Arg-gingipain A DNA vaccine prevents alveolar bone loss in mice.

One major pathogenic factor of Porphyromonas gingivalis is Arg-gingipain (Rgp), an arginine-specific cysteine proteinase. To clarify the effect of rgpA DNA vaccine, we immunized BALB/c mice via the abdomen with a Gene Gun or via the nasal cavity weekly for 6 weeks. After immunization, the mice were challenged orally with P. gingivalis. Immunization elicited IgG responses against P. gingivalis in both groups. Nasal immunization also induced sIgA against P. gingivalis, although Gene Gun immunization did not. Reduction of alveolar bone loss was observed in both groups at 42 days following initial infection. This effect was more pronounced in the intranasal immunization group than in the Gene Gun group. The results of this study suggest that immunization with rgpA DNA vaccine via the nasal cavity is an effective method for preventing alveolar bone loss incurred by infection with P. gingivalis.

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.

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.

Correlation between telomerase activity and telomeric-repeat binding factors in gastric cancer.

Telomeres of a specific length are essential for continuous cell proliferation. The length of telomeres must be maintained by telomerase action and the telomeric DNA-repeat binding protein must be protected. Therefore, there seems to be a relationship between cell immortality due to telomerase activity and telomeric DNA-repeat binding protein. We examined telomerase activity and the expression of telomeric-repeat binding factor 1 and 2 (TRF1 and TRF2) in gastric cancer. Telomerase activity was semi-quantified using the f-TRAP technique in 53 cancerous and non-cancerous gastric tissue specimens. TRF1 and TRF2 were also studied using an immunohistochemical method to determine the frequency of these factors in cell nuclei. Telomerase activity was observed in 79.2% of the cancerous tissue and in 39.6% of the non-cancerous tissue. The average semi-quantitative values for telomerase activity were 67.3 total product generated (TPG) unit/microg protein in cancerous tissue and 6.0 TPG unit/microg protein in non-cancerous tissue. Moreover, T0/1 tumor had the same incidence of telomerase activity as T2 or deeper tumors. These results indicated that the activation of telomerase begins at an early stage of carcinogenesis. TRF1 and TRF2 were detected in 45.1% and 42.9% of the cancerous tissue and in 70.6% and 65.6% of the non-cancerous tissue, respectively. In addition, low positive staining ratios were found for TRF1 and TRF2 when cancer had more deeply invaded. However, telomerase activity did not correlate with either TRF1 or TRF2. These findings suggest that optimal conditions for efficient telomerase are produced as cancer progresses, via suppression of TRFs.

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.

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.

[Detection of anti-nuclear antibodies in liver diseases by indirect immunofluorescence method and enzyme-linked immunosorbent assay].

Detection of anti-nuclear antibodies (ANA) is essential for diagnosing autoimmune diseases including autoimmune liver diseases. An indirect immunofluorescence (IIF) method with a cell line (HEp-2) derived from human laryngeal carcinoma has been used as a standard substrate. Recently, an enzyme-linked immunosorbent assay (ELISA) using multiple solid-phase antigens has been developed. We assayed sera from 272 cases of chronic liver diseases, 91 cases of healthy subjects and studied clinical significance of ANA. The sensitivity of IIF method in detection of ANA (fluorescence-ANA: FANA) and that of ELISA (ELISA-ANA: EANA) were 19.2% and 17.3% in chronic hepatitis B (CH-B), 16.7% and 17.3% in chronic hepatitis C (CH-C), 84.2% and 50.9% in primary biliary cirrhosis (PBC), 100% and 85.7% in autoimmune hepatitis (AIH) and 15.4% and 18.7% in healthy subjects. The sensitivity of EANA was considerably lower than that of FANA in PBC and AIH, but the sensitivity was the same in CH-C, CH-B, and healthy subjects. Because the solid-phase target antigens do not include nuclear antigen components recognized only by patients with PBC or AIH, ELISA can not detect all the species of ANA. This accounts for the low sensitivity of EANA in PBC and AIH. In conclusion, the current EANA is useful for screening of ANA, but FANA should be performed when PBC or AIH is suspected.

[An immunohistochemical study on microtubule associated protein-1 of normal and malignant human gastric tissue].

Microtubule Associated Protein-1 (MAP-1) plays an important role for polymerization of tublin to microtubules. Indirect immunohistochemistry using anti-MAP-1 monoclonal antibody was performed in this study. Difference of internuclear MAP-1 staining patterns between normal and malignant human gastric tissues was investigated. Normal gastric tissue had localized MAP-1 positive staining nucleus in corresponding area of generative cell zone of gastric gland. On the other hand, the 75% cases of gastric cancer showed numerous labeled nuclei in most of the cancer tissues, and 25% cases did not show positive staining of nuclei in cancer tissue. The nuclei staining were classified as negative (-), weak granular (+) and strong punctate (++). The tissue staining pattern was fallen into two patterns, uniform and ununiform stained groups. Positive nuclei staining for MAP-1 and staining pattern had no correlation with histological type of gastric cancer, vessel invasion and lymph node metastasis. But post operative 5-years survival rares were 77.3% in uniform stained group and 30.0% in ununiform stained group. Thus, there were relationship between the post operative survival ratio and the staining pattern of nuclei. Internuclear MAP-1 staining pattern was considered to be one of effective parameters on prognostic sign in gastric cancer.

[Two cases of rheumatoid arthritis presenting autoimmune hepatic diseases].

We report on two cases of rheumatoid arthritis (RA) presenting autoimmune hepatic diseases. The first patient, who had been diagnosed as RA at the age of 63, was hospitalized in order to undergo surgery for total left knee replacement at the age of 69. She acquired acute serum hepatitis as a result of blood transfusion she received during the operation. Five years later, she visited our clinic suffering from polyarthritis. She was found to have hyper-alkaline phosphatase (ALP) and hyper rGTP, but no AMA. The second patient, a 60-year-old female whose onset of RA was at the age of 45, complained of general fatigue, and was admitted to the hospital because of persistent liver dysfunction. When corticosteroid was administered to these patients, ALP and rGTP levels in the first case, and AST and ALT levels in the second case were reduced to values in the normal range. ANA in the first case continued to register negative, but ANA in the second case became positive after the patient developed acute hepatitis. Both patients were found to have anti-p25 triplet liver/kidney microsome antibody. We discuss the clinical significance of this antibody.

Coexistence of CREST syndrome and primary biliary cirrhosis. Serological studies of two cases.

Two patients with characteristic features of CREST syndrome and primary biliary cirrhosis are reported. Sera of both patients contained autoantibodies to centromere and mitochondrial antigens. Immunodiffusion analysis identified the specificities of precipitating antibodies to mitochondria of the first case as anti-M-A and M-C, and of the second case as anti-M-A and M-B antibodies. Simultaneous occurrence of 2 marker antibodies in an individual patient indicates that the 2 patients reported here have CREST syndrome and primary biliary cirrhosis, both of which are considered as a distinct clinical entity.

Precipitating antimitochondrial antibodies in Japanese patients with primary biliary cirrhosis.

The prevalence of three precipitating antibodies to mitochondria A (M-A), mitochondria B (M-B), and mitochondria C (M-C), reacting with the antigens in the mitochondrial fraction of sonicated rat liver was studied in Japanese patients with primary biliary cirrhosis and other liver diseases. Antibodies to M-A and M-B were found in 12 of 22 (54%) and 11 of 22 (50%) patients, respectively. Antibodies to M-C were found in only one of 22 (4%) patients. The titers of antibodies to M-A and M-B correlated with the titers of mitochondrial immunofluorescence staining on unfixed mouse kidney section (r = 0.71 and 0.81, respectively). These antibodies were not present in liver cirrhosis (20 patients), chronic active hepatitis (20), acute viral hepatitis (10), and hepatoma (10). However, the titers of these antibodies did not correlate with amount of immunoglobulin, immune complexes, and the severity of disease. This work confirms that the antibodies to M-A and M-B are also marker antibodies for Japanese patients with primary biliary cirrhosis.