Dr K. Miyachi, MD, Health Sciences Research Institute, Inc., 106 Godo-cho, Hodogaya-ku, Yokohama, Kanagawa 240-0005, Japan. E-mail: email@example.com
We previously reported that 12.5% of primary biliary cirrhosis (PBC) sera reacted with a 95 kDa cytosol protein (p95c) that was subsequently identified as a p97/valosin-containing protein (VCP). The clinical features and course of the six anti-p97/VCP-positive PBC patients with Scheuer's stage 1 and 2 liver biopsies were monitored for an average of 15 years. This group was compared with 50 PBC patients that did not have detectable anti-VCP. Autoantibodies to a full-length recombinant p97/VCP were assayed by immunoprecipitation. All six PBC patients with anti-VCP had antibodies to the mitochondrial pyruvate dehydrogenase complex-E2 antigen as measured by an addressable laser bead immunoassay. The first was a male with no evidence of liver failure that died of cerebral infarction at the age of 85. The second was a 73-year-old female with Hashimoto's thyroiditis who has remained clinically stable without ursodeoxycolic acid (UDCA) treatment. Although the third had no HCV antibodies, he developed hepatocellular carcinoma at the age of 76 and died of renal failure at 78. The fourth was a 50-year-old female who remained clinically stable during follow-up and the fifth with Hashimoto's thyroiditis and stable liver function following UCDA treatment. The sixth was a male patient presenting a mild clinical course. The clinical course of these patients was in contrast to the 50 comparison group PBC patients who did not have anti-p97/VCP. As the six PBC patients with anti-p97/VCP antibodies had slowly progressive liver disease and no mortality related to autoimmune liver disease, our observations suggest that this autoantibody might be an indicator of a favourable prognosis.
Patients with autoimmune diseases produce a variety of autoantibodies . Antimitochondrial antibodies (AMA), are a serological hallmark and are found in 85% of the primary biliary cirrhosis (PBC) patients, but they are not considered to be pathogenic [2, 3]. In addition to AMA, other autoantibodies that are detected in PBC patients include anticentromere , anti-SP100 , antibodies to nuclear envelope components gp210, p62 complex and Tpr [6–8] and the high mobility group (HMG) proteins HMG-1 and -2 . Except for anti-HMG-1 and -2, the prevalence of these antibodies in PBC sera is less than 30%. Although there is no evidence that these antibodies are responsible for pathogenesis, they are considered helpful in the diagnosis of PBC in the subset AMA-negative PBC patients . Of interest in the context of the present study, antibodies to the nuclear pore complex proteins gp210 and/or p62 are reported to be a marker for progressive PBC and a relatively poor prognosis [10, 11].
In 1990, Klein and Berg  reported that the anti-M9 antibodies were a marker for mild and slowly progressive PBC. In that study, the anti-M9 autoantigen was estimated to have a molecular mass of 97 kDa by immunoblot and was reported to be identical to glycogen phosphorylase. Subsequently, we studied a PBC serum and immunoprecipitated a cytosolic 95 kDa autoantigen (p95c) from HeLa cell lysates . As the p95c autoantigen was not detected by immunoblot, we concluded that p95c was not identical to M9 and that anti-p95c antibodies reacted with a conformational epitope of the cognate cytosolic antigen .
More recently, we became aware that p97/VCP (valosin-containing protein), a member of the extended AAA ATPase family of proteins, plays an important role in homotypic assembly of endoplasmic reticulum (ER), the Golgi complex and the nuclear membrane [15, 16]. In addition, it has been shown that p97 also participates in endoplasmic reticulum-associated degradation (ERAD) of aberrant proteins in the secretory pathway . Based on observations that human anti-p95c antibodies inhibited in vitro nuclear assembly in a Xenopus oocyte assay and immunoprecipitated human recombinant p97/VCP, we concluded that p95c was identical to p97/VCP . In the present study, we report on the clinical significance of anti-p97/VCP found in PBC patients that were followed for an average of 15 years. Our study suggests that anti-p97/VCP is a marker for a more favourable clinical course in PBC patients.
Materials and methods
Patients. In an earlier study, 30 PBC patients were identified with anti-p95c/p97/VCP autoantibodies, but only 13 were initially available for detailed analysis , of which, six patients had liver biopsies. However, the detailed histopathological report could not be retrieved in one patient, and she was excluded from the analysis. The remaining five patients were followed for a longer period (9–21 years, mean 14 years), and adequate sera and detailed clinical and laboratory data were available. In addition, one patient (no. 14)  became available for this study after the patient's consent was received from the attending physician. One patient with a Scheuer stage 1 biopsy was symptomatic. As a comparison group, we included a random cohort of 50 PBC patients from a speciality hepatology clinic who had been followed for a minimum of 1 year. All patients were evaluated routinely as dictated by standards of clinical practice and had documented clinical features and outcomes.
Immunoassays: immunodiffusion, immunoprecipitation and addressable laser bead immunoassay. The Ouchterlony double immunodiffusion method was used to screen for anti-p97/VCP antibodies [13, 14, 18]. Briefly, the prototype serum and 20 mg of rabbit thymus extract or rat liver supernatant were used as antigen source to detect autoantibodies as previously described [13, 14, 18].
HeLa cells were cultured in monolayers in methionine-free medium, radiolabelled for 20 h with 370 kBq/ml of [35S]-methionine (ICN Radiochemicals, Irvine, CA, USA) and reactive proteins were immunoprecipitated from the cell lysates and identified as previously published [19, 20].
The cDNA representing the full-length VCP (p97/VCP: accession number CAA78412; a gift from Dr Graham Warren, Yale University, New Haven, CT, USA) was used as a template for the in vitro transcription and translation reactions (TnT, Promega, Madison, WI, USA) in the presence of [35S]-methionine as previously described [21, 22]. TnT incubations were conducted at 30 °C for 1.5–2 h, and the presence of the translation products was confirmed by subjecting 2–5 μl samples to SDS-PAGE and analysis by autoradiography. The in vitro translated products were then used as antigen source. Immunoprecipitation (IP) reactions were prepared by combining 100 μl 10% protein A-Sepharose beads (Sigma, St Louis, MO, USA, catalogue #p-3391), 10 μl human serum, 500 μl NET2 buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA, 0.5% Nonidet p-40, 0.5% deoxycholic acid, 0.1% SDS, 0.02% sodium azide) and 5–10 μl of labelled recombinant protein obtained from the TnT reaction. After 1 h of incubation at 4–8 °C, the beads were washed five times in NET2, and the proteins eluted in 10 μl of SDS-PAGE sample buffer. The reactive proteins were then analysed by 10 or 12.5% SDS-PAGE.
An addressable laser bead immunoassay (ALBIA, developed by INOVA, San Diego, CA, USA) was used to test for antibodies to pyruvate dehydrogenase (M2), soluble liver antigen (SLA), chromatin and liver kidney microsome (LKM) antigen.
Nuclar assembly assay. Demembranated sperm chromatin was prepared  and stored at − 80 °C at a concentration of 40,000/μl. Xenopus laevis eggs were collected, dejellied and lysed to prepare an interphase extract . Nuclear envelope assembly assays were then performed essentially as described by Smythe and Newport  and published in our previous report .
The heterogeneity and specificity of autoantibodies found in patients with PBC were first identified by IP using [35S]-methionine-labelled HeLa lysates (Fig. 1). All six sera immunoprecipitated an approximately 95 kDa protein, which was previously shown to be identical to p97/VCP  and three sera (lanes 3–6) also immunoprecipitated a 74 kDa protein that comigrated with markers for pyruvate dehydrogenase complex (PDC)-E2. One serum (lane 4) bound an approximately 40 kDa protein, which is presumed to be PDC-E1α.
IP of the full-length radiolabelled recombinant p97/VCP protein was used to confirm the presence of anti-p97/VCP in the six sera (Fig. 2). Serum from the index PBC patient  was used as positive control (lane 7), and a normal human serum was included as a negative control (lane 8). Five sera immunoprecipitated the recombinant p97/VCP protein (lanes 1–4, 6) and although the fifth serum (lane 5) exhibited a precipitin line of identity with the index serum in the immunodiffusion assay and immunoprecipitated p95c from HeLa cell lysates (Fig. 1, lane 5), it did not immunoprecipitate the recombinant protein.
Clinical and serological features of 6 patients with anti-p97/VCP antibodies (Tables 1 and 2)
Table 1. Clinical and serological features of five patients with anti-p97/VCP
AMA M2 (pyruvate dehydrogenase complex) antibodies detected by ALBIA expressed as median fluorescence units (normal = <124); anti-p97/VCP detected by immunodiffusion using liver cytosol extracts as antigen source; anti-VCP detected by IP of recombinant VCP.
Table 2. Comparison of clinical features of six PBC patients with anti-VCP and a cohort of 50 PBC patients without anti-VCP antibodies
Anti-VCP(+) (n = 6)
Anti-VCP(–) (n = 50)
ACA, anticentromere antibodies detected by indirect immunofluorescence; AMA, antimitochondrial antibodies detected by indirect immunofluorescence; VCP, valosin-containing protein.
Number of symptomatic/number of asymptomatic
Follow-up year/mean year
Number of deaths because of hepatic failure number (%)
Number of deaths because of other cause (%)
Number of AMA positive (%)
Number of ACA positive (%)
Among the six patients with anti-p97/VCP antibodies, there were three females and three males who had an age range of 58–84 years (mean 72 years). Coincident morbidity included two patients with Hashimoto thyroiditis and two with Sjögren's syndrome. All the six patients had elevated levels of antibodies to pyruvate dehydrogenase (M2, range 840–4000 mean fluorescence units) but did not have antibodies to SLA, LKM or chromatin as measured by ALBIA. All patients had liver histopathology findings consistent with a diagnosis of PBC. The liver biopsy specimens obtained from cases 1 and 2 showed bile duct destruction compatible with PBC Scheuer stage 2. Another specimen revealed chronic nonsuppurative destructive cholangitis (CNSDC), compatible with Scheuer stage 1. The titre of serum anti-p97/VCP from case 5 as determined by double immunodiffusion was relatively low and may explain why it did not immunoprecipitate recombinant p97/VCP. The remaining four sera all demonstrated moderate (++) to strong (+++) IP reactions. Sera from the five patients demonstrated varying degrees of inhibition of nuclear envelope assembly (19–82%) in the in vitro assay, which was variably correlated with the titre of anti-p97/VCP antibodies (Table 1). The lack of complete correlation between these assays must take into account the different nature of the two assays (IP versus functional inhibition) and the possibility that other related antibodies may also participate in the INA reaction.
Longitudinal laboratory findings of six patients with anti-p97/VCP antibody (Fig. 3, Table 1)
Case 1 was a 69-year-old male previously diagnosed with obstructive liver disease in 1978. AMA was detected by indirect immunofluorescence at a dilution of 1:160, but could not be detected by double immunodiffusion using a rat liver mitochondrial fraction. This serum did exhibit a strong immunoprecipitin reaction when tested against rat liver supernatant. Abnormal liver functions as determined in 1983 included GOT 60 IU/l, GPT 55 IU/l, ALP 1766 IU/l, γ-GTP 614 IU/l and IgM 915 mg/dl. His liver biopsy specimen obtained in 1985 revealed typical chronic NSDC with histiocytes, including a multinucleated one around a damaged bile duct that demonstrated focal condensation of collagen at sites where the duct was probably present. The limiting plate showed segmental erosion and early short radiating septum, suggesting PBC at Scheuer stage 2 (Fig. 4). Administration of ursodeoxycolic acid (UDCA) (300–600 mg/day) was effective in maintaining liver function, but at the age of 84, he died of cerebral thrombosis (Table 1).
Case 2 was a 59-year-old female who visited the hospital in 1990 for evaluation and had liver function tests with results as follows: GOT 49 IU/l, GPT 30 IU/l, ALP 798 IU/l, γ-GTP 180 IU/l and IgM 1360 mg/ml. A liver biopsy performed in April 1990 showed Scheuer stage 1–2 CNSDC and the follicles were judged to be normal. Although this patient was not treated with UDCA, her health and liver status has remained stable (Table 1).
Case 3, a 57-year-old male, initially visited the hospital in 1984 complaining of symptoms of liver dysfunction and laboratory evaluation revealed GOT 42 IU/l, GPT 44 IU/l, ALP 563 IU/l, γ-GTP 194 IU/l and IgM 997 mg/ml. A liver biopsy was performed in February 1986, and histology revealed marked portal inflammation with numerous foci of piecemeal necrosis. There were also a few foci of destructive duct lesions, compatible with florid Scheuer stage 1 duct lesion. The patient was treated with UDCA (150 mg/day) from 1989 to 1998 when the dosage was increased to 600 mg/day. HCV antibodies and HBsAg have been absent since 1990 onwards but hepatocellular carcinoma was detected in July 2003 (Table 1).
The fourth patient initially visited the hospital for treatment of hypertension in 1982 at the age of 54. At that time, she complained of morning stiffness of her fingers and xeropthalmia. She had a positive AMA but did not have any clinical features of liver dysfunction. A liver biopsy revealed CNSDC, and attending physicians elected not to treat her with UDCA until October 1996, when laboratory testing revealed an elevated IgM of 733 mg/dl. She has since moved her residence and has been lost to follow-up (Table 1).
The fifth case was a 53-year-old woman who was suspected of having chronic liver disease since 1983 following an employee health check-up. She visited a medical clinic in March 1998, complaining of struma and pruritus, and exhibited clinical and laboratory features consistent with obstructive liver disease: γ-GTP 250 IU/l, ALP 454 IU/l and IgM 671 mg/ml. Her liver biopsy revealed mild fatty deposition in liver cells, the limiting plate was not destroyed and there was marked mononuclear cell infiltration into one of the Glisson's areas, where CNSDC was noted. Following administration of UDCA (300 mg/day) in July 1998, the liver function tests returned to within the normal range (γ-GTP 29 IU/l, ALP 243 IU/l, IgM 108 mg/ml). The patient currently maintains a stable clinical condition (Table 1).
The sixth case was a man who presented with epigastric pain and icterus at the age of 53 years in 1984. He was suspected of having cholelithiasis and the presence of AMA suggested a diagnosis of PBC. Following surgical removal of a common bile duct stone, obstructive liver impairment continued. His liver biopsy done at 55 years revealed an early stage of PBC. Although recent IgM levels performed at the present age of 72 years had increased from 569 mg/dl on the first visit to 881 mg/dl, the AST (33 IU/l), ALT (46 IU/l), ALP (261 IU/l) and γ-GTP (99 IU/l) were not significantly changed.
Comparison with 50 patients without anti-VCP
The background of 50 PBC patients without anti-VCP is summarized and compared with the five study patients in Table 2. Forty-three were females and seven were males with an age range of 42–77 years (median 60 years). The ratio of symptomatic to asymptomatic patients (1:5) was nearly identical to that of the anti-VCP group. Of note, 8/50 (16%) patients without anti-VCP died because of hepatic failure compared with none with anti-VCP. This is remarkable considering that the follow-up period of the PBC patients without anti-VCP was shorter than the anti-VCP group (mean 7 versus 15 years). Thirty-three percent of death due to other causes in the p97/VCP-positive patients was higher than 5% in the p97/VCP-negative patients. However, this was because the average age of the p97/VCP patients (72 years) was remarkably higher than that of the PBC patients (60 years) without anti-p97/VCP. In this comparison group, 39/50 (78%) patients had AMA and 16/50 (32%) had anticentromere antibodies.
Previously, we reported a novel antibody in autoimmune liver disease sera that reacted with a conformational epitope of a cytosolic 95 kDa protein, which we tentatively named p95c . These antibodies were found in 12.5% of PBC and 9.8% of autoimmune hepatitis sera. Recently, we have provided evidence that the p95c antigen is identical to p97/VCP based on in vitro experiments which showed anti-p95c antibody inhibiting nuclear assembly, the major function of p97/VCP, and reacting with the 97 kDa radiolabelled recombinant protein . The clinical features and outcome of patients became of interest when it was noted that they might have a more favourable clinical course. We originally gathered 30 sera over the past 10 years containing anti-p97/VCP. However, most sera were sent from the small hospital and private clinics and detailed clinical information could not be obtained. A few sera were gathered from large hospitals, but the patients' consent could not be obtained and as a result of which, only sera of six patients were available for inclusion in this study.
The p97/VCP protein was first associated with the AAA (ATPase associated with diverse cellular activities) family of proteins and accounts for as much as 1% of the cellular protein . The AAA family of proteins contain ∼230 amino acid residues and common ATPase domains (i.e. Walker motif) and a characteristic second region of homology [27, 28]. The crystal structure of p97/VCP has been reported  and was shown to play a key role in postmitotic assembly of the ER, Golgi apparatus and nuclear membrane . Furthermore, as an unfoldlase, it is associated with degradation of misfolded proteins in the ER that are released into the cytoplasm (ERAD) . Another important function of p97/VCP has been found to be augmentation of signals to nuclear factor kappa B (NF-κB) in carcinoma cells such as gastric cancer and hepatocellular carcinoma [30, 31].
Using conventional diagnostic assays, AMA are found in at least 85% of patients with PBC and is generally regarded as a diagnostic hallmark for this disease [2, 3]. It has been noted, however, that the clinical manifestations of PBC patients with AMA are almost the same as those of PBC patients without AMA. In 1990, Klein and Berg  proposed that anti-M4 was a marker for a poor prognosis of PBC and that anti-M9 indicated a good prognosis. However, these conclusions have not been widely validated.
In general, asymptomatic primary biliary cirrhosis (a-PBC) patients have a longer life expectancy than those who are symptomatic and have pruritus or/and scleral icterus. For example, in one cohort of 250 patients followed for 24 years, the median survival for a-PBC and symptomatic primary biliary cirrhosis (s-PBC) was 16 and 7.5 years, respectively . In Japan, the 5-year survival rates following diagnosis with s-PBC and only pruritus, and s-PBC with both pruritus and icterus, were calculated as 88 and 53%, respectively .
In this study, we have reported that six PBC patients with anti-p97/VCP antibodies had an overall survival rate of 67% over a 15-year period. Notably, the first and third patients who died during this period did not succumb to autoimmune liver failure. In contrast, the survival rate of the 50 comparison PBC patients was 74%, with 16% dying of liver failure. It is not clear why the PBC patients with anti-p97/VCP appeared to have a milder clinical course but because our number of patients is admittedly small, multicentre studies will be required to validate these impressions. Unfortunately, liver biopsies were performed only at clinical presentation in each of our six patients, which makes it difficult to confirm the histopathological status of relevant tissues later in their clinical course. It was noted that ALP, γ-GTP and IgM levels were more stable and the clinical course was thus considered to be less aggressive than patients without anti-p97/VCP.
In light of our observation that patients with anti-p97/VCP have a stable clinical course, it is intriguing to postulate that anti-p97/VCP antibodies have a protective role Most autoantibodies are not able to bind the cognate intracellular antigen of living cells. However, the anti-p97/VCP antibody could be unique in another way by gaining entry into certain cells through receptor-mediated endocytosis and a clathrin-binding protein such as Vsp25/SKD . If this occurs in some mononuclear cells that infiltrate and surround the small bile ducts, obstructive liver impairment may be diminished or delayed via mechanisms as previously described [35, 36]. Relevant to these considerations is our observation that anti-p97/VCP antibodies were detected at presentation and at an early stage of PBC. Currently, we are trying to explore this hypothesis in cell culture assays.
We thank Hiroshi Chiba for technical assistance at Health Sciences Research Institute. This research was supported in part by a grant from the Canadian Institutes for Health Research (#10884) to M. J. F.