OBJECTIVETo explore the clinical and pathological features of primary biliary cirrhosis (PBC) patients with negative anti-mitochondria antibody (AMA).

METHODSTwo hundreds and eight PBC patients were enrolled. The clinical and histological data of the negative AMA cases were compared with the AMA/AMA-M2 positive cases.

RESULTS30 out of the 208 cases (14.4%) were AMA negative patients in our study. The general status, biochemical tests and histological findings between the two groups had no significant difference (P > 0.05). The Gamma-globulin, IgG, IgM and IgA levels of AMA/AMA-M2 positive PBC patients were higher than that of the AMA negative cases (P < 0.05). The abnormal rate of cholesterol in AMA negative PBC patients was 65.4% as compared to 50.4% in AMA/AMA-M2 positive cases, no significant difference existed between (P > 0.05). Anti-nuclear antibody (ANA) was observed in 29 (96.7%) AMA negative PBC patients, including 14 (48.3%) with granular pattern, 8 (27.6%) with nuclear membrane pattern, 6 (20.7%) with kinetochore pattern and 1 (3.4%) with homogeneous pattern. AMA negative PBC patients had elevated serum ALP, GGT, IgM and cholesterol levels, and decreased serum AST, IgG and IgA levels as compared with that of autoimmune hepatitis patients (P < 0.05, respectively).

CONCLUSIONIn cholestatic patients with elevated IgM and cholesterol levels, ANA positive with non-homogeneous pattern, the diagnosis of PBC should be suspected, albeit AMA negative. The clinical, biochemical and histological features of the AMA negative PBC patients were similar to classic PBC patients, but quite different from autoimmune hepatitis.

Adult ; Antibodies, Antinuclear ; analysis ; Female ; Humans ; Liver Cirrhosis, Biliary ; immunology ; pathology ; Male ; Middle Aged ; Mitochondria ; immunology ; gamma-Globulins ; metabolism

OBJECTIVETo construct a replicative anti-tumor DNA vaccine PSCK-2PFcGB based on Semliki Forest Virus (SFV) replicon vector and observe its expression in vivo and in vitro.

METHODSThe plasmid pVAX1-2PFcGB was digested with Nhe I, and the digestion product was blunted prior to further digestion with BssH II to obtain the fragment 2PFcGB, a fusion gene containing the multitarget complex antigen 2PAG encoding both the most cytotoxic T lymphocyte epitopes of human survivin and chorionic gonadotropin β chain-CTP37 of human and monkey. The 2PFcGB fragment was inserted into the PSCK vector digested by Sma I. The products with the expected size were extracted and ligated, and the positive clones were screened by kanamycin and amplified. The recombinant PSCK-2PFcGB, following identification by colony PCR and restriction endonuclease Nde I, was transfected into 293T cells via lipofectamine 2000 and its expression was detected. The recombinant plasmid was also transfected into mouse quadriceps femoris muscle to observe its expression in vivo by immunohistochemistry.

RESULTSNde I digestion resulted in a fragment of the expected size. Transfection with the recombinant plasmid PSCK-2PFcGB resulted in successful expression of the antigen and adjuvant molecular protein in 293T cells, with the positivity rates of 5.70% and 19.75%, respectively. The fusion tumor antigen survivin and hCGβ-CTP37 were also detected in the muscular tissues of the mice.

CONCLUSIONA novel replicative anti-tumor DNA vaccine PSCK-2PFcGB has been successfully constructed and can be expressed in 293T cells and in the muscular tissues of immunized mice, which provide a basis for further studies of the antitumor activity and immunological mechanism of the DNA vaccine.

Animals ; Antibodies, Antinuclear ; immunology ; Cancer Vaccines ; biosynthesis ; immunology ; Gene Expression ; Genetic Vectors ; HEK293 Cells ; Humans ; Mice ; Muscle, Skeletal ; metabolism ; Plasmids ; Semliki forest virus ; genetics ; Vaccines, DNA ; biosynthesis ; immunology

OBJECTIVETo examine the levels of protein oxidation in systemic lupus erythematosus (SLE) and to evaluate the relation between oxidative protein damage and disease activity index.

METHODSPlasma was collected from SLE patients and healthy subjects as controls. Protein-bound carbonyls, protein thiols, superoxide dismutase (SOD), myeloperoxidase (MPO) and total antioxidant capacity (TAOC) were determined by spectrophotometry. Levels of anti-double-stranded DNA (anti-dsDNA) and antinuclear antibody (ANA) were quantified by enzyme-linked immunosorbent assay.

RESULTSWhen comparing with the control subjects, SLE patients exhibited elevated levels of protein carbonyls [(0.101 +/- 0.033) nmol/mg, (0.061 +/- 0.019)nmol/mg, P < 0.01], degraded levels of protein thiols [(3.911 +/- 0.968) nmol/mg, (4.655 +/- 0.798) nmol/mg, P < 0.01] and activities of T-SOD [(67.01 +/- 12.22) U/ml, (97.35 +/- 14.11) U/ml, P < 0.01]. Levels of plasma protein thiols and activities of T-SOD were lower in SLE patients positive for anti-dsDNA antibody, compared with patients negative for anti-dsDNA antibody.

CONCLUSIONWe found that the elevated levels of multiple markers of protein oxidation and degraded activities of antioxidant enzymes in plasma from SLE patients existed when comparing with the controls, and the all the levels were correlated with disease activity. Our findings suggested that protein oxidation might play a role in the pathogenesis of chronic organ damage in SLE.

Adult ; Antibodies, Antinuclear ; metabolism ; DNA ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Lupus Erythematosus, Systemic ; blood ; metabolism ; Male ; Middle Aged ; Oxidation-Reduction ; Peroxidase ; metabolism ; Proteins ; metabolism ; Superoxide Dismutase ; metabolism ; Young Adult

OBJECTIVETo evaluate the feasibility and stability of chemically conjugating IgM on collagen films.

METHODSIgM was labeled with 125I using the chloramine-T method. Six collagen films were randomly divided into two groups. In chemical coupling group 125I-labeled IgM was chemically coupled with the films through N-succinmiclyl-3- (2-pyridyl-dithio) propionate reaction. In control group 125I-labeled IgM was absorbed onto collagen films. The amount of IgM on the collagen films and the amount of IgM remained on the films after extensive rinsing with phosphate buffered saline were monitored by counting the radioactivity of 125I.

RESULTSThe amount of antibodies loaded onto collagen films in the chemical coupling group was 15 times higher than that on the control films, showing significant statistical difference (P < 0.01). And the stability of conjugation antibodies on collagen films was significantly better than the control films.

CONCLUSIONChemical coupling is an effective approach to immobilize antibodies on collagen for further plasmid DNA tethering.

Angioplasty, Balloon, Coronary ; instrumentation ; Animals ; Antibodies, Antinuclear ; metabolism ; Cattle ; Coated Materials, Biocompatible ; chemistry ; metabolism ; Collagen ; chemistry ; metabolism ; Genetic Vectors ; Immunoglobulin M ; metabolism ; In Vitro Techniques ; Mice ; Protein Binding ; Stents ; Surface Properties

We report here a case of a 59-yr-old man with CD4+ T-cell large granular lymphocytic leukemia (T-LGL). Peripheral blood examination indicated leukocytosis (45x10(9) cells/L) that consisted of 34% neoplastic lymphoid cells. Other laboratory results indicated no specific abnormalities except for serum antinuclear antibody titer (1:640), glucose (1.39 g/L), and hemoglobin A1c (7.7%) levels. Computed tomography indicated multiple small enlarged lymph nodes (<1 cm in diameter) in both the axillary and inguinal areas, a cutaneous nodule (1.5 cm in diameter) in the left suboccipital area, and mild hepatosplenomegaly. Bone marrow examination revealed hypercellular marrow that consisted of 2.4% neoplastic lymphoid cells. The neoplastic lymphoid cells exhibited a medium size, irregularly shaped nuclei, a moderate amount of cytoplasm, and large granules in the cytoplasm. Immunohistochemical analysis indicated CD3+, CD4+, T-cell receptor betaF1+, granzyme B+, and TIA1+. Flow cytometric analysis of the neoplastic lymphoid cells revealed CD3+, cytoplasmic CD3+, CD4+, and CD7+. Cytogenetic analysis indicated an abnormal karyotype of 46,XY,inv(3)(p21q27),t(12;17)(q24.1;q21),del(13)(q14q22)[2]/46,XY[28]. The patient was diagnosed with CD4+ T-LGL and received chemotherapy (10.0 mg methotrexate). This is the second case of CD4+ T-LGL that has been reported in Korea.
Antibodies, Antinuclear/analysis ; Blood Glucose/analysis ; Bone Marrow Cells/metabolism/pathology ; Hemoglobin A, Glycosylated/metabolism ; Humans ; Immunohistochemistry ; Immunophenotyping ; Karyotyping ; Leukemia, Large Granular Lymphocytic/*diagnosis/pathology/radiography ; Lymph Nodes/pathology ; Male ; Middle Aged ; Neoplastic Cells, Circulating/metabolism/pathology ; Tomography, X-Ray Computed

We report here a case of a 59-yr-old man with CD4+ T-cell large granular lymphocytic leukemia (T-LGL). Peripheral blood examination indicated leukocytosis (45x10(9) cells/L) that consisted of 34% neoplastic lymphoid cells. Other laboratory results indicated no specific abnormalities except for serum antinuclear antibody titer (1:640), glucose (1.39 g/L), and hemoglobin A1c (7.7%) levels. Computed tomography indicated multiple small enlarged lymph nodes (<1 cm in diameter) in both the axillary and inguinal areas, a cutaneous nodule (1.5 cm in diameter) in the left suboccipital area, and mild hepatosplenomegaly. Bone marrow examination revealed hypercellular marrow that consisted of 2.4% neoplastic lymphoid cells. The neoplastic lymphoid cells exhibited a medium size, irregularly shaped nuclei, a moderate amount of cytoplasm, and large granules in the cytoplasm. Immunohistochemical analysis indicated CD3+, CD4+, T-cell receptor betaF1+, granzyme B+, and TIA1+. Flow cytometric analysis of the neoplastic lymphoid cells revealed CD3+, cytoplasmic CD3+, CD4+, and CD7+. Cytogenetic analysis indicated an abnormal karyotype of 46,XY,inv(3)(p21q27),t(12;17)(q24.1;q21),del(13)(q14q22)[2]/46,XY[28]. The patient was diagnosed with CD4+ T-LGL and received chemotherapy (10.0 mg methotrexate). This is the second case of CD4+ T-LGL that has been reported in Korea.
Antibodies, Antinuclear/analysis ; Blood Glucose/analysis ; Bone Marrow Cells/metabolism/pathology ; Hemoglobin A, Glycosylated/metabolism ; Humans ; Immunohistochemistry ; Immunophenotyping ; Karyotyping ; Leukemia, Large Granular Lymphocytic/*diagnosis/pathology/radiography ; Lymph Nodes/pathology ; Male ; Middle Aged ; Neoplastic Cells, Circulating/metabolism/pathology ; Tomography, X-Ray Computed

We concentrated ourselves to evaluate the prognostic significance of the p53 gene mutations, its protein expression and MIB-1 index as a proliferative marker in canine mammary tumors. In the present study, a total of 20 cases were examined, among which there were 5 malignant mixed tumors, 4 mammary gland adenocarcinomas, 1 papillary adenocarcinoma, 8 benign mixed tumors and 2 mammary gland adenomas. Positive immunostaining for p53 with PAb240 antibody was found in 2 benign (20%) and 3 malignant (30%) tumors. However, PAb421 antibody did not give positive result at all. In Western blot analysis, the p53 expression in benign and malignant tumors was detected in 4 and 3 cases, respectively. p53 mutations were found in 6 cases out of the cases with detected p53 protein expression. The MIB-1 index in benign and malignant tumors were 17.6+/-20.8% and 29.0+/-27.2%, respectively and there was no significant difference between tumor types. There was a significant correlation between p53 mutations and p53 overexpression (correlation coefficient = 0.5, p < 0.05). In Kaplan-Meier survival analysis, the p53 index was associated with significantly shortened survival time (p < 0.01). In multivariate analysis, p53 overexpression was only an independent factor for indicator of worse prognosis in canine mammary tumors (p = 0.01). These results demonstrated that p53 gene mutations and protein overexpression using the PAb240 anti-p53 antibody were useful predictors of increased malignant potential and poor prognosis in canine mammary tumors.
Animals ; Antibodies, Antinuclear/metabolism ; Antibodies, Monoclonal/metabolism ; Blotting, Western/veterinary ; Dog Diseases/genetics/*metabolism ; Dogs ; Female ; Genes, p53/*genetics ; Immunohistochemistry/veterinary ; Ki-67 Antigen/metabolism ; Mammary Neoplasms, Animal/genetics/*metabolism ; *Mutation ; Predictive Value of Tests ; Proportional Hazards Models ; Tumor Suppressor Protein p53/*biosynthesis/genetics

OBJECTIVEGene vaccine security is of concern because of the possibility of insertion mutagenesis resulting in inactivation of tumor suppressor or activation of oncogene. The purpose of this study was to examine the potential of anti-caries DNA vaccine pcDNA3-gtfB integrating into the host cell genome.

METHODSAnti-caries DNA vaccine pcDNA3-gtfB was constructed by the previous study. The gtfB gene(904-4,578 bp, genebank M17361) was cloned from Streptococcus mutans GS-5. 36 Wistar rats were divided into 2 groups: submandibular gland-targeted injection(SGT) group and control group. Rats in SGT group were injected with 100 micrograms of plasmid pcDNA3-gtfB, rats in control group with PBS solution. Genomes from submandibular gland, kidney, heart, liver, lung, and brain tissues were isolated later in 12 weeks. Genomes from different tissues were purified by low-melting agarose electrophoresis. Using the purified genomes as template, plasmid integration were examined by PCR(upper primer: 5'-ATATGGTACCATGACCGAAGCGACATCTAAGCAAGA-3', lower primer: 5'-ACTACTCGAGTTAGAACCATTGACCCTG AGCATTGC-3'). The sensitivity level of PCR was determined by adding gradient plasmid copies into genomes in control group.

RESULTSThe examination of 6 tissues failed in revealing any evidence of integration at the sensitivity level that could detect 1 copy integration in 10,000 nuclei.

CONCLUSIONThe potential frequency of plasmid pcDNA3-gtfB integration into host cell genome would not exceed that of the spontaneous mutation. It was indicated that pcDNA3-gtfB was genetically safe as a promising anti-carious DNA vaccine.

Animals ; Antibodies, Antinuclear ; genetics ; immunology ; Cloning, Molecular ; Dental Caries ; prevention & control ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; Glucosyltransferases ; genetics ; Plasmids ; genetics ; immunology ; Rats ; Rats, Wistar ; Recombination, Genetic ; Streptococcal Vaccines ; genetics ; Streptococcus mutans ; genetics ; immunology ; Vaccines, DNA ; adverse effects ; genetics ; immunology

OBJECTIVETo investigate the role of IL-17 in the overproduction of autoantibodies and IL-6 overexpression by peripheral blood mononuclear cells (PBMC) of lupus nephritis (LN) patients.

METHODSFifteen consecutively hospitalized LN patients were selected as subjects and 15 healthy adults as normal controls. PBMC were obtained by Ficoll density gradient centrifugation. IgG, anti-dsDNA antibody and IL-6 protein levels were assessed using enzyme-linked immunosorbent assays (ELISA) on the supernatant of cultured PBMC of LN patients or normal controls. IL-6 mRNA levels in PBMC were measured using reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSIn medium culture, IgG, anti-dsDNA and IL-6 protein levels of the supernatant of PBMC from LN patients were significantly higher than those from normal controls (1492.1 +/- 73.2 ng/ml vs 636.7 +/- 51.9 ng/ml for IgG, 306.6 +/- 53.7 IU/ml vs 95.8 +/- 11.6 IU/ml for anti-dsDNA and 50.92 +/- 15.92 ng/ml vs 1.77 +/- 0.73 ng/ml for IL-6, all P < 0.001). In LN patients, IgG, anti-dsDNA and IL-6 protein levels were higher in the supernatants of PBMC in the IL-17-stimulated culture than the medium culture, but in normal controls, only the IL-6 protein levels were significantly higher. The increase in IgG, anti-dsDNA and IL-6 protein levels induced by IL-17 was dose-dependent and could be completely blocked by IL-17 monoclonal antibody mIgG(28) and partially blocked by dexamethasone. Similarly, IL-6 mRNA overexpression of PBMC in LN patients or normal controls induced by IL-17 was both dose- and time-dependent. During medium culture, IL-6 mRNA levels in LN patients were significantly higher than those in normal controls (1.80 +/- 0.11 vs 0.36 +/- 0.07). During stimulation with IL-17, IL-6 mRNA levels in LN patients were higher than those in normal controls (3.21 +/- 0.24 vs 1.30 +/- 0.14, P < 0.05) and also significantly higher when comparing the stimulated culture with the medium culture either in LN patients or normal control.

CONCLUSIONSIL-17 may play an important role in the pathogenesis of LN through the induction of IgG, anti-dsDNA overproduction and IL-6 overexpression of PBMC in LN patients.

Adolescent ; Adult ; Antibodies, Antinuclear ; biosynthesis ; Autoantibodies ; biosynthesis ; Female ; Humans ; Immunoglobulin G ; biosynthesis ; Interleukin-17 ; pharmacology ; Interleukin-6 ; biosynthesis ; genetics ; Leukocytes, Mononuclear ; metabolism ; Lupus Nephritis ; immunology ; Male ; RNA, Messenger ; analysis

Cardiac resynchronization therapy (CRT) has been proven its value in adult patients with congestive heart failure of low ejection fraction and wide QRS duration. Contrast to adult patients, CRT has been rarely applied for young patients. We report on a 9-yr-old boy with progressive left ventricular (LV) dilatation and dysfunction following chronic VVI pacemaker therapy for congenital complete atrioventricular block associated with maternal anti-SSA/Ro and SSB/La antibody. His LV dysfunction was improved after epicardially established CRT.
Antibodies, Antinuclear/metabolism ; Atrioventricular Block/congenital/therapy ; Cardiac Pacing, Artificial/*adverse effects ; *Cardiac Resynchronization Therapy ; Child ; Chronic Disease ; Electrocardiography ; Heart Ventricles ; Humans ; Male ; Natriuretic Peptide, Brain/blood ; Sjogren's Syndrome/immunology ; Ventricular Dysfunction, Left/etiology/radiography/*therapy