Anti-p97/VCP antibodies: an autoantibody marker for a subset of primary biliary cirrhosis patients with milder disease?

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.

Cutaneous immunological activation elicited by a low-fluence pulsed dye laser.

BACKGROUND: Three years ago, the nonablative wrinkle reduction laser (a 585-nm laser, Chromogenex V3; Chromogenex Light Technologies, Llanelli, U.K.) was developed, and there have already been several reports about its clinical effectiveness. The Chromogenex V3 laser has also been reported to be effective in treating acne and atopic dermatitis. These results suggest that the Chromogenex V3 laser has some immunological role. In this study, we investigated immunological changes elicited by laser irradiation at the ultrastructural level and by analysis of interleukin (IL)-2 and IL-4 mRNA in skin homing T lymphocytes. MATERIALS AND METHODS: Eight healthy adult volunteers (mean age 56.3 years, range 25-66 years) were recruited for this study. Ultrastructural analysis was done 3 h after the laser irradiation, as well as 1 day, 3 days, 1 week, 2 weeks, 4 weeks and 5 weeks later. IL-2 and IL-4 mRNAs in skin homing T cells cultured for 6 weeks were semiquantitatively measured using reverse transcriptase-polymerase chain reaction. RESULTS: Ultrastructural observations revealed that at 3 h after laser therapy, neutrophils, monocytes and mast cells could already be seen in the extravascular dermis. These dermal acute inflammatory changes were observed also at 1 week after laser treatment. Two weeks after laser treatment, the capillaries showed an almost normal structure. Four weeks after laser treatment, many lymphocytes and fibroblasts were observed. The numbers of these lymphocytes increased further at 5 weeks after the laser treatment. One week after the laser irradiation, all subjects were positive for IL-2 mRNA and for IL-4 mRNA. The level of IL-4 mRNA was larger compared with that of IL-2 mRNA in all subjects. CONCLUSION: The Chromogenex V3 is a 585-nm visible light laser, and it may affect the skin not only by selective photothermolysis but also by direct cutaneous immunological activation.

Autoantibodies from primary biliary cirrhosis patients with anti-p95c antibodies bind to recombinant p97/VCP and inhibit in vitro nuclear envelope assembly.

We have reported previously that p95c, a novel 95-kDa cytosolic protein, was the target of autoantibodies in sera of patients with autoimmune hepatic diseases. We studied 30 sera that were shown previously to immunoprecipitate a 95 kDa protein from [(35)S]-methionine-labelled HeLa lysates and had a specific precipitin band in immunodiffusion. Thirteen sera were available to test the ability of p95c antibodies to inhibit nuclear envelope assembly in an in vitro assay in which confocal fluorescence microscopy was also used to identify the stages at which nuclear assembly was inhibited. The percentage inhibition of nuclear envelope assembly of the 13 sera ranged from 7% to 99% and nuclear envelope assembly and the swelling of nucleus was inhibited at several stages. The percentage inhibition of nuclear assembly was correlated with the titre of anti-p95c as determined by immunodiffusion. To confirm the identity of this autoantigen, we used a full-length cDNA of the p97/valosin-containing protein (VCP) to produce a radiolabelled recombinant protein that was then used in an immunoprecipitation (IP) assay. Our study demonstrated that 12 of the 13 (93%) human sera with antibodies to p95c immunoprecipitated recombinant p97/VCP. Because p95c and p97 have similar molecular masses and cell localization, and because the majority of sera bind recombinant p97/VCP and anti-p95c antibodies inhibit nuclear assembly, this is compelling evidence that p95c and p97/VCP are identical.

Efficacy of imatinib mesylate (STI571) treatment for a patient with rheumatoid arthritis developing chronic myelogenous leukemia.

We report on an 80-year-old man with rheumatoid arthritis (RA) who presented with chronic myelogenous leukemia (CML). Five years after the onset of RA, the CML diagnosis was made. The patient was treated for CML with 300 mg of imatinib mesylate (STI; signal transduction inhibitor 571) for 8 weeks. Laboratory tests showed that the C-reactive protein level, percentage of cells exhibiting the Philadelphia chromosome (Ph1), WBC count, and Lansbury index for RA all dropped respectively from 7.5 mg/dl to 1.0 mg/dl, 74.9% to 1%, 25, 100/microl to 9900/microl, and 51% to 14%. Administration of imatinib mesylate is felt to be effective in treating not only CML but also RA in the active stage.

A male patient who developed late-onset primary biliary cirrhosis presenting with antinuclear envelope antibodies.

Abstract An 81-year-old man who had previously shown high levels of alkaline phosphatase (ALP), γ-glutamyltransferase (GTP), and total bilirubin presented with acute liver damage. He was positive for serum anti-gp210 and anti-p62 antibodies, but negative for serum antimitochondrial antibody. A liver biopsy revealed massive interstitial fibrosis and pseudolobulus, which were compatible with a diagnosis of primary biliary cirrhosis (PBC) at Scheuer's stage 4. He was given ursodeoxycolic acid at 600 mg/day. However, his condition deteriorated, and he eventually died of hepatic insufficiency in a state of malnutrition. We hypothesize that the presence of anti-gp210 and anti-p62 complex protein antibodies, rather than that of antimitochondrial antibodies, was correlated with the progression of PBC in this particular case.

Prospective study of a systemic sclerosis/dermatomyositis overlap patient presenting with anti-Ku and anti-Ki antibodies.

Abstract A 60-year-old woman visited the Keigu Clinic in January 1998 complaining of morning stiffness and flexion contracture of the distal interphalangeal joint. Blood tests showed the presence of antinuclear antibody at a 1 : 40 dilution with speckled staining. She was suspected of having Heberden's node. Nine months later, she developed Raynaud's phenomenon, sclerodactyly, and Gottron's sign, and was diagnosed with systemic sclerosis/dermatomyositis (SSc/DM) overlap. Blood tests revealed the presence of antinuclear antibody at a 1 : 5120 dilution, along with high titer of anti-Ku and anti-Ki antibodies. Subsequently, the patient developed interstitial pneumonia in January 2000. It is thought that the appearance of antinuclear antibody and development of other immunological events played an important role in determining this patient's limited SSc/DM overlap.

Differences in anti-LKM-1 autoantibody immunoreactivity to CYP2D6 antigenic sites between hepatitis C virus-negative and -positive patients.

Anti-liver kidney microsome type 1 autoantibodies (anti-LKM-1) are known to be present in sera of autoimmune hepatitis type II and a subset of chronic hepatitis C patients. The autoantigen to anti-LKM-1 has been identified to be cytochrome P450 IID6 (CYP2D6) and the most frequently cited CYP2D6 antigenic sites of anti-LKM-1 in sera from autoimmune hepatitis type II patients spans the region aa 256-269. Other antigenic sites on CYP2D6 exist and have been identified in the two patient groups. However, most of these sites are concentrated on the carboxyl-terminal side of the protein, and the amino-terminal region has not been thoroughly investigated. Here, we have studied the antigenicity of the CYP2D6 amino region and compared reactivities between hepatitis C virus (HCV)-negative and -positive Japanese patient groups. A total of 34 anti-LKM-1-positive sera (eight with autoimmune hepatitis type II and 26 with chronic hepatitis C) were included. The immunoreactivity of patients' sera was examined against four conformational and one linear CYP2D6 peptide fragments. A defined antigenic site spanning aa 181-245 was found to react with 88% (7/8) of autoimmune hepatitis type II patients, as opposed to only 38% (10/26) of chronic hepatitis C patients. This was a significant difference (P< 0.043). Among these positively reacting samples, five of the seven autoimmune hepatitis type II sera and four of the ten chronic hepatitis C sera also reacted with a synthetic peptide spanning aa 256-269. Anti-LKM-1 thus may be able to recognize simultaneously at least two antigenic sites on the CYP2D6 protein, and reactivities against individual epitopes may differ according to HCV infectivity status.

Autoantibodies to a 68/48 kDa protein in chronic fatigue syndrome and primary fibromyalgia: a possible marker for hypersomnia and cognitive disorders.

OBJECTIVE: To identify antinuclear antibodies (ANA) specific for chronic fatigue syndrome (CFS), and in related conditions such as fibromyalgia (FM) or psychiatric disorders. METHODS: One hundred and fourteen CFS patients and 125 primary and secondary FM patients were selected based on criteria advocated by the Centers for Disease Control and Prevention and by the American College of Rheumatology, respectively. As controls, healthy subjects and patients with either various psychiatric disorders or diffuse connective tissue diseases were included. Autoantibodies were examined by immunoblot utilizing HeLa cell extracts as the antigen. RESULTS: Autoantibodies to a 68/48 kDa protein were present in 13.2 and 15.6% of patients with CFS and primary FM, respectively. In addition, autoantibodies to a 45 kDa protein were found in 37.1 and 21.6% of the patients with secondary FM and psychiatric disorders, respectively. Meanwhile, these two autoantibodies were not found at all in connective tissue disease patients without FM, nor in healthy subjects (P<0.05). As a group, the anti-68/48 kDa-positive CFS patients presented more frequently with hypersomnia (P<0.005), short-term amnesia (P<0.07) or difficulty in concentration (P<0.05) than those CFS patients without the antibodies. CONCLUSIONS: The presence of the anti-68/48 kDa protein antibodies in a portion of both CFS and primary FM patients suggests the existence of a common immunological background. These antibodies may find utility as possible markers for a clinicoserological subset of CFS/FM patients with hypersomnia and cognitive complaints.

Immunoreactivity to various human cytochrome P450 proteins of sera from patients with autoimmune hepatitis, chronic hepatitis B, and chronic hepatitis C.

Numerous human Cytochrome P450 enzymes (CYPs) associated with 'phase I' drug metabolism have been identified. Among them, CYP2D6 is thought to be the major target autoantigen to anti-liver kidney microsome (LKM)-1 autoantibody, a characteristic feature of autoimmune hepatitis (AIH) type II. In this study, we were able to clone CYP2D6 cDNA from a human liver cDNA library and express the CYP2D6 recombinant protein, and also to prepare four other representative human CYP proteins (CYP1A2, 2C9, 2E1, and 3A4). These preparations were used to assay the immunoreactivity of patients with AIH type I (n=35) and type II (n=9). As comparison groups, sera from patients with chronic hepatitis B (n=15), chronic hepatitis C (n=55; 24 anti-LKM-1-positive, 31 anti-LKM-1-negative), and from normal controls (n=30) were included. The five CYP proteins did not react with sera from normal controls nor from patients with chronic hepatitis B. CYP2D6 reacted with sera from 100% (9/9) of AIH type II patients, 79% (19/24) of patients with anti-LKM-1-positive chronic hepatitis C, and 6.5% (2/31) of patients with anti-LKM-1-negative chronic hepatitis C. In contrast, CYP1A2 reacted with serum from one patient with AIH type I, CYP2E1 reacted with sera from two patients with AIH type I, one patient with anti-LKM-1-positive chronic hepatitis C, and two patients with anti-LKM-1-negative chronic hepatitis C, and CYP3A4 reacted with sera from one patient with AIH type II and one patient with anti-LKM-1-positive chronic hepatitis C. CYP2C9 did not react with any of the sera included in this study. From these results, it is suggested that CYPs other than CYP2D6 can function as immunotargets in certain disease conditions.

Differences in antigenic sites, recognized by anti-liver-kidney microsome-1 (LKM-1) autoantibody, between HCV-positive and HCV-negative sera in Japanese patients.

Anti-liver-kidney microsome-1 (LKM-1), which reacts with cytochrome P450 IID6 (CYP2D6), is an autoantibody present in autoimmune hepatitis type II, which affects primarily young patients. Recently, it has been shown some adult patients with chronic hepatitis C are also positive for anti-LKM-1. Thus, anti-LKM-1-positive patients can be classified into two subgroups: (1) those with autoimmune hepatitis type II and (2) those with chronic hepatitis C. We investigated the antigenic epitopes of CYP2D6 with which each of these two anti-LKM-1-positive subgroups reacted. Multiple deletion mutants of CYP2D6 were constructed from a human liver cDNA library and five recombinant fusion proteins expressed. Antigenic epitopes were determined by immunoblot analysis using these proteins. Anti-LKM-1 present in HCV-negative sera recognized at least two peptide regions of aa213-280 and aa341-477 of human CYP2D6. In contrast, anti-LKM-1 present in HCV-positive sera recognized only a single region of aa341-477. Thus, the sera of patients with autoimmune hepatitis type II and patients with chronic hepatitis C recognize different antigenic epitopes of the CYP2D6 molecule. To our knowledge, this is the first time LKM-1 autoantigens have been analyzed at the molecular level in Japanese patients.

A novel antibody directed against a three-dimensional configuration of a 95-kDa protein in patients with autoimmune hepatic diseases.

Sera from patients with primary biliary cirrhosis recognize various cellular components, such as mitochondria, centromere, nuclear envelope, and multiple nuclear dot antigens. There also appears to be a novel antibody reacting with a particular protein in these sera. The presence of this antibody was investigated by double immunodiffusion using rat liver cytoplasmic antigens, by immunoprecipitation of [35S]-methionine labelled HeLa cell extracts, and by immunoblot using disrupted HeLa cell extracts. Test sera were obtained from 491 patients with various liver diseases. Nine of the 491 sera were found to react with a 95-kDa protein as determined by immunoprecipitation of [35S]-methionine labelled HeLa cell extracts and by double immunodiffusion using a rat liver microsomal preparation. However, these same nine sera showed no reaction in the immunoblot assay. On the basis of its molecular mass and its presence in the cytoplasmic fraction, this antigen was named p95 C. This anti-p95 C antibody was detected in six of 50 (12%) sera from patients with primary biliary cirrhosis, and in three of 31 (9.7%) sera from patients with autoimmune hepatitis, but not in any of the remaining 410 sera obtained from patients with other hepatic diseases. It is concluded that anti-p95 C antibody reacts primarily with the native form of the 95-kDa protein, and represents another possible analyte for diagnosing autoimmune liver diseases.

Mechanism of influenza virus transcription inhibition by matrix (M1) protein.

The mechanism by which influenza virus matrix (M1) protein inhibits viral RNA (vRNA) transcription was investigated. Evidence has been generated that M1 protein inhibits the steps of vRNA transcription initiation and reinitiation more effectively than that of RNA chain elongation. The vRNA-associated nucleocapsid protein (NP) appears to be critical for this inhibition, implying that M1 protein binds to the ribonucleoprotein complex (RNP) through NP.

Monoclonal antibody analysis of influenza virus matrix protein epitopes involved in transcription inhibition.

Influenza virus M1 protein has been shown to inhibit viral RNA transcription, and in this study the epitopes on M1 critical for this function were localized. When a battery of 15 monoclonal anti-M1 antibodies were reacted with chemically cleaved fragments of M1 on a western blot, five distinct banding patterns were observed. A representative antibody was selected from each banding group, and its ability to reverse M1-effected transcription inhibition was measured. From these data, the sites on M1 critical for transcription inhibition were deduced. It appears now that the regions on M1 in the vicinity of amino acid residues #70 and #140 are critical for inhibition. Furthermore, by taking into account the hydropathicity and secondary structure, it is hypothesized that amino acids #70 and #140 are physically close together in the final three-dimensional conformation of M1 protein and that the residues in between form a loop and are thus removed from the functional site.

Objectives for a system of health care delivery for HIV infected people.

This paper assumes that the effectiveness and efficiency of an integrated community system of care for HIV infected people may depend to a large extent on common perceptions of the objectives of such a system among three sets of actors--the patient, the professional care manager, and the continual care giver. It discusses a decision analytic inquiry into that concurrence on objectives. The conclusion is that within the community studied there is strong evidence of a significant lack of such common purpose.

Western blot analyses of measles virus antibody in normal persons and in patients with multiple sclerosis, subacute sclerosing panencephalitis, or atypical measles.

A version of the Western blot was developed to detect serum antibodies against measles virus polypeptides. With this technique, a seroepidemiological survey of antibodies to the several measles virus proteins in diverse measles-related conditions was conducted. The sera were obtained from individuals with a recent or long-past history of natural measles, from persons with a history of immunization with live attenuated measles vaccine, and from patients with multiple sclerosis, subacute sclerosing panencephalitis, or atypical measles. The findings indicated that live attenuated measles vaccine elicits an antibody response qualitatively resembling that of a natural infection. In addition, multiple sclerosis patients made less antibody to the measles virus M protein than did individuals with a long-past history of natural measles. Thus, the immunological reaction of multiple sclerosis patients to measles virus is qualitatively, as well as quantitatively, different from that of normal persons. Finally, persons with subacute sclerosing panencephalitis and atypical measles mounted abnormally high antibody responses to measles virus polypeptides, in particular the P protein.