Medium conditioned by Chinese hamster ovary (CHO) cells transfected with the simian pre-pro-TGF beta 1 cDNA contains high levels of latent TGF beta 1. The amino-terminal region of the TGF beta 1 precursor is secreted and can be detected in the conditioned medium by immunoblotting using peptide antibodies specific for amino-terminal peptides. Chemical cross-linking of CHO-conditioned medium using bis-(sulfosuccinimidyl)-suberate (BS3) followed by immunoblot analyses indicates that latent recombinant TGF beta 1 contains both the cleaved amino-terminal glycopeptide and mature TGF beta 1 polypeptide in a noncovalent association and that this association confers latency. The data presented here do not support the involvement of a unique TGF beta binding protein(s) in latent recombinant TGF beta 1. Plasmin treatment of CHO-conditioned medium resulted in the appearance of TGF beta competing activity. In addition, immunoblot analysis of plasmin-treated CHO-conditioned medium indicates that the amino-terminal glycopeptide is partially degraded and that mature TGF beta 1 is released. Thus, activation of latent TGF beta 1 may occur by proteolytic nicking within the amino-terminal glycopeptide thereby causing a disruption of tertiary structure and noncovalent bonds, which results in the release of active, mature TGF beta 1. Acid activation of latent TGF beta, in comparison, appears to be due to dissociation of the amino-terminal glycopeptide from the mature polypeptide.

A goal of cartilage tissue engineering is the production of cell-laden constructs possessing sufficient mechanical and biochemical features to enable native tissue function. This study details a systematic characterization of a serum-free (SF) culture methodology employing transient growth factor supplementation to promote robust maturation of tissue-engineered cartilage. Bovine chondrocyte agarose hydrogel constructs were cultured under free-swelling conditions in serum-containing or SF medium supplemented continuously or transiently with varying doses of transforming growth factor beta 3 (TGF-beta3). Constructs were harvested weekly or bi-weekly and assessed for mechanical and biochemical properties. Transient exposure (2 weeks) to low concentrations (2.5-5 ng/mL) of TGF-beta3 in chemically defined medium facilitated robust and highly reproducible construct maturation. Constructs receiving transient TGF-beta3 exposure achieved native tissue levels of compressive modulus (0.8 MPa) and proteoglycan content (6-7% of wet weight) after less than 2 months of in vitro culture. This maturation response was far superior to that observed after continuous growth factor supplementation or transient TGF-beta3 treatment in the presence of serum. These findings represent a significant advance in developing an ex vivo culture methodology to promote production of clinically relevant and mechanically competent tissue-engineered cartilage constructs for implantation to repair damaged articular surfaces.

Traumatic brain injury (TBI) survivors experience long-term post-traumatic morbidities. In diffuse brain-injured rats, a chronic sensory sensitivity to whisker stimulation models the agitation of TBI survivors and provides anatomical landmarks across the whisker-barrel circuit to evaluate post-traumatic neuropathology. As a consequence of TBI, acute and chronic microglial activation can contribute to degenerative and reparative events underlying post-traumatic morbidity. Here we hypothesize that a temporal sequence of microglial activation states contributes to the circuit pathology responsible for post-traumatic morbidity, and test the hypothesis by examining microglial morphological activation and neuroinflammatory markers for activation states through gene expression and receptor-binding affinity. Adult male, Sprague-Dawley rats were subjected to a single moderate midline fluid percussion injury (FPI) or sham injury. Microglial activation was determined by immunohistochemistry, quantitative real-time PCR and receptor autoradiography in the primary somatosensory barrel field (S1BF) and ventral posterior medial nucleus (VPM) of the thalamus at 7 and 28 days following FPI. Morphological changes indicative of microglial activation, including swollen cell body with thicker, shrunken processes, were evident in S1BF and VPM at 7 and 28 days post-injury. Principally at 7 days post-injury in VPM, general inflammatory gene expression (major histocompatibility complex I, major histocompatibility complex II, translocator protein 18 kDa [TSPO]) is increased above sham level and TSPO gene expression confirmed by receptor autoradiography. Further, CD45, a marker of classical activation, and TGF-βI, an acquired deactivation marker, were elevated significantly above sham at 7 days post-injury. Daily administration of the anti-inflammatory ibuprofen (20mg/kg, i.p.) significantly reduced the expression of these genes. Evidence for alternative activation (arginase 1) was not observed. Thus, these data demonstrate concomitant classical activation and acquired deactivation phenotypes of microglia in diffuse TBI in the absence of overt contusion or cavitation. Anti-inflammatory treatment may further alleviate the neuropathological burden of post-traumatic inflammation.

Bi-allelic-inactivating mutations of the VHL tumor suppressor gene are found in the majority of clear cell renal cell carcinomas (VHL(-/-) RCC). VHL(-/-) RCC cells overproduce hypoxia-inducible genes as a consequence of constitutive, oxygen-independent activation of hypoxia inducible factor (HIF). While HIF activation explains the highly vascularized nature of VHL loss lesions, the relative role of HIF in oncogenesis and loss of growth control remains unknown. Here, we report that HIF plays a central role in promoting unregulated growth of VHL(-/-) RCC cells by activating the transforming growth factor-alpha (TGF-alpha)/epidermal growth factor receptor (EGF-R) pathway. Dominant-negative HIF and enzymatic inhibition of EGF-R were equally efficient at abolishing EGF-R activation and serum-independent growth of VHL(-/-) RCC cells. TGF-alpha is the only known EGF-R ligand that has a VHL-dependent expression profile and its overexpression by VHL(-/-) RCC cells is a direct consequence of HIF activation. In contrast to TGF-alpha, other HIF targets, including vascular endothelial growth factor (VEGF), were unable to stimulate serum-independent growth of VHL(-/-) RCC cells. VHL(-/-) RCC cells expressing reintroduced type 2C mutants of VHL, and which retain the ability to degrade HIF, fail to overproduce TGF-alpha and proliferate in serum-free media. These data link HIF with the overproduction of a bona fide renal cell mitogen leading to activation of a pathway involved in growth of renal cancer cells. Moreover, our results suggest that HIF might be involved in oncogenesis to a much higher extent than previously appreciated.

The effect of human mast cells on fibroblast activity was studied using an organotypic skin-equivalent culture system. Human mast cell-1 (HMC-1) cells were embedded in a collagen gel with neonatal dermal fibroblasts at a ratio of 1:4; keratinocytes then were allowed to stratify above this composite culture. Analysis of type a1(I) procollagen mRNA synthesis by in situ hybridization revealed a substantial increase in mRNA levels in the presence of mast cells and especially following degranulation, induced by calcium ionophore A23187. Tryptase, a major product of human mast cells, could substitute for mast cells in this culture system, up-regulating procollagen mRNA synthesis. Tryptase pretreated with the specific protease inhibitor bis(5-amidino-2-benzimidazo-lyl)methane (BABIM) markedly attenuated the collagen mRNA up-regulation. Further studies revealed HMC-1 cell sonicates stimulated fibroblast chemotaxis and procollagen mRNA synthesis. Inhibition of HMC-1 sonicates with either BABIM or a neutralizing mAb against tryptase resulted in significant reduction of fibroblast chemotaxis and procollagen mRNA, implying that tryptase accounted for the majority of HMC-1 sonicate activity. Tryptase directly stimulated fibroblast chemotaxis with optimal concentrations between 10 pM and 1 nM. The maximal response of optimal concentrations of tryptase was comparable with the known fibrogenic factor, TGF-beta. Inhibition of tryptase with BABIM resulted in approximately 50% reduction in chemotactic activity. Additional studies revealed that tryptase (0.3-3 nM) stimulated procollagen mRNA synthesis in confluent monolayers of dermal fibroblasts.

We performed a phase I trial of FANG vaccine, an autologous tumor-based product incorporating a plasmid encoding granulocyte-macrophage colony-stimulating factor (GMCSF) and a novel bifunctional short hairpin RNAi (bi-shRNAi) targeting furin convertase, thereby downregulating endogenous immunosuppressive transforming growth factors (TGF) β1 and β2. Patients with advanced cancer received up to 12 monthly intradermal injections of FANG vaccine (1 × 10(7) or 2.5 × 10(7) cells/ml injection). GMCSF, TGFβ1, TGFβ2, and furin proteins were quantified by enzyme-linked immunosorbent assay (ELISA). Safety and response were monitored. Vaccine manufacturing was successful in 42 of 46 patients of whom 27 received ≥1 vaccine. There were no treatment-related serious adverse events. Most common grade 1, 2 adverse events included local induration (n = 14) and local erythema (n = 11) at injection site. Post-transfection mean product expression GMCSF increased from 7.3 to 1,108 pg/10(6) cells/ml. Mean TGFβ1 and β2 effective target knockdown was 93.5 and 92.5% from baseline, respectively. Positive enzyme-linked immunospot (ELISPOT) response at month 4 was demonstrated in 9 of 18 patients serially assessed and correlated with survival duration from time of treatment (P = 0.025). Neither dose-adverse event nor dose-response relationship was noted. In conclusion, FANG vaccine was safe and elicited an immune response correlating with prolonged survival. Phase II assessment is justified.

alpha-Tocopherol modulates two major signal transduction pathways centered on protein kinase C and phosphatidylinositol 3-kinase. Changes in the activity of these key kinases are associated with changes in cell proliferation, platelet aggregation, and NADPH-oxidase activation. Several genes are also regulated by tocopherols partly because of the effects of tocopherol on these two kinases, but also independently of them. These genes can be divided in five groups: Group 1. Genes that are involved in the uptake and degradation of tocopherols: alpha-tocopherol transfer protein, cytochrome P450 (CYP3A), gamma-glutamyl-cysteine synthetase heavy subunit, and glutathione-S-transferase. Group 2. Genes that are implicated with lipid uptake and atherosclerosis: CD36, SR-BI, and SR-AI/II. Group 3. Genes that are involved in the modulation of extracellular proteins: tropomyosin, collagen-alpha-1, MMP-1, MMP-19, and connective tissue growth factor. Group 4. Genes that are connected to adhesion and inflammation: E-selectin, ICAM-1 integrins, glycoprotein IIb, IL-2, IL-4, IL-1b, and transforming growth factor-beta (TGF-beta). Group 5. Genes implicated in cell signaling and cell cycle regulation: PPAR-gamma, cyclin D1, cyclin E, Bcl2-L1, p27, CD95 (APO-1/Fas ligand), and 5a-steroid reductase type 1. The transcription of p27, Bcl2, alpha-tocopherol transfer protein, cytochrome P450 (CYP3A), gamma-glutamyl-cysteine sythetase heavy subunit, tropomyosin, IL-2, and CTGF appears to be upregulated by one or more tocopherols. All the other listed genes are downregulated. Gene regulation by tocopherols has been associated with protein kinase C because of its deactivation by alpha-tocopherol and its contribution in the regulation of a number of transcription factors (NF-kappaB, AP1). A direct participation of the pregnane X receptor (PXR) / retinoid X receptor (RXR) has been also shown. The antioxidant-responsive element (ARE) and the TGF-beta-responsive element (TGF-beta-RE) appear in some cases to be implicated as well.

Delta is a major transmembrane ligand for Notch receptor that mediates numerous cell fate decisions. The Notch signaling pathway has long been thought to be mono-directional, because ligands for Notch were generally believed to be unable to transmit signals into the cells expressing them. However, we showed here that Notch also supplies signals to neighboring mouse neural stem cells (NSCs). To investigate the Notch-Delta signaling pathway in a bi-directional manner, we analyzed functional roles of the intracellular domain of mouse Delta like protein 1 (Dll1IC). In developing mouse NSCs, Dll1IC, which is released from cell membrane by proteolysis, is present in the nucleus. Furthermore, we screened for transcription factors that bind to Dll1IC and demonstrated that Dll1IC binds specifically to transcription factors involved in TGF-beta/Activin signaling--Smad2, Smad3 and Smad4--and enhances Smad-dependent transcription. In addition, the results of the present study indicated that over-expression of Dll1IC in embryonic carcinoma P19 cells induced neurons, and this induction was blocked by SB431542, which is a specific inhibitor of TGF-beta/Activin signaling. These observations strongly suggested that Dll1IC mediates TGF-beta/Activin signaling through binding to Smads and plays an important role for bi-directional Notch-Delta signaling pathway.

Experimental autoimmune encephalomylitis (EAE), an animal mode of multiple sclerosis (MS), was previously considered that be mediated by Th1 cells. However, a number of recent studies provided strong evidence that T helper cells that produce IL-17 play a dominant role in the pathogenesis of EAE. Curcumin (1,7-Bis 94-hydroxy-3-methoxyphenyl)-1,6 heptadiene-3, 5-di-one) is a naturally occurring polyphenolic phytochemical isolated from the rhizome of the medicinal plant Curcuma longa. It has been strongly implicated as an anti-inflammatory agent, but the precise mechanisms of its action are largely unknown. In the present study, we have investigated the efficacy and mechanism of curcumin against EAE. The treatment of Lewis rats with curcumin significantly reduced the clinical severity of EAE, and had a dramatic reduction in the number of inflammatory cells infiltration in the spinal cord. The proliferation of the MBP-reaction lymphocyte also was reduced in a curcumin dose-dependent manner. Furthermore, the mRNA expression of the cytokine profiles was assessed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), revealing the dramatic decrease of IL-17, TGF-beta, IL-6, IL-21, STAT3, and RORgammat expression in curcumin-treated groups and STAT3-phosphorylation also was inhibited. These findings indicated that curcumin amelioration EAE was, to a large extent, due to inhibit differentiation and development of Th17 cells depends on down-regulating expression of IL-6, IL-21, RORgammat signaling and inhibition STAT3-phosphorylation, suggests it is useful in the treatment of MS and other Th17 cell-mediated inflammatory diseases.

One of the hallmarks of progressive renal disease is the development of tubulointerstitial fibrosis. This is frequently preceded by macrophage infiltration, raising the possibility that macrophages relay fibrogenic signals to resident tubulointerstitial cells. The aim of this study was to investigate the potentially fibrogenic role of interleukin-1beta (IL-1beta), a macrophage-derived inflammatory cytokine, on cortical fibroblasts (CFs). Primary cultures of human renal CFs were established and incubated for 24 hours in the presence or absence of IL-1beta. We found that IL-1beta significantly stimulated DNA synthesis (356.7% +/- 39% of control, P <.003), fibronectin secretion (261.8 +/- 11% of control, P <.005), collagen type 1 production, (release of procollagen type 1 C-terminal-peptide, 152.4% +/- 26% of control, P <.005), transforming growth factor-beta (TGF-beta) secretion (211% +/- 37% of control, P <.01), and nitric oxide (NO) production (342.8% +/- 69% of control, P <.002). TGF-beta (1 ng/mL) and the phorbol ester phorbol 12-myristate 13-acetate (PMA, 25 nmol/L) produced fibrogenic effects similar to those of IL-1beta. Neither a NO synthase inhibitor (N(G)-methyl-l-arginine, 1 mmol/L) nor a protein kinase C (PKC) inhibitor (bis-indolylmaleimide 1, 1 micromol/L) altered the enhanced level of fibronectin secretion or DNA synthesis seen in response to IL-1beta treatment. However, addition of a TGF-beta-neutralizing antibody significantly reduced IL-1beta-induced fibronectin secretion (IL-1beta + IgG, 262% +/- 72% vs IL-1beta + alphaTGF-beta 156% +/- 14%, P <.02), collagen type 1 production (IL-1beta + IgG, 176% +/- 28% vs IL-1beta + alphaTGF-beta, 120% +/- 14%, P <.005) and abrogated IL-1beta-induced DNA synthesis (245% +/- 49% vs 105% +/- 21%, P <.005). IL-1beta significantly stimulated CF DNA synthesis and production of fibronectin, collagen type 1, TGFbeta, and NO. The fibrogenic and proliferative action of IL-1beta on CF appears not to involve activation of PKC or production of NO but is at least partly TGFbeta-dependent.

We present a strategy to conjugate TGF-β1 into fibrin hydrogels to mimic the in vivo presentation of the growth factor in a 3D context. To this end, we engineered fusion proteins between TGF-β1 and a bi-functional peptide composed of a Factor XIII domain and a plasmin cleavage site. In another version the protease cleavage site was omitted to examine whether the growth factor that could not be released from the scaffold by cells had different effects on tissue constructs. The optimal insertion site which yielded correctly processed, functional protein was found between the latency associated peptide and mature TGF-β1 domains. In solution the fusion proteins exhibited similar biological activity as native TGF-β1 as evidenced by inhibition of cell proliferation and promoter activity assays. Immunoprecipitation experiments demonstrated that the fusion TGF-β1 protein bound to fibrinogen in a Factor XIII dependent manner and could be released from the peptide by the action of plasmin. In contrast to bolus delivery, immobilized TGF-β1 induced sustained signaling in fibrin-embedded cells for several days as evidenced by Smad2 phosphorylation. Prolonged pathway activation correlated with enhanced contractile function of vascular constructs prepared from hair follicle mesenchymal stem cells or bone marrow derived smooth muscle cells. Our results suggest that fibrin-immobilized TGF-β1 may be used to enhance the local microenvironment and improve the function of engineered tissues in vitro and potentially also after implantation in vivo where growth factor delivery faces overwhelming challenges.

Scatchard analyses of the binding of transforming growth factor-beta (TGF beta) to membranes from rat ventral prostate revealed the presence of high affinity (Kd = 140 pM) saturable binding sites for [125I]TGF beta. The binding of [125I]TGF beta to prostatic membranes, while displaced in the presence of excess unlabeled TGF beta, is unaffected by epidermal growth factor, nerve growth factor, fibroblast growth factor, or insulin, indicating the specificity of binding. Affinity labeling of these membrane receptors by covalent attachment to [125I]TGF beta with bis-(sulfosuccinimidyl)suberate and subsequent electrophoretic analysis of the labeled complexes revealed the specific binding of [125I]TGF beta to a macromolecule that predominantly migrates as a 260,000 mol wt band in 7.5% acrylamide gels. Castration-induced androgen deprivation produced a significant increase in [125I]TGF beta binding to prostatic membranes with no apparent change in the affinity of membrane receptors for TGF beta. TGF beta receptor levels per total gland increased approximately 2-fold by 3 days after castration, reached a peak value by day 4, and then declined during the subsequent 10 days. Androgen administration to 4-day castrated animals decreased the number of TGF beta receptors to a value similar to that in the intact controls. These results demonstrate the presence of specific binding sites for TGF beta in the rat ventral prostate. Furthermore, the TGF beta receptor levels seem to be under negative androgenic regulation, indicating a potential role for this growth factor in the mechanism of activation of castration-induced death of androgen-dependent epithelial cells in the ventral prostate.

OBJECTIVE

Crosstalk between cardiomyocytes and fibroblasts in physiological conditions and during disease remains poorly defined. Previous studies have shown that fibroblasts and myocytes interact via paracrine communication, but several experimental confounding factors, including the use of immature myocytes and the induction of alpha-smooth muscle actin (α-SMA) expression in fibroblasts by prolonged culture, have hindered our understanding of this phenomenon. We hypothesize that fibroblasts and myofibroblasts differentially affect cardiomyocytes viability, volume, and Ca(2+) handling via soluble mediators. More specifically here: (i) we compare the effects of freshly isolated fibroblasts and cultured fibroblasts from normal rat hearts on adult cardiomyocytes; (ii) we compare the effects of (freshly isolated) normal fibroblasts and myofibroblasts from pressure-overloaded hearts; and (iii) we study the contribution of TGF-β and the importance of the crosstalk between the two cell types.

RESULTS

We used co-culture methods and conditioned medium to investigate paracrine interaction between fibroblasts and cardiomyocytes. All fibroblast types reduce cardiomyocyte viability and increase cardiomyocyte volume but α-SMA-negative fibroblasts increase cardiomyocyte Ca(2+) transient amplitude, whereas cultured fibroblasts and myofibroblasts from pressure-overloaded hearts decrease Ca(2+) transient amplitude. In turn, cardiomyocytes release soluble mediators that affect fibroblast proliferation. Using SB431542 to block TGF-β type 1 receptors, we determined that TGF-β directly causes cardiomyocyte hypertrophy and participates in bi-directional regulatory signalling between fibroblasts and cardiomyocytes.

CONCLUSIONS

Fibroblasts have different roles during physiology and disease in regulating myocardial function via soluble mediators. A crosstalk between fibroblasts and cardiomyocytes, controlled by TGF-β, is crucial in this interaction.

Eight integrin α-β heterodimers recognize ligands with an Arg-Gly-Asp (RGD) motif. However, the structural mechanism by which integrins differentiate among extracellular proteins with RGD motifs is not understood. Here, crystal structures, mutations and peptide-affinity measurements show that αVβ6 binds with high affinity to a RGDLXXL/I motif within the prodomains of TGF-β1 and TGF-β3. The LXXL/I motif forms an amphipathic α-helix that binds in a hydrophobic pocket in the β6 subunit. Elucidation of the basis for ligand binding specificity by the integrin β subunit reveals contributions by three different βI-domain loops, which we designate specificity-determining loops (SDLs) 1, 2 and 3. Variation in a pair of single key residues in SDL1 and SDL3 correlates with the variation of the entire β subunit in integrin evolution, thus suggesting a paradigmatic role in overall β-subunit function.

Bone morphogenetic proteins (BMPs) are multi-functional cytokines, which belong to the transforming growth factor-β (TGF-β) family. In some cancer tissues, aberrant expression of various BMP signal components has been detected. Here, we describe the divergent roles of BMPs during the progression of cancer. BMPs exhibit various effects on both cancer cells and on tumor microenvironments. BMPs inhibit the proliferation of cancer cells, with some exceptions. BMPs also induce the differentiation of certain cancer stem cells, and attenuate their aggressiveness. In parallel, BMPs play a critical role in the regulation of tumor angiogenesis and the metastasis of cancer cells. Some mouse xenograft models have revealed that cancer metastases are prevented by the inhibition of BMP signaling. Together, these findings imply that BMPs function as both suppressors and promoters of tumors in a context dependent manner. The bi-directional characteristics of BMPs in cancer are similar to those of TGF-β, which was previously described as a molecular 'Jekyll and Hyde.'

BACKGROUND

Because the outcomes and sequelae after different types of brain injury (BI) are variable and difficult to predict, investigations on whether enhanced expressions of BI-associated biomarkers (BIABs), including transforming growth factor beta1 (TGF-beta1), S100B, glial fibrillary acidic protein (GFAP), neurofilament light chain (NF-L), tissue transglutaminases (tTGs), beta-amyloid precursor proteins (AbetaPP), and tau are present as well as whether impairment of the ubiquitin-proteasome system (UPS) is present have been widely used to help delineate pathophysiological mechanisms in various BIs. Larvae of Toxocara canis can invade the brain and cause BI in humans and mice, leading to cerebral toxocariasis (CT). Because the parasitic burden is light in CT, it may be too cryptic to be detected in humans, making it difficult to clearly understand the pathogenesis of subtle BI in CT. Since the pathogenesis of murine toxocariasis is very similar to that in humans, it appears appropriate to use a murine model to investigate the pathogenesis of CT.

METHODS

BIAB expressions and UPS function in the brains of mice inoculated with a single dose of 250 T. canis embryonated eggs was investigated from 3 days (dpi) to 8 weeks post-infection (wpi) by Western blotting and RT-PCR.

RESULTS

Results revealed that at 4 and 8 wpi, T. canis larvae were found to have invaded areas around the choroid plexus but without eliciting leukocyte infiltration in brains of infected mice; nevertheless, astrogliosis, an indicator of BI, with 78.9~142.0-fold increases in GFAP expression was present. Meanwhile, markedly increased levels of other BIAB proteins including TGF-beta1, S100B, NF-L, tTG, AbetaPP, and tau, with increases ranging 2.0~12.0-fold were found, although their corresponding mRNA expressions were not found to be present at 8 wpi. Concomitantly, UPS impairment was evidenced by the overexpression of conjugated ubiquitin and ubiquitin in the brain.

CONCLUSIONS

Further studies are needed to determine whether there is an increased risk of CT progression into neurodegenerative disease because neurodegeneration-associated AbetaPP and phosphorylated tau emerged in the brain.

The contemporary use of nanomedicines for cancer treatment has been largely limited to serving as carriers for existing therapeutic agents. Here, we provide definitive evidence that, the metallofullerenol nanomaterial Gd@C82(OH)22, while essentially not toxic to normal mammary epithelial cells, possesses intrinsic inhibitory activity against triple-negative breast cancer cells. Gd@C82(OH)22 blocks epithelial-to-mesenchymal transition with resultant efficient elimination of breast cancer stem cells (CSCs) resulting in abrogation of tumour initiation and metastasis. In normoxic conditions, Gd@C82(OH)22 mediates these effects by blocking TGF-β signalling. Moreover, under hypoxic conditions found in the tumour microenvironment, cellular uptake of Gd@C82(OH)22 is facilitated where it functions as a bi-potent inhibitor of HIF-1α and TGF-β activities, enhancing CSC elimination. These studies indicate that nanomaterials can be engineered to directly target CSCs. Thus, Gd-metallofullerenol is identified as a kind of non-toxic CSC specific inhibitors with significant therapeutic potential.

Protective immunity against Mycobacterium tuberculosis (MTB) in animal models is based on cell-mediated immunity (CMI), involving bi-directional interactions between T cells and cells of the monocyte/macrophage (MO/MA) lineage. Key factors include MO-derived interleukin (IL)-12 and tumor necrosis factor (TNF)-alpha as well as T cell derived IL-2 and interferon (IFN)-gamma. These cytokines appear particularly crucial in the induction of MA-mediated elimination of mycobacteria. Several lines of evidence indicate that similar mechanisms are operating in humans. During active pulmonary tuberculosis (PTB), signs of both immune depression and immune activation are concomitantly present. Decreased tuberculin skin test reactivity in vivo and deficient IFN-gamma production by MTB-stimulated mononuclear cells in vitro are observed. On the other hand, the serum levels of several cytokines, including TNF, and other inflammatory mediators are increased and circulating MO and T cell show phenotypic and functional evidence of in vivo activation. In this review, we will discuss the evidence for three models, which could explain this apparent paradox: 1. Stimulation of the T cell-suppressive function from MO/MA; 2. Intrinsic T cell refractoriness, possibly associated with tendency to apoptosis (programmed cell death), and 3. Compartmentalization and redistribution of immune responses to the site of disease. The opportunistic behavior of MTB during human immunodeficiency virus (HIV) infection can be explained by suppression of type-1 responses at the level of antigen-presenting cells, CD4 T cells and effector macrophages. The ominous prognostic significance of intercurrent PTB during HIV infection seems primarily due to prolonged activation of HIV replication in macrophages. Supportive immune therapy during PTB could aim at correcting the type-1 deficiency either by IFN-gamma inducers (e.g. IL-12, IL-18) or by neutralizing the suppressive cytokines transforming growth factor beta (TGF-beta) and IL-10. Alternatively, inflammatory over-activity could be reduced by neutralizing TNF. Finally, anti-apoptotic therapies (e.g. IL-15) might be considered.

Brain colonization by metastatic tumor cells offers a unique opportunity to investigate microenvironmental influences on the neoplastic process. The bi-directional interplay of breast cancer cells (mesodermal origin) and brain cells (neuroectodermal origin) is poorly understood and rarely investigated. In our patients undergoing neurosurgical resection of breast-to-brain metastases, specimens from the tumor/brain interface exhibited increased active gliosis as previously described. In addition, our histological characterization revealed infiltration of neural progenitor cells (NPCs) both outside and inside the tumor margin, leading us to investigate the cellular and molecular interactions between NPCs and metastases. Since signaling by the TGF-β superfamily is involved in both developmental neurobiology and breast cancer pathogenesis, we examined the role of these proteins in the context of brain metastases. The brain-metastatic breast cancer cell line MDA-MB-231Br (231Br) expressed BMP-2 at significantly higher levels compared to its matched primary breast cancer cell line MDA-MB-231 (231). Co-culturing was used to examine bi-directional cellular effects and the relevance of BMP-2 overexpression. When co-cultured with NPCs, 231 (primary) tumor cells failed to proliferate over 15 days. However, 231Br (brain metastatic) tumor cells co-cultured with NPCs escaped growth inhibition after day 5 and proliferated, occurring in parallel with NPC differentiation into astrocytes. Using shRNA and gene knock-in, we then demonstrated BMP-2 secreted by 231Br cells mediated NPC differentiation into astrocytes and concomitant tumor cell proliferation in vitro. In xenografts, overexpression of BMP-2 in primary breast cancer cells significantly enhanced their ability to engraft and colonize the brain, thereby creating a metastatic phenotype. Conversely, BMP-2 knockdown in metastatic breast cancer cells significantly diminished engraftment and colonization. The results suggest metastatic tumor cells create a permissive neural niche by steering NPC differentiation toward astrocytes through paracrine BMP-2 signaling.

What mechanism that determines microglia accomplishing destructive or constructive role in CNS remains nebulous. We report here that intracranial priming and rechallenging with Toxoplasma gondii in mice elicit neurotoxic CCR9+ Irg1+ (immunoresponsive gene 1) microglia, which render resistance to apoptosis and produce a high level of TNF-alpha; priming and rechallenging with lymphocytic choriomeningitis virus elicit neurosupportive CXCR3+ Irg1- microglia, which are sensitive to apoptosis and produce a high level of IL-10 and TGF-beta. Administration of CCR9 and/or Irg1 small interfering RNA alters the frequency and functional profiles of neurotoxic CCR9+ Irg1+ and neurosupportive CXCR3+ Irg1- microglia in vivo. Moreover, by using a series of different neurotropic pathogens, including intracellular parasites, chronic virus, bacteria, toxic substances, and CNS injury to intracranially prime and subsequent rechallenge mice, the bi-directional elicitation of microglia has been confirmed as neurotoxic CCR9+ Irg1+ and neurosupportive CXCR3+ Irg1- cells in these mouse models. These data suggest that there exist two different types of microglia, providing with a novel insight into microglial involvement in neurodegenerative and neuroinflammatory pathogenesis such as Alzheimer's disease and AIDS dementia.

MMH (Met murine hepatocyte) liver cells derived from transgenic mice expressing a truncated constitutively active form of human c-Met are non-transformed immortalized cell lines. We have previously shown that they harbor: (1) epithelial cells that express the liver-enriched transcription factors HNF4 and HNF1(alpha), and that can be stably induced by FGF1 to express liver functions, and (2) fibroblast-like bi-potential palmate cells that can differentiate into bile duct-like structures in Matrigel cultures, or into epithelial cells competent to express hepatic functions. Low concentrations of TGF(beta) have been found to inhibit growth and differentiation of MMH cells. The factor stabilized the palmate cell phenotype, and it provoked epithelial cells to acquire palmate-like morphological characteristics, in parallel with down-regulation of expression of HNF4 and HNF1(alpha) and activation of Snail transcripts. The effects of TGF(beta) were dominant if it was added with FGF1, but the effects on differentiation were abrogated if cells had been pre-treated with FGF1. This work identifies TGF(beta) as a factor that could be implicated in maintaining bi-potential precursor cells in the liver, FGF1 as one that could over-ride the TGF(beta) effects and Snail as a candidate for mediation of the signal.

Embryonic stem (ES) cells present an excellent system for addressing the relevance of our current knowledge about how cell fate is determined and how cells integrate multiple signals into a single outcome as a function of time. Many of the factors that mediate these phenomena have been discovered through classical embryological experiments and are organized into several major signal transduction pathways including TGF-beta/BMP, Jak-STAT, Hedgehog, Wnt, Notch and FGF/MAPK. This review will summarize the current understanding of TGF-beta signaling in ES and focus on early embryological roles of the TGF-beta member, GDF-3. GDF-3 is associated with the undifferentiated state of ES cells and two recent and contradictory reports examined the function and mechanism of GDF-3 in the context of both stem cells and early embryonic differentiations. While Levine and Brivanlou found that GDF-3 inhibits its own subfamily members (the BMPs), Chen and colleagues found that GDF-3 acts as a nodal-like TGF-beta ligand. These combined findings raise the intriguing possibility that GDF-3 acts as a bi-functional protein, to regulate the balance between the two modes of TGF-beta signaling.

Pulmonary vascular disease plays a major role in morbidity and mortality in infant and adult lung diseases in which increased levels of transforming growth factor (TGF)-alpha and its receptor EGFR have been associated. The aim of this study was to determine whether overexpression of TGF-alpha disrupts pulmonary vascular development and causes pulmonary hypertension. Lung-specific expression of TGF-alpha in transgenic mice was driven with the human surfactant protein (SP)-C promoter. Pulmonary arteriograms and arterial counts show that pulmonary vascular development was severely disrupted in TGF-alpha mice. TGF-alpha mice developed severe pulmonary hypertension and vascular remodeling characterized by abnormally extensive muscularization of small pulmonary arteries. Pulmonary vascular development was significantly improved and pulmonary hypertension and vascular remodeling were prevented in bi-transgenic mice expressing both TGF-alpha and a dominant-negative mutant EGF receptor under the control of the SP-C promoter. Vascular endothelial growth factor (VEGF-A), an important angiogenic factor produced by the distal epithelium, was decreased in the lungs of TGF-alpha adults and in the lungs of infant TGF-alpha mice before detectable abnormalities in pulmonary vascular development. Hence, overexpression of TGF-alpha caused severe pulmonary vascular disease, which was mediated through EGFR signaling in distal epithelial cells. Reductions in VEGF may contribute to the pathogenesis of pulmonary vascular disease in TGF-alpha mice.

We investigated the influence of transforming growth factor-beta (TGF-beta) on DNA synthesis in human fetal fibroblasts, as measured by the incorporation of [3H]thymidine and cell replication. In serum-free medium, without additional peptide growth factors, TGF-beta had no action on thymidine incorporation. However, in the presence of 0.1% v/v fetal calf serum, TGF-beta exhibited a bi-functional action on the cells. A dose-dependent stimulation of [3H]thymidine incorporation, and an increase in cell number, occurred with fibroblasts established from fetuses under 50 g body weight, with a maximum stimulation seen at 1.25 ng/ml. For fibroblasts from fetuses of 100 g or greater body weight, TGF-beta caused a dose-related decrease in thymidine uptake with a maximal inhibition at 2.5 ng/ml, and a small decrease in cell number. When DNA synthesis was stimulated by the addition of somatomedin-C/insulin-like growth factor I, epidermal growth factor, or platelet-derived growth factor, their actions were potentiated by the presence of TGF-beta on cells derived from fetuses under 50 g body weight, but inhibited on cells obtained from the larger fetuses weighing more than 100 g. Similar results were found for changes in cell number in response to TGF-beta when stimulated by SM-C/IGF I. The ability of TGF-beta to modulate [3H] thymidine incorporation did not involve a change in the time required for growth-restricted cells to enter the S phase of the replication cycle. These data suggest that TGF-beta may exert either a growth-promoting or growth-inhibiting action on human fetal connective tissues in the presence of other peptide growth factors, which is dependent on fetal age and development.