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Keratinocytes stimulated by microbial organisms secrete not only a variety of cytokines, chemokines and growth factors, but also antimicrobial peptides such as beta-defensins (HBDs) such as HBD-2 and HBD-3. AV119, a patented blend of avocado sugar, triggers the up-regulation of HBD-2 in skin epithelia upon contact with AV119, but the signalling mechanisms involved are not completely understood. The purpose of this study was to determine if AV119 was able to induce also the expression of HBD-3 in human keratinocytes. In addition, the receptor and intracellular pathways involved in the AV119 up-regulation of HBD-2 and HBD-3 were investigated. Our results demonstrated that AV119 induces a significantly increase of the expression of HBD-3. In addition, the HBD-2 and HBD-3 AV119-induced gene expression and release are TLR-2 dependent. Finally, we demonstrated that AV119 induced ERK/MAPK phosphorylation in human keratinocytes, thus providing evidence that HBD-2 and HBD-3 secretion is through the same transductional pathway. The ability of AV119 to induce also HBD-3 may amplify its therapeutic potential against a broader spectrum of bacterial and yeast strains responsible for human skin disorders.

Keratinocytes play an active role in innate immune responses by secreting a variety of cytokines and chemokines. The release of critical proinflammatory cytokines, which are necessary to activate the immune response, is induced by the stimulation of Toll-like receptors (TLRs) by molecules present on pathogenic micro-organisms such as lipopolysaccharide (LPS). AV119, a patented blend of avocado sugars, induced the aggregation of Malassezia furfur, a dimorphic, lipid-dependent yeast that is part of the normal human cutaneous commensal flora and inhibited its penetration into the keratinocytes. In the present study, the anti-inflammatory effects of AV119 were investigated in LPS-induced inflammation of human keratinocytes. In particular, we analysed the modulation of the LPS-induced expression of proinflammatory cytokines and heat shock protein 70 (HSP70) by AV119 and the involvement of TLR-4. Our data show that AV119 is able to modulate significantly the proinflammatory response in human keratinocytes, blocking the NF-kB activation in human keratinocytes.

Skin keratinocytes constitute a protective mechanical barrier against invading microorganisms. Stimulated keratinocytes produce endogenous peptides such as the beta-defensins that have direct antimicrobial activity against a broad spectrum of pathogens, including most bacteria, certain fungi and enveloped viruses. In particular, human beta-defensin 2 (HBD-2) is virtually absent in normal skin and its expression in human keratinocytes requires stimulation by cytokines or bacteria. AV119, a patented avocado sugar, triggers the up-regulation of HBD-2, but the signalling mechanisms involved in this up-regulation in stimulated keratinocytes are not fully understood. In the present study, we examined the intracellular signalling pathways and nuclear responses in skin keratinocytes that contribute to HBD-2 gene expression upon stimulation with AV119. Our data provide information on signalling pathways in which the activation of protein tyrosine kinases (PTKs) and protein kinase C (PKC) takes place and leads to AP-1 and HBD-2 gene activation.

Introduction: The aims of this study were, first, to investigate the in vivo effects of treatment with avocado/soybean unsaponifiables on the development of osteoarthritic structural changes in the anterior cruciate ligament dog model and, second, to explore their mode of action., Methods: Osteoarthritis was induced by anterior cruciate ligament transection of the right knee in crossbred dogs. There were two treatment groups (n = 8 dogs/group), in which the animals received either placebo or avocado/soybean unsaponifiables (10 mg/kg per day), which were given orally for the entire duration of the study (8 weeks). We conducted macroscopic and histomorphological analyses of cartilage and subchondral bone of the femoral condyles and/or tibial plateaus. We also conducted immunohistochemical analyses in cartilage for the following antigens: inducible nitric oxide synthase, matrix metalloproteinase (MMP)-1, MMP-13, a disintegrin and metalloproteinase domain with thrombospondin motifs (ADAMTS)4 and ADAMTS5., Results: The size of macroscopic lesions on the tibial plateaus was decreased (P = 0.04) in dogs treated with the avocado/soybean unsaponifiables. Histologically, in these animals the severity of cartilage lesions on both tibial plateaus and femoral condyles, and the cellular infiltration in synovium were significantly decreased (P = 0.0002 and P = 0.04, respectively). Treatment with avocado/soybean unsaponifiables also reduced loss of subchondral bone volume (P < 0.05) and calcified cartilage thickness (P = 0.01) compared with placebo. Immunohistochemical analysis of cartilage revealed that avocado/soybean unsaponifiables significantly reduced the level of inducible nitric oxide synthase (P < 0.05) and MMP-13 (P = 0.01) in cartilage., Conclusions: This study demonstrates that treatment with avocado/soybean unsaponifiables can reduce the development of early osteoarthritic cartilage and subchondral bone lesions in the anterior cruciate ligament dog model of osteoarthritis. This effect appears to be mediated through the inhibition of inducible nitric oxide synthase and MMP-13, which are key mediators of the structural changes that take place in osteoarthritis.

AV119 is a patented blend of two sugars from avocado that can induce human beta-defensin-2 production by normal human keratinocytes. In this study, we analysed the effect of AV119 on growth and invasiveness of Malassezia furfur, a dimorphic, lipid-dependent yeast that is part of the normal human cutaneous commensal flora. The ability to modulate the expression of the proinflammatory and immunomodulatory cytokines in normal human keratinocytes was also investigated. Microbiological assay demonstrated that this sugar induced the aggregation of yeast cells and inhibited the invasiveness of M. furfur, without affecting its growth. Real-time PCR analysis demonstrated that AV119 was able to modulate the HBD-2 response in treated keratinocytes, reaching a maximum after 48-h treatment, and to induce the recovery of a satisfactory proinflammatory response in human keratinocytes. As AV119 can induce aggregation of yeast cells, thus inhibiting their penetration into the keratinocytes, the sugar could be used in the preparation of cosmetics or pharmacological drugs to inhibit colonization of the skin by pathogenic strains of M. furfur.

Objective: To determine the effects of avocado/soybean unsaponifiables (ASU) on osteoblast-induced dysregulation of chondrocyte metabolism., Methods: Human chondrocytes were isolated from osteoarthritis (OA) cartilage and cultured in alginate beads for 4 or 10 days in the absence or presence of osteoblasts isolated from nonsclerotic (NSC) or sclerotic (SC) zones of OA subchondral bone plate in monolayer. Before co-culture, osteoblasts were incubated or not with 10 microg/ml ASU for 72 hours. Aggrecan, type II collagen, matrix metalloproteinase-3 (MMP-3) and MMP-13, tissue inhibitor of metalloproteinase (TIMP-1), transforming growth factor-beta1 (TGF-beta1) and TGF-beta3, inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2) mRNA levels in chondrocytes were quantified by RT-PCR. Aggrecan, osteocalcin, TGF-beta1, interleukin 1beta (IL-1beta), and IL-6 production were assayed by immunoassays., Results: In co-culture, SC osteoblasts induced a significant inhibition of matrix protein production and a significant increase of MMP synthesis by chondrocytes. In contrast, SC osteoblasts did not modify TIMP-1, TGF-beta1 and TGF-beta3, iNOS, or COX-2 mRNA levels in chondrocytes. The pretreatment of SC osteoblasts with ASU fully prevented the inhibitory effects of SC osteoblasts on matrix component production, and even significantly increased type II collagen mRNA level over the control (chondrocytes alone) value. In contrast, pretreatment of SC osteoblasts with ASU did not significantly modify the expression of MMP, TIMP-1, TGF-beta1, TGF-beta3, iNOS, or COX-2 gene by chondrocytes., Conclusion: ASU prevent the osteoarthritic osteoblast-induced inhibition of matrix molecule production, suggesting that this compound may promote OA cartilage repair by acting on subchondral bone osteoblasts. This finding constitutes a new mechanism of action for this compound, known for its beneficial effects on cartilage.

Objective: To investigate the effects of avocado (A)/soybean (S) unsaponifiables on the metabolism of human osteoarthritic (OA) chondrocytes cultured in alginate beads over 12 days., Methods: Enzymatically isolated OA chondrocytes were cultured in alginate beads in a well defined culture medium for 12 days, in the presence or not of 10-10 M interleukin 1beta (IL-1beta). DNA content was measured using a fluorometric method. Production of aggrecan (AGG), stromelysin-1 (MMP-3), tissue inhibitor of metalloproteinases-1 (TIMP-1), macrophage inflammatory protein-1beta (MIP-1beta), IL-6, and IL-8 were assayed by specific enzyme amplified sensitivity immunoassays. Prostaglandin (PG) E2 was measured by a specific radioimmunoassay and nitrite by a spectrophotometric method based on the Griess reaction. A commercial avocado and soybean mixture of unsaponifiables (A1S2) and each component separately were tested in a range of 0.625 to 40.0 micro g/ml., Results: After 12 days' incubation, A1S2 increased AGG synthesis and accumulation in alginate beads in a dose and time dependent manner. A1S2 promoted the recovery of aggrecan synthesis after 3 days of IL-1beta treatment. A1S2 was a potent inhibitor of basal and IL-1beta stimulated MMP-3 production. The procedure also weakly reversed the inhibitory effect of IL-1beta on TIMP-1 production. A1S2 inhibited basal production of MIP-1beta, IL-6, IL-8, NO*, and PGE2 by OA chondrocytes and partially counteracted the stimulating effect of IL-1 on PGE2. Compared to avocado or soybean added separately, the mixture had a superior effect on NO* and IL-8 production., Conclusion: A1S2 stimulated aggrecan production and restored aggrecan production after IL-1beta treatment. In parallel, A1S2 decreased MMP-3 production and stimulated TIMP-1 production. These results suggest A1S2 could have structure-modifying effects in OA by inhibiting cartilage degradation and promoting cartilage repair.