Modulation of cutaneous inflammation induced by ticks in contrasting phenotypes of infestation in bovines

Tick saliva contains molecules that are inoculated at the site of attachment on their hosts in order to modulate local immune responses and facilitate a successful blood meal. Bovines express heritable, contrasting phenotypes of infestations with the cattle tick, Rhipicephalus (Boophilus) microplus: breeds of Bos taurus indicus are significantly more resistant than those of Bos taurus taurus. Tick saliva may contain molecules that interfere with adhesion of leukocytes to endothelium and resistant hosts may mount an inflammatory profile that is more efficient to hamper the tick's blood meal. We show in vitro that adhesion of peripheral blood mononuclear cells to monolayers of cytokine-activated bovine umbilical endothelial cells was significantly inhibited by tick saliva. The inflammatory response to bites of adults of R. microplus mounted by genetically resistant and susceptible bovine hosts managed in the same pasture was investigated in vivo. The inflammatory infiltrates and levels of message coding for adhesion molecules were measured in biopsies of tick-bitten and control skin taken when animals of both breeds were exposed to low and high tick infestations. Histological studies reveal that cutaneous reactions of resistant hosts to bites of adult ticks contained significantly more basophils and eosinophils compared with reactions of the susceptible breed. Expression of the adhesion molecules – intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and P-selectin – was higher in adult-infested skin of susceptible hosts undergoing low infestations compared to resistant hosts; when host was exposed to high infestations expression of these adhesion molecules was down-regulated in both phenotypes of infestations. Expression of leukocyte adhesion glycoprotein-1 (LFA-1) was higher in skin from susceptible hosts undergoing low or high infestations compared to resistant hosts. Conversely, higher levels of E-selectin, which promotes adhesion of memory T cells, were expressed in skin of resistant animals. This finding may explain the resistant host's ability to mount more rapid and efficient secondary responses that limit hematophagy and infestations. The expression profiles observed for adhesion molecules indicate that there are differences in the kinetics of the inflammatory reactions mounted by resistant and susceptible hosts and the balance between tick and host is affected by the number of tick bites a host receives. We show that the contrasting phenotypes of infestations seen in bovines infested with R. microplus are correlated with differences in the cellular and molecular composition of inflammatory infiltrates elicited by bites with adult ticks.     

Induction of inflammatory host immune responses by organisms belonging to the genera Chlamydia/Chlamydophila

Chlamydia/Chlamydophila are a family of intracellular gram-negative bacteria that infect their hosts primarily via mucosal epithelia. Chronic disease associated with bacterial persistence, inflammation and tissue damage are common sequelae of infection with these organisms. Human epithelial cell lines respond to infection by releasing pro-inflammatory cytokines and chemokines such as interleukin (IL)-6 and IL-8, and upregulating the expression of mRNA encoding Iκ-B, the endogenous inhibitor of NF-κB. However, Iκ-B is not upregulated in response to bacterial lipopolysaccharide (LPS). The failure of epithelial cells to respond to LPS is associated with the absence of surface expression of CD14. Identification of the components of Chlamydia/Chlamydophila that can induce pro-inflammatory mediators coupled with the mechanisms by which epithelial cells detect infection and respond accordingly will advance the development of preventative strategies.     

The effect of methyl sulphonyl methane supplementation on biomarkers of oxidative stress in sport horses following jumping exercise

Background

Exercise induces changes in several organs and tissues, and this process might be due to oxidative damage caused by free radicals and inflammatory mediators. Methyl Sulphonyl Methane, better known as MSM, is a naturally occurring sulphur compound with well-known antioxidant properties. On the other hand, Vitamin C is important in limiting free radical damage in the aqueous phase of the cell, and cellular vitamin C status may be linked to the mechanisms involved in quenching cellular reactive oxygen species. The aim of this study was to determine if supplementation with MSM and vitamin C could alleviate exercise-induced oxidative stress in horses undergoing jumping competition.

Methods

Twenty four jumping horses involved in competition were used. Horses were given the following three treatment diets: control (without supplementation), MSM 8 mg/kg, and combined supplements (MSM 8 mg/kg + Vit-C 5 mg/kg). EDTA blood samples were collected before exercise, upon arrived to the schooling area (control), and each week after last show. Nitric oxide, carbon monoxide, lipid hydroperoxides and the antioxidant enzymes, glutathione peroxidase, glutathione transferase and glutathione reductase, plasma levels were determined.

Results

Competition induced a significant increase in lipid peroxidation, nitric oxide and carbon monoxide. By contrary, reduced glutathione as well as antioxidant enzyme activities, were decreased. MSM administration significantly ameliorated all these exercise-related changes, and this effect was potentiated by Vit C reaching values in some of the parameters similar to those found before competition.

Conclusion

These results suggest that jumping exercise could induce harmful effects on horses, probably due to an increase in oxidative damage and proinflammatory molecules. In addition, we have demonstrated that MSM could exert some protective effect on oxidative and inflammatory exercise-induced injury.     

Chrysoporthe puriensis causing canker and mortality in Pleroma mutabile in the Atlantic Forest,Brazil

Fungi of the Cryphonectriaceae family are globally known to be tree pathogens. In Brazil, several species of Chrysoporthe have been found causing stem and branch cankers in Pleroma (= Tibouchina) spp. Recently, Chrysoporthe puriensis was described as a new species causing stem and branch cankers in Pleroma granulosum, Pleroma candolleanum, and Pleroma heteromallum, all native species of the Melastomataceae family. During an investigation to collect isolates of the Cryphonectriaceae family in an important Brazilian biome, the Atlantic Forest, in Serra do Mar, structures typical of Chrysoporthe species were found in a different host, Pleroma mutabile. Fruiting bodies present in the bark of these trees were collected and isolated. The isolates were submitted on morphological characterization and phylogenetic analyses of the internal transcribed spacer (ITS) and β-tubulin gene regions using Maximum likelihood, Bayesian inference and Maximum parsimony methods. The isolates collected, for this study, were identified as C. puriensis. Pathogenicity tests on seedlings of Pleroma species and Eucalyptus clones revealed C. puriensis can infect and cause canker in these plant species as mortality. The results demonstrate the importance of delimiting the C. puriensis collection range to track its dissemination in other hosts. No host specificity was observed in the inoculation tests, suggesting this is an important finding, the pathogen causes diseases and mortality in several plants of the Atlantic Forest. Additionally, the pathogen can affect others hosts, such as Eucalyptus clones in commercial plantations.     

Three-colour flow cytometric detection of PrP in ovine leukocytes

PrP(c) (cellular prion protein, CD230) expression by subpopulations of lymphoid cells has been widely investigated in a variety of species, possibly because of the possible link between transmissible spongiform encephalopathies (TSE) transmission and blood transfusion. However, the role of the immune cells in the transmission of the disease is still unclear. Here we describe the optimisation and standardisation of a three-colour staining procedure to detect PrP in association with phenotypic and activation markers in ovine immune cells. We demonstrate a reproducible, flexible and sensitive method and that the combination of isotype-specific antibodies and Fab fragments is feasible. To our knowledge, this is the first report of such labelling of ovine cells. Using this method, we were able to detect differences in levels of PrP expression between blood and lymph node cells of the same animal, and considerable variability between animals. Moreover, we were able to explore possible associations between PrP expression and cellular activation and to identify cell subsets with different labelling patterns. We are currently employing this approach to evaluate variations in immunological parameters during experimental infection in sheep.     

Functional properties of spice extracts obtained via supercritical fluid extraction

In the present study the antioxidant, anticancer, and antimycobacterial activities of extracts from ginger (Zingiber officinale Roscoe), rosemary (Rosmarinus officinalis L.), and turmeric (Curcuma longa L.) were evaluated. The extracts were obtained using supercritical CO(2) with and without ethanol and/or isopropyl alcohol as cosolvent. The extracts' antioxidant power was assessed using the reaction between beta-carotene and linolenic acid, the antimycobacterial activity against M. tuberculosis was measured by the MABA test, and their anticancer action was tested against nine human cancer ancestries: lung, breast, breast resistant, melanoma, colon, prostate, leukemia, and kidney. The rosemary extracts exhibited the strongest antioxidant and the lowest antimycobacterial activities. Turmeric extracts showed the greatest antimycobacterial activity. Ginger and turmeric extracts showed selective anticancer activities.     

Ultrasensitive quantitative determination of paraquat: application to river, ground, and drinking water analysis in an agricultural area

The water specimens were collected from wells and irrigation ditches in the agricultural area to the south of Milan and from Olona River and Mantua Lake and analyzed for paraquat detection. The assay was performed using a specific polyclonal antibody raised in sheep and rabbit anti-sheep IgG conjugated with a chelating molecule 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid complexed with Eu3+ as a fluorescent marker. Bovine serum albumin conjugated with 5-(1'-methyl[4,4']bipyridinyl-1-yl)pentanoic acid was used in solid phase preparation. The sensitivity achieved was 20 ng L(-1). The recovery in samples spiked with three different PQ concentrations was between 88 and 108%.