Titration of non-replicating adenovirus as a vector for transducing active TGF-beta1 gene expression causing inflammation and fibrogenesis in the lungs of C57BL/6 mice

Investigators have shown that interstitial pulmonary fibrosis (IPF) can be induced in rats by overexpressing transforming growth factor beta1 (TGF-beta1) through a replication-deficient recombinant adenovirus vector instilled into the lungs (Sime et al. 1997). We have shown that this vector induces IPF in fibrogenic-resistant tumour necrosis factor alpha-receptor knockout (TNF-alphaRKO) mice (Liu et al. 2001). The object of our studies is to understand how peptide growth factors, such as TGF-beta1, mediate interstitial lung disease (ILD). To do so, we must be able to manipulate the dose of the factor and sort out its effects on multiple other mediators in the lung parenchyma. As a step in this complex process, in the studies reported here, we have determined the concentrations of the recombinant adenovirus vector carrying the gene for porcine active TGF-beta1 (AVTGFbeta1) that have little apparent effect, cause clear induction of disease, or severe disease. The disease largely resolves by 28 days in all cases, thus providing a valuable model to understand the mechanisms of the IPF that is mediated, at least in part, by TGF-beta1. The findings here show that 10(6) plaque-forming units (pfu) of AVTGFbeta1, provide essentially a 'no-effect' dose, but even this amount of TGF-beta1 causes a significant increase in whole-lung collagen by day 28 after treatment. In contrast, 10(8) and 10(9) pfu cause severe IPF in 4 days, whereas 10(7) and 5 x 10(7) are intermediate for all parameters studied, i.e. TGF-beta protein, inflammatory cells, cell proliferation, pro-alpha 1(I) collagen gene expression and whole-lung collagen accumulation, and expression of growth factors such as TGF-beta1, TNF-alpha and PDGF-A and -B. Interestingly enough, TGF-beta1, as a potent blocker of epithelial cell proliferation, appears to suppress airway epithelial cell growth that would be expected during the inflammatory phase of IPF. Thus, this model system helps us to understand some quantitative aspects of TGF-beta1 biological activity and allows us to manipulate this potent factor as a mediator of interstitial fibrogenesis.     

Heterogeneous response of nasal and lung fibroblasts to transforming growth factor-β1

Background Nasal polyposis is characterized by marked oedema, sparse extracellular matrix (ECM) and proliferating blood vessels. Pulmonary fibrosis is characterized by inflammatory cells accumulation, considerable ECM deposition and vascular abnormalities. Although lung fibrosis is not only and necessarily an inflammatory disorder, we hypothesized that the difference between nasal polyposis and pulmonary fibrosis may, in part, be due to the heterogeneity between nasal and lung fibroblasts. Fibroblasts participate in the inflammatory response by releasing ECM proteins and cytokines. TGF‐β is thought to participate in chronic inflammation and fibrosis. Myofibroblasts are the activated form of fibroblasts. A phenotypic hallmark of myofibroblasts is the expression of smooth muscle α‐actin (SMA). Objective We examined whether there is any heterogeneity between nasal and lung fibroblasts upon stimulation with TGF‐β₁ with regard to the synthesis of SMA, pro‐collagen type I and vascular endothelial growth factor (VEGF) as well as translocation of Smad proteins. Methods Fibroblasts lines were established from human biopsy tissue. The expression of SMA, pro‐collagen type I, VEGF mRNA was evaluated by reverse transciptase RT‐PCR. The amount of pro‐collagen type I and VEGF was measured by ELISA. By immunocytochemistry, we analysed the expression of SMA and Smad2, 3, 4 in cultured fibroblasts. Results TGF‐β₁ induced SMA and pro‐collagen type I synthesis in lung, but not in nasal fibroblasts. By contrast, TGF‐β₁ induced VEGF synthesis in both lung and nasal fibroblasts. After stimulation with TGF‐β₁, Smad2, 3, 4 were translocated from the cytoplasm to the nucleus in lung fibroblasts, whereas only Smad3 was translocated in nasal fibroblasts. Conclusion These results establish the heterogeneous responsiveness of fibroblast populations in the airways to TGF‐β₁ and that such a heterogeneity may contribute, at least in part, to the different pathological outcomes of inflammation in the upper and lower airways.     

Soluble HLA‐I‐mediated secretion of TGF‐β1 by human NK cells and consequent down‐regulation of anti‐tumor cytolytic activity

Soluble HLA class I (sHLA‐I) molecules can regulate survival of NK cells and their anti‐tumor killing activity. Herein, we have analysed whether interaction of sHLA‐I with CD8 and/or different isoforms of killer Ig‐like receptors (KIR) induced secretion of transforming growth factor (TGF)‐β1. CD8⁺KIR^? NK cell clones secreted TGF‐β1 upon the interaction of sHLA‐I with CD8 molecule. sHLA‐Cw4 or sHLA‐Cw3 alleles engaging inhibitory isoforms of KIR, namely KIR2DL1 or KIR2DL2, strongly downregulated TGF‐β1 production elicited through CD8. On the other hand, sHLA‐Cw4 or sHLA‐Cw3 alleles induced secretion of TGF‐β1 by ligation of stimulatory KIR2DS1 or KIR2DS2 isoforms. TGF‐β1 strongly reduced NK cell‐mediated tumor cell lysis and production of pro‐inflammatory cytokines such as TNF‐α and IFN‐γ. Also, TGF‐β1 inhibited NK cell cytolysis induced by the engagement of stimulatory receptors including NKG2D, DNAM1, 2B4, CD69, NKp30, NKp44 and NKp46. The IL‐2‐dependent surface upregulation of some of these receptors was prevented by TGF‐β1. Furthermore, TGF‐β1 hampered IL‐2‐induced NK cell proliferation but not IL‐2‐mediated rescue from apoptosis of NK cells. Depletion of TGF‐β1 restored all the NK cell‐mediated functional activities analysed. Taken together these findings suggest that sHLA‐I antigens may downregulate the NK cell‐mediated innate response by inducing TGF‐β1 release.     

The effects of triptolide on airway remodelling and transforming growth factor-β₁/Smad signalling pathway in ovalbumin-sensitized mice

Airway remodelling contributes to increased morbidity and mortality in asthma. We have reported that triptolide, the major component responsible for the immunosuppressive and anti‐inflammatory effects of Tripterygium wilfordii Hook F, inhibited pulmonary inflammation in patients with steroid‐resistant asthma. In the present study, we investigated whether triptolide inhibits airway remodelling in a mouse asthma model and observed the effects of triptolide on the transforming growth factor‐β₁ (TGF‐β₁)/Smad pathway in ovalbumin (OVA) ‐sensitized mice. BALB/c mice were sensitized to intraperitoneal OVA followed by repetitive OVA challenge for 8 weeks. Treatments included triptolide (40 μg/kg) and dexamethasone (2 mg/kg). The area of bronchial airway (WAt/basement membrane perimeter) and smooth muscle (WAm/basement membrane perimeter), mucus index and collagen area were assessed 24 hr after the final OVA challenge. Levels of TGF‐β₁ were assessed by immunohistology and ELISA, levels of TGF‐β₁ mRNA were measured by RT‐PCR, and levels of pSmad2/3 and Smad7 were assessed by Western blot. Triptolide and dexamethasone significantly reduced allergen‐induced increases in the thickness of bronchial airway and smooth muscle, mucous gland hypertrophy, goblet cell hyperplasia and collagen deposition. Levels of lung TGF‐β₁, TGF‐β₁ mRNA and pSmad2/3 were significantly reduced in mice treated with triptolide and dexamethasone, and this was associated with a significant increase in levels of Smad7. Triptolide may function as an inhibitor of asthma airway remodelling. It may be a potential drug for the treatment of patients with a severe asthma airway.     

Emodin ameliorates glucose-induced fibronectin synthesis in human peritoneal mesothelial cells by inhibiting PKC-α activation

Introduction Peritoneal dialysis (PD) is recognized as an effective treatment for patients with end‐stage renal failure. Conventional PD solutions are unphysiological, as they contain bio‐incompatible concentrations of glucose to provide the osmotic drive ( Popovich et al. 1978 ) Thus, with increasing duration on PD, the peritoneum undergoes progressive structural and functional deterioration. Diabetiform alterations of the peritoneum, for example, the reduplication of the basement membrane, and increased matrix accumulation and deposition within the submesothelium are commonly observed in PD patients. These changes are mediated in part through activation of PKC and induction of TGF‐β1 ( Ha et al. 2001 ). Emodin (3‐methyl‐1,6,8 trihydroxyanthraquinone) has previously been demonstrated to reduce cell proliferation and fibronectin synthesis in cultured mesangial cells ( Kuo et al. 2001 ; Yao et al. 1994 ). How emodin modulates glucose‐induced fibronectin synthesis in human peritoneal mesothelial cells (HPMCs) has not been elucidated and thus constitutes the aim of this study. Materials and methods Confluent growth‐arrested HPMCs were cultured under physiological (5 mm) or elevated (30 mm) d‐glucose concentrations in the presence or absence of emodin (20 µg/ml) for periods up to 72 h. PKC‐α activation in HPMCs cultured under control and experimental conditions was determined by Western blot analysis of cytosolic and cell membrane extracts. Synthesis of fibronectin and TGF‐β1 was investigated by a combination of ELISAs, immunohistochemistry and Western blot analysis. Mannitol was used as the hexose control. Results Exposure of HPMCs to 30 mm d‐glucose resulted in the activation of PKC‐α commencing 12 h after stimulation, as demonstrated by the translocation of PKC‐α from the cytosol to the cell membrane. Maximum induction of PKC‐α was observed 48 h after stimulation and increased 1.4‐fold and 3.7‐fold in the cytosolic and membrane fractions, respectively, compared to 5 mm d‐glucose. Activation of PKC‐α was accompanied by the induction of TGF‐β1 secretion in a time‐dependent manner (3.72 ± 0.29 and 4.30 ± 0.50 pg/µg cellular protein at 24 h and 48 h, respectively, for 30 mm d‐glucose compared to 2.13 ± 0.23 and 2.65 ± 0.32 pg/µg cellular protein at 24 h and 48 h, respectively, for 5 mm d‐glucose, P < 0.001 at both time points) and increased fibronectin synthesis. Whilst emodin had no effect on constitutive PKC‐α, TGF‐β1 or fibronectin synthesis, emodin abrogated elevated glucose stimulation of PKC‐α, and decreased TGF‐β1 secretion and fibronectin synthesis to basal levels. Discussion Our findings demonstrate that emodin can ameliorate the undesirable effects of concentrated glucose on HPMCs through the suppression of PKC‐α activation. This study suggests that emodin may have therapeutic potential in the prevention or treatment of glucose‐induced structural and functional abnormalities in the peritoneal membrane.     

Transforming growth factor beta 1 plays an important role in inducing CD4+CD25+forhead box P3+ regulatory T cells by mast cells

The role of mast cells (MCs) in the generation of adaptive immune responses especially in the transplant immune responses is far from being resolved. It is reported that mast cells are essential intermediaries in regulatory T cell (T_reg) transplant tolerance, but the mechanism has not been clarified. To investigate whether bone marrow‐derived mast cells (BMMCs) can induce T_regs by expressing transforming growth factor beta 1 (TGF‐β1) in vitro, bone marrow cells obtained from C57BL/6 (H‐2^b) mice were cultured with interleukin (IL)‐3 (10 ng/ml) and stem cell factor (SCF) (10 ng/ml) for 4 weeks. The purity of BMMCs was measured by flow cytometry. The BMMCs were then co‐cultured with C57BL/6 T cells at ratios of 1:2, 1:1 and 2:1. Anti‐CD3, anti‐CD28 and IL‐2 were administered into the co‐culture system with (experiment groups) or without (control groups) TGF‐β1 neutralizing antibody. The percentages of CD4⁺CD25⁺forkhead box P3 (FoxP3)⁺ T_regs in the co‐cultured system were analysed by flow cytometry on day 5. The T_reg percentages were significantly higher in all the experiment groups compared to the control groups. These changes were deduced by applying TGF‐β1 neutralizing antibody into the co‐culture system. Our results indicated that the CD4⁺ T cells can be induced into CD4⁺CD25⁺FoxP3⁺ T cells by BMMCs via TGF‐β1.     

CCR2+ monocytic myeloid‐derived suppressor cells (M‐MDSCs) inhibit collagen degradation and promote lung fibrosis by producing transforming growth factor‐β1

Monocytes infiltrating scar tissue are predominantly viewed as progenitor cells. Here, we show that tissue CCR2⁺ monocytes have specific immunosuppressive and profibrotic functions. CCR2⁺ monocytic cells are acutely recruited to the lung before the onset of silica‐induced fibrosis in mice. These tissue monocytes are defined as monocytic myeloid‐derived suppressor cells (M‐MDSCs) because they significantly suppress T‐lymphocyte proliferation in vitro. M‐MDSCs collected from silica‐treated mice also express transforming growth factor (TGF)‐β1, which stimulates lung fibroblasts to release tissue inhibitor of metalloproteinase (TIMP)‐1, an inhibitor of metalloproteinase collagenolytic activity. By using LysMCreCCR2^loxP/loxP mice, we show that limiting CCR2⁺ M‐MDSC accumulation reduces the pulmonary contents of TGF‐β1, TIMP‐1 and collagen after silica treatment. M‐MDSCs do not differentiate into lung macrophages, granulocytes or fibrocytes during pulmonary fibrogenesis. Collectively, our data indicate that M‐MDSCs contribute to lung fibrosis by specifically promoting a non‐degrading collagen microenvironment. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Submandibular gland morphogenesis: Stage‐specific expression of TGF‐α/EGF,IGF, TGF‐β, TNF,and IL‐6 signal transduction in normal embryonic mice and the phenotypic effects of TGF‐β2, TGF‐β3, and EGF‐r null mutations

Branching morphogenesis of the mouse submandibular gland (SMG) is dependent on cell‐cell conversations between and within epithelium and mesenchyme. Such conversations are typically mediated in other branching organs (lung, mammary glands, etc.) by hormones, growth factors, cytokines, and the like in such a way as to translate endocrine, autocrine, and paracrine signals into specific gene responses regulating cell division, apoptosis, and histodifferentiation. We report here the protein expression in embryonic SMGs of four signal transduction pathways: TGF‐α/EGF/EGF‐R; IGF‐II/IGF‐IR/IGF‐IIR; TGF‐βs and cognate receptors; TNF, IL‐6, and cognate receptors. Their in vivo spatiotemporal expression is correlated with specific stages of progressive SMG development and particular patterns of cell proliferation, apoptosis, and mucin expression. Functional necessity regarding several of these pathways was assessed in mice with relevant null mutations (TGF‐β2, TGF‐β₃, EGF‐R). Among many observations, the following seem of particular importance: (1) TGF‐α and EGF‐R, but not EGF, are found in the Initial and Pseudoglandular Stages of SMG development; (2) ductal and presumptive acini lumena formation was associated with apoptosis and TNF/TNF‐R1 signalling; (3) TGF‐β2 and TGF‐β3 null mice have normal SMG phenotypes, suggesting the presence of other pathways of mitostasis; (4) EGF‐R null mice displayed an abnormal SMG phenotype consisting of decreased branching. These and other findings provide insight into the design of future functional studies. Anat Rec 256:252–268, 1999. © 1999 Wiley‐Liss, Inc.     

Regulation of PTC phenotype and function by TGF-β1: implications for transdifferentiation

Introduction There is now increasing evidence that proximal tubular cells (PTCs) contribute to renal interstitial fibrosis by alteration of matrix turnover and by the generation of pro‐fibrotic cytokines such as TGF‐β1. Recent studies suggest that, through a process of transdifferentiation, the PTCs are one source of the interstitial myofibroblasts that directly drive the fibrotic process. The aim of this work was to examine the role and mechanism by which TGF‐β1 may regulate PTC phenotype and function. Methods Experiments were performed using both primary‐cultures of PTC and the human PTC cell line HK2. All experiments were performed on growth‐arrested cells in the absence of serum. Results TGF‐β1 altered cell phenotype, assessed by light microscopy, with cells appearing elongated and spindle‐shaped. This was associated with loss of cell–cell contact and rearrangement of the actin cytoskeleton, increased formation of stress fibres and focal adhesions. Disruption of the actin cytoskeleton with cytochalasin‐D prevented phenotypic alterations following addition of TGF‐β1. Transient transfection with Smad‐2/‐4 or Smad‐3/‐4 expression vectors did not alter cell phenotype. Previously, we have demonstrated β‐catenin translocation to PTC nuclei and its association with Smad proteins following addition of TGF‐β1, suggesting the possibility that TGF‐β1 may modulate Wnt signalling. Wnt‐responsive Xtwn‐reporter construct was, however, silent in response to TGF‐β1. Similarly, a second Wnt‐/LEF‐1‐regulated element Toplflash, which does not contain Smad‐binding sites, was insensitive to TGF‐β1 signalling. In contrast, phenotypic changes in response to TGF‐β1 were abrogated by inhibitors of the RhoA downstream target ROCK, which also prevented loss of cell–cell contact and adherens junction disassembly. Removal of TGF‐β1 and addition of 1% FCS, however, reverted cell phenotype to a typical cobblestone epitheliod appearance, suggesting that TGF‐β1 did not result in terminal PTC transdifferentiation. Cells grown on tissue culture dishes coated with either type‐I or type‐III collagen also acquired an elongated fibroblastic phenotype; this effect was exaggerated by the addition of TGF‐β1. In contrast to the cells stimulated with TGF‐β1 alone, following stimulation by both TGF‐β1 and exposure to interstitial collagens, cell phenotype was stable in that it was not reversed upon removal of TGF‐β1 and addition of FCS. Addition of TGF‐β1 to cells grown on type‐IV collagen had no greater effect than TGF‐β1 alone. Addition of TGF‐β1 alone had little effect on the expression of α‐SMA. In contrast, cells grown on either type‐I or type‐III collagen, following addition of TGF‐β1, demonstrated marked increased expression of α‐SMA, which appeared to be incorporated into the cell cytoskeleton. Similarly, the combination of interstitial collagen (either type‐I or type‐III) and TGF‐β1 had synergistic effect on the relocation and down‐regulation of the epithelial markers E‐cadherin and cytokeratin. Finally, the results demonstrated synergistic effects of coating with interstitial collagen (either type‐I or type‐III), on cell ‘fibroblastic’ cell function as assessed by cell migration and by the synthesis of type‐III and type‐IV collagen. Conclusion The results of these in vitro experiments suggest that terminal transdifferentiation of proximal tubular epithelial cells is the result of a combination of the effects of the pro‐fibrotic cytokine TGF‐β1 and exposure of the cells to components of the interstitial extra‐cellular matrix to which the cells are not exposed in the absence of damage to the tubular basement membrane.     

The calpain inhibitor calpeptin prevents bleomycin-induced pulmonary fibrosis in mice

Pulmonary fibrosis is characterized by progressive worsening of pulmonary function leading to a high incidence of death. Currently, however, there has been little progress in therapeutic strategies for pulmonary fibrosis. There have been several reports on cytokines being associated with lung fibrosis, including interleukin (IL)‐6 and transforming growth factor (TGF)‐β1. We reported recently that two substances (ATRA and thalidomide) have preventive effects on pulmonary fibrosis by inhibiting IL‐6‐dependent proliferation and TGF‐β1‐dependent transdifferentiation of lung fibroblasts. Rheumatoid arthritis is a chronic autoimmune disorder, and its pathogenesis is also characterized by an association with several cytokines. It has been reported that calpain, a calcium‐dependent intracellular cysteine protease, plays an important role in the progression of rheumatoid arthritis. In this study, we examined the preventive effect of Calpeptin, a calpain inhibitor, on bleomycin‐induced pulmonary fibrosis. We performed histological examinations and quantitative measurements of IL‐6, TGF‐β1, collagen type Iα1 and angiopoietin‐1 in bleomycin‐treated mouse lung tissues with or without the administration of Calpeptin. Calpeptin histologically ameliorated bleomycin‐induced pulmonary fibrosis in mice. Calpeptin decreased the expression of IL‐6, TGF‐β1, angiopoietin‐1 and collagen type Iα1 mRNA in mouse lung tissues. In vitro studies disclosed that Calpeptin reduced (i) production of IL‐6, TGF‐β1, angiopoietin‐1 and collagen synthesis from lung fibroblasts; and (ii) both IL‐6‐dependent proliferation and angiopoietin‐1‐dependent migration of the cells, which could be the mechanism underlying the preventive effect of Calpeptin on pulmonary fibrosis. These data suggest the clinical use of Calpeptin for the prevention of pulmonary fibrosis.     

Transforming growth factor‐β/Smad ‐ signalling pathway and conjunctival remodelling in vernal keratoconjunctivitis

Background Vernal keratoconjunctivitis (VKC) is a chronic ocular allergic inflammation characterized by corneal complications and the formation of giant papillae. Sma‐ and Mad‐related proteins (Smad) modulate extracellular matrix gene expression during wound healing, inflammation and tissue remodelling. Objective To investigate the relationship between allergic inflammation and TGF‐β/Smad signalling pathway, expression in VKC patients and in primary cultured conjunctival fibroblasts exposed to mediators found previously over‐expressed in VKC. Methods Smad‐2, ‐3, ‐7, phospho‐(p)Smads, TGF‐β1 and ‐β2 were evaluated in the conjunctiva of normal subjects (CT) and VKC patients by immunohistochemistry. The expression of Smads, pro‐collagen I (PIP), TGF‐β1, ‐β2, mitogen‐activated protein kinase (p38/MAPK), c‐Jun N‐terminal kinase (JNK) and extracellular signal‐regulated kinase (ERK1/2) were also determined in conjunctival fibroblast cultures exposed to histamine, IL‐4, ‐13, TGF‐β1, IFN‐γ and TNF‐α using immunostaining or RT‐PCR. Results Immunostaining for Smad‐2, ‐3, pSmad‐2, ‐3, TGF‐β1, ‐β2 and PIP was significantly increased in VKC stroma compared with CT. In conjunctival fibroblast cultures, Smad‐3 and PIP were stimulated by histamine, IL‐4, ‐13 and TGF‐β1 exposure, while PIP was reduced by IFN‐γ, and TNF‐α mRNA expression of Smad‐3 was increased by histamine, while Smad‐7 was reduced by IL‐4. In addition, histamine, IL‐4 and TNF‐α increased JNK and ERK1/2 expression. Conclusion and Clinical Relevance The TGF‐β/Smad signalling pathway is over‐expressed in VKC tissues and modulated in conjunctival fibroblasts by histamine, IL‐4, TGF‐β1 and TNF‐α. These mechanisms may be involved in fibrillar collagen production, giant papillae formation and tissue remodelling typical of VKC and might provide new therapeutic targets for its treatment. Cite this as: A. Leonardi, A. Di Stefano, L. Motterle, B. Zavan, G. Abatangelo and P. Brun, Clinical & Experimental Allergy, 2011 (41) 52–60.     

The complex pathophysiology of acquired aplastic anaemia

Immune‐mediated destruction of haematopoietic stem/progenitor cells (HSPCs) plays a central role in the pathophysiology of acquired aplastic anaemia (aAA). Dysregulated CD8⁺ cytotoxic T cells, CD4⁺ T cells including T helper type 1 (Th1), Th2, regulatory T cells and Th17 cells, natural killer (NK) cells and NK T cells, along with the abnormal production of cytokines including interferon (IFN)‐γ, tumour necrosis factor (TNF)‐α and transforming growth factor (TGF)‐β, induce apoptosis of HSPCs, constituting a consistent and defining feature of severe aAA. Alterations in the polymorphisms of TGF‐β, IFN‐γ and TNF‐α genes, as well as certain human leucocyte antigen (HLA) alleles, may account for the propensity to immune‐mediated killing of HSPCs and/or ineffective haematopoiesis. Although the inciting autoantigens remain elusive, autoantibodies are often detected in the serum. In addition, recent studies provide genetic and molecular evidence that intrinsic and/or secondary deficits in HSPCs and bone marrow mesenchymal stem cells may underlie the development of bone marrow failure.     

Attenuated TGF‐β1 responsiveness of dendritic cells and their precursors in atopic dermatitis

The responsiveness of DCs and their precursors to transforming growth factor beta1 (TGF‐β1) affects the nature of differentiating DC subsets, which are essential for the severity of atopic dermatitis (AD). To evaluate TGF‐β signaling in monocytes and monocyte‐derived DCs of AD patients compared with that of controls, in vitro generated Langerhans cell (LC) like DCs, expression of TGF‐β receptors, phospho‐Smad2/3 and Smad7 were evaluated. Furthermore, TNF‐α expression and synergistic effects of TNF‐α upon TGF‐β signaling and DC generation were evaluated. We found LC‐like DC differentiation of monocytes from AD patients in response to TGF‐β1 was remarkably reduced and TGF‐β1 receptor expression was significantly lower compared with that of healthy controls. Attenuated TGF‐β1 responsiveness mirrored by lower phospho‐Smad2/3 expression after TGF‐β1 stimulation and higher expression of inhibitory Smad7 was observed in monocytes from AD patients. During DC generation, mRNA expression of Smad7 was relatively higher in LC‐like DCs of AD patients. Lower TNF‐α expression of monocytes from AD patients might further contribute to attenuated TGF‐β signaling in the disease since TNF‐α had synergistic effects on TGF‐β1 signaling and LC generation through mediating the degradation of Smad7. Our results demonstrate alleviated TGF‐β1 signaling together with the amount of soluble co‐factors might direct the nature of differentiating DCs.     

Smad-3 as a mediator of the fibrotic response

Introduction Smad‐3, a key cytoplasmic mediator of transforming growth factor‐β (TGF‐β) signalling, mediates many of its inflammatory and fibrotic effects in vivo ( Roberts et al. 2001 ). Smad‐3 null mice are protected against cutaneous injury induced by ionizing irradiation ( Flanders et al. 2002 ). Here, we report on our continuing studies on radioprotection as well as protection against tubulointerstitial fibrosis following unilateral ureteral obstruction (UUO) in Smad‐3 null mice. Methods For radioprotection studies, the flank skin of Smad‐3^+/+ wild‐type (WT) and Smad‐3^–/– knockout (KO) mice was exposed to 30 Gy of localized γ‐irradiation and analysed for histology and gene expression at various times post irradiation. In the UUO model, the right proximal ureter of WT and KO mice was ligated, and 1–2 weeks later kidneys were analysed for inflammation, fibrosis and gene expression. Results Six weeks after exposure to irradiation, skin from KO mice shows less epidermal acanthosis and influx of mast cells, macrophages and neutrophils than skin of WT mice. Paradoxically, at 6–8 h post irradiation, KO skin shows a significantly greater number of neutrophils. Irradiated KO skin also exhibits less immunoreactive TGF‐β, fewer myofibroblasts and less scarring than does WT. Smad‐3 null dermal fibroblasts do not respond to the chemotactic effects of TGF‐β and show less induction of fibrogenic cytokines when treated with irradiation plus TGF‐β compared to WT cells. Following UUO, normal kidney architecture is preserved in KO mice, while kidneys from WT mice are enlarged with an influx of mononuclear cells and increased expression of collagen and TGF‐β1. Additionally, renal tubules in obstructed kidneys of KO mice remain positive for E‐cadherin without expression of α‐smooth muscle actin, while the opposite expression pattern is seen in obstructed kidneys of WT mice. TGF‐β treatment of primary cultures of WT renal tubular epithelial cells results in a phenotypic change from a cobblestone pattern to a spindle‐shaped fibroblastic appearance, while KO cells treated with TGF‐β maintain their original appearance. Conclusion Smad‐3 plays an important role in mediating pathogenic inflammation and fibrosis in several model systems and is also essential for TGF‐β1‐induced epithelial–mesenchymal transition in renal tubular epithelial cells. Inhibitors of the Smad‐3 pathway may have clinical applications in the treatment of a number of fibrotic conditions.     

Gene polymorphism in transforming growth factor‐beta codon 10 is associated with susceptibility to Giardiasis

Secretory immunoglobulin A (S‐IgA) antibodies have a central role in anti‐Giardial defence. It has been demonstrated that transforming growth factor‐beta1 (TGF‐β1) stimulates B lymphocytes to produce and secrete S‐IgA. We sought to determine the association between TGF‐β1 polymorphism (T+869C) with susceptibility to Giardiasis. The TGF‐β1 genotypes and levels of salivary (S‐IgA) were analysed in individuals with Giardiasis (97 symptomatic and 57 asymptomatic) and controls (n = 92). Individuals with symptomatic Giardiasis had the lowest levels of S‐IgA compared to individuals in asymptomatic Giardiasis and control groups (97%, 73% and 43%, <1 g L^?1, respectively, P = 0.002). The frequency of allele C and CC genotypes of TGF‐β1 polymorphism was significantly higher among symptomatic patients than asymptomatic and control groups. Logistic regression analysis demonstrated that the individuals homozygous for allele C of TGF‐β1 had a significantly higher risk for symptomatic Giardiasis with odds ratio of 2.76 (95% CI: 3.88, 1.71, P = 0.007). Among the participants with TT genotype per cent of individuals with S‐IgA level of more than 1 g L^?1 was almost twice the percentage in CC genotype individuals (14% versus 7% respectively P = 0.01). Our data suggest that CC genotype of TGF‐β1 polymorphism at codon 10 is associated with occurrence of Giardiasis.     

Hepatoprotectivity of Panduratin A against liver damage: In vivo demonstration with a rat model of cirrhosis induced by thioacetamide

This experiment evaluated Panduratin A (PA), a chalcone isolated from Boesenbergia rotunda rhizomes, for its hepatoprotectivity. Rats were subjected to liver damage induced by intra‐peritoneal injection of thioacetamide (TAA). PA was tested first for its acute toxicity and then administered by oral gavage at doses 5, 10, and 50 mg/kg to rats. At the end of the 8^th week, livers from all rats were excised and evaluated ex vivo. Measurements included alkaline phosphatase (AP), alanine transaminase (ALT), aspartate transaminase (AST) and gamma‐glutamyl transferase (GGT), serum platelet‐derived growth factor (PDGF) and transforming growth factor (TGF‐β1), and hepatic metalloproteinase enzyme (MMP‐2) and its inhibitor extracellular matrix protein (TIMP‐1). Oxidative stress was measured by liver malondialdehyde (MDA) and nitrotyrosine levels, urinary 8‐hydroxy 2‐ deoxyguanosine (8‐OH‐dG), and hepatic antioxidant enzyme activities. The immunohistochemistry of TGF‐β₁ was additionally performed. PA revealed safe dose of 250 mg/kg on experimental rats and positive effect on the liver. The results suggested reduced hepatic stellate cells (HSCs) activity as verified from the attenuation of serum PDGF and TGF‐β1, hepatic MMP‐2 and TIMP‐1, and oxidative stress. The extensive data altogether conclude that PA treatment could protect the liver from the progression of cirrhosis through a possible mechanism inhibiting HSCs activity.     

Polymorphism R25P in the gene encoding transforming growth factor‐beta (TGF‐β1) is a newly identified risk factor for proliferative diabetic retinopathy

Associations of the genetic polymorphisms in the promoter region and the signal peptide sequence of the transforming growth factor‐beta (TGF‐β₁) gene with proliferative diabetic retinopathy (PDR) in patients with non‐insulin‐dependent diabetes mellitus (NIDDM) were studied. A total of 245 Caucasian subjects comprised the two groups: NIDDM patients with PDR (n = 73) and NIDDM patients without PDR (n = 172). Allele frequencies of common TGF‐β₁ polymorphisms (at positions ?988C/A, ?800G/A, ?509C/T, +869T/C (L10P), and +915G/C (R25P)) were determined by PCR‐based methodology. All polymorphisms were in strong linkage disequilibrium (P < 10^?2). Significantly higher frequencies of both the L allele and the R allele of the signal sequence polymorphisms in PDR subjects were found (after a correction for multiple comparisons, P_corr < 10^?2 and P_corr < 10^?4, respectively). Calculated odds ratios (ORs) for the LL and RR genotypes were 2.89 (95% confidence interval (CI), 1.6–5.1) and 19.73 (95% CI, 2.6?146.8), respectively. No significant differences between groups were found for the ?800G/A and ?509C/T polymorphisms. The ?988A allele was not represented in our sample. Multiple logistic regression identified age, diabetes duration, and R25P polymorphism as significant predictors (P = 0.002, P = 0.000003, and P = 0.007, respectively). The frequencies of genotype combinations of the ?800G/A, ?509C/T, L10P, and R25P TGF‐β₁ polymorphisms were significantly different between the PDR and non‐PDR groups (χ² = 37.83, df = 20, P < 10^?2). The frequency of haplotype consisting of majority alleles was found significantly associated with PDR (P < 0.03). The presented data indicate that the R25P polymorphisms in the TGF‐β₁ gene could be regarded as a strong genetic risk factor for PDR. © 2002 Wiley‐Liss, Inc.     

Transforming growth factor‐β1 in asthmatic airway smooth muscle enlargement: is fibroblast growth factor‐2 required?

Enlargement of airway smooth muscle (ASM) tissue around the bronchi/bronchioles is a histopathological signature of asthmatic airway remodelling and has been suggested to play a critical role in the increased lung resistance and airway hyperresponsiveness seen in asthmatic patients. The pleiotropic cytokine, TGF‐β1, is believed to contribute to several aspects of asthmatic airway remodelling and is known to influence the growth of many cell types. Increased TGF‐β1 expression/signalling and ASM growth have been shown to occur concurrently in animal models of asthma. Abundant studies further substantiate this association by showing that therapeutic strategies that reduce or prevent TGF‐β1 overexpression/signalling lead to a parallel decrease or prevention of ASM enlargement. Finally, recent findings have supported a direct link of causality between TGF‐β1 overexpression/signalling and the overgrowth of ASM tissue. To follow‐up on these in vivo studies, many investigators have pursued detailed investigation of ASM in cell culture conditions, assessing the direct role of TGF‐β1 on cellular proliferation and/or hypertrophy. Inconsistencies among the in vitro studies suggest that the effect of TGF‐β1 on ASM cell proliferation/hypertrophy is contextual. A hypothesis focusing on fibroblast growth factor‐2 is presented at the end of this review, which could potentially reconcile the apparent discrepancy between the conflicting in vitro findings with the consistent in vivo finding that TGF‐β1 is required for ASM enlargement in asthma. Cite this as: Y. Bossé, J. Stankova and M. Rola‐Pleszczynski, Clinical & Experimental Allergy, 2010 (40) 710–724.