Objective To investigate the effect of insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) on the proliferation, migration and tumor immune microenvironment of colorectal cancer cells and its possible molecular mechanism. Methods The Cancer Genome Atlas (TCGA) database was used to analyze the expression levels of IGF2BP2 and MYC in colorectal cancer and adjacent tissues. The expression of IGF2BP2 in HCT-116 and SW480 human colorectal cancer cells was silenced by RNA interference (RNAi), and the silencing effect was detected by quantitative real-time PCR. After knocking down IGF2BP2, colony formation assay, CCK-8 assay and 5-ethynyl-2'-deoxyuridine (EdU) assay were employed to detect cell colony formation and proliferation ability. Transwell^TM assay was used to detect cell migration ability. Quantitative real-time PCR was used to detect the mRNA expression of IGF2BP2, MYC, tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β) and interleukin-10 (IL-10). The protein expression of IGF2BP2 and MYC was detected by western blot. The binding ability of IGF2BP2 and MYC in HCT-116 cells was detected by quantitative real-time PCR after RNA immunoprecipitation. Results The results of TCGA database showed that the expression of IGF2BP2 and MYC in colorectal cancer tissues was significantly higher than that in adjacent tissues, and the survival time of colorectal cancer patients with high expression of IGF2BP2 was shorter. After silencing IGF2BP2, the viability, proliferation and migration of HCT-116 and SW480 cells were decreased. The mRNA expression of MYC, TGF-β and IL-10 in IGF2BP2 knockdown group was significantly decreased, while the expression of TNF-α mRNA was increased. The expression of MYC protein and the stability of MYC mRNA were significantly decreased. RIP-qPCR results showed that IGF2BP2 could bind to MYC mRNA. Conclusion Knockdown of IGF2BP2 inhibits colorectal cancer cell proliferation, migration and promotes tumor immunity by down-regulating MYC expression.
Humans ; Cell Line, Tumor ; Cell Movement/genetics* ; Cell Proliferation/genetics* ; Colorectal Neoplasms/metabolism* ; Gene Expression Regulation, Neoplastic ; Interleukin-10/metabolism* ; RNA, Messenger ; RNA-Binding Proteins/metabolism* ; Transforming Growth Factor beta/genetics* ; Tumor Microenvironment/immunology* ; Tumor Necrosis Factor-alpha/metabolism* ; Proto-Oncogene Proteins c-myc/metabolism*

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

Dendritic cells (DCs), which are regarded as the most potent antigen-presenting cells, are involved in innate and adaptive immunity. Upon uptake of pathogens, DCs express cell surface markers and secrete cytokines. In this study, we analyzed production of cytokines and found that interleukin (IL)-10 and transforming growth factor (TGF)-beta production significantly increased in bone marrow-derived DCs and a mouse DC line, DC2.4, after treatment with crude antigen (CA) from liver fluke, Clonorchis sinensis. However, expression patterns of several activation molecules did not change. In addition, following treatment of DC2.4 cells with antigen from the lung fluke, Paragonimus westermani, production of IL-10 and TGF-beta significantly increased compared with groups treated with other parasite antigens, Spirometra erinacei plerocercoid CA and Echinococcus granulosus hydatid cystic fluid. We also found that treatment of DC2.4 cells with C. sinensis CA resulted in rapid and significant phosphorylation of extracellular signal-regulated kinase 1/2, a mitogen-activated protein kinase. Following treatment of DC2.4 cells with C. sinensis CA, treatment with an inhibitor specific to an extracellular signal-regulated kinase inhibited production of IL-10 and TGF-beta. Our results suggest that CA from C. sinensis has a role in the anti-inflammatory function of DC cells by inducing IL-10 and TGF-beta through activation of extracellular signal-regulated kinase 1/2.
Animals ; Antigens, Helminth/*pharmacology ; Cells, Cultured ; Clonorchis sinensis/*immunology ; Dendritic Cells/drug effects/*metabolism ; Interleukin-10/genetics/*metabolism ; MAP Kinase Signaling System ; Mice ; Transforming Growth Factor beta/genetics/*metabolism

OBJECTIVETo observe the effect of Huanglian Jiedu IJecoction (HJU) on systemic and vascular immune responses of high fat diet fed apoE deficient (apoE(-/-)) mice.

METHODSEight wild type C57BL6 mice were recruited as the wild type common food group. Totally 24 apoE(-/-) mice were randomly divided into the ApoE'common food group, the ApoE(-/-) hyperlipidemia group, and the ApoE(-/-) hyperlipidemia plus HJD group, 8 in each group. In the present study, the common food mice and high fat fed mice were fed with a chow diet or a high cholesterol diet for 4 weeks. HJD was given to mice in the ApoE(-/-) hyperlipidemia plus HJD group at the daily dose of 5 g/kg by gastrogavage, while equal volume of pure water was given to mice in the rest groups by gastrogavage. Four weeks later, the plasma levels of blood lipids, the ratio of peripheral blood mononuclear cells, and expressions of Toll-like receptor 4 (TLR-4) and CD36 on the monocytes were detected. The pathological changes and expressions of cytokines in local aorta were detected. The plasma cytokine levels in response to lipopolysaccharide (LPS) were analyzed. Results (1) Compared with the wild type common food group, TO, TG, and LDL-O significantly increased in the ApoE(-/-) common food group (P < 0. 05, P < 0.01). Compared with the ApoE(-/-) common food group, TC and LDL-C significantly increased in the hyperlipidemia group (P < 0. 05). There was no statistical difference in each index between the ApoE(-/-) hyperlipidemia group and the ApoE(-/-) hyperlipidemia plus HJD group (P > 0.05). (2) Compared with the wild type common food group, no obvious change of the ratio of peripheral blood mononuclear cells happened, the TLR4 expression level significantly increased in the ApoE'common food group (P < 0. 05). Compared with the ApoE common food group, the ratio of peripheral blood mononuclear cells and the TLR4 expression level significantly increased in the ApoE' hyperlipidemia group (P < 0.05). Compared with the ApoE(-/-) hyperlipidemia group, the ratio of peripheral blood mononuclear cells and the TLR4 expression level significantly decreased. Besides, the CD36 expression level also significantly decreased (P<0.05). (3) After stimulated by LPS for 3 h, compared with the wild type common food group, plasma TNF-ct and IL-b expressions significantly increased in the ApoE(-/-) common food group (P < 0.05). Compared with the ApoE(-/-) common food group, plasma expressions of IL-12, TNF-alpha, MCP-1, and IL-10 increased, but with no statistical difference in the ApoE(-/-) hyperlipidemia group (P > 0.05). After 4-week intervention of HJD, compared with the ApoE(-/-) hyperlipidemia group, the MCP-1 expression was significantly down-regulated, while the IL-10 expression significantly increased, showing statistical difference (P < 0.05). Compared with the wild type common food group, mRNA expression levels of IFN-gamma, MCP-1 , TNF-alpha, IL-10, and IL-1beta significantly increased (P < 0. 05, P < 0.01). Compared with the ApoE(-/-) common food group, not only mRNA expression levels of IFN-gamma, MCP-1, TNF-alpha, and IL-1beta, further significantly increased, but also IL-12, IL-10, and TGF-beta significantly increased (P < 0. 05, P < 0. 01). After 4-week intervention of HJD, compared with the ApoE(-/-) hyperlipidemia group, mRNA expression levels of MCP-1, TNF-alpha, IL-1beta, and IL-12 significantly decreased in the ApoE(-/-) hyperlipidemia plus HJD group (P < 0.05, P < 0.01).

CONCLUSIONSHigh fat diet induced systemic reaction and inflammatory reactions of local vessels. The local inflammatory response of vessels exceeded systemic inflammatory response. Intervention of HJD could attenuate inflammatory response, especially in local arteries. Meanwhile, it enhanced systemic anti-inflammatory reactions.

Animals ; Aorta ; pathology ; Apolipoproteins E ; genetics ; CD36 Antigens ; metabolism ; Chemokine CCL2 ; metabolism ; Dietary Fats ; adverse effects ; Drugs, Chinese Herbal ; pharmacology ; Female ; Hyperlipidemias ; blood ; etiology ; immunology ; Inflammation ; Interleukin-10 ; blood ; Interleukin-12 ; blood ; Interleukin-1beta ; blood ; Leukocytes, Mononuclear ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Systemic Inflammatory Response Syndrome ; blood ; etiology ; immunology ; Toll-Like Receptor 4 ; metabolism ; Transforming Growth Factor beta ; blood ; Tumor Necrosis Factor-alpha ; blood

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

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

Transforming growth factor-beta (TGF-beta) and its receptors have been suggested to play key roles in the pathogenesis of asthma. The aim of this study was to evaluate the effects of genetic variations in the TGF-beta receptor type III (TGFBR3) on asthma and on its related phenotypes in the general population. A cohort of 2,118 subjects aged from 10 to 18 years responded to a questionnaire concerning asthma symptoms and risk factors. Methacholine airway hyperresponsiveness (AHR), skin test responses to common aeroallergens, and serum total IgE levels were evaluated in the cohort. A total of 19 SNPs for TGFBR3 were found using direct re-sequencing in 24 healthy adults. Of these, informative SNPs [+44T>C (S15F) and +2753G>A at 3'UTR] were selected and scored using the high throughput single base extension method. Atopy was identified in subjects with 44T>C allele [P = 0.04, OR (95% CI) = 0.79 (0.62-0.99)] and in subjects with Ht1 (CG) more frequently than in subjects with other haplotypes [P = 0.04, OR (95% CI) = 1.27 (1.01-1.59)]. The A allele in 2753G>A was more common in subjects with non-atopic asthma [OR (95% CI) = 1.76 (1.01-3.05)]. A significant association was found between non-atopic asthma and 44T_2753A [OR (95% CI) = 2.16 (1.22-3.82)]. Genetic variations in TGFBR3 appear to be associated with a genetic predisposition to development of asthma and to phenotypes of asthma. Also, the minor allele 2753G and the haplotype TA in the TGFBR3 gene were associated with a pathogenesis of non-atopic asthma.
Adolescent ; Adult ; Asian Continental Ancestry Group/*genetics ; Asthma/ethnology/*genetics/immunology ; Case-Control Studies ; Child ; Cohort Studies ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; *Genetic Variation/physiology ; Genetics, Population ; Genome-Wide Association Study ; Genotype ; Humans ; Immunoglobulin E/immunology ; Linkage Disequilibrium ; Male ; Proteoglycans/*genetics ; Receptors, Transforming Growth Factor beta/*genetics

OBJECTIVETo investigate the antitumor effect of natural killer (NK) cells on human colorectal cancer cells HT-29 in vitro by blocking transforming growth factor-&beta; (TGF-&beta;) signaling in NK cells transfected with vector containing dominant negative TGF-&beta; type 2 receptor (DNT&beta;R2).

METHODSTGF-&beta;1 was added at the final concentration of 10 ng/ml for HT-29 cells. Primary NK cells were transfected with recombinant plasmid pIRES2-AcGFP-DNT&beta;R2 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-DNT&beta;R2 and 35.28% for the control plasmid pIRES2-AcGFP. The expression of DNT&beta;R2 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-&beta;1 than that without TGF-&beta;1 (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 DNT&beta;R2 vector was significantly higher than that with control vector against HT-29 cells cultured with 10 ng/ml TGF-&beta;1 (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 DNT&beta;R2 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 whether the CD4+CD25+Foxp3+ regulatory T cells are associated with serum TGF beta 1 in patients with hepatitis B.

METHODSPatients with chronic hepatitis B (CHB), chronic asymptomatic carriers (AsC), normal subjects (NS) and the resolved from HBV infection (Resolved) were recruited in this study. Flow cytometric analysis was used to detect the frequency and phenotype of peripheral CD4+CD25+Foxp3+ T cells, and Foxp3 gene expression were examined by real time PCR. Serum TGF beta 1 levels were measured by ELISA (enzyme-linked immunosorbent assay).

RESULTSPatients with CHB or AsC exhibited significantly higher frequency of CD4+CD25+Foxp3+ T cells compared to healthy controls. CD4+CD25+ T cells derived from patients with CHB and AsC expressed higher level of Foxp3-mRNA. Furthermore, the frequency of CD4+CD25+Foxp3+ regulatory T cells was correlated with serum HBV DNA copy numbers in patients with CHB and AsC. Our results indicated that the serum TGF beta was increased in CHB and AsC patients compared to control patients, and that serum TGF beta was correlated with the expression of Foxp3-mRNA and the frequency of CD4+CD25+Foxp3+ regulatory T cells in patients with CHB and AsC.

CONCLUSIONSThe findings have important implication in the understanding of the role and mechanism of aberrant CD4+CD25+Foxp3+ regulatory T cells in the maintenance of chronicity in hepatitis B patients.

Adolescent ; Adult ; CD4 Antigens ; immunology ; metabolism ; Carrier State ; blood ; immunology ; Female ; Flow Cytometry ; Forkhead Transcription Factors ; immunology ; metabolism ; Hepatitis B Surface Antigens ; blood ; immunology ; Hepatitis B, Chronic ; blood ; immunology ; Humans ; Interleukin-2 Receptor alpha Subunit ; immunology ; metabolism ; Male ; Phenotype ; Polymerase Chain Reaction ; RNA, Messenger ; genetics ; metabolism ; T-Lymphocytes, Regulatory ; immunology ; virology ; Transforming Growth Factor beta ; blood ; Viral Load ; Young Adult

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