Quantitative analysis of transforming growth factor beta 1 mRNA in patients with alcoholic liver disease

AIM: To investigate the expression of the transforming growth factor beta 1(TGF-beta 1) mRNA in different stages of alcoholic liver disease (ALD) and its clinical value. METHODS: One hundred and seven male alcoholics were grouped by clinical findings into four groups: alcohol abusers without liver impairment (n =22), alcoholic steatosis (n =30); alcoholic hepatitis (n=31); and alcoholic cirrhosis(n=24). Using peripheral blood mononuclear cells (PBMC) as samples the gene expression of TGF-beta 1 was examined quantitatively by reverse transcription polymerase chain reaction (RT-PCR) and dot blot. There are 34 healthy subjects served as control. RESULTS: The expression of TGF-beta 1 from all ALD patients was significantly greater than that in controls (1.320 +/- 1.162 vs 0.808 +/- 0.276, P【0.001). The differences of the expressions were significant between the patients from each groups (alcoholic steatosis, alcoholic hepatitis and alcoholic cirrhosis) and the controls (1.168 +/- 0.852, 1.462 +/- 1.657, 1.329 +/- 0.610 vs 0.808 +/- 0.276, P【0.050). No significant differences of TGF -beta 1 mRNA expression were observed between alcohol abusers without liver impairment and controls. The expressions in patients with alcoholic hepatitis and alcoholic cirrhosis were significantly greater than that in alcohol abusers respectively (1.462 +/- 1.657, 1.329 +/- 0.610 vs 0.841 +/- 0.706, P【0.050). No significant differences of TGF-beta 1 mRNA expression were observed between alcoholic fatty liver men and alcohol abusers. CONCLUSION: TGF-beta 1 expression level can be a risk factor for alcoholic liver disease and might be related to the inflammatory activity and fibrosis of the liver in patients.

Roles of Smad3 and Smad7 in rat pancreatic stellate cells activated by transforming growth factor-beta 1

BACKGROUND: Pancreatic stellate cells (PSCs) play a major role in promoting pancreatic fibrosis. Transforming growth factor beta 1 (TGF-beta 1) is a critical mediator of this process. This study aimed to determine the expression of the Smad3 and Smad7 genes in the process of PSC activation, and explore the mechanisms of chronic pancreatitis. METHODS: The expressions of Smad3 and Smad7 in PSCs before and after TGF-beta 1 treatment were detected by reverse transcription-polymerase chain reaction and Western blotting analysis. Smad3 expression was detected in PSCs after treatment with 5 ng/ml of TGF-beta 1 for 24 hours. RESULTS: Smad7 expression was decreased in TGF-beta 1 -activated PSCs (P<0.05) in a dose-dependent manner. When TGF-beta 1 concentration reached 10 ng/ml, the expression of p-Smad3, Smad3, and Smad7 was inhibited (P<0.05). CONCLUSIONS: TGF-beta 1 promotes the expression of Smad3 and inhibits the expression of Smad7 during the activation of PSCs. In contrast, high-dose TGF-beta 1 downregulates the expression of Smad3 in completely activated PSCs.

Effect of transforming growth factor beta and bone morphogenetic proteins on rat hepatic stellate cell proliferation and transdifferentiation

AIM: To explore different roles of TGF-β (transforming growth factor beta) and bone morphogenetic proteins (BMPs)in hepatic stellate cell proliferation and trans-differentiation.METHODS: Hepatic stellate cells were isolated from male Sprague-Dawley rats. Sub-cultured hepatic stellate cells were employed for cell proliferation assay with WST-1 reagent and Western blot analysis with antibody against smooth muscle alpha actin (SMA).RESULTS: The results indicated that TGF-β1 significantly inhibited cell proliferation at concentration as low as 0.1 ng/ml, but both BMP-2 and BMP-4 did not affect cell proliferation at concentration as high as 10 ng/ml. The effect on hepatic stellate cell trans-differentiation was similar between TGFβ1 and BMPs. However, BMPs was more potent at transdifferentiation of hepatic stellate cells than TGF-β1. In addition, we observed that TGF-β1 transient reduced the abundance of SMA in hepatic stellate cells.CONCLUSION: TGF-β may be more important in regulation of hepatic stellate cell proliferation while BMPs may be the major cytokines regulating hepatic stellate cell transdifferentiation.

Interference of Y-27632 on the signal transduction of transforming growth factor beta type 1 in ocular Tenon capsule fibroblasts

AIM: To investigate the interfering effect of Y-27632, a ROCK-I selective inhibitor, on the signal transduction pathway of transforming growth factor-beta 1 (TGF-beta 1) in ocular Tenon capsule fibroblasts (OTFS) in vitro. METHODS: After OTFS from passages 4 to 6 47 vitro were induced by TGF-beta 1 and then treated by Y-27632, the changes of the OTFS cell cycles were analyzed via flow cytometry, and the proteins expression of the alpha -smooth muscular actin (alpha -SMA), connective tissue growth factor (CTGF), collagen I were calculated by Western blot. After OTFS treated by the different concentrations of Y-27632, the expression levels of the alpha -SMA, CTGF and collagen I mRNA were assayed by RT-PCR. RESULTS: Y-27632 had no markedly effect on the OTFS cell cycles. After treated by TGF-beta 1, OTFS in G1 period significantly increased. The cell cycles distribution by both TGF-beta 1 and Y-27632 had no remarkable difference from that in control group. Y-27632 significantly inhibited the proteins expressions of both alpha -SMA and CTGF, while to some extent inhibited that of collagen I. TGF-beta 1 significantly promoted the proteins expressions of alpha -SMA, CTGF and collagen I. After OTFS treated by both TGF-beta 1 and Y-27632, of alpha -SMA, the protein expression was similar with that in control group (P=0.066>0.05), but the protein expression of CTGF or collagen I, respectively, was significantly different from that in control group (P=0.000<0.01). The differences of expressions of the alpha -SMA, CTGF and collagen I mRNA in 30, 150, 750 mu mol/L Y-27632 group were statistically significant, compared with those in control group, respectively (alpha -SMA, P=0.002, 0.000, 0.000; CTGF, P=0.014, 0.002, 0.001; collagen I,P=0.003, 0.002, 0.000). CONCLUSION: Blocking the Rho/ROCK signaling pathway by using of Y-27632 could inhibit the cellular proliferation and the expression of both CTGF and alpha -SMA whatever OTFS induced by TGF-beta 1 or not. Y-27632 suppressed the expression of collagen I mRNA without induction.

Establishment of a real-time PCR for quantifying transforming growth factor beta1 in blood of hepatocellular carcinoma patients

Background: The carcinogenesis of hepatocellular carcinoma (HCC) is a multi-factorial, multistep and complex process. Its prognosis is poor and early detection is of the utmost importance. Transforming growth factor β1 (TGF-β1) message RNA (mRNA) has been reported to be elevated in HCC patients using Northern blotting. However, little work has been done about the detection of TGF-β1 mRNA levels in peripheral blood of patients with HCC using the real-time polymerase chain reactions (PCR) method. Objective: To assess the prognostic value of quantitative levels of TGF-β1 mRNA in peripheral blood of patients with HCC, and to investigate the relationship between the expression of TGF-β1 mRNA in peripheral blood and many diagnostic and pathological factors. Methods: We developed an optimized Taqman real-time PCR to quantify TGF-β1 mRNA in peripheral blood of 53 patients with HCC and 44 healthy volunteers. In addition, blood was collected from patients with HCC for measuring levels of total bilirubin (TBil), prealbumin, albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyltranspeptidase (GGT), alpha-L-fucosidase (AFU), alpha fetoprotein (AFP), carcino-embryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), viral load and platelet counts. Statistical analysis was performed using the SPSS software system (SPSS 10.0). Results: In real-time PCR, fluorescence was detectable in all blood specimens from patients with HCC and healthy volunteers. The levels of TGF-β1 mRNA expression in patients with HCC were significantly higher compared to that in healthy volunteers (P<0.000 1), suggesting an association of the activated TGF-β1 gene transcription with hepato- carcinogenesis. Patients with HCC were divided into 2 groups according to their TGF-β1 mRNA above (group A, n=28)or below (group B, n=25) the mean level. Statistical results demonstrated that TGF-β1 mRNA expression level was correlated with patients age, serum levels of CEA, CA19-9 and viral copy number (P<0.05). Conclusion: Although this is a small sample size pilot study these findings imply that quantitative measurement of TGF-β1 mRNA level in peripheral blood may be a complementary serologic marker of HCC.

Clinical impact of plasma TGF-β1 and circulating TGF-β1 mRNA in diagnosis of hepatocellular carcinoma

BACKGROUND:Transforming growth factor-β(TGF-β) plays an important role in the regulation of cell growth and differentiation,angiogenesis,extracellular matrix formation,immunosuppression and cancer development. In this study,we investigated the levels of TGF-β1 and TGF-β1 mRNA expression,their relationship with HBV replication,and their diagnostic value for hepatocellular carcinoma(HCC). METHODS:Total RNAs were extracted from HCC samples and matched non-tumor tissues,and from peripheral blood mononuclear cells in HCC patients.TGF-β1 mRNA was amplified by RT-PCR and confirmed by DNA sequencing. The distribution of TGF-β1 expression was assessed by immunohistochemistry.The clinical characteristics were analyzed between TGF-β1 and HBV replication.The diagnostic value of circulating TGF-β1 and TGF-β1 mRNA levels were investigated in HCC patients. RESULTS:The incidence of hepatic TGF-β1 expression was 83.3%in HCC samples,43.3%in the surrounding tissues, 94.7%in the HBV DNA-positive group,and 63.6%in the HBV DNA-negative group.Liver TGF-β1 expression was associated with the degree of HCC differentiation and the status of HBV replication,but not with the size or number of tumors.Circulating TGF-β1 level and incidence of TGF-β1 mRNA were significantly higher in the HCC groupthan in any group of patients with benign liver disease, with a higher sensitivity of 89.5%and a specificity of 94.0% for HCC diagnosis when circulating TGF-β1 levels were >1.2μg/L.No significant correlation was found between TGF-β1 expression and AFP level or tumor size.Combining TGF-β1 level and serum AFP raised the detection rate to 97.4%. CONCLUSIONS:Abnormal expression of hepatic TGF-β1 is associated with the degree of HCC differentiation and HBV replication.Both circulating TGF-β1 and TGF-β1 mRNA can be used as sensitive biomarkers for the diagnosis and prognosis of HBV-induced HCC.

GFAP promoter directs lacZ expression specifically in a rat hepatic stellate cell line

AIM： The GFAP was traditionally considered to be a biomarker for neural gila （mainly astrocytes and nonmyelinating Schwann cells）. Genetically, a 2.2-kb human GFAP promoter has been successfully used to target astrocytes in vitro and in vivo. More recently, GFAP was also established as one of the several makers for identifying hepatic stellate cells （HSC）. In this project, possible application of the same 2.2-kb human GFAP promoter for targeting HSC was investigated. METHODS： The GFAP-lacZ transgene was transfected into various cell lines （HSC, hepatocyte, and other nonHSC cell types）. The transgene expression specificity was determined by X-gal staining of the β-galactosidase activity. And the responsiveness of the transgene was tested with a typical pro-fibrotic cytokine TGF-β1. The expression of endogenous GFAP gene was assessed by real-time RT-PCR, providing a reference for the transgene expression. RESULTS： The results demonstrated for the first time that the 2.2 kb hGFAP promoter was not only capable of directing HSC-specific expression, but also responding to a known pro-fibrogenic cytokine TGF-β1 by upregulation in a doseand time-dependent manner, similar to the endogenous GFAP. CONCLUSION： In conclusion, these findings suggested novel utilities for using the GFAP promoter to specifically manipulate HSC for therapeutic purpose.

ROLE OF TRANSFORMING GROWTH FACTOR β(TGF-β)IN REPAIRING OF BONE DEFECTS

TGF-β is a multifunctional cytokine that regulates many aspects of cellular function, including periosteal mesenchymal cell proliferation, differentiation. This experiment is to study its effects on bone defect repair. A rabbit radial bone defect model was used to evaluate the effect of TGF-β, which was extracted and purified from bovine blood platelets, on the healing of a large segmental osteoperiosteal defect. A 1. 5-centimeter segmental defect was created in the mid-upper part of the radial shaft of adult rabbits. The defect was filled with implant containing TGF-β that consisted of carrier and bovine TGF-β. Limbs served as controls received carrier alone. The defectswere examined radiographically and histologically at 4, 8,12 , 16 and 20 weeks after implantation. The results showed that in TGF-β implant group . the defect areas at 12 weeks post operation were bridged by uniform new bone and the cut ends of cortex could not be seen;while in control group, the defects remained clear. Only a small amount of new bone formed as a cap on the cut bone ends. In the experimental group, new lamellar and woven bone formed in continuity with the cut ends of the cortex. An early medullar canal appears to be forming and contained normal-appearancing marrow elements; while the control group displayed entirely fibrous tissue within the defect site. Remnants of the cancellous bone carrier were observed in the control specimen. These data demonstrate that exogenous TGF-β initiate osteogenesis and stimulate the bone defects repair in animal model.

Inhibitory effects of rapamycin on the different stages of hepatic fibrosis

AIM: To investigate and compare the inhibitory effects of rapamycin in the different stages of liver fibrosis.

Inhibitory Mechanism of Inteferon-gamma on Human Fibroblasts from Tenon's Capsule

Summary: The inhibitory mechanism of interferon-gamma (IFN-γ) on the fibroblasts from Tenon's capsule was studied. By using immunohistochemical SP method and pathological image system, the inhibitory effects of IFN-γ on the expression of transforming growth factor beta receptor I in the in vitro cultured fibroblasts from Tenon's capsule were quantitatively analyzed. The results showed that IFN-γ could reduce the expression of transforming growth factor beta receptor I in the fibroblasts with the following dose-effect relationship: Y=1937.5-134.2 Igx (r=-0.971, P<0.01). It was concluded that IFN-γ could inhibit the expression of transforming growth factor beta receptor I in the fibroblasts from Tenon's capsule. The modulation of the transforming growth factor beta receptor I expression by IFN-γ may be beneficial to the alleviation of the hyperplasia of scar after trabeculectomy.

Association of Down-regulation of CD109 Expression with Up-expression of Smad7 in Pathogenesis of Psoriasis

Transforming growth factor（TGF）-β signaling plays an important role in the pathogenesis of psoriasis. CD109, a novel TGF-β co-receptor, which inhibits TGF-β signaling by enhancing Smad7-dependent degradation of TGF-β type Ⅰ receptor（TGF-β RⅠ）, is abnormally expressed in psoriasis. To date, the expression of Smad7 and the correlation between CD109 and Smad7 expression in psoriasis have not been fully elucidated. This study was designed to investigate the expression and the correlation of CD109 and TGF-β signaling associated proteins in psoriasis and their roles in the pathogenesis of psoriasis. Thirty-two psoriasis specimens were subjected to immunohistochemical staining for CD109, Smad7, TGF-β RⅠ and Ki67. Ten normal skin（NS） specimens served as controls. The positive expression rate（% positive cells） of Smad7 and Ki67 in psoriasis was significantly higher than in NS（62.6%±19.9% vs. 17.2%±4.4%, and 50.7%±14.3% vs. 19.5%±3.2%, respectively, P〈0.001）, and the expression levels of CD109 and TGF-β RⅠ were reduced significantly in psoriasis as compared with NS（8.1%±6.7% vs. 35.8%±6.7% and 27.3%±3.4% vs. 3.0%±3.4%, respectively, P〈0.001）. There were significantly negative correlations between CD109 and Smad7（r=-0.831, P〈0.01）. These findings indicated that CD109 might play a certain role in the pathogenesis of psoriasis. Lower expression of CD109 and TGF-β RⅠ was highly correlated with higher expression of Smad7 and Ki67, suggesting that CD109 may induce the pathogenesis of psoriasis through Smad7-mediated degradation of TGF-β RⅠ, and lead to the termination of TGF-β signaling.

Tranilast inhibits TGF-β-induced collagen gel contraction mediated by human corneal fibroblasts

AIM： To determine if tranilast affects human corneal fibroblast（HCFs） contraction.METHODS： HCFs cultured in a three-dimensional type I collagen gel were treated with or without transforming growth factor beta（TGF-β） or tranilast. Gel diameter was measured as an indicator for collagen contraction. Immunoblot was performed to evaluate myosin light chain（MLC） and paxillin phosphorylation. Confocal microscopy was employed to examine the focal adhesions and actin stress fiber formation. Immunoblot analysis and gelatin zymography were performed to detect tissue inhibitors of metalloproteinases and matrix metalloproteinases（MMPs） in supernatant.RESULTS： The inhibitory effect of tranilast on HCFsmediated collagen gel contraction induced by TGF-β was dose-dependent. The significant effect of tranilast was started from 100 μmol/L and maximized at 300 μmol/L. The peak effect of 300 μmol/L tranilast also relied on the duration of treatment, which showed statistical significance from day 2. TGF-β-induced paxillin and MLC phosphorylation, stress fiber formation, focal adhesions, and MMP-1, MMP-2, and MMP-3 secretion in HCFs were also inhibited by tranilast.CONCLUSION： Tranilast suppresses the HCFs-cultured collagen gel contraction induced by TGF-β. It attenuates actin stress fibers formation, focal adhesions, and the secretion of MMPs, with these actions likely contributing to the inhibitory effect on HCF contractility. By attenuating the contractility of corneal fibroblasts, tranilast treatment may inhibit corneal scarring.

The involvement of p38 MAPK in transforming growth factor β1-induced apoptosis in murine hepatocytes

We reported in this manuscript that TGF-beta1 induces apoptosis in AML12 murine hepatocytes, which is associated with the activation of p38 MAPK signaling pathway. SB202190, a specific inhibitor of p38 MAPK, strongly inhibited the TGF-beta1-induced apoptosis and PAI-1 promoter activity. Treatment of cells with TGF-beta1 activates p38. Furthermore, over-expression of dominant negative mutant p38 also reduced the TGF-beta1-induced apoptosis. The data indicate that the activation of p38 is involved in TGF-beta1-mediated gene expression and apoptosis.

The expression and antigenicity identification of recombinant rat TGF-β1 in bacteria

In order to study structure-function details of TGF-beta1, the recombinant mature form of rat TGF-beta1 was expressed in bacteria. Synthesis of the 112 amino-acid carboxyl-terminal part of TGF-beta1 (amino acid 279-390) was controlled by an inducible gene expression system based on bacteriophage T7 RNA polymerase. This system allowed an active and selective synthesis of recombinant TGF-beta1. The molecular weight of expressed TGF-alpha1 monomer determined on SDS-polyacrylamide gel under reducing conditions was about 13 kD. Serial detergent washes combined with a single gel-filtration purification step were sufficient to purify the expression product to homogeneity. Amino-terminal sequencing revealed that the N-terminal of the recombinant protein was identical to the published data. In Western blot analysis the recombinant polypeptide showed excellent antigenicity against polyclonal TGF-beta1 antibody. The mature recombinant rat TGF-beta1 expressed in this study provides a useful tool for future detailed structural and functional studies.

Resveratrol downregulates TGF-β1 and Smad3 expression and attenuates oxidative stress in CCl_4-induced kidney damage in rats

Objective:To evaluate the effect of resveratrol against CCl4-induced nephrotoxicity.Methods:Forty-two male Wistar rats were divided into seven groups randomly.After six weeks,kidney weight,body weight,blood urea,serum creatinine,oxidative stress markers,and gene expression of renal transforming growth factor-beta1(TGF-β1),TGF-βreceptor type 1(TGF-βR1)and Smad3 were determined.In addition,the protein level of TGF-β1 in the tissue lysate was measured.Results:Resveratrol had a protective role in renal tissue by the improvement of antioxidant balance and reduction of renal parameters such as creatinine and urea(P<0.001).In addition,the renal m RNA level of TGF-β1,TGF-βR1,Smad3,as well as the protein level of TGF-β1 were decreased in rats treated with resveratrol(P<0.001),compared to the CCl4 group.Conclusions:Overall,resveratrol shows a protective effect against nephrotoxicity in CCl4 treated rats by reducing oxidative stress status and modulating the TGF-βsignaling.

Experimental Study of Plasmid TGF-β1 DNA Gene Transfer with Lipofectamine into Rabbit Corneal Epithelial Cells In Vitro

To investigate whether the TGF β1 plasmid DNA carried by lipofectamine could be introduced into cultured rabbit corneal epithelial cells, specific expression of the plasmid pMAM TGF β1 in the cultured corneal epithelial cells was studied. Two days after 12 h of transfection of pMAMTGF β1 mediated by lipofectamine into the cultured corneal epithelial cells, the TGF β1 protein expression specific for pMAMTGF β1 in the cells was detected by means of immunohistochemical staining and the positive rate was 23.37 %. The results suggested that foreign plasmid DNA could be effectively delivered into cultured rabbit corneal epithelial cells by means of lipofectamine, and this will provide a promising method of studying TGF β1 on the mechanism of physiology and pathology concerned with corneal epithelial cells.

Dynamic Expression of bFGF and TGFβ2 in Glomus Cell Grafts of Carotid Body in Rat Model of Parkinson Disease

To investigate the changes in the expression of basic fibroblast growth factor (bFGF) and transforming growth factor beta 2 (TGFβ2) in glomus cell grafts of carotid body in the rat model of 6 hydroxydopamine induced Parkinson disease, immunohistochemical staining of bFGF and TGFβ2 in the sections of striate body was done on the 2nd, 4th and 12th week after transplantation. The results showed that on the 2nd week after transplantation, bFGF annd TGFβ2 were not detectable in the glumous cell grafts. On the 4th week after graft, bFGF and TGFβ2 immunoreactivity was increased within the grafts and at the graft host interface but was restricted only to astrocytes. In the striatum surrounding the graft, bFGF was expressed persistently, while TGFβ2 showed transient expression. It was suggested that the transient expression of TGFβ2 was likely due more to the trauma imposed by the graft procedure than to an intrinsic. The deficiency in astrocytic bFGF early after graft may be responsible for the poor survival of grafted glomus cells of carotid body.

RhoA/ROCK signaling in chondrogenic differentiation of MSCs induced by TGF-β1

Objective： To investigate the role of RhoA/ROCK in the process of chondrogenic differentiation of MSCs in vitro induced by TGF-β1. Methods ： MSCs were isolated from rat bone marrow, cultured and passaged. The cultured MSCs at the 3rd passage were induced to differentiate into chondrocytes in induction medium containing TGF-β1, the expressions of RhoA and ROCKI/2 in the induced MSCs cells were detected by Western-blot and RT-PCR. Results： In parallel to chondrogenic marker gene expressions of collagen Ⅱand aggrecan, ROCK inhibition by Y27632 in these cells caused a significant decrease in mRNA level of collagen Ⅱ. Conclusion： The results suggest that RhoA/ROCK pathway may play an important and complex role in regulation of chondrogenic differentiation and gene expression.

Role of activin receptor-like kinase 1 in vascular development and cerebrovascular diseases

Activin receptor-like kinase 1(ALK1)is a transmembrane serine/threonine receptor kinase of the transforming growth factor beta(TGFβ)receptor superfamily.ALK1 is specifically expressed in vascular endothelial cells,and its dynamic changes are closely related to the proliferation of endothelial cells,the recruitment of pericytes to blood vessels,and functional differentiation during embryonic vascular development.The pathophysiology of many cerebrovascular diseases is today understood as a disorder of endothelial cell function and an imbalance in the proportion of vascular cells.Indeed,mutations in ALK1 and its co-receptor endoglin are major genetic risk factors for vascular arteriovenous malformation.Many studies have shown that ALK1 is closely related to the development of cerebral aneurysms,arteriovenous malformations,and cerebral atherosclerosis.In this review,we describe the various roles of ALK1 in the regulation of angiogenesis and in the maintenance of cerebral vascular homeostasis,and we discuss its relationship to functional dysregulation in cerebrovascular diseases.This review should provide new perspectives for basic research on cerebrovascular diseases and offer more effective targets and strategies for clinical diagnosis,treatment,and prevention.

Oral encapsulated transforming growth factorβ1 reduces endogenous levels:Effect on inflammatory bowel disease

BACKGROUND TreXTAM®is a combination of the key regulatory cytokine transforming growth factor beta(TGFβ)and all trans retinoic acid(ATRA)microencapsulated for oral delivery to immune structures of the gut.It is in development as a novel treatment for inflammatory bowel disease(IBD).AIM To measure TGFβlevels in blood and tissue after oral administration of encapsulated TGFβ.METHODS Animals were orally administered encapsulated TGFβby gavage.Levels of drug substance in blood and in gut tissues at various times after administration were measured by ELISA.RESULTS We made the surprising discovery that oral administration of TreXTAM dramatically(approximately 50%)and significantly(P=0.025)reduced TGFβlevels in colon,but not small intestine or mesenteric lymph nodes.Similarly,levels in rat serum after 25 d of thrice weekly dosing with either TreXTAM,or microencapsulated TGFβalone(denoted as TPX6001)were significantly(P<0.01)reduced from baseline levels.When tested in the SCID mouse CD4+CD25-adoptive cell transfer(ACT)model of IBD,oral TPX6001 alone provided only a transient benefit in terms of reduced weight loss.CONCLUSION These observations suggest a negative feedback mechanism in the gut whereby local delivery of TGFβresults in reduced local and systemic levels of the active form of TGFβ.Our findings suggest potential clinical implications for use of encapsulated TGFβ,perhaps in the context of IBD and/or other instances of fibrosis and/or pathological TGFβsignaling.