Anti-Inflammatory Performance of Lactose-Modified Chitosan and Hyaluronic Acid Mixtures in an In Vitro Macrophage-Mediated Inflammation Osteoarthritis Model.

The development and progression of osteoarthritis (OA) is associated with macrophage-mediated inflammation that generates a broad spectrum of cytokines and reactive oxygen species (ROS). This study investigates the effects of mid-MW hyaluronic acid (HA) in combination with a lactose-modified chitosan (CTL), on pro-inflammatory molecules and metalloproteinases (MMPs) expression, using an in vitro model of macrophage-mediated inflammation. METHODS: To assess chondrocyte response to HA and CTL in the presence of macrophage derived inflammatory mediators, cells were exposed to the conditioned medium (CM) of U937 activated monocytes and changes in cell viability, pro-inflammatory mediators and MMPs expression or ROS generation were analysed. RESULTS: CTL induced changes in chondrocyte viability that are reduced by the presence of HA. The CM of activated U937 monocytes (macrophages) significantly increased gene expression of pro-inflammatory molecules and MMPs and intracellular ROS generation in human chondrocyte cultures. HA, CTL and their combinations counteracted the oxidative damage and restored gene transcription for IL-1ß, TNF-α, Gal-1, MMP-3 and MMP-13 to near baseline values. CONCLUSIONS: This study suggests that HA-CTL mixture attenuated macrophage-induced inflammation, inhibited MMPs expression and exhibited anti-oxidative effects. This evidence provides an initial step toward the development of an early stage OA therapeutic treatment.

Atmospheric-Pressure Cold Plasma Induces Transcriptional Changes in Ex Vivo Human Corneas.

BACKGROUND: Atmospheric pressure cold plasma (APCP) might be considered a novel tool for tissue disinfection in medicine since the active chemical species produced by low plasma doses, generated by ionizing helium gas in air, induces reactive oxygen species (ROS) that kill microorganisms without substantially affecting human cells. OBJECTIVES: In this study, we evaluated morphological and functional changes in human corneas exposed for 2 minutes (min) to APCP and tested if the antioxidant n-acetyl l-cysteine (NAC) was able to inhibit or prevent damage and cell death. RESULTS: Immunohistochemistry and western blotting analyses of corneal tissues collected at 6 hours (h) post-APCP treatment demonstrated no morphological tissue changes, but a transient increased expression of OGG1 glycosylase that returned to control levels in 24 h. Transcriptome sequencing and quantitative real time PCR performed on different corneas revealed in the treated corneas many differentially expressed genes: namely, 256 and 304 genes showing expression changes greater than ± 2 folds in the absence and presence of NAC, respectively. At 6 h post-treatment, the most over-expressed gene categories suggested an active or enhanced cell functioning, with only a minority of genes specifically concerning oxidative DNA damage and repair showing slight over-expression values (<2 folds). Moreover, time-related expression analysis of eight genes up-regulated in the APCP-treated corneas overall demonstrated the return to control expression levels after 24 h. CONCLUSIONS: These findings of transient oxidative stress accompanied by wide-range transcriptome adjustments support the further development of APCP as an ocular disinfectant.

Disinfection of ocular cells and tissues by atmospheric-pressure cold plasma.

BACKGROUND: Low temperature plasmas have been proposed in medicine as agents for tissue disinfection and have received increasing attention due to the frequency of bacterial resistance to antibiotics. This study explored whether atmospheric-pressure cold plasma (APCP) generated by a new portable device that ionizes a flow of helium gas can inactivate ocular pathogens without causing significant tissue damage. METHODOLOGY/PRINCIPAL FINDINGS: We tested the APCP effects on cultured Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Candida albicans, Aspergillus fumigatus and Herpes simplex virus-1, ocular cells (conjunctival fibroblasts and keratocytes) and ex-vivo corneas. Exposure to APCP for 0.5 to 5 minutes significantly reduced microbial viability (colony-forming units) but not human cell viability (MTT assay, FACS and Tunel analysis) or the number of HSV-1 plaque-forming units. Increased levels of intracellular reactive oxygen species (ROS) in exposed microorganisms and cells were found using a FACS-activated 2',7'-dichlorofluorescein diacetate probe. Immunoassays demonstrated no induction of thymine dimers in cell cultures and corneal tissues. A transient increased expression of 8-OHdG, genes and proteins related to oxidative stress (OGG1, GPX, NFE2L2), was determined in ocular cells and corneas by HPLC, qRT-PCR and Western blot analysis. CONCLUSIONS: A short application of APCP appears to be an efficient and rapid ocular disinfectant for bacteria and fungi without significant damage on ocular cells and tissues, although the treatment of conjunctival fibroblasts and keratocytes caused a time-restricted generation of intracellular ROS and oxidative stress-related responses.

Extracellular ATP signaling during differentiation of C2C12 skeletal muscle cells: role in proliferation.

Evidence shows that extracellular ATP signals influence myogenesis, regeneration and physiology of skeletal muscle. Present work was aimed at characterizing the extracellular ATP signaling system of skeletal muscle C2C12 cells during differentiation. We show that mechanical and electrical stimulation produces substantial release of ATP from differentiated myotubes, but not from proliferating myoblasts. Extracellular ATP-hydrolyzing activity is low in myoblasts and high in myotubes, consistent with the increased expression of extracellular enzymes during differentiation. Stimulation of cells with extracellular nucleotides produces substantial Ca(2+) transients, whose amplitude and shape changed during differentiation. Consistently, C2C12 cells express several P2X and P2Y receptors, whose level changes along with maturation stages. Supplementation with either ATP or UTP stimulates proliferation of C2C12 myoblasts, whereas excessive doses were cytotoxic. The data indicate that skeletal muscle development is accompanied by major functional changes in extracellular ATP signaling.

Cutting edge issues in polymyositis.

Skeletal muscle is the target tissue of immunoflogistic processes in patients affected with idiopathic inflammatory myopathies (IIM). IIM are classified into three major forms: polymyositis (PM), dermatomyositis (DM), and inclusion body myositis. Recent data suggest that, in the major subsets of myositis, antigens in muscles drive a B-cell antigen-specific immune response. Moreover, some non-immunological mechanisms have been advocated. In this regard, an increased expression of Jo-1 and Mi-2 in muscle biopsies from PM and DM patients compared to normal muscle has been demonstrated; these candidate autoantigens in myositis are expressed at high levels in regenerating muscle cells rather than in mature myotubes. Myositis autoantigen upregulation has also been observed in neoplastic tissues, thus representing a potential link between cancer and autoimmunity in myositis. Myositis-specific autoantibodies (MSA) are disease markers and target intracellular proteins involved in key processes such as translocation and nuclear transcription. Myositis target antigens encompass aminoacyl-tRNA synthetases, the Mi-2 helicase/histone deacetylase protein complex, the signal recognition particle ribonucleoprotein, together with novel target antigens including p155/140, CADM-140, and SAE. Despite their high specificity for autoimmune myositis, MSA target non-muscle restricted proteins ubiquitary to all cell types, making the specific muscle involvement difficult to explain. Non-immunological mechanisms also seem to contribute to the pathogenesis of IIM; activation of endoplasmic reticulum stress response due to muscle regeneration and inflammation but independent to MHC-1 up-regulation has been recently reported in patients with myositis.

A transient antioxidant stress response accompanies the onset of disuse atrophy in human skeletal muscle.

It is presently unknown whether oxidative stress increases in disused skeletal muscle in humans. Markers of oxidative stress were investigated in biopsies from the vastus lateralis muscle, collected from healthy subjects before [time 0 (T0)], after 1 wk (T8), and after 5 wk (T35) of bed rest. An 18% decrease in fiber cross-sectional area was detected in T35 biopsies (P<0.05). Carbonylation of muscle proteins significantly increased about twofold at T35 (P<0.02) and correlated positively with the decrease in fiber cross-sectional area (P=0.04). Conversely, T8 biopsies showed a significant increase in protein levels of heme oxygenase-1 and glucose-regulated protein-75 (Grp75)/mitochondrial heat shock protein-70, two stress proteins involved in the antioxidant defense (P<0.05). Heme oxygenase-1 increase, which involved a larger proportion of slow fibers compared with T0, appeared blunted in T35 biopsies. Grp75 protein level increased threefold in T8 biopsies and localized especially in slow fibers (P<0.025), to decrease significantly in T35 biopsies (P<0.05). Percent change in Grp75 levels positively correlated with fiber cross-sectional area (P=0.01). Parallel investigations on rat soleus muscles, performed after 1-15 days of hindlimb suspension, showed that Grp75 protein levels significantly increased after 24 h of unloading (P = 0.02), i.e., before statistically significant evidence of muscle atrophy, to decrease thereafter in relation to the degree of muscle atrophy (P=0.03). Therefore, in humans as in rodents, disuse muscle atrophy is characterized by increased protein carbonylation and by the blunting of the antioxidant stress response evoked by disuse.

The T-tubule membrane ATP-operated P2X4 receptor influences contractility of skeletal muscle.

Evidence indicates that extracellular ATP may have relevant functions in skeletal muscle, even though the physiological role and distribution of specific signaling pathway elements are not well known. The present work shows that P2X4 receptor, an extracellular ATP-regulated cell membrane channel permeable to Ca2+, is expressed in several tissues of the rat, including skeletal muscle. A specific antibody detected a protein band of approximately 60 kDa. Immunofluorescence demonstrated that P2X4 has an intracellular localization, and confocal analysis revealed that the receptor colocalizes with the T-tubule membrane DHP receptor. Considering that the natural agonist of P2X4 is ATP, we explored if changes of extracellular ATP levels could occur in contracting skeletal muscle to regulate the channel. In vitro experiments showed that substantial ATP is released and rapidly hydrolyzed after electrical stimulation of rat muscle fibers. Results show that the presence of ATP-degrading enzymes (hexokinase/apyrase), inhibitors of P2X receptors or Ca2+-free conditions, all abolished the progressive twitch tension potentiation produced in soleus muscle by low-frequency (0.05 Hz) stimulation. These data reveal that ATP-mediated Ca2+ entry, most likely through P2X4 receptor, may play an important role in modulating the contractility of skeletal muscle.

Pregnancy, cytokines, and disease activity in systemic lupus erythematosus.

OBJECTIVE: To evaluate levels of selected cytokines and soluble receptors involved in the humoral immune response during pregnancy in systemic lupus erythematosus (SLE) patients. METHODS: Seventeen consecutive SLE patients and 8 matched healthy controls were prospectively studied during pregnancy. Sera were obtained within the last 3 months prior to pregnancy; at 9, 17, and 29 weeks of pregnancy; and at 1 month after delivery. Serum levels of interleukin-10 (IL-10), interleukin-6 (IL-6), and soluble tumor necrosis factor receptors p55 (sTNFR I) and p75 (sTNFR II) were evaluated. SLE activity was measured by the European Consensus Lupus Activity Measurement score modified for pregnancy. RESULTS: IL-10 serum levels were found to be higher (P <0.0001) in patients than in controls before conception, and still higher (P <0.0001) in SLE patients during gestation, without intertrimester changes. In SLE patients, IL-6 serum levels did not increase in the third trimester of pregnancy, as was observed in controls (P=0.011). No significant differences between SLE patients and controls were found in either sTNFR I or II levels or profiles before and during pregnancy. IL-10 and sTNFR I levels were significantly higher during pregnancy and postpartum in SLE patients with active disease (P=0.03 and P=0.01, respectively). CONCLUSION: The levels of some cytokines involved in the humoral immune response seem to be modified in the peripheral circulation of pregnant SLE patients. The most relevant modifications are the lower than expected increase of IL-6 in the third trimester of gestation and persistently high levels of IL-10 during pregnancy.