dL vs. 3201 { 247 mg/dL, P Å .05) than seronegative patients and they also had HLA DR4 more commonly than other patients (83% vs. 41%, P Å .006) and normal subjects (83% vs. 30%, P Å .00007). Patients seropositive for anti-dsDNA by either assay were also less frequently HLA DR3-positive/HLA DR4-negative than patients seronegative for anti-dsDNA (6% vs. 44%, P Å .006). Both groups were otherwise indistinguishable by clinical criteria. The 18 patients with anti-dsDNA by ELISA were indistinguishable from seronegative patients by clinical or laboratory findings at presentation (Table 2). HLA DR4, however, occurred more commonly in these patients than in seronegative counterparts (81% vs. 45%, P Å .03) and in normal subjects (81% vs. 30%, P Å .0004) (Table 2). ELISA-positive patients were HLA DR3-positive/HLA DR4-negative less frequently than ELISA-negative patients (6% vs. 41%, P Å .02) (Table 2) but the frequencies of this phenotype in patients and normal subjects were not statistically significant (6% vs. 14%, P Å .7). The 12 patients who had anti-dsDNA by the Crithidiabased assay had higher serum concentrations of g-globulin and immunoglobulin G at presentation than seronegative counterparts (Table 2). They were also more commonly HLA DR4-positive than seronegative patients (92% vs. 54%, P Å .007) and normal subjects (92% vs. 30%, P Å .00007) (Table 2). Heterozygosity for HLA DR3-DR4 was common compared with seronegative counterparts (42% vs. 6%, P Å .01) and normal subjects (42% vs. 3%, P Å .0003) (Table 2) whereas the HLA DR3-positive/HLA DR4-negative phenotype was rare compared with other patients (0% vs. 39%, P Å .01) and normal subjects (0% vs. 14%, P Å .3) (Table 2). Patients with anti-ssDNA had no distinctive laboratory features or HLA phenotype (Table 2). Associations Between Treatment Outcomes and Anti-dsDNA.
Treatment outcomes were assessed in the 51 patients who had received conventional therapies. The 20 patients with anti-dsDNA by either assay entered remission as commonly (13 of 19 patients vs. 29 of 32 patients, P Å .06) and experienced treatment failure as often (4 of 19 patients vs. 1 of 32 patients, P Å .06) as patients seronegative by either assay. The frequencies of death from hepatic failure (0 of 9 patients vs. 1 of 32 patients, P ú .9) and liver transplantation (0 of 19 patients vs. 2 of 32 patients, P Å .5) were also similar in each group. Importantly, the 17 patients with anti-dsDNA by ELISA failed corticosteroid treatment more often (24% vs. 3%, P Å .04) than ELISA-negative patients (Table 3). A similar distinction was not found between the patients seropositive or seronegative by the Crithidia-based assay (Table 3). The serum concentrations of anti-dsDNA by ELISA did not characterize patients with different treatment outcomes. Indeed, the mean serum levels of antibody were similar between patients who failed therapy and those who entered remission (56 { 14 IU/mL vs. 73 { 19 IU/mL, P Å .8).
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Clinical Features
Age (yr) Female:Male Immune disease AST (NR:°31 U/L) Bilirubin (NR°1.1 mg/dL) g-Globulin (NR:0.7-1.6 g/dL) IgG (NR:700-1,500 mg/dL) Anti-dsDNA by ELISA Anti-dsDNA by Crithidia HLA DR3 HLA DR3//DR40 HLA DR4 HLA DR4//DR30 HLA DR3-DR4 A1-B8-DR3
Anti-ssDNA
ELISA/ (n Å 18)
ELISA0 (n Å 35)
Crithidia/ (n Å 12)
Crithidia0 (n Å 41)
ELISA/ (n Å 45)
ELISA0 (n Å 8)
50 { 3 13:5 8 (44) 650 { 110 4.1 { 1.2 3.8 { 0.2 3964 { 290
51 { 3 25:10 16 (46) 443 { 54 4.0 { 0.9 3.4 { 0.2 3252 { 239
55 { 3 9:3 4 (33) 622 { 135 5.8 { 1.9 4.2 { 0.2\ 4454 { 333‡ 10 (83)*
49 { 3 29:12 20 (49) 481 { 56 3.5 { 0.7 3.4 { 0.2\ 3236 { 207‡ 8 (20)*
10 (56)* 5/16 (31) 1/16 (6)\ 13/16 (81)Ø 9/16 (56) 4/16 (25) 3/16 (19)
2 (6)* 15/29 (52) 12/29 (41)\ 13/29 (45)Ø 10/29 (34) 3/29 (10) 12/29 (41)
5/12 0/12 11/12 6/12 5/12 3/12
15/33 13/33 18/33 13/33 2/33 12/33
51 { 2 32:13 21 (47) 551 { 57 4.5 { 0.8 3.6 { 0.1 3618 { 205 18 (40)# 12 (27) 16/37 (43) 9/37 (24) 23/37 (62) 16/37 (43) 7/37 (19) 12/37 (32)
48 { 7 6:2 3 (38) 297 { 120 1.5 { 0.3 3.2 { 0.3 2589 { 373 0 (0)# 0 (0) 4/8 (50) 4/8 (50) 3/8 (38) 3/8 (38) 0/8 (0) 3/8 (38)
(42) (0)§ (92)† (40) (42)§ (25)
(45) (39)§ (54)† (39) (6)§ (36)
NOTE. Significantly different from each other at level of *P Å .0001, †P Å .007, ‡P Å .008, §P Å .01, \P Å .02, ØP Å .03, and #P Å .04. Numbers in parentheses are percentages. Abbreviation: NR, normal.
Long-term outcomes, including development of cirrhosis, death from hepatic failure, and need for liver transplantation, were similar in patients with and without anti-dsDNA by ELISA (Table 3). Follow-up, however, was significantly longer in the ELISA-negative patients and a comparable longterm outcome in the ELISA-positive patients could not be ensured (Table 3). Patients with anti-dsDNA by ELISA who were seronegative by the Crithidia-based assay also had a poor immediate treatment outcome. Three of seven patients deteriorated on corticosteroid treatment compared with only 2 of 44 other patients (43% vs. 4%, P Å .01). Long-term outcomes were similar between the 7 patients with anti-dsDNA by ELISA but not by the Crithidia-based assay and the 44 others but follow-up was shorter in the former group (mean follow-up, 70 { 18 mo vs. 108 { 11 mo, P Å .2). Associations Between Treatment Outcomes and Anti-ssDNA. Antibodies to ssDNA did not characterize patients with different treatment outcomes (Table 3). These antibodies were present in 25 of 33 patients seronegative for anti-dsDNA by either
assay (76%). Thirty-two of 33 patients received corticosteroid therapy and patients with anti-ssDNA as their only marker entered remission (22 of 24 patients vs. 7 of 8 patients, P ú .9) and failed corticosteroid therapy (0 of 24 patients vs. 1 of 8 patients, P Å .2) as commonly as others. Additionally, they were not distinguished from seronegative patients by HLA DR3, DR4, DR3-DR4, or A1-B8-DR3. DISCUSSION
Our findings reconfirm that anti-dsDNA by ELISA or by the indirect immunofluorescence assay based on Crithidia luciliae occur commonly in patients with ANA-positive type 1 autoimmune hepatitis and that these serologic markers are not restricted to individuals with SLE.8-10 Seropositivity for anti-dsDNA by the Crithidia-based assay is less frequent than by ELISA (23% vs. 34%) but the former assay may have greater specificity for these antibodies because Crithidia luciliae is a pure source of dsDNA.3,7 Indeed, using the Crithidiabased assay as the gold standard, the ELISA assay had only an 80% specificity for anti-dsDNA. Alternatively, the Crithidia-
TABLE 3. Associations Between Treatment Outcomes and Antibodies to dsDNA and ssDNA Anti-dsDNA
Anti-ssDNA
Treatment Outcome
ELISA/ (n Å 17)
ELISA0 (n Å 34)
Crithidia/ (n Å 12)
Crithidia0 (n Å 39)
ELISA/ (n Å 43)
ELISA0 (n Å 8)
Remission Relapse Treatment failure Developed cirrhosis Death Hepatic death Liver transplant Hepatic death or transplant Treatment duration (mo) Follow-up (mo)
12 (71) 10/12 (83) 4 (24)‡ 4/12 (33) 1 (6) 0 (0) 0 (0) 0 (0) 13 { 2 58 { 11*
30 (88) 23/30 (77) 1 (3)‡ 7/21 (33) 4 (12) 1 (3) 2 (6) 3 (9) 21 { 3 124 { 12*
9 (75) 7/9 (78) 1 (8) 2/8 (25) 1 (8) 0 (0) 0 (0) 0 (0) 13 { 2 58 { 16†
33 (85) 26/33 (79) 4 (10) 9/25 (36) 4 (10) 1 (3) 2 (5) 3 (8) 20 { 3 116 { 11†
35 (81) 27/35 (77) 4 (9) 9/30 (30) 5 (12) 1 (2) 2 (5) 3 (7) 18 { 3 102 { 11
7 (88) 6/7 (86) 1 (12) 2/3 (67) 0 (0) 0 (0) 0 (0) 0 (0) 18 { 3 104 { 16
NOTE. Significantly different from each other at levels of *P Å .0009, †P Å .01, and ‡P Å .04. Numbers in parentheses are percentages.
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WBS: Hepatology
HEPATOLOGY Vol. 26, No. 3, 1997
CZAJA ET AL.
based assay had a 94% specificity for anti-dsDNA, using ELISA as the gold standard. Because the Crithidia-based assay binds only the most avid antibodies,5,11 its specificity may be enhanced at the expense of sensitivity. In our study, the sensitivity of the Crithidia-based assay was low compared with ELISA (55% vs. 83%) and it may thereby underestimate the frequency and importance of low avidity antibodies.5,11 Our study suggests this latter possibility because the presence of anti-dsDNA by ELISA had a greater prognostic significance than those detected by the Crithidia-based assay (Table 3). Patients with anti-dsDNA by ELISA had a significantly higher frequency of treatment failure than ELISA-negative patients whereas similar differences in treatment outcome were not observed if seropositivity was defined by the Crithidia-based assay (Table 3). These observations suggest that anti-dsDNA by ELISA have a greater prognostic significance than those detected by the Crithidia-based assay, either as surrogate markers of increased disease severity or as pathogenic factors. Pathogenic anti-dsDNA have been described in SLE and these antibodies are capable of binding to cell surfaces and migrating to nuclei where they can interfere with essential nuclear functions.6,24-26 Such interactions in autoimmune hepatitis may facilitate hepatocyte damage and enhance the clinical consequences of the disease. Antibodies detected by the Crithidia-based assay may be highly specific for anti-dsDNA but less sensitive to the pertinent antibody species that affects nuclear function. The high frequency of HLA DR4 in our patients with antidsDNA by either the ELISA or Crithidia-based assays (Table 2) suggests that there is a genetic basis for their production. This genetic association implies that anti-dsDNA reflect a particular immunopathic process rather than a random event because of liver cell destruction and release of nuclear antigens.18,19,27 Future studies must compare the binding characteristics of the antibodies in autoimmune hepatitis with those in SLE. Our findings extend the earlier observations of Tsuchiya and colleagues who also ascribed a prognostic significance to the presence of anti-dsDNA (9). Our studies indicate for the first time that anti-dsDNA detected by ELISA identify patients with ANA-positive type 1 autoimmune hepatitis who respond less well to corticosteroids. We also indicate that the expression of anti-dsDNA in these patients is associated with HLA DR4 and that the detection of clinically pertinent antibodies is assay-dependent. Unfortunately, the durations of follow-up in our study groups were not comparable and we could not assess the long-term outcomes of the seropositive patients. Eighty-five percent of our ANA-positive patients were seropositive for anti-ssDNA and 47% had anti-ssDNA in the absence of anti-dsDNA. Seropositivity for anti-ssDNA, however, was not associated with distinctive clinical or laboratory features or the DR3 and DR4 antigens (Table 2). Furthermore, patients with anti-ssDNA had responses to corticosteroid therapy that were comparable with seronegative patients (Table 3). These findings indicated that anti-ssDNA lacked clinical and prognostic value and they strengthened the hypothesis that anti-dsDNA by ELISA had distinctive implications. In summary, anti-dsDNA are commonly found in patients with ANA-positive type 1 autoimmune hepatitis by ELISA or by indirect immunofluorescence using the Crithidia luciliae
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substrate and their expression is associated with HLA DR4. Patients with anti-dsDNA by ELISA have a poorer immediate response to corticosteroid therapy than seronegative patients and immunoreactivity by this assay may identify individuals who require closer monitoring and treatment adjustments. Antibodies to dsDNA detected by the Crithidia-based assay lack prognostic significance possibly because the assay lacks sensitivity or it does not detect collateral immunoreactivities associated with liver cell destruction. Antibodies to ssDNA are present in most patients and they do not have clinical relevance. Acknowledgment: The authors ackowledge Linda Grande for her secretarial assistance. REFERENCES 1. Tan EM, Schur PH, Carr RI, Kunkel HG. Deoxyribonucleic acid (DNA) and antibodies to DNA in the serum of patients with systemic lupus erythematosus. J Clin Invest 1966;45:1732-1740. 2. Koffler D, Carr R, Agnello V, Thoburn R, Kunkel HG. Antibodies to polynucleotides in human sera: antigenic specificity and relation to disease. J Exp Med 1971;134:294-312. 3. Sontheimer RD, Gilliam JN. An immunofluorescence assay for doublestranded DNA antibodies using the Crithidia luciliae kinetoplast as a double-stranded DNA substrate. J Clin Lab Med 1978;91:550-558. 4. Stollar BD. Antibodies to DNA. CRC Crit Rev Biochem 1986;20:1-36. 5. Sontheimer RD, McCauliffe DP, Zappi E, Targoff I. Antinuclear antibodies: clinical correlations and biologic significance. In: Callen JP, Dahl MV, Golitz LE, Greenway HT Jr, Schachner LA, eds. Advances in Dermatology. St. Louis: Mosby Year Book, Inc., 1992:3-53. 6. Reichlin M, Hahn B, Koren E. Characterization of anti-dsDNA antibodies: cross-reaction with SnRNP polypeptides and cell-bonding abilities. The Immunologist 1995;3:84-88. 7. Gurian LE, Rogoff TM, Ware AJ, Jordan RE, Combes B, Gilliam JN. The immunologic diagnosis of chronic active ‘‘autoimmune’’ hepatitis: distinction from systemic lupus erythematosus. HEPATOLOGY 1985;5: 397-402. 8. Wood JR, Czaja AJ, Beaver SJ, Hall S, Ginsburg WW, Kaufman DK, Markowitz H. Frequency and significance of antibody to doublestranded DNA in chronic active hepatitis. HEPATOLOGY 1986;6:976980. 9. Tsuchiya K, Kiyosawa K, Imai H, Sodeyama T, Furuta S. Detection of anti-double and anti-single stranded DNA antibodies in chronic liver disease: significance of anti-double stranded DNA antibody in autoimmune hepatitis. J Gastroentol 1994;29:152-158. 10. Leggett BA, Collins RJ, Cooksley WGE, Prentice RL, Powell LW. Evaluation of the Crithidia assay to distinguish between auto-immune chronic active hepatitis and systemic lupus erythematosus. J Gastroenterol Hepatol 1987;2:205-211. 11. Werle E, Blazek M, Fiehn W. The clinical significance of measuring different anti-dsDNA antibodies by using the Farr assay, an enzyme immunoassay and a Crithidia luciliae immunofluorescence test. Lupus 1992;1:369-377. 12. Johnson PJ, McFarlane IG, Alvarez F, Bianchi FB, Bianchi L, Burroughs A, Chapman RW, et al. Meeting Report. International Autoimmune Hepatitis Group. HEPATOLOGY 1993;18:998-1005. 13. Czaja AJ, Manns MP. The validity and importance of subtypes of autoimmune hepatitis: a point of view. Am J Gastroenterol 1995;90:12061211. 14. Hall S, Czaja AJ, Kaufman DK, Markowitz H, Ginsburg WW. How lupoid is lupoid hepatitis? J Rheumatol 1986;13:95-98. 15. Czaja AJ, Carpenter HA, Santrach PJ, Moore SB. Genetic predispositions for the immunological features of chronic active hepatitis. HEPATOLOGY 1993;18:816-822. 16. Czaja AJ. Diagnosis and therapy of autoimmune liver disease. Med Clin N Amer 1996;80:973-994. 17. Czaja AJ, Carpenter HA. Sensitivity, specificity and predictability of biopsy interpretations in chronic hepatitis. Gastroenterology 1993;105: 1824-1832. 18. Czaja AJ, Nishioka M, Morshed SA, Hachiya T. Patterns of nuclear immunofluorescence and reactivities to recombinant nuclear antigens in autoimmune hepatitis. Gastroenterology 1994;107:200-207.
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19. Czaja AJ, Cassani F, Cataleta M, Valentini P, Bianchi FB. Frequency and significance of antibodies to actin in type 1 autoimmune hepatitis. HEPATOLOGY 1996;24:1068-1073. 20. Czaja AJ, Manns MP, Homburger HA. Frequency and significance of antibodies to liver/kidney microsome type 1 in adults with chronic active hepatitis. Gastroenterology 1992;103:1290-1295. 21. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning. A laboratory manual. New York: Cold Spring Harbor Laboratory Press, 1989. 22. Bidwell JL, Bidwell EA, Savage DA, Middleton D, Klouda PT, Bradley BA. A DNA-RFLP typing system that positively identifies serologically well-defined and ill-defined HLA-DR and DQ alleles, including Drw10. Transplantation 1988;45:640-646. 23. Czaja AJ, Strettel MDJ, Thomson LJ, Santrach PJ, Moore SB, Donaldson PT, Williams R. Associations between alleles of the major histocompati-
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26. 27.
bility complex and type 1 autoimmune hepatitis. HEPATOLOGY 1997; 25:317-323. Okudaira K, Yoshizawa H, Williams RC Jr. Monoclonal murine antiDNA antibody interacts with living mononuclear cells. Arthritis Rheum 1987;30:669-678. Vlahakos D, Foster MH, Ucci AA, Barrett KJ, Datta SK, Madaio MP. Murine monoclonal anti-DNA antibodies penetrate cells, bind to nuclei, and induce glomerular proliferation and proteinuria in vivo. J Am Soc Nephrol 1992;2:1345-1354. Ohnishi K, Ebling FM, Mitchell B, Singh RR, Hahn BH, Tsao BP. Comparison of pathogenic and non-pathogenic murine antibodies to DNA: antigen binding and structural characteristics. Int Immunol 1994;6:817-830. Czaja AJ. Autoantibodies. Bailliere’s Clinical Gastroenterology. 1995;9: 723-744.
hepas
WBS: Hepatology