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In this report, we present a 63-year-old woman who had limited cutaneous systemic sclerosis and subsequently developed typical primary biliary cirrhosis after an acute myocardial infarction. The patient initially developed Raynaud's phenomenon, and 4 years later visited the clinic in 1994 complaining of abdominal distress, xerostomia, and xerophthalmia. A diagnosis of limited cutaneous systemic sclerosis was based on Raynaud's phenomenon, sclerodactyly and anti-centromere antibodies. She was also found to have anti-inositol 1,4,5-trisphosphate receptor 3 (IP(3)R3) antibodies, but anti-mitochondrial antibodies were only weakly positive. Seven years later, she developed vertigo and nausea, and was hospitalized due to complaints of an oppressive sensation of the anterior chest. Electrocardiogram results showed a reduction of R waves and ST segment elevation in II, III, and aVf leads. Coronary angiography showed 99% obstruction of the left anterior descending artery and 50% of stenosis of the right coronary artery. Three years later, the patient was noted to have anti-mitochondrial antibodies. Retrospective analysis of the patient's sera showed that IP(3)R3 antibodies were decreasing. Since myocardium is particularly rich in mitochondria, it is thought that myocardial infarction may have been the triggering event that initiated antigen-presenting cells to selectively induce an anti-mitochondrial antibody response.

SUMMARYWe 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 [35S]-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.

Topical antibiotics, isotretinoin or systemic antibiotics are usually used for acne therapy. However, isotretinoin cannot be used during pregnancy because it can cause significant birth defects while systemic antibiotics can have adverse side effects such as gastrointestinal irritation, photosensitivity and tetracycline sensitivity. Describe here is a high-intensity, narrow-band, blue light (ClearLight) system, and its therapeutic clinical effect is investigated on acne using cutaneous measurements, bacterial observations and ultrastructural changes.A total of 28 adult healthy volunteers with facial acne (mean age 28.1 years, range 16-56 years) were recruited for this study. They were treated with a total of eight serial biweekly 15-minute treatment sessions. Clinical counts of acne, as well as moisture, sebum and pH measurements were taken between each session. Nine of the 28 patients were followed for 2-3 months after the last treatment. Detection of bacteria in acne pustules was analyzed by culture and by polymerase chain reaction (PCR). Ultrastructural changes were examined in eight patients after four sessions of the light therapy.All patients completed the study. Overall, there was a 64.7% improvement in acne lesions. There were no bacterial changes before or after the therapy, although damaged Propionibacterium acnes were observed at the ultrastructural level.ClearLight performed eight times over 4 weeks can be useful in the treatment of acne. Further investigation will be needed to elucidate the mechanism of action of ClearLight.

Primary biliary cirrhosis (PBC) sera contain antibodies which recognize various nuclear envelope proteins of which antibody against gp210 has been proven to be diagnostic for disease. In contrast, the clinical significance of another nuclear envelope antibody, anti-p62 antibody has not been well investigated. In the present study, we have analyzed anti-nuclear envelope antibodies by indirect immunofluorescence and immunoblot using rat liver nuclear envelope proteins and wheat germ agglutinin-bound fraction. Test sera were obtained from 175 patients with PBC and from 120 controls. Anti-gp210, anti-lamina associated polypeptide 2, anti-lamin B receptor, and anti-p62 complex antibodies were detected with a frequency of 26% (46 of 175), 6% (11 of 175), 9% (16 of 175), and 13% (15 of 115), respectively. The confirmation of Scheuer's stage IV was made with a frequency of 27% (4 of 15) in PBC patients with anti-p62 complex antibody, in contrast to only 2% (2 of 100) in PBC patients without anti-p62 complex antibody. This difference was found to be statistically significant. The presence of anti-p62 complex antibody may be related with the progressive or advanced state of PBC.

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.

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.

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.

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.

Amantadine is not thought to be effective for the treatment of swine-origin influenza virus (S-OIV) based on an analysis of genetic sequences of the M2 protein. However, the actual clinical efficacy of amantadine has not been well documented. Here, we were able to compare the efficacies of amantadine and neuraminidase inhibitors. Subjects consisted of 428 patients, including 144 with seasonal influenza (flu) identified between 2008 and 2009, and 284 with S-OIV identified between July 1 and November 30, 2009. Diagnosis of flu was established using a rapid diagnostic kit obtained commercially in Japan. Body temperature sheets were obtained from 95% of the S-OIV patients. Times required to recover normal body temperature were compared among subjects using different antiviral drugs. Genetic abnormalities in the M2 protein were also investigated in 66 randomly selected subjects from within the patient pool. Overall, the average hours required to recover normal body temperature in S-OIV patients treated with amantadine (160 cases), with oseltamivir (59 cases), or with zanamivir (65 cases) were 33.9 ± 20.7, 31.7 ± 16.0, or 36.3 ± 21.6, respectively. These differences were not statistically significant. The N31S abnormality was found in all 14 samples taken from the H3N2 patients and in all of the 23 samples taken from in S-OIV patients. However, this abnormality was not found in any of the 30 samples taken from seasonal H1N1 patients. Amantadine was found to be equally effective in treating S-OIV patients as neuraminidase inhibitors. The genetic abnormality resulting in S31N amino acid conversion identified in some of the H3N2 and S-OIV patients is thought to alter the function of M2 protein only mildly.

In 2003, the ISO 15189 international standardization program on the quality and competence of the clinical reference laboratory was introduced. To date, 46 facilities have committed themselves to providing a higher level of medical service by incorporating a quality management system (QMS) and acquiring accreditation. QMS is defined as "setting up a policy and goals pertaining to quality, and adopting an appropriate system," and is a scheme that includes all managerial and technical factors that can affect test results. Regarding the Health Sciences Research Institute Group, 4 facilities have previously received the accreditation described above, but in the process of implementing the QMS, a number of problems have been identified. Here, we report on the effectiveness of adopting such a QMS based on the results of employee questionnaires, internal audits, customer complaint analyses, and external audits by the Japan Accreditation Board for Conformity Assessment (JAB), the official inspection body for accreditation.

Background Prenatal human immunodeficiency virus (HIV) testing is essential for the prevention of mother-to-child transmission. However, false-positive results of screening testing are a concern as they may cause unnecessary emotional stress to pregnant women waiting for confirmatory test results. In regions with an extremely low prevalence, the positive predictive values of screening are unacceptably low rate. Here, we propose a HIV screening algorithm consisting of serial two fourth-generation enzyme immunoassays to reduce the number of false-positive screening results. Methodology/Principal Findings When 6461 pregnant women presenting to two maternity hospitals located in the Tokyo metropolitan area of Japan from September, 2004 to January, 2006 were tested using Enzygnost HIV Integral as a first screening test, 27 showed positive reactions. When these positive reaction samples were tested using VIDAS HIV DUO Quick as a second screening test, only one of them had a positive reaction, and the remaining 26 were nonreactive. Confirmatory Western blots and nucleic acid amplification test also showed that one was positive and the remaining 26 were negative; the subject who was positive with the confirmatory tests was identical to the subject who was positive with the second screening test. Thus, by adding the second screening test, the false-positive rate was improved from 0.4% to 0%, and the positive predictive value from 3.7% to 100%, compared with the single screening test. Conclusion By applying our serial screening algorithm to HIV testing in maternity hospitals, many uninfected pregnant women would not need to receive confirmatory tests and be subjected to emotional turmoil while waiting for their confirmatory test results. This algorithm would be suitable for HIV testing of pregnant women living in low prevalence regions such as Japan.

IP(3)R2 and IP(3)R3 double knock-out mice present with exocrine dysfunctions such as secretion deficits of saliva and pancreatic juice. Therefore, we investigated whether the presence of antibodies to IP(3)Rs could be found in patients with Sjögren's syndrome, and the location of the epitopes. Subjects included 35 primary Sjögren's syndrome, 39 secondary Sjögren's syndrome, 144 rheumatoid arthritis, and 96 other connective tissue disease patients. As controls, 33 healthy subjects were included. Immunoblot was conducted using recombinant proteins IP(3)R1, IP(3)R2, and IP(3)R3 made from full-length cDNA, and core, T604, and EL for epitope mapping. Antibodies to IP(3)R1 in sera from patients with primary Sjögren's syndrome, secondary Sjögren's syndrome, and rheumatoid arthritis were found to be positive in 17 of 35 (48.6%), 13 of 39 (33%), and 34 of 124 (27.4%) cases, respectively. These frequencies were significantly higher than the 1 of 33 (3.0%) found in normal healthy subjects. The frequency of anti-IP(3)R2 antibodies in rheumatoid arthritis was found to be higher than those found in Sjögren's syndrome, systemic lupus erythematosus, and systemic sclerosis. Anti-IP(3)R2 antibodies found in rheumatoid arthritis primarily recognized residues 578-2171 of the internal coupling and regulatory domain. On the other hand, anti-IP(3)R1 found in Sjögren's syndrome recognized residues 224-604 of the core protein IP(3)R1. Anti-IP(3)R1 antibodies were present in 48.6% of primary Sjögren's syndrome and in 3.0% of normal healthy subjects. Anti-IP(3)R2 antibodies were detected most frequently in rheumatoid arthritis. Locations of the antigenic epitopes were found to differ among the disease conditions.

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.

Summary 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.

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.

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.

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.