Clin. exp. Immunol. (1978) 33, 204-210.

DNA-binding antibodies and hepatitis B markers in acute and chronic liver disease J. G. C. KINGHAM, SUHA RASSAM,* N. K. GANGULY, M. J. MCGUIRE, BUSHRA NASRATt S. T. HOL GATE, D. R. TR I GER & R. WRIGH T Profrssorial Medical Unit, Level F, Centre Block, Southampton General Hospital, Southampton, *Department of Medicine, Medical City, Baghdad, Iraq and tDepartment of Pathology, College oj Medicine, University of Baghdad, Iraq.

(Received 13 March 1978)

SUMMARY

A Farr technique has been used to assay antibodies to double-stranded DNA in the serum of patients with acute and chronic liver disease and carriers of HBsAg from the United Kingdom and Iraq. These antibodies were found in all groups from both countries. The highest levels were found in chronic active hepatitis and cirrhosis. In the Iraqi patients there was a strongly positive correlation between DNA-binding antibody levels and the presence of hepatitis B markers but not with disease activity. In the patients from the United Kingdom there was little correlation with disease activity and none with autoantibodies. Ninety-five per cent of asymptomatic carriers of HBsAG had elevated DNA-binding antibodies. It is suggested that hepatitis B-specific DNA might be one trigger to DNA antibody formation, though in liver disease a variety of factors are clearly operative.

INTRODUCTION Antibodies to double-stranded DNA (DNA Ab) are found in high titre in the sera of patients with systemic lupus erythematosus (SLE) (Pincus et al., 1969) and in New Zealand black mice (Steinberg, Pincus & Talal, 1969), a naturally occurring animal model of SLE. These antibodies were initially thought to be specific for SLE (Hughes, 1971); however, it is now recognised that anti-double-stranded DNA antibodies are found in other disorders, such as myasthenia gravis (Gershwin et al., 1976) and liver disease. Anti-double-stranded DNA antibodies in liver disease were first documented by Davis & Read (1975), who studied HBsAg-negative chronic active hepatitis (CAH) because of its many similarities to SLE. Although they detected high DNA-binding capacity in the sera ofthis group of patients, they reported normal binding in other forms of liver disease. Jain et al. (1976) confirmed the high DNA- binding capacity in CAH, but also found it in other forms of liver disease. In view of the possible aetiological role of viral DNA in the production of double-stranded DNA antibodies, we have assessed anti-DNA antibodies by serum DNA-binding capacity in a wide spectrum of liver disease, with particular reference to Hepatitis B virus (HBVt) infection. PATIENTS AND METHODS Two populations were studied, one from the United Kingdom, essentially white and British-born, the other Iraqi, essentially Arab. United Kingdom patients. Chronic active hepatitis. Forty-three patients, thirty-five female and eight male, with CAH (41 HBsAg-negative) diagnosed by clinical, serological and histological criteria (Sherlock, 1974). Age range 12-84 years (mean + s.d. 50 + 22 years). Correspondence: Professor R. Wright, Professorial Medical Unit, Level F, Centre Block, Southampton General Hospital, Southampton S09 4XY. 0099-9104/78/0800-0204$02.00 (C 1978 Blackwell Scientific Publications

204

DNA antibodies and HBV markers

205

Primary biliary cirrhosis. Twenty-three patients, eighteen female and five male, with PBC diagnosed by clinical, serological and histological criteria (Sherlock & Scheuer, 1973). Age range 41-80 years (mean ± s.d. = 64 ± 11 years). Akoholic cirrhosis. Twenty-one patients, eight female and thirteen male, with alcoholic cirrhosis. Age range 44-75 years (mean + s.d. = 60 ± 7 years). Alcoholic steatosis ± hepatitis. Fifteen patients, five female and ten male, with alcoholic fatty liver or alcoholic hepatitis. Age range 39-80 years (mean i s.d. = 54 ± 11 years). Acute viral hepatitis. Thirty-nine patients, sixteen female and twenty-three male, with acute viral hepatitis (seventeen HBsAg-positive, four female, thirteen male; twenty-two HBsAg-negative, eleven female, eleven male). Obstructive jaundice. Sixteen patients, six female and ten male, with obstructive jaundice. Age range 49-87 years (mean ± s.d.

=

65 ± 14 years).

HBsAg carriers.Thirty-two normal, healthy, asymptomatic carriers of HBsAg, twenty female and twelve male. Age range 19-56 years (mean ± s.d. = 34 + 12 years). Negative controls. Fifty normal, healthy controls (twenty-seven female and twenty-three male). Age range 17-66 years (mean + s.d. = 45 + 14 years). Positive controls. Twenty patients with systemic lupus erythematosus (SLE) having three or more of the American Rheumatoid Association critieria for diagnosis. Age range 13-73 years (mean ± s.d. = 31 ± 19). Iraqi patients. A total of 146 patients with chronic liver disease, seventy-four of these associated with HBV infection, and fifty normal, healthy controls were studied. Chronic active hepatitis. Forty patients with CAH with or without cirrhosis diagnosed clinically, serologically and histologically by accepted criteria (Sherlock, 1974). Twenty seven were HBsAg and anti-HBc-positive and a further two were positive for anti-HBc alone. Thus twenty-nine of forty (73%) were HBV associated, three female, twenty-six male. Age range 8-60 years (mean ± s.d. = 40 + 14 years). Eleven were HBV-negative, four female, seven male. Age range 2-64 years (mean ± s.d. = 28 ± 23 years). Cirrhosis. Twenty-six patients with clinical and histological cirrhosis, fourteen HBsAg and anti-HBc-positive and a further three positive for anti-HBc alone. Thus seventeen of twenty-six (65%) were HBV-associated, four female, thirteen male. Age range lk-65 years (mean ± s.d. = 39 + 20 years). Nine were HBV-negative, four female, five male. Age range 8-65 years (mean ± s.d. = 42 + 19 years). Twenty-two patients with clinical cirrhosis without histological confirmation, twelve HBsAg and anti-HBc-positive. None were positive for anti-HBc alone. Thus twelve of twenty-two (55%) were HBV-associated, four female, eight male. Age range 27-70 years (mean ± s.d. = 46 ± 14 years).Ten were HBV-negativc, three female, seven male. Age range 4-70 years (mean ± s.d. 37 ± 21 years). Combining the two groups of cirrhotics, twenty-nine out of forty-eight (60%) had markers of HBV infection. Miscellaneous liver disease. Fifty-eight patients with hepatomegaly, with or without splenomegaly. Liver biopsy was either normal or showed non-specific changes of mild inflammatory cell infiltration or steatosis. Nine were HBsAg and antiHBc-positive and a further seven positive for anti-HBc alone.Thus sixteen out of fifty-eight (28%) were HBV-associated, ten female, six male. Age range 17-61 years (mean ± s.d. 37 ± 15 years). Forty-two were HBV-negative, twenty-six female, sixteen male. Age range 15-70 years (mean i s.d. = 39 ± 15 years). Controls. Fifty normal, healthy subjects, nineteen female and thirty-one male, from a socioeconomic background similar to that of the patients with liver disease. Age range 15-51 years (mean ± s.d. = 31 + 11 years). Methods. The DNA-binding capacity of serum was used as a measure of anti-double-stranded DNA antibodies. DNAbinding was measured by the Farr technique, using a modification of the method of Carr et al. (1969). Double-stranded calf thymus DNA (Sigma) was labelled extrinsically with 3H actinomycin D (Radio Chemicals, Amersham). 50 p.1 of test serum with 750 gal of borate-buffered saline at pH 8-3 were heat-inactivated at 560C for 30 min. After cooling to 40C, 200 [L1 of labelled DNA were added and a sample incubated at 4VC for 18 hr in the dark. 1 0 ml of saturated ammonium sulphate was added and the precipitated globulin fraction was spun down at 1800 g for 1 hr. The precipitate was washed with half saturated ammonium sulphate and then dissolved in 600 p. of distilled water. The dissolved precipitate was dispersed in 4 0 ml of toluene-based scintillation fluid and its 3H activity measured in a Wallach 81000 liquid scintillation beta counter. DNA-binding was calculated by the percentage of the total 3H activity recovered in the precipitate. HBsAg was measured by passive haemagglutination (Hepatest, Burroughs Wellcome) and radioimmunoassay (AusriaAbbott Laboratories). Anti-HBc was measured by complement fixation in doubling dilutions in a Microtitre system. Core antigen for the assay was extracted from the liver of an immuno-suppressed patient with chronic HBV infection.

RESULTS Mean DNA-binding in normal healthy controls was 8-9 + 1-4% in fifty subjects from the United Kingdom and 8*6 + 1-7% in fifty subjects from Iraq. The normal range (mean + 2 s.d.) was thus taken as 5-12%0. The range of DNA-binding in twenty patients with SLE was 13-66% (mean ± s.d. = 307 + 16-1 %). The DNA-binding antibodies were predominantly of the IgG or IgM class.

206

5. G. C. Kinsgham et al.

United Kingdom patients DNA-binding was elevated in all groups of liver disease studied, and in asymptomatic carriers of HBsAg. The highest binding was seen in the patients with HBsAG-negative CAH; the range was 9-2-37.9,o1 (mean ± s.d. 22-3 + 699"), with only two results falling within the normal range (Fig. 1). Thirty-six of the results fell into the SLE range, mostly in the lower part, though four were greater than the mean SLE level of 30.70,0. 40

4

Mean SLE

30 00- -

o

8

0

0

30Q7+ 16-1%

00

0~~~~~~~~~~~~~~ 0~~~~~~~~~~~ 0~~~~~~~~~~~~

8

20

~~~88 0 80 0.0 °

COO

O

0

0~~~~~~~~~~~~~~~ noa a

0

Iaue fo nomladSEseaaeidctd.()HsgpsiierniHepoiiem ~ ag-

(Hig.~~~

n e g tai7e

~~~ 1).

though Chrons acshigyeg rhosas Piary bhmiany AIJ laiaee choelihepratitiws Act 9-28i Obstrjc(mea Choi +622~) Hssd 1though one thermean 692. sLE nTh rangyier 1 + onepwasas (maundc cariesd. hepatis Inth egopofit alc holasocae ie iestet-n ith cirrhosis and faityeeenneatths alcoholaicen Inathesgorou ifteen wih alchoi nofmalcohol-associatedaliverdisease.(0 posisn eangerthangh ftwety-h Ofpatheisor rivee patientsrmy ith ABc n hadtwenty-poneithve DNA-binding cnirrh leavsre virm normwal hepatitieo ftwety-liveralbue patients w t

had DNA-binding aboevhenoma nnte

greangethoug nonemas,

as high as the mean SLuE level (Fig. 1).

The cirrhotics had higher levels (mean ± s.d. 18-5 ± 3-60,) than those with hepatitis or fatty liver alone (mean ± s.d. 15-9 ± 3 -2 %) (Fig. 1). The difference was significant at the 10% level. The range of DNA-binding in thirty-nine patients with acute viral hepatitis was 7-1-30 ) (mean ± s.d. = 15-3 ± 5.31)0) (Fig. 1). Thirty per cent of these fell into the upper end of the normal range, although the greater proportion were elevated to levels similar to the alcoholic hepatitic group. Patients with hepatitis B (seventeen) had higher DNA-binding (mean + s.d. = 16&3 ± 6.4%) than those with non-B hepatitis (twenty-two) mean + s.d. 145 ± 4-3o/0), though the difference was not =

significant. The DNA-binding levels in the patients with obstructive jaundice (sixteen) were greater than normal,

207

DNA antibodies and HBV markers

the range being 8-0-17-0% (mean + s.d. = 12-7 ± 2.7%), but lower than all other groups under study (Fig. 1). Of the thirty-two healthy asymptomatic carriers of HBsAg, all but three had high DNA-binding though the levels were not as high as those found in active liver disease. The range was 11 2-17-3 0,, (mean + s.d. = 14-1 ± 1-7%) (Fig. 1). None of these patients had abnormal liver function tests. Seven had liver biopsies, with findings of chronic persistent hepatitis (3), non-specific inflammatory cell infiltration (3) or no abnormality (1). There was no correlation between biopsy findings and DNAbinding levels. TABLE 1. Mean and standard deviation, significance of difference from control and correlation with other laboratory findings (autoantibodies, AsT, bilirubin, alkaline phosphatase, albumin, globulin) of DNA-binding capacity in patients with liver disease from the United Kingdom n

% DNA-binding i 1 s.d.

CAH PBC

43 23

22.3±6.9* 18.6+6.2*

Alc. cirrhosis Alc. steatosis ± hepatitis Acute viral hepatitis Obstructive jaundice Carriers of HBsAg

21 15 39 16 32

18.5±3-6* 15.9±3.2* 15-3±5.3* 12.7±2-7* 14.1±1.7*

Correlation with laboratory findings

AsT P

DNA-binding antibodies and hepatitis B markers in acute and chronic liver disease.

Clin. exp. Immunol. (1978) 33, 204-210. DNA-binding antibodies and hepatitis B markers in acute and chronic liver disease J. G. C. KINGHAM, SUHA RASS...
810KB Sizes 0 Downloads 0 Views