C3f is a potential tool for the staging of osteoarthritis.

An attractive method for osteoarthritis (OA) staging is the measurement of biochemical markers in biological fluids, which could reflect dynamic and quantitative changes in joint remodeling and therefore disease progression. Proteome analysis has been recognized as one of the most effective tools to explore biomarkers as it can furnish a wealth of information in both diagnosis and prognosis of diseases. We have recently described an innovative tool for peptidome and lipidome profiling of fluids based on mesoporous aluminosilicate (MPAS) and Matrix-Assisted Laser Desorption/Ionization time-of-flight Mass Spectrometry (MALDI-TOF MS). The aim of this study was to analyze peptide profiles of human synovial fluid in patients with different grade of OA using MALDI-TOF-MS technique in order to identify potential markers of disease progression. Twenty-five patients older than 50 years and affected by primary knee OA diagnosed according to clinical and radiological criteria were enrolled. For each patient a synovial fluid sample was aspirated from the affected knee and analyzed using MALDI-TOF-MS technique. A statistically significant difference in the normalized area of two peaks (m/z=1865 and m/z=2021) was detected among different stages of OA. The 2 peaks were identified as Complement C3 peptide fragments: C3f and C3f Des-Arg. The expression levels of these two peptides (m/z=1865 and 2021) decreased with the progression of OA degrees severity (ρs=-0.434, p=0.03, and ρs=-0.532, p=0.006, respectively). This marker may be a useful tool for assessing the severity of knee OA and it may be a novel target for drug discovery, specifically for the development of disease modifying OA drugs. However further studies are required to clarify the role of C3f in OA pathogenesis.

The effects of nonsteroidal anti-inflammatory drugs on clinical outcomes, synovial fluid cytokine concentration and signal transduction pathways in knee osteoarthritis. A randomized open label trial.

OBJECTIVE: We investigated the effects of celecoxib, diclofenac, and ibuprofen on the disease-specific quality of life, synovial fluid cytokines and signal transduction pathways in symptomatic knee osteoarthritis (OA). DESIGN: Ninety patients scheduled for a total knee arthroplasty (TKA) were randomized to six groups that were treated with low and high dosages of celecoxib, diclofenac or ibuprofen. At the time of the first admission (T0) and at surgery (T1 = 14 days after beginning of the nonsteroidal anti-inflammatory drugs (NSAIDs)), samples of knee synovial fluid were obtained from each patient for analysis. During the surgery the synovial tissue was harvested from the knee of patients. The Western Ontario and McMaster universities (WOMAC) score was used to evaluate the patient disease-specific quality of life at T0 and T1. Microarray tests performed at T0 and T1 were used to evaluate the effects of NSAIDs on Tumor necrosis factor (TNF)-alpha, Interleukin-6 (IL-6), IL8 and Vascular endothelial growth factor (VEGF) concentration in the synovial fluid. Western blot assays evaluated the effects of NSAIDs on MAP kinase (MAPK) signal transduction pathway in the synovial membrane. RESULTS: NSAID treatment induced a statistically significant improvement in the WOMAC score and a statistically significant decrease in the IL-6, VEGF and TNF-alpha concentration in the synovial fluid. Higher dosages of NSAIDs provided a greater improvement in the disease-specific quality of life of patients and lower concentrations of pro-inflammatory cytokines in the synovial fluid. Inhibition of MAPKs was noted after NSAID treatment. CONCLUSION: Short-term NSAID treatment improves the patient disease-specific quality of life with a parallel decrease in pro-inflammatory synovial fluid cytokine levels in knee OA. Signal transduction pathways may be involved in regulating the anti-inflammatory effects of NSAIDs. ClinicalTrial.gov: NCT01860833.

Effects of simvastatin and rosuvastatin on RAS protein, matrix metalloproteinases and NF-κB in lung cancer and in normal pulmonary tissues.

OBJECTIVES: In this study, we have evaluated effects of 24-hour treatments with simvastatin or rosuvastatin on RAS protein, NF-κB and MMP expression in LC tissues obtained from 12 patients undergoing thoracic surgery. MATERIALS AND METHODS: Normal and lung tumour tissues obtained from each sample were exposed to simvastatin (2.5-30 µm) or rosuvastatin (1.25-30 µm) and western blot analysis was then performed. RESULTS: We documented increased expression of proteins, MMP-2, MMP-9 and NF-κB-p65 in LC tissues, with respect to normal tissues (P < 0.01). In the malignant tissues, simvastatin and rosuvastatin significantly (P < 0.01) and dose-dependently reduced RAS protein, MMP-2/9 and NF-κB-p65 expression. CONCLUSIONS: In conclusion, our results suggest that simvastatin and rosuvastatin could play a role in LC treatment by modulation of RAS protein, MMP-2/9 and NF-κB-p65.

Characteristics and clinical implications of the pharmacokinetic profile of ibuprofen in patients with knee osteoarthritis.

BACKGROUND AND OBJECTIVE: Ibuprofen is a non-selective cyclo-oxygenase (COX)-1/COX-2 inhibitor used to treat pain conditions and inflammation. Limited data have been published concerning the pharmacokinetic profile and clinical effects of ibuprofen in patients with osteoarthritis (OA). In this paper we compared the pharmacokinetic and clinical profile of ibuprofen (at a dosage of from 800 mg/day to 1800 mg/day) administered in patients affected by severe knee OA. METHODS: Ibuprofen was administered for 7 days to patients who were scheduled to undergo knee arthroplasty due to OA. After 7 days, the ibuprofen concentration in plasma and synovial fluid was measured through both high-performance liquid chromatography (HPLC)-UV and gas chromatography-mass spectroscopy (GC/MS), while clinical effects were evaluated through both visual analogue scale (VAS) and Western Ontario and McMaster Universities (WOMAC) scores. The Naranjo scale and the WHO causality assessment scale were used for estimating the probability of adverse drug reactions (ADRs). The severity of ADRs was assessed by the modified Hartwig and Siegel scale. RESULTS: Ibuprofen showed a dose-dependent diffusion in both plasma and synovial fluid, which was related to the reduction of pain intensity and improvement of health status, without the development of ADRs. CONCLUSION: Ibuprofen at higher dosages can be expected to provide better control of OA symptoms as a result of higher tissue distribution.

Effects of statins and farnesyl transferase inhibitors on ERK phosphorylation, apoptosis and cell viability in non-small lung cancer cells.

OBJECTIVE: 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) can affect post-translational processes, thus being responsible for decreased farnesylation and geranylgeranylation of intracellular small G proteins such as Ras, Rho and Rac, essential for cell survival and proliferation. In this regard, recent in vitro and in vivo studies suggest a possible role for both statins and farnesyl transferase inhibitors in the treatment of malignancies. Within such a context, the aim of our study was to investigate effects of either simvastatin (at concentrations of 1, 15, and 30 µm) or the farnesyl transferase inhibitor R115777 (at concentrations of 0.1, 1, and 10 µm), on two cultures of human non-small lung cancer cells, adenocarcinoma (GLC-82) and squamous (CALU-1) cell lines. In particular, we evaluated actions of these two drugs on phosphorylation of the ERK1/2 group of mitogen-activated protein kinases and on apoptosis, plus on cell numbers and morphology. MATERIALS AND METHODS: Western blotting was used to detect ERK phosphorylation, and to assess apoptosis by evaluating caspase-3 activation; apoptosis was also further assessed by terminal deoxynucleotidyl-mediated dUTP nick end labelling (TUNEL) assay. Cell counting was performed after trypan blue staining. RESULTS AND CONCLUSION: In both GLC-82 and CALU-1 cell lines, simvastatin and R115777 significantly reduced ERK phosphorylation; this effect, which reached the greatest intensity after 36 h treatment, was paralleled by a concomitant induction of apoptosis, documented by significant increase in both caspase-3 activation and TUNEL-positive cells, associated with a reduction in cell numbers. Our results thus suggest that simvastatin and R115777 may exert, in susceptible lung cancer cell phenotypes, a pro-apoptotic and anti-proliferative activity, which appears to be mediated by inhibition of the Ras/Raf/MEK/ERK signalling cascade.

Four proteins governing overangiogenic endothelial cell phenotype in patients with multiple myeloma are plausible therapeutic targets.

Bone marrow (BM) angiogenesis has an important role in the initiation and progression of multiple myeloma (MM). We looked at novel mechanisms of vessel formation in patients with MM through a comparative proteomic analysis between BM endothelial cells (ECs) of patients with active MM (MMECs) and ECs of patients with monoclonal gammopathy of undetermined significance (MGECs) and of subjects with benign anemia (normal ECs). Four proteins were found overexpressed in MMECs: filamin A, vimentin, α-crystallin B and 14-3-3ζ/δ protein, not yet linked to overangiogenic phenotype. These proteins gave a typical distribution in the BM of MM patients and in MMECs versus MGECs, plausibly according to a different functional state. Their expression was enhanced by vascular endothelial growth factor, fibroblast growth factor 2, hepatocyte growth factor and MM plasma cell conditioned medium in step with enhancement of MMEC angiogenesis. Their silencing RNA knockdown affected critical MMEC angiogenesis-related functions, such as spreading, migration and tubular morphogenesis. A gradual stabilization of 14-3-3ζ/δ protein was observed, with transition from normal ECs to MGECs and MMECs that may be a critical step for the angiogenic switch in MMECs and maintenance of the cell overangiogenic phenotype. These proteins were substantially impacted by anti-MM drugs, such as bortezomib, lenalidomide and panobinostat. Results suggest that these four proteins could be new targets for the antiangiogenic management of MM patients.

Self-rewetting fluids with suspended carbon nanostructures.

Thermal management is very important in modern electronic systems. Recent researches have been dedicated to the study of the heat transfer performances of binary or multi-component heat transfer fluids with peculiar surface tension properties and in particular to "self-rewetting fluids," i.e., liquids with a surface tension increasing with temperature and concentration. Thermophysical properties like surface tension, wettability and thermal conductivity, at different temperatures, have been measured not only for binary mixtures, but also for a number of ternary aqueous solutions with relatively low freezing point and for nanoparticles suspensions (so called nanofluids). Some of them interestingly exhibit the same anomalous positive surface tension gradient with temperature as binary self-rewetting solutions. Since in the course of liquid/vapour phase change, self-rewetting fluids behaviour induces a rather strong liquid inflow (caused by both temperature and concentration gradients) from the cold region (where liquid condensates) to the hot evaporator region, several interesting applications may be envisaged, e.g., the development of advanced wickless heat pipes for utilization in reduced gravity environments. The present work is dedicated to the study of the thermophysical properties of nanofluids based on water/alcohol solutions with suspended carbon nanostructures, in particular single-wall carbon nanohorns (SWNH), synthesised by an homemade apparatus with an AC arc discharge in open air. The potential interest of the proposed studies stems from the large number of possible industrial applications, including space technologies and terrestrial applications, such as cooling of electronic components.

Effects of budesonide on P38 MAPK activation, apoptosis and IL-8 secretion, induced by TNF-alpha and Haemophilus influenzae in human bronchial epithelial cells.

Non-typeable Haemophilus influenzae (NTHi) is one of the most frequently involved pathogens in bacterial exacerbations of chronic obstructive pulmonary disease (COPD). In the airways, the main tissue target of NTHi is bronchial epithelium, where this pathogen can further amplify the inflammatory and structural changes induced by proinflammatory cytokines such as tumour necrosis factor-alpha (TNF-alpha). Therefore, the aim of this study is to investigate, in primary cultures of human bronchial epithelial cells, the effects of NTHi on signal transduction pathways, apoptotic events and chemokine production activated by TNF-alpha. Moreover, we also evaluated the effects exerted on such cellular and molecular phenomena by a corticosteroid drug. p38 mitogen-activated protein kinase (MAPK) phosphorylation was analyzed by Western blotting, using an anti-phospho-p38 MAPK monoclonal antibody. Apoptosis was assayed by active caspase-3 expression. Interleukin-8 (IL-8/CXCL8) was detected in cell-free culture supernatants by ELISA. TNF-alpha induced a significant increase in p38 MAPK phosphorylation. NTHi was able to potentiate the stimulatory actions of TNF-alpha on caspase-3 expression and, to a lesser extent, on IL-8 secretion. These effects were significantly (P less than 0.01) inhibited by a pharmacological pre-treatment with budesonide. These results suggest that TNF-alpha is able to stimulate, via activation of p38 MAPK signalling pathway, IL-8 release and airway epithelial cell apoptosis; the latter effect can be markedly potentiated by NTHi. Furthermore, budesonide can be very effective in preventing, through inhibition of p38 MAPK phosphorylation, both structural and proinflammatory changes elicited in bronchial epithelium by TNF-alpha and NTHi.

Interleukin-6 receptor superantagonist Sant7 inhibits TGF-beta-induced proliferation of human lung fibroblasts.

OBJECTIVES: Both interleukin-6 (IL-6) and transforming growth factor-beta (TGF-beta) are crucially involved in fibrotic events that characterize interstitial lung diseases (ILD). Therefore, the aim of this study was to investigate in primary cultures of normal and fibrotic human lung fibroblasts (HLF), exposed to either IL-6 or TGF-beta1, the effects on phosphorylation of mitogen-activated protein kinases (MAPK) and cell growth of IL-6 signalling inhibition, performed by the IL-6 receptor superantagonist Sant7. MATERIALS AND METHODS: MAPK phosphorylation was detected by Western blotting, HLF viability and proliferation were evaluated using the trypan blue staining and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, respectively. RESULTS: Sant7, at a concentration of 1 microg/mL, was capable of significantly inhibiting HLF proliferation and MAPK phosphorylation induced by cell exposure to IL-6 (100 ng/mL) or TGF-beta1 (10 ng/mL), whose actions were more evident in fibrotic cells. CONCLUSIONS: These findings suggest that, in HLFs derived from patients with ILDs, the proliferative mechanisms activated by TGF-beta1 are at least in part mediated by an increased release of IL-6, leading to phosphorylation-dependent MAPK activation. Such preliminary findings may thus open new therapeutic perspectives for fibrogenic ILDs, based on inhibition of signal transduction pathways stimulated by the IL-6 receptor.

Microcirculation as a novel marker of membrane biocompatibility.

BACKGROUND: It is possible to consider microcirculation as a kind of OwitnessO of the complex biological reactions triggered by the dialytic treatment. The reactivity of microcirculation to the dialytic stress may represent a measure of the overall biocompatibility of the membrane. In this study we tested the hypothesis that different synthetic membranes may have different biological effects, particularly related to microcirculation. SUBJECTS AND METHODS: In this crossover study, we observed 16 chronically hemodialyzed patients. All patients were treated with the EVAL membrane; we recorded the TcPO2 during the second treatment of the week. All patients were then switched to the hf-PS membrane. During the study observation we did not change the dialytic prescription or the pharmacologic treatment. RESULTS: From the beginning of the session until 90O, the behavior of TcPO2 is similar for both the membranes. From 120O to the end of the treatment in sessions with the EVAL membrane, the TcPO2 values come back to the starting level, whereas in the treatments with hf-PS the TcPO2, the values remain at a lower level; there was a significant difference between EVAL and hf-PS in the values recorded. Arterial blood gas values of paO2 and paCO2 are quite similar in the treatments with both the membranes, without any significant difference. CONCLUSIONS: The analysis of microcirculation by means of TcPO2 measurement is a useful tool to obtain a OclinicalO measure of biocompatibility of the dialytic treatment and different membranes may have different impacts on TcPO2.

The fluid-dynamic disturbances induced on the ISS, based on the first acceleration measurements on board the space station.

The different acceleration components on the ISS that are responsible for the generation of convective motions in a fluid cell either in the presence of density gradients or in quasi-isodense processes, are analyzed. The NASA measurements of the quasi-steady and periodic acceleration on the ISS are considered and their effects on fluid-dynamic experiments are computed and discussed under different assumptions. In particular, numerical simulations are carried out to identify the relative importance of linear and pendular accelerations, due to possible rotations of the P/L around its center of mass. The effects caused by variable accelerations created by an isolation mount that exhibits an attenuation factor not constant within the payload volume, caused by the reaction forces of the umbilicals, are computed and analyzed.

The IL-6 receptor antagonist SANT-7 overcomes bone marrow stromal cell-mediated drug resistance of multiple myeloma cells.

The bone marrow micro-environment produces a number of different survival factors that are important for the malignant growth and drug resistance of multiple myeloma (MM) cells. One of the main factors reported to be essential for survival and growth of MM cells in some experimental systems is IL-6. Therefore, the development and testing of substances that interfere with IL-6 or IL-6 receptor (IL-6R) function might have therapeutic value for the treatment of MM. We analyzed the effect of the IL-6R antagonist SANT-7 on growth and survival of the IL-6--dependent MM cell lines INA-6 and XG-1 as well as primary MM cells from 7 patients co-cultured with bone marrow stromal cells (BMSCs). In particular, we were interested in whether SANT-7 enhances the growth-inhibitory effects of dexamethasone (Dex) and all-trans-retinoic acid (ATRA). None of the drugs when tested as a single substance, including SANT-7, induced major growth inhibition if MM cells were co-cultured with primary human BMSCs. However, when Dex and ATRA were given in combination with SANT-7, strong growth inhibition was achieved in cell lines and primary MM cells. This effect was due to cell-cycle arrest and induction of apoptosis.

An improved helper-dependent adenoviral vector allows persistent gene expression after intramuscular delivery and overcomes preexisting immunity to adenovirus.

Helper-dependent adenoviral vectors deleted of all viral coding sequences have shown an excellent gene expression profile in a variety of animal models, as well as a reduced toxicity after systemic delivery. What is still unclear is whether long-term expression and therapeutic dosages of these vectors can be obtained also in the presence of a preexisting immunity to adenovirus, a condition found in a high proportion of the adult human population. In this study we performed intramuscular delivery of helper-dependent vectors carrying mouse erythropoietin as a marker transgene. We found that low doses of helper-dependent adenoviral vectors can direct long-lasting gene expression in the muscles of fully immunocompetent mice. The best performance-i.e., 100% of treated animals showing sustained expression after 4 months-was achieved with the latest generation helper-dependent backbones, which replicate and package at high efficiency during vector propagation. Moreover, efficient and prolonged transgene expression after intramuscular injection was observed with limited vector load also in animals previously immunized against the same adenovirus serotype. These data suggest that human gene therapy by intramuscular delivery of helper-dependent adenoviral vectors is feasible.

In vivo neutralization of human IL-6 (hIL-6) achieved by immunization of hIL-6-transgenic mice with a hIL-6 receptor antagonist.

Neutralization of IL-6 represents an attractive therapeutic option in several diseases, including B cell neoplasia, osteoporosis, and autoimmunity. Therapeutic attempts in humans have shown that administration of injectable doses of a mAb to IL-6 does not provide efficient neutralization of the cytokine in vivo. Therefore, alternative approaches are needed. In this study, we evaluated whether the Ab response to human IL-6 (hIL-6) elicited by vaccination with Sant1 (a hIL-6 variant with seven amino acid substitutions) was able to fully correct in vivo the clinical and biological effects of a chronic endogenous overproduction of hIL-6 in the hIL-6-transgenic NSE/hIL-6 mice. Because of the overexpression of hIL-6, occurring since birth, with circulating levels in the nanogram per milliliter range, NSE/hIL-6 mice have a marked decrease in growth rate, associated with decrease in insulin-like growth factor I levels, and represent an animal model of the growth impairment associated with human chronic inflammatory diseases. Following immunization with Sant1, but not with hIL-6, NSE/hIL-6 mice developed high titers of polyclonal Abs to hIL-6. The Abs, acquired by transplacental transfer, effectively neutralized IL-6 activities in vivo as shown by the complete correction of the growth defect and normalization of insulin-like growth factor levels in the hIL-6-transgenic offspring. Immunization with Sant1 could therefore represent a novel and simple therapeutic approach for the specific neutralization of IL-6 in humans.

Prolonged expression and effective readministration of erythropoietin delivered with a fully deleted adenoviral vector.

Helper-dependent (HD) adenoviral (Ad) vectors, in which all viral coding sequences are deleted, have been generated. We show here that intravenous delivery of a mouse EPO (mEPO) expression cassette cloned in an HD vector in immunocompetent mice is effective and long lasting, but not permanent. A precise dose-response relationship between the dose of injected virus and stable EPO serum levels was observed, together with a 100-fold increase in gene expression per infectious particle when compared with a first-generation Ad vector bearing the same cassette. As a direct consequence, therapeutic increases in hematocrit that lasted more than 6 months were achieved with minute amounts of virus, which caused no detectable production of neutralizing antibodies. Intravenous readministration of the HD-mEPO vector in the same mice was as effective as in naive animals without any need for prior immunosuppression. Finally, HD-mEPO injection in subtotally nephrectomized rats improved the anemic status induced by surgery. HD Ad vectors are thus excellent tools for EPO gene therapy.

Optimization of the helper-dependent adenovirus system for production and potency in vivo.

Helper-dependent (HD) adenoviral vectors devoid of all viral coding sequences provide for safe and highly efficient gene transfer with long-lasting transgene expression. High titer stocks of HD vectors can be generated by using the cre-recombinase system. However, we have encountered difficulties with this system, including rearranged HD vectors and variable efficiency of HD vector rescue. These problems represent a major hindrance, particularly with regard to large-scale production. To overcome these limitations, we have modified the system in two ways: We constructed a new helper virus with a modified packaging signal and enhanced growth characteristics. We also redesigned the vector backbones by including noncoding adenovirus sequences adjacent to the right inverted terminal repeat and by incorporated a number of different segments of noncoding DNA of human origin as "stuffer." Comparison of these vectors showed that the nature of the stuffer sequence affects replication of the HD vector. Optimization of the system resulted in a more robust and consistent production of HD vectors with low helper contamination and high in vivo potency.

Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation.

We show that an electric treatment in the form of high-frequency, low-voltage electric pulses can increase more than 100-fold the production and secretion of a recombinant protein from mouse skeletal muscle. Therapeutical erythopoietin (EPO) levels were achieved in mice with a single injection of as little as 1 microgram of plasmid DNA, and the increase in hematocrit after EPO production was stable and long-lasting. Pharmacological regulation through a tetracycline-inducible promoter allowed regulation of serum EPO and hematocrit levels. Tissue damage after stimulation was transient. The method described thus provides a potentially safe and low-cost treatment for serum protein deficiencies.