CD8 membrane expression is down-regulated by transforming growth factor (TGF)-beta 1, TGF-beta 2, and prostaglandin E2.

PROBLEM: CD8 T-cells are present at a lower frequency in human decidua than in peripheral blood. Because transforming growth factor (TGF)-beta 2 down-regulates CD4 membrane expression, its contribution, as well as the contribution of TGF-beta 1 and prostaglandin (PG) E2, to the modulation CD8 expression was studied using human peripheral blood lymphocytes (PBLs). METHOD OF STUDY: PBLs were cultured with TGF-beta 1, TGF-beta 2, PGE 2, PGI 2, or day-12 rabbit blastocoelic fluid (BF) that was or was not depleted of TGF-beta 2 and/or PGE 2. Quantum Simply Cellular Microbeads were then used to evaluate CD8 membrane expression levels. RESULTS: This study is the first demonstration that treatment of PBLs with TGF-beta 1, TGF-beta 2, and PGE 2 leads to a dose-dependent decrease in CD8 expression. A significant inhibition was observed at 2.5 mg/mL for TGF-beta 2, 5 ng/mL for TGF-beta 1, and 10 ng/mL for PGE 2. In contrast, PGI 2 had no effect. Treatment of PBLs with BF day-12 decreased CD8 expression. This effect, however, was not observed when BF was depleted of TGF-beta 2 and/or PGE 2. CONCLUSIONS: Our results suggest that TGF-beta s and PGE 2 are important modulators of CD8 membrane expression in human lymphocytes. Because TGF-beta 1, TGF-beta 2, and PGE 2 are produced by the conceptus and by uterine cells and because the effect is observed after only 3 days of treatment, the present data suggest that these substances can locally modulate the phenotype of lymphocytes at the fetomaternal interface. Such modulation may explain, at least partly, the changes observed in the population of decidual lymphocytes during pregnancy.     

Age-dependent decrease in serum transforming growth factor (TGF)-beta 1 in healthy Japanese individuals; population study of serum TGF-beta 1 level in Japanese

Transforming growth factor-beta1 (TGF-beta1), a multi-functional cytokine, is involved in regulating a variety of cellular activities and the serum/plasma TGF-beta1 level is altered with various diseases. However, most published reports have described adult patients, and so we investigated the clinical significance of serum TGF-beta1 level in pediatric patients. The diagnostic application of the measurement of serum TGF-beta1 level depends critically on the control value, however, there is no information on the control value of serum TGF-beta1 for children. In the present study, we determined the serum TGF-beta1 level of healthy Japanese children as a control value with enzyme-linked immunosorbent assay (ELISA). The serum TGF-beta1 level of children (0-14 years old) was significantly higher than that of adults (over 15 years old) (p < 0.01). Thus, it is recommended that when the serum TGF-beta1 levels of patients are evaluated, they should be compared with those of age-matched controls.     

The effect of transforming growth factor (TGF)-beta1 and (TGF)-beta2 on nasal polyp fibroblast activities involved upper airway remodeling: modulation by fluticasone propionate

Transforming growth factor (TGF)-beta may play a significant role in nasal polyposis pathogenesis, possibly through fibroblast activation. We studied the effects of two TGF-beta isoforms (TGF-beta1 and TGF-beta2) on nasal polyposis fibroblasts by evaluating cell proliferation and differentiation into myofibroblasts. In addition, the inhibitory activity of different concentrations of fluticasone propionate (F.P.) was tested in this in vitro system. Primary nasal polyp tissue-derived fibroblasts were stimulated with different concentrations (1, 10 and 20 ng/ml) of TGF-beta1 and TGF-beta2 for different incubation periods (24, 48 and 72 h) and cell proliferation [3H thymidine ([3H]TdR) incorporation] and alpha-smooth muscle actin (alpha-SMA) expression (immunocytochemistry) was evaluated. The lowest concentration of TGF-beta1 (1 ng/ml) induced a significant increase in [3H]TdR incorporation at 48 and 72 h (p<0.05, each comparison), while in the presence of TGF-beta (10 ng/ml) and TGF-beta2 (1 ng/ml) the enhancement in cell proliferation was significant only after 48 h (p<0.05, each comparison with the unstimulated cells). In contrast, a significant increase in alpha-SMA expression was observed in the presence of the two highest concentration of both TGF-beta isoforms, at 48 and 72 h for TGF-beta1 (p<0.05, each comparison), but only at 72 h for TGF-beta2 (<0.05, each comparison). Finally, at all concentrations tested, F.P. significantly inhibited the TGF-beta1 and TGF-beta2-induced 3HTdR incorporation (p<0.01, each comparison) and the alpha-SMA expression (p<0.05, each comparison). Thus, in vitro different concentrations of TGF-beta1 and TGF-beta2 appear to sequentially stimulate primary nasal polyp tissue-derived fibroblast proliferation and myofibroblast differentiation. These activities are effectively inhibited by F.P.     

Expression of transforming growth factor beta 1 (TGF beta 1) and angiogenesis in broncho-pulmonary carcinoids

TGF beta 1 commonly produced by normal and neoplastic human cells, has capacity to regulate new blood vessel formation, to establish and maintain the vessel wall integrity; was found to have some significance in the lung cancer prognosis. Tumour angiogenesis is an important factor for tumour growth and metastasis. The purpose of this study was to find, if the immunoexpression of TGF beta 1 has any significance in determination of the histologic subtypes of carcinoids?; to find, if TGF beta 1 has any role and relation to carcinoids angiogenesis?; and to explore TGF beta 1 expression and angiogenesis with respect to metastatic potential of carcinoids. The study was performed on 48 resected broncho-pulmonary carcinoids: 35 typical (TC) and 13 atypical (AC), classified according to the WHO. Semiquantitative analysis for TGF beta 1 was performed. Sections stained using monoclonal antibody against TGF beta 1 were scored in scale from 0 to 4, according to the percentage of positively stained cells (pc) plus percentage of positively stained stroma (ps). The microvessels stained with CD34 monoclonal antibody, were counted in 0.75 mm2 field (microvessel density--MD), using the computerised image analysis system SAMBA 2005 (the morphometric software). The histologic subtype of carcinoids was related to age of the pts (AC occurred in older pts than TC, p = 0.027), to the tumour size (AC were larger than TC: respectively--3.25 cm and 2.4 cm, p = 0.009). Lymph node metastases were significantly more frequent in AC than in TC (38% vs 13%, p = 0.025). 85% carcinoids showed TGF beta 1 expression with various intensity, mainly in the stroma. There was no significant correlation between TGF beta 1 expression and tumour size, the histologic subtype nor the lymph node metastases. The angiogenesis expressed as MD, was not related to histology, nor to the presence of lymph node metastases. There was no correlation between TGF beta 1 expression and angiogenesis. Shown in our study, lack of relation between TGF beta 1 expression and angiogenesis, could support some of the published data indicating indirect action of TGF beta 1 on the angiogenesis. The rich vascularity found in carcinoids morphology could result from TGF beta 1, commonly expressed by the tumoural stroma. The angiogenesis nor TGF beta 1 expression do not determinate the carcinoids histology.     

Kinetics and distribution of transforming growth factor (TGF)-beta 1 mRNA in the dorsal skin of hypotrichotic WBN/ILA-Ht rats following topical application of T-2 toxin.

Depression of basal cell proliferating activity and subsequent induction of basal cell apoptosis in the epidermis and infiltration of inflammatory cells including mast cells in the dermis were observed in the dorsal skin of hypotrichotic WBN/ILA-Ht rats following the topical application of T-2 toxin in our previous study (ALBARENQUE et al. 1999). In the present study, kinetics of TGF-beta 1 mRNA was investigated using the same experimental system. The level of TGF-beta 1 mRNA of the whole skin tissue measured by competitive RT-PCR method showed a slight elevation from 6 to 12 hours after treatment (HAT) and reached the significantly higher level at 24HAT compared with the control skin. The increase in signals of TGF-beta 1 mRNA detected by in situ hybridization method started at 3HAT in the epidermis and progressed thereafter both in the epidermis and in the dermis. These results suggest that the elevated level of TGF-beta 1 mRNA may have a close relation to the induction of epidermal basal cell apoptosis as well as to the intradermal infiltration of mast cells and fibroblasts following the topical application of T-2 toxin.     

Inhibition of immune reactions in vivo by liposome associated transforming growth factor (TGF) type beta 1.

In view of its potent inhibitory capacity on immune cells in culture, we wished to determine the ability of transforming growth factor (TGF) beta 1 to down-regulate immune responses in vivo. Preliminary experiments suggested that, at the doses used, systemic injection of soluble TGF beta 1 could not affect bacterial-induced spleen enlargement in mice. Therefore, we sought to utilize a physiochemical property of this molecule, namely its high pI, to determine possible association between the ligand and preformed liposomes possessing an opposite charge. TGF beta 1 was preferentially associated with negatively charged, but not with neutral, liposomes. These TGF beta 1 associated liposomes were able to deliver a suppressive signal to indicator cells in vitro. Intravenous injection of TGF beta 1, associated with liposomes possessing an opposite charge, into mice immunized with heat-killed Corynebacterium parvum significantly reduced the size of the spleen as well as the number of splenocytes. Systemically administered TGF beta 1 associated liposomes could also inhibit delayed type hypersensitivity reactions to Listeria monocytogenes. These data suggest that appropriately administered, TGF beta 1 can inhibit immune responses in vivo.     

Mechanisms involved in valvuloseptal endocardial cushion formation in early cardiogenesis: roles of transforming growth factor (TGF)-beta and bone morphogenetic protein (BMP)

Endothelial-mesenchymal transformation (EMT) is a critical event in the generation of the endocardial cushion, the primordia of the valves and septa of the adult heart. This embryonic phenomenon occurs in the outflow tract (OT) and atrioventricular (AV) canal of the embryonic heart in a spatiotemporally restricted manner, and is initiated by putative myocardially derived inductive signals (adherons) which are transferred to the endocardium across the cardiac jelly. Abnormal development of endocardial cushion tissue is linked to many congenital heart diseases. At the onset of EMT in chick cardiogenesis, transforming growth factor (TGFbeta)-3 is expressed in transforming endothelial and invading mesenchymal cells, while bone morphogenetic protein (BMP)-2 is expressed in the subjacent myocardium. Three-dimensional collagen gel culture experiments of the AV endocardium show that 1) myocardially derived inductive signals upregulate the expression of AV endothelial TGFbeta3 at the onset of EMT, 2) TGFbeta3 needs to be expressed by these endothelial cells to trigger the initial phenotypic changes of EMT, and 3) myocardial BMP2 acts synergistically with TGFbeta3 in the initiation of EMT.     

Analysis of interleukin (IL)-1 beta and transforming growth factor (TGF)-beta-induced signal transduction pathways in IL-2 and TGF-beta secretion and proliferation in the thymoma cell line EL4.NOB-1

In the present study we investigated the interleukin (IL)-1beta and transforming growth factor-beta1 (TGF-beta1)-mediated proliferation, and production of IL-2 and TGF-beta, in the murine T-cell line, EL4.NOB-1. This cell line is resistant to TGF-beta concerning growth arrest but not autoinduction or suppression of IL-1-induced IL-2 production. When cocultured with IL-1beta, TGF-beta showed growth-promoting activity that could be antagonized by adding the phosphatidyl choline-dependent phospholipase C (PC-PLC) inhibitor, D609. Using specific enzyme inhibitors of protein kinases (PK) C and A, mitogen-activated protein kinase (MAPK), phospholipase A2 (PLA2), phosphatidylinositol-dependent (PI)-PLC and PC-PLC, we showed that IL-1beta-induced IL-2 synthesis was dependent on all investigated kinases and phospholipases, except PC-PLC. TGF-beta1 was able to inhibit IL-2 synthesis by the activation of PKA and MAPK. The same kinases are involved in TGF-beta autoinduction that is accompanied by a secretion of the active but not the latent growth factor and is antagonized by IL-1beta. Addition of the PI-PLC inhibitor, ET 18OCH3, or the PLA2 inhibitor (quinacrine) alone, resulted in secretion of latent TGF-beta and, in the case of ET 18OCH3, active TGF-beta. These data implicate a role for PI-PLC and PLA2 in the control of latency and secretion. Analysis of specific tyrosine activity and c-Fos expression showed synergistic but no antagonistic effects. These events are therefore not involved in IL- and TGF-beta-regulated IL-2 and TGF-beta production, but might participate in IL-1/TGF-beta-induced growth promotion.     

Expression of transforming growth factor-beta1, -beta2 and -beta3 in normal and diseased canine mitral valves

The pathogenesis of chronic valvular disease (CVD) in dogs remains unclear, but activation and proliferation of valvular stromal cells (VSC) and their transdifferentiation into myofibroblast-like cells has been described. These alterations may be influenced by transforming growth factor-beta (TGF-beta), a cytokine involved in extracellular matrix (ECM) regulation and mesenchymal cell differentiation. The present study investigates immunohistochemically the expression of TGF-beta1, -beta2, -beta3 and smooth muscle alpha actin (alpha-SMA) in normal canine mitral valves (MVs) (n=10) and in the valves of dogs with mild (n=7), moderate (n=14) and severe (n=9) CVD. In normal mitral valves there was no expression of alpha-SMA but VSC displayed variable expression of TGF-beta1 (10% of VSC labelled), TGF-beta2 (1-5% labelled) and TGF-beta3 (50% labelled). In mild CVD the affected atrialis contain activated and proliferating alpha-SMA-positive VSC, which strongly expressed TGF-beta1 and -beta3, but only 10% of these cells expressed TGF-beta2. In unaffected areas of the leaflet there was selective increase in expression of TGF-beta1 and -beta3. In advanced CVD the activated subendothelial VSC strongly expressed alpha-SMA, TGF-beta1 and -beta3. Inactive VSC within the centre of the nodules had much less labelling for TGF-beta1 and -beta3. TGF-beta1 labelling was strong within the ECM. These data suggest that TGF-beta plays a role in the pathogenesis of CVD by inducing myofibroblast-like differentiation of VSC and ECM secretion. Changed haemodynamic forces and expression of matrix metalloproteinases (MMPs) may in turn regulate TGF-beta expression.     

Development of skeletal muscles in transforming growth factor-beta 1 (TGF-beta1) null-mutant mice.

Fetal transforming growth factor-beta 1 (TGF-beta1) has been postulated to regulate the onset of myotube formation and/or pattern formation in developing skeletal muscles. In apparent contradiction of these hypotheses, the development of the extensor digitorum longus and soleus in TGF-beta1 null-mutant muscle was normal. The onset of secondary myotube formation, the numbers of myotubes formed, the proportion of fast and slow fibers, and the patterns of fiber types and connective tissues were essentially identical in TGF-beta1(+/+) and TGF-beta1(-/-) mice. A portion of the TGFbeta1 in skeletal muscles is derived from the mother, via the placenta. This maternal-derived TGF-beta1 was also not essential for the development of skeletal muscles, as the characteristics of pups born to a TGF-beta1(-/-) mother were normal TGF-beta1(-/-) mice die at weaning due to a generalized autoimmune attack. This postnatal death was circumvented by breeding the TGF-beta1 null mutation into nude mice (Whn(-/-)). Like many other strains of TGF-beta1(-/-) mice, extensive loss of Whn(-/-), TGF-beta1(-/-) embryos occurred in utero. However, a portion of the Whn(-/-), TGF-beta1(-/-) mice survived past weaning, remained healthy, and were fertile. The TGF-beta1(-/-) x Whn(-/-) mouse thus represents a valuable tool for the study of the function of TGF-beta1 in the adult, including its putative role as a pregnancy-related hormone.     

Receptors for transforming growth factor-beta (TGF-beta) on rat lung fibroblasts have higher affinity for TGF-beta 1 than for TGF-beta 2.

Most cell types have receptors for transforming growth factor-beta (TGF-beta) and respond similarly to TGF-beta 1 and TGF-beta 2. We have demonstrated the presence of a single class of high-affinity receptors (approximately 10,000 sites/cell) for TGF-beta 1 (Kd = 23 pM) and TGF-beta 2 (Kd = 41 pM) on early-passage rat lung fibroblasts (RLF). Incubation with unlabeled TGF-beta 1 and TGF-beta 2 resulted in concentration-dependent inhibition of binding of 15 pM [125I]TGF-beta 1 (ED50, 20 and 28 pM, respectively) and [125I]TGF-beta 2 (ED50, 36 and 56 pM, respectively). TGF-beta receptors affinity-cross-linked with 100 pM [125I]TGF-beta 1 or [125I]TGF-beta 2 were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and exhibited labeled protein bands of 68, 88, and 286 kD. Densitometric analysis of the resulting autoradiograms showed that the different molecular weight TGF-beta binding proteins exhibited separate affinities for the two forms of TGF-beta. Both TGF-beta 1 and TGF-beta 2 altered the morphology and cytoskeleton of RLF in a similar manner, but TGF-beta 1 was more potent than TGF-beta 2 in the inhibition of RLF growth and colony formation, with 50% inhibition by 0.12 pM TGF-beta 1 and 4.4 pM TGF-beta 2. Different affinities for the TGF-beta s may indicate selectivity among the receptor subtypes with regard to the biologic responsiveness of RLF to TGF-beta s. We believe this to be the first demonstration of biologically responsive TGF-beta receptors with different affinities for TGF-beta 1 and TGF-beta 2 on cells derived from normal, nonimmortal RLF. In establishing the basic mechanisms of pulmonary fibrosis, it will be essential to understand the biology and biochemistry of the receptors that may control cell division and production of extracellular matrix components by fibroblasts.     

The effect of transforming growth factor beta1 (TGF-beta1) on the regenerate bone in distraction osteogenesis

Distraction osteogenesis is a well established clinical treatment for limb length discrepancy and skeletal deformities. Transforming growth factor beta 1 (TGF-beta1) is a multifunctional peptide which controls proliferation and expression of cells specific to bone like chondrocytes, osteoblasts, osteoclasts including mesenchymal precursor cells. To decrease the external fixation time with increasing the strength of regenerate (newly formed bone after distraction) we tested the effect of locally applied transforming growth factor beta 1 on distraction osteogenesis. A total of 28 mature female white New zealand rabbits weighing 3,5 kg-4,5 kg were studied. 10 animals were belonging to biomechanical testing group (5 for the study and 5 for the control subgroups), and the others were to histology group. In biomechanical group after tibial osteotomy TGF-beta1 was applied subperiosteally for 5 days just proximal to osteotomy site. Control group received only the solvent. Seven days after tibial osteotomy distraction was started at a rate of 0.25 mm/12 hours for 3 weeks with a unilateral fixator. Rabbits were sacrificed at the end of a consolidation period 8 week after tibial osteotomy. We assessed density of the elongation zone of rabbit tibial bones with the computed tomography. Then biomechanical parametres were assessed using the torsional testing using the material testing machine. In histology group rabbits were classified as control and study (rabbits that were given TGF-beta1). Rabbits were sacrificed at the end of first week, second week and fourth week also at the end of consolidation period 8 week after tibial osteotomy. Immunohistochemical and histologic parameters were examined. Biomechanical testing was applied as torsional testing. These values are used in determination of maximal loading, stiffness and energy absorbed during testing (brittleness). The histomorphometric examination looked for the differences between the study and control groups in terms of bone formation pattern, bone quality and quantity. The immunohistochemical studies investigated the mechanism of TGF-beta1, and its presence in different cell types. The results of this study suggest that locally applied TGF-beta1 improves the mineral density of distraction gap and load to failure(energy absorbed during testing). Though there is no significant histomorphometric difference between the study and control groups, there is an increased bone mineral density and an according maximum energy absorbance in the study group. This effect can be explained by the following mechanism: TGF-beta1 exerts its effect on two different receptor types (Type 1 and 2). Type 1 receptors are localized to bone matrix and type 2 receptors are localized to the intracellular space. The specific stains utilized in the current experiment are specific to type 2 receptors. They have been shown to be down-regulated by exogenous TGF-beta1 injections. Most probably, type 1 receptors are up-regulated by this exogenous administration, but unfortunately, there is currently no specific stain on tha market to display type 1 receptors and to prove this explanation.     

Localization of recognition site between transforming growth factor-beta1 (TGF-beta1) and TGF beta receptor type II: possible implications in breast cancer

Although overexpression of TGF-beta1 protein has been demonstrated in advanced breast cancer (BC) patients, as well as in other solid tumours, the molecular mechanism of this process remains obscure. This paper proposes that a genetic/epigenetic alteration might occur in the TGF-beta1 gene, within the region coding for the recognition site with TGFbeta receptor type II, leading to a disruption of the ligand-receptor interaction and triggering the TGF-beta1 cascade-related BC progression. To establish the operational framework for this hypothesis, in the present study, this recognition site was identified by the Informational Spectrum Method (ISM) to comprise two TGF-beta1 peptides (positions 47-66 aa and 83-112 aa) and one receptor peptide at positions 112-151 aa of the extracellular domain of the receptor (TbetaRIIM). The TbetaRIIM locus was further evaluated by ISM-derived deletion analysis of the TbetaRII sequences. To provide experimental support for the proposed model, a pilot study of plasma TGF-beta1 analysis was performed in advanced BC patients (n = 8). Two commercial ELISA assays, one with specific alphaTGF-beta1 MAb (MAb) and other with TbetaRIIM as the immobilized phase, revealed pronounced differences in the pattern of plasma TGF-beta1 elevation. In MAb-profile, the TGF-beta1 increase was detected in 7 of 8 patients, whereas analogous TbetaRIIM-profile revealed the elevation in 3 of 8 patients, taking a 50% of maximal elevation as the cut-off value. These findings are consistent with the proposed aberration of TGF-beta1 ligand within the TbetaRII recognition site. Summarizing, this model system is a good starting point for further genetic studies, particularly on genetic/epigenetic alterations of sequences involved in TGF-beta1 and TbetaRIIM interaction, with putative prognostic value for breast cancer.     

The inhibitory effects of transforming growth factor-beta-1 (TGF-beta1) in autoimmune diseases

The importance of transforming growth factor-beta-1 (TGF-beta1) in immunoregulation and tolerance has been increasingly recognized. It is now proposed that there are populations of regulatory T cells (T-reg), some designated T-helper type 3 (Th3), that exert their action primarily by secreting this cytokine. Here, we emphasize the following concepts: (1) TGF-beta1 has multiple suppressive actions on T cells, B cells, macrophages, and other cells, and increased TGF-beta1 production correlates with protection and/or recovery from autoimmune diseases; (2) TGF-beta1 and CTLA-4 are molecules that work together to terminate immune responses; (3) Th0, Th1 and Th2 clones can all secrete TGF-beta1 upon cross-linking of CTLA-4 (the functional significance of this in autoimmune diseases has not been reported, but TGF-beta1-producing regulatory T-cell clones can produce type 1 inflammatory cytokines); (4) TGF-beta1 may play a role in the passage from effector to memory T cells; (5) TGF-beta1 acts with some other inhibitory molecules to maintain a state of tolerance, which is most evident in immunologically privileged sites, but may also be important in other organs; (6) TGF-beta1 is produced by many cell types, is always present in the plasma (in its latent form) and permeates all organs, binding to matrix components and creating a reservoir of this immunosuppressive molecule; and (7) TGF-beta1 downregulates adhesion molecules and inhibits adhesion of leukocytes to endothelial cells. We propose that rather than being passive targets of autoimmunity, tissues and organs actively suppress autoreactive lymphocytes. We review the beneficial effects of administering TGF-beta1 in several autoimmune diseases, and show that it can be effectively administered by a somatic gene therapy approach, which results in depressed inflammatory cytokine production and increased endogenous regulatory cytokine production.     

Temporal and spatial expression of connective tissue growth factor (CCN2; CTGF) and transforming growth factor beta type 1 (TGF-beta1) at the utero-placental interface during early pregnancy in the pig.

AIMS: To determine the localisation and distribution of connective tissue growth factor (CCN2; CTGF) and transforming growth factor beta type 1 (TGF-beta1) in uterine tissues from cycling and early pregnant pigs. METHODS: In situ hybridisation and immunohistochemistry were used to localise CCN2 (CTGF) or TGF-beta1 in uteri obtained from gilts on days 0, 5, 10, 12, 15, and 18 of the oestrous cycle or days 10, 12, 14, 16, 17, and 21 of gestation. RESULTS: In cycling animals, CCN2 (CTGF) mRNA and protein were abundant in luminal epithelial cells (LECs) and glandular epithelial cells (GECs), with lesser amounts in stromal fibroblasts and little or none in endothelial cells. A similar pattern of staining was seen up to day 10 of pregnancy, except that overall staining intensities for CCN2 (CTGF) mRNA or protein were higher and that stromal and endothelial cells were CCN2 (CTGF) positive. However, on days 12-17 there was a striking decrease in the amount of CCN2 (CTGF) in LECs at the utero-conceptus interface, which was associated with maternal stromal matrix reorganisation and the onset of subepithelial neovascularisation. This differential distribution of CCN2 (CTGF) was localised to those LECs that were in close proximity to or in apposition with trophoblast cells. This decrease in CCN2 (CTGF) staining was transient in nature and high amounts of CCN2 (CTGF) were again apparent in LECs on days 17-21, when endometrial neovascularisation and matrix remodelling were complete. The expression of uterine TGF-beta1 was comparable to that of CCN2 (CTGF) at most stages of the oestrous cycle or early pregnancy. Pre-elongation blastocysts recovered on day 10 were positive for both CCN2 (CTGF) and TGF-beta1 in the extra-embryonic trophectoderm, endoderm, and inner cell mass. On day 12, trophectoderm expressed low amounts of TGF-beta1 mRNA and non-detectable amounts of TGF-beta1 protein or CCN2 (CTGF) mRNA or protein. By days 17-21, the expression of both growth factors in the extra-embyronic/placental membranes increased and frequently exceeded that seen in LECs. CONCLUSIONS: The pattern of CCN2 (CTGF) production during the initial attachment phase supports a role for this factor in stromal remodelling and neovascularisation, although alternative functions at later stages such as epithelial-epithelial interactions are also possible. In most major cell types in the uterus or utero-placental unit, CCN2 (CTGF) expression was highly correlated with that of TGF-beta(1), indicating that CCN2 (CTGF) may mediate some of the functions of TGF-beta in the reproductive tract during the oestrous cycle and pregnancy. The data further highlight epithelium as an important source of CCN2 (CTGF) in the regulation of uterine function.     

Sandwich enzyme-linked immunosorbent assays (SELISAs) quantitate and distinguish two forms of transforming growth factor-beta (TGF-beta 1 and TGF-beta 2) in complex biological fluids

We have developed sandwich enzyme-linked immunosorbent assays (SELISAs) for TGF-beta 1 and TGF-beta 2 using both turkey and rabbit neutralizing polyclonal antibodies against native TGF-beta s. Each assay is based on the binding of two different antibodies to distinct epitopes of a single TGF-beta molecule. With these assays, TGF-beta types 1 and 2 can each be specifically quantitated in complex biological fluids, with detection limits of 2-5 pg. TGF-beta 3 and TGF-beta 5 either do not cross-react or cross-react very poorly in these assays. TGF-beta 1.2 heterodimer, although 50-80% neutralized by either TGF-beta 1 or TGF-beta 2 antibodies, shows only a 1.5 and 3.7% cross-reactivity in the TGF-beta 1 and TGF-beta 2 SELISAs, respectively. The SELISAs reported here represent the most specific, rapid, and precise assays for TGF-beta 1 and TGF-beta 2 reported thus far.     

Immune checkpoint molecules are regulated by transforming growth factor (TGF)-β1-induced epithelial-to-mesenchymal transition in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer with a high mortality rate. Epithelial-to-mesenchymal transition (EMT) confers cancer cells with immune evasive ability by modulating the expression of immune checkpoints in many cancers. Thus, the aim of our study is to examine the interplay between EMT and immune checkpoint molecules in HCC. A reversible EMT model was utilised with transforming growth factor (TGF)-β1 as an EMT inducer for HCC cell lines Hep3B and PLC/PRF/5. HCC cells were treated with TGF-β1 for 72 h and the EMT status and immune checkpoint expression were examined. In addition, the migratory ability of HCC cells were examined using wound healing and transwell migration assays in the reversible EMT model. siRNA-mediated knockdown of immune checkpoint molecule, B7-H3, was further utilised to validate the association between TGF-β1-mediated EMT and immune checkpoint expression in HCC. In addition, a web-based platform, SurvExpress, was utilised to evaluate the association between expression of TGF-β1 in combination with immune checkpoint molecules and overall survival in HCC patients. We observed induction of EMT upon treatment of HCC cells with TGF-β1 revealed by reduced expression of epithelial markers along with increased expression of mesenchymal markers. Withdrawal of TGF-β1 reversed the process of EMT with elevated expression of epithelial markers and reduced expression of mesenchymal markers. TGF-β1 treatment elevated the migratory potential of HCC cells which was reversed following reversal assay. Notably, during TGF-β1-induced EMT, there was upregulation of immune checkpoint molecules PD-L1 and B7-H3. However, the reversal of EMT decreased the expression of PD-L1 and B7-H3. In addition, TGF-β1 driven EMT was reversed following knockdown of B7-H3 in both HCC cells further validating the interplay between TGF-β1-mediated EMT and immune checkpoint expression in HCC. Furthermore, the coordinate expression of TGF-β1 with PD-L1 (p=0.01487) and B7-H3 (p=0.009687) was correlated with poor overall survival in 422 HCC patients. Our study has demonstrated a close association between TGF-β1-mediated EMT and regulation of immune checkpoints in HCC.