anti-TGF Beta Research Articles

Antisense Strategies in Therapy of Gliomas

The malignant brain tumour, glioblastoma, is invariably fatal with an average survival of 8-11 months (recent pharmacology has increased the median survival to 15, and rarely to 18 months). Different growth factors participate in the malignant development of the brain, especially IGF-1, EGF, TGF-beta and VEGF. Among the strategies applied to target and arrest the expression of growth factors, their receptors (tyrosine kinase) and down stream signalling pathway elements (i.e. PKC, Bcl-2, glycogen synthase) involved in cancer processes, the use of different inhibitors and especially of antisense technique has been among the leading technology proposed. Recent clinical results of glioblastomas are especially promising in situations in which antisense was used either as gene therapy (antisense anti TGF beta oligodeoxynucleotides), or as cellular gene therapy (transfection with antisense anti IGFI expression vector), following a classical surgery, radio- and chemotherapy. Using anti - IGF-I approach, median survival in glioblastoma patients has reached 21 months. The immune anti-tumour response in treated patients was confirmed by an increase of CD8+ T level in peripheral blood lymphocytes.

Suppression of cyclosporine a nephrotoxicity in vivo by transforming growth factor beta receptor-immunoglobulin G chimeric protein.

Transforming growth factor (TGF)beta is implicated in the pathogenesis of cyclosporine A (CsA) nephrotoxicity. We examined the efficacy of TGF beta receptor (R)II/immunoglobulin (Ig)G Fc, a soluble chimeric protein consisting of the extracellular domain of human TGF beta RII and IgG1 Fc, on CsA nephrotoxicity in mice. Subcutaneous injection of CsA (25 mg/kg/d) was given daily to mice maintained on a low-sodium diet. On days 1 and 7, an expression vector carrying cDNA for either TGF beta RII/IgG Fc or beta-galactosidase was transfected into the skeletal muscles by electroporation. At 2 or 3 weeks of CsA administration, plasma and renal TGF beta 1 levels, and tubulointerstitial injury and fibrosis were evaluated. After 2 weeks of CsA administration, plasma and renal TGF beta 1 levels increased to the maximum and then declined toward the baseline levels. Renal TGF beta 1 mRNA remained elevated until 3 weeks. Tubulointerstitial alterations became appreciable in 2 weeks and intensified by 3 weeks. At 2 weeks, the TGF beta RII/IgG Fc intervention abolished the increase in plasma TGF beta 1, attenuated the increase in renal TGF beta 1 by 50%, and markedly suppressed the histologic alterations. At 3 weeks, the histologic alterations remained markedly suppressed by the intervention, with no appreciable effects on the renal TGF beta 1 mRNA and protein. The introduction of TGF beta RII/IgG Fc by gene transfer effectively abrogated CsA-induced tubulointerstitial alterations. Suppression of tubulointerstitial changes was evident at 3 weeks when renal TGF beta 1 mRNA and protein were comparable to those with CsA alone, indicating that early anti-TGF beta intervention is effective in suppressing the progression of CsA nephrotoxicity despite persistent increases in renal TGF beta 1 expression.

Mechanisms of tolerance induced by TGF beta-treated APC: CD4 regulatory T cells prevent the induction of the immune response possibly through a mechanism involving TGF beta.

Transforming growth factor beta (TGF beta)-treated antigen-presenting cells (APC) pulsed with antigen induce tolerance in mice, i.e. inhibition of IFN-gamma production and delayed type hypersensitivity response. Although evidence suggests that regulatory T cells are involved, their mechanism of action is currently unknown and is the subject of the present study. Both CD4 and CD8 splenic T cells from mice injected i.v. with adherent thioglycolate-elicited peritoneal exudate cells cultured with TGF beta(2) and antigen (TGF beta-treated APC) transferred tolerance to naive recipients. Interestingly, TGF beta-treated APC from class II knockout mice were unable to induce tolerance in wild-type mice, whereas wild-type TGF beta-treated APC could induce tolerance in CD8 knockout mice. TGF beta was detected in cultures of lymphoid cells from mice injected with TGF beta-treated APC, and treatment with anti-TGF beta antibody in vivo impaired tolerance induction. TGF beta appeared to be involved in both the development of CD4 regulatory T cells and the effector function of the CD4 regulatory T cells. In summary, the important findings in this study are that CD4, and not CD8, regulatory T cells are required for tolerance induced by TGF beta-treated APC in naive mice, and tolerance appears to be mediated by a mechanism involving TGF beta.

Les interférons ont-ils une action antifibrosante ?Le point de vue de l'interniste

Purpose. -The antifibrotic action of interferon alpha on the liver is now established in hepatitis C (cf. article by T. Poynard). Numerous in vitro experimental evidences may lead people into believing in the efficacy of interferon gamma in patients with mucous membrane and pulmonary fibrosis. Current knowledge and key points. -A randomized study has confirmed the efficacy of interferon gamma in the idiopathic pulmonary fibrosis, probably by an anti TGF beta action. Several open studies have shown the efficacy of interferon gamma in patients with post-radiation cutaneous fibrosis (Chernobyl survivors, sequelae of radiotherapy for cancer). In systemic sclerosis, several teams have shown that interferon gamma reduced collagen synthesis by sclerodermic fibroblasts in vitro. Four open studies have also confirmed the efficacy of interferon gamma in systemic sclerosis. In our experience concerning 20 patients with diffuse systemic sclerosis, the five years survival with long term therapy was 85% and we observed an improvement in the cutaneous suppleness in 40% of these cases. There was no serious side effect. Future prospects and projects. -These encouraging results need to be confirmed by large randomized studies in pulmonary fibrosis and systemic sclerosis. Other indications should be assessed, particularly extra-cutaneous radiation sequelae and in systemic fibrosis (retroperitoneal, mediastinal and cervical).

A novel minimized fixed-bed cultivation system for hematopoietic cells

Abstract Ex-vivo expansion of primary human hematopoietic progenitor and stem cells is an important technique for clinical applications in cancer and gene therapies. The importance of stromal cells for the cultivation of hematopoietic cells has been shown in numerous studies. Therefore we developed a cultivation system for immobilized co-culture of hematopoietic and stromal cells 1) and for screening purposes a miniaturized parallel system, the so-called miniaturized loop-bioreactor 2) . These cultivation systems are based on the mimikry of the natural compartment of the hematopoieses, the bone marrow. The in vivo -like co-cultivation with stromal cells obtains important biological effects like cell contact and the production of cytokines and extracellular matrix. The immobilization of the cells on macroporous collagen microcarriers simulates the three-dimensional structure of bone marrow. And the optimal feeding of the cells is ensured by the continuous perfusion in the reactor. The miniaturized loop-bioreactor consists of a special stainless steel insert and a magnetic stirbar that are placed in a conventional 12-well culture plate. The microcarriers are placed as a fixed bed in this insert and are flown through with medium by the agitation of the magnetic stirbar. It is possible to investigate in this system twelve parallel experiments. By co-culturing CD34 + cells purified from umbilical cord blood with the irradiated murine stromal cell line S1/S1 in the miniaturized loop-bioreactor we have optimized different parameters for the co-cultivation. The experiments were performed over 7 days under standardized conditions with serum containing medium (IMDM) supplemented with IL-3, TPO, FLT-3-ligand, SCF and anti TGF-beta. In first experiments different cell densities on the carrier were analyzed. The optimal cells density ranged from 2.5* 10 4 to 1* 10 5 CD34 + cells per reactor system. The cell ratio between CD34 + and stromal cells plays an important role in the support of hematopoiesis. The optimal ratio of 1:10 between these cell types was determined by expansion of the CAFC. Another relevant cultivation parameter is the feeding rate. Frequent feeding improved significantly the expansion of MNC, whereas the highest rate of 1/2 reactor volume per day, showed the best results. Based on this parameter optimization we have cultivated CD34 + cells in co-culture with S1/S1 (ratio 1:10) and without stroma, both with a feeding rate of 1/2 medium exchange per day for two weeks in the miniaturized loop-reactor. The positive effect of the stromal cells is demonstrated by a strong increase in the expansion of MNC (454 fold vs 180 fold stroma free) and CFC (26 fold day 10). The stroma containing system generated also the greater CAFC output compared to the stroma free system (4.5 fold vs 2 fold). These results indicate that the novel miniaturized loop-reactor is a very suitable system for the cultivation of primary human hematopoietic cells and for the screening of various culture parameters. Using this system the positive effect of a stromal based and frequently fed cultivation strategy was proven. 1) P. Meissner, B. Schroder, C. Herfuth, M. Biselli. Development of a fixed bed bioreactor for the expansion of human hematopoietic progenitor cells. Cytotechnology, Vol. 30, pp. 227-234, 1999. 2) Schmidt, S., Jelinek, N., Biselli, M.; patient application DE: 198 22 050 A1.

Phorbol ester-induced production of cytostatic factors by normal and oncogenic Ha- ras -transformed human breast cell lines

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on cell cycle progression were examined in the human breast cell line MCF10A-Neo and a derivative line which expresses a Ha-ras oncogene (MCF10A-NeoT cells). Exposure of MCF10A-Neo cultures to TPA induced a G(1) arrest that lasted approximately 16-24 h (IC(50) approximately 0.5 nM). TPA-treated cultures produced a cytostatic conditioned medium. Cytostatic activity was detectable within 1 h of TPA treatment, peaked 3-7 h later and disappeared between 16 and 24 h post-treatment. However, cytostatic conditioned medium could be quickly regenerated by re-feeding previously treated cultures with new medium. Removal of latent transforming growth factor beta (TGF beta) from the culture medium, supplementing the culture medium with anti-TGFbeta or soluble TGF beta(II) receptor, or pre-absorption of conditioned medium with anti-TGF beta all reduced the cytostatic effects of TPA or conditioned medium on MCF10A-Neo proliferation by approximately 50%. Co-treatment with the serine protease inhibitors aprotinin or plasminogen activator inhibitor-1 also suppressed the cytostatic activity of TPA approximately 50%. Conditioned medium isolated from TPA-treated MCF10A-Neo cultures was transiently cytostatic to MCF10A-NeoT cells. The proliferation of MCF10A-NeoT cultures, in contrast to MCF10A-Neo cells, was suppressed at least 72 h following TPA exposure. Conditioned medium isolated from TPA-treated MCF10A-NeoT cultures also suppressed MCF10A-NeoT proliferation for approximately 72 h, but suppressed MCF10A-Neo proliferation for <24 h. These studies suggest that TPA quickly activates proteolytic processes in MCF10A-Neo cells leading to the activation of latent TGF beta supplied by the serum in the culture medium. TPA also stimulates the production of an additional cytostatic factor(s) which signals via a mechanism not involving the TGF beta(II) receptor. Lastly, expression of an activated Ha-ras oncogene alters both the types of cytostatic factors produced following TPA treatment and responsiveness to these factors.

Overexpression of c-Met/hepatocyte growth factor receptors in human prostatic adenocarcinoma.

Hepatocyte growth factor (HGF) and c-met proto-oncogene product (c-Met) have varied biological functions in different tissues and have been implicated in mitogenic, motogenic and morphogenic responses in both organ regeneration and carcinogenesis. Some studies have suggested that the overexpression of c-Met and epidermal growth factor receptor (EGFR) are associated with growth advantage, while transforming growth factor-beta receptor II (TGF beta R II) is associated with growth disadvantage of human prostatic adenocarcinoma. However, it is unclear if the expression of c-Met correlates with the expression of EGFR and TGF beta R II, and with the proliferative status of human prostatic adenocarcinoma. Using immunohistochemical staining with anti-c-Met (C-12), anti-EGFR (NCL-EGFR) and anti-TGF beta R II (L-21) antibodies, we determined the frequency of expression of c-MET, EGFR, and TGF beta R II respectively in a series of 134 radical prostatectomy specimens. We evaluated the relationship between the expression of these receptors and clinicopathological characteristics. Overall, c-Met immunostaining was detected in 54 of 134 (40.3%) cases, EGFR in 45 (33.6%) and TGF beta R II in 64 (48.4%). The overexpression of c-Met was significantly more common in poorly differentiated (P < 0.0001) and in the diffusely infiltrated specimens (P < 0.0005). In contrast, TGF beta R II was significantly overexpressed in the well differentiated specimens (P < 0.0001) and associated negatively with c-Met (P < 0.0001). Overall, these data suggest that c-Met/HGF receptor and TGF beta R II overexpression may be involved in the differentiation of human prostatic adenocarcinoma, c-Met with de-differentiation and TGF beta R II with differentiation.

Cytokine-mediated regulation of 92-kilodalton type IV collagenase, tissue inhibitor or metalloproteinase-1 (TIMP-1), and TIMP-3 messenger ribonucleic acid expression in human endometrial stromal cells.

Interleukin-1 (IL-1) is expressed in human endometrium and has been shown to play an integral role in local cellular interactions during implantation. In addition, the matrix metalloproteinase (MMP) and its inhibitor, the tissue inhibitor of metalloproteinase (TIMP), are crucial during implantation, mediating in vitro trophoblast penetration, and are regulated by several cytokines expressed by trophoblast cells. We have investigated the roles of IL-1 beta and transforming growth factor-beta (TGF beta) in regulating TIMP-1, TIMP-3, and 92-kDa type IV collagenase messenger ribonucleic acid (mRNA) expression in human endometrial stromal cells using quantitative competitive PCR. Confluent stromal cell cultures treated with progesterone and estradiol for 9 days were stimulated with IL-1 beta, IL-1 beta plus anti-IL-1 beta antibody, TGF beta, and TGF beta plus anti-TGF beta antibody for an additional 24 h. Competitive complementary DNA fragments were constructed by deletion of a defined fragment from each of the target complementary DNA sequences and coamplified in quantitative competitive PCR as an internal standard. TIMP-1 and TIMP-3, but not 92-kDa type IV collagenase mRNA, were expressed in stromal cells. The 92-kDa type IV collagenase mRNA was only expressed after stimulation with IL-1 beta. IL-1 beta both augmented 92-kDa type IV collagenase mRNA expression and decreased TIMP-1 and TIMP-3 mRNA expression in a dose-dependent manner. Conversely, TGF beta augmented TIMP-1 and TIMP-3 mRNA expression, but did not affect 92-kDa type IV collagenase expression. IL-1 and TGF beta-mediated changes were both neutralized by specific antibodies. These results provide indirect evidence that IL-1 and TGF beta may play crucial roles at the embryo-maternal interface during trophoblast invasion by regulating stromal cell expression of TIMP-1, TIMP-3, and 92-kDa type IV collagenase, all of which are known to be important in trophoblast invasion.

Molecular and cellular basis of radiation fibrosis

Purpose: Recent data from the literature and the experimental work of the authors clearly indicate that TGF- beta 1 is a key modulator of cellular events, for example, induction of terminal differentiation, resulting in radiation-induced fibrosis. Therefore, the present study analysed which cellular processes induced by exogeneously added TGF- beta could be responsible for the induction, development and manifestation of the fibrotic phenotype in culture. Materials and methods: Rat lung fibroblast cultures (passage 1) were used. As a function of treatment with TGF- beta and/or anti-TGF beta -antibody, the clonogenic activity and differentiation pattern were analysed by colony-formation assays. Results: It could be demonstrated that treatment of rat lung progenitor fibroblasts with TGF- beta 1 resulted in a pronounced shift in the differentiation pattern, i.e. induction of post-mitotic fibrocytes. This TGF- beta 1-dependent terminal differentiation could be abolished by simultaneous treatment with a neutralizing antibody directed against TGF- beta 1. Conclusions: The data presented indicate that TGF- beta 1 is one major candidate mediating the accelerated terminal differentiation of progenitor fibroblasts to post-mitotic functional fibrocytes, which results in the fibrotic phenotype of this cell system.

Shear stress as an inhibitor of vascular smooth muscle cell proliferation. Role of transforming growth factor-beta 1 and tissue-type plasminogen activator.

We examined whether shear stress can inhibit vascular smooth muscle cell (VSMC) proliferation in vitro directly. Human VSMCs were exposed to fluid flow for 24 hours using a cone-plate apparatus, and their proliferation was inhibited significantly by shear stresses of 1.4 and 2.8 Pa (14 and 28 dyne/cm2), according to the magnitude. Next, we investigated whether transforming growth factor-beta 1 (TGF beta 1), which is known to be an important cytokine that suppresses VSMC proliferation, is the predominant mediator of shear-induced inhibition of VSMC growth. After exposure of VSMCs to shear stress (2.8 Pa) for 24 hours, gene expression of TGF beta 1 and, interestingly, tissue-type plasminogen activator, which converts plasminogen to plasmin, an activator of TGF beta 1, increased twofold and fivefold, respectively. The levels of both latent and active forms of TGF beta 1 in conditioned media of VSMCs exposed to fluid flow increased significantly. An anti-TGF beta 1 antibody reversed shear-induced inhibition of VSMC growth significantly. We concluded that shear stress inhibited VSMC proliferation in vitro and this inhibition was mediated predominantly by TGF beta 1 in an autocrine manner. These data suggest that shear stress plays an important role as an inhibitor of atherogenesis in endothelium-desquamated lesions.

Transurethral Bladder Tumor Resection Alters Fibronectin Expression in Transitional Carcinoma Cell Lines

To examine the effect of TGF beta 1 and post-transurethral resection of bladder tumor (TURBT) urine on fibronectin (FN) expression by transitional carcinoma (TCC) cell lines. Northern analysis and rtPCR for mRNA transcripts, in combination with ELISA and immunohistochemistry for protein, were used to quantify the effect of TGF beta 1 and post-TURBT urine on cellular expression of FN in two TCC lines, 253J and 647V. The TGF beta 1-dependent nature of the response seen in post-TURBT urine was confirmed using exogenous TGF beta 1 or blocking antibodies to TGF beta 1. Active and latent levels of TGFb1 were measured in post-TURBT urine and urine "spiked" cell culture media. Baseline FN expression was low in the poorly differentiated, metastatic 253J line and high in the moderately differentiated 647V line. TGF beta 1 and post-TURBT urine increased FN expression at both the mRNA and protein levels in the 253J line. Anti-TGF beta 1 antibodies inhibited the effect of post-TURBT urine on FN expression in 253J cells. High levels of TGF beta 1 decreased FN expression in 647V cells. Only latent TGF beta 1 was present in samples of urine and media. FN production in low expressing TCC lines can be augmented by exogenous TGF beta 1. TURBT increases latent urinary TGF beta 1 which in turn affects TCC FN expression. Altered tumor expression of FN following TURBT may influence the effectiveness of intravesical BCG.

Inhibition of Corneal Fibrosis by Topical Application of Blocking Antibodies to TGF?? in the Rabbit

Previous studies have shown that TGF beta 1 induces activation and myofibroblast transformation of cultured rabbit corneal keratocytes. To determine whether TGF beta has a similar function in vivo, we evaluated the effect of TGF beta-blocking antibodies on corneal fibrosis after lamellar keratectomy (LK) in the rabbit. A total of 51 rabbits received standard LK wounds, and eyes were treated with 50 microliters of Celluvisc/PBS, containing 10, 50, or 100 micrograms of 1D11, a mouse monoclonal anti-TGF beta-blocking antibody. Control wounds received either 100 micrograms of an irrelevant mouse monoclonal antibody or vehicle alone. At days 14, 28, 42, and 56, eyes were evaluated by in vivo confocal microscopy (CM) and the mice were killed for light microscopy (LM) and immunostaining with antibodies to human fibronectin. In vivo CM of LK wounds clearly identified a disorganized layer that contained irregularly arranged fibroblasts and reflective extracellular matrix overlying normal corneal stroma. In a subset of 11 eyes stained with 5-(4,6-dichlorotriazinyl) aminofluorescein (DTAF) immediately after injury, the thickness of the disorganized layer identified by in vivo CM significantly correlated with both anterior corneal fibrosis (r = 0.627; p < 0.025) and depth of keratocyte activation (r = 0.8980; p < 0.0005), indicating that in vivo CM can be used quantitatively to assess anterior stromal fibrosis. In eyes treated with an irrelevant monoclonal antibody, in vivo corneal fibrosis averaged 100 +/- 26 microns thick at day 14, whereas treatment with 10, 50, and 100 micrograms anti-TGF beta significantly reduced (p < 0.0005) the anterior disorganization in a dose-dependent fashion to 101 +/- 32, 45 +/- 11, and 56 +/- 18 microns, respectively. Semiquantitative measurement of anti-fibronectin staining within the wound revealed that anti-TGF beta significantly reduced the intensity of anti-fibronectin staining in the anterior 50 microns of the corneal stroma (p < 0.003). These findings indicate that TGF beta plays an important in vivo role in keratocyte activation and myofibroblast transformation. Furthermore, the in vivo use of TGF beta-blocking antibody effects may allow modulation of corneal fibrosis after refractive surgery.

ACTH angiotensin II and TGF beta participate in the regulation of steroidogenesis in bovine adrenal glomerulosa cells.

Bovine zona glomerulosa cells, on the first day of culture, produce aldosterone as their major steroid with no detectable cortisol secretion. Continuous incubation with ACTH had no effect on aldosterone production nor on aldosterone synthase activity. This treatment resulted in a dose and time dependent rise in 17 alpha-hydroxylase activity, in parallel with an increase in cytochrome P-450(17 alpha) (CYP17) protein and mRNA. We have previously shown that TGF beta 1 is a potent inhibitor of differentiated functions of bovine fasciculata-reticularis cells and that CYP17 and AII receptors are the major targets explaining this effect. The present study examined whether 17 alpha-hydroxylase activity in glomerulosa cells could be regulated by angiotensin II (AII) and transforming growth factor-beta 1 (TGF beta 1). AII inhibits the induction of CYP17 by ACTH in a dose dependent manner. TGF beta 1 also blocks almost completely the stimulatory effect of ACTH. In order to suppress the endogenous action of TGF beta 1, incubations were performed with an anti-TGF beta antibody. This specific antibody induces the expression of CYP17 resulting in increased activity and mRNA levels. These results show that AII is able to modulate the expression of CYP17 in adrenal glomerulosa cells following ACTH stimulation. Furthermore, TGF beta 1 exerts an autocrine effect on the differentiation of glomerulosa cells through a regulatory loop repressing CYP17 activity.

Increased renal tubular expression of transforming growth factor beta in human allografts correlates with cyclosporine toxicity

Cyclosporine A (CsA) is a potent immunosuppressive drug that inhibits the transcription of several proinflammatory cytokines including interleukin-2. In contrast, CsA stimulates transcription of the pleuripotent cytokine, transforming growth factor-beta (TGF beta). Since the effect of CsA in transplant recipients is unpredictable, we examined whether tissue levels of TGF beta protein in renal allografts correlate with in vivo CsA responsiveness. Intra-allograft TGF beta protein content was assessed in renal biopsies by immunohistochemical means using the mouse anti-TGF beta monoclonal antibody (Mab), 1D11. We studied 68 specimens: 21 with acute CsA toxicity (ACT), 11 with acute tubular necrosis (ATN) and 36 with acute cellular rejection (ACR). Intensity of TGF beta immunostaining was evaluated in a blinded fashion using a scale from 0 to 3+. In biopsies with histological evidence of CsA toxicity, 77% demonstrated intense (2 to 3+) TGF beta immunostaining. TGF beta protein was detected in both proximal and distal tubules but was either absent or present in low levels within glomeruli and interstitium. In contrast, only one of the 11 biopsies with ATN had minimal staining (1+) for TGF beta. The remaining 10 biopsies with ATN were negative for TGF beta immunostaining. In biopsies with ACR, the levels of renal TGF beta were more variable with 36% showing intense (2 to 3+) staining and 64% having minimal or no (0 to 1+) tubular TGF beta. Within the first 18 months post-transplantation, patients with intense TGF beta staining and ACR underwent an average of 4.1 +/- 1.8 allograft biopsies and suffered 33% graft losses. During the same period of time, the patients with ACR and absent or low (0 to 1+) TGF beta levels underwent only 2.1 +/- 1.2 biopsies, maintained better late renal function and suffered 4% graft losses. In conclusion, we demonstrate that TGF beta protein levels in renal allografts correlate with CsA effect and differentiate ACT from ATN. In CsA treated patients who develop ACR, TGF beta levels predict the subsequent clinical course and graft function. Therefore, evaluating tissue levels of TGF beta may offer unique diagnostic and prognostic benefits in the care of patients receiving CsA based immunosuppression.

Hypertrophy of rabbit proximal tubule cells is associated with overexpression of TGF beta.

The expression of transforming growth factor beta (TGF beta) in proximal tubule cells from rabbit kidney cortex after uninephrectomy (UNX) was investigated. Cell protein and [3H]leucine incorporation in these cells were significantly increased, while cell number was decreased, at two weeks following UNX. At this time period after UNX, we found that proximal tubule cells showed a dramatic increase of cytoplasmic immunostaining with a pan-specific anti-TGF beta antibody. This was accompanied by a 3-fold increase in TGF beta 1 mRNA expression in these cells. Furthermore, proximal tubule cells from two-week uninephrectomized rabbits secrete about 2-fold higher TGF beta bioactivity to the cell conditioned medium compared to cells from sham-operated animals. Addition of anti-TGF beta 1, beta 2, beta 3 neutralizing antibody increased the growth of the former cells, and it abolished cell hypertrophy. These results indicate that hypertrophy of proximal tubule cells during compensatory renal growth is associated with overexpression of TGF beta.

Effects of Basic FGF and TGF??1 on F-Actin and ZO-1 Organization During Cat Endothelial Wound Healing

Previous studies suggest the existence of two separate and distinct mechanisms of endothelial wound healing (i.e., cell migration and cell spreading), which may be controlled by unique, injury-dependent, wound-related factors. The purpose of our study was to evaluate potential biologic mediators regulating healing of the growth arrested cat endothelium by using an ex vivo, organ-culture model. Three buttons were punched from each cornea of 11 cats with a 6-mm trephine. A 1- to 2-mm diameter endothelial scrape injury (SI) was made, and buttons were cultured in (a) serum-free media (SFM), (b) serum plus media (20% fetal calf serum), (c) SFM plus basic fibroblast growth factor (bFGF), (d) SFM plus bFGF and heparin, (e) SFM plus transforming growth factor-beta 1 (TGF beta 1), or (f) SFM plus TGF beta 1 and anti-TGF beta 1. At various times from 8-48 h after injury, buttons were stained with phalloidin and anti-ZO-1, and imaged by using laser scanning confocal microscopy. Evaluation of SI in cat corneal buttons under serum-free conditions showed maintenance of normal endothelial differentiation, indicating that the organ-culture SI model mimics in vivo SI. Addition of TGF beta 1 produced a dramatic reorganization of apical F-actin and development of stress fibers, as well as the loss of normal cell border-associated ZO-1 distribution. The effects of TGF beta 1 were blocked by the neutralizing antibodies to TGF beta 1. Addition of serum or bFGF produced much less pronounced changes in F-actin and ZO-1 distribution. These results suggest that TGF beta 1 may play a critical role in modulating the wound-healing response of the corneal endothelium.

Inhibition and prevention of monosodium urate monohydrate crystal–induced acute inflammation in vivo by transforming growth factor β1

We investigated the effects of transforming growth factor beta 1 (TGF beta 1) on monosodium urate monohydrate (MSU) crystal-induced acute inflammation in vivo. One hour after MSU crystal-induced acute inflammation was produced in the rat subcutaneous air pouch model, the effects of recombinant human TGF beta 1 (rHuTGF beta 1; 10-100 pg/animal) and ultrapure TGF beta 1 (UPTGF beta 1; 100 and 500 pg/animal) were assessed, based on absolute and differential white blood cell counts in the exudate. The effects of 10 pg of rHuTGF beta 1 preincubated with a specific anti-TGF beta antibody, and the effects of coinjection of crystals and rHuTGF beta 1, were also studied. UPTGF beta 1 and rHuTGF beta 1 markedly reduced MSU crystal-induced inflammation. Recombinant human TGF beta 1 also reduced inflammation when administered concomitantly with MSU crystals. Moreover, rHuTGF beta 1 and UPTGF beta 1, injected 1 hour after MSU crystal injection, reduced the inflammatory response in a dose-dependent manner. Injection of rHuTGF beta 1 (100 pg/animal) resulted in a > 90% reduction in the maximal white blood cell count, achieved 6 hours after crystal injection. Preincubation of rHuTGF beta 1 with a specific anti-TGF beta 1 antibody significantly (P < 0.01) reversed the inhibitory effect of rHuTGF beta 1 on the inflammatory response. Consistent with the regulation of inflammatory cell recruitment into the joint, the percentage of monocytes markedly decreased (P < 0.01) following local injection with rHuTGF beta 1 6 hours after MSU crystal injection. Exogenous TGF beta 1 prevents and inhibits MSU crystal-induced acute inflammation in vivo. Its role in the self-limitation of gouty attacks deserves consideration, among the various other factors involved.

Modulation of B16 melanoma growth and metastasis by anti-transforming growth factor beta antibody and interleukin-2.

Earlier evidence suggests that transforming growth factor beta (TGF beta) plays a significant role in tumor progression and metastasis. The most likely mechanism of the action of TGF beta is induction of immunosuppression in the host, allowing for unchecked tumor growth and metastasis. We attempted to test that hypothesis and to compare antitumor effects of anti-TGF beta antibody alone and in combination with interleukin-2 (IL-2). Six- to 8-week-old female C57B1-6 mice were induced with murine B16 melanoma by tail vein injection. Therapy was started 48 h after tumor injections. Monoclonal anti-TGF beta antibody (2G7) was administered intraperitoneally (i.p.) at 500 micrograms every other day, and IL-2 at 10,000 U i.p. twice daily, for 21 days. A threefold decrease in the number of lesions in the anti-TGF beta/IL-2 treatment group compared with the control group was observed, a highly significant statistical difference (p = 0.002). No statistically significant differences were seen between the control group and other studied groups (IL-2 alone, anti-TGF beta alone). Analysis of TGF beta levels in plasma by the TGF beta-1 Quantikine assay indicated normal levels in the control and IL-2 groups, and significantly diminished levels in the two groups that received TGF beta antibody. However, acid-ethanol extraction of plasma (to reverse antibody binding before assay) showed normal plasma TGF beta levels in all groups, suggesting that the antibody may alter the availability of TGF beta in vivo. Microscopic analysis of metastases revealed a decrease in the average size of lesions in the groups treated with IL-2. Thus, combination therapy using anti-TGF beta antibody and IL-2 may be a novel, less toxic approach to tumor immunotherapy.

TGF-beta contributes to the shift toward Th2-type responses through direct and IL-10-mediated pathways in tumor-bearing mice.

In previous reports, we showed that tumor-derived TGF-beta induced overproduction of IL-10, and these suppressive cytokines caused macrophage suppression in EL4-bearing mice. Proliferation of T cells from EL-4, but not IL-2, whereas T cells from normal mice were responsive to IL-2. A balance between Th1- and Th2-type cytokine production in EL4-T in response to anti-CD3 Ab or phorbor myristate acetate plus A23187 shifted toward the Th2 dominant pattern. The prevention of TGF-beta and IL-10 activates in vivo by administration of anti-IL-10 Ab (anti-IL-10) or anti TGF-beta Ab (anti-TGF-beta) resulted in the reduction in EL4-T of both IL-4 dependent proliferation and Th2-dominant cytokine production induced by anti-CD-3 stimulation. In addition, the anti-TGF-beta treatment resulted in complete restoration in EL4-T of suppressed IL-2 responsiveness, IL-2R expression, and Th1-type cytokine production, whereas the anti-IL-10 treatment produced partial recovery. These results lead us to conclude that TGF-beta drives the shift in the Th1/Th2 balance toward Th2 via IL-10-mediated development of the Th2 responses and via inhibition of the Th1-type responses directly in EL4-bearing mice.

Restoration of interleukin-2 production in tumor-bearing rats through reducing tumor-derived transforming growth factor beta by treatment with bleomycin.

We studied mechanisms of immunosuppression caused by tumor-derived transforming growth factor-beta (TGF beta) and restoration of the immune response by treatment with bleomycin in rats bearing KDH-8 hepatoma. Interleukin-2 (IL-2) production from splenocytes of KDH-8-tumor-bearing rats progressively decreased as the KDH-8 tumor grew. IL-2 production from concanavalin-A-stimulated normal rat splenocytes was significantly inhibited by in vitro cultured KDH-8-tumor-cell-conditioned medium; this inhibition could be blocked by neutralizing the conditioned medium with anti-TGF beta antibody. TGF beta activities were found in KDH-8-tumor-tissue-conditioned medium without acid treatment and were found in tumor-cell-conditioned medium after acid treatment; TGF beta mRNA and TGF beta protein were found in cultured KDH-8 tumor cells. These results suggested that the KDH-8-tumor-derived TGF beta might be involved in the inhibition of IL-2 production from splenocytes. To determine whether bleomycin chemotherapy could reduce tumor-derived TGF beta and restore the immune responses, we treated KDH-8 tumor-bearing rats with bleomycin (5 mg/kg, one shot) at an appropriate time (before the occurrence of immunosuppression) resulting in a significant reduction of TGF beta activity in KDH-8 tumor tissues and restoration of IL-2 production from splenocytes of tumor-bearing rats; KDH-8 tumor growth ultimately regressed. In vitro experiments also showed that TGF beta activity, mRNA expression, and protein synthesis in KDH-8 tumor cells were reduced by bleomycin treatment, and that bleomycin-treated-KDH-8-tumor-cell-conditioned medium did not inhibit IL-2 production from normal rat splenocytes. These results suggest that bleomycin treatment restored IL-2 production in tumor-bearing rats through reducing the tumor-derived TGF beta.