Titanium surfaces with nanotopography modulate cytokine production in cultured human gingival fibroblasts

Implant topography is an important factor that influences many cell types. To understand the role of topography in the inflammatory events, we evaluated the response of human gingival fibroblasts (HGFs) by the release pattern of cytokines. HGFs were cultured on Ti discs for 24 and 48 h. Four different surface treatments were used: machining method (turned), blasting followed by an acid-etching method (BAE), oxidative nanopatterning (ON) method, and an association of blasting followed by an acid-etching plus oxidative nanopatterning (BAE+ON) method. Extracellular levels of IL-6, IL-8, transforming growth factor beta (TGF-beta), IL-4, and IL-10 were measured by enzyme-linked immunosorbant assay. Increased levels of IL-6 and IL-8 were observed in all surfaces after 24 h which decreased after 48 h. BAE, ON, and BAE+ON surfaces showed a reduction in IL-6 levels compared with the turned after 48 h (p < 0.05). On one hand, IL-8 production was lower in BAE+ON in comparison to the turned surface (p < 0.05). On the other hand, IL-4 showed increased levels with 48 h, which were significantly different between turned, BAE, and ON surfaces, but not with BAE+ON. Additionally, TGF-beta and IL-10 production were not detected. This study indicates that nanotopography might be important in the modulation of the inflammatory response in cultured HGFs. (c) 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A 100A:2629-2636, 2012.

Increased fibroblast telomerase expression precedes myofibroblast alpha-smooth muscle actin expression in idiopathic pulmonary fibrosis

OBJECTIVE: This study sought to identify the relationship between fibroblast telomerase expression, myofibroblasts, and telomerase-mediated regulatory signals in idiopathic pulmonary fibrosis. METHODS: Thirty-four surgical lung biopsies, which had been obtained from patients with idiopathic pulmonary fibrosis and histologically classified as usual interstitial pneumonia, were examined. Immunohistochemistry was used to evaluate fibroblast telomerase expression, myofibroblast alpha-smooth muscle actin expression and the tissue expression of interleukin-4, transforming growth factor-beta, and basic fibroblast growth factor. The point-counting technique was used to quantify the expression of these markers in unaffected, collapsed, mural fibrosis, and honeycombing areas. The results were correlated to patient survival. RESULTS: Fibroblast telomerase expression and basic fibroblast growth factor tissue expression were higher in collapsed areas, whereas myofibroblast expression and interleukine-4 tissue expression were higher in areas of mural fibrosis. Transforming growth factor-beta expression was higher in collapsed, mural fibrosis and honeycombing areas in comparison to unaffected areas. Positive correlations were found between basic fibroblast growth factor tissue expression and fibroblast telomerase expression and between interleukin-4 tissue expression and myofibroblast alpha-smooth muscle actin expression. Negative correlations were observed between interleukin-4 expression and basic fibroblast growth factor tissue expression in areas of mural fibrosis. Myofibroblast alpha-smooth muscle actin expression and interleukin-4 tissue expression in areas of mural fibrosis were negatively associated with patient survival. CONCLUSION: Fibroblast telomerase expression is higher in areas of early remodeling in lung tissues demonstrating typical interstitial pneumonia, whereas myofibroblast alpha-smooth muscle actin expression predominates in areas of late remodeling. These events seem to be regulated by basic fibroblast growth factor and interleukin-4 tissue expression, respectively.

Immune Regulatory Properties of Allogeneic Adipose-Derived Mesenchymal Stem Cells in the Treatment of Experimental Autoimmune Diabetes

Adipose-derived mesenchymal stem cells (ADMSCs) display immunosuppressive properties, suggesting a promising therapeutic application in several autoimmune diseases, but their role in type 1 diabetes (T1D) remains largely unexplored. The aim of this study was to investigate the immune regulatory properties of allogeneic ADMSC therapy in T cell-mediated autoimmune diabetes in NOD mice. ADMSC treatment reversed the hyperglycemia of early-onset diabetes in 78% of diabetic NOD mice, and this effect was associated with higher serum insulin, amylin, and glucagon-like peptide 1 levels compared with untreated controls. This improved outcome was associated with downregulation of the CD4(+) Th1-biased immune response and expansion of regulatory T cells (Tregs) in the pancreatic lymph nodes. Within the pancreas, inflammatory cell infiltration and interferon-gamma levels were reduced, while insulin, pancreatic duodenal homeobox-1, and active transforming growth factor-beta 1 expression were increased. In vitro, ADMSCs induced the expansion/proliferation of Tregs in a cell contact-dependent manner mediated by programmed death ligand 1. In summary, ADMSC therapy efficiently ameliorates autoimmune diabetes pathogenesis in diabetic NOD mice by attenuating the Th1 immune response concomitant with the expansion/proliferation of Tregs, thereby contributing to the maintenance of functional beta-cells. Thus, this study may provide a new perspective for the development of ADMSC-based cellular therapies for T1D. Diabetes 61:2534-2545, 2012

Coffee and Caffeine Protect against Liver Injury Induced by Thioacetamide in Male Wistar Rats

Coffee intake has been inversely related to the incidence of liver diseases, although there are controversies on whether these beneficial effects on human health are because of caffeine or other specific components in this popular beverage. Thus, this study evaluated the protective effects of coffee or caffeine intake on liver injury induced by repeated thioacetamide (TAA) administration in male Wistar rats. Rats were randomized into five groups: one untreated group (G1) and four groups (G2G5) treated with the hepatotoxicant TAA (200 similar to mg/kg b.w., i.p.) twice a week for 8 similar to weeks. Concomitantly, rats received tap water (G1 and G2), conventional coffee (G3), decaffeinated coffee (G4) or 0.1% caffeine (G5). After 8 similar to weeks of treatment, rats were killed and blood and liver samples were collected. Conventional and decaffeinated coffee and caffeine intake significantly reduced serum levels of alanine aminotransferase (ALT) (p similar to<similar to 0.001) and oxidized glutathione (p similar to<similar to 0.05), fibrosis/inflammation scores (p similar to<similar to 0.001), collagen volume fraction (p similar to<similar to 0.01) and transforming growth factor beta-1 (TGF-beta 1) protein expression (p similar to=similar to 0.001) in the liver from TAA-treated groups. In addition, conventional coffee and caffeine intake significantly reduced proliferating cellular nuclear antigen (PCNA) S-phase indexes (p similar to<similar to 0.001), but only conventional coffee reduced cleaved caspase-3 indexes (p similar to<similar to 0.001), active metalloproteinase 2 (p similar to=similar to 0.004) and the number of glutathione S-transferase placental form (GST-P)-positive preneoplastic lesions (p similar to<similar to 0.05) in the liver from TAA-treated groups. In conclusion, conventional coffee and 0.1% caffeine intake presented better beneficial effects than decaffeinated coffee against liver injury induced by TAA in male Wistar rats.

Inhibition allergenspezifischer Immunantworten im Mausmodell der Typ-I Allergie nach biolistischer Vakzinierung mit allergenkodierenden Plasmiden in Kombination mit für immunmodulatorische Moleküle kodierender Plasmid-DNA

Die Induktion von Toleranz spielt bei der Inhibition allergischer Immunreaktionen eine wichtige Rolle. Hierbei ist die Induktion regulatorischer T Zellen (Treg) von großer Bedeutung. Da zu einer erfolgreichen Behandlung von allergischen Erkrankungen bisher nur wenige Therapiemöglichkeiten zur Verfügung stehen wie die spezifische Immuntherapie (SIT), die allerdings nicht immer zum Erfolg führt, ist es wichtig neue Therapieformen zu entwickeln. rnIn dieser Arbeit wurde daher die biolistische DNA-Immunisierung mit Kombinations-Vakzinen bestehend aus einem allergenkodierenden Plasmid (βGalaktosidase (βGal)) in Kombination mit einem Plasmid, welches für ein immunmodulatorisches Molekül kodiert (Indolamin-2,3-Dioxygenase (IDO), Transforming Growth Factor beta (TGF-β) oder Interleukin-10 (IL-10)), durchgeführt und im Mausmodell der allergeninduzierten IgE-vermittelten Atemwegsinflammation auf ihre Wirksamkeit untersucht. Die Expression des Allergens zusammen mit dem immunregulatorischen Molekül in transfizierten Dendritischen Zellen (DCs) sollte zu einer Induktion von Treg führen und somit eine Suppression der Immunantwort bewirken. rnIn den Versuchen wurde zunächst der Effekt einer Transgenexpression unter der Kontrolle des ubiquitären CMV-Promotors mit dem der Transgenexpression unter der Kontrolle des Fascin-Promotors, der eine Genxpression spezifisch in DCs erlaubt, verglichen. Hierbei stellte sich heraus, dass es wichtig ist die Expression des Antigens mit Hilfe des Fascin-Promotors auf DCs zu beschränken. Einzig in diesem Fall konnte nach der Vakzinierung ein inhibitorischer Effekt auf die Entwicklung einer Atemwegshyperreaktivität durch Expression des Immunmodulatoren IDO beobachtet werden. Es zeigte sich auch, dass es von Vorteil ist, wenn das immunregulatorische Molekül unter Verwendung des CMV-Promotors in allen transfizierten Zellen exprimiert wird. Dies bewirkt, dass IDO in ausreichenden Konzentrationen vorhanden ist. rnDie Expression von βGal unter der Kontrolle des Fascin-Promotors (pFascin-βGal) in Kombination mit der Expression der Moleküle IL-10, TGF-β oder IDO unter Kontrolle des CMV-Promotors (pCMV-IL-10, pCMV-TGFβ, pCMV-IDO) bewirkte eine Immunsupprimierung, die sich in einer inhibierten Produktion antigenspezifischer Antikörper, einer verminderten Zytokin-Produktion, einer reduzierten Induktion zytotoxischer T-Zellen und in einer Inhibition der allergeninduzierten Atemwegshyperreaktivität zeigte, im Vergleich zu einer Vakzinierung mit pFascin-βGal in Kombination mit einem Kontroll-Plasmid. Bei nachfolgender Proteinsensibilisierung blieben diese Effekte jedoch nicht bestehen. Einzig durch Vakzinierung mit IL-10-kodierenden Plasmiden konnte eine moderate Verminderung der Atemwegsreaktivität nachgewiesen werden. rnIn einem therapeutischen Modell der Atemwegsinflammation, in dem die Mäuse vor der DNA-Immunisierung mit dem Protein sensibilisiert wurden, wurde demonstriert, dass im Vergleich zu Mäusen, die nur mit dem Protein sensibilisiert wurden, eine DNA-Immunisierung mit pFascin-βGal aber auch mit pCMV-βGal einen inhibierenden Einfluss auf die Entwicklung einer Atemwegsinflammation hat. Eine weitere Reduktion der Atemwegsreaktivität durch eine kombinierte Vakzinierung mit pCMV-IDO wurde nur erreicht, wenn βGal unter der Kontrolle des Fascin-Promotors exprimiert wurde, nicht aber unter Kontrolle des CMV-Promotors.rn

Nail Lichen Planus: Successful Treatment with Etanercept

BACKGROUND: Etanercept is a fully human tumor necrosis factor a receptor fusion protein that binds tumor necrosis factor a with greater affinity than natural receptors. Biologics are widely used in the treatment of psoriasis and psoriasis arthritis and may represent a new therapeutic option for some patients with psoriatic nail disease. CASE REPORT: We report a case of lichen planus limited to the toe nails successfully treated with etanercept monotherapy. CONCLUSION: The significant improvement of our case suggests that etanercept is an effective treatment modality for lichen planus limited particularly to the nails. Further controlled studies are needed to establish the effectiveness and therapeutic regimes.

Differential response of Human Bone Marrow Stromal Cells to either TGF-β1 or to rhGDF-5

Cell therapy along with growth factor injection is currently widely investigated to restore the intervertebral disc. However, there is increasing evidence that transplanted unconditioned bone marrow-derived stromal cells (BMSCs) cannot thrive in the intervertebral disc "niche". Moreover, uncertainty exists with respect to the cell phenotype that would be suitable to inject. The intervertebral disc cell phenotype only recently has been started to be characterised using transcriptomics profiling. Recent findings suggest that cytokeratin 19 (KRT-19) could be used as a potential candidate marker for the intervertebral disc, or more specifically the nucleus pulposus cell (NPC) phenotype. We present in vitro cell culture data using alginate bead culture of primary human BMSCs exposed to the standard chondrogenic stimulus, transforming growth factor beta-1 (TGF-β), the growth and differentiation factor 5 and/or bovine NPCs to induce a potential "discogenic" pathway. Chondrogenic induction via TGF-β pathway provoked down-regulation of KRT-19 gene expression in four out of five donors after 18 days of culture, whereas KRT-19 expression remained unchanged in the "discogenic" groups. In addition, the ratio of aggrecan/collagen II gene expression showed a remarkable difference (of at least 3 magnitudes) between the chondrogenic stimulus (low ratio) and the discogenic stimulus (high ratio). Therefore, KRT-19 and aggrecan/collagen II ratio may be potential markers to distinguish chondrogenic from "discogenic" differentiation.

TNFR1 is essential for CD40, but not for lipopolysaccharide-induced sickness behavior and clock gene dysregulation

Autoimmune and infectious diseases are associated with behavioral changes referred to as sickness behavior syndrome (SBS). In autoimmunity, the generation of anti-self T lymphocytes and autoantibodies critically involves binding of CD40 ligand on T-cells to its receptor CD40 on B-cells, dendritic cells and macrophages. Activation of CD40 leads to production of proinflammatory cytokines and, as shown here, induces SBS. Here we report that these behavioral changes depend on the expression of tumor necrosis factor alpha receptor 1 (TNFR1), but not on interleukin-1 receptor 1 or interleukin-6. Moreover, the intensity of SBS correlates with suppression of E-box controlled clock genes, including Dbp, and upregulation of Bmal1. However, the absence of TNFR1 does not interfere with the development of SBS and dysregulation of clock genes in mice treated with lipopolysaccharide. Thus, our results suggest that TNFR1 mediates SBS and dysregulation of clock genes in autoimmune diseases.

Novel agents targeting the IGF-1R/PI3K pathway impair cell proliferation and survival in subsets of medulloblastoma and neuroblastoma

The receptor tyrosine kinase (RTK)/phosphoinositide 3-kinase (PI3K) pathway is fundamental for cancer cell proliferation and is known to be frequently altered and activated in neoplasia, including embryonal tumors. Based on the high frequency of alterations, targeting components of the PI3K signaling pathway is considered to be a promising therapeutic approach for cancer treatment. Here, we have investigated the potential of targeting the axis of the insulin-like growth factor-1 receptor (IGF-1R) and PI3K signaling in two common cancers of childhood: neuroblastoma, the most common extracranial tumor in children and medulloblastoma, the most frequent malignant childhood brain tumor. By treating neuroblastoma and medulloblastoma cells with R1507, a specific humanized monoclonal antibody against the IGF-1R, we could observe cell line-specific responses and in some cases a strong decrease in cell proliferation. In contrast, targeting the PI3K p110α with the specific inhibitor PIK75 resulted in broad anti-proliferative effects in a panel of neuro- and medulloblastoma cell lines. Additionally, sensitization to commonly used chemotherapeutic agents occurred in neuroblastoma cells upon treatment with R1507 or PIK75. Furthermore, by studying the expression and phosphorylation state of IGF-1R/PI3K downstream signaling targets we found down-regulated signaling pathway activation. In addition, apoptosis occurred in embryonal tumor cells after treatment with PIK75 or R1507. Together, our studies demonstrate the potential of targeting the IGF-1R/PI3K signaling axis in embryonal tumors. Hopefully, this knowledge will contribute to the development of urgently required new targeted therapies for embryonal tumors.

Cause or effect of arteriogenesis: compositional alterations of microparticles from CAD patients undergoing external counterpulsation therapy

Recently, a clinical study on patients with stable coronary artery disease (CAD) showed that external counterpulsation therapy (ECP) at high (300 mmHg) but not at low inflation pressure (80 mmHg) promoted coronary collateral growth, most likely due to shear stress-induced arteriogenesis. The exact molecular mechanisms behind shear stress-induced arteriogenesis are still obscure. We therefore characterized plasma levels of circulating microparticles (MPs) from these CAD patients because of their ambivalent nature as a known cardiovascular risk factor and as a promoter of neovascularization in the case of platelet-derived MPs. MPs positive for Annexin V and CD31CD41 were increased, albeit statistically significant (P<0.05, vs. baseline) only in patients receiving high inflation pressure ECP as determined by flow cytometry. MPs positive for CD62E, CD146, and CD14 were unaffected. In high, but not in low, inflation pressure treatment, change of CD31CD41 was inversely correlated to the change in collateral flow index (CFI), a measure for collateral growth. MPs from the high inflation pressure group had a more sustained pro-angiogenic effect than the ones from the low inflation pressure group, with the exception of one patient showing also an increased CFI after treatment. A total of 1005 proteins were identified by a label-free proteomics approach from MPs of three patients of each group applying stringent acceptance criteria. Based on semi-quantitative protein abundance measurements, MPs after ECP therapy contained more cellular proteins and increased CD31, corroborating the increase in MPs. Furthermore, we show that MP-associated factors of the innate immune system were decreased, many membrane-associated signaling proteins, and the known arteriogenesis stimulating protein transforming growth factor beta-1 were increased after ECP therapy. In conclusion, our data show that ECP therapy increases platelet-derived MPs in patients with CAD and that the change in protein cargo of MPs is likely in favor of a pro angiogenic/arteriogenic property.

Sialic acid binding immunoglobulin-like lectins may regulate innate immune responses by modulating the life span of granulocytes

The regulation of cell death is a key element in building up and maintaining both innate and adaptive immunity. A critical role in this process plays the tumor necrosis factor (TNF)/nerve growth factor (NGF) receptor family of death receptors. Recent work suggests that sialic acid binding immunoglobulin (Ig) -like lectins (Siglecs) are also empowered to transmit death signals, at least into myeloid cells. Strikingly, death induction by Siglecs is enhanced when cells are exposed to proinflammatory survival cytokines. Based on these recent insights, we hypothesize that at least some members of the Siglec family regulate immune responses via the activation of caspase-dependent and caspase-independent cell death pathways.

Grover's disease induced by cetuximab

A 71-year-old man exhibited an acute acneiform rash affecting the face and the upper trunk about 2 weeks after starting cetuximab, an epidermal growth factor (EGF) receptor antagonist treatment for metastatic colon cancer. The skin eruption faded after stopping cetuximab and applying topical corticosteroids. The reexposure to cetuximab 3 weeks later provoked a more extended relapse of the skin rash, which then clinically and histologically corresponded to transient acantholytic dermatosis . While the acneiform cutaneous side effects of the EGF receptor antagonists are interpreted as a result of the direct interference with pilosebaceous follicle homeostasis, in this case an acrosyringium-related pathogenesis might be postulated. Applying topical corticosteroids and emollients, the cetuximab therapy could be pursued.

Differential expression of TGFbeta-stimulated clone 22 in normal prostate and prostate cancer

The transforming growth factor-beta (TGFbeta) superfamily and its downstream effector genes are key regulators of epithelial homeostasis. Altered expression of these genes may be associated with malignant transformation of the prostate gland. The cDNA array analysis of differential expression of the TGFbeta superfamily and functionally related genes between patient-matched noncancerous prostate (NP) and prostate cancer (PC) bulk tissue specimens highlighted two genes, namely TGFbeta-stimulated clone-22 (TSC-22) and Id4. Verification of their mRNA expression by real-time PCR in patient-matched NP and PC bulk tissue, in laser-captured pure epithelial and cancer cells and in NP and PC cell lines confirmed TSC-22 underexpression, but not Id4 overexpression, in PC and in human PC cell lines. Immunohistochemical analysis showed that TSC-22 protein expression in NP is restricted to the basal cells and colocalizes with the basal cell marker cytokeratin 5. In contrast, all matched PC samples lack TSC-22 immunoreactivity. Likewise, PC cell lines do not show detectable TSC-22 protein expression as shown by immunoblotting. TSC-22 should be considered as a novel basal cell marker, potentially useful for studying lineage determination within the epithelial compartment of the prostate. Conversely, lack of TSC-22 seems to be a hallmark of malignant transformation of the prostate epithelium. Accordingly, TSC-22 immunohistochemistry may prove to be a diagnostic tool for discriminating benign lesions from malignant ones of the prostate. The suggested tumour suppressor function of TSC-22 warrants further investigation on its role in prostate carcinogenesis and on the TSC-22 pathway as a candidate therapeutic target in PC.

Identification and in silico analyses of novel TGFBR1 and TGFBR2 mutations in Marfan syndrome-related disorders

Very recently, heterozygous mutations in the genes encoding transforming growth factor beta receptors I (TGFBR1) and II (TGFBR2) have been reported in Loeys-Dietz aortic aneurysm syndrome (LDS). In addition, dominant TGFBR2 mutations have been identified in Marfan syndrome type 2 (MFS2) and familial thoracic aortic aneurysms and dissections (TAAD). In the past, mutations of these genes were associated with atherosclerosis and several human cancers. Here, we report a total of nine novel and one known heterozygous sequence variants in the TGFBR1 and TGFBR2 genes in nine of 70 unrelated individuals with MFS-like phenotypes who previously tested negative for mutations in the gene encoding the extracellular matrix protein fibrillin-1 (FBN1). To assess the pathogenic impact of these sequence variants, in silico analyses were performed by the PolyPhen, SIFT, and Fold-X algorithms and by means of a 3D homology model of the TGFBR2 kinase domain. Our results showed that in all but one of the patients the pathogenic effect of at least one sequence variant is highly probable (c.722C > T, c.799A > C, and c.1460G > A in TGFBR1 and c.773T > G, c.1106G > T, c.1159G > A, c.1181G > A, and c.1561T > C in TGFBR2). These deleterious alleles occurred de novo or segregated with the disease in the families, indicating a causative association between the sequence variants and clinical phenotypes. Since TGFBR2 mutations found in patients with MFS-related disorders cannot be distinguished from heterozygous TGFBR2 mutations reported in tumor samples, we emphasize the importance of segregation analysis in affected families. In order to be able to find the mutation that is indeed responsible for a MFS-related phenotype, we also propose that genetic testing for sequence alterations in TGFBR1 and TGFBR2 should be complemented by mutation screening of the FBN1 gene.

Airway remodelling in children with cystic fibrosis

BACKGROUND: The relationship between airway structural changes and inflammation is unclear in early cystic fibrosis (CF) lung disease. A study was undertaken to determine changes in airway remodelling in children with CF compared with appropriate disease and healthy controls. METHODS: Bronchoalveolar lavage and endobronchial biopsy were performed in a cross-sectional study of 43 children with CF (aged 0.3-16.8 years), 7 children with primary ciliary dyskinesia (PCD), 26 with chronic respiratory symptoms (CRS) investigated for recurrent infection and/or cough and 7 control children with no lower airway symptoms. Inflammatory cells, cytokines, proteases and matrix constituents were measured in bronchoalveolar lavage fluid (BALF). Reticular basement membrane (RBM) thickness was measured on biopsy specimens using light microscopy. RESULTS: Increased concentrations of elastin, glycosaminoglycans and collagen were found in BALF from children with CF compared with the CRS group and controls, each correlating positively with age, neutrophil count and proteases (elastase activity and matrix metalloproteinase-9 (MMP-9) concentration). There were significant negative correlations between certain of these and pulmonary function (forced expiratory volume in 1 s) in the CF group (elastin: r = -0.45, p<0.05; MMP-9:TIMP-1 ratio: r = -0.47, p<0.05). Median RBM thickness was greater in the CF group than in the controls (5.9 microm vs 4.0 microm, p<0.01) and correlated positively with levels of transforming growth factor-beta(1) (TGF-beta(1); r = 0.53, p = 0.01), although not with other inflammatory markers or pulmonary function. CONCLUSIONS: This study provides evidence for two forms of airway remodelling in children with CF: (1) matrix breakdown, related to inflammation, proteolysis and impaired pulmonary function, and (2) RBM thickening, related to TGF-beta(1) concentration but independent of other markers of inflammation.