Transforming growth factor β2 (TGF-β2)-induced connective tissue growth factor (CTGF) expression requires sphingosine 1-phosphate receptor 5 (S1P5) in human mesangial cells

Transforming growth factor β2 (TGF-β2) is well known to stimulate the expression of pro-fibrotic connective tissue growth factor (CTGF) in several cell types including human mesangial cells. The present study demonstrates that TGF-β2 enhances sphingosine 1-phosphate receptor 5 (S1P5) mRNA and protein expression in a time and concentration dependent manner. Pharmacological and siRNA approaches reveal that this upregulation is mediated via activation of classical TGF-β downstream effectors, Smad and mitogen-activated protein kinases. Most notably, inhibition of Gi with pertussis toxin and downregulation of S1P5 by siRNA block TGF-β2-stimulated upregulation of CTGF, demonstrating that Gi coupled S1P5 is necessary for TGF-β2-triggered expression of CTGF in human mesangial cells. Overall, these findings indicate that TGF-β2 dependent upregulation of S1P5 is required for the induction of pro-fibrotic CTGF by TGF-β. Targeting S1P5 might be an attractive novel approach to treat renal fibrotic diseases.

RNA interference screening identifies a novel role for autocrine fibroblast growth factor signaling in neuroblastoma chemoresistance

Chemotherapeutic drug resistance is one of the major causes for treatment failure in high-risk neuroblastoma (NB), the most common extra cranial solid tumor in children. Poor prognosis is typically associated with MYCN amplification. Here, we utilized a loss-of-function kinome-wide RNA interference screen to identify genes that cause cisplatin sensitization. We identified fibroblast growth factor receptor 2 (FGFR2) as an important determinant of cisplatin resistance. Pharmacological inhibition of FGFR2 confirmed the importance of this kinase in NB chemoresistance. Silencing of FGFR2 sensitized NB cells to cisplatin-induced apoptosis, which was regulated by the downregulation of the anti-apoptotic proteins BCL2 and BCLX(L). Mechanistically, FGFR2 was shown to activate protein kinase C-δ to induce BCL2 expression. FGFR2, as well as the ligand fibroblast growth factor-2, were consistently expressed in primary NB and NB cell lines, indicating the presence of an autocrine loop. Expression analysis revealed that FGFR2 correlates with MYCN amplification and with advanced stage disease, demonstrating the clinical relevance of FGFR2 in NB. These findings suggest a novel role for FGFR2 in chemoresistance and provide a rational to combine pharmacological inhibitors against FGFR2 with chemotherapeutic agents for the treatment of NB.Oncogene advance online publication, 1 October 2012; doi:10.1038/onc.2012.416.

Type I IFN induction in response to Listeria monocytogenes in human macrophages: evidence for a differential activation of IFN regulatory factor 3 (IRF3)

Listeria monocytogenes is a prototypic bacterium for studying innate and adaptive cellular immunity as well as host defense. Using human monocyte-derived macrophages, we report that an infection with a wild-type strain, but not a listeriolysin O-deficient strain, of the Gram-positive bacterium L. monocytogenes induces expression of IFN-beta and a bioactive type I IFN response. Investigating the activation of signaling pathways in human macrophages after infection revealed that a wild-type strain and a hemolysin-deficient strain of L. monocytogenes activated the NF-kappaB pathway and induced a comparable TNF response. p38 MAPK and activating transcription factor 2 were phosphorylated following infection with either strain, and IFN-beta gene expression induced by wild-type L. monocytogenes was reduced when p38 was inhibited. However, neither IFN regulatory factor (IRF) 3 translocation to the nucleus nor posttranslational modifications and dimerizations were observed after L. monocytogenes infection. In contrast, vesicular stomatitis virus and LPS triggered IRF3 activation and signaling. When IRF3 was knocked down using small interfering RNA, a L. monocytogenes-induced IFN-beta response remained unaffected whereas a vesicular stomatitis virus-triggered response was reduced. Evidence against the possibility that IRF7 acts in place of IRF3 is provided. Thus, we show that wild-type L. monocytogenes induced an IFN-beta response in human macrophages and propose that this response involves p38 MAPK and activating transcription factor 2. Using various stimuli, we show that IRF3 is differentially activated during type I IFN responses in human macrophages.

Vacuum-assisted closure therapy increases local interleukin-8 and vascular endothelial growth factor levels in traumatic wounds

BACKGROUND: Clinical observations are suggesting accelerated granulation tissue formation in traumatic wounds treated with vacuum-assisted closure (VAC). Aim of this study was to determine the impact of VAC therapy versus alternative Epigard application on local inflammation and neovascularization in traumatic soft tissue wounds. METHODS: Thirty-two patients with traumatic wounds requiring temporary coverage (VAC n = 16; Epigard n = 16) were included. At each change of dressing, samples of wound fluid and serum were collected (n = 80). The cytokines interleukin (IL)-6, IL-8, vascular endothelial growth factor (VEGF), and fibroblast growth factor-2 were measured by ELISA. Wound biopsies were examined histologically for inflammatory cells and degree of neovascularization present. RESULTS: All cytokines were found to be elevated in wound fluids during both VAC and Epigard treatment, whereas serum concentrations were negligible or not detectable. In wound fluids, significantly higher IL-8 (p < 0.001) and VEGF (p < 0.05) levels were detected during VAC therapy. Furthermore, histologic examination revealed increased neovascularization (p < 0.05) illustrated by CD31 and von Willebrand factor immunohistochemistry in wound biopsies of VAC treatment. In addition, there was an accumulation of neutrophils as well as an augmented expression of VEGF (p < 0.005) in VAC wound biopsies. CONCLUSION: This study suggests that VAC therapy of traumatic wounds leads to increased local IL-8 and VEGF concentrations, which may trigger accumulation of neutrophils and angiogenesis and thus, accelerate neovascularization.

Leukemia inhibitory factor favours neurogenic differentiation of long-term propagated human midbrain precursor cells

There is a lot of excitement about the potential use of multipotent neural stem cells for the treatment of neurodegenerative diseases. However, the strategy is compromised by the general loss of multipotency and ability to generate neurons after long-term in vitro propagation. In the present study, human embryonic (5 weeks post-conception) ventral mesencephalic (VM) precursor cells were propagated as neural tissue-spheres (NTS) in epidermal growth factor (EGF; 20 ng/ml) and fibroblast growth factor 2 (FGF2; 20 ng/ml). After more than 325 days, the NTS were transferred to media containing either EGF+FGF2, EGF+FGF2+heparin or leukemia inhibitory factor (LIF; 10 ng/ml)+FGF2+heparin. Cultures were subsequently propagated for more than 180 days with NTS analyzed at various time-points. Our data show for the first time that human VM neural precursor cells can be long-term propagated as NTS in the presence of EGF and FGF2. A positive effect of heparin was found only after 150 days of treatment. After switching into different media, only NTS exposed to LIF contained numerous cells positive for markers of newly formed neurons. Besides of demonstrating the ability of human VM NTS to be long-term propagated, our study also suggests that LIF favours neurogenic differentiation of human VM precursor cells.

TGF-ß1 enhances the BMP-2-induced chondrogenesis of bovine synovial explants and arrests downstream differentiation at an early stage of hypertrophy

BACKGROUND Synovial explants furnish an in-situ population of mesenchymal stem cells for the repair of articular cartilage. Although bone morphogenetic protein 2 (BMP-2) induces the chondrogenesis of bovine synovial explants, the cartilage formed is neither homogeneously distributed nor of an exclusively hyaline type. Furthermore, the downstream differentiation of chondrocytes proceeds to the stage of terminal hypertrophy, which is inextricably coupled with undesired matrix mineralization. With a view to optimizing BMP-2-induced chondrogenesis, the modulating influences of fibroblast growth factor 2 (FGF-2) and transforming growth factor beta 1 (TGF-ß1) were investigated. METHODOLOGY/PRINCIPAL FINDINGS Explants of bovine calf metacarpal synovium were exposed to BMP-2 (200 ng/ml) for 4 (or 6) weeks. FGF-2 (10 ng/ml) or TGF-ß1 (10 ng/ml) was introduced at the onset of incubation and was present either during the first week of culturing alone or throughout its entire course. FGF-2 enhanced the BMP-2-induced increase in metachromatic staining for glycosaminoglycans (GAGs) only when it was present during the first week of culturing alone. TGF-ß1 enhanced not only the BMP-2-induced increase in metachromasia (to a greater degree than FGF-2), but also the biochemically-assayed accumulation of GAGs, when it was present throughout the entire culturing period; in addition, it arrested the downstream differentiation of cells at an early stage of hypertrophy. These findings were corroborated by an analysis of the gene- and protein-expression levels of key cartilaginous markers and by an estimation of individual cell volume. CONCLUSIONS/SIGNIFICANCE TGF-ß1 enhances the BMP-2-induced chondrogenesis of bovine synovial explants, improves the hyaline-like properties of the neocartilage, and arrests the downstream differentiation of cells at an early stage of hypertrophy. With the prospect of engineering a mature, truly articular type of cartilage in the context of clinical repair, our findings will be of importance in fine-tuning the stimulation protocol for the optimal chondrogenic differentiation of synovial explants.

Influence of root canal disinfectants on growth factor release from dentin

INTRODUCTION During dentinogenesis, growth factors become entrapped in the dentin matrix that can later be released by demineralization. Their effect on pulpal stem cell migration, proliferation, and differentiation could be beneficial for regenerative endodontic therapies. However, precondition for success, as for conventional root canal treatment, will be sufficient disinfection of the root canal system. Various irrigation solutions and intracanal dressings are available for clinical use. The aim of this study was 2-fold: to identify a demineralizing solution suitable for growth factor release directly from dentin and to evaluate whether commonly used disinfectants for endodontic treatment will compromise this effect. METHODS Dentin disks were prepared from extracted human teeth and treated with EDTA or citric acid at different concentrations or pH for different exposure periods. The amount of transforming growth factor-β1 (TGF-β1), fibroblast growth factor 2, and vascular endothelial growth factor were quantified via enzyme-linked immunosorbent assay and visualized by gold labeling. Subsequently, different irrigation solutions (5.25% sodium hypochloride, 0.12% chlorhexidine digluconate) and intracanal dressings (corticoid-antibiotic paste, calcium hydroxide: water-based and oil-based, triple antibiotic paste, chlorhexidine gel) were tested, and the release of TGF-β1 was measured after a subsequent conditioning step with EDTA. RESULTS Conditioning with 10% EDTA at pH 7 rendered the highest amounts of TGF-β1 among all test solutions. Fibroblast growth factor 2 and vascular endothelial growth factor were detected after EDTA conditioning at minute concentrations. Irrigation with chlorhexidine before EDTA conditioning increased TGF-β1 release; sodium hypochloride had the opposite effect. All tested intracanal dressings interfered with TGF-β1 release except water-based calcium hydroxide. CONCLUSIONS Growth factors can be released directly from dentin via EDTA conditioning. The use of disinfecting solutions or medicaments can amplify or attenuate this effect.

Enhanced proliferation and dopaminergic differentiation of ventral mesencephalic precursor cells by synergistic effect of FGF2 and reduced oxygen tension

Effective numerical expansion of dopaminergic precursors might overcome the limited availability of transplantable cells in replacement strategies for Parkinson's disease. Here we investigated the effect of fibroblast growth factor-2 (FGF2) and FGF8 on expansion and dopaminergic differentiation of rat embryonic ventral mesencephalic neuroblasts cultured at high (20%) and low (3%) oxygen tension. More cells incorporated bromodeoxyuridine in cultures expanded at low as compared to high oxygen tension, and after 6 days of differentiation there were significantly more neuronal cells in low than in high oxygen cultures. Low oxygen during FGF2-mediated expansion resulted also in a significant increase in tyrosine hydroxylase-immunoreactive (TH-ir) dopaminergic neurons as compared to high oxygen tension, but no corresponding effect was observed for dopamine release into the culture medium. However, switching FGF2-expanded cultures from low to high oxygen tension during the last two days of differentiation significantly enhanced dopamine release and intracellular dopamine levels as compared to all other treatment groups. In addition, the short-term exposure to high oxygen enhanced in situ assessed TH enzyme activity, which may explain the elevated dopamine levels. Our findings demonstrate that modulation of oxygen tension is a recognizable factor for in vitro expansion and dopaminergic differentiation of rat embryonic midbrain precursor cells.

Unique modifications of translation elongation factors

Covalent modifications of proteins often modulate their biological functions or change their subcellular location. Among the many known protein modifications, three are exceptional in that they only occur on single proteins: ethanolamine phosphoglycerol, diphthamide and hypusine. Remarkably, the corresponding proteins carrying these modifications, elongation factor 1A, elongation factor 2 and initiation factor 5A, are all involved in elongation steps of translation. For diphthamide and, in part, hypusine, functional essentiality has been demonstrated, whereas no functional role has been reported so far for ethanolamine phosphoglycerol. We review the biosynthesis, attachment and physiological roles of these unique protein modifications and discuss common and separate features of the target proteins, which represent essential proteins in all organisms.

Antibacterial and antiinflammatory kinetics of curcumin as a potential antimucositis agent in cancer patients

The antiinflammatory agent curcumin (diferuloylmethane) has a potential to mitigate cancer therapy-induced mucositis. We assessed the in vitro extent of its bactericidal activity and determined the kinetics of its antiinflammatory effect on pharyngeal cells. Bactericidal activity was assessed using the LIVE/DEAD® Kit after 4 h of exposure to curcumin (50-200 μM) in 18 oropharyngeal species commonly associated with bacteremia in febrile neutropenia. Moraxella catarrhalis or its outer membrane vesicles were used to determine the inhibitory effect of curcumin on bacteria-induced proinflammatory activity as determined by cytokine release into the supernatant of Detroit 562 pharyngeal cells using the Luminex® xMAP® technology. Curcumin exerted a concentration-dependent bactericidal effect on all 18 species tested. After 4 h at 200 μM, 12 species tested were completely killed. Preincubation of Detroit cells with 200 μM curcumin for 5 to 60 min resulted in complete suppression of the release of tumor necrosis factor-α, interleukin (IL)-6, IL-8, monocyte chemoattractant protein 1, granulocyte macrophage-colony stimulating factor, and vascular endothelial growth factor. Fibroblast growth factor-2 and interferon-γ were not affected. Repetitive exposure to curcumin resulted in repetitive suppression of cytokine/chemokine expression lasting from 4 to 6 h. Through reduction of oral microbial density as well as suppression of inflammation cascades curcumin may prevent cancer therapy-induced oral mucositis, e.g., when applied as multiple daily mouth washes.

Evaluation of magnetic resonance colonography at 3.0 Tesla regarding diagnostic accuracy and image quality

OBJECTIVES: To assess magnetic resonance (MR)-colonography (MRC) for detection of colorectal lesions using two different T1w three-dimensional (3D)-gradient-recalled echo (GRE)-sequences and integrated parallel data acquisition (iPAT) at a 3.0 Tesla MR-unit. MATERIALS AND METHODS: In this prospective study, 34 symptomatic patients underwent dark lumen MRC at a 3.0 Tesla unit before conventional colonoscopy (CC). After colon distension with tap water, 2 high-resolution T1w 3D-GRE [3-dimensional fast low angle shot (3D-FLASH), iPAT factor 2 and 3D-volumetric interpolated breathhold examination (VIBE), iPAT 3] sequences were acquired without and after bolus injection of gadolinium. Prospective evaluation of MRC was performed. Image quality of the different sequences was assessed qualitatively and quantitatively. The findings of the same day CC served as standard of reference. RESULTS: MRC identified all polyps >5 mm (16 of 16) in size and all carcinomas (4 of 4) correctly. Fifty percent of the small polyps 0.6). CONCLUSIONS: MRC using 3D-GRE-sequences and iPAT is feasible at 3.0 T-systems. The high-resolution 3D-FLASH was slightly preferred over the 3D-VIBE because of better image quality, although both used sequences showed no statistical significant difference.

Unique posttranslational modifications in eukaryotic translation factors and their roles in protozoan parasite viability and pathogenesis.

Protozoan parasites are one of the major causes of diseases worldwide. The vector transmitted parasites exhibit complex life cycles involving interactions between humans, protozoa, and arthropods. In order to adapt themselves to the changing microenvironments, they have to undergo complex morphological and metabolic changes. These changes can be brought about by expressing a new pool of proteins in the cell or by modifying the existing repertoire of proteins via posttranslational modifications (PTMs). PTMs involve covalent modification and processing of proteins thereby modulating their functions. Some of these changes may involve PTMs of parasite proteins to help the parasite survive within the host and the vector. Out of many PTMs known, three are unique since they occur only on single proteins: ethanolamine phosphoglycerol (EPG) glutamate, hypusine and diphthamide. These modifications occur on eukaryotic elongation factor 1A (eEF1A), eukaryotic initiation factor 5A (eIF5A) and eukaryotic elongation factor 2 (eEF2), respectively. Interestingly, the proteins carrying these unique modifications are all involved in the elongation steps of translation. Here we review these unique PTMs, which are well conserved in protozoan parasites, and discuss their roles in viability and pathogenesis of parasites. Characterization of these modifications and studying their roles in physiology as well as pathogenesis will provide new insights in parasite biology, which may also help in developing new therapeutic interventions.

Phenotype of capillaries in skeletal muscle of nNOS-knockout mice

Because neuronal nitric oxide synthase (nNOS) has a well-known impact on arteriolar blood flow in skeletal muscle, we compared the ultrastructure and the hemodynamics of/in the ensuing capillaries in the extensor digitorum longus (EDL) muscle of male nNOS-knockout (KO) mice and wild-type (WT) littermates. The capillary-to-fiber (C/F) ratio (-9.1%) was lower (P ≤ 0.05) in the nNOS-KO mice than in the WT mice, whereas the mean cross-sectional fiber area (-7.8%) and the capillary density (-3.1%) varied only nonsignificantly (P > 0.05). Morphometrical estimation of the area occupied by the capillaries as well as the volume and surface densities of the subcellular compartments differed nonsignificantly (P > 0.05) between the two strains. Intravital microscopy revealed neither the capillary diameter (+3% in nNOS-KO mice vs. WT mice) nor the mean velocity of red blood cells in EDL muscle (+25% in nNOS-KO mice vs. WT mice) to significantly vary (P > 0.05) between the two strains. The calculated shear stress in the capillaries was likewise nonsignificantly different (3.8 ± 2.2 dyn/cm² in nNOS-KO mice and 2.1 ± 2.2 dyn/cm² in WT mice; P > 0.05). The mRNA levels of vascular endothelial growth factor (VEGF)-A were lower in the EDL muscle of nNOS-KO mice than in the WT littermates (-37%; P ≤ 0.05), whereas mRNA levels of VEGF receptor-2 (VEGFR-2) (-11%), hypoxia inducible factor-1α (+9%), fibroblast growth factor-2 (-14%), and thrombospondin-1 (-10%) differed nonsignificantly (P > 0.05). Our findings support the contention that VEGF-A mRNA expression and C/F-ratio but not the ultrastructure or the hemodynamics of/in capillaries in skeletal muscle at basal conditions depend on the expression of nNOS.

Phospholipid oxidation generates potent anti-inflammatory lipid mediators that mimic structurally related pro-resolving eicosanoids by activating Nrf2.

Exposure of biological membranes to reactive oxygen species creates a complex mixture of distinct oxidized phospholipid (OxPL) species, which contribute to the development of chronic inflammatory diseases and metabolic disorders. While the ability of OxPL to modulate biological processes is increasingly recognized, the nature of the biologically active OxPL species and the molecular mechanisms underlying their signaling remain largely unknown. We have employed a combination of mass spectrometry, synthetic chemistry, and immunobiology approaches to characterize the OxPL generated from the abundant phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) and investigated their bioactivities and signaling pathways in vitro and in vivo. Our study defines epoxycyclopentenones as potent anti-inflammatory lipid mediators that mimic the signaling of endogenous, pro-resolving prostanoids by activating the transcription factor nuclear factor E2-related factor 2 (Nrf2). Using a library of OxPL variants, we identified a synthetic OxPL derivative, which alleviated endotoxin-induced lung injury and inhibited development of pro-inflammatory T helper (Th) 1 cells. These findings provide a molecular basis for the negative regulation of inflammation by lipid peroxidation products and propose a novel class of highly bioactive compounds for the treatment of inflammatory diseases.