Interleukin 17 (IL-17)-producing helper T cells (T(H)17 cells) are often present at the sites of tissue inflammation in autoimmune diseases, which has led to the conclusion that T(H)17 cells are main drivers of autoimmune tissue injury. However, not all T(H)17 cells are pathogenic; in fact, T(H)17 cells generated with transforming growth factor-β1 (TGF-β1) and IL-6 produce IL-17 but do not readily induce autoimmune disease without further exposure to IL-23. Here we found that the production of TGF-β3 by developing T(H)17 cells was dependent on IL-23, which together with IL-6 induced very pathogenic T(H)17 cells. Moreover, TGF-β3-induced T(H)17 cells were functionally and molecularly distinct from TGF-β1-induced T(H)17 cells and had a molecular signature that defined pathogenic effector T(H)17 cells in autoimmune disease.

BACKGROUND

Cancer-associated fibroblasts (CAFs) activated by tumour cells are the predominant type of stromal cells in breast cancer tissue. The reciprocal effect of CAFs on breast cancer cells and the underlying molecular mechanisms are not fully characterised.

METHODS

Stromal fibroblasts were isolated from invasive breast cancer tissues and the conditioned medium of cultured CAFs (CAF-CM) was collected to culture the breast cancer cell lines MCF-7, T47D and MDA-MB-231. Neutralising antibody and small-molecule inhibitor were used to block the transforming growth factor-β (TGF-β) signalling derived from CAF-CM, which effect on breast cancer cells.

RESULTS

The stromal fibroblasts isolated from breast cancer tissues showed CAF characteristics with high expression levels of α-smooth muscle actin and SDF1/CXCL12. The CAF-CM transformed breast cancer cell lines into more aggressive phenotypes, including enhanced cell-extracellular matrix adhesion, migration and invasion, and promoted epithelial-mesenchymal transition (EMT). Cancer-associated fibroblasts secreted more TGF-β1 than TGF-β2 and TGF-β3, and activated the TGF-β/Smad signalling pathway in breast cancer cells. The EMT phenotype of breast cancer cells induced by CAF-CM was reversed by blocking TGF-β1 signalling.

CONCLUSIONS

Cancer-associated fibroblasts promoted aggressive phenotypes of breast cancer cells through EMT induced by paracrine TGF-β1. This might be a common mechanism for acquiring metastatic potential in breast cancer cells with different biological characteristics.

It is estimated worldwide that over 6 million people per annum experience a burn injury. Despite advances in management and improved survival rates, the incidence of hypertrophic scarring remains high. These scars are particularly common after burns and are often raised, red, hard and may cause abnormal sensations. Such pathological scarring can lead to severe functional impairment, psychological morbidity, and costly long term healthcare. Wound healing is an inherent process which restores the integrity of the skin after injury and although scarring is a frequent by-product, the scarless wound healing observed in early human gestational fetuses suggests that it is not an essential component of the response. This has lead to a large body of research attempting to understand the mechanisms behind scarring and in turn prevent it. One of the main focuses of recent research has been the role played by the growth factor TGF-β in the process of both wound healing and scar formation. The three isoforms (TGF-β1, TGF-β2 and TGF-β3) appear to have overlapping functions and predominantly mediate their effects through the intracellular SMAD pathway. Initial research suggested that TGF-β1 was responsible for the fibrotic scarring response whereas the scarless wound healing seen in fetal wounds was due to increased levels of TGF-β3. However, the reality appears to be far more complex and it is unlikely that simply altering the ratio of TGF-β isoforms will lead to scarless wound healing. Other aspects of the TGF-β system that appear promising include the downstream mediator CTGF, the proteoglycan decorin and the binding protein p311. Other putative mechanisms which may underlie the pathogenesis of hypertrophic scars include excessive inflammation, excessive angiogenesis, altered levels of matrix metalloproteinases, growth factors, and delayed apoptosis of fibrotic myofibroblasts either due to p53 genetic alterations or tensile forces across the wound. If an effective treatment for hypertrophic scars following burns injury is to be developed then further work must be carried out to understand the basic mechanisms of pathological scarring.

Chondrogenic differentiation of mesenchymal stem cells (MSCs) is accurately regulated by essential transcription factors and signaling cascades. However, the precise mechanisms involved in this process still remain to be defined. MicroRNAs (miRNAs) regulate various biological processes by binding target mRNA to attenuate protein synthesis. To investigate the mechanisms for miRNAs-mediated regulation of chondrogenic differentiation, we identified that miR-145 was decreased during transforming growth factor beta 3 (TGF-β3)-induced chondrogenic differentiation of murine MSCs. Subsequently, dual-luciferase reporter gene assay data demonstrated that miR-145 targets a putative binding site in the 3'-UTR of SRY-related high mobility group-Box gene 9 (Sox9) gene, the key transcription factor for chondrogenesis. In addition, over-expression of miR-145 decreased expression of Sox9 only at protein levels and miR-145 inhibition significantly elevated Sox9 protein levels. Furthermore, over-expression of miR-145 decreased mRNA levels for three chondrogenic marker genes, type II collagen (Col2a1), aggrecan (Agc1), cartilage oligomeric matrix protein (COMP), type IX collagen (Col9a2) and type XI collagen (Col11a1) in C3H10T1/2 cells induced by TGF-β3, whereas anti-miR-145 inhibitor increased the expression of these chondrogenic marker genes. Thus, our studies demonstrated that miR-145 is a key negative regulator of chondrogenic differentiation by directly targeting Sox9 at early stage of chondrogenic differentiation.

Mesenchymal stem cells (MSCs) are being recognized as a viable cell source for cartilage repair and members of the transforming growth factor-beta (TGF-β) superfamily are a key mediator of MSC chondrogenesis. While TGF-β mediated MSC chondrogenesis is well established in in vitro pellet or hydrogel cultures, clinical translation will require effective delivery of TGF-βs in vivo. Here, we investigated the co-encapsulation of TGF-β3 containing alginate microspheres with human MSCs in hyaluronic acid (HA) hydrogels towards the development of implantable constructs for cartilage repair. TGF-β3 encapsulated in alginate microspheres with nanofilm coatings showed significantly reduced initial burst release compared to uncoated microspheres, with release times extending up to 6 days. HA hydrogel constructs seeded with MSCs and TGF-β3 containing microspheres developed comparable mechanical properties and cartilage matrix content compared to constructs supplemented with TGF-β3 continuously in culture media, whereas constructs with TGF-β3 directly encapsulated in the gels without microspheres had inferior properties. When implanted subcutaneously in nude mice, constructs containing TGF-β3 microspheres resulted in superior cartilage matrix formation when compared to groups without TGF-β3 or with TGF-β3 added directly to the gel. However, calcification was observed in implanted constructs after 8 weeks of subcutaneous implantation. To prevent this, the co-delivery of parathyroid hormone-related protein (PTHrP) with TGF-β3 in alginate microspheres was pursued, resulting in partially reduced calcification. This study demonstrates that the controlled local delivery of TGF-β3 is essential to neocartilage formation by MSCs and that further optimization is needed to avert the differentiation of chondrogenically induced MSCs towards a hypertrophic phenotype.

BACKGROUND

Platelet-rich plasma (PRP) is nowadays widely applied in different clinical scenarios, such as orthopedics, ophthalmology and healing therapies, as a growth factor pool for improving tissue regeneration. Studies into its clinical efficiency are not conclusive and one of the main reasons for this is that different PRP preparations are used, eliciting different responses that cannot be compared. Platelet quantification and the growth factor content definition must be defined in order to understand molecular mechanisms behind PRP regenerative strength. Standardization of PRP preparations is thus urgently needed.

METHODS

PRP was prepared by centrifugation varying the relative centrifugal force, temperature, and time. Having quantified platelet recovery and yield, the two-step procedure that rendered the highest output was chosen and further analyzed. Cytokine content was determined in different fractions obtained throughout the whole centrifugation procedure.

RESULTS

Our method showed reproducibility when applied to different blood donors. We recovered 46.9 to 69.5% of total initial platelets and the procedure resulted in a 5.4-fold to 7.3-fold increase in platelet concentration (1.4 × 10(6) to 1.9 × 10(6) platelets/μl). Platelets were highly purified, because only <0.3% from the initial red blood cells and leukocytes was present in the final PRP preparation. We also quantified growth factors, cytokines and chemokines secreted by the concentrated platelets after activation with calcium and calcium/thrombin. High concentrations of platelet-derived growth factor, endothelial growth factor and transforming growth factor (TGF) were secreted, together with the anti-inflammatory and proinflammatory cytokines interleukin (IL)-4, IL-8, IL-13, IL-17, tumor necrosis factor (TNF)-α and interferon (IFN)-α. No cytokines were secreted before platelet activation. TGF-β3 and IFNγ were not detected in any studied fraction. Clots obtained after platelet coagulation retained a high concentration of several growth factors, including platelet-derived growth factor and TGF.

CONCLUSIONS

Our study resulted in a consistent PRP preparation method that yielded a cytokine and growth factor pool from different donors with high reproducibility. These findings support the use of PRP in therapies aiming for tissue regeneration, and its content characterization will allow us to understand and improve the clinical outcomes.

Proper activation of macrophages (Mφ) in the inflammatory phase of acute wound healing is essential for physiological tissue repair. However, there is a strong indication that robust Mφ inflammatory responses may be causal for the fibrotic response always accompanying adult wound healing. Using a complementary approach of in vitro and in vivo studies, we here addressed the question of whether mesenchymal stem cells (MSCs)-due to their anti-inflammatory properties-would control Mφ activation and tissue fibrosis in a murine model of full-thickness skin wounds. We have shown that the tumor necrosis factor-α (TNF-α)-stimulated protein 6 (TSG-6) released from MSCs in co-culture with activated Mφ or following injection into wound margins suppressed the release of TNF-α from activated Mφ and concomitantly induced a switch from a high to an anti-fibrotic low transforming growth factor-β1 (TGF-β1)/TGF-β3 ratio. This study provides insight into what we believe to be a previously undescribed multifaceted role of MSC-released TSG-6 in wound healing. MSC-released TSG-6 was identified to improve wound healing by limiting Mφ activation, inflammation, and fibrosis. TSG-6 and MSC-based therapies may thus qualify as promising strategies to enhance tissue repair and to prevent excessive tissue fibrosis.

Transforming growth factor β (TGF-β) has long been implicated in fibrotic diseases, including the multisystem fibrotic disease systemic sclerosis (SSc). Expression of TGF-β-regulated genes in fibrotic skin and lungs of patients with SSc correlates with disease activity, which points to this cytokine as the central mediator of pathogenesis. Patients with SSc often develop pulmonary arterial hypertension (PAH), a particularly lethal complication caused by vascular dysfunction. Several genetic diseases with vascular features related to SSc, such as familial PAH and hereditary haemorrhagic telangiectasia, are caused by mutations in the TGF-β-sensing ALK-1 signalling pathway. These observations suggest that increased TGF-β signalling causes both vascular and fibrotic features of SSc. The question of how latent TGF-β becomes activated in local SSc tissues is, therefore, central to the understanding of SSc. Both TGF-β1 and TGF-β3 can be activated by integrins αvβ6 and αvβ8, whose upregulation in bronchial epithelial cells can activate TGF-β in SSc lungs. Other αv integrins, thrombospondin-1 or altered TGF-β sequestration by matrix proteins might be important in other target tissues. How the immune system triggers this process remains unclear, although links between inflammation and TGF-β activation are emerging. Together, these observations provide an increasingly secure framework for understanding TGF-β in SSc pathogenesis.

Type 1 diabetes (T1D) is an autoimmune disease that is caused by the destruction of insulin-producing beta cells. Viral infections induce immune responses that can damage beta cells and promote T1D or on the other hand prevent the development of the disease. However, the opposing roles of viral infections in T1D are not understood mechanistically. We report here that viruses that do not inflict damage on beta cells provided protection from T1D by triggering immunoregulatory mechanisms. Infection of prediabetic NOD mice with Coxsackie virus B3 or lymphocytic choriomeningitis virus (LCMV) delayed diabetes onset and reduced disease incidence. Delayed T1D onset was due to transient upregulation of programmed cell death-1 ligand 1 (PD-L1) on lymphoid cells, which prevented the expansion of diabetogenic CD8+ T cells expressing programmed cell death-1 (PD-1). Reduced T1D incidence was caused by increased numbers of invigorated CD4+CD25+ Tregs, which produced TGF-beta and maintained long-term tolerance. Full protection from T1D resulted from synergy between PD-L1 and CD4+CD25+ Tregs. Our results provide what we believe to be novel mechanistic insight into the role of viruses in T1D and should be valuable for prospective studies in humans.

TTF-1 [thyroid transcription factor-1; also known as Nkx2.1, T/EBP (thyroid-specific-enhancer-binding protein) or TITF1] is a homeodomain-containing transcription factor essential for the morphogenesis and differentiation of the thyroid, lung and ventral forebrain. TTF-1 controls the expression of select genes in the thyroid, lung and the central nervous system. In the lung, TTF-1 controls the expression of surfactant proteins that are essential for lung stability and lung host defence. Human TTF-1 is encoded by a single gene located on chromosome 14 and is organized into two/three exons and one/two introns. Multiple transcription start sites and alternative splicing produce mRNAs with heterogeneity at the 5' end. The 3' end of the TTF-1 mRNA is characterized by a rather long untranslated region. The amino acid sequences of TTF-1 from human, rat, mouse and other species are very similar, indicating a high degree of sequence conservation. TTF-1 promoter activity is maintained by the combinatorial or co-operative actions of HNF-3 [hepatocyte nuclear factor-3; also known as FOXA (forkhead box A)], Sp (specificity protein) 1, Sp3, GATA-6 and HOXB3 (homeobox B3) transcription factors. There is limited information on the regulation of TTF-1 gene expression by hormones, cytokines and other biological agents. Glucocorticoids, cAMP and TGF-beta (transforming growth factor-beta) have stimulatory effects on TTF-1 expression, whereas TNF-alpha (tumour necrosis factor-alpha) and ceramide have inhibitory effects on TTF-1 DNA-binding activity in lung cells. Haplo-insufficiency of TTF-1 in humans causes hypothyroidism, respiratory dysfunction and recurring pulmonary infections, underlining the importance of optimal TTF-1 levels for the maintenance of thyroid and lung function. Recent studies have implicated TTF-1 as a lineage-specific proto-oncogene for lung cancer.

Scar formation, with persistent alteration of the normal tissue structure, is an undesirable and significant result of both wound healing and fibrosing disorders. There are few strategies to prevent or to treat scarring. The transforming growth factor beta (TGF-β) superfamily is an important mediator of tissue repair. Each TGF-β isoform may exert a different effect on wound healing, which may be context-dependent. In particular, TGF-β1 may mediate fibrosis in adults' wounds, while TGF-β3 may promote scarless healing in the fetus and reduced scarring in adults. Thus, TGF-β3 may offer a scar-reducing therapy for acute and chronic wounds and fibrosing disorders.

OBJECTIVE

When used as an alternative substrate following bare sclera removal of pterygium and other ocular surface diseases, amniotic membrane transplantation can reduce scarring on the reconstructed conjunctival surface. This study was carried out to determine if the amniotic membrane (AM) suppresses the expression of the TGFb signaling system in cultured normal conjunctival (HCF) and pterygial body fibroblasts (PBF).

METHODS

HCF and PBF were cultured on AM and plastic wells in serum-containing and serum-free DMEM with or without TGF-beta1. Total RNA was extracted and subjected to Northern hybridization with probes of TGF-beta1, b2 and b3; TGF-beta receptors (TGF- beta R) type I, II and III; a-smooth muscle actin (alpha-SM), b1-integrin, CD44, fibroblast growth factor receptor 1 (FGF-R1/ flg) and platelet-derived growth factor receptor b (PDGFR-beta); and GAPDH as a loading control. MTT assay was used for cell proliferation.

RESULTS

Amniotic membrane markedly suppressed the transcript expression of TGF-beta2, b3 and all three types of TGF-beta receptors by both fibroblasts as compared to their cultures on plastic surface. In addition, expression of CD44 transcript was also markedly suppressed while that of b1 integrin, a-SM actin, and FGFR1/flg was mildly suppressed. In contrast, expression of TGF-beta1 and PDGFR-beta remained largely unchanged. The cell proliferation of HCF and PBF grown on AM was also significantly suppressed.

CONCLUSIONS

Amniotic membrane matrix uniquely suppresses TGF- beta signaling in both types of fibroblasts. It may also suppress signaling via CD44, b1 integrin and FGFR1/flg. As a result, the phenotype may become less mitogenic, contractile and fibrogenic. These data support in part why amniotic membrane transplantation has an anti-scarring effect for conjunctival surface reconstruction.

Following myocardial infarction, tissue repair is mediated by the recruitment of monocytes and their subsequent differentiation into macrophages. Recent findings have revealed the dynamic changes in the presence of polarized macrophages with pro-inflammatory (M1) and anti-inflammatory (M2) properties during the early (acute) and late (chronic) stages of cardiac ischemia. Mesenchymal stem cells (MSCs) delivered into the injured myocardium as reparative cells are subjected to the effects of polarized macrophages and the inflammatory milieu. The present study investigated how cytokines and polarized macrophages associated with pro-inflammatory (M1) and anti-inflammatory (M2) responses affect the survival of MSCs. Human MSCs were studied using an in vitro platform with individual and combined M1 and M2 cytokines: IL-1β, IL-6, TNF-α, and IFN-γ (for M1), and IL-10, TGF-β1, TGF-β3, and VEGF (for M2). In addition, polarization molecules (M1: LPS and IFN-γ; M2: IL-4 and IL-13) and common chemokines (SDF-1 and MCP-1) found during inflammation were also studied. Indirect and direct co-cultures were conducted using M1 and M2 polarized human THP-1 monocytes. M2 macrophages and their associated cytokines supported the growth of hMSCs, while M1 macrophages and their associated cytokines inhibited the growth of hMSCs in vitro under certain conditions. These data imply that an anti-inflammatory (M2) environment is more accommodating to the therapeutic hMSCs than a pro-inflammatory (M1) environment at specific concentrations.

Secondary palate formation is a complex process that is frequently disturbed in mammals, resulting in the birth defect cleft palate. Gene targeting has identified components of cytokine/growth factor signalling systems such as Tgf-alpha/Egfr, Eph receptors B2 and B3 (Ephb2 and Ephb3, respectively), Tgf-beta2, Tgf-beta3 and activin-betaA (ref. 3) as regulators of secondary palate development. Here we demonstrate that the mouse orphan receptor 'related to tyrosine kinases' (Ryk) is essential for normal development and morphogenesis of craniofacial structures including the secondary palate. Ryk belongs to a subclass of catalytically inactive, but otherwise distantly related, receptor protein tyrosine kinases (RTKs). Mice homozygous for a null allele of Ryk have a distinctive craniofacial appearance, shortened limbs and postnatal mortality due to feeding and respiratory complications associated with a complete cleft of the secondary palate. Consistent with cleft palate phenocopy in Ephb2/Ephb3-deficient mice and the role of a Drosophila melanogaster Ryk orthologue, Derailed, in the transduction of repulsive axon pathfinding cues, our biochemical data implicate Ryk in signalling mediated by Eph receptors and the cell-junction-associated Af-6 (also known as Afadin). Our findings highlight the importance of signal crosstalk between members of different RTK subfamilies.

BACKGROUND

Uterine leiomyomas (fibroids) are the most common benign estrogen-dependent tumors of premenopausal women. TGF-β3 up-regulates the synthesis of many of extracellular matrix proteins that are associated with tissue fibrosis.

OBJECTIVE

To examine the effect of 1,25-dihydroxyvitamin D(3) (vitamin D(3)) on TGF-β3-induced fibrosis-related protein expression in immortalized human uterine leiomyoma (HuLM) cells.

METHODS

HuLM cells were treated with TGF-β3 with or without vitamin D(3). Western blot analyses were employed to test the effect of vitamin D(3) on TGF-β3-induced protein expression of collagen type 1, fibronectin, and plasminogen activator inhibitor-1 proteins. Western blots as well as immunofluorescence analyses were used to verify the effect of vitamin D(3) on TGF-β3-induced Smad activation involved in extracellular matrix protein synthesis and deposition, which ultimately lead to tissue fibrosis.

RESULTS

We observed that TGF-β3 induced fibronectin and collagen type 1 protein expression in HuLM cells, and that effect was suppressed by vitamin D(3). TGF-β3 also induced protein expression of plasminogen activator inhibitor-1, an important TGF-β target, in HuLM cells, which was also inhibited by vitamin D(3). Additionally, TGF-β3 induced phosphorylation of Smad2 as well as nuclear translocation of Smad2 and Smad3 in HuLM cells, whereas vitamin D significantly reduced all these TGF-β3-mediated effects. Therefore, our results suggest that vitamin D(3) has consistently reduced TGF-β3 effects that are involved in the process of fibrosis in human leiomyoma cells.

CONCLUSIONS

Vitamin D(3) is an antifibrotic factor that might be potentially useful as a novel therapeutic for nonsurgical treatment of benign uterine fibroids.

Corneal tissue engineering has attracted the attention of many researchers over the years, in part due to the cornea's avascularity and relatively straightforward structure. However, the highly organized and structured nature of this optically clear tissue has presented a great challenge. We have previously developed a model in which human corneal fibroblasts (HCFs) are stimulated by a stable vitamin C (VitC) derivative to self-assemble an extracellular matrix (ECM). Addition of TGFβ1 enhanced the assembly of ECM; however, it was accompanied by the upregulation of specific fibrotic markers. In this study, we tested the effects of all three TGFβ isoforms (-β1, -β2 and -β3) on ECM production, as well as expression of fibrotic markers. HCFs were grown in four media conditions for 4 weeks: control, VitC only; T1, VitC + TGFβ1; T2, VitC + TGFβ2; and T3, VitC + TGFβ3. The cultures were analysed with western blots, TEM and indirect immunofluorescence (IF). Compared to controls, all TGFβ isoforms stimulated matrix production by about three-fold. IF showed the presence of type III collagen and smooth muscle actin (SMA) in T1 and T2; however, T3 showed little to no expression. In western blots, T3 stimulated a lower type III:type I collagen ratio when compared to the other conditions. In addition, TEM indicated that T3 stimulated a higher level of matrix alignment and organization. HCFs stimulated by VitC and TGFβ3 appear to generate a matrix that mimics the normal adult or developing human cornea, whereas TGF-β1 and -β2 drive the constructs towards a more fibrotic path.

Despite advances in surgical technique, rotator cuff repairs are plagued by a high rate of failure. This failure rate is in part due to poor tendon-to-bone healing; rather than regeneration of a fibrocartilaginous attachment, the repair is filled with disorganized fibrovascular (scar) tissue. Transforming growth factor beta 3 (TGF-β3) has been implicated in fetal development and scarless fetal healing and, thus, exogenous addition of TGF-β3 may enhance tendon-to-bone healing. We hypothesized that: TGF-β3 could be released in a controlled manner using a heparin/fibrin-based delivery system (HBDS); and delivery of TGF-β3 at the healing tendon-to-bone insertion would lead to improvements in biomechanical properties compared to untreated controls. After demonstrating that the release kinetics of TGF-β3 could be controlled using a HBDS in vitro, matrices were incorporated at the repaired supraspinatus tendon-to-bone insertions of rats. Animals were sacrificed at 14-56 days. Repaired insertions were assessed using histology (for inflammation, vascularity, and cell proliferation) and biomechanics (for structural and mechanical properties). TGF-β3 treatment in vivo accelerated the healing process, with increases in inflammation, cellularity, vascularity, and cell proliferation at the early timepoints. Moreover, sustained delivery of TGF-β3 to the healing tendon-to-bone insertion led to significant improvements in structural properties at 28 days and in material properties at 56 days compared to controls. We concluded that TGF-β3 delivered at a sustained rate using a HBDS enhanced tendon-to-bone healing in a rat model.

Regarding cartilage repair, tissue engineering is currently focusing on the use of adult mesenchymal stem cells (MSC) as an alternative to autologous chondrocytes. The potential of stem cells from various tissues to differentiate towards the chondrogenic phenotype has been investigated and it appears that the most common and studied sources are bone marrow (BM) and adipose tissue (AT) for historical and easy access reasons. In addition to three dimensional environment, the presence of member(s) of the transforming growth factor (TGF-β family and low oxygen tension have been reported to promote the in vitro differentiation of MSCs. Our work aimed at characterizing and comparing the degree of chondrogenic differentiation of MSCs isolated from BM and AT cultured in the same conditions. We also further aimed at and at determining whether hypoxia (2% oxygen) could affect the chondrogenic potential of AT-MSCs. Cells were first expanded in the presence of FGF-2, then harvested and centrifuged to allow formation of cell pellets, which were cultured in the presence of TGF-β3 and/or Bone Morphogenetic Protein-2 (BMP-2) and with 2 or 20% oxygen tension, for 24 days. Markers of the chondrocyte (COL2A1, AGC1, Sox9) and hypertrophic chondrocyte (COL10A1, MMP-13) were monitored by real-time PCR and/or by immunohistological staining. Our data show that BMP-2/TGF-β3 combination is the best culture condition to induce the chondrocyte phenotype in pellet cultures of BM and AT-MSCs. Particularly, a switch in the expression of the pre-chondrogenic type IIA form to the cartilage-specific type IIB form of COL2A1 was observed. A parallel increase in gene expression of COL10A1 and MMP-13 was also recorded. However when AT-MSCs were cultured in hypoxia, the expression of markers of hypertrophic chondrocytes decreased when BMP-2/TGF-β3 were present in the medium. Thus it seems that hypoxia participates to the control of AT-MSCs chondrogenesis. Altogether, these cellular model systems will help us to investigate further the potential of different adult stem cells for cartilage engineering.

Removal of injured/damaged meniscus, a vital fibrocartilaginous load-bearing tissue, impairs normal knee function and predisposes patients to osteoarthritis. Meniscus tissue engineering solution is one option to improve outcomes and relieve pain. In an attempt to fabricate knee meniscus grafts three layered wedge shaped silk meniscal scaffold system was engineered to mimic native meniscus architecture. The scaffolds were seeded with human fibroblasts (outside) and chondrocytes (inside) in a spatial separated mode similar to native tissue, in order to generate meniscus-like tissue in vitro. In chondrogenic culture in the presence of TGF-b3, cell-seeded constructs increased in cellularity and extracellular matrix (ECM) content. Histology and Immunohistochemistry confirmed maintenance of chondrocytic phenotype with higher levels of sulfated glycosaminoglycans (sGAG) and collagen types I and II. Improved scaffold mechanical properties along with ECM alignment with time in culture suggest this multiporous silk construct as a useful micro-patterned template for directed tissue growth with respect to form and function of meniscus-like tissue.

CONCLUSIONS

Wound healing is an intricate biological process in which the skin, or any other tissue, repairs itself after injury. Normal wound healing relies on the appropriate levels of cytokines and growth factors to ensure that cellular responses are mediated in a coordinated manner. Among the many growth factors studied in the context of wound healing, transforming growth factor beta (TGF-β) is thought to have the broadest spectrum of effects.

UNASSIGNED

Many of the molecular mechanisms underlying the TGF-β/Smad signaling pathway have been elucidated, and the role of TGF-β in wound healing has been well characterized. Targeting the TGF-β signaling pathway using therapeutic agents to improve wound healing and/or reduce scarring has been successful in pre-clinical studies.

RESULTS

Although TGF-β isoforms (β1, β2, β3) signal through the same cell surface receptors, they display distinct functions during wound healing in vivo through mechanisms that have not been fully elucidated. The challenge of translating preclinical studies targeting the TGF-β signaling pathway to a clinical setting may require more extensive preclinical research using animal models that more closely mimic wound healing and scarring in humans, and taking into account the spatial, temporal, and cell-type-specific aspects of TGF-β isoform expression and function.

CONCLUSIONS

Understanding the differences in TGF-β isoform signaling at the molecular level and identification of novel components of the TGF-β signaling pathway that critically regulate wound healing may lead to the discovery of potential therapeutic targets for treatment of impaired wound healing and pathological scarring.

Scar formation is an intractable medical problem that appears after skin wounds have healed. Recent research has shown that exosomes secreted by human adipose mesenchymal stem cells (ASC-Exos) can benefit wound healing. To further explore the therapeutic potential of ASC-Exos, we investigated their effects on mitigating scar formation, and the underlying mechanisms of these effects. We found that intravenous injection of ASC-Exos decreased the size of scars and increased the ratio of collagen III to collagen I in murine incisional wounds. Exosome treatment also prevented the differentiation of fibroblasts into myofibroblasts and increased the ratio of transforming growth factor-β3 (TGF-β3) to TGF-β1 in vivo. Additionally, we found that ASC-Exos increased the matrix metalloproteinases-3 (MMP3) expression of skin dermal fibroblasts by activating the ERK/MAPK pathway, leading to a high ratio of MMP3 to tissue inhibitor of matrix metalloproteinases-1 (TIMP1), which is also beneficial for the remodelling of extracellular matrix (ECM). In conclusion, our results demonstrated that ASC-Exos promote ECM reconstruction in cutaneous wound repair by regulating the ratios of collagen type III: type I, TGF-β3:TGF-β1 and MMP3:TIMP1, and by regulating fibroblast differentiation to mitigate scar formation. Therefore, the application of ASC-Exos may be a novel therapeutic approach for scarless wound repair.

TRIM28 is a component of heterochromatin complexes whose function in the immune system is unknown. By studying mice with conditional T cell-specific deletion of TRIM28 (CKO mice), we found that TRIM28 was phosphorylated after stimulation via the T cell antigen receptor (TCR) and was involved in the global regulation of CD4(+) T cells. The CKO mice had a spontaneous autoimmune phenotype that was due in part to early lymphopenia associated with a defect in the production of interleukin 2 (IL-2) as well as incomplete cell-cycle progression of their T cells. In addition, CKO T cells showed derepression of the cytokine TGF-β3, which resulted in an altered cytokine balance; this caused the accumulation of autoreactive cells of the T(H)17 subset of helper T cells and of Foxp3(+) T cells. Notably, CKO Foxp3(+) T cells were unable to prevent the autoimmune phenotype in vivo. Our results show critical roles for TRIM28 in both T cell activation and T cell tolerance.

OBJECTIVE

To investigate the effects of human gut micro-organisms on cytokine production by human intestinal cell lines.

RESULTS

Quantitative real-time PCR assays were developed to measure the production of pro-inflammatory (IL-1α, IL-6, IL-18 and TNFα) and anti-inflammatory (TGF-β1, TGF-β2, TGF-β3, IL-4 and IL-10) cytokines in HT-29 and Caco-2 cell lines. They were co-cultured with a range of mucosal bacteria isolated from ulcerative colitis patients, together with lactobacilli and bifidobacteria obtained from healthy people. HT-29 cells were also co-cultured with Campylobacter jejuni, enterotoxigenic Escherichia coli (ETEC), enteropathogenic E. coli and Salmonella typhimurium. The majority of commensal bacteria tested suppressed the expression of anti-inflammatory cytokine mRNA, increased IL-18, reduced IL-1α, and with the exception of nonpathogenic E. coli, reduced TNF-α. All overtly pathogenic species increased both pro-inflammatory and anti-inflammatory cytokine mRNA.

CONCLUSIONS

Commensal and pathogenic species induced fundamentally different cytokine responses in human intestinal epithelial cell lines.

CONCLUSIONS

Interactions between commensal bacteria tested in this study and the innate immune system were shown to be anti-inflammatory in nature, in contrast to the pathogenic organisms investigated. These data contribute towards our understanding of how potential probiotic species can be used to suppress the pro-inflammatory response in inflammatory bowel disease.

The intricate interaction between protein endocytosis, transcytosis, recycling and endosome- or ubiquitin-mediated protein degradation determines the junction integrity in epithelial cells including Sertoli cells at the blood-testis barrier (BTB). Studies have shown that androgens and cytokines (e.g., TGF-β3) that are known to promote and disrupt BTB integrity, respectively, accelerate protein endocytosis at the BTB. We hypothesized that testosterone-induced endocytosed proteins are transcytosed and recycled back to the Sertoli cell surface, whereas cytokine-induced endocytosed proteins are degraded so that androgens and cytokines have opposing effects on BTB integrity. Herein, we report that both testosterone and TGF-β3 induced the steady-state level of clathrin, an endocytic vesicle protein. Testosterone and TGF-β3 also induced the association between internalized occludin (a BTB integral membrane protein) and clathrin, as well as early endosome antigen-1 (EEA-1). Interestingly, testosterone, but not TGF-β3, also induced the levels of proteins that regulate protein transcytosis (e.g., caveolin-1) and recycling (e.g., Rab11), and their association with internalized occludin and N-cadherin from the cell surface. In contrast, TGF-β3, but not testosterone, induced the level of ubiquitin-conjugating enzyme E2 J1 (Ube2j1), a protein crucial to the intracellular protein degradation pathway, and its association with internalized occludin. Based on these findings and recent reports in the field, we hypothesize that the concerted effects of testosterone and TGF-β3 likely facilitate the transit of preleptotene spermatocytes at the BTB while maintaining the immunological barrier in that testosterone induces the assembly of "new" tight junction (TJ)-fibrils below migrating spermatocytes via protein transcytosis and recycling before cytokines induce the disassembly of "old" TJ-fibrils above spermatocytes via endocytic vesicle-mediated degradation of internalized proteins. This thus provides a unique mechanism in the testis to facilitate the transit of preleptotene spermatocytes, many of which are connected in "clones" via cytoplasmic bridges, at the BTB while maintaining the immunological barrier during stage VIII of the seminiferous epithelial cycle of spermatogenesis.