Cell mediated immune responses play a prominent role in syphilis, which is caused by Treponema pallidum. The role of dendritic cells (DC) in the syphilitic infection is not well understood in human. In the present study, we studied interaction of T. pallidum with DC, generated from human peripheral blood mononuclear cells with GM-CSF and IL-4. After adding T. pallidum for 16 hours to immature DC at culture day 7, the change of surface antigens on DC was monitored by flow cytometry, the amount of IL-12 in culture supernatant of DC was measured by ELISA and T cell stimulatory capacity of DC was checked in mixed lymphocyte reaction (MLR). We have observed an efficient phagocytosis of T. pallidum by electron microscopy as early as 2 hours after addition of T. pallidum to DC. Interaction of DC with T. pallidum resulted in increased surface expression of CD83 which was proportionally increased according to the number of T. pallidum. Expressions of CD80, CD86 and HLA-DR on DC were slightly increased. The amount of IL-12 in the culture supernatant of DC was increased (1, 099pg/ml) after the addition of T. pallidum. T. pallidum-infected DC also displayed enhanced T cell stimulatory capacity in MLR. As seen from the above, we observed phagocytosis of T. pallidum by DC as early as 2 hours after addition of T. pallidum to DC and found that T. pallidum can stimulate DC maturation which mean that DC modulate an protective immune response during T. pallidum infection.
Cells, Cultured ; Dendritic Cells/cytology/*immunology/*microbiology ; Human ; Interleukin-12/metabolism/secretion ; Lymphocyte Culture Test, Mixed ; Microscopy, Electron ; Phagocytosis/immunology ; Receptors, Cell Surface/immunology/metabolism ; Support, Non-U.S. Gov't ; Syphilis/*immunology ; T-Lymphocytes/immunology ; Treponema pallidum/*immunology/ultrastructure

OBJECTIVE: To investigate the expression and role of Interleukin-33 (IL-33) and ST2 in the nasal polyps of human Eosinophilic and non-Eosinophilic chronic rhinosinusitis with nasal polyps (ECRS and non-ECRS). METHOD: IL-33 and ST2 protein expression in nasal polyps of ECRS and non-ECRS as well as in seemingly normal mucosa of the inferior turbinate tissue was investigated by immunohistochemical staining and messenger RNA (mRNA) expression of IL-33 and ST2 was assessed by realtime polymerase chain reaction (PCR) in 27 subjects with ECRS, 33 subjects with non-ECRS, and 11 control subjects. RESULT: (1) The ST2 was found both in nasal polyps of ECRS and non-ECRS,especially in ECRS, yet hardly found in the normal mucosa of the inferior turbinate tissue; (2) The expression of ST2 mRNA in nasal polyps of ECRS was higher than that in non-ECRS and normal inferior turbinate tissue, and the difference was both prominent in statistics (P<0.01); (3) The expression patterns of IL-33 at both mRNA and protein levels were not significantly different among the three groups (P>0.05). CONCLUSION The IL-33 and its receptor ST2 were both expressed in human nasal polyps including ECRS and non-ECRS, meanwhile the expression patterns of ST2 at both mRNA and protein levels were significantly higher in nasal polyps of ECRS. The current study suggests that IL-33 and its receptor ST2 may play important roles in the pathogenesis of chronic rhinosinusitis with nasal polyps, especially in ECRS through the increased expression of ST2 in Eosinophils as a hypothesis.
Chronic Disease ; Eosinophils ; immunology ; Humans ; Interleukin-1 Receptor-Like 1 Protein ; Interleukin-33 ; metabolism ; Nasal Mucosa ; metabolism ; Nasal Polyps ; immunology ; RNA, Messenger ; Real-Time Polymerase Chain Reaction ; Receptors, Cell Surface ; metabolism ; Rhinitis ; immunology ; Sinusitis ; immunology ; Turbinates ; metabolism

Mammalian zona pellucida 3(ZP3) plays an important role in the induction of capacitating sperm acrosome reaction. In this study, we obtained the soluble mZP3 fusion protein and identified its immunoreactivity. mZP3 cDNA was cloned into plasmid pMAL-p2x, and the recombinant plasmid was transformed into Escherichia coli BL21. To get the soluble mZP3 fusion protein, we tried to optimize the expression conditions, including additives, IPTG concentrations, temperatures and induction duration. Then, Western blotting and ELISA were used to identify the immunoreactivity of the purified protein. Based on the optimization experiments, we concluded that the best soluble expression conditions for the mZP3 fusion protein involved incubation to an A600 of 0.6, addition of glucose to a final concentration of 0.02 mol/L, addition of IPTG to a final concentration of 0.6 mmol/L and then further incubation for 4 h at 25 degrees C. Western blotting and ELISA showed that the mZP3 fusion protein retained immunoreactivity. The fusion protein can be used as solubility antigens for developing the immunocontraception vaccines of mZP3 and detecting the immune effects of the vaccine.
Animals ; Egg Proteins ; biosynthesis ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Female ; Membrane Glycoproteins ; biosynthesis ; genetics ; immunology ; Mice ; Receptors, Cell Surface ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Solubility ; Vaccines, Contraceptive ; immunology ; Zona Pellucida Glycoproteins

Analysis of the T-cell receptor (TCR) repertoire of innate CD4+ T cells selected by major histocompatibility complex (MHC) class II-dependent thymocyte-thymocyte (T-T) interaction (T-T CD4+ T cells) is essential for predicting the characteristics of the antigens that bind to these T cells and for distinguishing T-T CD4+ T cells from other types of innate T cells. Using the TCRmini Tg mouse model, we show that the repertoire of TCRalpha chains in T-T CD4+ T cells was extremely diverse, in contrast to the repertoires previously described for other types of innate T cells. The TCRalpha chain sequences significantly overlapped between T-T CD4+ T cells and conventional CD4+ T cells in the thymus and spleen. However, the diversity of the TCRalpha repertoire of T-T CD4+ T cells seemed to be restricted compared with that of conventional CD4+ T cells. Interestingly, the frequency of the parental OT-II TCRalpha chains was significantly reduced in the process of T-T interaction. This diverse and shifted repertoire in T-T CD4+ T cells has biological relevance in terms of defense against diverse pathogens and a possible regulatory role during peripheral T-T interaction.
Amino Acid Sequence ; Animals ; Antigens, Surface/metabolism ; CD4-Positive T-Lymphocytes/cytology/*immunology/*metabolism ; Cell Communication ; Cell Differentiation/genetics/immunology ; Clonal Evolution ; Histocompatibility Antigens Class II/*immunology ; *Immunity, Innate ; Immunophenotyping ; Lymphocyte Count ; Mice ; Mice, Knockout ; Mice, Transgenic ; Peptide Fragments/chemistry ; Phenotype ; Receptors, Antigen, T-Cell/chemistry/*genetics/metabolism ; Receptors, Antigen, T-Cell, alpha-beta/chemistry/genetics ; Spleen/cytology ; Thymocytes/cytology/immunology/metabolism

IL-4 and IL-13 are closely related cytokines that are produced by Th2 cells. However, IL-4 and IL-13 have different effects on the development of asthma phenotypes. Here, we evaluated downstream molecular mechanisms involved in the development of Th2 type asthma phenotypes. A murine model of Th2 asthma was used that involved intraperitoneal sensitization with an allergen (ovalbumin) plus alum and then challenge with ovalbumin alone. Asthma phenotypes, including airway-hyperresponsiveness (AHR), lung inflammation, and immunologic parameters were evaluated after allergen challenge in mice deficient in candidate genes. The present study showed that methacholine AHR and lung inflammation developed in allergen-challenged IL-4-deficient mice but not in allergen-challenged IL-13-deficient mice. In addition, the production of OVA-specific IgG2a and IFN-gamma-inducible protein (IP)-10 was also impaired in the absence of IL-13, but not of IL-4. Lung-targeted IFN-gamma over-expression in the airways enhanced methacholine AHR and non-eosinophilic inflammation; in addition, these asthma phenotypes were impaired in allergen-challenged IFN-gamma-deficient mice. Moreover, AHR, non-eosinophilic inflammation, and IFN-gamma expression were impaired in allergen-challenged IL-12Rbeta2- and STAT4-deficient mice; however, AHR and non-eosinophilic inflammation were not impaired in allergen-challenged IL-4Ralpha-deficient mice, and these phenomena were accompanied by the enhanced expression of IL-12 and IFN-gamma. The present data suggest that IL-13-mediated asthma phenotypes, such as AHR and non-eosinophilic inflammation, in the Th2 type asthma are dependent on the IL-12-STAT4-IFN-gamma axis, and that these asthma phenotypes are independent of IL-4Ralpha-mediated signaling.
Allergens/immunology ; Animals ; Asthma/complications/*immunology/pathology/physiopathology ; Bronchial Hyperreactivity/complications/immunology/pathology ; Disease Models, Animal ; Interferon-gamma/*immunology ; Interleukin-12/*immunology ; Interleukin-12 Receptor beta 2 Subunit/metabolism ; Interleukin-13/deficiency/*immunology ; Interleukin-4/deficiency ; Methacholine Chloride ; Mice ; Mice, Transgenic ; Models, Immunological ; Organ Specificity ; Pneumonia/complications/immunology/pathology ; Receptors, Cell Surface/metabolism ; STAT4 Transcription Factor/*metabolism ; Signal Transduction/*immunology ; Th2 Cells/*immunology

OBJECTIVETo explore the role of CD(8)(+)CD(28)(-) T regulatory cells (Tr) in the immunological pathogenesis of acute infection with Epstein-Barr virus in children.

METHODSThe present study enrolled 25 children with infectious mononucleosis (IM) and 25 age-matched healthy children. Flow cytometric analysis was performed to detect the percentage of CD(3)(+), CD(3)(+)CD(4)(+), CD(3)(+)CD(8)(+), CD(8)(+)CD(28)(+) by determining the ratio of positive cells in lymphocytes. Reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR were used to analyze IL-6, IL-10, IFN-gamma expression in CD(8)(+)CD(28)(-) Tr cells and ILT-3, ILT-4 expression in monocytes/macrophages.

RESULTSThe proportions of CD(8)(+)CD(28)(-)T cells in children with acute-phase IM was significantly higher than those in the controls (P < 0.01). The expression level of IL-6, IL-10, IFN-gamma, ILT-3, ILT-4 mRNA significantly increased compared to those of the controls (P < 0.01).

CONCLUSIONThe CD(28) expressed on CD(8)(+) T cells in vivo is gradually lost with age and CD(8)(+)CD(28)(-) cells increase up 50% to adult. EBV can directly infect B cells, trigger CD(8)(+) CTL response and destroy the target cells to cause serious immunopathological lesion. Therefore we speculate that the expansion of CD(8)(+)CD(28)(-) Tr cells in children with IM may be an adaptive immune response to avoid serious inflammation and autoimmune reactions.

CD28 Antigens ; immunology ; CD8-Positive T-Lymphocytes ; immunology ; Case-Control Studies ; Child ; Child, Preschool ; Cytokines ; immunology ; Epstein-Barr Virus Infections ; immunology ; Female ; Flow Cytometry ; Herpesvirus 4, Human ; Humans ; Infectious Mononucleosis ; immunology ; virology ; Male ; Membrane Glycoproteins ; metabolism ; Receptors, Cell Surface ; metabolism ; Receptors, Immunologic ; metabolism ; T-Lymphocytes, Regulatory ; immunology

OBJECTIVETo explore the role of lung interstitial dendritic cells in immunodissonance and organ injury in multiple organ dysfunction syndrome (MODS).

METHODSAnimal model of MODS was established by injecting zymosan into the peritoneal cavity of C57BL/6 mice. The mice were randomly divided into groups of normal, 3 - 6 hours, 12 - 48 hours, 5 - 7 days, 10 - 12 days post injection. Pathological changes of lung and interstitial dendritic cells were studied by light and transmission electron microscope. Immunohistochemistry, RT-PCR and flow cytometry analyses were used to document status of biomarkers, including specific surface markers (CD205 and CD11c), costimulatory molecules (CD80 and CD86), SLC and its receptor CCR7 in lung, CD4+ and CD8+ T lymphocyte subtypes in peripheral blood.

RESULTSAt early stage of injury, interstitial dendritic cells showed an increase in proliferation with expression of low level of CD80 and CD86. In contrast, the expression of SLC and its receptor CCR7 in lung were increased. The ratio of CD4+/CD8+ declined in peripheral blood. At the stage of SIRS, interstitial dendritic cells continued to proliferate with high expressions of CD80 and CD86. SLC and CCR7 in lung also increased. The ratio of CD4+/CD8+ declined markedly in peripheral blood. At the MODS stage, interstitial dendritic cells further proliferated, but the expression of CD80 and CD86 declined to a very low level. Although the level of SLC increased consistently, the level of CCR7 continued to decrease, along with a markedly decreased CD4+/CD8+ ratio in peripheral blood.

CONCLUSIONSAlterations of lung interstitial dendritic cells are likely to influence the course of immunological dysfunction of MODS. The level of CCR7 may serve as an indicator of the migration activity of interstitial dendritic cells and systemic immune response.

Animals ; Antigens, CD ; metabolism ; B7-1 Antigen ; metabolism ; B7-2 Antigen ; metabolism ; CD11c Antigen ; metabolism ; CD4-CD8 Ratio ; Cell Proliferation ; Chemokine CCL21 ; metabolism ; Dendritic Cells ; immunology ; metabolism ; ultrastructure ; Disease Models, Animal ; Lectins, C-Type ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Minor Histocompatibility Antigens ; Multiple Organ Failure ; chemically induced ; immunology ; metabolism ; pathology ; Random Allocation ; Receptors, CCR7 ; metabolism ; Receptors, Cell Surface ; metabolism ; Zymosan

OBJECTIVETo investigate the expression of indoleamine 2, 3-dioxygenase (IDO) in breast cancer and its correlation with clinicopathologic factors and prognosis.

METHODSThe expression of IDO, CD31, CD105 proteins in 40 specimens of breast cancer were assessed by immunohistochemistry.

RESULTSThe overexpression rate of IDO in breast cancer was 67.5% (27/40), and expression of IDO was closely associated with clinical stage and lymph nodes metastasis. The disease-free survival rate in patients with IDO overexpression was not significantly lower than that in patients with negative or low expression of IDO (P > 0.05). Moreover, the expression of IDO was positively correlated with CD105-labeled microvessel density (r = 0.659, P < 0.05).

CONCLUSIONSExpression of IDO is associated with clinical stage and lymph nodes metastasis, and microvessel densitty. IDO expression may promote the growth and metastasis of breast cancer, probably via the increased agiogenesis. A larger sample study is needed to verify whether the prognosis of beast cancer is significantly correlated with IDO expression.

Adenocarcinoma ; enzymology ; immunology ; pathology ; Adult ; Aged ; Antigens, CD ; metabolism ; Breast Neoplasms ; enzymology ; immunology ; pathology ; Carcinoma, Ductal, Breast ; enzymology ; immunology ; pathology ; Carcinoma, Medullary ; enzymology ; immunology ; pathology ; Disease-Free Survival ; Endoglin ; Female ; Follow-Up Studies ; Humans ; Immunohistochemistry ; Indoleamine-Pyrrole 2,3,-Dioxygenase ; metabolism ; Lymphatic Metastasis ; Microvessels ; enzymology ; immunology ; Middle Aged ; Neoplasm Staging ; Platelet Endothelial Cell Adhesion Molecule-1 ; metabolism ; Receptors, Cell Surface ; metabolism ; Survival Rate

To obtain the recombinant pZP3alpha protein for the study of the contraceptive vaccines, the DNA sequence (446-1423) encoding purified pZP3alpha was inserted into a vector--pPICZalphaA. The recombinant plasmid pPICZalphaA-pZP3alpha was linearized and then transformed into Pichia pastoris GS115 by electroporation. Engineering strains were attained by screening with zeocin and induced to produce rpZP3alpha in high-density fermentation. Then rpZP3alpha was purified by Cu2+ metal affinity column chromatography from the separated and concentrated fermentative supernatants. The purified rpZP3alpha was identified by SDS-PAGE and Western blot, and the quantity, purity and rate of recovery of the rpZP3alpha were analyzed by Quantity One software. One male rabbit was immunized with the Cu-NTA-purified rpZP3alpha. The antibody responses against rpZP3alpha and porcine ZP were detected by ELISA and the indirect immunofluorescence. Engineering strains expressing rpZP3alpha in secretion were constructed. A 46kD component named rpZP3alpha which can react with anti-pZP3 antibody was purified from fermentative supernatants of engineering strains and the average yield of purified rpZP3alpha obtained from fermentative supernatants was 8mg/L. The purity and the rate of recovery were up to 92% and 63% respectively. The anti-rpZP3alpha antiserum was prepared by immunization of a male rabbit with purified rpZP3alpha. This anti-rpZP3alpha antiserum could react with rpZP3alpha and purified pZP3 in ELISA and bind to porcine zona pellucida which produced bright green fluorescence in the indirect immunofluorescence. The rpZP3alpha (46kD) protein could be successfully expressed in the Pichia pastoris expression system. And this protein retained the immunogenic activity of natural pZP3.
Animals ; Egg Proteins ; genetics ; metabolism ; Electroporation ; Fermentation ; Immunization ; Male ; Membrane Glycoproteins ; genetics ; metabolism ; Pichia ; genetics ; metabolism ; Rabbits ; Receptors, Cell Surface ; genetics ; metabolism ; Recombinant Proteins ; genetics ; immunology ; secretion ; Swine ; Zona Pellucida Glycoproteins

OBJECTIVETo explore the anti-inflammatory mechanisms of high density lipoprotein (HDL) by observing the effects of apoprotein (apo)AI, a major protein component of HDL, on the inflammatory macrophage cell polarity.

METHODSCultured mice marrow-derived macrophages were stimulated with lipopolysaccharide and interferon after 10 µg/ml of apoAI were added to the macrophages for 24 hours. The expression of membrane molecules CD16/32, CD206 were detected by fluorescence activated cell sorting (FACS). ELISA was used to detect the secretion of IL-10 and IL-12. Real-time quantitative PCR was used to detect the mRNA expression of TLR4, MyD88 and IRF5.

RESULTSCompared to macrophages stimulated by interferon and lipopolysaccharide but without pretreatment with apoAI, pre-incubation with apoAI significantly downregulated the expression of CD16/32 (91.17% ± 1.99% vs.50.47% ± 1.02%, P < 0.05), IL-12 [(747.27 ± 3.74)pg/ml vs. (73.80 ± 4.56)pg/ml, P < 0.05], upregulated the expression of CD206(0.33% ± 0.12% vs. 3.00% ± 0.36%, P < 0.05), IL -10 expression [(23.56 ± 4.30) pg/ml vs.(32.91 ± 2.47) pg/ml, P < 0.05], and reduced the mRNA expression of TLR4 (1.000 ± 0.025 vs.0.708 ± 0.003, P < 0.05) , MyD88 (1.591 ± 0.005 vs. 1.341 ± 0.005, P < 0.05) , IRF5 (0.954 ± 0.005 vs. 0.463 ± 0.003, P < 0.05) .

CONCLUSIONApoAI enhances the switch of inflammatory macrophages to anti-inflammatory macrophages possibly through inhibiting TLR4-MyD88-IRF5 pathway.

Animals ; Apolipoprotein A-I ; pharmacology ; Cell Line ; Female ; Interferon Regulatory Factors ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-12 ; metabolism ; Lectins, C-Type ; metabolism ; Macrophages ; drug effects ; immunology ; metabolism ; Mannose-Binding Lectins ; metabolism ; Mice ; Mice, Inbred C57BL ; Myeloid Differentiation Factor 88 ; metabolism ; Receptors, Cell Surface ; metabolism ; Receptors, IgG ; metabolism ; Toll-Like Receptor 4 ; metabolism