The n-acetyl phenylalanine glucosamine derivative attenuates the inflammatory/catabolic environment in a chondrocyte-synoviocyte co-culture system

Osteoarthritis (OA), the most prevalent degenerative joint disease, still lacks a true disease-modifying therapy. The involvement of the NF-κB pathway and its upstream activating kinases in OA pathogenesis has been recognized for many years. The ability of the N-acetyl phenylalanine glucosamine derivative (NAPA) to increase anabolism and reduce catabolism via inhibition of IKKα kinase has been previously observed in vitro and in vivo. The present study aims to confirm the chondroprotective effects of NAPA in an in vitro model of joint OA established with primary cells, respecting both the crosstalk between chondrocytes and synoviocytes and their phenotypes. This model satisfactorily reproduces some features of the previously investigated DMM model, such as the prominent induction of ADAMTS-5 upon inflammatory stimulation. Both gene and protein expression analysis indicated the ability of NAPA to counteract key cartilage catabolic enzymes (ADAMTS-5) and effectors (MCP-1). Molecular analysis showed the ability of NAPA to reduce IKKα nuclear translocation and H3Ser10 phosphorylation, thus inhibiting IKKα transactivation of NF-κB signalling, a pivotal step in the NF-κB-dependent gene expression of some of its targets. In conclusion, our data confirm that NAPA could truly act as a disease-modifying drug in OA

Hyaluronic acid reduces bacterial fouling and promotes fibroblasts’ adhesion onto chitosan 2D-wound dressings

Wound healing is a dynamic process that can be seriously delayed by many factors including infectious complications. The development of dressings with intrinsic wound healing activity and/or releasing bioactive compounds may help with addressing such an issue. In this study, hyaluronic acid (HA) at different percentages (1–35%) was used to modify chitosan (CS) biological and physico-chemical properties in order to obtain 2D-matrices able to promote healing and protect from infection. HA incorporation in the CS matrix decreased film transparency and homogeneity, but improved film water uptake and surface wettability. The water vapor transmission rate (WVTR) increased up to a 5% HA content, where it reached the highest value (672 g/m2 day), and decreased for higher HA contents. At all of the tested HA concentrations, HA affected mechanical properties providing matrices more flexible than pure CS with benefit for wound care. Pure CS films permitted S. epidermidis adhesion and biofilm formation. That was not true for CS/HA matrices, where HA at concentrations equal to or greater than 5% was able to avoid S. epidermidis adhesion. Fibroblasts adhesion also took benefit from the HA presence in the film, especially at 5% content, where the best adhesion and proliferation was found

Nanostructured tic layer is highly suitable surface for adhesion, proliferation and spreading of cells

Cell culture is usually performed in 2D polymer surfaces; however, several studies are conducted with the aim to screen functional coating molecules to find substrates more suitable for cell adhesion and proliferation. The aim of this manuscript is to compare the cell adhesion and cytoskeleton organization of different cell types on different surfaces. Human primary fibroblasts, chondrocytes and osteoblasts isolated from patients undergoing surgery were seeded on polystyrene, poly-d-lysine-coated glass and titanium carbide slides and left to grow for several days. Then their cytoskeleton was analyzed, both by staining cells with phalloidin, which highlights actin fibers, and using Atomic Force Microscopy. We also monitored the production of Fibroblast Growth Factor-2, Bone Morphogenetic Protein-2 and Osteocalcin, using ELISA, and we highlighted production of Collagen type I in fibroblasts and osteoblasts and Collagen type II in chondrocytes by immunofluorescences. Fibroblasts, chondrocytes and osteoblasts showed both an improved proliferative activity and a good adhesion ability when cultured on titanium carbide slides, compared to polystyrene and poly-d-lysine-coated glass. In conclusion, we propose titanium carbide as a suitable surface to cultivate cells such as fibroblasts, chondrocytes and osteoblasts, allowing the preservation of their differentiated state and good adhesion properties

PLA and PBAT-based electrospun fibers functionalized with antibacterial bio-based polymers

Antimicrobial fibers based on biodegradable polymers, poly(lactic acid) (PLA), and poly(butylene adipate-co-terephthalate) (PBAT) are prepared by electrospinning. For this purpose, a biodegradable/bio-based polyitaconate containing azoles groups (PTTI) is incorporated at 10 wt.% into the electrospinning formulations. The resulting fibers functionalized with azole moieties are uniform and free of beads. Then, the accessible azole groups are subjected to N-alkylation, treatment that provides cationic azolium groups with antibacterial activity at the surface of fibers. The positive charge density, roughness, and wettability of the cationic fibers are evaluated and compared with flat films. It is confirmed that these parameters exert an important effect on the antimicrobial properties, as well as the length of the alkylating agent and the hydrophobicity of the matrix. The quaternized PLA/PTTI fibers exhibit the highest efficiency against the tested bacteria, yielding a 4-Log reduction against S. aureus and 1.7-Log against MRSA. Then, biocompatibility and bioactivity of the fibers are evaluated in terms of adhesion, morphology and viability of fibroblasts. The results show no cytotoxic effect of the samples, however, a cytostatic effect is appreciated, which is ascribed to the strong electrostatic interactions between the positive charge at the fiber surface and the negative charge of the cell membranes

Is a phenylalanine-glucosamine derivative able to affect the main responsible for IκB protein phosphorylation?

Nuclear factor κB (NFkB) is an ubiquitous transcription factor well known for its role in the innate immune response. In fact, it is involved as activator of inflammatory mediators such as cytokines. It consists of subunit dimers including p65, RelB, c-Rel, p50 and p52. Under normal conditions, they are bound in the cytosol by an inhibitory protein known as inhibitor of NFkB (IkB). The responsible for IkB phosphorylation is IkB kinase (IKK) complex, consisting of two catalytic subunits (IKKα and IKKβ) and a regulatory one. When activated, IKK phosphorylates IkB, thus tagging it for proteasomal degradation and freeing the NFkB subunit dimers which translocate to the nucleus and bind to specific DNA sequences within the promoter region of vast array of genes. Previous in vivo and in vitro studies in our laboratory demonstrate that Glucosamine (GlcN) and its N-acetyl phenylalanine derivative (NAPA) are able to counteract effects induced by inflammatory cytokines, particularly modulating some genes, under the control of NFkB, upregulated by TNF-α. So, the aim of this study is to better understand whether these two molecules may affect the activity of IKKα and IKKβ through some specific interactions. HTB-94, treated respectively with TNF-α and single molecules, are grown, lysated, incubated with anti-IKKα antibody and eventually treated with magnetic beads. This assay is performed to immunoprecipitate the activated IKK complex. Subsequently, an in vitro kinase assay is performed using a recombinant protein as substrate while in presence and absence of both molecules, to analyze the immunoprecipitated complex (IP-IKK). An immunocytochemistry is performed to visualize if IKKα re-localization is affected by GlcN and NAPA. Owing to immunoprecipitated complex formation, molecules can directly interact with kinases because they don’t need to cross cell membrane. In presence of GlcN, IP-IKK can phosphorylate the recombinant substrate, whileas in presence of NAPA 0.5mM the phosphorylation is inhibited. Further studies are performed evaluating IKK inhibition activity by GlcN and NAPA on recombinant protein using recombinant IKKα and IKKβ molecules. As previously observed, NAPA strongly inhibits IKKα activity on itself and on the recombinant substrate, but no effect is observed on IKKβ. On the contrary, GlcN cannot inhibit either IKKα or IKKβ at any concentrations used. In immunocytochemistry IKKα is mainly cytoplasmic in untreated cells, but it accumulates in nucleus under TNF-α stimulus. Pre-treatment with both molecules shows a mainly cytoplasmic IKKα localization. In conclusion, it is observed that both molecules affect IKKα localization, but only NAPA is an effective IkB phosphorylation inhibitor explaining how both molecules modulate expression level gene under NFkB control. Scotto d'Abusco A; Politi A; Giordano C; Scandurra R¸ Arthritis Research & Therapy 2010; doi: 10.1186/ar2920

Activity and role of BFT, an enterotoxin produced by Bacteroides fragilis

[No abstract available

Glucosamine and its N-acetylphenyl-alanine derivative own regulate metalloprotease production in chondrocytes by affecting MAP kinase phosphrylation.

Glucosamine (GlcN), a symptom-control drug used in the treatment of osteoarthritis (OA), has been proved effective in slowing OA progression (Reginster et al., 2001). OA is characterized by a progressive degradation of cartilage due to an imbalance between synthesis and degradation of the extracellular matrix component. Production of proinflammatory cytokines stimulates chondrocytes to secrete an excess of proteases. We report the effects of GlcN and its N-acetyl phenylalanine derivative (NAPA) on metalloprotease (MMP) production in immortalized chondrocyte cell line, LBPVA55, stimulated with proinflammatory cytokine interleukin (IL)-1 β. We analyzed, by quantitative-real time-PCR, MMP-1, -2, -3, -8, -9, 13 mRNA expression level in cells treated with 2.5 and10 mM GlcN or with 2.5 and 10 mM NAPA, after stimulation with 10 ng/ml IL-1β. We found MMP-1, -3, and -13 upregulated by IL-1β stimulation and brought back by GlcN and NAPA. Same results were obtained when MMP-1, -3, and -13 protein levels were analyzed by ELISA. Mengshol et al. (2000), previously demonstrated that IL-1β induction of MMPs requires MAP kinases activation. To verify if GlcN and NAPA could affect p38, JNK, and ERK MAP kinases activity, we analyzed the whole cell extracts by Western blot analysis, using antiphospho-antibodies. We found phospho-p38, phospho-JNK, and phospho-ERK increased by IL-1β;phospho-JNK and phospho p38, but not phospho-ERK, were downregulated by GlcN and NAPA. Finally, we discovered that GlcN and NAPA, by affecting p38 and JNK kinases whose phosphorylation is requested to activate AP1 transcription factor, downregulated MMP-1, -3, and -13 production