Ian C. Locke

Cytotoxicity of clove (Syzygium aromaticum) oil and its major components to human skin cells.

Abstract.  The essential oil extracted from clove (Syzygium aromaticum) is used as a topical application to relieve pain and promote healing in herbal medicine and also finds use in the fragrance and flavouring industries. Clove oil has two major components, eugenol and β‐caryophyllene, which constitute 78% and 13% of the oil, respectively. Clove oil and these components are generally recognized as ‘safe’, but the in‐vitro study here demonstrates cytotoxic properties of both the oil and eugenol, towards human fibroblasts and endothelial cells. Clove oil was found to be highly cytotoxic at concentrations as low as 0.03% (v/v) with up to 73% of this effect attributable to eugenol. β‐caryophyllene did not exhibit any cytotoxic activity, indicating that other cytotoxic components may also exist within the parent oil.

CYTOTOXICITY OF LAVENDER OIL AND ITS MAJOR COMPONENTS TO HUMAN SKIN CELLS

Abstract.  Lavender (Lavandula angustifolia) oil, chiefly composed of linalyl acetate (51%) and linalool (35%), is considered to be one of the mildest of known plant essential oils and has a history in wound healing. Concerns are building about the potential for irritant or allergenic skin reactions with the use of lavender oil. This study has demonstrated that lavender oil is cytotoxic to human skin cells in vitro (endothelial cells and fibroblasts) at a concentration of 0.25% (v/v) in all cell types tested (HMEC‐1, HNDF and 153BR). The major components of the oil, linalyl acetate and linalool, were also assayed under similar conditions for their cytotoxicity. The activity of linalool reflected that of the whole oil, indicating that linalool may be the active component of lavender oil. Linalyl acetate cytotoxicity was higher than that of the oil itself, suggesting suppression of its activity by an unknown factor in the oil. Membrane damage is proposed as the possible mechanism of action.

The corticotrophin-releasing factor-like peptide urocortin reverses key deficits in two rodent models of Parkinson's disease.

The potential neuroprotective action of the corticotrophin‐releasing factor‐related peptide urocortin (UCN) was investigated in the rat 6‐hydroxydopamine (6‐OHDA) and lipopolysaccharide (LPS) paradigms of Parkinsons disease. UCN (20 fmol) was either given at the same time as (T = 0) or 7 days after (T = +7) intracerebral 6‐OHDA or LPS injection. At 14 days after 6‐OHDA or LPS injection, circling behaviour was measured following apomorphine challenge. Circling was significantly lower in rats given UCN at either T = 0 or T = +7 compared with animals given 6‐OHDA or LPS and vehicle. Sham‐treated rats showed no circling. Consistent with these observations, striatal dopamine concentrations were markedly higher in 6‐OHDA/LPS + UCN rats vs. 6‐OHDA/LPS + vehicle groups. Additionally, l‐dihydroxyphenylalanine production by tyrosine hydroxylase was greatly reduced in the striata of 6‐OHDA/LPS + vehicle rats, whereas this was not the case in rats coadministered UCN. Finally, the numbers of tyrosine hydroxylase‐positive cells recorded in the substantia nigra of 6‐OHDA/LPS + vehicle‐treated animals were markedly lower than those of sham‐operated or 6‐OHDA/LPS + UCN rats. Critically, UCN was effective in reversing lesion‐induced deficits when given either at the same time as or 7 days after the neurotoxic insult. To our knowledge, this is the first time that such an effect has been demonstrated in vivo. The apparent ability of UCN to arrest the progression of or even reverse nigral lesions once established suggests that pharmacological manipulation of this system could have substantial therapeutic utility.

A comparison of the characteristics of circulating anti-myeloperoxidase autoantibodies in vasculitis with those in non-vasculitic conditions

Although circulating anti‐neutrophil cytoplasmic antibodies (ANCA) specific for myeloperoxidase (MPO) are strongly associated with the presence of vasculitis, they have been described in sera from patients with other conditions. High levels of anti‐MPO antibodies can also persist in sera from patients with vasculitis despite the achievement of clinical remission. One possible interpretation is that a potentially pathogenic subset of anti‐MPO antibodies exists, which is only present in patients with active vasculitis. We therefore compared the characteristics of anti‐MPO antibodies in sera from patients with active vasculitis (n = 18) with those present in remission (n = 9) and in a disease control group (n = 10) without clinical evidence of vasculitis. The class, subclass and ability of anti‐MPO antibodies from the three groups of patients to recognize three different conformational epitopes were analysed using ELISA‐based techniques. The expression of an idiotope, designated 9G4, was also examined. Epitope recognition by anti‐MPO antibodies from all patients tested was found to be similar. Sera from patients with active vasculitis showed an over‐representation of IgG4 subclass anti‐MPO antibodies and a more frequent presence of IgM class anti‐MPO antibodies. In disease controls, IgG1 anti‐MPO antibodies were predominant. In vitro, neutrophil activation by ANCA has been shown to be dependent on engagement of neutrophil FcγRIIa receptors following binding of these autoantibodies to surface‐expressed ANCA antigens. We found that active vasculitis may be associated with the presence of circulating anti‐MPO antibodies which do not significantly bind this receptor, suggesting that mechanisms other than those dependent on FcγRIIa binding should be explored. In addition, the expression of the 9G4 idiotope on anti‐MPO antibodies in 60% (12/18) of patients with active vasculitis and 20% (2/10) of disease control patients may indicate a common origin for anti‐MPO antibodies in different individuals.

In situ development of self-defensive antibacterial biomaterials: phenol-g-keratin-EC based bio-composites with characteristics for biomedical applications

Recently, the development of highly inspired biomaterials with multi-functional characteristics has gained considerable attention, especially in biomedical and other health-related areas of the modern world. It is well known that the lack of antibacterial potential has significantly limited biomaterials for many challenging applications such as infection-free wound healing and/or tissue engineering. In this perspective, a series of novel bio-composites with natural phenols as functional entities and keratin-EC as a base material were synthesised by laccase-assisted grafting. Subsequently, the resulting composites were removed from their respective casting surfaces and critically evaluated for their antibacterial and biocompatibility features. Information is also given on their degradation profile in soil. In situ synthesised phenol-g-keratin-EC bio-composites possess strong anti-bacterial activity against the Gram-positive and Gram-negative bacterial strains, i.e., B. subtilis NCTC 3610, S. aureus NCTC 6571, E. coli NTCT 10418 and P. aeruginosa NCTC 10662. More specifically, 10HBA-g-keratin-EC and 20T-g-keratin-EC composites were 100% resistant to colonisation by all of the aforementioned bacterial strains, whereas 15CA-g-keratin-EC and 15GA-g-keratin-EC showed almost negligible colonisation. Moreover, at various phenolic concentrations, it was clear that the newly synthesised composites remained cytocompatible with human keratinocyte-like HaCaT, as an obvious cell ingrowth tendency was observed during the neutral red dye uptake assay. From the degradation point of view, an increase in the degradation rate was recorded during the soil burial analyses. Our investigations could encourage greater utilisation of natural materials in the development of bio-composites with novel and sophisticated characteristics for potential applications.

Urocortin protects chondrocytes from NO-induced apoptosis: a future therapy for osteoarthritis?

Osteoarthritis (OA) is characterized by a loss of joint mobility and pain resulting from progressive destruction and loss of articular cartilage secondary to chondrocyte death and/ or senescence. Certain stimuli including nitric oxide (NO) and the pro-inflammatory cytokine tumor necrosis factor α (TNF-α have been implicated in this chondrocyte death and the subsequent accelerated damage to cartilage. In this study, we demonstrate that a corticotrophin releasing factor (CRF) family peptide, urocortin (Ucn), is produced by a human chondrocyte cell line, C-20/A4, and acts both as an endogenous survival signal and as a cytoprotective agent reducing the induction of apoptosis by NO but not TNF-α when added exogenously. Furthermore, treatment with the NO donor S-nitroso-N-acetyl-D-L-penicillamine upregulates chondrocyte Ucn expression, whereas treatment with TNF-α does not. The chondroprotective effects of Ucn are abolished by both specific ligand depletion (with an anti-Ucn antibody) and by CRF receptor blockade with the pan-CRFR antagonist α-helical CRH(9-41). CRFR expression was confirmed by reverse transcription-PCR with subsequent amplicon sequence analysis and demonstrates that C-20/A4 cells express both CRFR1 and CRFR2, specifically CRFR1α and CRFR2β. Protein expression of these receptors was confirmed by western blotting. The presence of both Ucn and its receptors in these cells, coupled with the induction of Ucn by NO, suggests the existence of an endogenous autocrine/paracrine chondroprotective mechanism against stimuli inducing chondrocyte apoptosis via the intrinsic/mitochondrial pathway.

Chondroprotective and anti-inflammatory role of melanocortin peptides in TNF-α activated human C-20/A4 chondrocytes

Melanocortin MC1 and MC3 receptors, mediate the anti‐inflammatory effects of melanocortin peptides. Targeting these receptors could therefore lead to development of novel anti‐inflammatory therapeutic agents. We investigated the expression of MC1 and MC3 receptors on chondrocytes and the role of α‐melanocyte‐stimulating hormone (α‐MSH) and the selective MC3 receptor agonist, [DTRP8]‐γ‐MSH, in modulating production of inflammatory cytokines, tissue‐destructive proteins and induction of apoptotic pathway(s) in the human chondrocytic C‐20/A4 cells.

Poly(3-hydroxybutyrate)-ethyl cellulose based bio-composites with novel characteristics for infection free wound healing application.

A series of bio-composites including poly3-hydroxybutyrate [P(3HB)] grafted ethyl cellulose (EC) stated as P(3HB)-EC were successfully synthesised. Furthermore, natural phenols e.g., p-4-hydroxybenzoic acid (HBA) and ferulic acid (FA) were grafted onto the newly developed P(3HB)-EC-based bio-composites under laccase-assisted environment without the use of additional initiators or crosslinking agents. The phenol grafted bio-composites were critically evaluated for their antibacterial and biocompatibility features as well as their degradability in soil. In particular, the results of the antibacterial evaluation for the newly developed bio-composites indicated that 20HBA-g-P(3HB)-EC and 15FA-g-P(3HB)-EC bio-composites exerted strong bactericidal and bacteriostatic activity against Gram(-)E. coli NTCT 10418 as compared to the Gram(+)B. subtilis NCTC 3610. This study shows further that at various phenolic concentrations the newly synthesised bio-composites remained cytocompatible with human keratinocyte-like HaCaT skin cells, as 100% cell viability was recorded, in vitro. As for the degradation, an increase in the degradation rate was recorded during the soil burial analyses over a period of 42 days. These findings suggest that the reported bio-composites have great potential for use in wound healing; covering the affected skin area which may favour tissue repair over shorter periods.

Development of bio-composites with novel characteristics: Evaluation of phenol-induced antibacterial, biocompatible and biodegradable behaviours

This paper describes a laccase-assisted grafting of gallic acid (GA) and thymol (T) as functional entities onto the previously developed P(3HB)-g-EC composite. GA-g-P(3HB)-g-EC and T-g-P(3HB)-g-EC bio-composites were prepared by laccase-assisted free radical-induced graft polymerisation of GA and T onto the P(3HB)-g-EC based composite using surface dipping and incorporation technique. The results of the antibacterial evaluation for the prepared composites indicated that 15GA-g-P(3HB)-g-EC, 15T-g-P(3HB)-g-EC and 20T-g-P(3HB)-g-EC composites possessed the strongest bacteriostatic and bactericidal activities against Gram-positive Bacillus subtilis NCTC 3610 and Staphylococcus aureus NCTC 6571 and Gram-negative Escherichia coli NTCT 10418 and Pseudomonas aeruginosa NCTC 10662 strains. In this study, we have also tested GA-g-P(3HB)-g-EC and T-g-P(3HB)-g-EC bio-composites for their ability to support and maintain multilineage differentiation of human keratinocyte-like (HaCaT) skin cells in-vitro. From the cytotoxicity results, the tested composites showed 100% viability and did not induce any adverse effect on a HaCaTs morphology. Finally, in soil burial evaluation, a progressive increase in the degradation rate of GA-g-P(3HB)-g-EC and T-g-P(3HB)-g-EC bio-composites was recorded with the passage of time up to 6 weeks. In summary, our current findings suggest that GA-g-P(3HB)-g-EC and T-g-P(3HB)-g-EC bio-composites are promising candidates for biomedical type applications such as skin regeneration, multiphasic tissue engineering and/or medical implants.

Melanocortin Peptide Therapy for the Treatment of Arthritic Pathologies

Arthritic pathologies are a major cause of morbidity within the western world, with rheumatoid arthritis affecting approximately 1% of adults. This review highlights the therapeutic potential of naturally occurring hormones and their peptides, in both arthritic models of disease and patients. The arthritides represent a group of closely related pathologies in which cytokines, joint destruction, and leukocytes play a causal role. Here we discuss the role of naturally occurring pro-opiomelanocortin (POMC)-derived melanocortin peptides (e.g., alpha melanocyte stimulating hormone [a-MSH]) and synthetic derivatives in these diseases. Melanocortins exhibit their biological efficacy by modulating proinflammatory cytokines and subsequent leukocyte extravasation. Their biological effects are mediated via seven transmembrane G-protein-coupled receptors, of which five have been cloned, identified, and termed MC1 to MC5. Adrenocorticotrophic hormone represents the parent molecule of the melanocortins; the first 13 amino acids of which (termed a-MSH) have been shown to be the most pharmacologically active region of the parent hormone. The melanocortin peptides have been shown to display potent anti-inflammatory effects in both animal models of disease and patients. The potential anti-inflammatory role for endogenous peptides in arthritic pathologies is in its infancy. The ability to inhibit leukocyte migration, release of cytokines, and induction of anti-inflammatory proteins appears to play an important role in affording protection in arthritic injury, and thus may lead to potential therapeutic targets.