A. Ferlazzo

Efficacy of treatment with glycosaminoglycans on experimental collagen-induced arthritis in rats

To evaluate the antioxidant activity of the glycosaminoglycans hyaluronic acid (HYA) and chondroitin-4-sulphate (C4S), we used a rat model of collagen-induced arthritis (CIA). Arthritis was induced in Lewis rats by multiple intradermal injections of 250 μl of emulsion containing bovine type II collagen in complete Freunds adjuvant at the base of the tail and into three to five other sites on the back. Rats were challenged again with the same antigen preparation 7 days later. Disease developed about 11 days after the second immunization. The effects of treatment in the rats were monitored by biochemical parameters and by macroscopic and histological evaluations in blood, synovial tissue and articular cartilage. Arthritis produced the following symptoms: severe periarticular erythema, edema and inflammation in the hindpaws; membrane peroxidation in the cartilage of the joints; endogenous antioxidant wasting; high tumour necrosis factor-α (TNF-α) plasma levels; and synovial neutrophil accumulation. Treatment with HYA and C4S, starting at the onset of arthritis for 10 days, limited the erosive action of the disease in the articular joints of knee and paw, reduced lipid peroxidation, restored the endogenous antioxidants reduced glutathione (GSH) and superoxide dismutase, decreased plasma TNF-α levels, and limited synovial neutrophil infiltration. These data confirm that erosive destruction of the joint cartilage in CIA is due at least in part to free radicals released by activated neutrophils and produced by other biochemical pathways. The beneficial effects obtained with the treatment suggest that HYA and C4S could be considered natural endogenous macromolecules to limit erosive damage in CIA or as a useful tool with which to study the involvement of free radicals in rheumatoid arthritis.

Evaluation of stress during transport

Domestic animals are transported for a variety of reasons including breeding, biomedical purposes, slaughter and, in the case of sporting horses, for competitions, pleasure activities or ceremonial proceedings. Studies to determine the amount of stress on farm animals during transport often have highly variable results and are difficult to interpret. The reaction of animals to stressors depends on the duration and intensity of the stressors, the animals previous experience, its physiological status and the immediate environmental restraints. Behavioural, haematological, haematochemical, physiological and neuro-hormonal (β-endorphin, ACTH, cortisol, iodothyronines) variables are discussed on the basis of handling, loading and transport procedures of animals.

Aromatic trap analysis of free radicals production in experimental collagen-induced arthritis in the rat: protective effect of glycosaminoglycans treatment.

Many findings demonstrated that Glycosaminoglycans (GAGs) and Proteoglycans (PGs) possess antioxidant activity. Collagen-induced arthritis (CIA) is an experimental animal model similar to human rheumatoid arthritis (RA) in which free radicals are involved. Sodium salicylate can be used as a chemical trap for hydroxyl radicals (OH ” ), the most damaging reactive oxygen species (ROS), yielding 2,5-dihydroxybenzoic acid), (2,5-DHBA) and 2,3-dihydroxybenzoic acid (2,3-DHBA). The measurement of these two acids in the plasma allows to indirectly assess the production of OH ” radicals. The aim of the study was to investigate the effect of hyaluronic acid (HYA) (30 mg/kg i.p.) or chondroitin-4-sulphate (C4S) (30 mg/kg i.p.), on free radical production in Lewis rats subjected to CIA. After the immunization with bovine collagen type II in complete Freunds adjuvant, rats developed an erosive hind paw arthritis, that produced high plasma OH ” levels assayed as 2,3-DHBA and 2,5-DHBA, primed lipid peroxidation, evaluated by analyzing conjugated dienes (CD) in the articular cartilage; decreased the concentration of endogenous vitamin E (VE) and catalase (CA) in the joint cartilage; enhanced macrophage inflammatory protein-2 (MIP-2) serum levels and increased elastase (ELA) evaluated as an index of activated leukocyte polymophonuclear (PMNs) accumulation in the articular joints. The administration of HYA and C4S starting at the onset of arthritis (day 11) for 20 days, limited inflammation and the clinical signs in the knee and paw, reduced OH ” production, decreased CD levels, partially restored the endogenous antioxidants VE and CA, reduced MIP-2 serum levels and limited PMNs infiltration. The results indicate that the GAGs HYA and C4S significantly reduce free radical production in CIA and could be used as a tool to investigate the role of antioxidants in RA.

Glycosaminoglycans reduce oxidative damage induced by copper (Cu+2), iron (Fe+2) and hydrogen peroxide (H2O2) in human fibroblast cultures.

Acid glycosaminoglycans (GAGs) antioxidant activity was assessed in a fibroblast culture system by evaluating reduction of oxidative system-induced damage.Three different methods to induce oxidative stress in human skin fibroblast cultures were used. In the first protocol cells were treated with CuSO4 plus ascorbate. In the second experiment fibroblasts were exposed to FeSO4 plus ascorbate. In the third system H2O2 was utilised.The exposition of fibroblasts to each one of the three oxidant systems caused inhibition of cell growth and cell death, increase of lipid peroxidation evaluated by the analysis of malondialdehyde (MDA), decrease of reduced glutathione (GSH) and superoxide dismutase (SOD) levels, and rise of lactate dehydrogenase activity (LDH).The treatment with commercial GAGs at different doses showed beneficial effects in all oxidative models. Hyaluronic acid (HA) and chondroitin-4-sulphate (C4S) exhibited the highest protection. However, the cells exposed to CuSO4 plus ascorbate and FeSO4 plus ascorbate were better protected by GAGs compared to those exposed to H2O2.These outcomes confirm the antioxidant properties of GAGs and further support the hypothesis that these molecules may function as metal chelators. Published in 2004.

Reduction of DNA Fragmentation and Hydroxyl Radical Production by Hyaluronic Acid and Chondroitin-4-sulphate in Iron Plus Ascorbate-induced Oxidative Stress in Fibroblast Cultures

Glycosaminoglycans (GAGs), components of extracellular matrix, are thought to play important roles in cell proliferation and differentiation in the repair process of injured tissue. Oxidative stress is one of the most frequent causes of tissue and cell injury and the consequent lipid peroxidation is the main manifestation of free radical damage. It has been found to play an important role in the evolution of cell death. Since several reports have shown that hyaluronic acid (HYA) and chondroitin-4-sulphate (C4S) are able to inhibit lipid peroxidation during oxidative stress, We investigated the antioxidant capacity of these GAGs in reducing oxidative damage in fibroblast cultures. Free radicals production was induced by the oxidizing system employing iron (Fe2+) plus ascorbate. We evaluated cell death, membrane lipid peroxidation, DNA damage, protein oxidation, hydroxyl radical (OH•) generation and endogenous antioxidant depletion in human skin fibroblast cultures. The exposition of fibroblasts to FeSO4 and ascorbate caused inhibition of cell growth and cell death, increased OH• production determined by the aromatic trap method; furthermore it caused DNA strand breaks and protein oxidation as shown by the DNA fragments analysis and protein carbonyl content, respectively. Moreover, it enhanced lipid peroxidation evaluated by the analysis of conjugated dienes (CD) and decreased antioxidant defenses assayed by means of measurement of superoxide dismutase (SOD) and catalase (CAT) activities. When fibroblasts were treated with two different doses of HYA or C4S a protective effect, following oxidative stress induction, was shown. In fact these GAGs were able to limit cell death, reduced DNA fragmentation and protein oxidation, decreased OH• generation, inhibited lipid peroxidation and improved antioxidant defenses. Our results confirm the antioxidant activity of HYA and C4S and this could represent a useful step in the understanding of the exact role played by GAGs in living organisms.

Improved high-performance liquid chromatographic method to estimate aminosugars and its application to glycosaminoglycan determination in plasma and serum

An improved isocratic high-performance liquid chromatography (HPLC) method for the analysis of L-(-)-fucose. D-(+)-galactosamine, D-(+)-glucosamine, D-(+)-galactose, obtained by hydrolysis of glycosaminoglycans (GAGs) and D-(+)-glucose and D-(+)-mannose is described. The presence in circulation of GAGs, acid polysaccharide sequences of alternate monosaccharide units, aminosugar and uronic acid (galactose in keratan sulfate), has been measured in terms of their sugar components. To evaluate concentration of these circulating sugars we considered blood samples obtained from healthy humans. Plasma or serum was filtered through weak anion-exchange Ecteola-cellulose either untreated or after mild alkaline treatment. GAGs adhering to resin were recovered by salt elution, and desalted on Bio-Gel P-2 resin. GAG fractionation by charge was carried out on a strong anion exchanger. GAG composition was evaluated in terms of galactose and aminosugars, measured in HPLC by the proposed procedure using anion-exchange resin and pulsed amperometric detection. The mobile phase consisted of 0.02 M NaOH and elution was carried out at flow-rate of 1.0 ml/min. The amperometric detector was set as follows: t1 (0.5 s), E1 (+0.1 V); t2 (0.09 s), E2 (+0.6 V); t3 (0.05 s), E3 (-0.6 V). The analysis required 14 min. Calibration standard curves for the six analytes were linear from 0.25 to 40 microM. RSD values for intra- and inter-day variabilities were < or = 5.3% at concentrations between 0.25 and 40 microM. Accuracy, expressed as percentage error, ranged from - 16 to 14%. The method was specific and sensitive with quantitation limits of 1 pmol for L-(-)-fucose, D-galactosamine and D-glucosamine, 3 pmol for D-(+)-galactose and D-(+)-glucose and 5 pmol for D-(+)-mannose. The results of the assay showed higher GAG concentrations in serum than in plasma.

Characteristics of the interactions between acid glycosaminoglycans and proteins in normal human plasma as revealed by the behaviour of the protein-polysaccharide complexes in ultrafiltration and Chromatographie procedures

Acid glycosaminoglycans were isolated from normal human plasma: (a) following fractionation of plasma (with protease inhibitors) on Sephadex G-200; (b) by ultrafiltration through membranes with retention of molecules above 50 kDa, with and without previous addition of NaCl, Triton X-100, urea, or guanidine HCl; (c) by filtering on Ecteola-cellulose either untreated plasma or after treatment with NaCl, urea, Triton X-100, papain or NaOH. More than 95% of plasma glycosaminoglycans interact with plasma proteins to give complexes that exhibit reproducible behaviour on Sephadex G-200 and are retained by ultrafiltration membranes, which the 12-20 kDa polysaccharide chains do filter. High charge plasma glycosaminoglycans show ionic interactions with proteins, while low charge glycosaminoglycan interactions are resistant to Ecteola charged groups, to 0.5% Triton and 4 M urea, while not to 4 M guanidine HC1. Some glycosaminoglycan-protein complexes appear resistant to proteolysis, suggesting that they may originate from lymphocytes. The simple method utilized for plasma GAG measurement may represent an useful tool in clinical practice.

Effects of competition experience and transportation on the adrenocortical and thyroid responses of horses

To evaluate whether the amount of experience of sport horses and the stress of transport affected their adrenocortical and thyroid responses, the plasma concentrations of total cortisol and total and free iodothyronine of 63 horses were studied before and after show jumping competitions. There were 14 trained inexperienced jumpers (group 1), 20 trained experienced jumpers (group 2), 10 trained inexperienced jumpers that had been transported just before the competition (group 3) and 19 trained experienced jumpers that had been transported just before the competition (group 4). The concentrations were measured under basal conditions and five and 30 minutes after the competition. There were significant increases relative to the basal values in the total cortisol concentrations of all four groups of horses at five and 30 minutes (P<0·001), but there were no significant differences between the groups. In contrast, there were no significant changes in the concentrations of triiodothyronine, thyroxine and free thyroxine after the competition and there were no significant differences between the groups. However, the horses in group 2 had significantly lower basal concentrations of free triiodothyronine than the horses in groups 1, 3 and 4 and the difference was maintained at five and 30 minutes after the competition.

TNF-α, IFN-γ, and IL−1β modulate hyaluronan synthase expression in human skin fibroblasts: Synergistic effect by concomital treatment with FeSO4 plus ascorbate

Several reports have shown that a number of cytokines such as tumor necrosis-α (TNF-α), interferon-γ (IFN-γ), and interleukin-β (IL-1β) are capable to induce hyaluronan sinthases (HASs) mRNA expression in different cell culture types. The obvious consequence of this stimulation is a marked increment in hyaluronan (HA) production. It has been also reported that oxidative stress, by itself, may increase HA levels. The aim of this study was to evaluate how TNF-α, IFN-γ,IL−1β, and exposition to oxidative stress may modulate HAS activities in normal human skin fibroblasts. Moreover, the effects on HAS mRNA expression of the concomitant treatment with cytokines and oxidants, and the HA concentrations after treatments, were studied. TNF-α, IFN-γ, and IL-1β were added to normal or/and exposed to FeSO4 plus ascorbate fibroblast cultures and HAS1, HAS2 and HAS3 mRNA content, by PCR-real time, was assayed 3,h later. HA levels were also evaluated after 24,h incubation. The treatment of fibroblasts with cytokines up-regulated HASs gene expression and increased HA production. IL-1β induced HAS mRNA expression and HA production more efficiently than TNF-α and IFN-γ. The exposition of the fibroblasts with the oxidant system markedly increased HAS activities while slightly HA production. The concomitant treatment of cells with the cytokines and the oxidant was able to further enhance, in a dose dependent way, with synergistic effect on HAS mRNA expression. On the contrary HA levels resulted unaffected by the concomitant treatment, and resemble those obtained with the exposure to FeSO4 plus ascorbate only. This lack in HA production could be due to the deleterious action of free radicals on the HA synthesis.

Effect of long-distance road transport on thyroid and adrenal function and haematocrit values in Limousin cattle: influence of body weight decrease.

The purpose of this study was to evaluate the effect of long-distance road transport as a relevant stressor on total and free iodothyronines, cortisol levels and haematocrit values in 10 male Limousin cattle. Serum T3,T4,fT3,fT4 and cortisol concentrations were analysed by immunoenzymatic assays. Serum cortisol levels and haematocrit modifications were also evaluated on the basis of percentage body weight decrease. The results showed a general increase of total and free iodothyronines and cortisol levels after short-and long-distance road transport and a decrease 15 days after transport, as compared to basal values. Significant positive correlations between T3 and T4, between T3 and fT3, and between T4 and fT4 were found. These results suggest that transport stress induces an increase in the activity of thyroid and adrenal function in Limousin cattle that is evident after even a short-distance road transport and continues to increase after long-distance transport.