TGF-beta Antibody Research Articles

Tumor necrosis factor-related apoptosis-inducing ligand enhances collagen production by human lung fibroblasts.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/APO-2L) is a member of the tumor necrosis factor family that induces apoptosis in a variety of transformed cell lines and in normal human hepatocytes and brain cells. Soluble TRAIL at high concentrations was found to induce apoptotic death in normal human lung fibroblasts, whereas at low concentrations it was found to stimulate collagen production by these cells. Collagen alpha2(I) mRNA expression was assessed by semiquantitative reverse transcriptase/polymerase chain reaction; total soluble collagen was measured in culture supernatants by the Sircol assay. Both alpha2(I) collagen mRNA level and total soluble collagen secretion were increased upon TRAIL stimulation, with peak response (> 4-fold increase in mRNA level) at 1 ng/ml TRAIL. Analysis of the transcriptional response in TRAIL-stimulated fibroblasts, using DNA microarray hybridization, revealed an augmented expression of a number of genes involved in tissue remodeling, including those related to the transforming growth factor-beta (TGF-beta) pathway. DNA microarray results for the increase in TGF-beta1 mRNA level were confirmed by Northern blot analysis and by measurements of total active TGF-beta1 in culture supernatants. In addition, pan-specific TGF-beta antibody was shown to inhibit TRAIL-stimulated collagen mRNA and protein expression. These data suggest that TRAIL can enhance extracellular matrix synthesis in fibroblasts by triggering TGF-beta production that acts in an autocrine manner.

High glucose and endothelial cell growth: novel effects independent of autocrine TGF-beta 1 and hyperosmolarity.

Human endothelial cells were exposed to 5 mM glucose (control), 25 mM (high) glucose, or osmotic control for 72 h. TGF-beta1 production, cell growth, death, and cell cycle progression, and the effects of TGF-beta1 and TGF-beta neutralization on these parameters were studied. High glucose and hyperosmolarity increased endothelial TGF-beta1 secretion (P < 0.0001) and bioactivity (P < 0.0001). However, high glucose had a greater effect on reducing endothelial cell number (P < 0.001) and increasing cellular protein content (P < 0.001) than the osmotic control. TGF-beta antibody only reversed the antiproliferative and hypertrophic effects of high glucose. High glucose altered cell cycle progression and cyclin-dependent kinase inhibitor expression independently of hyperosmolarity. High glucose increased endothelial cell apoptosis (P < 0.01), whereas hyperosmolarity induced endothelial cell necrosis (P < 0.001). TGF-beta antibody did not reverse the apoptotic effects observed with high glucose. Exogenous TGF-beta1 mimicked the increased S phase delay but not endoreduplication observed with high glucose. High glucose altered endothelial cell growth, apoptosis, and cell cycle progression. These growth effects occurred principally via a TGF-beta1 autocrine pathway. In contrast, apoptosis and endoreduplication occurred independently of this cytokine and hyperosmolarity.

Mifepristone-induced secretion of transforming growth factor β1-induced apoptosis in prostate cancer cells

Successful therapy should induce apoptosis in prostate cancer cells irrespective of their androgen response. We have investigated the possibility of utilizing Mifepristone and Tamoxifen as treatment options for prostate cancer cells. Because preliminary results demonstrated induction of apoptosis by these drugs, the mechanism of induction of apoptosis was investigated. LNCaP-C4 prostate cancer cells were treated with Mifepristone and/or Tamoxifen. To confirm cytotoxic effects, nude mice with LNCaP-C4 xenografts were treated with Mifepristone and Tamoxifen. Cell viability was assayed using Sulforhodamine B (SRB) assay and DNA fragmentation was measured by ELISA. Culture media from vehicle- and drug-treated cells were collected and secretion of transforming growth factor beta1 (TGFbeta1) was estimated by ELISA. Role of TGFbeta1 was confirmed by inhibiting its function using TGFbeta1 antibody or M6P, which blocked activation of TGFbeta1. Apoptotic effects were determined by immunoblots of cytochrome c levels in cytosol and by in vitro colorimetric assay of caspase-3 activity. Results showed that although both drugs induced apoptosis in LNCaP-C4 cells, Mifepristone was more effective. The effects of these drugs on xenografts confirmed in vitro results. It was hypothesized that drug-induced secretion of TGFbeta1 may be responsible for induction of apoptosis. Neutralization of TGFbeta1 with an antibody or blocking the activation of TGFbeta1 by M6P abrogated the effects of Mifepristone and Tamoxifen confirming our hypothesis. Furthermore, treatment with Mifepristone and/or Tamoxifen released cytochrome c into the cytoplasm and induced activity of caspase-3, providing evidence that the drug-stimulated secretion of TGFbeta1 was responsible for induction of apoptosis in these cells. In conclusion, both Mifepristone and Tamoxifen induced apoptosis mediated through TGFbeta1. However, no critical advantage was noted by the addition of Tamoxifen to Mifepristone treatment.

Human helper T cell activation and differentiation is suppressed by porcine small intestinal submucosa.

A cell-free biomaterial derived from porcine small intestinal submucosa (SIS) has been used successfully in many models as a xenogeneic scaffolding material without generating immune-mediated inflammatory reactions. We investigated whether this absence of inflammation is due to the presence of porcine transforming growth factor beta (TGF-beta) activity found in SIS that may have immunosuppressive properties on helper T (Th) cell subset activation and differentiation. We used in vitro models for the generation of human Th1 and Th2 cells to investigate the influence of SIS. We found that SIS partially suppressed Th1 cell expansion and secretion of interleukin 12 (IL-12) and interferon gamma (IFN-gamma) in a TGF-beta-dependent manner, but Th1 cell expansion and IFN-gamma secretion could be fully overcome by addition of recombinant IL-12. The suppression by SIS of Th cell activation also involved the induction of Th cell apoptosis. In addition, SIS completely abolished the generation of Th2 cells in vitro, but this effect of SIS was not reversed by neutralizing TGF-beta antibodies. Our results indicate the presence in SIS of factors that can suppress Th cell activation through both the inhibition of IL-12 secretion and the induction of Th cell apoptosis. We established further that these factors include TGF-beta and at least one other factor.

Up-regulation of Sodium-dependent Glucose Transporter by Interaction with Heat Shock Protein 70

Heat shock stress induces some heat shock proteins, including Hsp70, and activates sodium-dependent glucose transport in porcine renal LLC-PK(1) cells, but its mechanisms have not been described in detail. We investigated whether sodium-dependent glucose transporter (SGLT1) interacts with Hsp70 to increase SGLT1 activity. Heat shock stress increased SGLT1 activity without changing SGLT1 expression. The increase of SGLT1 activity was completely inhibited by an anti-transforming growth factor-beta1 (TGF-beta1) antibody. Instead of heat shock stress, TGF-beta1 increased SGLT1 activity dose- and time-dependently without changing SGLT1 expression. We found that the amount of Hsp70 immunoprecipitated from TGF-beta1-treated cells with an anti-SGLT1 antibody was higher than that of the control cells. Transfection of an anti-Hsp70 antibody into the cells inhibited the increase of SGLT1 activity. With confocal laser microscopy, both SGLT1 and Hsp70 was localized near the apical membrane in the TGF-beta1-treated cells, and an anti-Hsp70 antibody disturbed this localization. Furthermore, we clarified that an anti-Hsp70 antibody inhibited interaction of SGLT1 with Hsp70 in vitro. These results suggest that Hsp70 forms a complex with SGLT1 and increases the expression level of SGLT1 in the apical membrane, resulting in up-regulation of glucose uptake.

Iron chelation-induced senescence-like growth arrest in hepatocyte cell lines: association of transforming growth factor beta1 (TGF-beta1)-mediated p27Kip1 expression.

Iron is essential for cellular proliferation in all organisms. When deprived of iron, the growth of cells is invariably inhibited. However, the mechanism involved remains largely unclear. In the present study, we have observed that subcytotoxic concentrations of desferroxamine mesylate (DFO), an iron chelator, specifically inhibited the transition from G1 to S-phase of Chang cells, a hepatocyte cell line. This was accompanied by the appearance of senescent biomarkers, such as enlarged and flattened cell morphology, senescence-associated beta-galactosidase activity and reduced expression of poly(ADP-ribose) polymerase. Concomitantly, p27Kip1 (where Kip is kinase-inhibitory protein) was induced markedly, whereas other negative cell-cycle regulators, such as p21Cip1 (where Cip is cyclin-dependent kinase-interacting protein), p15INK4B and p16INK4A (where INK is inhibitors of cyclin-dependent kinase 4), were not, implying its association in the G1 arrest. Furthermore, the induction of p27Kip1 was accompanied by an increased level of transforming growth factor beta1 (TGF-beta1) mRNA. When neutralized with an anti-(TGF-beta1) antibody, p27Kip1 induction was completely abolished, indicating that TGF-beta1 is the major inducer of p27Kip1. Finally, DFO-induced senescence-like arrest was found to be independent of p53, since cell-cycle arrest was still observed with two p53-negative cell lines, Huh7 and Hep3B cells. In conclusion, DFO induced senescence-like G1 arrest in hepatocyte cell lines and this was associated with the induction of p27Kip1 through TGF-beta1, but was independent of p53.

Temporal localization of immunoreactive transforming growth factor beta1 in normal equine skin and in full-thickness dermal wounds.

To describe the localization of immunoreactive transforming growth factor (TGF)-beta1 in both normal skin and full-thickness dermal wounds of the limb and the thorax of the horse. Six full-thickness excisional wounds were created on the lateral aspect of one metacarpal region and on the midthoracic area of each horse. Sequentially collected tissue specimens from wound margins were assessed for TGF-beta1 expression by immunohistochemistry. Four horses (2 to 4 years of age). A neutralizing monoclonal anti-human TGF-beta1 antibody was used to detect the spatial expression of TGF-beta1 protein by immunohistochemical localization in biopsies obtained before wounding and at 12 and 24 hours, and 5, 10, and 14 days. No differences in localization of immunoreactive TGF-beta1 were detected between limb and thorax, for either intact skin or wounds. Unwounded epidermis stained moderately for TGF-beta1 protein throughout all layers, whereas the dermis was relatively devoid of immunoreactivity. During the acute stage of repair, migrating epithelium lost its stain, whereas cells of epidermal appendages remained strongly immunoreactive. The epithelium recovered its TGF-beta1 immunoreactivity during wound remodeling, although cells of the stratum corneum remained negative. Macrophages of the inflammatory exudate had positive cytoplasmic staining that diminished with time. Immunoreactivity of granulation tissue fibroblasts was evident early on and increased throughout the repair process. TGF-beta1 is constitutively expressed in normal, unwounded equine epithelium. Its expression is upregulated within the skin on injury and is associated with the cells involved in wound repair. A more precise understanding of the temporal and spatial expression of TGF-beta1 during wound repair in horses should provide the groundwork for possible future manipulations of both normal and aberrant tissue repair.

Phorbol Ester Activation of a Proteolytic Cascade Capable of Activating Latent Transforming Growth Factor-β: A PROCESS INITIATED BY THE EXOCYTOSIS OF CATHEPSIN B

12-O-Tetradecanoylphorbol-13-acetate (TPA) suppresses the proliferation of the human breast epithelial cell line MCF10A-Neo by initiating proteolytic processes that activate latent transforming growth factor (TGF)-beta in the serum used to supplement culture medium. Within 1 h of treatment, cultures accumulated an extracellular activity capable of cleaving a substrate for urokinase-type plasminogen activator (uPA) and tissue plasminogen activator (tPA). This activity was inhibited by plasminogen activator inhibitor-1 or antibodies to uPA but not tPA. Pro-uPA activation was preceded by dramatic changes in lysosome trafficking and the extracellular appearance of cathepsin B and beta-hexosaminidase but not cathepsins D or L. Co-treatment of cultures with the cathepsin B inhibitors CA-074 or Z-FA-FMK suppressed the cytostatic effects of TPA and activation of pro-uPA. In the absence of TPA, exogenously added cathepsin B activated pro-uPA and suppressed MCF10A-Neo proliferation. The cytostatic effects of both TPA and cathepsin B were suppressed in cells cultured in medium depleted of plasminogen/plasmin or supplemented with neutralizing TGF-beta antibody. Pretreatment with cycloheximide did not suppress the exocytosis of cathepsin B or the activation of pro-uPA. Hence, TPA activates signaling processes that trigger the exocytosis of a subpopulation of lysosomes/endosomes containing cathepsin B. Subsequently, extracellular cathepsin B initiates a proteolytic cascade involving uPA, plasminogen, and plasmin that activates serum-derived latent TGF-beta.

Transforming growth factor-beta1 suppresses atopic dermatitis-like skin lesions in NC/Nga mice.

Atopic dermatitis is a chronic, relapsing inflammatory disorder characterized by pruritic and eczematous skin lesions. Transforming growth factor (TGF)-beta1 has been implicated in the suppression of inflammatory responses. The purpose of this study is to determine whether TGF-beta1 suppresses skin lesions in a mouse model of atopic dermatitis. We used the NC/Nga strain of mice as an in vivo model of atopic dermatitis. The effects of exogenous TGF-beta1 on atopic dermatitis-like skin lesions in NC/Nga mice were evaluated clinically, histologically and immunologically. Subcutaneous injection of recombinant TGF-beta1 macroscopically suppressed eczematous skin lesions in NC/Nga mice associated with reduced serum immunoglobulin E (IgE) levels. Histological analysis showed that TGF-beta1 significantly inhibited the infiltration of inflammatory cells such as mast cells and eosinophils into the skin of NC/Nga mice. Spontaneous interferon (IFN)-gamma production from splenocytes of NC/Nga mice was down-regulated by the treatment with TGF-beta1 and neutralizing antibody against IFN-gamma inhibited skin lesions in NC/Nga mice. The inhibitory effect of TGF-beta1 on the skin lesions lasted at least 1 week after cessation of the treatment. These findings indicate that TGF-beta1 suppressed atopic dermatitis-like skin lesions in NC/Nga mice at least in part through down-regulation of IFN-gamma. These results suggest that TGF-beta1 may have a therapeutic potential for atopic dermatitis.

Renoprotective effects of valsartan and enalapril in STZ-induced diabetes in rats

Effects of the angiotensin II type 1 (AT1) receptor antagonist valsartan and the angiotensin-converting enzyme (ACE) inhibitor enalapril were studied in streptozotocine (STZ)-induced diabetes in rats on the basis of microalbuminuria (Ma) and renal morphology. Five groups of Wistar rats were used, one group was the non-diabetic control, one group consisted of untreated STZ-diabetics and 3 groups of STZ-diabetics were treated with either enalapril and/or valsartan for 30 days. Blood glucose (BG) and Ma levels, body and kidney weight and glomerular size were measured. Immunohistochemical staining with an anti-transforming growth factor-beta1 (TGF-beta1) antibody was performed as well. In STZ-diabetics, BG and Ma levels were significantly increased when compared with the non-diabetic group. Although Ma levels in the valsartan-treated group was found to be higher than those in the non-diabetics group after 15 days of treatment, in all treated diabetic groups Ma levels were significantly decreased as compared with STZ-diabetics at the end of the experiment. Thickening of the glomerular and tubular basement membranes, increased mesangial matrix and glomerular size were found in the untreated diabetic group. All these changes were less in the treated groups. A significant increase in TGF-beta1 immunoreactivity was found in glomeruli of untreated STZ-diabetics as compared with non-diabetics. Again, TGF-beta1 expression was decreased in the treated groups as compared with untreated STZ-diabetics. We conclude that valsartan and enalapril have renoprotective effects in diabetic nephropathy. A combined therapy has an advantage because lower dosages of these drugs can be used. Their beneficial effects are related to a blockade of the renin-angiotensin system (RAS) and a decrease in TGF-beta1 expression in glomeruli.

TGF-beta isoform release and activation during in vitro bronchial epithelial wound repair.

Restitution of an epithelial layer after environmental or biological damage is important to maintain the normal function of the respiratory tract. We have investigated the role of transforming growth factor (TGF)-beta isoforms in the repair of layers of 16HBE 14o(-) bronchial epithelial-derived cells after damage by multiple scoring. ELISA showed that both latent TGF-beta1 and TGF-beta2 were converted to their active forms 2 h after wounding. Time-lapse microscopy showed that the addition of TGF-beta1, but not TGF-beta2, progressively increased the rate of migration of damaged monolayers at concentrations down to 250 pg/ml. This increase was blocked by addition of a neutralizing TGF-beta1 antibody. Phase-contrast microscopy and inhibition of proliferation with mitomycin C showed that proliferation was not required for migration. These results demonstrate that conversion of latent to active TGF-beta1 and TGF-beta2 during in vitro epithelial wound repair occurs quickly and that TGF-beta1 speeds epithelial repair. A faster repair may be advantageous in preventing access of environmental agents to the internal milieu of the lung although the production of active TGF-beta molecules may augment subepithelial fibrosis.

Neurons modulate oxytocin receptor expression in rat cultured astrocytes: involvement of TGF-beta and membrane components.

We examined the effect of neurons on oxytocin (OT) receptors (OTR) and OTR gene expression in cultured astrocytes. The addition of neuron-conditioned medium induced an increase of both OTR binding and OTR mRNA level. This effect was enhanced after the medium was boiled or acidified. As it is known that transforming growth factor-beta (TGF-beta) can be released from carrier proteins by acid or heat, TGF-beta1 and 2 were tested and found to induce an increase of OTR binding. Furthermore, TGF-beta antibody abolished the stimulatory effect of normal or acidified neuron-conditioned medium. Neurons added to cultured astrocytes without contact mimicked the stimulatory effect of the conditioned medium. In contrast, neurons added with contact, induced a decrease in OTR binding and an increase of mRNA level, whereas neuronal membranes induced a decrease of both OTR binding and mRNA levels. In conclusion, the present data demonstrate that in vitro, neurons are able to modulate astrocytic OTR expression at the level of both protein and mRNA. They stimulate this expression through their release of TGF-beta and inhibit it by the action of unknown membrane components.

Role of TGF-beta in the retinoic acid-induced inhibition of proliferation and melanin synthesis in chick retinal pigment epithelial cells in vitro.

The purpose of this study was to investigate the effects of all-trans retinoic acid (RA) on the induction of transforming growth factor-beta (TGF-beta) that is concerned with the proliferation and melanin synthesis of chick retinal pigment epithelial (RPE) cells in vitro. Chick RPE cells were cultured in the presence or absence of RA and anti-TGF-beta antibody for 7 days. The effects of RA and pan-specific TGF-beta antibody on RPE cell proliferation were assessed by counting the number of cells, and their effects on melanin synthesis were evaluated by measuring the melanin content of the cells. TGF-beta activity in the culture supernatant of RPE cells was measured using CCL-64 cells. RA significantly inhibited RPE cell proliferation and increased melanin synthesis. The addition of pan-specific TGF-beta antibody to the culture blocked the inhibition of RPE cell proliferation and the increased melanin synthesis. RA induced TGF-beta production in the culture supernatant of RPE cells. These findings indicate that RA regulates the proliferation and melanin synthesis of RPE cells via induction of TGF-beta.

The role of transforming growth factor-beta in the modulation of mouse cranial suture fusion.

Transforming growth factor-beta (TGF-beta) is actively expressed during mouse calvarial suture fusion. However, the role TGF-beta plays in this process remains unclear. The present study was performed to investigate whether modulation of suture fusion can be achieved by blocking the bioavailability of TGF-beta. Both in vitro and in vivo models were studied. For the in vitro model, the posterior frontal sutures from 24-day-old mice were harvested and cultured for 2, 3, or 4 weeks in the presence of 20 microg/ml of pan-specific TGF-beta polyclonal antibody or rabbit IgG as a control. Culture media were changed every 48 hours and fresh antibody or rabbit IgG was added during each media change. Suture fusion was evaluated by histometric analysis. For the in vivo model, TGF-beta antisense plasmid DNA complexed with lipofectamine was injected into the subgaleal layer along the frontal suture of 22-day-old mice under anesthesia. For control groups, empty vector plasmid DNA+ complexed with lipofectamine was used. The posterior frontal sutures were harvested at various time points and examined by histometric analysis and reverse transcription and polymerase chain reaction for the detection of messenger RNA. The in vitro studies demonstrated that the presence of TGF-beta antibody in culture media delayed posterior frontal suture fusion. By 3 weeks in culture, new bone area was only 20 percent of that in control groups as determined by histometric analysis. By the end of the fourth week, suture fusion was only 25 percent completed compared with controls. The in vivo studies demonstrated that, compared with the control, posterior frontal suture fusion was significantly delayed in the animals injected with antisense plasmid DNA. Suture fusion was complete in control animals by postnatal day 45. There was a 70 percent inhibition of suture fusion (new bone area) in antisense groups measured with histometric analysis. Four days after antisense plasmid DNA injection (age of 26 days), messenger RNA expression for TGF-beta 1 was 77 percent lower than in the control group. We have demonstrated that modulation of TGF-beta affects calvarial suture formation in vitro and in vivo with TGF-beta antibody and TGF-beta1 antisense plasmid DNA treatment, respectively. These data indicate that TGF-beta may play a causative role in mouse posterior frontal suture fusion. Modulation TGF-beta 1 gene expression in vivo can alter the natural history of cranial suture fusion.

Transforming growth factor-beta1 expression in early biopsy specimen predicts long-term graft function following pediatric renal transplantation.

The main cause of late graft loss or declining long-term graft function is chronic allograft nephropathy (CAN), characterized by progressive interstitial fibrosis. Transforming growth factor (TGF)-beta1 plays a key role in fibrogenesis. We immunohistochemically investigated whether the degree of TGF-beta1 expression in early biopsy specimens routinely obtained from stable allografts at 100 d could predict fibrosis and graft dysfunction in the late phase. Patients were children with grafts from related donors. We immunohistochemically determined intracellular and extracellular expression of TGF-beta1 in the graft using LC antibody (LC) for intracellular TGF-beta1 and CC antibody (CC) for extracellular TGF-beta1. The change in creatinine clearance between 100 d and 3 yr after transplantation (DeltaCcr) was used as an index of long-term graft function. We also used image analysis to calculate the relative area involved by interstitial fibrosis in the trichrome-stained section of graft biopsy specimens at 100 d and 3 yr, designating the change as DeltaFI. DeltaCcr was -4.2+/-9.4 mL/min in subjects with minimal early immunoreactivity for CC and -20.5+/-15.9 mL/min in subjects with strong reactivity (p<0.05). DeltaCcr was -14.5+/-18.6 mL/min in subjects with minimal early immunoreactivity for LC and -11.7+/-12.8 mL/min in those with strong reactivity. DeltaFI in subjects with minimal CC reactivity (1.28+/-4.11%) tended to be lower than that in subjects with strong reactivity (8.45+/-15.47%). Neither fibrosis at 100 d nor DeltaFI differed between subjects with minimal and strong LC reactivity. Thus, strong extracellular TGF-beta1 expression in grafts at 100 d after transplantation is associated with a long-term decline in graft function and tends to be associated with increased graft fibrosis at 3 yr.

The expression and antigenicity identification of recombinant rat TGF-beta1 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.

Suppression of asthma-like responses in different mouse strains by oral tolerance.

In this study we examined the effect of oral antigen (Ag) administration on the development of experimental asthma in different mouse strains. We selected BALB/c, BP2, CBA/Ca interleukin (IL)-5 transgenic, and BALB/c T-cell receptor-delta-deficient mouse strains because they exhibit different aspects of the asthma syndrome. Mice exposed to 1% ovalbumin (OVA), dissolved in the drinking water for 5 consecutive days, became unresponsive to subsequent immunogenic OVA challenges. This regimen of OVA administration induced Ag-specific unresponsiveness in all mouse strains tested, including gammadelta-deficient mice that are said to be resistant to tolerance induction. The Ag-specific unresponsiveness was characterized by reduced (almost absent) airway eosinophilic inflammation, airway hyperreactivity, and mucus production; also by low levels of T helper (Th) 2-type cytokines in bronchoalveolar lavage fluid, and decreased immunoglobulin (Ig) G1 and IgE OVA-specific antibody production. The unresponsive state was not associated with increased levels of the suppressive cytokines IL-10 and transforming growth factor (TGF)-beta or with immune deviation toward the Th1 pathway due to increased levels of interferon-gamma and IL-12. Moreover, treatment with anti- TGF-beta antibodies did not abrogate oral tolerance. Oral Ag administration was quite effective in suppressing the development of key features of asthma when initiated after primary immunization (Day 0) or after booster (Day 7), but not after challenge (Day 14) when it increased allergic responses. Collectively, our findings show for the first time the beneficial and detrimental effects of oral Ag administration on the development of experimental asthma.

Are stable postoperative biliary atresia patients really stable?

Transforming growth factor-beta 1 (TGF beta-1) is an important mediator of liver-cell proliferation and replication that is implicated in hepatic fibrosis (HF). Hepatic stellate cells (HSC) are activated by TGF beta-1 and are the main precursor cells involved in fibrogenesis. The correlation between serum TGF beta-1, activated HSC in liver-biopsy specimens, and liver biochemistry was investigated to determine the value of TGF beta-1 as an indicator of clinical status in postoperative biliary atresia (BA) patients. Thirty-two postoperative BA patients (mean age 11.2 +/- 2.8 years) and 13 normal controls (mean age 10.3 +/- 3.7 years) were studied. Based on average liver function test (LFT) results over a 3-month period immediately prior to this study, the BA patients were divided into group I (anicteric, normal LFT; n = 10); group II (anicteric, elevated liver transaminases; n = 12), and group III (jaundiced end-stage liver fibrosis awaiting liver transplantation; n = 10). Serum TGF beta-1 was determined using ELISA. Liver-biopsy specimens were examined with antibody against TGF beta-1 and alpha-smooth muscle actin (SMA) antibody for detection of activated HSC. Serum TGF beta-1 was significantly higher in groups I (11.4 +/- 3.7 ng/ml; P < 0.01) and II (23.3 +/- 11.3 ng/ml; P < 0.001) than in group III (3.0 +/- 1.5 ng/ml) and controls (4.5 +/- 2.5 ng/ml) despite normal LFT in group I. The 3 subjects with the highest serum TGF beta-1 in group II had bile lakes. Biopsies from groups I and II were strongly positive for TGF beta-1 in hepatocytes and Kupffer cells and for activated HSC detected by SMA compared with group III and controls. Because serum TGF beta-1 and activated HSC are only present during active fibrosis, we conclude that there is progressive fibrogenesis even in seemingly normal postoperative BA patients. In particular, bile lakes should be regarded as a key sign of progressive HF, the presence of which should be regarded with extreme caution. We suggest that serum TGF beta-1 could be used as an accurate indicator of progressive fibrogenesis in postoperative BA patients.

TGF-beta in uveal melanoma.

Uveal melanoma is the most common primary ocular cancer among adults and patients with distant metastases seldom survive longer than a year. Melanomas of the eye have the advantage of growing in the special environment of an immune privileged site and it has long been shown, that the special immunosuppressive properties of the intraocular microenvironment are strongly mediated by cytokines, especially transforming growth factor-beta (TGF-beta). Here, we sought to investigate the presence of TGF-beta in surgically removed uveal melanoma specimens using immunohistochemical methods to verify possible autocrine mechanisms. Immunocytochemistry for pan-TGF-beta and TGF-beta(2) was performed on 13 melanoma specimens using an alkaline phosphatase labeling procedure. Melanocytic origin of the tumors was confirmed by HMB-45 staining. All tissue samples exhibited positive staining using either pan-TGF-beta or TGF-beta(2) antibody regardless of cell-type, size of the tumor, or tumor location. The intensity of staining did not vary significantly within a given tumor. All tumors stained positive against the HMB-45 antibody. Many cytokines have been found to act on melanoma tumors. The presence of the TGF-beta(2) isoform in all specimens points to progressive tumor-growth as has been shown for melanomas of the skin. Based on our immunohistochemical findings and the immunosuppressive properties of TGF-beta, we suppose that ocular melanomas should be able to create their own immunosuppressive environment even in the uvea, which might be a non-privileged site.

Phosphatidylinositol 3-Kinase Function Is Required for Transforming Growth Factor β-mediated Epithelial to Mesenchymal Transition and Cell Migration

We have studied the role of phosphatidylinositol 3-OH kinase (PI3K)-Akt signaling in transforming growth factor beta (TGFbeta)-mediated epithelial to mesenchymal transition (EMT). In NMuMG mammary epithelial cells, exogenous TGFbeta1 induced phosphorylation of Akt at Ser-473 and Akt in vitro kinase activity against GSK-3beta within 30 min. These responses were temporally correlated with delocalization of E-cadherin, ZO-1, and integrin beta(1) from cell junctions and the acquisition of spindle cell morphology. LY294002, an inhibitor of the p110 catalytic subunit of PI3K, and a dominant-negative mutant of Akt blocked the delocalization of ZO-1 induced by TGFbeta1, whereas transfection of constitutively active p110 induced loss of ZO-1 from tight junctions. In addition, LY294002 blocked TGFbeta-mediated C-terminal phosphorylation of Smad2. Consistent with these data, TGFbeta-induced p3TP-Lux and p(CAGA)(12)-Lux reporter activities were inhibited by LY294002 and transiently expressed dominant-negative p85 and Akt mutants in NMuMG and 4T1 cells. Dominant-negative RhoA inhibited TGFbeta-induced phosphorylation of Akt at Ser-473, whereas constitutively active RhoA increased the basal phosphorylation of Akt, suggesting that RhoA in involved in TGFbeta-induced EMT. Finally, LY294002 and neutralizing TGFbeta1 antibodies inhibited ligand-independent constitutively active Akt as well as basal and TGFbeta-stimulated migration in 4T1 and EMT6 breast tumor cells. Taken together, these data suggest that PI3K-Akt signaling is required for TGFbeta-induced transcriptional responses, EMT, and cell migration.