High TGF-beta Research Articles

M6A-Related lncRNAs Are Potential Prognostic Biomarkers of Cervical Cancer and Affect Immune Infiltration.

The correlation of m6A-related lncRNAs with the prognosis and immune microenvironment of cervical cancer is not yet clear. In this study, we identified 7 m6A-related prognostic lncRNAs by Pearson correlation and univariate Cox regression analyses based on TCGA-cervical cancer dataset. Then, patients were divided into two clusters by consensus clustering based on the 7 m6A-related prognostic lncRNA expression. Cluster 1 was characterized by survival and stage disadvantage, enrichment of immunosuppressive and carcinogenic activation pathways. Besides, cluster 1 had higher immunosuppressive factor TGFbeta and lower immune cell infiltration compared with cluster 2. According to the expression of 7 m6A-related lncRNA, a 6-m6A-related lncRNA risk score model was established in the training set by LASSO regression analysis. The high-risk group had worse overall survival than the low-risk group. No matter in the training or validation sets, the m6A-related lncRNA risk score was an independent prognostic factor for overall survival. Meanwhile, we validated the independent prognostic value of risk score in the disease-specific survival and progression-free survival by multivariate Cox analysis. The high-risk group was characterized by higher TGFbeta and regulatory T cell and was rich in malignant pathways. Additionally, we also detected and compared the expression levels of four m6A-related prognostic lncRNA in 9 tumor samples and 9 normal tissues using quantitative real-time polymerase chain reaction assay. In conclusion, the novel m6A-related lncRNA risk score is a potential prognostic predictor of cervical cancer patients. These 6 m6A-related lncRNAs might serve as key mediators of the immune microenvironment and represent promising therapeutic targets for improving cervical cancer prognosis.

Abstract 929: Transcriptomic analysis of pre-treatment tissue samples to predict clinical benefit to durvalumab in HIV-infected cancer patients

Abstract Introduction: Treatment with anti PD-1/PD-L1 antibodies has demonstrated clinical activity in different types of solid tumors, but only 20 to 30% of patients (pts) respond to these immune checkpoint inhibitors (ICIs). Therefore, predictive biomarkers of response that can assist in pt selection are urgently needed. Single biomarker expression, like PD-L1, may not provide enough information about cancer cells and the tumor microenviroment. Novel technologies, analyzing hundreds of genes at the same time, are needed to yield better predictive gene signatures of ICI response. The DURVAST trial analyzed the feasibility of durvalumab treatment in HIV-infected cancer pts, which are usually excluded from ICI clinical trials. The trial included 20 patients with different tumor types and yielded a disease control rate of 56%. Methods: Pre-ICI-treatment FFPE tumor tissue samples from 14 HIV-infected cancer pts (including 11 lung, 1 melanoma, 1 anal and 1 bladder cancer) were analyzed using the nCounter NanoString platform with the IO360 panel, including 770 genes involved in tumor biology, microenvironment and immune response. Gene expression results were correlated with clinical benefit (CB) (objective response and stable disease of more than 24 weeks by RECIST1.1 criteria), and compared to other predictive markers. Results: Exploratory analysis of pre-treatment gene expression profiles (GEPs) revealed differentially expressed genes (DEGs) between the pts with- and without CB. Panel-incorporated biological signatures related to tumor and immune activities were evaluated and some of the most DEGs (based on higher log2FC values and nominal p-values ≤0.05) were shown to be involved in cytokine and chemokine signaling. Although not significant, pts without CB tend to have lower expression of genes involved in cytokine and chemokine signaling (p = 0.097). In contrast, pts without CB tended to have a higher TGF beta signature scores (p = 0.318). When combining both signatures, we obtained an aggregated signature score that was significantly different between pts with- and without CB (p = 0.017). While the positive predictive values were the same for all tests, our signature score outperformed PD-L1 expression positivity by immunohistochemistry and PD-L1 RNA expression as predictors for clinical benefit with a two-fold higher sensitivity and negative predictive value. Conclusion: Gene expression analysis of pre-treatment tumor samples revealed distinct GEPs between HIV-infected cancer pts with- and without CB, where combined high baseline recruitment and activation of immune cells by cytokine and chemokine signaling pathways and low immunosuppressive TGF beta signaling pathways predict CB from durvalumab treatment. This predictive score outperformed other predictive markers of CB. These findings need further validation in an external and non-HIV infected pt cohort, in addition to a pt cohort treated with other ICIs. Citation Format: Jillian Wilhelmina Bracht, Maria Gonzalez-Cao, Teresa Moran, Judith Dalmau, Javier Garcia-Corbacho, Reyes Bernabe, Oscar Juan, Javier de Castro, Ana Gimenez-Capitan, Remedios Blanco, Erika Aldeguer, Sonia Rodriguez, Ana Drozdowskyj, Jordi Argilaguet, Julian Blanco, Julia Prado, Christian Brander, Jorge Carrillo, Bonaventura Clotet, Bartomeu Massuti, Mariano Provencio, Chung-Ying Huang, Clara Mayo de las Casas, Monica Garzon, Andres Felipe Cardona, Oscar Arrieta, Andreas Meyerhans, Miguel Angel Molina-Vila, Javier Martinez-Picado, Rafael Rosell. Transcriptomic analysis of pre-treatment tissue samples to predict clinical benefit to durvalumab in HIV-infected cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 929.

Abstract P3-10-18: Elucidating the role of functional signal transduction pathway activity in sensitivity and response of triple negative breast cancer to PI3K inhibition

Abstract Introduction The PI3K signaling pathway is frequently active in triple negative breast cancer (TNBC), but response to PI3K inhibition is still poorly understood. To gain insights in therapy response and resistance, it is important not only to assess genetic alterations of these tumors, but also their phenotypic characteristics. Furthermore, not only is the functional status of the PI3K pathway important to know, but also other signal transduction pathways that may drive tumor growth, as they may explain primary or acquired therapy resistance. Material & Method We analyzed 17 patient derived xenograft (PDX) models of TNBC with varying response to buparlisib treatment. Long-term response (˜30 days) was assessed by comparing growth rates between buparlisib and vehicle treated PDXs. For characterization, mRNA expression analysis was performed on tumor material post 3 days of vehicle or buparlisib treatment of each PDX model. Functional pathway activity was determined using computational Bayesian networks that look at mRNA levels of pathway target genes resulting from activation (Verhaegh et al, Cancer Res 2014), amongst others for the FOXO-PI3K (van Ooijen et al, Am J Pathol, in press), ER, AR, Hedgehog, TGFbeta and Wnt pathways. These computational networks were calibrated on samples with known pathway activity, and biologically validated on various healthy and diseased cell and tissue types. Results On an initial set of 6 PDX models, pathway activity clearly varied between different TNBC PDXs, and between vehicle and buparlisib treatment. ER pathway activity was low in all samples, as expected in TNBC. Two PDX models with the most growth reduction by buparlisib showed high PI3K activity, of which one based on low FOXO activity and the other on oxidative stress. The former, best responding PDX showed a clear reduction in PI3K activity (restoring FOXO activity), when comparing 3 days buparlisib to vehicle treatment. Of the two PDX models with least growth reduction, one had low PI3K activity, while the other one, carrying a PIK3CA mutation, did show high PI3K activity (low FOXO), but this remained after treatment. Other differences in pathway activity that were found, included slightly elevated AR and Wnt activity in one PDX with good response, and somewhat higher TGFbeta activity in four PDXs (good, medium and poor response). Analysis of the remaining 11 PDXs is ongoing, including other signal transduction pathways, to investigate the variation in pathway activity across the entire panel and to shed more light on the differences in tumor biology between the PDX models. Conclusion Our computational Bayesian networks measured differences in functional activity of signal transduction pathways across a collection of TNBC PDX models, which may be expected due to the large variation between TNBC tumors. Functional PI3K activity was related to growth inhibition by buparlisib treatment, and reduction of PI3K pathway activity was observed upon treatment in responding PDX models. Other pathways also showed variation across PDXs. Further clinical evaluation of our signal transduction pathway activity measurement is ongoing. RT-qPCR based analysis is available, optimized for FFPE tissue and small samples. Citation Format: Guo Z, Verhaegh W, Hoog J, Rajput S, van Strijp D, van de Stolpe A, Li S, Ma C. Elucidating the role of functional signal transduction pathway activity in sensitivity and response of triple negative breast cancer to PI3K inhibition [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-10-18.

Inhibiting TGF-Beta Signaling in the Tumor Micro-Environment Enhances the Cytotoxic Function of Adoptive NK Cells

Introduction: The tumor micro-environment poses a limitation to the efficacy of adoptive NK cell therapy due to several immunosuppressive cytokines. TGF-beta, produced in excess by tumor cells, regulatory T cells and stromal cells, facilitates epithelial to mesenchymal transformation thereby promoting metastasis and fibrosis. This high TGF-beta milieu in cancer patients also impairs innate natural killer (NK) cell mediated cancer immunity through various mechanisms.Adoptive transfer of healthy donor NK cells will have limited clinical efficacy when highly activated NK cells are introduced into the immunosuppressive TGF-beta rich tumor microenvironment of cancer patients. We are testing several small molecule inhibitors of the TGF-beta receptor in combination with healthy donor NK cells with a goal of enhancing adoptive NK cell therapy in various disease models.Methods: Human NK cells isolated from healthy donor peripheral blood were expanded over a 21-day period in co-culture with irradiated K562 cells genetically modified to express membrane-bound IL-21. (Somanchi et al. 2011 JoVE 48. doi: 10.3791/2540). Cell culture media was supplemented with IL-2 (50mU/mL).For in vitro assays, at the beginning of expansion week 3 the culture conditions for NK cells was continued unchanged, supplemented with TGF-beta 1 ligand (TGF-B1) at 5ng/mL or 10ng/mL either alone or in combination with EW7197 (a TGF-beta Type 1 receptor inhibitor) or with EW7197 alone. These NK cells were tested with Calcein-AM release cytotoxicity assays at various time points from addition of TGF-B1 (0h, 36h, 72h and 96h). Briefly, NK cells were co-cultured with OCI-AML cells labeled with Calcein-AM at a ratio of 5NK:1 OCI-AML. At the end of 4 hours co-incubation, cytotoxicity was measured by relative fluorescence of calcein release into the culture supernatant compared with Triton-X induced complete target cell lysis.In a murine liver metastases model using the colon cancer cell line HCT116, 105 HCT116 cells were surgically implanted into NSG mouse spleens (following hemi-splenectomy). Mice in 2 groups (3/group) subsequently received 5 x 106 NK cells each (tail vein), weekly for two weeks, starting 10 days post-op. Another control group received vehicle infusions alone while a fourth control group received the TGF-beta inhibitor LY2157299 by oral gavage twice daily at 75mg/kg for two weeks. One group of mice receiving NK cells also received twice daily LY2157299 for two weeks starting with the first NK cell infusion. Mice receiving NK cells also received IL2 (75,000U IP) three times a week for two weeks.Results: At TGF-beta levels similar to that found in AML patients (5ng/ml), NK cell killing of OCI cells was markedly impaired progressively from 36h to 96h exposure. This reduced cytotoxic activity correlated with a 3-4 fold decrease in expression of NKG2D most marked at 96h(Figure 1). CD16 expression was also significantly reduced by 2-3 fold on TGF-beta exposed NK cells after 96h. The TGF-beta inhibitor EW7197 maintained NK cell killing as well as NKG2D and CD16 expression in the presence of TGF-B1 (Figure 1).At 32 days post-splenic implantation, vehicle mice (those that received HCT116 either alone or in combination with LY2157299 alone) appeared moribund and had grossly distended abdomens with ascites. All mice were autopsied at this time point. All control mice had grossly enlarged livers with significant metastases and absence of viable liver tissue (Fig 2). In contrast, mice treated with two weekly infusions of NK cells and LY2157299 for 4 weeks had no ascites and predominantly healthy livers. All mice that received NK cells alone had some evidence of liver metastases with various degrees of disease burden. Composite H&E stains of liver sections from 3 mice/group showed <10% liver metastases microscopically in mice treated with a combination of NK cells and LY2157299. This was significantly reduced compared to 80-90% in the untreated or LY2157299 alone mice and 60-70% in the NK cell alone group (Fig 3).Discussion: Our preliminary results indicate that TGF-beta inhibition in combination with adoptive NK cell therapy can mitigate the immunosuppressive tumor microenvironment conferred by TGF-beta signaling. We are in the process of validating this approach in other preclinical models of both hematologic malignancies and solid tumors. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Immunostaining Protocol: P-Smad2 (Xenograft and Mice)

Metastasis depends on a gene program expressed by the tumor microenvironment upon TGF-beta stimulation. CRC (Colorectal cancer) cell lines did not induce robust stromal TGF-beta responses when injected into nude mice as shown by lack of p-SMAD2 accumulation in tumor-associated stromal cells. To enforce high TGF-beta signaling in xenografts, we engineered CRC cell lines to secrete active TGF-beta. Subcutaneous tumors obtained from HT29-M6TGF-β, KM12L4aTGF-β cells and SW48TGF-β cells contained abundant p-SMAD2+ stromal cells.

TGF-beta1 is associated with the progression of intracranial deep white matter lesions: a pilot study with 5 years of magnetic resonance imaging follow-up

Objectives: Elevated expression of transforming growth factor (TGF)-beta1 has been reported in hereditary cerebral small-vessel (HCSV) disease. The aim of this study was to clarify whether TGF-beta1 is a risk factor for intracranial deep white matter lesions (DWLs) and their progression in a general elderly population.Methods: The subjects included 81 participants (Groups DWL, DWLP, and C) who had voluntarily undergone a health examination and brain magnetic resonance imaging (MRI) in 2003 and 2008 and 43 age-matched patients with previous symptomatic brain infarctions. Deep white matter lesions were graded from Grade 0 to 3 according to the Fazekas classification. Group DWL (23 subjects) was defined as DWLs with no progression in the grade level, and Group DWLP (progression of DWL) (12 subjects) was defined as DWLs with an increase in one or more grade number and an apparent worsening of Grade 3.Forty-six age-matched control subjects with consistent normal brain MRI were included in Group C. The associations between DWLs and various vascular risk factors, including peripheral blood TGF-beta1 levels, were examined.Results: In addition to the classical risk factors, the highest TGF-beta1 levels were found in Group DWLP. The TGF-beta1 levels were significantly higher in Group DWLP than in Group DWL, and DWLP was significantly correlated with elevated TGF-beta1 levels (odds ratio [OR] = 1·72).Conclusions: The present data suggest that TGF-beta1 may be important in the pathogenesis and progression of DWLs, and it is expected to be useful as a clinical indicator reflecting the presence of intracranial white matter lesions.

AB0094 Association of TGF-beta1 - 509c/t polymorphism and TGF-beta1 serum levels with systemic lupus erythematosus

BackgroundRecent evidences suggest a role of transforming growth factor-beta1 (TGF-beta1) in preventing autoimmune diseases, such as systemic lupus erythematosus (SLE).ObjectivesThe aim of this study was to investigate the relationship of...

272 MIR-338-3P IS DOWN-REGULATED BY HEPATITIS B VIRUS X AND INHIBITS CELL PROLIFERATION BY TARGETING CYCLIND1 MAINLY AT 2397-2403NT OF CYCLIND1 3 UTR

epithelial-to-mesenchymal transition (EMT), which converts it in a tumor promoter [2]. TGF-beta in culture induces apoptosisresistant hepatocytes to undergo EMT [3], coincident with the acquisition of stem cell properties [4]. The aim of this work was to assess and compare expression of well-known stem markers in hepatocellular carcinoma cell lines with different epithelial/mesenchymal phenotype and autocrine expression of TGF-beta. Methods: In vitro cell culture of HepG2, Hep3B, PLC-PRF-5, SNU449. In vivomodels of subcutaneous implanted Hep3B and HepG2 cells. Results: Using immunofluorescence and flow cytometry, we found closely inexistent expression of the stem-markers Thy-1 and c-Kit, but we identified a noteworthy pattern for EpCAM and CD44 in differently aggressive cell lines. This expression patterns were also found in tumor samples obtained from xenografts by immunohistochemistry. CD44 expression, a marker linked to metastatic spreading [5] was only present in Snu449 cell line, which showed, among the used lines, the highest TGF-beta expression levels and the most mesenchymal profile, concordant with a late TGF-beta signature [6]. Pericellular EpCAM was found in all other cell lines, coincident with a more epithelial phenotype and an early TGF-beta signature. No expression was found in Snu449 cells. Although EpCAM is missing in differentiated hepatocytes, its presence in progenitor cells during regeneration is linked to a hepatic lineage commitment process. Furthermore, its cleavage is necessary for its stem-related gene promotion [7]. Conclusion: CD44/EpCAM phenotype is an interesting trait to look for in HCC, in order to correlate its presence with a dedifferentiated and invasive phenotype. Acknowledgements: Grants: MCINN, Spain (BFU2009-07219 and ISCIII-RTICC: RD06/0020) and AGAUR-Generalitat de Catalunya (2009SGR-312). ISCIII-RTICC “Short-stay” grant was fundamental for cooperating with University of Extremadura.

The kinetics of mRNA transforming growth factor beta1 expression and its serum concentration in graft-versus-host disease after allogeneic hemopoietic stem cell transplantation for myeloid leukemias

SummaryBackgroundGraft-versus-host disease (GVHD) is still a major complication following allogeneic hematopoietic stem cell transplantation (alloHSCT). Recent data indicates that transforming growth factor beta1 (TGF-β1) may play a role in development of GVH reaction.Material/MethodsForty patients with acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) were included. Quantitative real time polymerase chain reaction (RT-qPCR) was performed to assess the expression of mRNA TGF-β1. TGF-β1 serum concentration was assessed using a commercial ELISA.ResultsIn all patients, a prompt decrease in TGF-β1 mRNA expression and its serum concentration was demonstrated after conditioning. In patients with acute GVHD, TGF-β1 mRNA expression and its serum concentration remained low until day +30 after transplant as compared to the day of transplant (p<0.03 and p<0.006, respectively). TGF-β1 mRNA expression and its serum concentration significantly increased on day +100 in patients who developed chronic GVHD as compared to the day of transplant (p<0.0009 and p<0.02, respectively).ConclusionsTGF-β1 seems to be an additional regulator of donor engraftment; its low levels probably being one of the factors contributing to the development of acute GVHD. On the other hand, chronic GVHD symptoms seem to correlate with high TGF-β1 mRNA expression and its serum concentration in patients who underwent bone marrow transplantation for myeloid leukemias. Nevertheless, further studies with greater numbers of patients are needed to establish the role of TGF-β1 in graft-versus-host disease pathophysiology.

Distribution of mRNAs encoding transforming growth factors‐β1, ‐2, and ‐3 in the intact rat brain and after experimentally induced focal ischemia

Transforming growth factors-beta1 (TGF-beta1), -2, and -3 form a small group of related proteins involved in the regulation of proliferation, differentiation, and survival of various cell types. Recently, TGF-betas were also demonstrated to be neuroprotective. In the present study, we investigated their distribution in the rat brain as well as their expression following middle cerebral artery occlusion. Probes were produced for all types of TGF-betas, and in situ hybridization was performed. We demonstrated high TGF-beta1 expression in cerebral cortex, hippocampus, central amygdaloid nucleus, medial preoptic area, hypothalamic paraventricular nucleus, substantia nigra, brainstem reticular formation and motoneurons, and area postrema. In contrast, TGF-beta2 was abundantly expressed in deep cortical layers, dentate gyrus, midline thalamic nuclei, posterior hypothalamic area and mamillary body, superior olive, areas of monoaminergic neurons, spinal trigeminal nucleus, dorsal vagal complex, cerebellum, and choroid plexus, and a high level of TGF-beta3 mRNA was found in cerebral cortex, hippocampus, basal amygdaloid nuclei, lateral septal nucleus, several thalamic nuclei, arcuate and supramamillary nuclei, superior colliculus, superior olive, brainstem reticular formation and motoneurons, area postrema, and inferior olive. Focal brain ischemia induced TGF-betas with markedly different expression patterns. TGF-beta1 was induced in the penumbral region of cortex and striatum, whereas TGF-beta2 and -beta3 were induced in different layers of the ipsilateral cortex. The expression of the subtypes of TGF-betas in different brain regions suggests that they are involved in the regulation of different neurons and bind to different latent TGF-beta binding proteins. Furthermore, they might have subtype-specific functions following ischemic attack.

Elevated Cyclic Stretch Induces Aortic Valve Calcification in a Bone Morphogenic Protein-Dependent Manner

Calcified aortic valve (AV) cusps have increased expression of bone morphogenic proteins (BMPs) and transforming growth factor-beta1 (TGF-beta1). Elevated stretch loading on the AV is known to increase expression of matrix remodeling enzymes and pro-inflammatory proteins. Little, however, is known about the mechanism by which elevated stretch might induce AV calcification. We investigated the hypothesis that elevated stretch may cause valve calcification via a BMP-dependent mechanism. Porcine AV cusps were cultured in a stretch bioreactor, at 10% (physiological) or 15% (pathological) stretch and 70 beats per minute for 3, 7, and 14 days, in osteogenic media supplemented with or without high phosphate (3.8 mmol/L), TGF-beta1 (1 ng/ml), as well as the BMP inhibitor noggin (1, 10, and 100 ng/ml). Fresh cusps served as controls. Alizarin red and von Kossa staining demonstrated that 15% stretch elicited a stronger calcification response compared with 10% stretch in a fully osteogenic medium containing high phosphate and TGF-beta1. BMP-2, -4, and Runx2 expression was observed after 3 days on the fibrosa surface of the valve cusp and was stretch magnitude-dependent. Cellular apoptosis was highest at 15% stretch. Tissue calcium content and alkaline phosphatase activity were similarly stretch-dependent and were significantly reduced by noggin in a dose dependent manner. These results underline the potential role of BMPs in valve calcification due to altered stretch.

Relationship and clinical significance of TGF-beta1 expression with Treg cell infiltration in hepatocellular carcinoma

There are few studies about origins of regulatory T (Treg) cells increased in primary hepatocellular carcinoma (HCC) tissue. Studies showed that Treg cells could be induced by transforming growth factor-beta1 (TGF-beta1), but the relation between TGF-beta1 expression and Treg cell infiltration is unclear in HCC tissue. This study was to investigate the expression of TGF-beta1 and correlations with the amount of Treg cells in HCC, and to evaluate their clinical values in predicting the prognosis of HCC. Envision immunohistochemistry was used to detect the expression of TGF-beta1 and Foxp3 in 102 specimens of HCC tissue and paired adjacent non-tumor liver tissue. Of the 102 specimens of HCC, 41 showed low TGF-beta1 expression and 61 (59.8%) showed high expression; of the 102 specimens of adjacent non-tumor tissue, 22 showed low TGF-beta1 expression and 80 (78.4%) showed high expression. The high expression rate of TGF-beta1 was significantly lower in HCC than in adjacent non-tumor tissues (P = 0.001). Average Foxp3+ cell density in HCC was 2.98 cells/HP, but there was very few or no expression of Foxp3 in adjacent non-tumor liver tissue. Expression of TGF-beta1 was positively correlated with expression of Foxp3 in HCC tissues (r = 0.228, P = 0.021). The expression of TGF-beta1 was significantly higher in HCC tissues with high preoperative AFP concentration than in those with low preoperative AFP concentration (P = 0.023). TGF-beta1 and Foxp3 expression had no correlations with tumor diameter, tumor capsule, liver cirrhosis, and so on. The 5-year survival rate was not different between HCC tissues with high and low TGF-beta1 expression (P = 0.790); however, it was significantly lower in HCC tissues with high Treg cell infiltration than in those low infiltration (25% vs. 44%, P = 0.007). Cox multivariate analysis showed that the number of Treg cells and tumor capsule were independent prognostic factors (P = 0.021, P = 0.001). Expression of TGF-beta1 may relate to the infiltration of Treg cells in HCC tissues, but the relation need to be further investigated. The number of Treg cells in HCC tissues could be used as a potential immunological prognostic indicator for HCC patients after resection.

Distinct Th1, Th2 and Treg cytokines balance in chronic periapical granulomas and radicular cysts

Periapical lesions are a host response that involves immune reaction to prevent dissemination of bacteria from an infected root canal. The purpose of this study was to evaluate the levels of nitric oxide (NO), IL-4, TGF-beta, tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) in chronic periapical lesions and to determine their possible association with clinical and radiographic parameters. Seventeen human radicular cysts and 30 periapical granulomas were used in this study. Cytokines and NO were assessed by enzyme-linked immunosorbent assay and by the Griess reaction respectively confirmed by immunohistochemical. TNF-alpha and IFN-gamma were detected in 10% of granulomas and in 41.2% and 70% of radicular cysts. IL-4 was reactive in 24% of cysts, and TGF-beta was positive in all samples. Patients with tenderness showed significantly higher levels of IFN-gamma and IL-4 (P < 0.05). Swelling was associated with high levels of TNF-alpha, IFN-gamma, and IL-4 (P < 0.05). Lesions presenting bone resorption were associated with high levels of NO (P < 0.05). Periapical granulomas display a regulatory environment characterized by high TGF-beta and low inflammatory cytokine levels, while radicular cysts has mist Th1 and Th2 inflammatory reaction with the presence of IFN-gamma, TNF-alpha, and IL-4.

Decreased IP-10 and elevated TGFbeta1 levels are associated with viral clearance following therapy in patients with hepatitis C virus.

The role of pro-fibrogenic cytokines in the outcome of infections with hepatitis C virus (HCV) and the response to treatment with pegylated interferon-alpha (pegIFNα) and ribavirin remains unclear. To address this issue, we assessed hepatic fibrosis and plasma markers pertinent to T-cell mediated fibrogenesis and inflammation at the start of treatment. Levels of soluble (s)CD30, interleukin-13 receptor alpha 2 (IL-13Rα2), total and active transforming growth factor-beta 1 (TGFβ1), interleukin-18 (IL-18) and interferon-gamma inducible protein-10 (IP-10, CXCL10) were correlated with the severity of fibrosis and with treatment outcome using multiple logistic regression modelling. The Hepascore algorithm was confirmed as a marker of fibrosis, but was a poor predictor of treatment outcome. Inclusion of all immunological markers improved prediction based on Hepascore alone (p = 0.045), but optimal prediction was achieved with an algorithm (“TIPscore”) based on TGFβ1 (total), IP-10, age, sex and HCV genotype (p = 0.003 relative to Hepascore). Whilst this was only marginally more effective than predictions based on HCV genotype age and sex (p = 0.07), it associates high TGFβ1 and low IP-10 levels with a failure of therapy.

Gap junction reduction in cardiomyocytes following transforming growth factor-β treatment and Trypanosoma cruzi infection

Gap junction connexin-43 (Cx43) molecules are responsible for electrical impulse conduction in the heart and are affected by transforming growth factor-beta (TGF-beta). This cytokine increases during Trypanosoma cruzi infection, modulating fibrosis and the parasite cell cycle. We studied Cx43 expression in cardiomyocytes exposed or not to TGF-beta T. cruzi, or SB-431542, an inhibitor of TGF-beta receptor type I (ALK-5). Cx43 expression was also examined in hearts with dilated cardiopathy from chronic Chagas disease patients, in which TGF-beta signalling had been shown previously to be highly activated. We demonstrated that TGF-beta treatment induced disorganised gap junctions in non-infected cardiomyocytes, leading to a punctate, diffuse and non-uniform Cx43 staining. A similar pattern was detected in T. cruzi-infected cardiomyocytes concomitant with high TGF-beta secretion. Both results were reversed if the cells were incubated with SB-431542. Similar tests were performed using human chronic chagasic patients and we confirmed a down-regulation of Cx43 expression, an altered distribution of plaques in the heart and a significant reduction in the number and length of Cx43 plaques, which correlated negatively with cardiomegaly. We conclude that elevated TGF-beta levels during T. cruzi infection promote heart fibrosis and disorganise gap junctions, possibly contributing to abnormal impulse conduction and arrhythmia that characterise severe cardiopathy in Chagas disease.

Glucosamine induces translocation of protein kinase C isoenzymes in mesangial cells.

Activation of protein kinase C (PKC) has been implicated in the high glucose-induced stimulation of matrix protein production in mesangial cells. Since we have found (Kolm-Litty et al., 1998) that glucosamine, similar to the PKC activator phorbol myristate acetate (PMA), mimicks high glucose-induced TGF-beta1 overexpression and subsequent matrix overproduction, the action of these agents on the translocation of PKC isoenzymes was studied in cultured mesangial cells. Exposure to 12 mM glucosamine resulted in rapid and specific translocation of PKC-isoenzymes in mesangial cells i.e. glucosamine caused an increased and sustained translocation of PKC-alpha, -beta and -epsilon while PKC-zeta was essentially unaffected. Comparison with PMA-induced translocation exhibited distinct differences. Exposure to high glucose concentrations of mesangial cells induced translocation of PKC-beta and down-regulation of PKC-epsilon while PKC-alpha and -zeta were essentially unaltered. Presence of azaserine an inhibitor of glutamine: fructose-6-phosphate amidotransferase, the key enzyme of the hexosamine pathway, attenuated the high glucose-induced effects on the membrane fraction of PKC-beta. Our results indicate that i) glucosamine is a potent stimulator of PKC-translocation exhibiting an isoenzyme specific translocation kinetic which is different from PMA-induced PKC-isoenzyme translocation ii) the hexosamine pathway may be possibly involved in the high glucose-induced activation of PKC.

Muco‐ciliary differentiation of nasal epithelial cells is decreased after wound healing in vitro

Epithelial damage and modifications of cell differentiation are frequent in airway diseases with chronic inflammation, in which transforming growth factor-beta1 (TGF-beta1) plays an important role. The aim of this study was to evaluate the differentiation of human nasal epithelial cells (HNEC) after wound healing and the potential effects of TGF-beta1. Basal, mucus, and ciliated cells were characterized by cytokeratin-14, MUC5AC, and betaIV tubulin immunodetection, respectively. Their expression was evaluated in situ in nasal polyps and in an in vitro model of wound healing in primary cultures of HNEC after wound closure, under basal conditions and after TGF-beta1 supplementation. Using RT-PCR, the effects of TGF-beta1 on MUC5AC and DNAI1 genes, specifically transcribed in mucus and ciliated cells, were evaluated. In situ, high TGF-beta1 expression was associated with low MUC5AC and betaIV tubulin expression. In vitro, under basal conditions, MUC5AC expression remained stable, cytokeratin-14 expression was strong and decreased with time, while betaIV tubulin expression increased. Transforming growth factor-beta1 supplementation downregulated MUC5AC and betaIV tubulin expression as well as MUC5AC and DNAI1 transcripts. After a wound, differentiation into mucus and ciliated cells was possible and partially inhibited in vitro by TGF-beta1, a cytokine that may be involved in epithelial remodeling observed in chronic airway diseases.

TGF-β signaling and collagen deposition in chronic rhinosinusitis

Chronic rhinosinusitis is an inflammatory disease with distinct cytokine and remodeling patterns. The objective was to analyze the presence of TGF-beta isoforms, receptors, intracellular signaling, and collagen deposition in chronic rhinosinusitis. Sinonasal mucosal samples obtained from chronic rhinosinusitis with nasal polyps (CRSwNP; n = 13), chronic rhinosinusitis without nasal polyps (CRSsNP; n = 13), and controls (n = 10) were analyzed for TGF-beta isoforms 1 and 2 by means of ELISA and IHC, and for TGF-beta R1, 2, and 3 by RT-PCR and IHC. As downstream proteins, phospho-Smad 2 (pSmad 2) and collagen were analyzed by performing immunostaining and picrosirius red staining, respectively. TGF-beta 1 and 2 protein concentrations, TGF-beta receptor (R) I and TGF-beta RIII mRNA expression, the number of pSmad 2-positive cells, and total collagen amount were significantly higher in CRSsNP versus controls. In CRSwNP, TGF-beta 1 protein concentration, TGF-beta RII and TGF-beta RIII mRNA expression, the number of pSmad 2-positive cells, and total collagen amount were significantly lower versus controls. Only TGF-beta 2 protein was found higher in CRSwNP versus controls. A high TGF-beta 1 protein expression, increased TGF-beta RI expression, and a high number of pSmad 2-positive cells all indicate an enhanced TGF-beta signaling in CRSsNP, whereas a low TGF-beta 1 protein concentration, a decreased expression of TGF-beta RII, and a low number of pSmad 2-positive cells in CRSwNP indicate a low level of TGF-beta signaling in CRSwNP. These findings are compatible with the remodeling patterns observed, reflected by a lack of collagen in CRSwNP, and excessive collagen production with thickening of the collagen fibers in the extracellular matrix in CRSsNP.

Local action of endogenous renal tubular atrial natriuretic peptide

Up-regulation of atrial natriuretic peptide (ANP) mRNA in the kidneys in several disorders has been demonstrated; however, evidence that ANP synthesized by the kidney exerts a local function has never been produced. Therefore, we investigated whether endogenous ANP could modulate high glucose-stimulated TGF-beta1, collagen type I and nuclear factor-kappaB (NF-kappaB) in NRK-52E cells using transfection of ANP and ANP small interfering RNA (siANP). NRK-52E cells were grown with or without transfection with ANP plasmid; cells were also transfected with ANP siRNA or control siRNA. These cells were then stimulated with a high glucose concentration to modulate ANP, TGF-beta1, collagen type I, NF-kappaB and IkappaB-alpha, and the results showed that ANP, TGF-beta1, collagen type I and NF-kappaB significantly increased in untransfected cells, and the transfection of ANP significantly attenuated high glucose-activated TGF-beta1, collagen I and NF-kappaB expression. ANP siRNA knocked-down ANP but significantly increased TGF-beta1 and collagen I under normal glucose conditions; ANP siRNA decreased IkappaB-alpha but strongly enhanced high glucose-activated TGF-beta1, collagen type I and NF-kappaB. In contrast, medium from ANP-transfected cells attenuated high glucose-activated TGF-beta1 and collagen type I expression in NRK-52E cells transfected with siANP. In conclusion, our results demonstrated that siANP increased activation of TGF-beta1, collagen type I and NF-kappaB in NRK-52E cells under high glucose conditions, and medium from ANP-transfected cells attenuated high glucose-activated TGF-beta1 and collagen type I. This is the first study to demonstrate the auto/paracrine action of endogenous ANP in renal tubular cells on the attenuation of hyperglycemia-activated TGF-beta1 and NF-kappaB expression. J. Cell. Physiol. 219: 776-786, 2009. (c) 2009 Wiley-Liss, Inc.

TGF-β1 genotype and phenotype in breast cancer and their associations with IGFs and patient survival

Transforming growth factor-β (TGF-β)-mediated signals play complicated roles in the development and progression of breast tumour. The purposes of this study were to analyse the genotype of TGF-β1 at T29C and TGF-β1 phenotype in breast tumours, and to evaluate their associations with IGFs and clinical characteristics of breast cancer. Fresh tumour samples were collected from 348 breast cancer patients. TGF-β1 genotype and phenotype were analysed with TaqMan® and ELISA, respectively. Members of the IGF family in tumour tissue were measured with ELISA. Cox proportional hazards regression analysis was performed to assess the association of TGF-β1 and disease outcomes. Patients with the T/T (29%) genotype at T29C had the highest TGF-β1, 707.9 pg mg−1, followed by the T/C (49%), 657.8 pg mg−1, and C/C (22%) genotypes, 640.8 pg mg−1, (P=0.210, T/T vs C/C and C/T). TGF-β1 concentrations were positively correlated with levels of oestrogen receptor, IGF-I, IGF-II and IGFBP-3. Survival analysis showed TGF-β1 associated with disease progression, but the association differed by disease stage. For early-stage disease, patients with the T/T genotype or high TGF-β1 had shorter overall survival compared to those without T/T or with low TGF-β1; the hazard ratios (HR) were 3.54 (95% CI: 1.21–10.40) for genotype and 2.54 (95% CI: 1.10–5.89) for phenotype after adjusting for age, grade, histotype and receptor status. For late-stage disease, however, the association was different. The T/T genotype was associated with lower risk of disease recurrence (HR=0.13, 95% CI: 0.02–1.00), whereas no association was found between TGF-β1 phenotype and survival outcomes. The study suggests a complex role of TGF-β1 in breast cancer progression, which supports the finding of in vitro studies that TGF-β1 has conflicting effects on tumour growth and metastasis.