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

The inhibitory mechanism of interferon-gamma (IFN-gamma) on the fibroblasts from Tenon's capsule was studied. By using immunohistochemical SP method and pathological image system, the inhibitory effects of IFN-gamma on the expression of transforming growth factor beta receptor I in the in vitro cultured fibroblasts from Tenon's capsule were quantitatively analyzed. The results showed that IFN-gamma could reduce the expression of transforming growth factor beta receptor I in the fibroblasts with the following dose-effect relationship: Y = 1937.5-134.2 Igx (r=-0.971, P<0.01). It was concluded that IFN-gamma could inhibit the expression of transforming growth factor beta receptor I in the fibroblasts from Tenon's capsule. The modulation of the transforming growth factor beta receptor I expression by IFN-gamma may be beneficial to the alleviation of the hyperplasia of scar after trabeculectomy.
Conjunctiva ; metabolism ; pathology ; Connective Tissue ; drug effects ; Fibroblasts ; metabolism ; pathology ; Filtering Surgery ; Glaucoma ; pathology ; surgery ; Humans ; Interferon-gamma ; biosynthesis ; genetics ; Receptors, Transforming Growth Factor beta ; analysis

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

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

To explore the difference of negative regulatory factors among T lymphocyte subsets in bone marrow (BM) of myelodysplastic syndromes (MDS) and their relations to apoptotic gene Fas, different lymphocyte subsets in BM were categorized by monoclonal antibodies with 3 color fluorescence using flow cytometry, and the intracellular cytokines such as tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) were determined following marrow cells culture. Then Fas mRNA of bone marrow mononuclear cells (BMMNC) were examined by RT-PCR. The results showed that TNF-alpha, IFN-gamma levels in BM of MDS both increased, the former produced by cells CD4+CD45RO+, CD8+CD45RO+, the latter by cells CD4+CD45RO+, CD8+CD45RO+, CD8+CD45RA+, in which the cells CD8+CD45RO+ were dominant. Fas mRNA expression had relationship with IFN-gamma produced by T cells but not with TNF-alpha. It is concluded that in hematopoietic microenvironment of MDS, not only the T lymphocyte subsets are in disorder, but also negative regulatory factors secreted by T lymphocyte increase. T lymphocytes play an important role in producing IFN-gamma in patients with MDS.
Adult ; Aged ; Bone Marrow Cells ; metabolism ; Female ; Humans ; Interferon-gamma ; biosynthesis ; Male ; Middle Aged ; Myelodysplastic Syndromes ; metabolism ; pathology ; RNA, Messenger ; analysis ; T-Lymphocyte Subsets ; metabolism ; Tumor Necrosis Factor-alpha ; biosynthesis ; fas Receptor ; genetics

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

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

Canine Interferon-gamma (CaIFN-gamma) cDNA was cloned from spleen T cells of dog by reverse transcription-polymerase chain reaction (RT-PCR). CaIFN-gamma cDNA were digested with Hind III and Not I, and inserted into pRc/CMV2 expression vector. The pRc/CMV2/CaIFN-gamma vector was sequenced, and predicted to produce a signal peptide of 23 amino acids and a mature protein of 143 amino acids with a molecular weight of 19 kD. Two potential N-glycosylation sites are located at positions 16 and 83 of the mature protein. Comparison of the CaIFN-gamma protein sequence with that of CaIFN-gamma reported from DDBJ/GenBank revealed a homology of 99%. To establish a long time expression system, pRc/CMV2/CaIFN-gamma vector was transfected into mouse SP2/0 cell line. The SP2/0 cells culture supernatants was harvested and the antiviral activity was measured following cytopathic-effect inhibition assay using Madin-Darby Canine Kidney (MDCK)-vesicular stomatitis virus(VSV) system. Initial transformants with G418 phenotype produced recombinant CaIFN-gamma titers ranging from 2,500 to 5,000 u/mL of culture medium.
Amino Acid Sequence ; Animals ; Base Sequence ; Bone Marrow Neoplasms ; metabolism ; Cattle ; Cell Line ; Cloning, Molecular ; DNA, Complementary ; analysis ; chemistry ; Dogs ; Interferon-gamma ; biosynthesis ; genetics ; pharmacology ; Mice ; Molecular Sequence Data ; Recombinant Proteins

To investigate the value of apoptosis of the allo-antigen specific T cells induced by Fas/FasL pathway in order to prevent GVHD in allo-transplant, the CD34(+) cells were transfected with FasL or not, used as effector cells, mixed with allo-antigen specific T lymphocytes with presence or absence of IFN-gamma or IL-2. After 5 days, apoptosis of T cells was detected by TdT nick end mediated dUTP labeling (TUNEL) and flow cytometry. The effects of IFN-gamma or IL-2 on apoptosis of CD34(+) cells of graft induced by Fas/FasL pathway observed as controls. The apoptosis incidence of T cells was (12.1 +/- 1.5)% when CD34(+) cells transfected with FasL were used as effector cells, that was much higher than that T cells with CD34(+) cells non-transfected (p < 0.01). In the presence of IFN-gamma or IL-2, apoptosis incidence reached to (20.1 +/- 2.3)% or (17.6 +/- 1.3)% respectively (p < 0.01). When sFasL was added to CD34(+) cells freshly isolated or induced with IFN-gamma or IL-2, the incidence or apoptosis of CD34(+) cells was (7.8 +/- 0.8)%, (18.7 +/- 1.6)% (p < 0.01) or (7.9 +/- 1.0)% (P > 0.05) respectively. The results suggest that it is possible to induce apoptosis of the allo-antigen specific T cells in grafts activated by allo-antigen by exogenous Fas ligand expressed on receptor cells and that may hopefully provide a new method to prevent GVHD
Animals ; Antigens, CD34 ; analysis ; Apoptosis ; Cell Communication ; physiology ; DNA, Complementary ; genetics ; Fas Ligand Protein ; Humans ; Interferon-gamma ; pharmacology ; Membrane Glycoproteins ; genetics ; physiology ; Mice ; T-Lymphocytes ; cytology ; physiology ; Transfection ; fas Receptor ; biosynthesis

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