OBJECTIVETo investigate the molecular mechanism of action of BET bromodomain inhibitor JQ1 in treating airway remodeling in asthmatic mice.

METHODSA total of 24 mice were randomly divided into control group, ovalbumin (OVA)-induced asthma group (OVA group), and JQ1 intervention group (JQ1+OVA group), with 8 mice in each group. OVA sensitization/challenge was performed to establish a mouse model of asthma. At 1 hour before challenge, the mice in the JQ1+OVA group were given intraperitoneal injection of JQ1 solution (50 μg/g). Bronchoalveolar lavage fluid (BALF) and lung tissue samples were collected at 24 hours after the last challenge, and the total number of cells and percentage of eosinophils in BALF were calculated. Pathological staining was performed to observe histopathological changes in lung tissue. RT-PCR and Western blot were used to measure the mRNA and protein expression of E-cadherin and vimentin during epithelial-mesenchymal transition (EMT).

RESULTSCompared with the control group, the OVA group had marked infiltration of inflammatory cells in the airway, thickening of the airway wall, increased secretion of mucus, and increases in the total number of cells and percentage of eosinophils in BALF (P<0.01). Compared with the OVA group, the JQ1+OVA group had significantly alleviated airway inflammatory response and significant reductions in the total number of cells and percentage of eosinophils in BALF (P<0.01). Compared with the control group, the OVA group had significant reductions in the mRNA and protein expression of E-cadherin and significant increases in the mRNA and protein expression of vimentin (P<0.01); compared with the OVA group, the JQ1+OVA group had significant increases in the mRNA and protein expression of E-cadherin and significant reductions in the mRNA and protein expression of vimentin (P<0.01); there were no significant differences in these indices between the JQ1+OVA group and the control group (P>0.05).

CONCLUSIONSMice with OVA-induced asthma have airway remodeling during EMT. BET bromodomain inhibitor JQ1 can reduce airway inflammation, inhibit EMT, and alleviate airway remodeling, which provides a new direction for the treatment of asthma.

Airway Remodeling ; drug effects ; Animals ; Asthma ; drug therapy ; Azepines ; pharmacology ; Cadherins ; analysis ; genetics ; Epithelial-Mesenchymal Transition ; Female ; Mice ; Nuclear Proteins ; antagonists & inhibitors ; Ovalbumin ; immunology ; RNA, Messenger ; analysis ; Transcription Factors ; antagonists & inhibitors ; Triazoles ; pharmacology ; Vimentin ; analysis ; genetics

OBJECTIVEOmenn syndrome is a rare autosomal recessive hereditary severe combined immunodeficiency. The purpose of this study was to understand clinical characteristics and genetic mutation type of Omenn syndrome and to improve the recognition of Omenn syndrome among pediatric clinicians.

METHODOne suspected case of severe combined immunodeficiency was found to have pneumonia repeatedly, intractable diarrhea, poor antibiotic treatment effect, lymphadenopathy, hepatosplenomegaly and erythroderma. The patient was diagnosed as having Omenn syndrome by RT-PCR, and the expression of RAG1/RAG2 and gene analysis of RAG1/RAG2 were performed.

RESULTThe classification of lymphocyte was CD3(+) cells (35.3%), CD19(+) cells (0.4%), CD16(+) cells (57.6%). After stimulation with phytohemagglutinin (PHA), lymphocyte proliferation of the child was extremely low. Genetic studies showed RAG1 homozygous deletion mutation (2302 del T). He had detectable activated T-lymphocytes with low circulating B-lymphocytes and no evidence of maternal T-cell engrafment as indicated by the short tandem repeat (STR) analysis.

CONCLUSIONOmenn syndrome is a severe combined immunodeficiency disease caused by mutations in the RAG1/RAG2 gene. The disease has been reported rarely in China. The clinical manifestations of the disease is early postnatal repeated infections and erythroderma. Mutation analysis of RAG1/RAG2 gene may help to confirm the diagnosis and may be useful in early immune reconstitution and genetic counseling.

Amino Acid Sequence ; Biomarkers ; blood ; DNA Mutational Analysis ; DNA-Binding Proteins ; genetics ; Genotype ; Homeodomain Proteins ; genetics ; Humans ; Infant ; Lymphocytes ; immunology ; pathology ; Male ; Microsatellite Repeats ; Mutation ; Nuclear Proteins ; genetics ; Phenotype ; Receptors, Antigen, T-Cell, alpha-beta ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Severe Combined Immunodeficiency ; diagnosis ; genetics ; pathology

Exosomes are small membrane vesicles secreted from various types of cells. Tumor-derived exosomes contain MHC class I molecules and tumor-specific antigens, receiving attention as a potential cancer vaccine. For induction of efficient anti-tumor immunity, CD4+ helper T cells are required, which recognize appropriate MHC class II-peptide complexes. In this study, we have established an MHC class II molecule-expressing B16F1 murine melanoma cell line (B16F1-CIITA) by transduction of the CIITA (Class II transactivator) gene. Exosomes from B16-CII cells (CIITA-Exo) contained a high amount of MHC class II as well as a tumor antigen TRP2. When loaded on dendritic cells (DCs), CIITA-Exo induced the increased expression of MHC class II molecules and CD86 than the exosomes from the parental cells (Exo). In vitro assays using co-culture of immunized splenocytes and exosome-loaded DCs demonstrated that CIITA-Exo enhanced the splenocyte proliferation and IL-2 secretion. Consistently, compared to B16-Exo, CIITA-Exo induced the increased mRNA levels of inflammatory cytokines such as TNF-alpha, chemokine receptor CCR7 and the production of Th1-polarizing cytokine IL-12. A tumor preventive model showed that CIITA-Exo significantly inhibited tumor growth in a dose-dependent manner. Ex vivo assays using immunized mice demonstrated that CIITA-Exo induced a higher amount of Th1-polarized immune responses such as Th1-type IgG2a antibodies and IFN-gamma cytokine as well as TRP2-specific CD8+ T cells. A tumor therapeutic model delayed effects of tumor growth by CIITA-Exo. These findings indicate that CIITA-Exo are more efficient as compared to parental Exo to induce anti-tumor immune responses, suggesting a potential role of MHC class II-containing tumor exosomes as an efficient cancer vaccine.
Animals ; Cancer Vaccines/genetics/immunology ; Cell Line, Tumor ; Cell Proliferation ; Dendritic Cells/immunology ; Exosomes/genetics/*metabolism ; Gene Expression Regulation ; Gene Transfer Techniques ; Immunity, Cellular/immunology ; Immunity, Humoral/immunology ; Immunotherapy ; Lymphocyte Activation/immunology ; Melanoma, Experimental/mortality/pathology/*physiopathology ; Mice ; Mice, Inbred C57BL ; Nuclear Proteins/*genetics/*metabolism ; Survival Analysis ; T-Lymphocytes/immunology/metabolism ; Trans-Activators/*genetics/*metabolism ; Transduction, Genetic

OBJECTIVETo separate the cell subpopulation with high tumorigenic ability and study the biological characteristics of this subpopulation in laryngeal carcinoma cells.

METHODSHuman laryngeal carcinoma cells were obtained by primary tissue culture technique. CD44 and CD133 molecules were used as markers to isolate the CD44(+), CD133(+), CD44(+)CD133(+) and CD44(+)CD133(-) cell subpopulations from the laryngeal carcinoma cells by flow cytometry. A nude mouse tumor xenograft model was developed for the study of the tumorigenic effects of the different cell populations. 1 x 10(6), 1 x 10(5), 1 x 10(4) and 1 x 10(3) cells were injected into the left flank of the mice, respectively. The mice were observed for palpable tumor formation and were sacrificed at 4 weeks later to assess the tumor formation rate, tumor volume and tumor weight. Boyden chamber migration assay was used to determine the migration ability and immunochemistry was used to detect the expression of stem cell antigen SCA-1 and beta1-integrin. Semi-quantities RT-PCR and Western blot analysis were performed to detect the expression level of Bmi-1 in the different cell subpopulations.

RESULTSThe growth of subcutaneous tumors in nude mice showed that a tumor can be generated with 1 x 10(3) CD44(+)CD133(+) cells. When the same dose of 1 x 10(6) CD44(+)CD133(+) cells was injected into the mice, both the average weight and volume of the tumors were significantly higher than those generated from other cell subpopulations. Boyden chamber migration assay showed that the invasion ability of CD44(+)CD133(+) cells was significantly higher than that of other cell subsets. The results of immunochemical analysis showed an abundant expression of stem cell antigen SCA-1 and beta1-integrin in the CD44(+)CD133(+) cells. Semi-quantitative RT-PCR and Western blot analysis provided strong evidence that the Bmi-1 expression in CD44(+)CD133(+) and CD133(+) cells was very significantly higher than that in CD44(+), CD44(+)CD133(-) and control cells (P < 0.01).

CONCLUSIONOur findings demonstrate that CD44(+)CD133(+) subset cells in laryngeal carcinoma posses some biological characteristics of tumor stem cells, which may be the original cells of laryngeal carcinoma and may become a new target of tumor therapy.

AC133 Antigen ; Animals ; Antigens, CD ; analysis ; Antigens, Ly ; metabolism ; Cell Adhesion ; Gene Expression Regulation, Neoplastic ; Glycoproteins ; analysis ; Humans ; Hyaluronan Receptors ; analysis ; Integrin beta1 ; metabolism ; Laryngeal Neoplasms ; immunology ; metabolism ; pathology ; Male ; Membrane Proteins ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Nuclear Proteins ; genetics ; metabolism ; Peptides ; analysis ; Polycomb Repressive Complex 1 ; Proto-Oncogene Proteins ; genetics ; metabolism ; RNA ; metabolism ; Repressor Proteins ; genetics ; metabolism ; Tumor Burden ; Tumor Cells, Cultured

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

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

Triple A syndrome is a rare genetic disorder caused by mutations in the achalasia-addisonianism-alacrima syndrome (AAAS) gene which encodes a tryptophan aspartic acid (WD) repeat-containing protein named alacrima-achalasia-adrenal insufficiency neurologic disorder (ALADIN). Northern blot analysis shows that the 2.1 kb AAAS mRNA is expressed in various tissues with stronger expression in testis and pancreas. We show that human ALADIN is a protein with an apparent molecular weight of 60 kDa, and expressed in the adrenal gland, pituitary gland and pancreas. Furthermore, biochemical analysis using anti-ALADIN antibody supports the previous finding of the localization of ALADIN in the nuclear membrane. The mutations S544G and S544X show that alteration of S544 residue affects correct targeting of ALADIN to the nuclear membrane.
Adrenal Insufficiency/*genetics ; Antibodies/immunology ; Cloning, Molecular ; DNA, Complementary/genetics ; Esophageal Achalasia/*genetics ; Gene Expression Profiling ; Hela Cells ; Humans ; Lacrimal Apparatus Diseases/*genetics ; Mutagenesis, Site-Directed ; Nerve Tissue Proteins/*analysis/*genetics/immunology ; Nuclear Pore/chemistry ; Nuclear Pore Complex Proteins/*analysis/*genetics/immunology ; RNA, Messenger/analysis/genetics ; Syndrome ; Tissue Distribution

Oxidative stress plays critical roles in airway inflammation that is usually accompanied by increased vascular permeability and plasma exudation. VEGF increases vascular permeability and leads to airway inflammation. In addition, VEGF has been shown to enhance receptor activator of NF-kappaB (RANK) expression in endothelial cells. An aim of the study was to determine the potential role of antioxidant in the regulation of RANK expression in murine model of asthma. We have used a C57BL/6 mouse model of allergic asthma to evaluate the effect of L-2-oxothiazolidine-4-carboxylic acid (OTC), a prodrug of cysteine, which acts as an antioxidant, and VEGF receptor inhibitor on RANK mRNA expression. The mice develop the following pathophysiological features of asthma in the lungs: increased expression of RANK mRNA, increased number of inflammatory cells of the airways, increased vascular permeability, and increased levels of VEGF. Administration of OTC and VEGF receptor inhibitor markedly reduced plasma extravasation and VEGF levels in allergen-induced asthmatic lungs. We also showed that the increased RANK mRNA expression at 72 h after ovalbumin inhalation were reduced by the administration of OTC or VEGF receptor inhibitor. The results indicate that OTC and VEGF receptor inhibitor which inhibit up-regulation of VEGF expression modulate RANK expression that may be in association with the regulation of vascular permeability, and suggest that VEGF may regulate the RANK expression. These findings provide a crucial molecular mechanism for the potential use of antioxidants to prevent and/or treat asthma and other airway inflammatory disorders.
Vascular Endothelial Growth Factor A/analysis/antagonists & inhibitors/metabolism ; Thiazolidines ; Thiazoles/*pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Receptors, Vascular Endothelial Growth Factor/antagonists & inhibitors ; Receptors, Tumor Necrosis Factor/genetics/*metabolism ; Receptors, Cytoplasmic and Nuclear/genetics/*metabolism ; Reactive Oxygen Species/metabolism ; RNA, Messenger/genetics/metabolism ; Pyrrolidonecarboxylic Acid ; Proto-Oncogene Proteins c-akt/metabolism ; Protein Kinase Inhibitors/pharmacology ; Prodrugs/pharmacology ; Phosphorylation/drug effects ; Ovalbumin/immunology ; Osteoprotegerin ; Mice, Inbred C57BL ; Mice ; Immunohistochemistry ; Glycoproteins/genetics/*metabolism ; Gene Expression/drug effects ; Female ; Capillary Permeability/drug effects ; Bronchoalveolar Lavage Fluid/chemistry/cytology ; Blotting, Western ; Asthma/*drug therapy/immunology/metabolism ; Antioxidants/*pharmacology ; Animals

OBJECTIVEThis paper studied the effect of RNaseP against CIITA on repressing class II MHC (MHCII) expression.

METHODSIt was constructed that M1-RNA with guide sequences (GS), recognizing the 629 site of CIITA (M1-629-GS), by PCR from pTK117 plasmid, then was cloned into psNAV (psNAV-M1-629-GS). CIITA target gene was obtained from Raji cell by RT-PCR, and then inserted into pGEM-7zf (+) (pGEM-800). psNAV-M1-629-GS and pGEM-800 were transcribed and then mixed up and incubated in vitro. Stable transfectants of hepatocyte with psNAV-M1-629-GS by nanometer were tested for MHCII induction by recombinant human interferon-gamma (IFN-gamma). mRNA abundance of CIITA was measured by RT-PCR.

RESULTSIt showed that M1-629-GS could exclusively cleave pGEM-800 that formed a base pair with the GS. When induced with IFN-gamma, the expression of HLA-DR, -DP, -DQ on psNAV-M1-629-GS+ hepatocyte was (1.01+/-0.51)%, (4.37+/-1.28)%, (1.98+/-0.42)% respectively, was down-modulated 90.65%, 89.11% and 65.32% compared with control, while the mRNA content of CIITA reduced significantly (P<0.01).

CONCLUSIONM1-629-GS could effectively repress MHCII expressing through cleaving CIITA mRNA. These results provided insight into the future application of it as a new nucleic acid drug against the rejection of hepatic transplantation.

Graft Rejection ; prevention & control ; Histocompatibility Antigens Class II ; analysis ; Humans ; Liver Transplantation ; immunology ; Nuclear Proteins ; genetics ; RNA, Messenger ; analysis ; Ribonuclease P ; pharmacology ; Trans-Activators ; genetics

The identification of primary location of a metastatic tumor is a difficult diagnostic problem and sometimes can be facilitated by the use of immunohistochemical markers. Thyroid transcription factor-1 (TTF-1) is a 38-kDa nuclear homeodomain transcription factor that is expressed specifically in lung or thyroid neoplasms. Cytokeratin 20 (CK20) is a 46-kDa low-molecular-weight cytokeratin that shows restricted expression in adenocarcinomas of the gastrointestinal tract (GIT) and transitional cell carcinomas of the urinary tract. We studied the immunohistochemical expression of TTF-1 and CK20 in 68 metastatic carcinomas in cervical lymph nodes. The primary sites were the lung in 29 cases, stomach in 13, colorectum in 3, and other sites in 23. TTF-1 expression was detected in 69.0% of metastatic lung carcinomas and none in metastatic GIT carcinomas, whereas CK20 expression was detected in 68.8% of metastatic GIT carcinomas and none of metastatic lung carcinomas. TTF-1 had a specificity of 0.95 and a sensitivity of 0.69 for metastatic lung carcinoma, whereas CK20 had a specificity of 1.00 and a sensitivity of 0.69 for metastatic GIT carcinoma. These results indicate that TTF-1 and CK20 should be the first choice as a component of antibody panel to prove or to exclude the lung and GIT origin, respectively, especially in patients presenting with metastatic carcinomas of unknown primary site.
Adenocarcinoma/chemistry/pathology/secondary ; Carcinoma/chemistry/pathology/*secondary ; Gastrointestinal Neoplasms/chemistry/pathology ; Homeodomain Proteins/analysis ; Humans ; Intermediate Filament Proteins/*analysis/immunology ; Keratin-20 ; Lung Neoplasms/chemistry/pathology ; Lymph Nodes/chemistry/pathology ; Lymphatic Metastasis/*diagnosis/pathology ; Neck ; Neoplasms, Unknown Primary/chemistry/pathology ; Nuclear Proteins/*analysis/immunology ; Sensitivity and Specificity ; Transcription Factors/*analysis/immunology ; Tumor Markers, Biological/analysis