Antimicrobial Efficacy of an Innovative Emulsion of Medium Chain Triglycerides against Canine and Feline Periodontopathogens

OBJECTIVES: To test the in vitro antimicrobial efficacy of a non-toxic emulsion of free fatty acids against clinically relevant canine and feline periodontopathogens 
METHODS: Antimicrobial kill kinetics were established utilising an alamarBlue® viability assay against 10 species of canine and feline periodontopathogens in the biofilm mode of growth at a concentration of 0·125% v/v medium chain triglyceride (ML:8) emulsion. The results were compared with 0·12% v/v chlorhexidine digluconate and a xylitol-containing dental formulation. Mammalian cellular cytotoxicity was also investigated for both the ML:8 emulsion and chlorhexidine digluconate (0·25 to 0·0625% v/v) using in vitro tissue culture techniques.
RESULTS: No statistically significant difference was observed in the antimicrobial activity of the ML:8 emulsion and chlorhexidine digluconate; a high percentage kill rate (>70%) was achieved within 5 minutes of exposure and was maintained at subsequent time points. A statistically significant improvement in antibiofilm activity was observed with the ML:8 emulsion compared with the xylitol-containing formulation. The ML:8 emulsion possessed a significantly lower (P<0·001) toxicity profile compared with the chlorhexidine digluconate in mammalian cellular cytotoxicity assays.
CLINICAL SIGNIFICANCE: The ML:8 emulsion exhibited significant potential as a putative effective antimi-crobial alternative to chlorhexidine- and xylitol- based products for the reduction of canine and feline periodontopathogens.

Effects of orally administered Bdellovibrio bacteriovorus on the well-being and Salmonella colonization of young chicks

Bdellovibrio bacteriovorus is a bacterium which preys upon and kills Gram-negative bacteria, including the zoonotic pathogens Escherichia coli and Salmonella. Bdellovibrio has potential as a biocontrol agent, but no reports of it being tested in living animals have been published, and no data on whether Bdellovibrio might spread between animals are available. In this study, we tried to fill this knowledge gap, using B. bacteriovorus HD100 doses in poultry with a normal gut microbiota or predosed with a colonizing Salmonella strain. In both cases, Bdellovibrio was dosed orally along with antacids. After dosing non-Salmonella-infected birds with Bdellovibrio, we measured the health and well-being of the birds and any changes in their gut pathology and culturable microbiota, finding that although a Bdellovibrio dose at 2 days of age altered the overall diversity of the natural gut microbiota in 28-day-old birds, there were no adverse effects on their growth and well-being. Drinking water and fecal matter from the pens in which the birds were housed as groups showed no contamination by Bdellovibrio after dosing. Predatory Bdellovibrio orally administered to birds that had been predosed with a gut-colonizing Salmonella enterica serovar Enteritidis phage type 4 strain (an important zoonotic pathogen) significantly reduced Salmonella numbers in bird gut cecal contents and reduced abnormal cecal morphology, indicating reduced cecal inflammation, compared to the ceca of the untreated controls or a nonpredatory ΔpilA strain, suggesting that these effects were due to predatory action. This work is a first step to applying Bdellovibrio therapeutically for other animal, and possibly human, infections.

Cigarette Smoke, Airway Epithelial Cells and Host Defence

In COPD inflammation driven by exposure to tobacco smoke results in impaired innate immunity in the airway and ultimately to lung injury and remodeling. To understand the biological processes involved in host interactions with cigarette derived toxins submerged epithelial cell culture is widely accepted as a model for primary human airway epithelial cell culture research. Primary nasal and bronchial epithelial cells can also be cultured in air-liquid interface (ALI) models. ALI and submerged culture models have their individual merits, and the decision to use either technique should primarily be determined primarily by the research hypothesis.

Cigarette smoke has gaseous and particulate matter, the latter constituent primarily represented in cigarette smoke extract (CSE). Although not ideal in order to facilitate our understanding of the responses of epithelial cells to cigarette smoke, CSE still has scientific merit in airway cell biology research. Using this model, it has been possible to demonstrate differences in levels of tight junction disruption after CSE exposure along with varied vulnerability to the toxic effects of CSE in cell cultures derived from COPD and control study groups.

Primary nasal epithelial cells (PNECs) have been used as an alternative to bronchial epithelial cells (PBECs). However, at least in subjects with COPD, PNECs cannot consistently substitute for PBECs. Although airway epithelial cells from patients with COPD exhibit a constitutional pro-inflammatory phenotype, these cells have a diminished inflammatory response to CSE exposure. COPD epithelial cells have an increased susceptibility to undergo apoptosis, and have reduced levels of Toll-like receptor-4 expression after CSE exposure, both of which may account for the reduced inflammatory response observed in this group.

The use of CSE in both submerged and ALI epithelial cultures has extended our understanding of the cellular mechanisms that are important in COPD, and helped to unravel important pathways which may be of relevance in its pathogenesis.

The progress in understanding and treatment of diabetic retinopathy

In most countries, diabetic retinopathy is the most frequently occurring complication of diabetes mellitus and remains a leading cause of vision loss globally. Its etiology and pathology have been extensively studied for half a century, yet there are disappointingly few therapeutic options. Although some new treatments have been introduced for diabetic macular edema (DME) (e.g. intravitreal vascular endothelial growth factor inhibitors ('anti-VEGFs') and new steroids), up to 50% of patients fail to respond. Furthermore, for people with proliferative diabetic retinopathy (PDR), laser photocoagulation remains a mainstay therapy, even though it is inherently a destructive procedure. This review summarizes the clinical features of diabetic retinopathy and its risk factors. It describes details of retinal pathology and the cell culture approaches and animal models that are used to mimic its key components, advance understanding of its pathogenesis, and enable identification of new therapeutic targets. We emphasise that although there have been significant advances, there is still a pressing need for a better understanding basic mechanisms to enable development of reliable and robust means to identify patients at highest risk, and to intervene effectively before vision loss occurs.

Label-free based quantitative proteomics analysis of primary neonatal porcine Leydig cells exposed to the persistent contaminant 3-methylsulfonyl-DDE

Evidence that persistent environmental pollutants may target the male reproductive system is increasing. The male reproductive system is regulated by secretion of testosterone by testicular Leydig cells, and perturbation of Leydig cell function may have ultimate consequences. 3-Methylsulfonyl-DDE (3-MeSO₂-DDE) is a potent adrenal toxicants formed from the persistent insecticide DDT. Although studies have revealed the endocrine disruptive effect of 3-MeSO₂-DDE, the underlying mechanisms at cellular level in steroidogenic Leydig cells remains to be established. The current study addresses the effect of 3-MeSO₂-DDE on viability, hormone production and proteome response of primary neonatal porcine Leydig cells. The AlamarBlue™ assay was used to evaluate cell viability. Solid phase radioimmunoassay was used to measure concentration of hormones produced by both unstimulated and Luteinizing hormone (LH)-stimulated Leydig cells following 48h exposure. Protein samples from Leydig cells exposed to a non-cytotoxic concentration of 3-MeSO₂-DDE (10μM) were subjected to nano-LC-MS/MS and analyzed on a Q Exactive mass spectrometer and quantified using label-free quantitative algorithm. Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA) were carried out for functional annotation and identification of protein interaction networks. 3-MeSO₂-DDE regulated Leydig cell steroidogenesis differentially depending on cell culture condition. Whereas its effect on testosterone secretion at basal condition was stimulatory, the effect on LH-stimulated cells was inhibitory. From triplicate experiments, a total of 6804 proteins were identified in which the abundance of 86 proteins in unstimulated Leydig cells and 145 proteins in LH-stimulated Leydig cells was found to be significantly regulated in response to 3-MeSO₂-DDE exposure. These proteins not only are the first reported in relation to 3-MeSO₂-DDE exposure, but also display small number of proteins shared between culture conditions, suggesting the action of 3-MeSO₂-DDE on several targeted pathways, including mitochondrial dysfunction, oxidative phosphorylation, EIF2-signaling, and glutathione-mediated detoxification. Further identification and characterization of these proteins and pathways may build our understanding to the molecular basis of 3-MeSO₂-DDE induced endocrine disruption in Leydig cells.

Effects of LL-37 Peptide Mimetics on Gingival Fibroblast Function

Host defence peptides, including the cathelicidin LL-37, play an important role in mucosal immunity, functioning as both antimicrobial agents and modulators of the inflammatory response. In the current climate of antibiotic resistance, the idea of using naturally occurring antimicrobial peptides, or their synthetic mimetics, to combat oral infection is particularly appealing. Objectives: The aim of this study was to investigate the effects of parent LL-37, and two peptide mimetics (KR-12 and KE-18), on cytokine expression and response to bacterial challenge by gingival fibroblasts. Methods: KR-12 and KE-18 are peptide mimetics of the biologically active, mid-region sequence of LL-37. The effects of commercially available LL-37, KR-12 and KE-18 on gingival fibroblast response to E coli and P gingivalis LPS challenge, analysed by IL-6 and IL-8 expression, were determined in cell culture by ELISA. The direct effects of each peptide on IL-6, IL-8, CXCL-1 and HGF expression were also determined by ELISA. The MTT assay was used to evaluate peptide effects on fibroblast viability. Results: LL-37 and KE-18, but not KR-12, inhibited LPS induction of inflammatory cytokine expression and directly stimulated CXCL-1 production by fibroblasts. All 3 peptides stimulated production of IL-8 and HGF. Neither LL-37 nor KE-12 affected cell viability, while KE-18, at higher concentrations, induced cell death. Conclusions: Shorter, peptide mimetics of LL-37, in particular KE-18, retain the immunomodulatory effects of the parent molecule and possess excellent potential as therapeutic agents in the treatment of oral infections including periodontal disease.