PURPOSE: To identify new immunogenic HLA-A*33;03-restricted epitopes from the human papillomavirus (HPV) 16 E7 protein for immunotherapy against cervical cancer. MATERIALS AND METHODS: We synthesized fourteen overlapping 15-amino acid peptides and measured intracellular interferon-γ (IFN-γ) production in PBMC and CD8+ cytotoxic T lymphocytes (CTLs) after sensitization with these peptides using flow cytometry and ELISpot assay. The immunogenicity of epitopes was verified using a ⁵¹Cr release assay with SNU1299 cells. RESULTS: Among the fourteen 15-amino acid peptides, E7₄₉₋₆₃ (RAHYNIVTFCCKCDS) demonstrated the highest IFN-γ production from peripheral blood mononuclear cells (PBMCs), and CD8+ CTLs sensitized with E7₄₉₋₆₃ showed higher cytotoxic effect against SNU1299 cells than did CD8+ CTLs sensitized with other peptides or a negative control group. Thirteen 9- or 10-amino acid overlapping peptides spanning E7₄₉₋₆₃, E7₅₀₋₅₉ (AHYNIVTFCC), and E7₅₂₋₆₁ (YNIVTFCCKC) induced significantly higher IFN-γ production and cytotoxic effects against SNU1299 cells than the other peptides and negative controls, and the cytotoxicity of E7₅₀₋₅₉- and E7₅₂₋₆₁-sensitized PBMCs was induced via the cytolytic effect of CD8+ CTLs. CONCLUSION: We identified E7₅₀₋₅₉ and E7₅₂₋₆₁ as novel HPV 16 E7 epitopes for HLA-A*33;03. CD8+ CTL sensitized with these peptides result in an antitumor effect against cervical cancer cells. These epitopes could be useful for immune monitoring and immunotherapy for cervical cancer and HPV 16-related diseases including anal cancer and oropharyngeal cancer.
Amino Acid Sequence ; CD8-Positive T-Lymphocytes/immunology/metabolism ; Epitopes/*immunology/therapeutic use ; Female ; *HLA-A Antigens ; Human papillomavirus 16/*immunology ; Humans ; *Immunotherapy ; Interferon-gamma/analysis/*biosynthesis ; Leukocytes, Mononuclear/immunology/metabolism ; T-Lymphocytes, Cytotoxic/immunology/metabolism ; Uterine Cervical Neoplasms/*therapy

Metastasis is the main cause of death in cancer patients. To improve the outcomes of patients undergoing a surgery, new adjuvant therapies that can effectively inhibit metastases have to be developed. Studies have shown that flavonoid naringenin, a natural product that is mainly present in grapes and citrus, may contribute to cancer prevention. It has many advantages compared to traditional chemotherapeutic drugs, such as low toxicity. To determine whether naringenin can also inhibit metastases, a breast cancer resection model that mimics clinical situations was established. We found that orally administered naringenin significantly decreased the number of metastatic tumor cells in the lung and extended the life span of tumor resected mice. Flow cytometry analysis revealed that T cells displayed enhanced antitumor activity in naringenin treated mice, with an increased proportion of IFN-γ and IL-2 expressing T cells. In vitro studies further demonstrated that relief of immunosuppression caused by regulatory T cells might be the fundamental mechanism of metastasis inhibition by naringenin. These results indicate that orally administered naringenin can inhibit the outgrowth of metastases after surgery via regulating host immunity. Thus, naringenin can be an ideal surgical adjuvant therapy for breast cancer patients.
Animals ; Anticarcinogenic Agents ; administration & dosage ; therapeutic use ; Antigens, CD ; analysis ; Breast Neoplasms ; drug therapy ; immunology ; pathology ; surgery ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Chemotherapy, Adjuvant ; Female ; Flavanones ; administration & dosage ; therapeutic use ; Humans ; Immunosuppressive Agents ; administration & dosage ; therapeutic use ; Interferon-gamma ; biosynthesis ; immunology ; Interleukin-2 ; biosynthesis ; immunology ; Lung Neoplasms ; drug therapy ; immunology ; prevention & control ; secondary ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; T-Lymphocytes, Regulatory ; drug effects ; immunology ; metabolism

OBJECTIVETo construct, express and identify the recombinant plasmid pcDNA3.1(+)/sflk1-IFN-gamma encoding bifunctional protein sflk1-IFN-gamma (soluble fetal liver kinase 1 and interferon-gamma).

METHODSsflk1 and IFN-gamma gene fragments were cloned by RT-PCR, and then inserted into pcDNA3.1(+) plasmid between BamHI-EcoRI and XhoI-XbaI restriction sites to form the recombinant plasmid pcDNA3.1(+)/sflk1-IFN-gamma. The recombinant sflk1-IFN-gamma transiently expressed in COS-7 cells was detected by ELISA and Western blotting. Bioactivities of sflk1-IFN-gamma fusion protein were identified by proliferation inhibition assay with H5V cells and NK activity assay.

RESULTSpcDNA3.1(+)/sflk1-IFN-gamma can be effectively expressed in COS-7 cells. Concentrations of sflk1 and IFN-gamma in culture supernatants of pcDNA3.1(+)/sflk1-IFN-gamma transfected COS-7 cells were (20.85+/-2.48) ng/ml and (1.08+/-0.09) ng/ml, respectively. Western blotting showed that the molecular weight of sflk1-IFN-gamma fusion protein was about 130 kDa, while that of sflk1 was 115 kDa. The supernatants of transfected cells significantly inhibited the proliferation of H5V cells stimulated by mouse VEGF 164 and enhanced the NK activity of splenocytes, demonstrating that sflk1-IFN-gamma fusion protein possessed the bioactivities of both sflk1 and IFN-gamma.

CONCLUSIONThe constructed plasmid pcDNA3.1(+)/sflk1-IFN-gamma can be effectively expressed in eukaryotes. The expressed sflk1-IFN-gamma fusion protein has the biological activities of both sflk1 and IFN-gamma.

Animals ; COS Cells ; Cercopithecus aethiops ; Female ; Interferon-gamma ; biosynthesis ; genetics ; Mice ; Mice, Inbred C57BL ; Plasmids ; Recombinant Proteins ; analysis ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; Vascular Endothelial Growth Factor Receptor-2 ; biosynthesis ; genetics

Angioplasty, Balloon, Coronary ; C-Reactive Protein ; analysis ; Coronary Restenosis ; prevention & control ; Drug-Eluting Stents ; Humans ; Inflammation ; complications ; etiology ; prevention & control ; Interferon-gamma ; blood ; Interleukin-6 ; blood ; Muscle, Smooth, Vascular ; immunology ; Sirolimus ; administration & dosage ; Tumor Necrosis Factor-alpha ; biosynthesis ; blood ; Tunica Intima ; pathology

In order to determine the role of Peyer's patch lymphocytes (PPL) in self-clearing of Cryptosporidium parvum infection in murine models, changes in PPL subsets, their cytokine expression, and in vitro IgG1 and IgA secretions by PPL were observed in primary- and challenge-infected C57BL/6 mice. In primary-infected mice, the percentages of CD4+ T cells, CD8+ T cells, sIgA+ B cells, IL-2+ T cells, and IFN-gamma+ T cells among the PPL, increased significantly (P < 0.05) on day 10 post-infection (PI). Secretion of IgG1 and IgA in vitro by PPL also increased on day 10 PI. However, all these responses, with the exception of IgG1 and IgA secretions, decreased in challenge-infected mice on day 7 post-challenge (= day 13 PI); their IgG1 and IgA levels were higher (P > 0.05) than those in primaryinfected mice. The results suggest that murine PPL play an important role in self-clearing of primary C. parvum infections through proliferation of CD4+, CD8+, IL-2+, and IFN-gamma+ T cells, and IgG1 and IgA-secreting B cells. In challenge infections, the role of T cells is reduced whereas that of B cells secreting IgA appeared to be continuously important.
Animals ; Antibodies, Protozoan/analysis/metabolism ; Cattle ; Cryptosporidiosis/*immunology/parasitology ; Cryptosporidium parvum/*immunology ; Feces/parasitology ; Female ; Humans ; Immunoglobulin A/analysis/biosynthesis ; Immunoglobulin G/analysis/biosynthesis ; Interferon-gamma/analysis/biosynthesis ; Interleukin-2/analysis/biosynthesis ; Lymphocytes/*immunology ; Male ; Mice ; Mice, Inbred C57BL ; Peyer's Patches/cytology/*immunology ; Specific Pathogen-Free Organisms

BACKGROUNDT cell immune abnormalities in patients with dilated cardiomyopathy (DCM) has been intensively studied over the past 10 years. Our previous study has suggested that immunization of mice with the peptides derived from human adenine nucleotide translocator (ANT) result in the production of autoantibodies against the ANT and histopathological changes similar to those in human DCM. The ANT peptides can induce autoimmune cardiomyopathy like DCM in Balb/c mice. In this study we aimed to focus on the molecular mechanism of T cells in the autoimmune cardiomyopathy mouse model by detecting the expression of the two T cell signaling molecules.

METHODSThe ANT peptides were used to cause autoimmune cardiomyopathy in Balb/c mice. Anti-L3T4 or rat anti-mouse IgG was administered to the mice (n = 6 in each group) simultaneously immunized with ANT. ELISA analysis was used to detect autoantibodies against the ANT peptides and the percentages of interferon-gamma and interleukin-4 producing cells among splenic CD4(+) lymphocytes was determined by using flow cytometry analysis. The expression of CD45 in spleen T cells was determined by immunohistochemistry and the mRNAs of T cell signaling molecules were detected by real-time PCR.

RESULTSTreatment of ANT immunized Balb/c mice with anti-CD4 mAb caused a reduction in the gene expression of P56lck and Zap-70 and a lower level of CD45 expression by spleen T cells. Also, a reverse of the Th1/Th2 ratio that results in the reduced production of antibodies against ANT was found in the anti-CD4 monoclonal antibodies (mAb) group. Whereas irrelevant antibody (rat anti-mouse IgG) did not suppress T cell signaling molecules nor inhibit CD45 expression, and control-antibody mice did not show any significant differences compared with the DCM group.

CONCLUSIONThe results show that anti-CD4 mAb is a powerful inhibitor of the early initiating events of T cell receptor (TCR) signal transduction in mouse autoimmune dilated cardiomyopathy.

Adenine Nucleotide Translocator 1 ; immunology ; Animals ; Antibodies, Monoclonal ; therapeutic use ; Autoantibodies ; blood ; Autoimmune Diseases ; therapy ; CD4 Antigens ; immunology ; Cardiomyopathy, Dilated ; immunology ; therapy ; Interferon-gamma ; biosynthesis ; Interleukin-4 ; biosynthesis ; Leukocyte Common Antigens ; analysis ; Mice ; Mice, Inbred BALB C ; Receptors, Antigen, T-Cell ; antagonists & inhibitors ; physiology ; Signal Transduction

OBJECTIVE: To evaluate the expression of hCTLA4-Ig and their biological function in newborn porcine islets (NPIs) transfected with AAV-hCTLA4-Ig. METHODS: Cultured NPIs were transfected with AAV-hCTLA4-Ig. The expression of CTLA4-Ig in these NPIs was assayed by RT-PCR and immunocytochemistry. The levels of IL-2, IFN-gamma, and TNF-alpha in the culture medium were assayed by ELISA after these cells the co-cultured with human. The response of glucose-stimulated insulin secretion was observed in the transgene group and the control group. RESULTS: The expressions of CTLA4-Ig mRNA and protein were detected in the transgene group. The levels of cytokines were obviously lower in the transgene group than those in the control group (P<0.01). There was no significant difference in the response of glucose-stimulated insulin release between the transgene group and the control group (P>0.05). CONCLUSION AAV mediated hCTLA4-Ig expression in NPIs could inhibit T lymphocyte to produce cytokines, while the endocrine functions of the NPIs were not significantly affected.
Animals ; Animals, Newborn ; Antigens, CD ; biosynthesis ; genetics ; Antigens, Differentiation ; biosynthesis ; genetics ; CTLA-4 Antigen ; Cells, Cultured ; Dependovirus ; genetics ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Humans ; Immunoglobulin Fc Fragments ; biosynthesis ; genetics ; Immunohistochemistry ; Interferon-gamma ; analysis ; Interleukin-2 ; analysis ; Islets of Langerhans ; cytology ; immunology ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Swine ; Transfection ; Tumor Necrosis Factor-alpha ; analysis

OBJECTIVETo explore the effect of Astragalus membranaceus (AM) on T-helper cell type 1 (Thl) specific transcription factor T-box expressed in T cells (T-bet) expression and Thl/Th2 equilibrium.

METHODSThe levels of T-bet mRNA in peripheral blood mononuclear cells (PBMCs) from 15 patients with asthma and 15 healthy subjects were determined by reverse transcription-polymerase chain reaction (RT-PCR). PBMCs in asthma patients were incubated with AM and then the concentration of interferon gamma (IFN-gamma) and interleukin-4 (IL-4) in the supernate before and after AM intervention were determined by ELISA. The numbers of CD4 + CCR3 + and CD4 + CCR5 + cells were counted by flow cytometry.

RESULTSThe expression of T-bet mRNA and the level of IFN-gamma were lower, but level of serum IL-4 was higher in asthma patients when compared with those in healthy subjects respectively. After AM (60 microg/ml) intervention, the former two parameters raised and showed a positive correlation between them, while the level of IL-4 was decreased. The mean percentage of CD4 + CCR3 + cells in asthma patients was significantly higher but that of CD4 + CCR5 + cells was lower when compared with those in healthy subjects respectively. After AM intervention, the abnormal change in the two indexes was improved to certain extent, showing a reversing status of Th2 polarization.

CONCLUSIONAM could increase the expression of T-bet mRNA and Thl cytokines such as IFN-Y, and might reverse the Th2 predominant status in asthma patients.

Adult ; Asthma ; drug therapy ; immunology ; Astragalus membranaceus ; Cell Polarity ; Cross-Sectional Studies ; Female ; Humans ; Interferon-gamma ; biosynthesis ; blood ; Interleukin-4 ; blood ; Male ; Middle Aged ; Phytotherapy ; RNA, Messenger ; analysis ; Receptors, CCR3 ; Receptors, CCR5 ; blood ; Receptors, Chemokine ; blood ; T-Box Domain Proteins ; genetics ; Th1 Cells ; drug effects ; immunology ; Up-Regulation

OBJECTIVETo study the changes of IL-12 produced by dendritic cells in peripheral blood in children with Henoch-Schonlein purpura (HSP), and to explore its influence on TH1/TH2 balance in order to elucidate its significance in the pathogenesis of HSP.

METHODSThe levels of interferon-gamma (IFN-gamma), interleukin-4 (IL-4) and interleukin-12 (IL-12) in plasma were determined by ELISA in 60 HSP children (HSP group) and 21 healthy children (Control group). Peripheral blood mononuclear cells (PBMC) of 22 HSP patients and 21 healthy children were cultured in vitro and then were transformed into dendritic cells. The levels of IL-12 in the supernatant were detected by ELISA and the positive expression rate of CD1a(+) was detected by indirect immunofluorescence procedure.

RESULTS1) The levels of IFN-gamma and the ratio of IFN-gamma/IL-4 in plasma of the HSP group were lower than those of the Control group (IFN-gamma 30.59 +/- 11.27 pg/mL vs 43.38 +/- 19.19 pg/mL; IFN-gamma/IL-4 ratio 0.70 +/- 0.28 vs 1.33 +/- 0.57) (P < 0.01). The levels of IL-12 in the HSP group were also lower than those of the Control group (153.95 +/- 91.88 pg/mL vs 323.06 +/- 162.34 pg/mL; P < 0.01). In contrast, the levels of IL-4 were higher than those of the Control group (45.08 +/- 9.19 pg/mL vs 32.95 +/- 7.10 pg/mL; P < 0.01). The plasma levels of IL-12 positively correlated with the IFN-gamma levels (r=0.52, P < 0.01) and the ratio of IFN-gamma/IL-4 (r=0.43, P < 0.01) in the HSP group. 2) The IL-12 levels in the supernatant of the HSP group were lower than those of the Control group (357.06 +/- 153.56 pg/mL vs 489.80 +/- 213.45 pg/mL; P < 0.05), and had a positive correlation with the plasma IL-12 levels (r=0.74, P < 0.01). 3) The positive expression rate of CD1a(+) of the HSP group was lower than that of the Control group [(27.42 +/- 10.75)% vs (35.68 +/- 12.18)%; P < 0.05], and positively correlated with the IL-12 levels in the supernatants (r=0.57, P < 0.01) and in plasma (r=0.68, P < 0.01).

CONCLUSIONSThere was an imbalance of TH1/TH2 in HSP children. The decrease of TH1 function had a positive correlation with the low levels of IL-12 in plasma, while the latter correlated closely with decreased number and / or function of dendritic cells, suggesting that the decreased number and / or function of dendritic cells in peripheral blood resulted in the imbalance of TH1/TH2 indirectly.

Adolescent ; Antigens, CD1 ; analysis ; Cells, Cultured ; Child ; Child, Preschool ; Dendritic Cells ; immunology ; Female ; Humans ; Interferon-gamma ; blood ; Interleukin-12 ; biosynthesis ; blood ; Interleukin-4 ; blood ; Male ; Purpura, Schoenlein-Henoch ; immunology ; Th1 Cells ; immunology ; Th2 Cells ; immunology

OBJECTIVETo investigate IFN-gamma producing-cells (IFN-gamma PCs) in allogeneic mixed lymphocyte reaction (MLR) and acute graft versus host disease (aGVHD) model of mice.

METHODSEnzyme linked immunospot assay (ELISPOT) was applied to study IFN-gamma PCs in MHC mismatched mice spleen cell MLR and aGVHD model of mice.

RESULTIFN-gamma PCs increased significantly in MLR after allogeneic mice spleen cell stimulation. In the experimental mice aGVHD model, IFN-gamma PCs were significantly higher in the severe aGVHD group than those in the moderate aGVHD. In the moderate aGVHD group, mice with GVHD prophylaxis regimen demonstrated significantly lower level of IFN-gamma PCs, compared with those without prophylaxis. IFN-gamma PCs were significantly correlated with the GVHD clinical scores in the group with moderate aGVHD and prophylaxis regimen.

CONCLUSIONELISPOT is a fast, sensitive and specific approach to evaluate alloresponse in allogeneic mice MLR and IFN-gamma PCs are correlated closely with the severity of aGVHD and prophylaxis regimen in the MHC-mismatched mice model.

Animals ; Enzyme-Linked Immunosorbent Assay ; methods ; Graft vs Host Disease ; immunology ; Interferon-gamma ; analysis ; biosynthesis ; genetics ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; T-Lymphocytes ; immunology ; metabolism