TGF-beta 1 modulates the homeostasis between MMPs and MMP inhibitors through p38 MAPK and ERK1/2 in highly invasive breast cancer cells

Background: Metastasis is the main factor responsible for death in breast cancer patients. Matrix metalloproteinases (MMPs) and their inhibitors, known as tissue inhibitors of MMPs (TIMPs), and the membrane-associated MMP inhibitor (RECK), are essential for the metastatic process. We have previously shown a positive correlation between MMPs and their inhibitors expression during breast cancer progression; however, the molecular mechanisms underlying this coordinate regulation remain unknown. In this report, we investigated whether TGF-beta 1 could be a common regulator for MMPs, TIMPs and RECK in human breast cancer cell models. Methods: The mRNA expression levels of TGF-beta isoforms and their receptors were analyzed by qRT-PCR in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential. The highly invasive MDA-MB-231 cell line was treated with different concentrations of recombinant TGF-beta 1 and also with pharmacological inhibitors of p38 MAPK and ERK1/2. The migratory and invasive potential of these treated cells were examined in vitro by transwell assays. Results: In general, TGF-beta 2, T beta RI and T beta RII are over-expressed in more aggressive cells, except for T beta RI, which was also highly expressed in ZR-75-1 cells. In addition, TGF-beta 1-treated MDA-MB-231 cells presented significantly increased mRNA expression of MMP-2, MMP-9, MMP-14, TIMP-2 and RECK. TGF-beta 1 also increased TIMP-2, MMP-2 and MMP-9 protein levels but downregulated RECK expression. Furthermore, we analyzed the involvement of p38 MAPK and ERK1/2, representing two well established Smad-independent pathways, in the proposed mechanism. Inhibition of p38MAPK blocked TGF-beta 1-increased mRNA expression of all MMPs and MMP inhibitors analyzed, and prevented TGF-beta 1 upregulation of TIMP-2 and MMP-2 proteins. Moreover, ERK1/2 inhibition increased RECK and prevented the TGF-beta 1 induction of pro-MMP-9 and TIMP-2 proteins. TGF-beta 1-enhanced migration and invasion capacities were blocked by p38MAPK, ERK1/2 and MMP inhibitors. Conclusion: Altogether, our results support that TGF-beta 1 modulates the mRNA and protein levels of MMPs (MMP-2 and MMP-9) as much as their inhibitors (TIMP-2 and RECK). Therefore, this cytokine plays a crucial role in breast cancer progression by modulating key elements of ECM homeostasis control. Thus, although the complexity of this signaling network, TGF-beta 1 still remains a promising target for breast cancer treatment.

Innovative treatments for shelf-life extension of whole and fresh-cut fruit

Maintaining the postharvest quality of whole and fresh-cut fruit during storage and distribution is the major challenge facing fruit industry. For this purpose, industry adopt a wide range of technologies to enable extended shelf-life. Many factors can lead to loss of quality in fresh product, hence the common description of these products as ‘perishable’. As a consequence normal factors such as transpiration and respiration lead ultimately to water loss and senescence of the product. Fruits and vegetables are living commodities and their rate of respiration is of key importance to maintenance of quality. It has been commonly observed that the greater the respiration rate of a product, the shorter the shelf-life. The principal problem for fresh-cut fruit industries is the relative shorter shelf-life of minimally processed fruit (MPF) compared to intact product. This fact is strictly connected with the higher ethylene production of fruit tissue stimulated during fresh-cut processing (peeling, cutting, dipping). 1-Methylcyclopropene (1-MCP) is an inhibitor of ethylene action and several researches have shown its effectiveness on the inhibition of ripening and senescence incidence for intact fruit and consequently on their shelf-life extension. More recently 1-MCP treatment has been tested also for shelf-life extension of MPF but discordant results have been obtained. Considering that in some countries 1-MCP is already a commercial product registered for the use on a number of horticultural products, the main aim of this actual study was to enhance our understanding on the effects of 1-MCP treatment on the quality maintenance of whole and fresh-cut climacteric and non-climacteric fruit (apple, kiwifruit and pineapple). Concerning the effects of 1-MCP on whole fruit, was investigated the effects of a semi-commercial postharvest treatment with 1-MCP on the quality of Pink Lady apples as functions of fruit ripening stage, 1-MCP dose, storage time and also in combination with controlled atmospheres storage in order to better understand what is the relationship among these parameters and if is possible to maximize the 1-MCP treatment to meet the market/consumer needs and then in order to put in the market excellent fruit. To achieve this purpose an incomplete three-level three-factor design was adopted. During the storage were monitored several quality parameters: firmness, ripening index, ethylene and carbon dioxide production and were also performed a sensory evaluations after 6 month of storage. In this study the higher retention of firmness (at the end of storage) was achieved by applying the greatest 1-MCP concentration to fruits with the lowest maturity stage. This finding means that in these semi-commercial conditions we may considerate completely blocked the fruit softening. 1-MCP was able to delay also the ethylene and CO2 production and the maturity parameters (soluble solids content and total acidity). Only in some cases 1-MCP generate a synergistic effect with the CA storage. The results of sensory analyses indicated that, the 1-MCP treatment did not affect the sweetness and whole fruit flavour while had a little effect on the decreasing cut fruit flavour. On the contrary the treated apple was more sour, crisp, firm and juicy. The effects of some treatment (dipping and MAP) on the nutrient stability were also investigated showing that in this case study the adopted treatments did not have drastic effects on the antioxidant compounds on the contrary the dipping may enhance the total antioxidant activity by the accumulation of ascorbic acid on the apple cut surface. Results concerning the effects of 1-MCP in combination with MAP on the quality parameters behaviour of the kiwifruit were not always consistent and clear: in terms of colour maintenance, it seemed to have a synergistic effect with N2O MAP; as far as ripening index is concerned, 1-MCP had a preservative effect, but just for sample packed in air.

Aspects of poriferan (Suberites domuncula) apoptosis and innate immune system and evolutionary implications

Survivin, a unique member of the family of inhibitors of apoptosis (IAP) proteins, orchestrates intracellular pathways during cell division and apoptosis. Its central regulatory function in vertebrate molecular pathways as mitotic regulator and inhibitor of apoptotic cell death has major implications for tumor cell proliferation and viability, and has inspired several approaches that target survivin for cancer therapy. Analyses in early-branching Metazoa so far propose an exclusive role of survivin as a chromosomal passenger protein, whereas only later during evolution the second, complementary antiapoptotic function might have arisen, concurrent with increased organismal complexity. To lift the veil on the ancestral function(s) of this key regulatory molecule, a survivin homologue of the phylogenetically oldest extant metazoan taxon (phylum Porifera) was identified and functionally characterized. SURVL of the demosponge Suberites domuncula shares significant similarities with its metazoan homologues, ranging from conserved exon/intron structures to the presence of localization signal and protein-interaction domains, characteristic of IAP proteins. Whereas sponge tissue displayed a very low steady-state level, SURVL expression was significantly up-regulated in rapidly proliferating primmorph cells. In addition, challenge of sponge tissue and primmorphs with cadmium and the lipopeptide Pam3Cys-Ser-(Lys)4 stimulated SURVL expression, concurrent with the expression of newly discovered poriferan caspases (CASL and CASL2). Complementary functional analyses in transfected HEK-293 revealed that heterologous expression of poriferan survivin in human cells not only promotes cell proliferation but also augments resistance to cadmium-induced cell death. Taken together, these results demonstrate both a deep evolutionary conserved and fundamental dual role of survivin, and an equally conserved central position of this key regulatory molecule in interconnected pathways of cell cycle and apoptosis. Additionally, SDCASL, SDCASL2, and SDTILRc (TIR-LRR containing protein) may represent new components of the innate defense sentinel in sponges. SDCASL and SDCASL2 are two new caspase-homolog proteins with a singular structure. In addition to their CASc domains, SDCASL and SDCASL2 feature a small prodomain NH2-terminal (effector caspases) and a remarkably long COOH-terminal domain containing one or several functional double stranded RNA binding domains (dsrm). This new caspase prototype can characterize a caspase specialization coupling pathogen sensing and apoptosis, and could represent a very efficient defense mechanism. SDTILRc encompasses also a unique combination of domains: several leucine rich repeats (LRR) and a Toll/IL-1 receptor (TIR) domain. This unusual domain association may correspond to a new family of intracellular sensing protein, forming a subclass of pattern recognition receptors (PRR).

SMO inhibitor specifically targets the Hedgehog Pathway and reverts the drug-resistance of Leukemic Stem Cells

Abnormal Hedgehog signaling is associated with human malignancies. Smo, a key player of that signaling, is the most suitable target to inhibit this pathway. To this aim several molecules, antagonists of Smo, have been synthesized, and some of them have started the phase I in clinical trials. Our hospital participated to one of these studies which investigated the oral administration of a new selective inhibitor of Smo (SMOi). To evaluate ex vivo SMOi efficacy and to identify new potential clinical biomarkers of responsiveness, we separated bone marrow CD34+ cells from 5 acute myeloid leukemia (AML), 1 myelofibrosis (MF), 2 blastic phases chronic myeloid leukemia (CML) patients treated with SMOi by immunomagnetic separation, and we analysed their gene expression profile using Affimetrix HG-U133 Plus 2.0 platform. This analysis, showed differential expression after 28 days start of therapy (p-value ≤ 0.05) of 1,197 genes in CML patients and 589 genes in AML patients. This differential expression is related to Hedgehog pathway with a p-value = 0.003 in CML patients and with a p-value = 0.0002 in AML patients, suggesting that SMOi targets specifically this pathway. Among the genes differentially expressed we observed strong up-regulation of Gas1 and Kif27 genes, which may work as biomarkers of responsiveness of SMOi treatment in CML CD34+ cells whereas Hedgehog target genes (such as Smo, Gli1, Gli2, Gli3), Bcl2 and Abca2 were down-regulated, in both AML and CML CD34+ cells. It has been reported that Bcl-2 expression could be correlated with cancer therapy resistance and that Hedgehog signaling modulate ATP-binding (ABC) cassette transporters, whose expression has been correlated with chemoresistance. Moreover we confirmed that in vitro SMOi treatment targets Hedgehog pathway, down-regulate ABC transporters, Abcg2 and Abcb1 genes, and in combination with tyrosine kinase inhibitors (TKIs) could revert the chemoresistance mechanism in K562 TKIs-resistant cell line.

Methods and strategies to identify the mode of action of phytoactive compounds

Small molecules affecting biological processes in plants are widely used in agricultural practice as herbicides or plant growth regulators and in basic plant sciences as probes to study the physiology of plants. Most of the compounds were identified in large screens by the agrochemical industry, as phytoactive natural products and more recently, novel phytoactive compounds originated from academic research by chemical screens performed to induce specific phenotypes of interest. The aim of the present PhD thesis is to evaluate different approaches used for the identification of the primary mode of action (MoA) of a phytoactive compound. Based on the methodologies used for MoA identification, three approaches are discerned: a phenotyping approach, an approach based on a genetic screen and a biochemical screening approach.rnFour scientific publications resulting from my work are presented as examples of how a phenotyping approach can successfully be applied to describe the plant MoA of different compounds in detail.rnI. A subgroup of cyanoacrylates has been discovered as plant growth inhibitors. A set of bioassays indicated a specific effect on cell division. Cytological investigations of the cell division process in plant cell cultures, studies of microtubule assembly with green fluorescent protein marker lines in vivo and cross resistant studies with Eleusine indica plants harbouring a mutation in alpha-tubulin, led to the description of alpha-tubulin as a target site of cyanoacrylates (Tresch et al., 2005).rnII. The MoA of the herbicide flamprop-m-methyl was not known so far. The studies described in Tresch et al. (2008) indicate a primary effect on cell division. Detailed studies unravelled a specific effect on mitotic microtubule figures, causing a block in cell division. In contrast to other inhibitors of microtubule rearrangement such as dinitroanilines, flamprop-m-methyl did not influence microtubule assembly in vitro. An influence of flamprop-m-methyl on a target within the cytoskeleton signalling network could be proposed (Tresch et al., 2008).rnIII. The herbicide endothall is a protein phosphatase inhibitor structurally related to the natural product cantharidin. Bioassay studies indicated a dominant effect on dark-growing cells that was unrelated to effects observed in the light. Cytological characterisation of the microtubule cytoskeleton in corn tissue and heterotrophic tobacco cells showed a specific effect of endothall on mitotic spindle formation and ultrastructure of the nucleus in combination with a decrease of the proliferation index. The observed effects are similar to those of other protein phosphatase inhibitors such as cantharidin and the structurally different okadaic acid. Additionally, the observed effects show similarities to knock-out lines of the TON1 pathway, a protein phosphatase-regulated signalling pathway. The data presented in Tresch et al. (2011) associate endothall’s known in vitro inhibition of protein phosphatases with in vivo-effects and suggest an interaction between endothall and the TON1 pathway.rnIV. Mefluidide as a plant growth regulator induces growth retardation and a specific phenotype indicating an inhibition of fatty acid biosynthesis. A test of the cuticle functionality suggested a defect in the biosynthesis of very-long-chain fatty acids (VLCFA) or waxes. Metabolic profiling studies showed similarities with different groups of VLCFA synthesis inhibitors. Detailed analyses of VLCFA composition in tissues of duckweed (Lemna paucicostata) indicated a specific inhibition of the known herbicide target 3 ketoacyl-CoA synthase (KCS). Inhibitor studies using a yeast expression system established for plant KCS proteins verified the potency of mefluidide as an inhibitor of plant KCS enzymes. It could be shown that the strength of inhibition varied for different KCS homologues. The Arabidopsis Cer6 protein, which induces a plant growth phenotype similar to mefluidide when knocked out, was one of the most sensitive KCS enzymes (Tresch et al., 2012).rnThe findings of my own work were combined with other publications reporting a successful identification of the MoA and primary target proteins of different compounds or compound classes.rnA revised three-tier approach for the MoA identification of phytoactive compounds is proposed. The approach consists of a 1st level aiming to address compound stability, uniformity of effects in different species, general cytotoxicity and the effect on common processes like transcription and translation. Based on these findings advanced studies can be defined to start the 2nd level of MoA characterisation, either with further phenotypic characterisation, starting a genetic screen or establishing a biochemical screen. At the 3rd level, enzyme assays or protein affinity studies should show the activity of the compound on the hypothesized target and should associate the in vitro effects with the in vivo profile of the compound.

Tissue engineering strategies for biomaterial-based anticancer drug delivery and enhancement of angiogenesis in tumor-resected bone

Patienten, die an Osteosarkom leiden werden derzeit mit intravenös applizierten krebstherapeutischen Mitteln nach Tumorresektion behandelt, was oftmals mit schweren Nebenwirkungen und einem verzögerten Knochenheilungsprozess einhergeht. Darüber hinaus treten vermehrt Rezidive aufgrund von verbleibenden neoplastischen Zellen an der Tumorresektionsstelle auf. Erfolgreiche Knochenregeneration und die Kontrolle von den im Gewebe verbleibenden Krebszellen stellt eine Herausforderung für das Tissue Engineering nach Knochenverlust durch Tumorentfernung dar. In dieser Hinsicht scheint der Einsatz von Hydroxyapatit als Knochenersatzmaterial in Kombination mit Cyclodextrin als Medikamententräger, vielversprechend. Chemotherapeutika können an Biomaterial gebunden und direkt am Tumorbett über einen längeren Zeitraum freigesetzt werden, um verbliebene neoplastische Zellen zu eliminieren. Lokal applizierte Chemotherapie hat diverse Vorteile, einschließlich der direkten zytotoxischen Auswirkung auf lokale Zellen, sowie die Reduzierung schwerer Nebenwirkungen. Diese Studie wurde durchgeführt, um die Funktionsfähigkeit eines solchen Arzneimittelabgabesystems zu bewerten und um Strategien im Bereich des Tissue Engineerings zu entwickeln, die den Knochenheilungsprozess und im speziellen die Vaskularisierung fördern sollen. Die Ergebnisse zeigen, dass nicht nur Krebszellen von der chemotherapeutischen Behandlung betroffen sind. Primäre Endothelzellen wie zum Beispiel HUVEC zeigten eine hohe Sensibilität Cisplatin und Doxorubicin gegenüber. Beide Medikamente lösten in HUVEC ein tumor-unterdrückendes Signal durch die Hochregulation von p53 und p21 aus. Zudem scheint Hypoxie einen krebstherapeutischen Einfluss zu haben, da die Behandlung sensitiver HUVEC mit Hypoxie die Zellen vor Zytotoxizität schützte. Der chemo-protektive Effekt schien deutlich weniger auf Krebszelllinien zu wirken. Diese Resultate könnten eine mögliche chemotherapeutische Strategie darstellen, um den Effekt eines zielgerichteten Medikamenteneinsatzes auf Krebszellen zu verbessern unter gleichzeitiger Schonung gesunder Zellen. Eine erfolgreiche Integration eines Systems, das Arzneimittel abgibt, kombiniert mit einem Biomaterial zur Stabilisierung und Regeneration, könnte gesunden Endothelzellen die Möglichkeit bieten zu proliferieren und Blutgefäße zu bilden, während verbleibende Krebszellen eliminiert werden. Da der Prozess der Knochengeweberemodellierung mit einer starken Beeinträchtigung der Lebensqualität des Patienten einhergeht, ist die Beschleunigung des postoperativen Heilungsprozesses eines der Ziele des Tissue Engineerings. Die Bildung von Blutgefäßen ist unabdingbar für eine erfolgreiche Integration eines Knochentransplantats in das Gewebe. Daher ist ein umfangreich ausgebildetes Blutgefäßsystem für einen verbesserten Heilungsprozess während der klinischen Anwendung wünschenswert. Frühere Experimente zeigen, dass sich die Anwendung von Ko-Kulturen aus humanen primären Osteoblasten (pOB) und humanen outgrowth endothelial cells (OEC) im Hinblick auf die Bildung stabiler gefäßähnlicher Strukturen in vitro, die auch effizient in das mikrovaskuläre System in vivo integriert werden konnten, als erfolgreich erweisen. Dieser Ansatz könnte genutzt werden, um prä-vaskularisierte Konstrukte herzustellen, die den Knochenheilungsprozess nach der Implantation fördern. Zusätzlich repräsentiert das Ko-Kultursystem ein exzellentes in vitro Model, um Faktoren, welche stark in den Prozess der Knochenheilung und Angiogenese eingebunden sind, zu identifizieren und zu analysieren. Es ist bekannt, dass Makrophagen eine maßgebliche Rolle in der inflammatorisch-induzierten Angiogenese spielen. In diesem Zusammenhang hebt diese Studie den positiven Einfluss THP-1 abgeleiteter Makrophagen in Ko-Kultur mit pOB und OEC hervor. Die Ergebnisse zeigten, dass die Anwendung von Makrophagen als inflammatorischer Stimulus im bereits etablierten Ko-Kultursystem zu einer pro-angiogenen Aktivierung der OEC führte, was in einer signifikant erhöhten Bildung blutgefäßähnlicher Strukturen in vitro resultierte. Außerdem zeigte die Analyse von Faktoren, die in der durch Entzündung hervorgerufenen Angiogenese eine wichtige Rolle spielen, eine deutliche Hochregulation von VEGF, inflammatorischer Zytokine und Adhäsionsmoleküle, die letztlich zu einer verstärkten Vaskularisierung beitragen. Diese Resultate werden dem Einfluss von Makrophagen zugeschrieben und könnten zukünftig im Tissue Engineering eingesetzt werden, um den Heilungsprozess zu beschleunigen und damit die klinische Situation von Patienten zu verbessern. Darüber hinaus könnte die Kombination der auf Ko-Kulturen basierenden Ansätze für das Knochen Tissue Engineering mit einem biomaterial-basierenden Arzneimittelabgabesystem zum klinischen Einsatz kommen, der die Eliminierung verbliebener Krebszellen mit der Förderung der Knochenregeneration verbindet.

Rapid assessment of internodal myelin integrity in central nervous system tissue

Monitoring pathology/regeneration in experimental models of de-/remyelination requires an accurate measure not only of functional changes but also of the amount of myelin. We tested whether X-ray diffraction (XRD), which measures periodicity in unfixed myelin, can assess the structural integrity of myelin in fixed tissue. From laboratories involved in spinal cord injury research and in studying the aging primate brain, we solicited "blind" samples and used an electronic detector to record rapidly the diffraction patterns (30 min each pattern) from them. We assessed myelin integrity by measuring its periodicity and relative amount. Fixation of tissue itself introduced +/-10% variation in periodicity and +/-40% variation in relative amount of myelin. For samples having the most native-like periods, the relative amounts of myelin detected allowed distinctions to be made between normal and demyelinating segments, between motor and sensory tracts within the spinal cord, and between aged and young primate CNS. Different periodicities also allowed distinctions to be made between samples from spinal cord and nerve roots and between well-fixed and poorly fixed samples. Our findings suggest that, in addition to evaluating the effectiveness of different fixatives, XRD could also be used as a robust and rapid technique for quantitating the relative amount of myelin among spinal cords and other CNS tissue samples from experimental models of de- and remyelination.

Differential effects of dexamethasone on the chondrogenesis of mesenchymal stromal cells: influence of microenvironment, tissue origin and growth factor

Mesenchymal stromal cells (MSCs), which reside within various tissues, are utilized in the engineering of cartilage tissue. Dexamethasone (DEX)--a synthetic glucocorticoid--is almost invariably applied to potentiate the growth-factor-induced chondrogenesis of MSCs in vitro, albeit that this effect has been experimentally demonstrated only for transforming-growth-factor-beta (TGF-β)-stimulated bone-marrow-derived MSCs. Clinically, systemic glucocorticoid therapy is associated with untoward side effects (e.g., bone loss and increased susceptibility to infection). Hence, the use of these agents should be avoided or limited. We hypothesize that the influence of DEX on the chondrogenesis of MSCs depends upon their tissue origin and microenvironment [absence or presence of an extracellular matrix (ECM)], as well as upon the nature of the growth factor. We investigated its effects upon the TGF-β1- and bone-morphogenetic-protein 2 (BMP-2)-induced chondrogenesis of MSCs as a function of tissue source (bone marrow vs. synovium) and microenvironment [cell aggregates (no ECM) vs. explants (presence of a natural ECM)]. In aggregates of bone-marrow-derived MSCs, DEX enhanced TGF-β1-induced chondrogenesis by an up-regulation of cartilaginous genes, but had little influence on the BMP-2-induced response. In aggregates of synovial MSCs, DEX exerted no remarkable effect on either TGF-β1- or BMP-2-induced chondrogenesis. In synovial explants, DEX inhibited BMP-2-induced chondrogenesis almost completely, but had little impact on the TGF-β1-induced response. Our data reveal that steroids are not indispensable for the chondrogenesis of MSCs in vitro. Their influence is context dependent (tissue source of the MSCs, their microenvironment and the nature of the growth-factor). This finding has important implications for MSC based approaches to cartilage repair.

Acquisition of complement inhibitor serine protease factor I and its cofactors C4b-binding protein and factor H by Prevotella intermedia

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with (125)I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases.

Treatment of soft tissue recessions at titanium implants using a resorbable collagen matrix: a pilot study

OBJECTIVES: To histologically assess the effectiveness of a porcine-derived collagen matrix (CM) and a subepithelial connective tissue graft (CTG) for the coverage of single mucosal recessions at osseointegrated dental implants. MATERIALS AND METHODS: Chronic-type mucosal Miller Class I-like recessions (mean clinical defect height: 0.67 ± 0.33-1.16 ± 0.19 mm) were established at the buccal aspect of titanium implants with platform switch in six beagle dogs. The defects were randomly allocated to either (1) coronally advanced flap surgery (CAF) + CM, (2) CAF + CTG or (3) CAF alone. At 12 weeks, histomorphometrical measurements were made (e.g.) between the implant shoulder (IS) and the mucosal margin (PM) and IS and the outer contour of the adjacent soft tissue (mucosal thickness [MT]). RESULTS: All treatment procedures investigated were associated with an almost complete soft tissue coverage of the defect area (i.e. coronal positioning of PM relative to IS). Mean IS-PM and MT values tended to be increased in both CAF + CM (1.04 ± 0.74 mm/0.71 ± 0.55 mm) and CAF + CTG (0.88 ± 1.23 mm/0.62 ± 0.66 mm) groups when compared with CAF (0.16 ± 0.28 mm/0.34 ± 0.23 mm) alone. These differences, however, did not reach statistical significance. CONCLUSIONS: Within the limits of this pilot study, it was concluded that all treatment procedures investigated were effective in covering soft tissue recessions at titanium implants.

Analysis of 3D Soft Tissue Changes After 1- and 2-Jaw Orthognathic Surgery in Mandibular Prognathism Patients

Purpose Orthognathic surgery has the objective of altering facial balance to achieve esthetic results in patients who have severe disharmony of the jaws. The purpose was to quantify the soft tissue changes after orthognathic surgery, as well as to assess the differences in 3D soft tissue changes in the middle and lower third of the face between the 1- and 2-jaw surgery groups, in mandibular prognathism patients. Materials and Methods We assessed soft tissue changes of patients who have been diagnosed with mandibular prognathism and received either isolated mandibular surgery or bimaxillary surgery. The quantitative surface displacement was assessed by superimposing preoperative and postoperative volumetric images. An observer measured a surface-distance value that is shown as a contour line. Differences between the groups were determined by the Mann-Whitney U test. The Spearman correlation coefficient was used to evaluate a potential correlation between patients' surgical and cephalometric variables and soft tissue changes after orthognathic surgery in each group. Results There were significant differences in the middle third of the face between the 1- and 2-jaw surgery groups. Soft tissues in the lower third of the face changed in both surgery groups, but not significantly. The correlation patterns were more evident in the lower third of the face. Conclusion The overall soft tissue changes of the midfacial area were more evident in the 2-jaw surgery group. In 2-jaw surgery, significant changes would be expected in the midfacial area, but caution should be exercised in patients who have a wide alar base.

Negative regulation of NF-κB by the ING4 tumor suppressor in breast cancer

Nuclear Factor kappa B (NF-κB) is a key mediator of normal immune response but contributes to aggressive cancer cell phenotypes when aberrantly activated. Here we present evidence that the Inhibitor of Growth 4 (ING4) tumor suppressor negatively regulates NF-κB in breast cancer. We surveyed primary breast tumor samples for ING4 protein expression using tissue microarrays and a newly generated antibody. We found that 34% of tumors expressed undetectable to low levels of the ING4 protein (n = 227). Tumors with low ING4 expression were frequently large in size, high grade, and lymph node positive, suggesting that down-regulation of ING4 may contribute to breast cancer progression. In the same tumor set, we found that low ING4 expression correlated with high levels of nuclear phosphorylated p65/RelA (p-p65), an activated form of NF-κB (p = 0.018). Fifty seven percent of ING4-low/p-p65-high tumors were lymph node-positive, indicating a high metastatic tendency of these tumors. Conversely, ectopic expression of ING4 inhibited p65/RelA phosphorylation in T47D and MCF7 breast cancer cells. In addition, ING4 suppressed PMA-induced cell invasion and NF-κB-target gene expression in T47D cells, indicating that ING4 inhibited NF-κB activity in breast cancer cells. Supportive of the ING4 function in the regulation of NF-κB-target gene expression, we found that ING4 expression levels inversely correlated with the expression of NF-κB-target genes in primary breast tumors by analyzing public gene expression datasets. Moreover, low ING4 expression or high expression of the gene signature composed of a subset of ING4-repressed NF-κB-target genes was associated with reduced disease-free survival in breast cancer patients. Taken together, we conclude that ING4 negatively regulates NF-κB in breast cancer. Consequently, down-regulation of ING4 leads to activation of NF-κB, contributing to tumor progression and reduced disease-free patient survival in breast cancer.

High-grade supraclavicular soft tissue sarcoma as secondary malignancy after successful treatment of acute myeloid leukemia: case report and literature review

It is well known that the treatment protocols for hematopoetic neoplasms carry a high risk of long-term oncogenicity. However, few reports have been published of sarcomas as secondary malignancies. An unusual case report of a soft tissue sarcoma appearing as a secondary cancer is presented, with a review of the published data. The present report involves a soft tissue sarcoma of the neck that occurred 18 years after curative treatment of acute myeloid leukemia by induction chemotherapy and bone marrow transplantation. Consecutive graft-versus-host disease affected the cervical skin. Soft tissue sarcomas appearing as secondary tumors are rare in oncology. The presented case describes the appearance of a sarcoma 18 years after curative treatment of acute myeloid leukemia. This is only the second case of this type reported in published studies.

Use and relevance of a bovine mammary gland explant model to study infection responses in bovine mammary tissue

Our aim was to develop an explant model to define more precisely the early response of bovine mammary epithelial cells to infection. Therefore we investigated the mRNA expression encoding for some soluble immunological factors in lipopolysaccharide (LPS)-treated bovine mammary gland explants. Explants were taken out from the mammary gland of eight lactating cows after slaughter then incubated with LPS (10 mug/ml) for 6 h. The mRNA expression of alpha-lactalbumin (alpha-la), various cytokines, tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, IL-8, and two immunoglobulin receptors, the neonatal Fc receptor (FcRn) and polymeric immunoglobulin receptor (pIGR), were assessed with qPCR before and after 3 h and 6 h of LPS challenge. Both immunoglobulin receptors and alpha-la increased at 3 h then recovered their initial level at 6 h whereas IL-1beta, IL-6 and IL-8 increased only after 6 h (P<0.05). Surprisingly, TNF-alpha transcripts did not show any regulation in response to the LPS treatment. We nevertheless concluded that our model was valid to examine the short-term response of mammary epithelial cell challenged with LPS.

The selective Cox-2 inhibitor Celecoxib suppresses angiogenesis and growth of secondary bone tumors: an intravital microscopy study in mice

BACKGROUND: The inhibition of angiogenesis is a promising strategy for the treatment of malignant primary and secondary tumors in addition to established therapies such as surgery, chemotherapy, and radiation. There is strong experimental evidence in primary tumors that Cyclooxygenase-2 (Cox-2) inhibition is a potent mechanism to reduce angiogenesis. For bone metastases which occur in up to 85% of the most frequent malignant primary tumors, the effects of Cox-2 inhibition on angiogenesis and tumor growth remain still unclear. Therefore, the aim of this study was to investigate the effects of Celecoxib, a selective Cox-2 inhibitor, on angiogenesis, microcirculation and growth of secondary bone tumors. METHODS: In 10 male severe combined immunodeficient (SCID) mice, pieces of A549 lung carcinomas were implanted into a newly developed cranial window preparation where the calvaria serves as the site for orthotopic implantation of the tumors. From day 8 after tumor implantation, five animals (Celecoxib) were treated daily with Celecoxib (30 mg/kg body weight, s.c.), and five animals (Control) with the equivalent amount of the CMC-based vehicle. Angiogenesis, microcirculation, and growth of A549 tumors were analyzed by means of intravital microscopy. Apoptosis was quantified using the TUNEL assay. RESULTS: Treatment with Celecoxib reduced both microvessel density and tumor growth. TUNEL reaction showed an increase in apoptotic cell death of tumor cells after treatment with Celecoxib as compared to Controls. CONCLUSION: Celecoxib is a potent inhibitor of tumor growth of secondary bone tumors in vivo which can be explained by its anti-angiogenic and pro-apoptotic effects. The results indicate that a combination of established therapy regimes with Cox-2 inhibition represents a possible application for the treatment of bone metastases.