Tacrolimus may induce the production of nucleolar anti-nuclear antibody in liver transplant patients.
ABSTRACT Immunosuppressive drugs have been used to prevent graft rejection in most allo-liver recipients and the immune state of these patients differs greatly before and after transplant operation. This study aims at evaluating the immune state of liver transplant patients treated with tacrolimus by investigating the production of anti-nuclear antibodies (ANA).
A hundred and eighty-eight serum samples from 94 allo-liver recipients treated with tacrolimus and from 94 patients with matched liver diseases were tested for ANA by indirect immunofluorescence assay with HEp-2 cells as substrate.
ANA were detected as positive in 20.2% of the liver transplant patients treated with tacrolimus, and in 12.8% of disease-matched control patients, but this difference was not statistically significant (P=0.17). However, the frequency of nucleolar ANA pattern in ANA-positive cases was significantly higher in the liver transplant patients (63.2%) than in the control group (16.7%) (P=0.01).
Tacrolimus may contribute to producing nucleolar ANA in liver transplant patients. The autoimmune disease susceptibility of allo-liver recipients treated with tacrolimus requires further studying.
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ABSTRACT: Liver cirrhosis and cholelithiasis are both familiar diseases in China. However, the rates of operative complications and death are still high in patients with these diseases. This study was designed to determine the operative indications as well as suitable procedures in the treatment of patients with cholelithiasis and liver cirrhosis. We studied retrospectively 60 patients with cholelithiasis and liver cirrhosis who had undergone operation from January 2000 to July 2006. We analyzed the loss of blood during operation, postoperative complications and death rate to determine the proper treatment. Fifty patients were cured and 10 (16.7%) died postoperatively, i.e., six patients died from hepatic-renal failure and multisystem organ dysfunction and 4 from massive bleeding in the gallbladder bed. The 10 patients were clearly correlated with the Child-Pugh classification: Child A (8%), Child B (20%) and Child C (30%). Postoperative bleeding occurred in 10 patients (16.7%), intraabdominal in 6 and gastrointestinal in 4. Seven of the 10 patients with bleeding died postoperatively. The proper perioperative management of patients with cholelithiasis and liver cirrhosis can decrease the mortality. Cholelithiasis should be managed first by emergency operation. It is safe for the patients of Child A to undergo laparoscopy. It is very safe for patients with cirrhosis and cholelithiasis to undergo devascularization and shunt operation followed by biliary tract surgery.Hepatobiliary & pancreatic diseases international: HBPD INT 11/2007; 6(5):479-82. · 1.08 Impact Factor
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ABSTRACT: With the technical advances and improvements in perioperative management and immunosuppressants, liver transplantation is the standard treatment for patients with end-stage liver diseases. In Asia, a shortage of deceased donor liver grafts is the universal problem to be faced with in all transplant centres. Many surgical innovations are then driven to counteract this problem. This review focuses on 3 issues that denote the development of liver transplantation in Asian countries. These include living donor liver transplantation (LDLT), split liver transplantation (SLT) and liver transplantation for hepatocellular carcinoma (HCC). Minimal graft weight, types of liver graft to donate and the inclusion of the middle hepatic vein with the graft are the main issues to be established in LDLT. The rapid growth and wide dissemination of LDLT has certainly alleviated the supply-and-demand problem of liver grafts in Asia. SLT is another attractive approach. Technical expertise, donor selection and graft allocation are the main determinants for its success. Liver transplantation plays a key role in the management of HCC in Asia. LDLT would be the main strategy in this aspect. The issue of extending the selection criteria for HCC patients for LDLT is still controversial. On the whole, future developments to increase the donor pool for the expanding recipient need in Asia would involve transplantation from non-heart beating donor and ABO incompatible transplantation.Annals of the Academy of Medicine, Singapore 05/2009; 38(4):322-10. · 1.25 Impact Factor
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ABSTRACT: Late graft dysfunction that does not result from recognised causes, such as rejection, infection, or vascular or biliary complications, can occur after liver transplantation. We investigated a particular type of unexplained graft dysfunction that is associated with autoimmune features in children who underwent transplantation at our unit between 1991 and 1996. Seven (4%) of 180 liver-transplant recipients developed an unexplained but characteristic form of graft dysfunction (five boys, two girls; median age at presentation 10.3 years, range 2.0-19.4). The median period after surgery was 24 months (6-45). The indications for transplantation had been extrahepatic biliary atresia (four patients), Alagille's syndrome (one), drug-induced acute liver failure (one), and alpha1-antitrypsin deficiency (one). Four patients were on triple immunosuppression with cyclosporin, azathioprine, and prednisolone; and three were on tacrolimus. Immunoglobulin measurements, autoantibody studies, serological studies, and HLA typing were undertaken. Liver-biopsy samples were taken. Infectious and surgical complications were excluded. Liver-biopsy samples showed the histological changes of chronic hepatitis, including portal and periportal hepatitis with lymphocytes and plasma cells, bridging collapse, and perivenular-cell necrosis without changes typical of acute or chronic rejection. All patients had high concentrations of IgG (median 22 g/L [range 17.2-34.4]) and high titres of autoantibodies. All but one patient responded to prednisolone 2 mg/kg daily and an increase in or addition of azathioprine (1.5 mg/kg daily) within a median of 32 days (7-316). One responder relapsed owing to poor compliance but went into remission after treatment was restored. All six respondents remain in remission on a reduced dose of prednisolone (5-10 mg/day) and 1.5 mg/kg daily azathioprine at a median of 283 days (range 108-730) follow-up. Our data show that symptoms of autoimmune hepatitis, which are responsive to the classical treatment for this condition, can appear in liver-transplant patients while they are on anti-rejection immunosuppression. Whether the liver damage in these patients is a form of rejection or the consequence of autoimmune attack has yet to be established.The Lancet 03/1998; 351(9100):409-13. · 38.28 Impact Factor
Received: 12.02.2011 Accepted: 30.06.2011
J Gastrointestin Liver Dis
September 2011 Vol. 20 No 3, 267-270
Address for correspondence:
Department of Laboratory Medicine
West China Hospital of
Tacrolimus may Induce the Production of Nucleolar Anti-
nuclear Antibody in Liver Transplant Patients
Yongkang Wu, Bei Cai, Jiangtao Tang, Yangjuan Bai, Lanlan Wang
Department of Laboratory Medicine, West China Hospital of Sichuan University, Chengdu, China
Background & Aims: Immunosuppressive drugs have
been used to prevent graft rejection in most allo-liver recipients
and the immune state of these patients differs greatly before
and after transplant operation. This study aims at evaluating
the immune state of liver transplant patients treated with
tacrolimus by investigating the production of anti-nuclear
antibodies (ANA). methods: A hundred and eighty-eight
serum samples from 94 allo-liver recipients treated with
tacrolimus and from 94 patients with matched liver diseases
were tested for ANA by indirect immunofluorescence assay
with HEp-2 cells as substrate. Results: ANA were detected
as positive in 20.2% of the liver transplant patients treated
with tacrolimus, and in 12.8% of disease-matched control
patients, but this difference was not statistically significant
(P=0.17). However, the frequency of nucleolar ANA pattern
in ANA-positive cases was significantly higher in the
liver transplant patients (63.2%) than in the control group
(16.7%) (P=0.01). Conclusion: Tacrolimus may contribute
to producing nucleolar ANA in liver transplant patients. The
autoimmune disease susceptibility of allo-liver recipients
treated with tacrolimus requires further studying.
Tacrolimus – liver transplant – nucleolar ANA.
About 8% of the Chinese population, nearly 100 million
patients, suffer from hepatitis B or are carriers of the hepatitis
B virus (HBV) . A definite association of HBV infection
and liver cirrhosis or liver cancer has been demonstrated
. Both liver cirrhosis and cancer could cause liver
dysfunction that severely impairs the quality of life of patients
. Liver transplantation nowadays is a widely accepted
treatment option for liver cirrhosis and cancer . After liver
transplantation, immunosuppressive treatment is necessary to
reduce the risk of subsequent graft rejection and to guarantee
a long-term survival of exogenous liver in recipients.
Although the immunosuppressive regimens for all solid
organ transplants are fairly similar, tacrolimus (also known
as FK506) is one of the most popular immunosuppressants
. As a potent suppressant of T lymphocytes, tacrolimus
is believed to regulate the immune state and balance of
recipients after an operation. So the diseases related to the
immune disorder, such as tumor and infection resulting from
lower immunity, have drawn the attention of clinicians when
applying a tacrolimus-based regime.
Up till now, the relationship between the tacrolimus
treatment and development of autoantibodies or autoimmune
diseases has not yet been deciphered. Since the 90s, several
groups reported the presence of de novo autoimmune
liver diseases as well as serum autoantibodies after liver
transplantation and administration of immunosuppressive
regimen [6-7]. However, the inducing effect of tacrolimus
on autoimmune disease occurrence after liver transplantation
remains to be clarified. Anti-nuclear antibodies (ANAs)
constitute a group of important autoantibodies acting against
the nucleus or cytoplasmic substances of nucleated cells.
The production of these autoantibodies mostly occurs in a
number of autoimmune disorders including systemic lupus
erythematosus and Sjögren’s syndrome [8-9]. The aim of the
current study is to evaluate the immune state of allo-liver
recipients by measuring ANA in the serum in two groups
of liver transplant patients with a long-term tacrolimus
treatment and disease-matched controls.
Patients and methods
This study has been approved by the Ethics Committee
of the West China Hospital of Sichuan University. Written
informed consent was obtained from each participant.
268 Wu et al
The hundred and eighty-eight liver transplant patients and
patients in a control group were recruited from in-patients
and out-patients of West China Hospital from 2007 to 2010.
Patients from the control group were matched by means of
age, gender, etiology, and disease. In the liver transplant
group, 91 (96.8%) cases were detected to be HBV-positive
with preoperative testing, while the control group counted
92 (97.9%) HBV-infected patients. All allo-recipients were
treated with tacrolimus alone as an immunosuppressive
agent. The patients with autoimmune liver disease such as
autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC)
and patients with hepatitis C were excluded from the study
due to a definitely high ANA prevalence.
The blood samples were collected in 4 ml BD Vacutainer
without anticoagulant and allowed to clot up to 1 hour
at room temperature. Within 2 hours after collection the
samples were centrifuged at 1500g for 10 min at 40C to
collect serum. The sera were stored at -800C and thawed at
40C overnight prior to analysis.
ANA were determined by indirect immunofluorescence
assay with commercialized ANA kit (EUROIMMUN Co.,
Germany) with HEp-2 cell lines as substrate at an initial serum
dilution of 1:100 according to the manufacturer’s instructions.
The serum was further diluted as 1:320, 1:1000, 1:3200 and
1:10000 subsequently, when a positive result was obtained with
the lower dilution. ANA fluorescence patterns were defined as
speckled, homogeneous, nucleolar (Fig. 1), centromere, nuclear
dots and cytoplasm staining, etc. ANA fluorescence pattern of
each sample was observed in double blind with a fluorescence
microscope (Model E6000 Nikon Co., Japan).
Quantitative data obeying a normal distribution were
described by their mean and standard deviation (SD). When
the distribution of data was skew, the median and range were
used instead. The Mann-Whitney U or Chi-square test was
applied for comparing both patient groups. A probability
value (P) inferior to 0.05 was considered statistically
significant. All data were analyzed with SPSS 16.0.
Age, gender, and disease
The characteristics of the subjects enrolled in the trial
are shown in Table I. There was no significant difference
between liver transplant and control patients in age, gender,
etiology, and disease distribution (P>0.05). This indicates
that the matching based on these 4 variables was adequate.
The incidence of ANA positivity was 20.2% and 12.8%
respectively, in the groups of liver transplant patients treated
with tacrolimus and the control patients. There was no
statistical difference between these two groups (P=0.17).
However, the frequency of nucleolar pattern, which reached
up to 63.2% in the ANA-positive liver transplant patients,
was only 16.7% in the control group, indicating a significant
difference between both groups (P=0.01) (Table II). For
all enrolled transplant patients, the median duration of
tacrolimus treatment was 42 months, and the mean plasma
concentration of the drug was 6.2±2.4 ng/ml.
The transplant patients were divided into a positive
subgroup with 19 cases and a negative subgroup with
75 patients based on the presence of ANAs. The median
durations of treatment with tacrolimus were 36 and 42
months, respectively in the positive and negative subgroups.
No significant difference was found between both subgroups
(P=0.58). The mean plasma concentrations of tacrolimus
in the positive and negative subgroups were 5.8±2.4 and
6.3±2.4 ng/ml, respectively. The difference in mean (0.5 ng/
ml) between both subgroups was not statistically significant
Because of the risk of rejection after liver transplantation,
immunosuppressive drugs are necessary for sustaining the
graft function and improving the life quality of recipients.
Tacrolimus, an effective calcineurin inhibitor, was approved
for clinical use based on the merit of its immunosuppressive
activity . Its action mechanism can be readily explained.
Tacrolimus binds to 12 kDa FK506-binding immunophilin
protein (FKBP12) of type-3 ryanodine receptor resulting
in a FK506-FKBP12 complex. This complex associates
with the calcineurin with high affinity and inhibits its
serine/threonine phosphatase activity, which results in
the inhibition of the transcription of interleukin 2 and
the blockade of Ca2+ dependent T lymphocyte activation
pathway. The immunosuppressive activity of tacrolimus
determines an impairment of T lymphocyte activation and
proliferation , so that cellular and humoral immunity of
liver transplant patients will be imbalanced after long-term
usage of the drug.
Fig 1. Example of nucleolar pattern of ANA detected
by indirect immunofluorescence assay with HEp-2
cells as substrate (40×amplified).
Nucleolar ANAs in allo-liver recipients treated with tacrolimus 269
Furthermore the transplanted liver as an exogenous
antigen could stimulate and activate the patients’ immune
responses . Consequently, the patients’ own immune
balance would be re-regulated because of the foreign
organ and tacrolimus usage. In order to achieve a new
immune equilibrium, the immune system including immune
molecules, cells, and organs would be extensively regulated
as compared with that before transplantation [13-15]. Since
the immunological tests, such as the ANA test, were not
performed for most patients before transplantation, it was
impossible to design a self-controlled study. Therefore, it
was decided to choose patients with matched liver diseases
as controls in this trial.
Antinuclear antibodies are one of the effective indicators
to evaluate the disorders of immune system resulting from
the imbalance of immunity against the own cellular antigens
. Normally, a positive result of ANA test may suggest
the emergence of auto-reactivity due to primary autoimmune
or drug-induced autoimmune disorders . Because of the
high prevalence of ANA in autoimmune liver diseases and
hepatitis C, patients with these disorders were excluded from
both groups of liver transplant and control patients in order
to diminish the non-specific influence of these diseases on
the ANA incidence studied in the current trial [18-19]. The
overall incidences and median titers of ANA in the liver
transplant group were comparable to those of the control
group. However, it could not be concluded that the immune
state of the liver transplant patients was similar to that of
the control patients.
Antinuclear antibodies represent a spectrum of
autoantibodies directed against various cellular components
. Different ANA patterns were determined according to
typical fluorescence staining patterns . The frequency
of the nucleolar pattern was significantly higher in liver
transplant patients compared with the control patients in this
study, although nucleolar pattern is reported as a rare pattern
of ANA, especially in autoimmune diseases . Despite the
well known association between nucleolar ANA pattern and
scleroderma [23, 24], no liver transplant patients treated with
tacrolimus had obvious scleroderma-related symptoms.
Since anti-smooth muscle antibody (SMA) and liver
kidney microsomal antibody (LKM) have also been detected
besides ANAs in some post-liver transplantation cases
with immunosuppressive regimens [6-7], a further study
concerning these autoantibodies and the related autoimmune
diseases in allo-liver recipients treated with tacrolimus would
be necessary to clarify the autoimmunity-inducing effect of
The current study demonstrates that the long-term
administration of tacrolimus in liver transplant patients
induces the production of nucleolar ANA. As a suggestion,
a set of autoantibodies, including ANA, should be regularly
monitored in liver transplant patients treated with tacrolimus
for monitoring the impact of this drug on disease prognosis
and, if necessary, in time decreasing the dose.
This study was supported by the National Natural Science
Foundation of China (Grant No. 30670819, 30900658,
30772051 and 30950010).
Conflicts of interest
None to declare.
1. Xu Q, Gu L, Wu ZY. Operative treatment for patients with
cholelithiasis and liver cirrhosis. Hepatobiliary Pancreat Dis Int
2. Leung N. HBV and liver cancer. Med J Malaysia 2005; 60 (Suppl
Table II. ANA prevalence and titer in liver transplant patients and controls
Group Number of
Median titer of
ANA positive (%)a
Median titer of
9419 (20.2%) * 100 (100~1000) **12/19 (63.2%) † 100 (100~320) †† 7/19 (36.8%)
Control9412 (12.8%) *100 (100~320) ** 2/12 (16.7%) †210 (100~320) †† 10/12 (83.3%)
Note: a including pure nucleolar pattern and nucleolar pattern mixed with others; b Other pattern including speckled, centromere,
cytoplasm and homogeneous pattern etc; *χ2 = 1.89, P = 0.17; ** Z = -0.26, P = 0.80; †χ2 = 6.42, P= 0.01; †† Z = -0.21, P= 0.83.
Table I. Characteristics of the subjects and disease distribution
Group Number of
Disease distribution (%)
Liver transplant patients 9479/1544 (24 ~ 70) 91 (96.8%)52.1%14.9%33.0%
Control 9479/1546 (24 ~ 78)92 (97.9%)55.3%14.9%29.8%
Note: a including severe hepatitis, liver failure, intrahepatic bile duct stone and severe liver rupture etc. excluding autoimmune liver
disease, AIH, PBC and Hepatitis C.
270 Wu et al
3. Canning C, O’Brien M, Hegarty J, O’Farrelly C. Liver Immunity
and tumour surveillance. Immunol Lett 2006; 107: 83-88.
4. Ng KK, Lo CM. Liver transplantation in Asia: past, present and
future. Ann Acad Med Singapore 2009; 38:322-331.
5. Holt CD, Ingle G, Sievers TM. Inhibitors of calcineurin. J Pharmacy
Practice 2003; 16:414-433.
6. Kerkar N, Hadzic N, Davies ET, et al. De-novo autoimmune hepatitis
after liver transplantation. Lancet 1998; 351:409-413.
7. Evans HM, Kelly DA, McKiernan PJ, Hubscher S. Progressive
histological damage in liver allografts following pediatric liver
transplantation. Hepatology 2006; 43:1109-1117.
8. Aganovic-Musinovic I, Prljaca-Zecevic L, Subasic D. The incidence
of ANA and ETI-dsDNA detected by enzyme immunoassays and
indirect immunofluorescence assay (IFA). Med Arh 2010; 64:68-70.
9. Huo AP, Lin KC, Chou CT. Predictive and prognostic value of
antinuclear antibodies and rheumatoid factor in primary Sjogren’s
syndrome. Int J Rheum Dis 2010; 13:39-47.
10. Sosa I, Reyes O, Kuffler DP. Immunosuppressants: neuroprotection
and promoting neurological recovery following peripheral nerve and
spinal cord lesions. Exp Neurol 2005; 195:7-15.
11. McConnell JL, Wadzinski BE. Targeting protein serine/threonine
phosphatases for drug development. Mol Pharmacol 2009; 75: 1249-
12 Kuwana M, Okazaki Y, Ikeda Y. Splenic macrophages maintain the
anti-platelet autoimmune response via uptake of opsonized platelets
in patients with immune thrombocytopenic purpura. J Thromb
Haemost 2009; 7:322-329.
13. Di Benedetto F, Di Sandro S, De Ruvo N, et al. Sirolimus monotherapy
in liver transplantation. Transplant Proc 2007; 39:1930-1932.
14. Canadas O, Sáenz A, Orellana G, Casals C. Equilibrium studies of a
fluorescent tacrolimus binding to surfactant protein A. Anal Biochem
15. Lilly LB, Grant D. Optimization of cyclosporine for liver
transplantation. Transplant Proc 2004; 36(2 Suppl):267S-270S.
16. Altintas A, Ozel A, Okur N, et al. Prevalence and clinical significance
of elevated anti-nuclear antibody test in children and adult patients
with idiopathic thrombocytopenic purpura. J Thromb Thrombolysis
17. Olsen NJ. Drug-induced autoimmunity. Best Pract Res Clin
Rheumatol 2004; 18:677-688.
18. Burak KW, Urbanski SJ, Swain MG. A case of coexisting primary
biliary cirrhosis and primary sclerosing cholangitis a new overlap
of autoimmune liver diseases. Dig Dis Sci 2001; 46: 2043-2047.
19. Zhang FK, Jia JD, Wang BE. Clinical evaluation of serum antibody-
negative primary biliary cirrhosis. Hepatobiliary Pancreat Dis Int
20. Hiemann R, Hilger N, Michel J, et al. Automatic analysis of
immunofluorescence patterns of HEp-2 cells. AnnN Y Acad Sci
21. Parker JC, Burlingame RW, Webb TT, Bunn CC. Anti-RNA
polymerase III antibodies in patients with systemic sclerosis detected
by indirect immunofluorescence and ELISA. Rheumatology (Oxford)
22. Zabek J, Palacz A, Pyka J, Krzewska I. Antinucleolar antibodies
in diagnostics of antiphospholipid syndrome. Pol Arch Med Wewn
23. Błaszczyk M, Jarzabek-Chorzelska M, Jabłońska S, et al.
Autoantibodies to nucleolar antigens in systemic scleroderma:
clinical correlations. Br J Dermatol 1990; 123:421-430.
24. Muro Y. Antinuclear antibodies. Autoimmunity 2005; 38: 3-9.