OBJECTIVETo investigate the antitumor effect of natural killer (NK) cells on human colorectal cancer cells HT-29 in vitro by blocking transforming growth factor-β (TGF-β) signaling in NK cells transfected with vector containing dominant negative TGF-β type 2 receptor (DNTβR2).

METHODSTGF-β1 was added at the final concentration of 10 ng/ml for HT-29 cells. Primary NK cells were transfected with recombinant plasmid pIRES2-AcGFP-DNTβR2 and control plasmid pIRES2-AcGFP using Amaxa Nucleofector technology respectively. The cytotoxicity of these two types of NK cells to HT-29 cells was detected and analyzed by cell counting kit-8.

RESULTSThe transfection efficiency of primary NK cells was 18.85% for the plasmid pIRES2-AcGFP-DNTβR2 and 35.28% for the control plasmid pIRES2-AcGFP. The expression of DNTβR2 in NK cells was confirmed by Western blotting and RT-PCR. Primary NK cells displayed significantly lower cytotoxicity against HT-29 cells incubated with TGF-β1 than that without TGF-β1 (effect-target cell ratio 10:1,14.40%∓ 2.00% vs. 26.14% ∓ 2.50%, P > 0.05; effect-target cell ratio 20:1, 19.18% ∓ 2.49% vs. 40.81% ∓ 3.50%, P > 0.05). The cytotoxicity of NK cells transfected with DNTβR2 vector was significantly higher than that with control vector against HT-29 cells cultured with 10 ng/ml TGF-β1 (effect-target cell ratio 10:1, 21.17% ∓ 2.49% vs. 11.48% ∓ 1.11% ,P > 0.05; and effect-target cell ratio 20:1, 35.30% ∓ 3.78% vs. 17.19% ∓ 2.29%, P > 0.05).

CONCLUSIONNK cells transfected with DNTβR2 vector show better antitumor effect, which may provide new method for NK-based adoptive immunotherapy for cancer.

HT29 Cells ; Humans ; Killer Cells, Natural ; immunology ; metabolism ; Plasmids ; genetics ; Receptors, Transforming Growth Factor beta ; genetics ; Transfection ; Transforming Growth Factor beta ; metabolism ; pharmacology

OBJECTIVETo investigate the different expression of transforming growth factor beta(1) (TGF-beta(1)) in lung cancer specimens of Gejiu miners, and non-miners in other regions.

METHODSThirty specimens of Gejiu miners' lung cancer and 30 specimens of non-miners' were observed in this experiment. The expression of TGF-beta(1) protein and TGF-beta(1) mRNA were detected by the methods of immunohistochemistry and in situ hybridization. The results were quantitatively analyzed using image analysis system.

RESULTSThe positive rate of TGF-beta(1) protein expression in Gejiu miners and non-miners was 75.39%and 44.78% respectively, and the positive rate of TGF-beta(1) mRNA was 63.96% and 34.07% respectively. There were significant differences between the two groups (P < 0.01).

CONCLUSIONThe expression of TGF-beta(1) in lung cancer of Gejiu miners was significantly higher than that of non-miners. The pathogenesis of lung cancer may be different between Gejiu miner and non-miners. High expression of TGF-beta(1) may be one of the reasons of high incidence of lung cancer in Gejiu miners.

Humans ; Immunohistochemistry ; In Situ Hybridization ; Lung Neoplasms ; genetics ; metabolism ; pathology ; RNA, Messenger ; genetics ; metabolism ; Transforming Growth Factor beta ; genetics ; immunology ; Transforming Growth Factor beta1

Humans ; Immunoglobulin Light Chains ; genetics ; immunology ; Immunoglobulin kappa-Chains ; genetics ; immunology ; Kidney ; immunology ; metabolism ; pathology ; Kidney Diseases ; immunology ; pathology ; therapy ; Mutation ; Transforming Growth Factor beta ; metabolism

OBJECTIVETo study the correlation between the expression of Egr-1 and NF-kappaB and the up-regulation of TNF-alpha and TGF-beta1 in macrophages after stimulation by silica in-vitro.

METHODSMacrophages were treated with antibodies against Egr-1 and NF-kappaB and antisense oligonucleotides. The level of TNF-alpha protein in the cell supernatant was then measured using enzyme-linked immunoadsorbent assay (ELISA). The expression of TGF-beta1 protein was detected by immunocytochemistry. The expression of TNF-alpha and TGF-beta1 mRNAs was also monitored by reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTSCompared with silica-stimulated macrophages untreated with antibodies, the cells treated with 10 micro g/ml of Egr-1 or NF-kappaB antibodies were associated with reduced expression of TNF-alpha and TGF-beta1 proteins and mRNAs (P < 0.05). Compared with silica-stimulated untransfected group, the antisense group was associated with obvious reduction in the expression of TNF-alpha and TGF-beta1 proteins and mRNAs (P < 0.05).

CONCLUSIONThe expression of TNF-alpha and TGF-beta1 mRNAs and proteins are associated with activation of Egr-1 and NF-kappaB in macrophages, after stimulation by silica. It is possible that the corresponding antibodies and antisense oligonucleotides may become a potential therapeutic tool in the management of silicosis in the future.

Animals ; Antibodies ; immunology ; Cells, Cultured ; DNA-Binding Proteins ; genetics ; immunology ; Early Growth Response Protein 1 ; Immediate-Early Proteins ; genetics ; immunology ; Macrophages ; cytology ; metabolism ; Mice ; NF-kappa B ; genetics ; immunology ; Oligonucleotides, Antisense ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Silicon Dioxide ; pharmacology ; Silicosis ; etiology ; Transcription Factors ; genetics ; immunology ; Transforming Growth Factor beta ; biosynthesis ; genetics ; Transforming Growth Factor beta1 ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics

OBJECTIVETo evaluate the changes of CD(4)(+)CD(25)(+) T cells in peripheral blood from patients with breast cancer.

METHODSSixty four patients with breast cancer, 15 patients with benign breast tumors and 9 healthy volunteers were included in this study. The proportion of CD(4)(+)CD(25)(+) T cells population in total T cells was evaluated by flow cytometric analysis. The cytokine production (TGF-beta1) was measured by ELISA.

RESULTSThe population of CD(4)(+)CD(25)(+) T cells in peripheral blood from patients with breast cancer accounted for (5.1 +/- 2.9)% of the total amount of T lymphocytes, and was significantly higher in comparison with that in patients with benign tumors and in healthy volunteers (P < 0.05). The CD(4)(+)CD(25)(+) T cells population in breast cancer patients was positively correlated with the cancer size and with TGF-beta1 level (r = 0.511 and r = 0.253, respectively), and negatively correlated with CD(8)(+)CD(28)(+) T cells and NK cells (r = -0.243 and r = -0.301, respectively).

CONCLUSIONThe CD(4)(+)CD(25)(+) regulatory T cells in peripheral blood of patients with breast cancer is significantly increased in comparison with that in patients with benign breast tumor and in healthy subjects. It may be responsible for immune suppression in breast cancer patients.

Adult ; Aged ; Breast Neoplasms ; immunology ; CD4-Positive T-Lymphocytes ; immunology ; Cell Count ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Interleukin-2 Receptor alpha Subunit ; immunology ; Middle Aged ; T-Lymphocyte Subsets ; immunology ; Transforming Growth Factor beta ; biosynthesis ; genetics

Human amniotic membrane-derived mesenchymal stem cells (hAM-MSCs) are capable of differentiating into several lineages and possess immunomodulatory properties. In this study, we investigated the soluble factor-mediated immunomodulatory effects of hAM-MSCs. Mitogen-induced peripheral blood mononuclear cell (PBMC) proliferation was suppressed by hAM-MSCs in a dose-dependent manner as well as hAM-MSC culture supernatant. Moreover, interferon-gamma and interleukin (IL)-17 production significantly decreased from PBMC, whereas IL-10 from PBMCs and transforming growth factor beta (TGF-beta) production from hAM-MSCs significantly increased in co-cultures of hAM-MSCs and PBMCs. Production of several MSC factors, including hepatocyte growth factor (HGF), TGF-beta, prostaglandin E2 (PGE2), and indoleamine 2, 3 dioxygenase (IDO), increased significantly in hAM-MSCs co-cultured with PBMCs. These results indicate that the immunomodulatory effects of hAM-MSCs may be associated with soluble factors (TGF-beta, HGF, PGE2, and IDO), suggesting that hAM-MSCs may have potential clinical use in regenerative medicine.
Amnion/cytology/*immunology ; Cell Differentiation/immunology ; Coculture Techniques ; Dinoprostone/genetics/immunology ; Female ; Hepatocyte Growth Factor/genetics/immunology ; Humans ; Immunologic Factors/*immunology ; Immunophenotyping ; Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics/immunology ; Interferon-gamma/immunology ; Interleukin-10/analysis/immunology ; Interleukin-17/analysis/immunology ; Leukocytes, Mononuclear/cytology/immunology ; Mesenchymal Stem Cells/cytology/*immunology ; Pregnancy ; RNA, Messenger/chemistry/genetics ; Regenerative Medicine/methods ; Reverse Transcriptase Polymerase Chain Reaction ; Transforming Growth Factor beta/genetics/immunology

Immunosuppressive regulatory T lymphocytes (Treg) expressing the transcription factor Foxp3 play a vital role in the maintenance of tolerance of the immune-system to self and innocuous non-self. Most Treg that are critical for the maintenance of tolerance to self, develop as an independent T-cell lineage from common T cell precursors in the thymus. In this organ, their differentiation requires signals from the T cell receptor for antigen, from co-stimulatory molecules, as well as from cytokine-receptors. Here we focus on the cytokines implicated in thymic development of Treg, with a particular emphasis on the roles of interleukin-2 (IL-2) and IL-15. The more recently appreciated involvement of TGF-β in thymic Treg development is also briefly discussed. Finally, we discuss how cytokine-dependence of Treg development allows for temporal, quantitative, and potentially qualitative modulation of this process.
Animals ; Cell Differentiation ; genetics ; Cytokines ; immunology ; Forkhead Transcription Factors ; genetics ; immunology ; Gene Expression Regulation ; Immune Tolerance ; genetics ; Interleukin-15 ; genetics ; immunology ; Interleukin-2 ; genetics ; immunology ; Mice ; Receptors, Antigen, T-Cell ; genetics ; immunology ; T-Lymphocytes, Regulatory ; immunology ; Transforming Growth Factor beta ; genetics ; immunology

OBJECTIVESTo examine the expression and purification of the TGFbeta1 vaccine from prokaryotic expression system and to determine the antigenicity of the fusion protein of recombinant vector pET28a/ HBcAg1-71-TGFbeta132-HBcAg89-144.

METHODSThe reconstructed vector pGEMEX-1/CTC was subcloned to pET28a and transformed into E. coli BL21 (DE3). The recombinant 6xHis- HBcAg1-71- TGFbeta132- HBcAg89-144 was to be expressed after induction by IPTG and purified with Ni-NTA-His affinity chromatography. The detection of the formation of core-like particles was done under an electron microscope and of their antigenity by using ELISA and Western blot.

RESULTSA 2.46 x 10(4) protein was obtained by optimizing the conditions for both expression and purification. The protein had the TGFbeta1 antigenicity but not a HBc antigenity and the formed core-like particles were bigger than natural core particles.

CONCLUSIONThe recombinant fusion protein in the prokaryotic expressed system can be used as an anti-TGFbeta1 vaccine to inhibit hepatic fibrosis.

Epitopes ; immunology ; Genetic Vectors ; Hepatitis B Core Antigens ; biosynthesis ; genetics ; Humans ; Liver Cirrhosis ; prevention & control ; Prokaryotic Cells ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection ; Transforming Growth Factor beta ; biosynthesis ; genetics ; Vaccines, Synthetic ; biosynthesis ; immunology

OBJECTIVETo investigate the regulation of Toll-like receptors (TLRs) on immune suppressive cytokines in situ colon cancer cells.

METHODSThe mRNA and protein expression spectrum of TLRs in HT-29 cells were determined by RT-PCR and Western blot respectively. The cytokines and chemokines levels of supernant of HT-29 stimulated by lipoplysaccharide(LPS) were detected with ELISA.

RESULTSTLR1-9 were expressed in HT-29 cells on mRNA level. After LPS stimulation, TLR4 mRNA and protein expressions were up-regulated in HT-29 cells, and TGF-beta, VEGF, IL-8, CCL20 and IL-6 levels increased significantly(all P<0.01). Except IL-6, up-regulation of the other cytokines was not suppressed by NF-kappa B inhibitor.

CONCLUSIONTLRs expressed on colon cancer cells may elevate the immune suppressive cytokines and chemokines, which promote the immune escape of cancer cells.

Chemokine CCL20 ; metabolism ; Colonic Neoplasms ; immunology ; metabolism ; Cytokines ; immunology ; HT29 Cells ; Humans ; Interleukin-6 ; metabolism ; NF-kappa B ; metabolism ; RNA, Messenger ; genetics ; Signal Transduction ; Toll-Like Receptor 4 ; immunology ; metabolism ; Transforming Growth Factor beta ; metabolism ; Up-Regulation

In fascioliasis, T-helper 2 (Th2) responses predominate, while little is known regarding early immune phenomenon. We herein analyzed early immunophenotype changes of BALB/c, C57BL/6, and C3H/He mice experimentally infected with 5 Fasciola hepatica metacercariae. A remarkable expansion of CD19+ B cells was observed as early as week 1 post-infection while CD4+/CD8+ T cells were down-regulated. Accumulation of Mac1+ cells with time after infection correlated well with splenomegaly of all mice strains tested. The expression of tumor necrosis factor (TNF)-alpha mRNA in splenocytes significantly decreased while that of IL-4 up-regulated. IL-1beta expression was down-modulated in BALB/c and C57BL/6 mice, but not in C3H/He. Serum levels of transforming growth factor (TGF)-beta were considerably elevated in all mice during 3 weeks of infection period. These collective results suggest that experimental murine fascioliasis might derive immune suppression with elevated levels of TGF-beta and IL-4 during the early stages of infection.
Animals ; B-Lymphocytes/immunology ; CD4-Positive T-Lymphocytes/immunology ; CD8-Positive T-Lymphocytes/immunology ; Down-Regulation ; Fasciola hepatica/*immunology ; Fascioliasis/*immunology ; Immunophenotyping ; Immunosuppression ; Interleukin-4/blood/genetics/*immunology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C3H ; Mice, Inbred C57BL ; Spleen/immunology ; Transforming Growth Factor beta/blood/genetics/*immunology