The immunology of liver disease

The Immunology of Liver Disease

SHEILA SHERLOCK,M.D. London, England

From the Department of Medicine. The Royal Free Hospital, London W.C. 1, England. This study was supported by grants from the Endowment Fund of the Royal Free Hospital and from the Davis Memorial Fund. Requests for reprints should be addressed to Dr. Sheila Sherlock.

Volume 49, November 1970

Serum immunoglobulin (IgG, IgA, IgM) values are not diagnostic of any one liver disease, although a high serum IgM in patients with chronic cholestasis makes obstruction to large main bile ducts unlikely. Changes in circulating antibodies are nonspecific and include positive antinuclear factor in some patients with chronic hepatitis and primary biliary cirrhosis. The antimitochondrial (M) test has proved useful in the differential diagnosis of primary biliary cirrhosis from surgical obstruction to main bile ducts. The antibody against smooth muscle is positive in about two thirds of patients with active chronic hepatitis. Delayed type hypersensitivity reactions are impaired in primary biliary cirrhosis, but this effect seems to be a consequence of the disease rather than related to etiology. A form of chronic hepatitis and cirrhosis is associated with persistence of Australia antigen in the serum. In some of these patients primary hepatocellular carcinoma may develop. The evaluation of "immunosuppressive" regimens in the treatment of liver disease, particularly active chronic hepatitis and primary biliary cirrhosis, has been impeded by lack of controlled prospective trials. However, recent controlled trials have suggested that prednisolone increases the life span of patients with active chronic hepatitis. Serum alpha-feto-globulin can be found in about 30 per cent of Caucasiansand 60 per cent of non-Caucasians suffering from primary hepatocellular carcinoma.

The last fifteen years has seen a great increase in our knowledge concerning the relationship of liver disease to disturbed immunologic processes. As always, they have depended on improved technology, both simple and advanced. Growing recognition of such disorders as acute active chronic hepatitis and primary biliary cirrhosis has led to greater efforts to study their pathogenesis and to evaluate them immunologically. Hepatic transplantation is now in its infancy, but its introduction has encouraged many well trained immunologists to enter the field of liver disease. Finally, by simple immunodiffusion methods, Australia antigen has been discovered, and the presence of serum (x-feto proteins has been reported in the blood of patients with hepatocellular carcinoma. This is a general review of all these aspects.

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IMMUNOLOGYOF LIVERDISEASE-- SHERLOCK

J o ON STEROIDS J

tially of slower gamma2 globulin [4,9]. Rarely, a monoclonal picture may be seen [10]. SERUM

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Serum IgG in chronic liver disease. From Feizi [12].

SERUM

GLOBULINS

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PATTERNS

Elevation of total serum globulin levels is a well known phenomenon accompanying most forms of liver disease. It was the basis of positivity of many of the seroflocculation tests which have now happily disappeared from the clinical scene. Recognition of different fractions of the total serum globulin concentration has been fruitful and has led to the first indications of an abnormal immune mechanism in patients with chronic liver disease. Since gamma globulin levels to a great extent reflect antibody-like protein levels, the hypergammaglobulinaemial which is associated with an increased turnover rate [1], is likely to be part of an immunologic response. Electrophoresis of the serum proteins shows a polyclonal (high gamma gamma) response in patients with advanced chronic hepatocellular disease. The increase in gamma globulin also involves fast gamma globulin [2] and leads to a characteristic filling in of the gap normally present between the beta and gamma gamma globulins [3] and also to lack of '~2 globulin (haptoglobulins and macroglobulins) synthesis, and these globulins may be impaired in the presence of liver disease [4]. Patients suffering from active chronic hepatitis (juvenile cirrhosis, lupoid hepatitis, plasma cell hepatitis) [ 5 - 8 ] show a particularly marked elevation in serum gamma globulin levels. The rise occurs in the presence of normal serum albumin and ~2 globulin levels, indicating well maintained hepatocellular function. The beta~gammagamma fusion is not seen but the gamma peak is narrower and more distinct and consists essen-

694

IMMUNOGLOBUMNS

Serum electrophoresis has been followed by estimation of the individual immunoglobulins, IgG, IgA, IgM, IgD and IgE, which are located in the electrophoretic regions beta and gamma; of these, only IgG, IgA and IgM have been well investigated in patients with liver disease (Figures 1, 2 and 3). The serum IgG level is particularly raised in active chronic hepatitis t1!] but also in a proportion of patients with other forms of chronic liver disease [12]. Elevation of serum IgA levels is said to be a feature of alcoholic disease [13-15], but equally high values are found in active chronic hepatitis [12]. The serum IgM level is raised in primary biliary cirrhosis, in general this is so, and, with few exceptions, a very high serum IgM level in the presence of cholestatic jaundice is strongly suggestive of primary biliary cirrhosis [16]. Normal results have been reported in extrahepatic biliary obstruction and in chlorpromazine-induced cholestasis [17]. Occassionally, however, high values are observed in chronic cholestasis and normal results in some patients with primary biliary cirrhosis [12,17]. Patients with acute viral hepatitis may have high IgM values [4], particularly in the first two weeks of the illness [18]. J O ON STEROIDS]

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Serum IgA in chronic liver disease. From Feizi [12].

The American Journal of Medicine

IMMUNOLOGY OF LIVER DISEASE- SHERLOCK

Depressed immunoglobulin levels (IgG, IgA and IgM) are said to occur in Wilson's disease [14], but this is not universal [12]. Patients with Wilson's disease may have very high total serum globulin levels, and the most common immunoglobulin change is an elevation of serum IgM levels [12]. The similarity in immunoglobulin patterns in chronic active hepatitis and cryptogenic cirrhosis is striking (Figure 4) and may represent a response to the same or similar antigenic stimuli in the two diseases [12]. When the similarity in the total serum gamma globulin levels extends to the degree of plasma cell infiltration and cell necrosis in the liver biopsy specimen, distinction can sometimes be difficult; these criteria may indeed merely represent variable individual immunologic responses. In many patients with chronic active hepatitis, cirrhosis ultimately develops. It is possible that cryptogenic cirrhosis may be the late stage of a subclinical active chronic hepatitis. Unfortunately serum immunoglobulin changes cannot be interpreted from the diagnostic viewpoint as reflecting any one type of hepatobiliary disease, but only as an indication of disturbed circulating globulins in the presence of almost any liver disorder. Immunoglobulins are not produced in the normal liver, but in chronic hepatic disease they have been identified in the lymphoid and plasma cells infiltrating portal zones and parenchyma. These

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Volume 49, November 1970

Cryptogenic c{rrhosis

Primary biliory cirrhosis

Alcoholic cirrhosis

Chronic cholestosis

Figure 4. Incidence of raised immunoglobu/in levers in active chronic hepatitis, cryptogenic cirrhosis, primary biliary cirrhosis, alcoholic cirrhosis and in chronic cholestasis due to biliary strictures or stones. From Feizi [12].

immunoglobulins are presumably manufactured by mesenchymal cells. The immunoglobulin-containing cells can be identified by a fluorescent antibody technic [19,20] (Figures 5 and 6). In patients with primary biliary cirrhosis serum IgM levels are high, and IgM.containing liver cells are found in large numbers in the liver, largely related to damaged bile ductules [17]. IgG appears to be the predominant immunoglobulin in the mesenchymal cells in active chronic hepatitis [19]. Over-all, in liver disease, however, IgA-containing cells predominate [20]. More cells are found in the presence of piecemeal necrosis of liver cells and lymphoid infiltration [20]. High serum immunoglobulin levels are not necessarily associated with high levels of immunoglobulin-containing cells in the liver [20]. CHANGES

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IN C I R C U L A T I N G

ANTIBODIES

The rise in serum immunoglobulin levels in patients with liver disease can be studied further by noting the individual antibodies contained within them. Antinuclear factor of both speckled and diffuse varieties is present in some half to one third of Patients with active chronic hepatitis and in 46 per cent of those with primary biliary cirrhosis or cryptogenic cirrhosis [21,22]. In 1953 the lupus erythematosus cell phenomenon was demonstrated in the blood of patients with active chronic hepatitis [23], and this led to application of the term "lupoid" hepatitis to the condition of active chronic hepatitis with lupus erythematosus cells in the blood [5], although the phenomenon can be shown in only 12 per cent of patients with this clinical picture [7]. In active chronic hepatitis, the titer of antinuclear

695

IMMUNOLOGY OF LIVER DISEASE -- SHERLOCK

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an autoimmune process has never been demonstrated. This possibility of autoaggression in liver disease now seems a little unlikely. Nevertheless, the demonstration of the various tissue autoantibodies in patients with liver disease has proved of considerable interest and has led to useful procedures which are valuable in diagnosis, if difficult to explain (Table I). Antimitochondrial (M) Antibody. Primary biliary cirrhosis is a diseasb characterized by slow progressive destruction of the septal and interlobular bile ducts [28,29]. It was therefore of considerable interest when a cytoplasmic antibody reacting with ductules was found in the serum of some 75 per cent of patients with this disease i-17]. This antibody seems to be organ-specific as it does not react with salivary duct cells. Unfortunately, it is also found in the serum of patients with other liver diseases, including 67 per cent of those with viral hepatitis and in 10 per cent of the normal population. However, a further more interesting immunofluorescent test has been described in primary biliary cirrhosis [22,30,311. Serum from patients with pri.

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Figure 5. Immunoglobulin.containing cells in the liver of pa. tients with hepatitis and cirrhosis. Ceil counts in excess of 0.5~high power field are joined by lines for the demonstration of the immunoglobulin pattern in each patient. From Hadziy. annis et al. [20].

antibody correlates with the serum gamma globulin level. In the 1950's growing interest in autoimmunity as an etiologic factor in disease, together with the demonstration of various immune reactions involving complement fixation with liver extracts, led to speculation that some types of liver disease would turn out to be related to autoimmunity. Active chronic hepatitis was an obvious choice, since many nonspecific antibodies had already been demonstrated in the serum of sufferers, and the marked lymphocytic infiltration of the liver suggested the forbidden clones of autoimmunity. It was suggested that, after viral or nutritional injury, liver cell components might become antigenic and so provoke an autoimmune reaction that was responsible for perpetuation of inflammation and progression to a macronodular cirrhosis [5,24-26]. However, although many investigators have applied the complement fixation reaction to studies of antibodies in liver disease, in no instance has there been evidence of an organ-specific liver antigen [ 2 2 , 2 4 27]. Destruction of liver cells as a response to

696

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Figure 6. Immunoglobulin containing ceils in the liver of patients with primary biliary cirrhosis, secondary biliary cirrhosis and other biliary diseases. Cell counts in excess of 0.5/high power field have been joined by lines as in Figure 5. From Hadziyannis etal. [20].

The American Journal of Medicine

IMMUNOLOGY OF LIVER DISEASE -- SHERLOCK

TABLE I

Smooth Muscle (SMA) and Mitochondrial (M) I m m u n o f l u o r e s c e n c e in V a r i o u s Diseases*

% Positive Disease Active chronic hepatitis Primary biliary cirrhosis, Systemic lupus erythematosus Extrahepatic biliary obstruction

No. SMA

No.

M

39 40 10 20

12 40 17 28

28 98 29 7

67 50 0 0

*Data f r o m Doniach et al. [ 2 2 ] .

mary biliary cirrhosis gives granular cytoplasmic staining in unfixed tissue sections, using a fluorescein-conjugate of antihuman gamma globulin and complement in the double-layer immunofluorescent test. The staining reactions are not organ- or species-specific, but cells known to be rich in mitochondria are preferentially stained. This M test can be adapted as a complement fixation test using liver or kidney. The antigen involved has been localized to the mitochondria. A further series of elegant studies showed that the complement fixing antigen was a component of the mitochondrial inner membrane [32-34]. The enzymes of the electron transport chain and a number of regularly extractable mitochondrial proteins are not associated with the fragment containing the antigen. There is some evidence that the antigen, which is a lipoprotein, may be a membrane transport protein [35]. Since inner membranes have structural similarities with the coat of some viruses or bacteria, there is a possibility that M antibodies indicate cross reaction with as yet unidentified microorganisms [35]. This M antibody was present in 187 of 213 (83 per cent) patients with primary biliary cirrhosis and in only two of 253 (0.8 per cent) patients with mechanical obstruction to main bile ducts [45]. This makes it a very useful and practical method of distinguishing primary biliary cirrhosis from forms of cholestasis in which surgical intervention might be useful. The M antibody is also present in active chronic hepatitis (23 per cent) and cryptogenic cirrhosis (25 per cent), but here the differentiation from surgical jaundice is not important. Low titers are usually found in these and other conditions such as virus hepatitis and cholestatic drug jaundice. It is negative in pericholangitis and sclerosing cholangitis secondary to ulcerative colitis, and also in alcoholic liver disease. Results for the M test show a fair correlation with the level of serum IgM [42] (Figure 7). Smooth Muscle Antibody. Active chronic hepatitis

Volume 49, November 1970

is a chronic disease of the liver affecting mainly young girls, but also subjects of all ages and both sexes. Associated diseases include ulcerative colitis, thyroiditis, diabetes, necrosing dermal arteriolitis, renal tubular lesions, fever and arthralgias. Insidious, usually mild, jaundice is associated with hepatosplenomegaly and the late development of liver failure. Serum transaminase and gamma globulin levels are markedly elevated. It has variously been called lupoid hepatitis [5], juvenile'cirrhosis [7] and plasma cell hepatitis [6]. Various, nonspecific serologic antibody tests are positive [21]. Johnson and coworkers [36] demonstrated a further antibody which reacted by immunofluorescence with smooth muscle in eight of ten patients with probable active chronic hepatitis, and this was confirmed by Whittingham and co-workers [37] in Melbourne. Smooth muscle antibody can be differentiated from other known antitissue antibodies demonstrated by immunofluorescence, including antinuclear factor, the skeletal muscle autoantibody of myasthenia gravis, and thyroid and gastric parietal cell antibody. It is not present in systemic lupus erythematosus with positive antinuclear fluorescence. This is strong evidence that active chronic hepatitis is not a variant of systemic lupus erythematosus. One patient with active chronic hepatitis and membranous glomerular nephritis had smooth muscle antibody in the serum and immunofluorescence revealed gamma globulin 800 700

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Figure 7. Correlation of serum IgM level and mitochondrial antibody titers in primary biliary cirrhosis. M ~- mitochondrial antibody test. From Hadziyannis et al. [42].

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IMMUNOLOGY OF LIVER DISEASE -- SHERLOCK

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[39].

Figure 9. Active chronic hepatitis. There is marked piecemeal necrosis and isolation of liver cells into isolated groups (rosettes). Plasma and lymphocytic infiltration is conspicuous. Hematoxylin and eosin stain, original magnification X 40.

Figure

10, Primary biliary cirrhosis. A granuloma with a giant cell adjoins damaged septal bile ducts. Cellular infiltration is conspicuous. Hematoxylin and eosin strain, original magnification X 40.

698

The American Journal of Medicine

IMMUNOLOGY OF LIVER DISEASE - - SHERLOCK

in the glomeruli [37]. This might be related to the mild glomerular lesions sometimes seen in patients with active chronic hepatitis [38]. The smooth muscle antibody test is helpful in confirming the diagnosis of active chronic hepatitis in about 70 per cent of cases [22,36,37]. This antibody is also present in the serum of some 50 per cent of patients with primary biliary cirrhosis and 28 per cent with cryptogenic cirrhosis, and in low titers in some patients with acute viral hepatitis [22]. The significance of this reaction is unknown. The antibody is not organ- or species-specific, and the relevant antigen has not been found in liver. It cannot therefore be regarded as an antibody against any component of the liver cell so far identified. DISTURBANCES OF CELL-BASED IMMUNITY (DELAYED-TYPE HYPERSENSITIVITY) Some liver diseases are marked by histologic changes very similar to those seen in the rejecting hepatic transplant. In both primary biliary cirrhosis and active chronic hepatitis there are lymphocyte accumulations, often in follicles, occasional granuIomas and many plasma cells, all features of the rejection reaction (Figures 8 and 9). Graft versus host (runting) rejection reactions are believed to be mediated predominantly by destructive mononuclear cells (lymphocytes), hence disturbances of cell-based (delayed-type hypersensitivity) immunity might be anticipated in these three chronic liver diseases; very little attention has been paid to this aspect of the problem. Some granulomas seem to be dependent upon immune processes. For instance, chronic detayed hypersensitivity induced in animals leads to formation of granulomas, and cellular immunity in response to certain infections is manifest in granulomatous reactions. The association of granulomas histologically indistinguishable from those of sarcoidosis in some patients with primary biliary cirrhosis is therefore of particular interest [29,31] (Figure 8). They were present in twenty-nine of sixty-nine patients in one series [39]. The granulomas are not only in relation to damaged bile ducts but can be found also in lymph nodes outside the liver in the hepatic hilum and in the omentum, also in the pulmonary lymph nodes [39]. Enlarged, fleshy lymph glands are well recognized in the porta hepatis and along the bile ducts in primary biliary cirrhosis. Whether or not the formation of granulomas in primary biliary cirrhosis is an indication of delayed hypersensitivity to unknown antigenic material remains unknown.

Volume 49, November 1970

Depression of delayed-type hypersensitivity in primary bi!iary cirrhosis can also be demonstrated by anergy to various skin test materials such as the chemical sensitizer 2,4-1 di-nitrochlorobenzene (DNCB). An initial application is made to the skin of an arm, followed one month later by the application of a dilute solution to another area of the skin. A positive reaction comprises erythema and induration at forty-eight hours; 94 per cent of control subjects have a positive reaction, compared with 46 per cent of those with primary biliary cirrhosis (P <0.01) [39]. Similarly a significantly higher percentage of patients with primarY biliary cirrhosis fail to react to tuberculin when compared with normal controls. In healthy subjects, lymphocyte transformation in response to phytohaemagglutin is striking and morphologic examination reveals that at seventy-two hours 40 to 80 per cent of the cells are large immunoblasts. In patients with primary biliary cirrhosis there is impaired transformation compared with healthy subjects or those with chronic cholestasis due to other causes (Figure 10). Tobias and coworkers [40] have shown that one of two patients with chronic active hepatitis and two of four with primary biliary cirrhosis gave positive lymphocyte transformation reactions using as antigen their own liver tissue obtained by needle biopsy. Three patients with alcoholic liver disease and one with colitis and pericholangitis gave negative results. These findings also provide evidence that immunologic reactions of the delayed hypersensitive type are abnormal in some forms of chronic liver disease. In another study, using phytohaemagglutin, it was shown that peripheral lymphocytes from patients with acute infectious hepatitis and chronic aggressive hepatitis could not be stimulated in culture [41]. Patients with alcoholic hepatitis showed normal results. The similar findings in virus hepatitis and aggressive hepatitis suggested that persistence of viral infection had led to aggressive chronic liver disease. The disturbed delayed-type hypersensitivity reaction seems to be unrelated to the raised levels of nonspecific circulating antibodies. In primary biliary cirrhosis almost all patients had positive antimitochondrial antibodies whereas only half were anergic by skin testing and lymphocyte transformation [39]. Similarly, although all the patients with chronic liver disease studied who showed a positive lymphocyte transformation also were found to have anticytoplasmic antibodies at some time in the course of their illness, the converse was not true [40]. In

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I M M U N O L O G Y OF LIVER DISEASE - - SHERLOCK

another study, in patients with primary biliary cirrhosis, no correlation was found between the histologic evidences of disturbance of cellular immunity (lymphoid and plasma cell aggregates with or without germinal centres and granulomas) and the level of serum M antibodies [42]. These results support the concept that the serum M antibody at least is a marker of disease and not quantitatively related to the disturbed cell immunity and to its severity. THE RELATIONSHIP BETWEEN ACTIVE CHRONIC HEPATITIS, PRIMARY BILIARY CIRRHOSIS AND CRYPTOGENIC CIRRHOSIS

The regular and consistent demonstration in high titer of antibody tests such as antinuclear fluorescence (ANF), smooth muscle antibody (SMA) and antimitochondrial (M) are virtually confined to three liver diseases, active chronic hepatitis, primary biliary cirrhosis and cryptogenic cirrhosis, particularly in women [43]. Results are negative in such conditions as alcoholic liver disease, Wilson's disease, sclerosing cholangitis and biliary atresia, even though the extent of hepatocellular or biliary damage may be as great. This suggests that the increases do not simply represent antibodies developing as a response to constituents of damaged liver cells or bile ducts. There is considerable overlap between the hepatic histology of active chronic hepatitis (Figure 8) and primary biliary cirrhosis (Figure 9). Both show marked mononuclear cell infiltration with accumulation into lymphoid follicles, but piecemeal necrosis is more conspicuous in active chronic hepatitis whereas bile duct injury is more characteristic of primary biliary cirrhosis. Moreover, I have under my care a young female patient with chronic liver disease in whom multiple liver biopsy specimens show the classic features of active chronic hepatitis in one and those of primary biliary cirrhosis in another. Hans Popper (personal communication) goes one better and owns a liver biopsy slide, one half of which shows classic active hepatitis and the other primary biliary cirrhosis. The similarity of the serum immunoglobulin patterns in active chronic and cryptogenic cirrhosis has already been discussed. This led to a suggestion that the three diseases result from a single process [!2,43]. The subclinical hepatitis with positive M test recently described in some patients with collagenoses might represent an early stage of active chronic hepatitis or primary biliary cirrhosis may progress toward damage to hepatocytes or to bile ductules [44]. It is also possible that primary biliary cirrhosis may occur in a

700

low grade form which remains nonprogressive. It is uncertain whether the inlurious process is related to autoimmunity, as suggested by Doniach and others [43], or whether the immune processes directed against tissue constituents are responsible for the pathogenesis of the lesions in the liver. Despite all the marked immunologic disturbances shown In active chronic hepatitis, primary biliary cirrhosis and some forms of cryptogenic cirrhosis, it is difficult to relate them to etiology and in particular to call these diseases autoimmune. Animal experiments have also failed to provide an autoimmune basis for liver injury. Immunization of animals with liver extracts has not given rise to a convincing form of hepatitis or progression to cirrhosis [46]. The fundamental difficulty remains that the various antibodies demonstrated are not hepatobiliary specific. Liver cell specific antibodies which could have provided the basis for such a localization have not been demonstrated. Similarly although antiductular antibodies have been found in primary biliary cirrhosis they have proved to be nonspecific, and the antimitochondrial antibody reacts with mitochondria from every tissue. At a cellular level the demonstration of altered delayed-type hypersensitivity in primary biliary cirrhosis seems to be a consequence rather than a cause of the disease. Why these three conditions should have histologic and serologic features in common remains unknown. AUSTRALIA (ASSOCIATED) ANTIGEN WITH CHRONIC HEPATITIS

Chronic persistent hepatitis, chronic aggressive he patitis and cirrhosis have all been reported in persons with persistent Australia antigen in their serum [47-50]. The aggressive hepatitis in these patients has many points in common with the chronic active (lupoid) hepatitis already discussed in which the test for Australia antigen is negative [50]. Hepatic histology may be identical, serum gamma globulin levels may be as high, and a history of a preceding attack of acute hepatitis is found in a similar number in both groups. Differences from classic active chronic hepatitis include the high incidence n older men, the occasional appearance of the liver biopsy specimen suggesting a recent hepatitis, less conspicuous splenomegaly and stigmata of chronic liver disease, and absence of the diseases commonly associated with classic active chronic hepatitis, such as arthralgias and fever. In view of the close similarity of the hepatic histologic appearances of the two

The American Journal of Medicine

IMMUNOLOGY

groups, plasma cells and lymphocytes abounding in both, serum antibody studies in the Australia antigen-associated group are of considerable interest. Smooth muscle antibody is present in 67 per cent of patients with classic active chronic hepatitis and is either absent or present in low titer in patients with the Australia antigen-associated disease. Of twelve such patients it was absent in nine, present in a titer of only 1 in 10 in two and 1 in 40 in one [50]. This is very similar to the figures reported during the actue stage of virus hepatitis [22]. In another study, nineteen of seventY-eight (24 per cent) patients with acute viral hepatitis had smooth muscle antibody. It was often present only intermittently, it occurred as frequently in patients with a posffive reaction to Australia antigen as in those with a negative reaction, and it could not be detected both early and late in the disease [51]. In nine it was present in high titer. In patients with chronic hepatitis, there seem to be an inverse ratio between the presence of Australia antigen and smooth muscle antibody, and this again suggests that these are etiologically distinct forms of the disease [51]. It was shown that smooth muscle antibody is not an antibody to the Australia antigen [51]. These results might indicate that the type of viral hepatitis with persistent Australia antigen does not initiate the type of chronic hepatitis predominantly affecting young girls and called active, chronic or lupoid. It does not exclude another type of viral hepatitis as the basis. Almeida and Waterson [52], in a preliminary communication, found by electron microscopy that Australia antigen-antibody complexes were present in the serum of patients with virus hepatitis and suggested that the state of the antigen and the presence of the antibody might be correlated with different responses of the host. One person, a carrier, is known to have had the antigen in his blood for at least twenty years and seems to have been unharmed by it. On the other hand, he produced no antibody to it. In a second patient with chronic active hepatitis, the serum contained both free antigen (i.e., unbound by antibody) and also antigen-antibody complexes. This is characteristic of a system with antigen excess. The host response is too poor to clear the antigen but adequate for the continued formation of antigen-antibody complexes. Such complexes, by analogy with acute nephritis, could be injurious to the liver cell. Dixon [53] has stated that the prolonged presence of immune complexes would lead to a situation as much degenerative as inflammatory. Such complexes would take up complement and would explain the anticomplementary

Volume 49, November 1970

OF L I V E R D I S E A S E - - S H E R L O C K

nature of 95 per cent of the serum specimens from patients with acute viral hepatitis [54]. In another series seven of sixteen serum samples with positive Australia antigen showed slight to moderate degrees of anticomplementary activity, suggesting the presence of antigen-antibody complexes [55]. The postulate that chronic hepatitis with positive Australia antigen is an immune complex disease is an interesting one but needs further investigation before it can be said to be proved. IMMUNOLOGIC CHANGES IN DRUG-RELATED LIVER DISEASE

Circulating antibodies are unusual in drug-related jaundice. However, they have been noted in a small proportion of cases. Antimitochondrial antibodies were found in seven of nine serum samples from patients with jaundice after halothane anaesthesia, and in four of seven with jaundice after chlorpromazine [56]. In another study, three of five patients with drug-related hepatitis, two halothanerelated and one following chlordiazepoxide, gave a positive M test by fluorescence, and in two the complement fixation test was positive [22]. The titer of M antibodies was usually low and transient. Seven patients with cholestatic drug jaundice gave a negative reaction by fluorescence. Rodriguez and co-workers [56] postulate that some drugs, such as halothane or chlorpromazine, might combine with mitochondria or produce mitochondrial damage and form stable drug-mitochondrial complexes to elicit an immunologic reaction. Positive results for antinuclear fluorescence and thyroid and gastric parietal cell antibodies were no greater than in matched controls [22]. Tests for Australia (hepatitis-associated) antigen are negative in drug-related jaundice [57,58], making it unlikely that the drug activates the virus and that the disease is a coincidental virus hepatitis. Halothane toxicity is particularly incriminated as a sensitization phenomenon. The latent period lessens with each exposure and fevers, rash and eosinophila may be associated [59]. In an anesthetist with recurrent hepatitis after halothane, eosinophilia developed after rechallenge [60]. Hepatic histology shows multiple granulomas, plasma cells and eosinophils [61]. Moreover, an increase of '~Hthymidine uptake into the DNA of lymphocytes incubated with halothane was noted using the plasma of six of eight patients with hepatitis after halothane exposure, but not in healthy control subjects or in three patients with chronic liver disease. The effect disappeared with recovery [58],

701

IMMUNOLOGY OF LIVER DISEASE -- SHERLOCK

T A B L E II

Trial of Prednisolone in Active Chronic Hepatitis--Royal Free Hospital 1963-1969"

Subjects

No.

Alive

Dead

Treated

22 27

19 12

3 15

Controls

p <0.01 *Data

of Cook et al. [65].

M antibodies were found in six of the eight patients and in general correlated with the lymphocyte results. This supports the view that the patients had become sensitized to halothane. This might be a useful method of differentiating halothane-related hepatitis from acute viral hepatitis, which it resembles so closely. IMMUNOSUPPRESSIVE

REGIMENS

Marked lymphoreticular hyperactivity, e v i d e n c e d by increases in immunoglobulins and lymphocytic tissue accumulation, is a feature of almost all forms of chronic liver disease, so it is not surprising that corticosteroid therapy has been used for treatment. In immunopathologic conditions, corticosteroids probably act by reducing the damaging effects of antigen-antibody interaction at or near cell surfaces, as well as by exerting a general nonspecific effect on the maintenance of cellular integrity. The resulting decreased release of antigen would break a vicious circle process. Diseases such as chronic active hepatitis and primary biliary cirrhosis, in which the immunologic disturbance is particularly marked, have been special candidates for prednisone treatment. Unfortunately, many of the clinical trials of its use have been uncontrolled or retrospective and so have been of limited value. The Copenhagen Study Group [62] report a prospective controlled trial on the effect of prednisone on the survival of patients with chronic liver disease. The patients were randomly allotted to the treated or the untreated group on the basis of their date of birth. A comparison was made between 159 prednisone-treated patients and 165 controls. Five centers in Denmark were involved, a very good example of cooperative effort. The death rate was the same in both groups. When female patients without ascites were considered alone, the death rate in the prednisone-treated cases was significantly reduced. It seems likely that many of these latter patients fell into the active chronic (lupoid)

702

hepatitis group. In this condition, it is generally agreed, prednisone induces well-being, improves appetite and lessens fatigue. These could simply be nonspecific effects of the drug. Biochemical changes are less constant although serum bilirubin, transaminase and gamma globulin levels usually fall [5-8,63]. The effect on hepatic histology is variable and unconvincing, but there is, unfortunately, a large sampling error when needle biopsy specimens are used to assess this condition. Certainly progression from the stage of chronic hepatitis to that of cirrhosis does not seem to be prevented [7]. Prospective randomized clinical trials are difficult to set up and carry through, and results may not be meaningful for many months or indeed years. A controlled clinical trial of prednisolone in active chronic hepatitis has been carried out at the Royal Free Hospital in London [64,65] (Table II). Sealed envelopes were used to assign patients to treated or control groups. After six years, analysis of the results showed a marked advantage for the prednisolone-treated patients: only three of twenty-two died, whereas seventeen of the twenty-seven control subjects died (p < 0.01). Cure of the disease or prevention of cirrhosis was not suggested, but early deaths, particularly in the first two years, were less. The results so greatly favored prednisolone therapy that it became unjustifiable to continue the trial, so patients in the control group were given prednisolone if there was no contraindication. Apart from any possible beneficial effects in altering immune responses, prednisolone had a marked effect on parameters which are apparently specific measures of hepatocellular function. In particular, the storage capacity for bromsulfalein was significantly better in the treated than in the control group, although 5.0

o E ,..=4.0

~ 3.0

2.0

;2

2'4

3'6

4'8

~o

7'2

TIHE {months)

Figure 11. Royal Free Hospital trial of prednisone treatment of active chronic hepatitis. 9 Corticosteroid. 9 controls. The mean • 1 standard deviation of the mean is shown for each time interval. Differences between the means are significant at 6 (t = 2.62; p < 0.02), 12 (t _~ 3.76, p < 0.001), 18 (t = 4.91, p < 0.001) and 24 (t ~ 4.13, p < 0.001) months. From Cook et al. [65].

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the transport maximum was unchanged [64]. Serum albumin concentration rose, so that in one year it had reached normal values, whereas in the untreated patients results were significantly lower (Figure 11). At three to five years after commencing treatment, this difference had disappeared, and the serum albumin concentration in both treated and control subjects was equal. These observations suggest that prednisolone might have a direct effect on hepatocellular function. Cortisol markedly increases serum albumin synthesis in the isolated perfused rat liver, and the absolute synthetic rate of albumin is also increased by prednisolone in patients with chronic liver disease [66,67]. Preliminary results have been reported from the Mayo Clinic [68] of a well planned double-blind trial of prednisolone therapy in chronic active liver disease. Patients were selected if the illness was of more than ten weeks' duration, if liver biopsy had been performed and if there was a persistent tenfold increase in serum glutamic oxalacetic transaminase (SGOT) or a fivefold elevation with a twofold increase in gamma globulin. There were fifteen patients in the group given prednisone (20 mg/day), thirteen in the group receiving placebo therapy. So far there has been one death in the latter group. Subjective improvement was the same in both groups. Currently, prednisolone shows significant advantages (P < 0.05) in decreasing serum bilirubin, SGOT and gamma globulin levels, and piecemeal necrosis. Azathioprine is an alternative method of treatment. It inhibits cell mitosis as an antimetabolite and reduces the production of antibody. In a clinical trial, Mackay [69] found that azathioprine (100 to 200 mg daily, depending upon the neutrophil leukocyte count) was as effective as prednisolone in improving biochemical tests and maintaining quiescence. There was no significant improvement in any index after the initial zero to three month period. The suppressive treatment served to maintain the initial improvement in liver function. In another study, Mistilis and Blackburn [70] gave 6-mercaptopurine or azathioprine to seventeen patients with active chronic hepatitis; in eight, toxic reactions included anorexia, jaundice, coma, thrombocytopenia and leukopenia. They seemed to be related to both the dose and the severity of the underlying liver disease. One hundred milligrams appeared to be excessive for many patients with active chronic hepatitis; in a smaller dose, fourteen patients were treated for up to four and a half years with complete freedom from toxicity. 6-Mercaptopurine was

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given to twenty-three patients with active chronic hepatitis in the pediatric age range, and the results compared with a previously published untreated series of patients [71]. A good therapeutic response was associated with positivity for four parameters, namely, onset of disease consistent with an attack of acute infective hepatitis, symptoms of disease in other systems, high serum levels of gamma globulin and suspicious or positive lupus erythematosus cell tests. In a Mayo Clinic trial [68] one group was given azathioprine in a dose of 100 mg daily; on the whole this group did not do as well as those receiving placebo or prednisolone therapy. There were four deaths among thirteen azathioprine-treated patients (none in prednisolone-treated, one in the placebotreated patients), and toxicity necessitated withdrawal of the drug in four patients. These investigators conclude that the therapeutic efficiency of azathioprine is currently suspect. A conclusion is difficult on the basis of these studies. The problems of controlled trials, together with the different criteria used by the various groups for inclusion, make results difficult to compare. It does, however, seem that the results of prednisolone therapy of chronic active liver disease are beneficial, especially in the early stages. It should be continued in a maintenance dose of 10 to 20 mg daily until results of biochemical tests are normal; this usually takes at least six months. Relapses necessitate further treatment. Azathioprine should be reserved for those in whom complications follow prednisolone therapy, when a coincidental condition such as diabetes precludes its use or when control is not achieved with prednisolone alone. Prednisolone is contraindicated in primary biliary cirrhosis because of the bone thinning which commonly complicates this condition [72]. So far, there is only one report of it [73]. Two patients were treated; one, nonicteric, showed a reduction in pruritus, serum alkaline phosphatase, bromsulfalein retention and liver size; the other was icteric and showed no change in liver size, although pruritus lessened and serum alkaline phosphatase and bilirubin levels decreased. Clearly, more patients must be assessed and a controlled trial of azathioprine therapy in primary biliary cirrhosis is now in progress at the Royal Free Hospital. HEPATIC HOMOTRANSPLANTATION

Currently (April 1970), there will have been 109 orthotopic hepatic homotransplantations performed by thirty-three teams. There are eight survivors, the

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IMMUNOLOGYOF LIVERDISEASE-- SHERLOCK

into the actively antimetabolic substance. It is obviously important, in the prevention of rejection, that there should be good matching of donor and recipient on the basis of tissue typing. In fact, there are many exceptions to the rule of good match, good result. Starzl's longest survivor (254 days) [76] had a particularly poor match. The transplanted liver has been shown to synthesize serum albumin [77]. In one patient with primary biliary cirrhosis, serum antimitochondrial antibodies fell when she received a new liver [77]. The transplanted liver, however, can make gamma globulin of the phenotype of the recipient [78]. SERUM ALPHA-FETO PROTEIN IN PATIENTS WITH LIVER DISEASE Figure 12. Immunoelectrophoretic patterns of fetal serum (f), two serum samples from patients with hepatocellular carcinoma (hl and h2), and a d u l t serum (a). The well contained rabbit anti-~,-fetoprotein (M7) or horse antihuman serum protein (H). Lines of precipitation, marked with arrows, were observed for f, h l and h2 against M7 but not for a. From Foil et al. [ 8 3 ] .

longest for twenty-six months. If the patient survives the operative period, death has usually resulted from rejection (with hepatic failure) or from infections consequent upon the large doses of antirejection drugs. Rejection usually commences about a week postoperatively and is characterized by increasing serum bilirubin and transaminase values. Hepatic histology shows marked infiltration with lymphoid and plasma cells, focal necrosis of liver cells, sometimes with acidophil bodies, and centrilobular cholestasis and hepatocellular dropout. The appearances resemble those of active chronic hepatitis. Later, varying degrees of fibrosis link central areas to central areas [74]. How much these changes are related to rejection and how much to the effect of antimetabolite therapy remains uncertain. The vascular aspect of rejection is also important. There is a spasm of the hepatic artery and portal vein, with ischemic liver damage. Gamma globulin fluorescence can be demonstrated in the hepatic artery and portal vein in the human liver after rejection [75]. Rejection is treated by increasing the dose of prednisolone and perhaps giving antilymphocyte serum. Increase in azathioprine dosage is not helpful as this requires good hepatocellular function for its transformation

704

The appearance of fetal antigens in patients with neoplastic diseases is well recognized. One such antigen is an e,-feto-protein (Figure 12). This protein, which is a normal component of plasma proteins in human fetuses older than six weeks, reaches maximal concentration between the twelfth and sixteenth week of fetal life. A few weeks after birth, the protein disappears from the circulation, and it is absent in healthy children and adults. The fetal serum protein reappears in patients with primary carcinoma of the liver [79,80], and it has been suggested that its presence is diagnostic of this condition [81,82]. It is also found in one third of embryonal tumors of the ovary and testis in childhood. There is considerable geographic variation in the frequency of positive results in patients with hepatocellular carcinoma. In.o~non-Caucasians, it is present in about 60 to 70 per cent, whereas in Caucasians it is about 30 per cent [83,84]. The positivity is related to size of tumor and to its anaplastic properties [84]. Survival is shorter in those with a positive test, the mean being 120 days in those with the fetal protein and 240 days in those without [84]. The etiology of primary liver cancer is not known, s o it is of considerable interest that five patients with cirrhosis complicated by primary liver cell cancer have also shown positive tests for serum Australia (hepatitis-associated) antigen [50]. Two of these also showed positive (x-fetoprotein tests. This might suggest that viral hepatitis, and the cirrhosis which rarely follows it, is a precancerous condition.

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IMMUNOLOGY OF LIVER DISEASE -- SHERLOCK

REFERENCES 1. Cohen S: "f-Globulin metabolism.Brit Med Bull 19: 202,1963. 2. Franklin M, Bean WB, Paul WD, Routh JI, De la Huerga J and Popper H: Electrophoretic studies in liver disease; gamma, globulin in chronic liver disease. J Clin Invest 30: 729, 1951. 3. Demeulenaere L, Wieme RJ: Special electrophoretic anomalies in the serum of liver patients. A report of 1,145 cases. Amer J Dig Dis 6: 661, 1961. 4. Hobbs JR: Serum proteins in liver disease. Proc Roy Soc Med 60: 1250, 1967. 5. Mackay IR, Taft LI, Cowling DC: Lupoid hepatitis. Lancet 2: 1323, 1956. 6. Page AR, Good RA: Plasma-cell hepatitis, with special attention to steroid therapy. J Dis Child 99: 288, 1960. 7. Read AE, Sherlock S, Harrison CV: Active juvenile cirrhosis considered as part of a systemic disease and the effect of corticosteroid therapy. Gut 4: 378, 1963. 8. Sherlock S: WaldenstrSm's chronic active hepatitis. Acta Med Scand 179 (supp 445): 426, 1966. 9. Tomasi TB Jr: Diseases of the liver, Immunological Diseases (Samter M, Alexander HL, ed), Boston, Little Brown, 1965, p 881. 10. Awadzki AZ, Edwards GA: Dysimmunoglobulinaemia associated with hepato-biliary disorders. Amer J Med 48: 196, 1970. 11. Schumacher K: Die 13edeutung immunologischer Reaktionen fiJr Diagnose und Pathogenese der chronischen Hepatitis. Kiln Wschr 47: 949, 1969. 12. Feizi T: Immunoglobulins in chronic liver disease. Gut9: 193, 1968. 13. Lee Fl: Immunoglobulins in viral hepatitis and active alcoholic liver-disease. Lancet 2: 1043, 1965. 14. McKelvey EM, Fahey JL: Immunoglobulin changes in disease: quantitation on the basis of heavy polypeptide chains, IgG (G), IgA (A), and IgM (M), a n d of light polypeptide chains type K (I) and type L (ll). J Clin Invest 44: 1778, 1965. 15. Gleichman E, Deicher H: Quantitative Immunoglobulin-Bestimmungen im Serum bei entzLindlichen Leberkrankheiten. Kiln Wschr 46: 793, 1968. 16. Bevan G, Baldus WP, Gleich GJ: Serum immunoglobulin levels in cholestasis. Gastroenterology 56: 1040, 1969. 17. Paronetto F, Schaffner F, Popper H: Immunocytochemical and serological observations in primary biliary cirrhosis. New Eng J Med 271: 1123, 1964. 18. Fateh-Moghadam A, Lamerz R, Eisenberg J, Knedel M: Die Bedeutung der Immunglobuline for die Diagnose und Verlaufsbeurteilung von Lebererkrankungen. Kiln Wschr 47: 129, 1969. 19. Paronetto F, Rubin E, Popper H: Local formation of-r-globulin in the diseased liver, and its relation to hepatic necrosis. Lab Invest 11: 150, 1962. 20. Hadziyannis S, Feizi T, Scheuer PJ, Sherk3ck S: Immunoglobulin-containing cells in the liver. Clin Exp Immun 5: 497, 1969. 21. Bouchier lAD, Rhodes K, Sherlock S: Serological abnorrealities in patients with liver disease. Brit Med J 1: 592, 1964. 22. Doniach D, Roitt IM, Walker JG, Sherlock S: Tissue antibodies in primary biliary cirrhosis, active chronic (lupoid)hepatitis, cryptogenic cirrhosis and other liver diseases and their clinical implications. Clin Exp Immun 1: 237, 1966.

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23. Saint EG, King WE, Joske RA, Finckh ES: Course of infectious hepatitis with special reference to prognosis and chronic stage. Aust Ann Med 2: 113, 1953. 24. Gajdusek DC: An autoimmune reaction against human tissue antigens in certain acute and chronic diseases. I. Serological investigations. Arch Intern Med (Chicago) 101: 9, 1958. 25. Asherson GL: Antibodies against nuclear and cytoplasmic cell constituents in system!c lupus erythematosus and other diseases. Brit J Exp Path 40: 209, 1959. 26. Muschel LH, Simonton LA, Wells PA, Fife EH Jr: Occurrence of complement-fixing antibodies reactive with normal tissueconstituents in normal and disease states. J Clin Invest 40: 517, 1961. 27. Pasnick L J, Beall GN, Vanarsdel PP Jr: Complement fixing reactions with liver tissue in human disease. Amer J Med 33: 774, 1962. 28. Sherlock S: Primary biliary cirrhosis (chronic intrahepatic obstructive jaundice). Gastroenterology 37: 574, 1959. 29. Rubin E, Schaffner F, Popper H: Primary biliary cirrhosis. Chronic non-suppurative destructive cholangitis. Amer J Path 46: 387, 1965. 30. Walker JG, Doniach D, Roitt IM, Sherlock S: Serological tests in diagnosis of primary biliary cirrhosis. Lancet 1: 827, 1965. 31. Goudie RB, MacSween RNM, Goldberg DM: Serological and histological diagnoses of primary biliary cirrhosis. J Clin Path 19: 527, 1966. 32. Berg PA, Doniach D, Roitt IM: Mitochondrial antibodies in primary biliary cirrhosis. 1. Localization of the antigen to mitochondrial membranes. J Exp Med 126: 277, 1967. 33. Berg PA, Muscatello U, Horne RW, Roitt IM, Doniach D: Mitochondrial antibodies in primary biliary cirrhosis. I1. The complement fixing antigen as a component of mitochondrial inner membranes. Brit J Exp Path 50: 200, 1969. 34. Berg PA, Doniach D, Roitt IM: Immunologische Ph~nomene bei Leberkrankheiten: die Bedeutung mitochondrialer Antik6rper. Kiln Wschr 47: 1297, 1969. 35. Berg PA, Roitt IM, Doniach D, Horne RW: Mitochondrial antibodies in primary biliary cirrhosis. II1. Characterization of the inner-membrane complement fixing antigen. Clin Exp Immun 4: 511, 1969. 36. Johnson GD, Holborow EJ, Glynn LE: Antibody to smooth muscle in patients with liver disease. Lancet 2: 878, 1965. 37. Whittingham S, Mackay IR, Irwin J: Autoimmune hepatitis. Immunofluorescence reactions with cytoplasm of smooth muscle and renal glomerular cells. Lancet 1: 1333, 1966. 38. Silva H, Hall EW, Hill KR, Shaldon S, Sherlock S: Renal involvement in active juvenile cirrhosis. J Clin Path 18" 157, 1965. 39. Fox RA, Scheuer PJ, James DG, Sharma O, Sherlock S: Impaired delayed hypersensitivity in primary biliary cirrhosis. Lancet 1: 959, 1969. 40. Tobias H, Safran AP, Schaffner F: Lymphocyte stimulation and chronic liver disease Lancet 1: 193, 1967. 41. Rossler R, Havemann K, Dolle W: Unterschiedliche Reaktion von Lymphocyten auf Phytoh~magglutinin (PHA) bei Lebererkrankungen. Kiln Wschr 47: 803, 1969. 42. Hadziyannis S, Scheuer PJ, Feizi T, Naccarato R, Doniach, D, Sherlock S: Immunological and histological studies in primary biliary cirrhosis. J Clin Path 23: 95, 1970. 43. Doniach D, Walker JG: A unified concept of autoimmune hepatitis. Lancet 1: 813, 1969.

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44. Walker JG, Doniach D, Doniach h Mitochondrial antibodies and subclinical liver disease. Quart J Med 39: 31, 1970. 45. Doniach D, Walker JG, Roitt IM, Berg PA: Current .oncepts: "autoallergic" hepatitis. New Eng J Med 282: 86, 1970. 46: Paronetto F, Popper H: Hetero-iso- and autoimmune phenomena in the liver, Textbook of Immunopathology, vol 2 (Miescher PA, Muller-Eberhard HJ, eds), New York, Grune & Stratton, 1969, p 562. 47. Wright R, McCollum RW, Klatskin G: Australia antigen in acute and chronic liver disease. Lancet 2: 117, 1969. 48. Gitnick GL, Gleich GJ, Schoenfield LJ, Baggenstoss AH, Sutnick AI, Blumberg BS, London WT, Summerskill WHJ: Australia antigen in chronic active liver disease with cirrhosis. Lancet 2: 285, 1969. 49. Mathews JD, Mackay IR: Australia antigen in chronic hepatitis in Australia. Brit Med J 1: 259, 1970. 50. Sherlock S, Fox RA, Niazi SP, Scheuer PJ: The association of chronic liver.dispase and primary liver cell cancer with Australia (hepatitis-associated) antigen. Lancet 1: 1243, 1970. 51. Wright R: Australia antigen and smooth-muscle antibody in acute and chronic hepatitis. Lancet 1: 521, 1970. 52. Almeida JD, Waterson AP: Immune complexes in hepatitis. Lancet 2: 983, 1969. 53. Dixon FJ: The role of antigen-antibody complexes in disease. Harvey Lect 58: 21, 1963. 54. Shulman NR, Barker LF: Virus-like antigen, antibody and antigen-antibody complexes in hepatitis measured by complement fixation. Science 165: 304, 1969. 55. Willems FTC, Kunst VAJM, Rosier JGMC: Complement-fixation test for Australia antigen. Lancet 1: 953, 1970. 56. Rodriguez M, Paronetto F, Schaffner F, Popper H: Antimitochondrial antibodies in jaundice following drug administration. JAMA 208: 148, 1969. 57. Fox RA, Niazi SP, Sherlock S: Hepatitis-associated antigen in chronic liver disease. Lancet 2: 609, 1969. 58. Paronetto F, Popper H: Lymphocyte stimulation induced by halothane in patients with post-halothane ,hepatitis. New Eng J Med 283: 277, 1970. 59. Popper H, Rubin E, Gardiol D, Schaffner F, Paronetto F: Drug-induced liver disease. A penalty for progress. Arch Intern Med (Chicago) 115: 128, 1965. 60. Klatskin G, Kimberg DV: Recurrent hepatitis attributable to halothane sensitization in an anesthetist. New Eng J Med 280: 515, 1969. 61. Klion FM, Schaffner F, Popper H: Hepatitis after exposure to halothane. Ann Intern Med 71: 467, 1969. 62. Copenhagen Study Group for Liver Diseases: effect of prednisolone on the survival of patients with cirrhosis of the liver. Lancet 1: 119, 1969. 63. Reynolds TB, Edmondson HA, Peters RL, Redeker A: Lupoid hepatitis. Ann Intern Med 61: 650, 1964. 64. Cook GC, Velasco M, Sherlock S: Effect of corticosteroid therapy on bromsulphthalein excretion in active chronic hepatitis. Gut 9: 270, 1968. 65. Cook GC, Mulligan R, Sherlock S: Controlled prospective trial

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of corticosteroid therapy in active chronic hepatitis. Quart J Med (in press). 65. John DW, Miller LL: Regulation of net biosynthesis of serum albumin and acute phase plasma proteins. Induction of enhanced net synthesis of fibrinogen, alpha,-acid glycoprotein, alpha2 (acute phase)-globulin and haptoglobin by amino acids and hormones during perfusion of the isolated normal rat liver. J Biol Chem 244: 6134, 1969. 67. Cain GD, Mayer G, Jones EA: Augmentation of albumin but not fibrinogen synthesis by corticosteroids in patients with hepatocellular disease. Gastroenterology (in press). 68. Soloway RD, Baggenstoss AH, EIveback LR, Schoenfield LJ, Stubbs BL, Summerskill WHJ: Are azathioprine and prednisone effective in th e early treatment of chronic active liver disease (CALD)? J. Clin Invest (in press). 69. Mackay IR: Chronic hepatitis: effect of prolonged suppressive treatment and comparison of azathioprine with prednisolone. Quart J Med 37: 379, 1968. 70. Mistilis SP, Blackburn CRB: The treatment of active chronic hepatitis with 6-mercaptopurine and azathioprine. Aust Ann Med 16: 305, 1967. 71. Page AR, Good RA, Pollara B: Long-term results of therapy in patients with chronic liver disease associated with hypergammaglobulinemia.Amer J Med 47: 765, 1969. 72. Sherlock S: Diseases of the Liver and Biliary System, 4th ed, Philadelphia, Davis, 1968, p 287. 73. Fischer JA, Schmid M: Treatment of primary biliary cirrhosis with azathioprine. Lancet 1: 421, 1967. 74. Porter KA: Pathology of the orthotopic homograft and heterograft, Experience in Hepatic Transplantation (Starzl TE, ed) London, WB Saunders, Ltd, 1969. 75. Najarian JS: Postgraduate Surgical Course, University of Minnesota 1970. Personal communication. 76. Starzl TE: Experience in Hepatic Transplantation, London, WB Saunders Ltd, 1969. 77. Williams R: Transplantation of the liver in man. Brit Med J 1: 585, 1970. 78. Kashiwagi N; Serum immunochemical studies, Experience in Hepatic Transplantation (Starzl TE, ed) Philadelphia, WB Saunders, 1969, p 394. 79. Abelev Gh Antigenic structure of chemically-induced hepatomas. Progr Exp Tumour Res 7: 104, 1965. 80. Tatarinov YS: Content of embryo specific alpha globulin in blood serum of human foetus, newborn and adult man in primary cancer of liver. Vop Med Khim 11: 20, 1965. 81. Uriel J, de Nechaud B, Stanislawski.Birencwajg M, Massayeff R, Leblanc L, Quenum C, Loisillie F, Grabar G: Antig~nes embryonnaires et cancer du foie chez I'homme: association de la alpha 1-foetoproteine s~rique avec I'hdpatome primarie. C R Acad Sci 265: 75, 1967. 82. Alpert ME, Uriel J and de Nechaud B: Alpha, feto-globulin in the diagnosis of human hepatoma. New Eng J Med 278: 984, 1968. 83. Foil AK, Sherlock S, Adinolfi M: Serum
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