Helix Transcription Factor Research Articles

Bmp4 regulates chick Ebf2 and Ebf3 gene expression in somite development

The chick Early B-cell Factor-2 and 3 (cEbf2 and cEbf3) genes are members of EBF family of helix loop helix transcription factors. The expression, regulation and importance of these genes have been extensively studied in lymphatic, nervous and muscular tissues. Recently, a new role for some members of EBF in bone development has been investigated. However, the expression profile and regulation in the axial skeleton precursor, the somite, have yet to be elucidated. Therefore, this study was aimed to investigate the expression and regulation of cEbf2 and cEbf3 genes in the developing chick embryo somite from HH4 to HH28. The spatiotemporal expression study revealed predominant localization of cEbf2 and cEbf3 in the lateral sclerotomal domains and later around vertebral cartilage anlagen of the arch and the proximal rib. Subsequently, microsurgeries, ectopic gene expression experiments were performed to analyze which tissues and factors regulate cEbf2 and cEbf3 expression. Lateral barriers experiments indicated the necessity for lateral signal(s) in the regulation of cEbf2 and cEbf3 genes. Results from tissue manipulations and ectopic gene expression experiments indicate that lateral plate-derived Bmp4 signals are necessary for the initiation and maintenance of cEbf2 and cEbf3 genes in somites. In conclusion, cEbf2 and cEbf3 genes are considered as lateral sclerotome markers which their expression is regulated by Bmp4 signals from the lateral plate mesoderm.

The Winged Helix Transcription Factor Foxa3 Regulates Adipocyte Differentiation and Depot-Selective Fat Tissue Expansion

Conversion of mesenchymal stem cells into terminally differentiated adipocytes progresses sequentially through regulated transcriptional steps. While it is clear that the late phases of adipocyte maturation are governed by the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ), less is known about the transcriptional control of the initial stages of differentiation. To identify early regulators, we performed a small interfering RNA (siRNA) screen of Forkhead-box genes in adipocytes and show here for the first time that the winged helix factor Foxa3 promotes adipocyte differentiation by cooperating with C/EBPβ and -δ to transcriptionally induce PPARγ expression. Furthermore, we demonstrate that mice with genetic ablation of Foxa3 have a selective decrease in epididymal fat depot and a cell-autonomous defect to induce PPARγ specifically in their visceral adipocytes. In obese subjects, FOXA3 is differentially expressed in visceral and subcutaneous adipose depots. Overall, our study implicates Foxa3 in the regulation of adipocyte differentiation and depot-selective adipose tissue expansion.

Unraveling the Role of FOXQ1 in Colorectal Cancer Metastasis

Malignant cell transformation, invasion, and metastasis are dependent on the coordinated rewiring of gene expression. A major component in the scaffold of these reprogramming events is one in which epithelial cells lose intercellular connections and polarity to adopt a more motile mesenchymal phenotype, which is largely supported by a robust transcriptional machinery consisting mostly of developmental transcription factors. This study demonstrates that the winged helix transcription factor, FOXQ1, contributes to this rewiring process, in part by directly modulating the transcription of TWIST1, itself a key mediator of metastasis that transcriptionally regulates the expression of important molecules involved in epithelial-to-mesenchymal transition. Forced expression and RNA-mediated silencing of FOXQ1 led to enhanced and suppressed mRNA and protein levels of TWIST1, respectively. Mechanistically, FOXQ1 enhanced the reporter activity of TWIST1 and directly interacted with its promoter. Furthermore, enhanced expression of FOXQ1 resulted in increased migration and invasion in colorectal cancer cell lines, whereas knockdown studies showed the opposite effect. Moreover, using the in vivo chicken chorioallantoic membrane metastasis assay model, FOXQ1 significantly enhanced distant metastasis with minimal effects on tumor growth. These findings reveal FOXQ1 as a modulator of TWIST1-mediated metastatic phenotypes and support its potential as a biomarker of metastasis.

Characterization of the transcriptional activity of the basic helix–loop–helix (bHLH) transcription factor Atoh8

The atonal-related Neurogenin/NeuroD family of basic helix–loop–helix (bHLH) transcription factors comprises potent inducers of neuronal and endocrine differentiation programs in the nervous and digestive system. Atonal homolog 8 (Atoh8) displays high similarity in the bHLH domain with NeuroD proteins. Yet, available evidences indicate that Atoh8 has distinctive features including a ubiquitous expression pattern in embryonic tissues and the ability to inhibit differentiation. To gain insights into Atoh8 function, we aimed at identifying Atoh8 targets and investigated the effects of Atoh8 on global gene expression patterns in pancreatic mPAC cells, a model of bHLH-dependent endocrine differentiation. Our data reveal that Atoh8 is a weak transcriptional activator and does not exhibit proendocrine activity. Conversely, it blocks the induction of a reduced group of gene targets of the atonal-related proendocrine factor Neurogenin3. We show that Atoh8 lacks a transactivation domain and possesses intrinsic repressor activity that depends on a conserved Proline-rich domain. Atoh8 binds the ubiquitous E protein E47 and its ability to repress transcription may partly result from its ability to inhibit E47/E47 and Neurogenin3/E47 dimer activities. These results reveal distinctive transcriptional properties of Atoh8 within the atonal-related bHLH family that may be associated with the acquisition of new biological functions.

Multisite Light-Induced Phosphorylation of the Transcription Factor PIF3 Is Necessary for Both Its Rapid Degradation and Concomitant Negative Feedback Modulation of Photoreceptor phyB Levels in Arabidopsis

Plants constantly monitor informational light signals using sensory photoreceptors, which include the phytochrome (phy) family (phyA to phyE), and adjust their growth and development accordingly. Following light-induced nuclear translocation, photoactivated phy molecules bind to and induce rapid phosphorylation and degradation of phy-interacting basic Helix Loop Helix (bHLH) transcription factors (PIFs), such as PIF3, thereby regulating the expression of target genes. However, the mechanisms underlying the signal-relay process are still not fully understood. Here, using mass spectrometry, we identify multiple, in vivo, light-induced Ser/Thr phosphorylation sites in PIF3. Using transgenic expression of site-directed mutants of PIF3, we provide evidence that a set of these phosphorylation events acts collectively to trigger rapid degradation of the PIF3 protein in response to initial exposure of dark-grown seedlings to light. In addition, we show that phyB-induced PIF3 phosphorylation is also required for the known negative feedback modulation of phyB levels in prolonged light, potentially through codegradation of phyB and PIF3. This mutually regulatory intermolecular transaction thus provides a mechanism with the dual capacity to promote early, graded, or threshold regulation of the primary, PIF3-controlled transcriptional network in response to initial light exposure, and later, to attenuate global sensitivity to the light signal through reductions in photoreceptor levels upon prolonged exposure.

Abstract A34: Nucleosome dynamics of cell differentiation

Abstract Nucleosome occupancy is fundamental for establishing chromatin architecture. However, little is known about the relationship between nucleosome dynamics and initial cell lineage specification. Here, we determine the mechanisms that control global nucleosome dynamics during embryonic stem (ES) cell differentiation into endoderm. Both nucleosome depletion and de novo occupation occur during the differentiation process, with higher overall nucleosome density after differentiation. The variant histone H2A.Z and the winged helix transcription factor Foxa2 both act to regulate nucleosome depletion and gene activation, thus promoting ES cell differentiation, whereas DNA methylation promotes nucleosome occupation and suppresses gene expression. Nucleosome depletion during ES cell differentiation is dependent on Nap1l1-coupled SWI/SNF and INO80 chromatin remodeling complexes. Thus, both epigenetic and genetic regulators cooperate to control nucleosome dynamics during ES cell fate decisions. Currently, we determine whether chromatin remodeling complexes are involved in nucleosome occupation. Citation Format: Zhaoyu Li. Nucleosome dynamics of cell differentiation. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A34.

Immunomodulatory effects of polysaccharopeptide in immunosuppressed mice induced by cyclophosphamide

Polysaccharopeptide (PSP) is well known for its immunoregulatory effects. In the present study, the effect of PSP on white blood cell (WBC) count, T lymphocyte subsets, B lymphocytes, Th1/Th2 balance and negative immune regulators was investigated using an immunosuppressed mouse model. The results demonstrated that the WBC count and the absolute number of CD3+CD4+ T cells, CD3+CD8+ T cells and CD3‑CD19+ B cells in the peripheral blood were increased in PSP‑treated groups as compared with the cyclophosphamide (Cy) group. In addition, PSP reduced interleukin (IL)‑4 and GATA binding protein 3 (GATA‑3) mRNA relative expression levels and elevated the ratios of IL‑2/IL‑4 and the transcription factors, T‑box‑containing protein/GATA‑3. The relative mRNA expression levels of the forkhead/winged‑helix transcription factor box protein 3 (Foxp3), programmed death‑1 (PD‑1) and IL‑10 were also downregulated by PSP. These observations indicate that the immunoregulatory effects of PSP are associated with restoration of WBC number, improving the absolute number of T lymphocyte subsets and B lymphocytes, inducing the Th1/Th2 response and downregulating the negative immune regulators, Foxp3, PD‑1 and IL‑10.

The bHLH transcription factor hand is required for proper wing heart formation in Drosophila

The Hand basic helix–loop–helix transcription factors play an important role in the specification and patterning of various tissues in vertebrates and invertebrates. Here, we have investigated the function of Hand in the development of the Drosophila wing hearts which consist of somatic muscle cells as well as a mesodermally derived epithelium. We found that Hand is essential in both tissues for proper organ formation. Loss of Hand leads to a reduced number of cells in the mature organ and loss of wing heart functionality. In wing heart muscles Hand is required for the correct positioning of attachment sites, the parallel alignment of muscle cells, and the proper orientation of myofibrils. At the protein level, α-Spectrin and Dystroglycan are misdistributed suggesting a defect in the costameric network. Hand is also required for proper differentiation of the wing heart epithelium. Additionally, the handC-GFP reporter line is not active in the mutant suggesting an autoregulatory role of Hand in wing hearts. Finally, in a candidate-based RNAi mediated knock-down approach we identified Daughterless and Nautilus as potential dimerization partners of Hand in wing hearts.

Changes of transcription factors Bcl-6, Foxp3 and RORγt in CD4(+) cells in bone marrow of immuno-related hematocytopenia

To evaluate the possible mechanism of transcription factors B cell lymphoma 6 (Bcl-6) , forkhead/winged helix transcription factor 3 (Foxp3) and retinoic acid related orphan receptor (RORγt) in CD4(+) T cells for immuno-related hematocytopenia (IRH). CD4(+) T cells were harvested from 40 IRH patients, 38 aplastic anemia subjects and 25 normal controls and separated by magnetic activated cell sorting (MACS). Then the expressions of transcription factors of Foxp3, RORγ and Bcl-6 in CD4(+) T cells were measured by real time fluorescent quantitative-polymerase chain reaction (QRT-PCR). Auto-antibody was detected on CD34(+) cells (67.5% (27/40) ), CD15(+) cells (65.0% (26/40)), GlyA(+) cells (75.0% (30/40) ), auto-antibody involving three, two or one myeloid cell were detected in 27.5% (11/40), 52.5% (21/40), 20.0% (8/40) of IRH patients. Compensatory increase of Foxp3 mRNA was found in IRH (0.124 (0.073-0.198) vs 0.071 (0.046-0.118), P < 0.05). The expression of Bcl-6 was higher (2.243 (0.854-4.544) vs 1.211 (0.131-2.816), P < 0.05). Compared to aplastic anemia, the expression of RORγt was lower in IRH (0.133 (0.068-0.189) vs 0.290 (0.138-0.480), P < 0.01) and the ratio of Treg/Th17 shifted to Th17 in patients with aplastic anemia (Foxp3/RORγt ratio,0.500 (0.240-0.795) vs 0.975 (0.483-1.416), P < 0.01). As one kind of bone marrow failures caused by autoantibody to bone marrow cells, IRH may occur due to a high expression of Bcl-6 in CD4(+) T cells, its immunopathogenesis is different from that of aplastic anemia.

Foxa1 and Foxa2 Are Required for the Maintenance of Dopaminergic Properties in Ventral Midbrain Neurons at Late Embryonic Stages

The maintained expression of transcription factors throughout the development of mesodiencephalic dopaminergic (mDA) neurons suggests multiple roles at various stages in development. Two members of the forkhead/winged helix transcription factor family, Foxa1 and Foxa2, have been recently shown to have an important influence in the early development of mDA neurons. Here we present data demonstrating that these genes are also involved in the later maintenance of the mDA system. We conditionally removed both genes in postmitotic mDA neurons using the dopamine transporter-cre mouse. Deletion of both Foxa1 and Foxa2 resulted in a significant reduction in the number of tyrosine hydroxylase (TH)-positive mDA neurons. The decrease was predominantly observed in the substantia nigra region of the mDA system, which led to a loss of TH+ fibers innervating the striatum. Further analysis demonstrated that the reduction in the number of TH+ cells in the mutant mice was not due to apoptosis or cell-fate change. Using reporter mouse lines, we found that the mDA neurons were still present in the ventral midbrain, but that they had lost much of their dopaminergic phenotype. The majority of these neurons remained in the ventral mesencephalon until at least 18 months of age. Chromatin immunoprecipitation suggested that the loss of the mDA phenotype is due to a reduction in the binding of the nuclear orphan receptor, Nurr-1 to the promoter region of TH. These results extend previous findings and demonstrate a later role for Foxa genes in regulating the maintenance of dopaminergic phenotype in mDA neurons.

Immunoregulatory effects of Glycyrrhizic acid exerts anti-asthmatic effects via modulation of Th1/Th2 cytokines and enhancement of CD4+CD25+Foxp3+ regulatory T cells in ovalbumin-sensitized mice

Ethnopharmacological relevanceGlycyrrhizic acid (GA) is the main bioactive ingredient of licorice (Glycyrrhiza glabra), and has been found to be associated with multiple therapeutic properties. Aim of the studyIn this study, we investigated immunoregulatory effects of glycyrrhizic acid on anti-asthmatic effects and underlying mechanisms. Materials and methodsAsthma model was established by ovalbumin-induced. A total of 60 mice were randomly assigned to six experimental groups: control, model, dexamethasone (2mg/kg) and GA (10mg/kg, 20mg/kg, 40mg/kg). Airway resistance (Raw) were measured by the forced oscillation technique, histological studies were evaluated by The hematoxylin and eosin (HE) staining, Th1/Th2 and Th17 cytokines were evaluated by enzyme-linked immunosorbent assay (ELISA), and CD4+CD25+Foxp3+ regulatory T cells (Tregs) was evaluated by Flow Cytometry (FCM), the forkhead/winged helix transcription factor (Foxp3) was evaluated by western blotting. ResultsOur study demonstrated that, compared with model group, GA inhibited OVA-induced increases in Raw and eosinophil count; interleukin (IL)-4, IL-5, IL-13 levels were recovered in bronchoalveolar lavage fluid compared; increased IFN-γ level in bronchoalveolar lavage fluid; histological studies demonstrated that GA substantially inhibited OVA-induced eosinophilia in lung tissue and airway tissue compared with model group. Flow cytometry studies demonstrated that GA substantially enhanced Tregs compared with model group. ConclusionThese findings suggest that GA may effectively ameliorate the progression of asthma and could be used as a therapy for patients with allergic asthma.

Abstract 758: Studies in cancer models with AGX51, a Direct Transcriptional RegulatorTM.

Abstract Studies in cancer models with AGX51, a Direct Transcriptional RegulatorTM The inhibitors of differentiation (Id) proteins (Id1, Id2, Id3, and Id4) are negative regulators of differentiation that act by sequestering basic helix loop helix (bHLH, e.g. E47) transcription factors. Ids are highly expressed during embryonic development but in adult tissues their expression is rare to absent. However, Id proteins are required for tumor angiogenesis, highly expressed in many cancers and as shown in many clinical studies, associated with an aggressive phenotype and a poor clinical outcome As a proof of concept, decreased angiogenesis and tumor load as a result of blocking Id expression by antisense and siRNA molecules demonstrated that Id is a compelling anti-cancer target. Despite these findings, the Ids are a heretofore unexplored target for the treatment of cancer. While no anti-Id agent has been studied clinically or has undergone preclinical development, AGX recently discovered and patented the first unique and potent anti-Id agent, AGX51. This small molecule is an inhibitor of the Id1-E47 interaction and was discovered through a systematic research effort using x-ray crystallography, gel shift (EMSA) assays, Matrigel evaluations and xenograft studies. AGX51 is the founding member of a new class of therapeutics: Direct Transcriptional RegulatorsTM. In preclinical studies: (1) At a dose below 7 mg/kg, bid, AGX51 significantly blocked the growth of tumors in mice with implanted human breast cancer cells when treated briefly with a taxane like Taxol®, paclitaxel, or Taxotere®, docetaxel. This effect of AGX51 was expected based on the discovery that endothelial cell production in the bone marrow is responsible for the rapid repair to damaged vasculature after administration of paclitaxel or vascular disrupters such as ZD6126 or AVE8062, through mobilization of endothelial precursor cells (EPCs) followed by homing of the EPCs to the damaged vasculature; (2) At a dose below 20 nM, AGX51 restored cell cycle control in DU-145 human prostate cancer cells; (3) At low micromolar concentrations, AGX51 blocked migration of DU-145 cancer cells in “scratch” assays; (4) As a single agent, AGX51 completely blocked tumor associated angiogenesis in nude mice implanted with MDA-MB231 human breast cancer cells at a dose of 60 mg/kg, bid; (5) As expected, AGX51 restored levels of p21 and p27, two important mediators of cell cycle regulation in both PC3 and DU145 cancer cells; (6) AGX51 caused no toxicity in multiple day dosing based on weight, clinical chemistry or hematology measurements; (7) AGX51 has a terminal elimination half-life in mice of approximately 6 hours, which is suggestive of ultimate bid and perhaps even qd dosing in man. In summary, a specific inhibitor of Id-E47 interaction that has anti-tumor activity is described for the first time. This research was supported by AngioGenex, Inc. Citation Format: William Garland, Richard Salvador, Jaideep Chaudhary. Studies in cancer models with AGX51, a Direct Transcriptional RegulatorTM. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 758. doi:10.1158/1538-7445.AM2013-758

Abstract 5455: Differential regulation of E2A (TCF3) by androgens in prostate cancer cells.

Abstract Introduction: E2A (TCF3) is a multifunctional basic helix loop helix (bHLH) transcription factor. E2A promotes cell differentiation, acts as a negative regulator of cell proliferation in normal cells and cancer cell lines and is required for normal B-cell development. Previous studies from our laboratory has shown that E2A expression is highly increased in prostate cancer as compared to normal prostate and that it acts as a tumor promoter in prostate cancer. Given the diverse biological pathways regulated/ influenced by E2A little is known about its regulation in prostate cancer. Experimental design: E2A expression in androgen sensitive LNCaP and insensitive C81 prostate cancer cell lines was determined by western blot after treatments with androgen receptor (AR) agonist R1881 and antagonist casodex. Putative Androgen Response Elements (ARE) were identified in the first intronic region of the E2A gene using some of the online bioinformatics tools and confirmed by Chromatin Immunoprecipitation (ChIP) with AR antibody and Luciferase reporter assays on the above mentioned cell lines after treatments with R1881 and casodex. Results: E2A expression was found to increase with the increasing aggrasiveness of prostate cancer cell lines as compared to normal prostate epithelial cell line RWPE1. When androgen responsive cell line LNCaP was treated with R1881 there was an increased expression of E2A which decreased upon treatment with antiandrogen casodex whereas the E2A expression remained unaltered upon similar treatments in androgen insensitive cell line C81. The first intronic region of the E2A gene was predicted to contain two putative ARE sites. ChIP after treatment of LNCaP and C81 cells with R1881 and casodex showed that the intronic region was bound by AR in LNCaP cells only in the presence of R1881, whereas C81 cells showed a pulldown with AR in presence as well as absence of R1881. Similar results were observed in luciferase reporter assays indicating that E2A is transactivated by AR in LNCaP cell lines whereas it is independent of androgens in C81 cell line. Furthermore, Luciferase reporter assays also confirmed that only one of the two predicted putative AREs was functionally active and responsible for the androgen mediated regulation of E2A. Conclusion: Our results indicate that E2A is differentially regulated in Prostate cancer cell lines. The increased expression of E2A and its role as a tumor promoter in prostate cancer cell lines may be contributed to its loss of androgen dependence as is evident in its progression from androgen dependent LNCaP to independent C81 cells. Acknowledgements: This study was supported by NIH/NCI RO1 CA128914 and NIH/NCRR/RCMI G12RR03062. Citation Format: Divya Patel, Jaideep Chaudhary. Differential regulation of E2A (TCF3) by androgens in prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5455. doi:10.1158/1538-7445.AM2013-5455

Abstract 2703: A direct regulation of Twist1 by FOXQ1 promotes colorectal cancer metastasis.

Abstract Abstract Malignant cell transformation, invasion and metastasis are dependent on the coordinated rewiring of gene expression. A major component in the scaffold of these reprogramming events is one in which epithelial cells lose intercellular connections and polarity and adopt a motile mesenchymal phenotype, one that is largely supported by a robust transcriptional machinery consisting mostly of developmental transcription factors. We investigated the winged helix transcription factor, FOXQ1, identified from an oligonucleotide microarray expression analysis, as being concomitantly deregulated in both epithelial and stromal compartments. We found that FOXQ1 contributes to epithelial to mesenchymal transition (EMT), in part by directly affecting the transcription of Twist1, itself a key mediator of metastasis that transcriptionally regulates the expression of important molecules involved in EMT. Reporter gene assays and chromatin immunoprecipitation experiments showed that FOXQ1 regulated and directly interacted with the Twist1 promoter, leading to a suppression of E-cadherin and increased expression of mesenchymal markers. Forced expression and RNAi mediated silencing of FOXQ1 led to increased and decreased levels of Twist1 mRNA and protein levels, respectively, with concomitant effects on migration, invasion and in vivo metastasis of colorectal cancer cells. Moreover, FOXQ1 mRNA and protein expression correlated in a series of resected patient samples. Conclusively our results show that FOXQ1 is a novel mediator of colorectal cancer metastasis and joins the network of transcription factors that orchestrate EMT. Citation Format: Mohammed Abba, Nitin Patil, Kabeer Rasheed, Laura D. Nelson, Giridhar Mudduluru, Jörg H. Leupold, Heike Allgayer. A direct regulation of Twist1 by FOXQ1 promotes colorectal cancer metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2703. doi:10.1158/1538-7445.AM2013-2703

Circulating autoantibody to FOXP3 may be a potential biomarker for esophageal squamous cell carcinoma

The present study was undertaken to develop a relatively quantitative enzyme-linked immunosorbent assay (ELISA) in-house using human leukocyte antigen class II-restricted epitopes in order to test circulating autoantibodies to human forkhead/winged helix transcription factor (FOXP3) as a biomarker for esophageal cancer. A total of 97 patients with esophageal squamous cell carcinoma (ESCC) and 227 healthy subjects were recruited for this study, and their plasma samples were collected for antibody analysis with the ELISA approach. Student's t test showed that the anti-FOXP3 IgG antibody levels were significantly higher in the patient group than the control group (t=6.23, P<0.0001). Based on a cutoff value determined by the mean+3SD of control IgG levels, the positive rate was 5.15 % in patients with ESCC as compared to 0.88 % in control subjects (X (2) =6.53, P=0.019, OR=5.85, 95 % CI 1.12-30.67), in which patients at stage I had the highest positivity (11.54 %, X (2) =12.15, P=0.0005, OR=13.10, 95 % CI 2.09-82.04). The sensitivity against >95 % specificity was 22.7 % for the IgG assay with an inter-assay deviation of 13.35 %. This work suggests that circulating IgG autoantibody to FOXP3 may be a potential biomarker for early diagnosis of esophageal cancer.

Concomitant Increase of OX40 and FOXP3 transcripts in peripheral blood of patients with breast cancer.

Regulatory T cells (T-regs) have an important role in cancer by suppression of protective antitumor immune responses. Regulatory T cells express the forkhead/winged helix transcription factor (FOXP3) and OX40 molecules which have important regulatory roles in the immune system. To evaluate FOXP3 and OX40 transcripts in the peripheral blood mononuclear cells of women with breast cancer. Blood samples from 40 women with histologically-confirmed infiltrating ductal carcinoma of the breast and 40 healthy volunteer women without a history of malignancy or autoimmune disorders were collected. The abundance of FOXP3 and OX40 gene transcripts were determined by quantitative real-time PCR (qRT-PCR). There was a significant positive correlation between FOXP3 and OX40 gene expression in women with breast cancer in a stage dependent manner. This finding emphasizes the importance of T-regs as predominant targets for breast cancer immunotherapy.

Impairment of CD4+ cytotoxic T cells predicts poor survival and high recurrence rates in patients with hepatocellular carcinoma

The role of CD4(+) cytotoxic T cells (CTLs) in hepatocellular carcinoma (HCC) remains obscure. This study characterized CD4(+) CTLs in HCC patients and further elucidated the associations between CD4(+) CTLs and HCC disease progression. In all, 547 HCC patients, 44 chronic hepatitis B (CHB) patients, 86 liver cirrhosis (LC) patients, and 88 healthy individuals were enrolled in the study. CD4(+) CTLs were defined by flow cytometry, immunohistochemistry, and lytic granule exocytosis assays. A multivariate analysis of prognostic factors for overall survival was performed using the Cox proportional hazards model. Circulating and liver-infiltrating CD4(+) CTLs were found to be significantly increased in HCC patients during early stage disease, but decreased in progressive stages of HCC. This loss of CD4(+) CTLs was significantly correlated with high mortality rates and reduced survival time of HCC patients. In addition, the proliferation, degranulation, and production of granzyme A, granzyme B, and perforin of CD4(+) CTLs were inhibited by the increased forkhead/winged helix transcription factor (FoxP3(+) ) regulatory T cells in these HCC patients. Further analysis showed that both circulating and tumor-infiltrating CD4(+) CTLs were independent predictors of disease-free survival and overall survival after the resection of the HCC. The progressive deficit in CD4(+) CTLs induced by increased FoxP3(+) regulatory T cells was correlated with poor survival and high recurrence rates in HCC patients. These data suggest that CD4(+) CTLs may represent both a potential prognostic marker and a therapeutic target for the treatment of HCC.

Regulatory T cells in skin lesions and blood of patients with bullous pemphigoid

Although regulatory T cells (Tregs) are affected in several autoimmune skin diseases, only two studies have been performed in patients with bullous pemphigoid (BP) with contrasting results. To characterize Tregs and to determine the serum levels of regulatory cytokines in patients with BP. In BP lesional skin, immunohistochemistry and confocal microscopy were performed for CD4(+) , CD25(+) , forkhead/winged helix transcription factor (FOXP3)(+) , transforming growth factor (TGF)-β(+) and interleukin (IL)-10(+) cells. In addition, the number of CD4(+) CD25(++) FOXP3(+) Tregs in peripheral blood was assessed by flow cytometry, and the levels of TGF-β and IL-10 were determined in serum samples by enzyme-linked immunosorbent assay before and after steroid therapy. Controls included patients with psoriasis, atopic dermatitis (AD) and healthy donors. The frequency of FOXP3(+) cells was significantly reduced in skin lesions from patients with BP (P<0.001) compared with psoriasis and AD. Moreover, the number of IL-10(+) cells was lower in BP than in psoriasis (P<0.001) and AD (P=0.002), while no differences were observed in the number of TGF-β(+) cells. CD4(+) CD25(++) FOXP3(+) Treg in the peripheral blood of patients with BP was significantly reduced compared with healthy controls (P<0.001), and augmented significantly after steroid therapy (P=0.001). Finally, TGF-β and IL-10 serum levels were similar in patients with BP compared with healthy controls. However, after therapy, BP patients showed significantly higher IL-10 serum levels than before therapy (P=0.01). These data suggest that the depletion of Tregs and of IL-10 in patients with BP may be an important factor in the pathogenesis of the disease.

FOXP3 genetic variant and risk of acute coronary syndrome in Chinese Han population

Coronary artery disease is the most common type of heart disease and a leading cause of morbidity and mortality all over the world. Acute coronary syndrome (ACS) is the most serious form of coronary artery disease. Recently, many studies indicated that genetic susceptibility may play a vital role in the pathogenesis of coronary heart disease including ACS. Forkhead/winged helix transcription factor (FOXP3) gene polymorphisms have been previously found to be associated with inflammatory diseases. To determine whether FOXP3 polymorphisms are associated with ACS, we examined the single nucleotide polymorphism rs3761548 of FOXP3 gene by polymerase chain reaction-polyacrylamide gel electrophoresis in 226 ACS patients and 259 unrelated healthy subjects. Our results showed that single nucleotide polymorphism rs3761548 had association with ACS in Chinese Han population. These data indicate that, for the first time, FOXP3 gene polymorphism may appear to play an important role in the susceptibility of ACS in Chinese Han population.

Significance of expression of forkhead/winged helix transcription factor in HBV-associated glomerulonephrititis

Significance of expression of forkhead/winged helix transcription factor in HBV-associated glomerulonephrititis