Antigens ; analysis ; Autoantigens ; analysis ; Breast Neoplasms ; metabolism ; Citrates ; Female ; Formaldehyde ; Hot Temperature ; Humans ; Hydrogen-Ion Concentration ; Immunohistochemistry ; methods ; Iodide Peroxidase ; analysis ; Iron-Binding Proteins ; analysis ; Paraffin Embedding ; Receptors, Progesterone ; analysis ; Thyroid Gland ; immunology ; Tissue Fixation

BACKGROUND/AIMS: Functional gastrointestinal disorders are those in which no abnormal metabolic or physical processes, which can account for the symptoms, can be identified. The irritable bowel syndrome (IBS) is a significant functional disorder, which affects 10-20 percent of the population worldwide. Predominant symptoms of IBS are abnormal defecation associated with abdominal pain, both of which may be exacerbated by psychogenic stress. Our study was designed to test a hypothesis that symptoms in a subset of patients with a diagnosis of IBS are associated with an autoimmune degenerative neuropathy in the enteric nervous system. METHODS: Serum was collected from Rome II-IBS patients and controls at the University of North Carolina Functional Gastrointestinal Diseases Center. Assay procedures were immunohistochemical localization of antibody binding to enteric neurons and human protein microarray assay for antigens recognized by antibodies in the sera. RESULTS: Eighty-seven percent of IBS sera and 59% of control sera contained anti-enteric neuronal antibodies. Antibody immunostaining was seen in the nucleus and cytoplasm of neurons in the enteric nervous system. Protein microarray analysis detected antibody reactivity for autoantigens in serum with anti-enteric neuronal antibodies and no reactivity for the same autoantigens in samples not containing anti-enteric neuronal antibodies in our immunostaining assay. Antibodies in sera from IBS patients recognized only 3 antigens out of an 8,000 immunoprotein array. The 3 antigens were: (1) a nondescript ribonucleoprotein (RNP-complex); (2) small nuclear ribonuclear polypeptide A; and (3) Ro-5,200 kDa. CONCLUSIONS: Results of the present study suggest that symptoms in a subset of IBS patients might be a reflection of enteric neuronal damage or loss, caused by circulating anti-enteric autoimmune antibodies.
Abdominal Pain ; Antibodies ; Autoantigens ; Cytoplasm ; Defecation ; Enteric Nervous System ; Gastrointestinal Diseases ; Humans ; Irritable Bowel Syndrome ; Neurons ; North Carolina ; Physical Processes ; Protein Array Analysis ; Ribonucleoproteins ; Rome

OBJECTIVETo identify thyroid peroxidase (TPO) gene mutations in 35 patients with congenital hypothyroidism.

METHODGenomic DNA was isolated from peripheral blood samples of 35 patients with congenital hypothyroidism. All of the 17 exons and flanking introns of TPO gene were amplified by PCR, then the PCR products were sequenced bi-directionally and were analyzed by restriction endonucleases.

RESULTOne patient had compound heterozygous mutations c.961A>G/c.2422delT, one was c.2268insT/c.1477G>A, and three was homozygous mutation c.2268insT. The TPO gene mutation c.961A>G [p. Thr321Ala] was one novel mutation.

CONCLUSIONHigh frequency mutation in TPO gene was detected in patients with congenital hypothyroidism.

Autoantigens ; genetics ; Case-Control Studies ; Child ; Child, Preschool ; Congenital Hypothyroidism ; genetics ; DNA Mutational Analysis ; Exons ; Female ; Humans ; Infant ; Iodide Peroxidase ; genetics ; Iron-Binding Proteins ; genetics ; Male ; Mutation

OBJECTIVETo investigate the expression of cancerous inhibitor of protein phosphatase 2A(CIP2A) in human colorectal cancer, and to examine the association of CIP2A expression with clinicopathology and prognosis.

METHODSCIP2A expression in colorectal cancer tissue microarray of 92 cases was detected by immunohistochemistry method.

RESULTSUp-regulated CIP2A expression was closely related with TNM staging, histological type, peritoneal seeding and liver metastasis (all P<0.05), but not related with gender, age, tumor location, CEA, family history and grade of differentiation. Overall survival rates of 1-, 3-, 5-, and 10-year in high CIP2A expression group were 97.1%, 71.4%, 59.2%, and 44.4% respectively, significantly lower than 98.2%, 85.7%, 80.3%, and 74.9% in low CIP2A expression group(P=0.021). Multivariate analysis showed that CIP2A was not an independent factor associated with prognosis(P=0.099, HR=1.982, 95%CI:0.879 to 4.469).

CONCLUSIONSUp-regulated CIP2A expression is closely related to clinicopathology of colorectal cancer. CIP2A may be used as a potential predictive marker of metastasis, prognosis and therapeutic target in colorectal cancer.

Autoantigens ; Biomarkers, Tumor ; metabolism ; Colorectal Neoplasms ; enzymology ; pathology ; Humans ; Immunohistochemistry ; Liver Neoplasms ; Membrane Proteins ; Neoplasm Staging ; Prognosis ; Protein Phosphatase 2 ; metabolism ; Survival Rate ; Tissue Array Analysis

Autoimmune sera have been used in the diagnosis of autoimmune diseases as well as the analysis of nuclear substructures. In an attempt to study the biological characteristics of the nuclear matrix, we screened human sera using immunofluorescent staining and immunoblot. We detected antibodies against nuclear matrix (NM), a remnant nonchromatin protein compartment after the treatment of detergent, salt and nuclease, in 212 out of 284 tested sera (74.6%) by immunoblot. Peptides with molecular weights of 70 kDa, 50 kDa and 25 kDa were detected in the order of frequency. Clinical informations of 198 out of 212 cases were available and went as follows: 38 cases were autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis; 132 non-autoimmune and non-neoplastic diseases; 16 neoplastic diseases and 12 cases unclassified. The immunofluorescent staining intensity by anti-nuclear matrix protein (NMP) antibodies decreased variably, but fibrillogranular, speckled and nucleolar immunolocalization patterns were retained after in situ fractionation. Ku70 and La protein were detected by anti-NMP antibodies. Immunolocalization by anti-NMP antibodies indicates that the NMPs constitute a variety of characteristic nuclear substructures and may serve as autoantigens in diverse human diseases. In addition, the presence of Ku70 and La protein as NMPs suggests that the NM can be functionally active in association with DNA or RNA.
Autoantigens/analysis* ; Autoimmune Diseases/immunology ; Autoimmune Diseases/blood ; Base Sequence ; DNA, Complementary ; DNA-Binding Proteins/analysis* ; Fluorescent Antibody Technique, Indirect ; Hela Cells ; Human ; Immunoblotting ; Molecular Sequence Data ; Nuclear Matrix/immunology ; Nuclear Proteins/analysis* ; Ribonucleoproteins/analysis* ; Tumor Cells, Cultured

Adult ; Aged ; Alanine Transaminase ; blood ; Aspartate Aminotransferases ; blood ; Autoantibodies ; analysis ; immunology ; Autoantigens ; immunology ; Biomarkers ; blood ; Biopsy, Needle ; Female ; Humans ; Immunohistochemistry ; Liver Cirrhosis, Biliary ; blood ; diagnosis ; immunology ; Male ; Middle Aged ; Mitochondria ; immunology ; Nuclear Pore Complex Proteins ; immunology ; Retrospective Studies

BACKGROUNDAngiogenesis is a prerequisite for tumor growth and plays an important role in rapidly growing tumors, such as malignant gliomas. A variety of factors controlling the angiogenic balance have been described, and among these, the endogenous inhibitor of angiogenesis, tumstatin, has drawn considerable attention. The current study investigated whether expression of tumstatin by glioma cells could alter this balance and prevent tumor formation.

METHODSWe engineered stable transfectants from human glioma cell line U251 to constitutively secrete a human tumstatin protein with c-myc and polyhistidine tags. Production and secretion of the tumstatin-c-myc-His fusion protein by tumstatin-transfected cells were confirmed by Western blotting analysis. In the present study, we identify the anti-angiogenic capacity of tumstatin using several in vitro and in vivo assays. Student's t-test and one-way analysis of variance (ANOVA) test were used to determine the statistical significance in this study.

RESULTSThe tumstatin transfectants and control transfectants (stably transfected with a control plasmid) had similar in vitro growth rates compared to their parental cell lines. However, the conditioned medium from the tumstatin transfected tumor cells significantly inhibits proliferation and causes apoptosis of endothelial cells. It also inhibits tube formation of endothelial cells on Matrigel. Examination of armpit tumors arising from cells overexpressing tumstatin repress the growth of tumor, accompanying the decreased density of CD31 positive vessels in tumors ((5.62 ± 1.32)/HP), compared to the control-transfectants group ((23.84 + 1.71)/HP) and wild type U251 glioma cells group ((29.33 + 4.45)/HP).

CONCLUSIONAnti-angiogenic gene therapy using human tumstatin gene may be an effective strategy for the treatment of glioma.

Animals ; Autoantigens ; genetics ; Brain Neoplasms ; blood supply ; therapy ; Cell Line, Tumor ; Cell Proliferation ; Collagen Type IV ; genetics ; Genetic Therapy ; Glioma ; blood supply ; pathology ; therapy ; Humans ; Mice ; Mice, Inbred BALB C ; Neovascularization, Pathologic ; prevention & control ; Platelet Endothelial Cell Adhesion Molecule-1 ; analysis ; Transfection

To study whether the antibody against the testis form of the nuclear autoantigenic sperm protein (tNASP) could result in reproductive failure, we successfully cloned and expressed a 339-bp cDNA fragment of mouse tNASP (mtNASP). Using mouse as a model, recombinant mtNASP (rmtNASP) and a synthetic peptide, human tNASP(393-408) (htNASP(393-408)), were investigated for their antifertility effect. Active immunization with rmtNASP or the synthesized peptide raised high antibody titers in the immunized mice. Sperm-egg binding and fusion assay were carried out in 8-10-week-old BALB/c mice. Sperm-egg binding and in vitro fertilization of mouse oocytes were inhibited by co-incubation of zona-free mouse oocytes with capacitated mouse spermatozoa in the presence of varying concentrations of the antisera against rmtNASP. There was a significant antifertility effect in animals immunized with rmtNASP or the synthesized peptide. The effect on fertility in the mice immunized with the synthesized peptide was reversible. Our data indicate that active immunization with rmtNASP antigen may induce a strong antibody response that causes an inhibition of fertility.
Adult ; Animals ; Autoantibodies ; administration & dosage ; immunology ; Autoantigens ; chemistry ; immunology ; pharmacology ; Contraception, Immunologic ; Female ; Fertility ; drug effects ; immunology ; Humans ; Immune Sera ; immunology ; pharmacology ; Male ; Mice ; Nuclear Proteins ; chemistry ; immunology ; pharmacology ; Rabbits ; Recombinant Proteins ; immunology ; Sequence Analysis, Protein ; Sperm Motility ; drug effects ; immunology ; Sperm-Ovum Interactions ; immunology ; Spermatozoa ; drug effects ; immunology ; Vaccines, Contraceptive ; immunology ; pharmacology

OBJECTIVESTo obtain prokaryotic expressed IA-2 recombinant protein and to identify its immunological activity.

METHODSThe complimentary DNA (cDNA) coding for the intracytoplasmic part of IA-2 (IA-2ic) was amplified from human fetal brain RNA, and was subcloned into the PinPoint Xa-1 T vector to construct recombinant expression plasmid, and was then expressed in E. coli JM109 cells as a fusion protein with a biotinylated peptide sequence at the aminoterminus. The biotinylated fusion protein was then purified by affinity chromatography and was subsequently dialyzed. Finally, its immunogenicity was evaluated by enzyme linked immunosorbent assay (ELISA).

RESULTSThe purified IA-2ic fusion protein resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a single Coomassie brilliant blue stained band with a molecular weight of 59 kDa and its immunogenicity was confirmed by ELISA.

CONCLUSIONSE. coli expressed IA-2ic fusion protein has immunological activity. It can be used for detection of IA-2 autoantibodies (IA-2A) and for further studies on type 1 diabetes in future.

Animals ; Autoantigens ; biosynthesis ; DNA, Complementary ; analysis ; Diabetes Mellitus, Type 1 ; immunology ; Escherichia coli ; genetics ; Humans ; Membrane Proteins ; biosynthesis ; genetics ; isolation & purification ; Plasmids ; Protein Tyrosine Phosphatase, Non-Receptor Type 1 ; Protein Tyrosine Phosphatases ; biosynthesis ; genetics ; isolation & purification ; Rabbits ; Receptor-Like Protein Tyrosine Phosphatases, Class 8 ; Recombinant Fusion Proteins ; biosynthesis ; immunology ; isolation & purification

OBJECTIVETo evaluate whether the D-3-phosphoglycerate dehydrogenase (Phgdh) correlative antibodies is crucial for AIH, we cloned Phgdh cDNA and constructed plasmid, then purified and identified the immunoreactivity of the recombinant protein, and established the enzyme linked immunosorbent assay (ELISA) to detect Phgdh autoantigen correlative antibodies in diagnosis of autoimmune hepatitis.

METHODSThe constructed plasmid was transformed into E. coli. BL21(D3). This fusion protein was purified by Ni-NTA chromatography and its immunoreactivity was identified by SDS-PAGE and Western blot. The ELISA with the fusion protein was established first, then, the Phgdh autoantigen correlative antibodies in serum of patients with AIH (65) and patients with PBC (122) as well as chronic hepatitis B (CHB) (56), chronic hepatitis C (CHC) (117), and normal controls (60) were detected.

RESULTSThe sequence of Phgdh autoantigen gene was the same as the sequence reported on the genebank. The fusion protein was found about 60kD strip on SDS-PAGE. Western blot analysis showed that the fusion protein had immunoreactivity. When analyzing the serum by ELISA, the immune reactivity to Phgdh was detected in 66.15% of patients with AIH, 21.42% of patients with PBC, 12.50% of patients with CHB, 6.83% of patients with CHC, and 3.30% of normal individuals. The differences of prevalence between AIH patients and healthy controls as well as other diseases were of statistical significance (P less than 0.01).

CONCLUSIONThe Phgdh cDNA is successfully cloned into E. coli BL21 (D3). The frequency of antibodies to Phgdh is much higher in patients with AIH than in patients with PBC, CHB, CHC and normal control. The antibodies to Phgdh may have utility in improved diagnosis of AIH.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Autoantibodies ; analysis ; genetics ; Autoantigens ; immunology ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; methods ; Escherichia coli ; genetics ; metabolism ; Female ; Gene Expression ; Genetic Vectors ; Hepatitis B, Chronic ; blood ; diagnosis ; Hepatitis, Autoimmune ; blood ; diagnosis ; Humans ; Liver Cirrhosis, Biliary ; blood ; diagnosis ; Male ; Middle Aged ; Phosphoglycerate Dehydrogenase ; genetics ; immunology ; Plasmids ; Recombinant Proteins ; genetics ; metabolism ; Young Adult