Evaluation of the antioxidant properties of carexanes in AGS cells transfected with the Helicobacter pylori's protein HspB.

Naturally derived compounds represent a potential source of pharmacologically active drugs able to contrast different diseases, including gastric cancer, a multifactorial disease, in which the important role played by H. pylori infection has been demonstrated. Carexanes, stilbene derivatives, isolated from plants of the Carex distachya Desf., are unusual secondary metabolites with a tetracyclic skeleton arising from a cyclization of prenylstilbenoid precursors. In this study we firstly showed the ability of three purified carexanes CxB, CxG, and CxI to enhance the antioxidant response of AGS cells and to contrast the effect of the H. pylori's protein HspB. Among them CxI was the molecule that best modified the expression of genes involved in the antioxidant response. In particular, CxI was able to reduce Keap-1 gene expression and induce NQO1 gene expression, both at 4 and 24 h in AGS cells, as showed by real time PCR. Nrf2 induction was evident only at 24 h. Interestingly, the effect of CxI was stronger in HspB-transfected AGS cells, where Keap-1 gene expression was nearly abrogated. Finally, we demonstrated that CxI was able to reduce also COX-2 gene expression in HspB-transfected AGS cells, compared with untreated HspB-transfected cells, both at 4 and 24 h. This study first report that carexanes might represent candidate molecules able to contrast the deleterious effect of HspB protein but also to reduce the inflammatory process induced by H. pylori infection.

Correlation between genetic variability and virulence factors in clinical strains of Malassezia pachydermatis of animal origin.

Malassezia pachydermatis is a yeast belonging to the microbiota of the skin and mucous membranes of dog and cat, but it can also act as pathogen, causing dermatitis. The aim of this work was to evaluate the genetic variability of M. pachydermatis strains isolated from symptomatic dogs and cats and determine a correlation between genotype and phenotype. For this purpose eleven strains of M. pachydermatis were molecularly classified by nested-polymerase chain reaction (nested-PCR) based on ITS-1 and ITS-2 regions, specific for fungal rRNA genes. Furthermore, random amplification of polymorphic DNA (RAPD) was applied for genetic typing of M. pachydermatis isolates identifying four different genotypes. Strains belonging to genotype 1 produced the highest amount of biofilm and phospholipase activity. The inflammatory response induced by M. pachydermatis strains in immortalized human keratinocytes (HaCat cells) was significantly different when we compared the results obtained from each strain. In particular, HaCat cells infected with the strains belonging to genotypes 1 and 2 triggered the highest levels of increase in TLR-2, IL-1ß, IL-6, IL-8, COX-2 and MMP-9 expression. By contrast, cells infected with the strains of genotype 3 and those of genotype 4 did not significantly induce TLR-2 and cytokines. The results obtained might suggest a possible association between genotype and virulence factors expressed by M. pachydermatis strains. This highlights the need for a more accurate identification of the yeast to improve the therapeutic approach and to monitor the onset of human infections caused by this emergent zoonotic pathogen.

In vitro growth versus inhibition of growth of Malassezia pachydermatis in the presence of the antibacterial drug gentamicin.

Malassezia pachydermatis is part of the normal cutaneous microbiota of most warm-blooded vertebrates and is associated with otitis externa and seborrhoeic dermatitis in dogs and cats. In this study, we evaluated the growth capacity of nine M. pachydermatis strains on Sabouraud medium in the presence of a high concentration of gentamicin. Strains of M. pachydermatis cultured on Sabouraud medium in the presence of 50 and 100 µg gentamicin ml(-1) displayed different growth patterns such as growth or lack of growth. We hypothesized that this difference in growth of M. pachydermatis strains was correlated with the different genotypes of the strains. Random amplification of polymorphic DNA (RAPD) was applied for genetic typing of M. pachydermatis isolates, derived from the external ears of house pet cats suffering from otitis externa. The M. pachydermatis strains were cultured on commercial or home-made Sabouraud medium supplemented or not with gentamicin. RAPD analysis demonstrated a genetic heterogeneity between each strain. In particular, five out of nine strains tested were able to form colonies in the presence of gentamicin. However, a correlation between M. pachydermatis genotype and growth capacity in the presence of gentamicin was not widely demonstrated.

Innate immune response in human keratinocytes infected by a feline isolate of Malassezia pachydermatis.

Malassezia pachydermatis is a normal inhabitant of canine and feline skin that can spread to other pets. The outer layer or epidermis is made up primarily of keratinocytes, which are capable of releasing various factors and expressing receptors that are significantly involved in the immune regulation. Little is known about the mechanism by which M. pachydermatis overcomes the natural barrier of the skin. The aim of this study was to evaluate the direct in vitro interaction between human keratinocytes and a clinical strain of live M. pachydermatis isolated as a pure culture from an otitic cat. Human keratinocytes (HaCat) were infected with M. pachydermatis to analyse the modulation of the innate immune response. Gene expression was analysed by real-time PCR. We demonstrated that M. pachydermatis invaded HaCat cells and modulated the expression of TLR2 after 24h infection, while HBD-2, IL-1ß TNF-α, IL-6 and IL-8 were modulated both at 24 and 48 h. Thus, our results demonstrated that M. pachydermatis is able to stimulate the innate immune response in infected human keratinocytes indicating a possible role of this yeast as a human opportunistic pathogen.