Response to steroids in de novo autoimmune hepatitis after liver transplantation

Response to Steroids in De Novo Autoimmune Hepatitis After Liver Transplantation 1 Margarita Rodrı´guez-Mahou,2 Emilio Alvarez,3 Jose Luis Vicario,4 ˜ Magdalena Salcedo,1 Javier Vaquero,1 Rafael Banares, ´ 1 Jose´ Luis R. Tı´scar,2 Diego Rincon, ´ 1 Sonia Alonso,1 Alejandro De Diego,1 and Gerardo Clemente1 Alicia Herna´ ndez-Albujar,

Graft dysfunction associated with autoimmune phenomena has been recently described in liver transplant recipients without previous autoimmune disease. However, the natural history, diagnostic criteria, and definitive therapeutic approach of de novo autoimmune hepatitis (de novo AIH) are poorly understood. We report 12 cases of de novo AIH 27.9 ⴞ 24.5 months after liver transplantation: the outcome of 7 patients treated with steroids is compared with a group of 5 nontreated patients. Nontreated patients lost the graft after 5.8 ⴞ 2.6 months from de novo AIH onset. All treated patients were alive after 48.4 ⴞ 14 (29-65) months from de novo AIH onset, and none of them lost the graft. However, 5 patients relapsed in relation to steroid tapering. All patients presented an atypical antiliver/ kidney cytosolic autoantibody, associated to classical autoantibodies in 10 cases. Histological study showed several degrees of lobular necrosis and inflammatory infiltrate. HLA antigen frequencies and matching were compared with 2 control groups (16 orthotopic liver transplantation [LTX] patients without de novo AIH and 929 healthy blood donors); de novo AIH patients showed a higher prevalence of HLA-DR3 (54.5% vs. 25.9%, P ⴝ .04) than healthy controls, which was not observed in LTX patients without de novo AIH. In conclusion, this new disease should be included in the differential diagnosis of unexplained graft dysfunction. In addition, treatment with steroids results in a dramatically improved outcome. However, maintenance therapy is usually required. (HEPATOLOGY 2002;35:349-356.)

O

rthotopic liver transplantation (LTX) is the standard therapeutic approach for treatment of end-stage acute and chronic liver diseases. Although long-term results of LTX have been improved in past years, late graft dysfunction with abnormalities of liver function tests may occur, usually associated with chronic rejection, recurrence of the original disease, or biliary and vascular complications. De novo autoimmune hepatitis (de novo AIH) has been recently described as a new type of graft dysfunction in pediatric patients receiving LTX.1 It is characterized by the development of graft dysfunction with features that resemble those of autoimmune hepatitis, in patients without a previous history of autoimmune disease (AD). In some of these patients, an atypical antiliver/kidney miAbbreviations: LTX, orthotopic liver transplantation; AD, autoimmune disease; de novo AIH: de novo autoimmune hepatitis; Atypical–LKC, atypical anti-liver/kidney cytosolic autoantibody; IIF, indirect immunofluorescence; ANA, antinuclear antibodies; SMA, anti-smooth muscle antibody; LKM-1, type 1 liver-kidney microsomal antibody; LC1, type 1 liver cytosol antibody; HBV, hepatitis B virus; HCV, hepatitis C virus; CMV, cytomegalovirus; AIH, autoimmune hepatitis. From the 1Liver Transplantation Unit, Gastroenterology and Hepatology Department; 2Autoimmunity Laboratory, Immunology Department; 3Pathology Department, Hospital General Universitario Gregorio Maran˜o´n, Universidad Complutense; 4HLA Laboratory, Regional Transfusion Center, Madrid, Spain. Received July 17, 2001; accepted November 5, 2001. Address reprint requests to: M. Salcedo Plaza, Unidad de Trasplante Hepa´tico, Servicio de Aparato Digestivo, Hospital General Universitario Gregorio Maran˜o´n, C/ Dr. Esquerdo 46, Madrid 28007, Spain. E-mail: [email protected]; fax: (34) 91-409-1498. Copyright © 2002 by the American Association for the Study of Liver Diseases. 0270-9139/02/3502-0014$35.00/0 doi:10.1053/jhep.2002.31167

crosomal antibody (atypical-LKM) was shown. Other groups have reported the same cause of graft dysfunction in adults with similar immunological findings.2 The outcome of de novo AIH remains largely unknown, but several cases with severe liver damage and hepatic failure leading to death have been described,2 indicating the need for specific management of this complication. Although the therapeutic approach of de novo AIH is not well established, increasing immunosuppression with steroids and azathioprine has been suggested. However, the schedule, duration of therapy and long-term graft evolution after treatment are not clearly elucidated. Although the pathogenesis of AIH is still not well known, several factors have been implicated in the development of this condition, namely the HLA status and also viral infections. The possible influence of all these factors in the development of de novo AIH has not been investigated yet. Therefore the aim of this study was, first, to evaluate the outcome of de novo AIH in liver transplant recipients after steroid treatment, and second, to analyze the possible existence of pathogenic factors of the disease.

Patients and Methods Between 1990 and 1999, 350 LTX in adult recipients were performed in our Liver Transplant Unit. Twelve cases (3.4%) of de novo AIH associated to atypical antiliver/kidney cytosolic autoantibodies (atypical-LKC) were diagnosed in 11 patients. The diagnosis of de novo AIH was based on a combination of histological and biochemical signs of graft hepatitis, associated to autoimmune phenomena consisting in significant titers of autoantibodies and hypergammaglobulinemia, and exclusion of other causes of graft dysfunction. The International Autoimmune Hepatitis Group 349

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Table 1. Indications for Transplantation, Previous Autoantibody Status, and Score of the International Autoimmune Hepatitis Group for Each Patient (Probable AIH 10-15, Definite AIH > 15 Before Treatment) Scoring System of AIH

Indication for LTX

Nontreated 1 2 3 4 5 Treated 1 2 3 4 5 6 7*

Ab Before LTX

Before LTX

At Diagnosis

Cryptogenic cirrhosis Alcoholic cirrhosis HCV cirrhosis PSC Alcoholic cirrhosis

Negative Negative Negative Negative Negative

6 5 ⫺3 7 9

17 16 11 19 15

Extrahepatic biliary atresia HCV cirrhosis HBV cirrhosis Alcoholic cirrhosis HCV cirrhosis Alcoholic cirrhosis Retransplantation

Negative

3

15

8 5 3 3 7 17

14 16 16 10 16 19

Negative Negative Negative ANA 1:40 ANA 1:40 ANA 1/160, SMA 1/160, atypical LKC 1/640

Abbreviations: LTX, orthotopic liver transplantation; Ab, autoantibodies; HCV, hepatitis C virus; PSC, primary sclerosing cholangitis; HBV, hepatitis B virus; ANA, anti-nuclear antibodies; SMA, anti-smooth muscle antibodies; Atypical LKC, atypical antiliver kidney cytosolic antibodies. *This is patient #1 of nontreated group, who was retransplanted because of liver failure secondary to de novo AIH in the first liver transplant.

score revisited in Chicago in 19983 was also applied before LTX and at the time of graft dysfunction. In the first 5 patients (nontreated group), the diagnosis was retrospectively established, and they did not receive any specific treatment for this condition. The remaining 7 cases, including a patient from the former group who required retransplantation and experienced recurrent AIH, were started on prednisone at 5.7 ⫾ 6.2 months (range, ⬍ 1-17) after de novo AIH diagnosis (induction dose, 1 mg/Kg tapered to the minimum maintenance). Indication for transplantation was similar in both groups (Table 1). Postoperative immunosuppression was based on cyclosporine, azathioprine, and prednisolone. The dose of cyclosporine was adjusted to maintain trough concentration levels of 250-350 ␮g/L during the first month, reducing thereafter to maintain them around 100 ␮g/L during the first year. Azathioprine was administered at a starting dose of 1.5 mg/kg daily. Most patients were on 25 mg/day of azathioprine at 12 months after LTX. Initial prednisolone dose was 200 mg IV, tapered to 10 mg/day at 3 months. Prednisolone was withdrawn during the first year in 10 patients. Histologically confirmed acute rejection episodes were treated with 3 to 5 boluses of 500 to 1000 mg of methyl-prednisolone. Patients were switched to tacrolimus in case of poor response to previous steroid treatment. Autoantibodies were determined in all patients. The immunological study was performed at de novo AIH diagnosis, except in 3 patients from the nontreated group in whom frozen stored serum obtained closely to liver dysfunction was used. Serum samples diluted 1:40 were screened by indirect immunofluorescence (IIF)

on frozen rat organ tissue sections (liver, kidney and stomach) according to the Roitt & Doniach method4 (MeDiCa Medical Diagnostics California, Carlsbad CA) and on a laryngeal carcinoma cell line HEp-2 (Meridian Diagnostics Europe D-61352, Bad Homburg, Germany) to detect antinuclear (ANA), antismooth muscle (SMA), antimitochondria (AMA-M2), antiliver/ kidney microsomal (LKM1), and antiliver cytosol type-1 (LC1) autoantibodies. Determinations were considered positive if the titer was 1:40 or higher; thereafter, the positive sera were titrated to extinction. Anti-dsDNA antibodies were detected by enzimolinked immunosorbent assay (ELISA) according to manufacturer’s instructions (ORGENTEC Diagnostika GmbA, Mainz, Germany). With regard to immunodiffusion, undiluted and diluted 1:2 patient serum samples were compared with anti-LC1 reference serum samples (Pr. RL. Humbel, CH Luxembourg) by identity reaction in the Outcherlony double immunodiffusion against rat liver cytosol, microsomal, and mitochondrial fractions, according to de Duve’s method, as described by Martini et al.5 Absorption test was performed with mitochondrial, microsomal, and cytosolic fractions. Immunoglobulin quantification was performed by immunonephelometry (Array R Beckman). Hepatitis B virus (HBV) was determined by a third generation of enzymatic immunoassay (Abbott AuszymeR Monoclonal, CorzymeR Germany). Hepatitis C virus (HCV) was determined by a third generation ELISA (Orthotm HCV 3.0 Elisa test system, Germany). The determination of HCV RNA was done by reversetranscription PCR (Amplicor, Roche Diagnostics, Welwyn Garden City, UK). Virological study of nonhepatotropic virus infection included: Cytomegalovirus (CMV) by capture CMV IgG Elisa (Wampole, Innogenetics and CMV IgM, Biome´rieux), Parvovirus B19 by IgM EIA (Biotrin, Innogenetics), Herpes group by IgG Elisa (Wampole, Innogenetics), Epstein-Barr virus by EBV Viral capsid Antigen (D-18), IgG Elisa (Wampole, Innogenetics), and by Monolatex test (Biokit SA, Spain). Liver biopsies were embedded automatically in paraffin wax, stained by hematoxylin eosin, Masson’s trichrome, and Pearl’s method, and evaluated by 2 different observers (E.A., A.H-A) in a blind manner. The presence of interface hepatitis, plasma cells in the inflammatory infiltrate, rosetting of periportal hepatocytes, and biliary changes according to the criteria of the International Autoimmune Hepatitis Group,3 were assessed in each case. Immunohistochemical techniques to exclude CMV, HBV, and EpsteinBarr virus were performed as appropriate. We studied HLA class I and II polymorphism by standard complement-dependent microcytotoxicity assays and/or by PCR with sequence-specific primers for the following antigens/alleles: A (1, 2, 3, 11, 23, 24, 25, 26, 29, 30, 31, 32, 33, 34, 66, 68, 69, 80), B (7, 8, 13, 18, 27, 35, 37, 38, 39, 41, 42, 44, 45, 47, 48, 49, 50, 51, 52, 53, 55, 56, 57, 58, 60, 61, 62, 63, 64, 65, 67, 70, 73), Cw (1, 2, 3, 4, 5, 6, 7, 8), DRB1 (1, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18), DRB3/4/5 (51, 52, 53), and DQB1 (2, 4, 5, 6, 7, 8, 9). We compared HLA matching (loci A, B, and DRB1) between donors and recipients of liver grafting in the 12 de novo AIH patients with the HLA matching in a control group of 16 patients without autoantibodies after LTX. Antigen frequencies of 929 unrelated healthy blood donors were subsequently compared with those obtained in the transplanted groups.

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Table 2. Baseline Characteristics of the Patients

Gender (male/female) Age at diagnosis (years) Clinical characteristics at diagnosis Jaundice Malaise ALT (U/L) Alkaline phosphatase (U/L) Bilirubin (mg/dL) Previous rejection Immunosuppression at de novo AIH onset Interval between LTX and de novo AIH onset (months)

Nontreated nⴝ5

Treated nⴝ7

P

4/1 47.6 ⫾ 7.7

4/3 46.4 ⫾ 14.7

.6 .8

3 3 615 ⫾ 313 595 ⫾ 244 6.3 ⫾ 7.9 3 CyA, Az: 3 CyA: 2

4 2 297 ⫾ 233 365.7 ⫾ 149 9.6 ⫾ 12.1 3 CyA, Az: 3 CyA, Az, Predn: 2 CyA, Predn: 2 19.2 ⫾ 10.7

40.2 ⫾ 34

.06 .07 .6 1

.24

Abbreviations: ALT, alanine aminotransferase; LTX, liver transplantation; CyA, cyclosporine; Az, azathioprine; Predn, prednisone.

Results are reported as mean ⫾ SD Quantitative variables were compared by Student’s t test or the Mann-Whitney test as appropriate. Categorical variables were compared using the two-tailed Fisher’s exact test. Wilcoxon signed rank test was used to compare nonparametric variables in related samples. A P value ⬍ .05 was considered significant.

Results The mean period between LTX and de novo AIH was 27.9 ⫾ 24.5 months (range 6-97). Baseline characteristics of patients are shown in Table 2. There were no significant differences between treated and nontreated patients regarding age, clinical presentation at diagnosis, interval between LTX and development of de novo AIH, or immunosuppressive regimen at de novo AIH onset. The blood trough levels of cyclosporine were also similar (nontreated: 142 ⫾ 73 ng/mL, treated: 157 ⫾ 46 ng/mL, N.S). When the International Autoimmune Hepatitis Group scoring system3 was applied, 8 patients had definite (⬎ 15) and 4 had probable AIH at the time of graft dysfunction (Table 1). In contrast, when the scoring system was used before LTX, no patient reached criteria of AIH (except the patient previously retransplanted because of de novo AIH). Histological Changes. Two different patterns of liver damage were recognized. One of them was characterized by portal and periportal hepatitis with inflammatory infiltrate with plasma cells and slight periportal necrosis (Fig. 1). This pattern was usually related to mild clinical and biological alterations. The second pattern was characterized by severe centrilobular necrosis involving most lobules, with more severe portal inflammation and periportal fibrosis with porto-central bridging (Fig. 2), usually related to severe graft dysfunction with lethargy, malaise, jaundice, and coagulation disturbances. The occurrence of the more severe pattern was similar between nontreated and treated patients (3 and 2 cases in each group, respectively). None of the patients showed histological findings of acute cellular rejection. Clinical and Biochemical Evolution. The clinical and biochemical evolution in nontreated and treated patients was very different. Nontreated patients had a poor outcome and developed

351

chronic liver disease leading to cirrhosis in all cases. Two patients underwent retransplantation, one of them dying in the postoperative course because of multiorgan failure. The remaining 3 patients of this group died due to severe and progressive liver failure before retransplantation could be performed. The average time between the onset of graft dysfunction and loss of the graft was short (5.8 ⫾ 2.6 months, range 3-9). ALT and bilirubin remained high or even increased in nontreated patients (Fig. 3). Moreover, ALT was significantly higher in nontreated patients compared with treated patients at 1, 3, and 6 months, respectively (387 ⫾ 239 UI/L vs. 115 ⫾ 71 UI/L, 573 ⫾ 25 UI/L vs. 67 ⫾ 34 UI/L, 653 ⫾ 349 UI/L vs. 129 ⫾ 62 UI/L); (P ⬍ .05 Mann-Whitney test). Treated patients, however, showed a dramatically different evolution after steroid therapy, with a significant and progressive decrease of ALT and bilirubin at 1 and 3 months after diagnosis (P ⬍ .05, Wilcoxon signed rank test) (Fig. 3). This significant improvement of liver function tests was accompanied by disappearance of lobular necrosis and a marked decrease of portal inflammatory infiltrate. No patient lost the graft nor died because of de novo AIH after a mean follow-up of 48.4 ⫾ 14 months (range, 29-65). Biopsy-proven relapse of de novo AIH occurred in 5 patients, and it was associated with withdrawal of steroids in one patient and a decrease of the maintenance dose of prednisone in the remaining. All of them had biochemical response after resuming previous doses of steroids. During follow-up, 3 patients had mild abnormalities in serum liver enzymes; 2 were related to histological signs of mild hepatitis C reinfection and the other to mild duct damage. In long term follow-up, 3 patients received combined therapy with steroids and azathioprine to minimize adverse events of high doses of steroids. Immunological Findings. At the time of graft dysfunction, all patients had significant titers of autoantibodies (Table 3). Interestingly, all patients were positive for an atypical antiliver/kidney cytosolic antibody (atypical-LKC) at time of diagnosis. AtypicalLKC antibody describes a staining pattern of rat liver hepatocyte cytoplasm (mainly in the centrilobular area) (Fig. 4A.) and distal and proximal tubules of rat kidney (Fig. 4B and C.), respectively. Immunodiffusion testing showed a precipitation line of this antibody with the cytosolic fraction. No precipitation line was observed neither with microsomal nor mitochondrial fractions. Moreover, no identity pattern with reference to antiliver cytosol LC-1 autoantibody was observed by immunodiffusion. Ten cases (83%) had also positive ANA and/or SMA autoantibodies. All patients were negative for LKM-1 and LC-1 autoantibodies. Atypical LKC antibody titers closely correlated with clinical activity of de novo AIH; titers were highest at onset and flares of de novo AIH, and decreased after steroid therapy following improvement of liver function tests. In 5 of the 7 treated patients, determinations of autoantibodies were available before the onset of de novo AIH, showing positivity at lower titers than those seen after diagnosis was established. All patients had polyclonal hypergammaglobulinemia, mainly because of a marked increase of serum IgG (2908 ⫾ 1727 mg/ dL, range 1220-6390; normal values 700-1600 mg/dL). Additionally, 4 patients had a Kappa monoclonal gammopathy, which disappeared after treatment (Table 3). Virologic Features. At diagnosis, all patients had antibodies against CMV of IgM class. In addition, one patient from the

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Fig. 1. Mild form. (A) Low power microphotograph before treatment shows the increase in portal surface resulting from the presence of mononuclear infiltrate, which penetrates in the periportal areas. No lobular lesion is apparent. H.E. 40⫻. (B) Same patient after steroid therapy. No portal infiltrate or lobular lesion can be seen. H.E. 200⫻.

nontreated group and 2 from the treated group showed IgM-antibodies against Parvovirus, and one nontreated patient had IgMantibody against Epstein-Barr virus. No patient, except those previously diagnosed, presented infection by HVC or HVB. HLA Typing. Liver transplant recipients with de novo AIH showed a higher prevalence of HLA-DR17 (DR3) (54.5% vs. 25.9%, P ⫽ .04) than healthy controls. However, LTX patients without de novo AIH did not show this association (31.3% vs. 25.9%, P ⫽ .4). HLA-B8 was also found more frequently in AIH patients, as well as B38, DR15, DR51, and DQ6, but this finding was not significant (Table 4). The same mismatching level (HLA-A, -B, -DRB1) was observed between both transplanted groups (mean 4.7 ⫾ 0.94 vs. 4.6 ⫾ 1.08, in AIH and controls, respectively).

Discussion Unexplained graft dysfunction despite extensive diagnostic procedures is not unusual after OLT. Recently, a new form of graft dysfunction associated with autoimmune phenomena in LTX patients without previous AIH has been reported in pediatric1,6 and adult2,7-9 recipients, without any apparent relationship with LTX

Fig. 2. Severe form. (A) Liver biopsy 3 years after transplantation. A marked expansion of portal tracts can be seen. The portal area shows a dense infiltration by mononuclear cells, which penetrate into the periphery of the hepatic lobule. H.E. 100⫻. (B) Same case. Centrolobular area showing the disappearance of liver cells and the inflammatory infiltrate. Plasma cells can be easily identified. H.E. 400⫻. (C) Liver biopsy after steroid therapy. A marked reduction of portal inflammatory infiltrate can be seen. A persistent lymphocytic infiltrate resulting from chronic VHC hepatitis can be seen. No lobular lesion is present. H.E. 40⫻.

indication. The outcome and specific treatment of this complication are not well known. Although steroid dose increase has been suggested for the treatment of this condition,1,2,7,8,10 there are no reports specifically addressed to this important issue. In this article we describe the evolution of de novo AIH in a representative series of patients treated and not treated with steroids. The most relevant finding of this report is that those patients who did not receive steroids had a very poor outcome with early loss of the graft, almost invariably leading to death if not retransplanted. By contrast, all steroid-treated patients markedly improved, with long-term survival without retransplantation. Furthermore, some of these patients relapsed after decreasing or withdrawal of steroid maintenance therapy; relapse improvement after achieving the previous doses of steroids further supports the autoimmune nature of the disorder. The poor outcome of nontreated patients highlights the importance of a correct diagnosis and an early aggressive therapy. Several reasons may explain why this goal is difficult to achieve. First and foremost, a high level of clinical suspicion is needed. In fact, the International Autoimmune Hepatitis Group scoring system revisited in Chicago in 19983 may be inappropiate for making the

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Fig. 3. Individual values of alanine aminotransferase (ALT) (A) and total bilirubin (BIL) (B) in nontreated (F) and treated patients (E) after starting therapy with steroids. Horizontal lines represent the mean values in nontreated (●●●) and treated patients (—) at each interval (expressed in months). (A) P ⫽ .04 in treated with respect to nontreated patients after 1 month of therapy († vs. †). P ⬍ .05 comparing ALT at diagnosis of AIH with respect to 1 and 3 months of therapy in treated patients (* vs. †, P ⫽ .01, † vs. ‡, P ⫽ .02). (B) P ⫽ .04 Bilirubin baseline vs. 1 month after therapy in treated patients (* vs. †).

diagnosis in the context of LTX because of the weight of the negative points of the score, such as viral liver disease, or the likely absence of female predominance in de novo AIH, and the potential effect of immunosuppression. The limitations of the classical AIH score has also been noted in the diagnosis of recurrent AIH after LTX, where the possibility of a modified score has been recently Table 3. Maximal Titration of Autoantibodies and Other Autoimmune-Associated Features Autoantibodies Atypical LKC

ANA

SMA

Nontreated 1

1/160

1/160

1/640

2

0

0

1/40

3 4

0 1/80

1/160 1/160

1/60 1/2560

5

1/40

0

1/640

Treated 1

1/80

1/80

1/640

2 3 4 5 6

1/160 1/40 1/640 1/320 0

0 1/40 0 0 0

1/160 1/640 1/2560 1/40 1/640

7*

0

1/160

1/80

Anti-dsDNA

Other Immunologic Features

87.7 IU/ml† 79.7 IU/ml‡ Kappa monoclonal gammopathy — Kappa monoclonal gammopathy Kappa monoclonal gammopathy

46 IU/ml†

Increased serum immunocomplexes — — — — Kappa monoclonal gammopathy Uveitis

Abbreviations: ANA, anti-nuclear antibodies; SMA, anti-smooth muscle antibodies; Atypical LKC, atypical anti-liver kidney cytosolic antibodies; anti-dsDNA, Anti-double stranded DNA. *This is patient #1 of Group A, who was retransplanted because of liver failure secondary to de novo AIH in the first transplant. †,‡Low and high avidity anti-dsDNA (normal value 10-30 IU/mL).

suggested.11,12 This is particularly evident for patients with HCV recurrence. However, HCV patients reported in this article had the most striking histological features, and all of them had a severe clinical presentation. Furthermore, when the scoring system was applied, all HCV patients reached scores of definite AIH. For these reasons, although the scoring system yielded diagnostic scores for the patients of our study, we propose the following items to be taken into special account in the evaluation of de novo AIH in LTX: (1) Allograft dysfunction not associated with other known causes of graft dysfunction, such as vascular or biliary disease, rejection, or viral hepatitis; (2) serum positivity to autoantibodies; (3) histological evidence of portal and periportal hepatitis with or without centrilobular necrosis, and lymphoplasmacytic portal tract infiltrate with a variable degree of plasma cells, in the absence of other ethiologically relevant histological findings; and (4) response to steroids. In addition, the concomitant seroconversion to nonhepatotropic viruses, the slightly different histological pattern compared with AIH in nontransplanted population, and the coexistence of other factors potentially implicated in liver damage may also make a correct and early diagnosis difficult. It is also possible that some opportunistic viral infections such as cytomegalovirus, EpsteinBarr virus, or parvovirus, might have acted as triggering factors of the disease. In this context, several viruses have been identified as triggers of AIH in the nontransplanted population.13-17 Likewise, the apparent paradox of developing AD in the context of immunocompromised patients can contribute to the low clinical suspicion of this entity. However, it is well known that some immunodeficiency conditions are associated with AD.18-20 The histological findings of de novo AIH may differ from the interface hepatitis usually found in AIH in nontransplanted population.3 In the most severe cases, liver biopsy showed severe centrilobular necrosis with marked portal inflammation and periportal fibrosis with porto-central bridging. Because some of these findings may remind in some way those secondary to ischemic

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Fig. 4. (A) Characteristic pattern of immunofluorescence with a diffuse and intense area 3 hepatocyte staining. Rat liver 400⫻. (B) Rat cortical renal area. Positive immunofluorescence of the epithelium of proximal renal tubules is shown. Both distal and glomerular renal tubules are negative; 200⫻. (C) Staining of the deep portion of the cortical-medular junction. Global immunofluorescence is shown, Henle loops are negative; 200⫻.

injury, Doppler and angiographic examinations were performed to exclude this condition. In less aggressive cases, the characteristic finding was the presence of an inflammatory infiltrate expanding to periportal areas, with variable degrees of lobular necrosis. The significance of the emergence of positive autoantibodies after bone marrow and solid organ transplantation is still an intriguing issue. Several studies have reported a very high prevalence of diverse autoantibodies in these settings,21-23 probably reflecting a disturbance of the immune system secondary to immunosuppressive therapy, the frequent bacterial and viral infections of these patients, or the exposure to new antigens of the grafted organ.24 Thus, just like in the nontransplanted population, autoantibodies cannot be considered neither pathogenic nor pathognomonic. However, they have occasionally been associated with the occurrence of diverse complications, namely renal,23 cardiac,25 and liver allograft rejection26,27 or autoimmune dysfunction.1,2 As far as de novo AIH is concerned, all of our patients showed seropositivity to autoantibodies. Furthermore, all patients were seropositive to a specific atypical-LKC pattern, and in 75% of them, typical autoantibodies could also be shown. Regardless of any possible pathogenic implications of atypical-LKC autoantibody, we observed a direct relationship between its titers and the severity of liver damage and also the level of atypical-LKC autoantibody significantly decreased after steroid treatment.

Table 4. HLA Antigens in Donors and Recipients: in de novo AIH Patients, in Liver Transplantation Control Group Without AD and Healthy Blood Donors

B8 B38 DR15 DR3 DR51 DQ6

De novo AIH (Recipient) n ⴝ 11

De novo AIH (Donor) n ⴝ 11

Control (Recipient) N ⴝ 16

Control (Donor) n ⴝ 16

Healthy Blood Donors n ⴝ 929

27.3 27.3 36.4 54.5* 45.5 63.6

0 18.2 9.1 0 18.2 27.3

25 6.3 18.8 31.3 18.8 43.8

6.3 6.3 25 0 25 37.5

10.9 7.6 18.2 25.9 21.6 36.2

NOTE. Data are expressed as percentage. Represented values are those approaching or showing statistical significance. *P ⬍ .05 AIH vs. healthy blood donors.

If the pathogenesis of AIH still remains unclear in nontransplanted patients, this is even more true for de novo AIH. Although clinical, serological, and histological features of both diseases possibly reflect common pathogenic pathways, a large amount of factors belonging to the liver transplant setting may itself influence the immune system regulation. Infections, frequently observed after LTX, may disrupt peripheral tolerance by the breakdown of vascular or cellular barriers and the occurrence of cell death by necrosis instead of apoptosis, exposing self antigens to the immune system. Bystander activation of macrophages and T lymphocytes, superantigen effects of bacterial products, and molecular mimicry processes are other factors that may occur during infection.24,28,29 A distinctive feature of LTX is the need of immunosuppression, usually achieved by the use of calcineurin inhibitors. The possible relationship between cyclosporine and autoimmune phenomena is supported by several clinical and experimental features. Thus, cyclosporine has been found to be responsible for the induction of a T-cell-mediated autoimmunity in certain situations, the so-called cyclosporine-induced autoimmunity. This entity describes an AD similar to graft-versus-host-disease that occurs on cessation of cyclosporine A treatment after syngenic bone marrow transplantation in humans and rodents.30-34 It has been related to a defective de novo T-cell development in the thymus, favoring the emergence of autoaggressive T-cell clones. In addition, the inhibition of the action of calcineurin mediated by cyclosporine may influence the recently described role of calcineurin in the apoptosis-signaling cascade.35-37 This fact is more relevant if we take into account the growing evidence of the important role that apoptosis plays in the pathogenesis of many AD.38-40 Susceptibility to AD is clearly influenced by genetic factors. In nontransplanted patients with AIH, HLA A1, B8, and DR3 haplotypes are associated with a more aggressive clinical course and poor response to treatment.41-43 An increased relative risk for type 1 AIH in patients with HLA DR3 and DR4 has been described. HLA DR3 patients experience higher rates of treatment failure, relapse, referral to LTX, and recurrent AIH after LTX,44 whereas HLA DR4 is associated with a better disease behavior.45,46 We have also found a significant increase in the prevalence of DR3 in de novo AIH patients, as well as a trend to higher frequencies for B8. Both antigens are known to be associated by strong linkage dis-

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equilibrium. Whether the recurrence of AIH is more frequent in HLA-DR3-positive recipients grafted with HLA-DR3-negative donors as suggested by Wright et al,47 could not be confirmed in our study because no donor was HLA-DR3-positive. Interestingly, we observed a trend toward higher frequencies of HLA-DR15, -DR51, and -DQ6 in recipients with de novo AIH than in the 2 control groups. These antigens are tightly linked constituting a class II haplotype that has been associated with 2 recognized AD, namely systemic lupus erythematosus and multiple sclerosis.48,49 The importance of this genetic background may partly explain the recurrent disease in one of our patients, who was DR3/DR15-, DR51/DR52-, and DQ2/DQ6-positive. Finally, the term of de novo AIH may also be controversial as it is possible that the immune response is initiated against nonnative proteins of the grafted liver. However, until a better knowledge of the true pathogenesis of this entity is accomplished, the overall clinical, histological, and immunological characteristics of these disorders strongly suggest autoimmune mechanisms. According to this, a recent article describing a similar series of patients has proposed the term “graft dysfunction mimicking AIH.”50 In conclusion, we present a series of adult liver transplant recipients who developed graft dysfunction related to autoimmunity, with variable degrees of severity, in which misdiagnosis may be fatal leading to death or retransplantation. The therapeutic approach with steroids, associated or not with azathiophrine, is accompanied by a marked improvement of the disease and avoids graft loss. In presence of patients with unexplained graft dysfunction it is very important to bear in mind this entity and to develop an adequate diagnostic strategy, including determination of serum autoantibodies. Future advances in the knowledge of triggering and conditioning factors will aid in the understanding of the pathogenesis of the disease and will allow a correct preventive and therapeutic approach.

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