Canine model for investigating the impact of oral enrofloxacin on commensal coliforms and colonization with multidrug-resistant Escherichia coli

A model was developed in dogs to determine the impact of oral enrofloxacin administration on the indigenous coliform population in the gastrointestinal tract and subsequent disposition to colonization by a strain of multidrug-resistant Escherichia coli (MDREC). Dogs given a daily oral dose of 5 mg enrofloxacin kg(-1) for 21 consecutive days showed a significant decline in faecal coliforms to levels below detectable limits by 72 In of administration. Subsequently, faecal coliforms remained suppressed throughout the period of enrofloxacin dosing. Upon termination of antibiotic administration, the number of excreted faecal coliforms slowly returned over an 8-day period, to levels comparable to those seen prior to antibiotic treatment. Enrofloxacin-treated dogs were more effectively colonized by MDREC, evidenced by a significantly increased count of MDREC in the faeces (7.1 +/- 1.5 log(10) g(-1)) compared with non-antibiotic-treated dogs (5.2 +/- 1.2; P = 0.003). Furthermore, antibiotic treatment also sustained a significantly longer period of MDREC excretion in the faeces (26.8 +/- 10.5 days) compared with animals not treated with enrofloxacin (8.5 +/- 5.4 days; P = 0.0215). These results confirm the importance of sustained delivery of an antimicrobial agent to maintain and expand the colonization potential of drug-resistant bacteria in vivo, achieved in part by reducing the competing commensal coliforms in the gastrointestinal tract to below detectable levels in the faeces. Without in vivo antimicrobial selection pressure, commensal coliforms dominated the gastrointestinal tract at the expense of the MDREC population. Conceivably, the model developed could be used to test the efficacy of novel non-antibiotic strategies aimed at monitoring and controlling gastrointestinal colonization by multidrug-resistant members of the Enterobacteriaceae that cause nosocomial infections.

Cryotherapy for treatment of oral lesions

Cryotherapy is the deliberate destruction of tissue by application of extreme cold. It is well received by patients due to a relative lack of discomfort, the absence of bleeding and minimal to no scarring after healing. It has many applications in oral medicine and clinical oral pathology, and is extremely useful in patients for whom surgery is contra-indicated due to either age or medical history. In this paper we outline the principles, mechanisms of action, and current applications of cryotherapy in the treatment of oral lesions, and present some clinical cases.

Effects of oral vitamin C on monocyte:Endothelial cell adhesion in healthy subjects

Monocyte recruitment and retention in the vasculature is influenced by oxidative stress and is involved in cardiovascular disease (CVD). Individuals with low plasma ascorbate are at elevated risk of CVD. It is unknown whether vitamin C supplementation affects monocyte adhesion to endothelial cells (ECs) in healthy non-smokers. In a randomised double-blind crossover study the effect of vitamin C supplementation (six weeks, 250 mg/day) was determined in subjects with normal (HIC) and below average (LOC) plasma vitamin C concentration at baseline (mean = 67μM, n = 20, mean = 32μM, n = 20, respectively). LOC subjects showed 30% greater monocyte adhesion to ECs. This was significantly reduced by 37% (P < 0.02) following vitamin C supplementation to levels of HIC monocyte adhesion. No differences in plasma malondialdehyde concentrations were observed between groups or after supplementation. In conclusion, vitamin C supplementation normalises monocyte adhesion in subjects with low plasma vitamin C (LOC). This process may be related to a direct effect on monocytes, independent of lipid peroxidation. © 2002 Elsevier Science (USA). All rights reserved.

Investigating the modulation of oral drug absorption using in vitro models

Dipeptides can be absorbed into cells via the dipeptide transporter (which also transported tripeptides and dipeptide derivatives). The optimum conditions for measuring the inhibition of Gly-Pro uptake in Caco-2 cells were identified. A number of structure-activity relationships were identified. These included the effects of increasing the amino-acid chain-length, and the presence of a thiol or hydroxyl group in the side-chain increased IC50 while the presence of a hydroxyl group did not. The benzyl esters had lower or equal IC50 values compared to the parent dipeptides while the methyl esters had higher values. These results indicated that while molecular properties did affect IC50, the size, charge and composition of three particular groups caused the most significant effects, supporting the structure-activity relationship identified. An assay was developed using calcein-AM to show the inhibition of p-glycoprotein activity. There was no significant change due to the presence of mannitol but there was in the presence of clyclosporin A (p<0.01). Incubating the cells with the test solution for 30 minutes before the addition of the ester resulted in a significant (p<0.001) difference. The assay was specific for p-glycoprotein, as the presence MRP inhibitors had no effect (p>0.05). The modified protocol allowed the identification of p-glycoprotein inhibitors quickly and simply using a cell suspension of unmodified cells. The clinically relevant buffering of grapefruit juice to pH 7 led to a four-fold increase in intracellular calcein and hence significant inhibition of p-glycoprotein. Buffered orange and lemon juices had no effect on the assay. Flavone derivatives had previously been found to be inhibitors of CYP3A4 yet neither naringin nor naringenin had any significant effect at concentrations found in grapefruit juice. Of the other (non-grapefruit) flavone derivatives tested, hesperidin, found in orange juice, had no significant effect, kaempferol and rutin also had no effect while genistein significantly inhibited p-glycoprotein (results that support previous studies). Hydroxycinnamic acids had no effect on p-glycoprotein. Studies on other compounds found that the balance between inhibiting p-glycoprotein and disrupting cell membranes depends on the compound containing an oxygen atom and the size of the negative charge on it, as well as three-dimensional arrangement of the atoms.

Oxygen tension modulates the cytokine response of oral epithelium to periodontal bacteria

Background: There is an inverse relationship between pocket depth and pocket oxygen tension with deep pockets being associated with anaerobic bacteria. However, little is known about how the host tissues respond to bacteria under differing oxygen tensions within the periodontal pocket. Aim: To investigate the effect of different oxygen tensions upon nuclear factor-kappa B (NF-?B) activation and the inflammatory cytokine response of oral epithelial cells when exposed to nine species of oral bacteria. Materials and Methods: H400 oral epithelial cells were equilibrated at 2%, 10% or 21% oxygen. Cells were stimulated with heat-killed oral bacteria at multiplicity of infection 10:1, Escherichia coli lipopolysaccharide (15 µg/ml) or vehicle control. Interleukin-8 (IL-8) and tumour necrosis factor-alpha (TNF-a) levels were measured by enzyme-linked immunosorbent assay and NF-?B activation was measured by reporter vector or by immunohistochemical analysis. Results: Tannerella forsythensis, Porphyromonas gingivalis and Prevotella intermedia elicited the greatest epithelial NF-?B activation and cytokine responses. An oxygen-tension-dependent trend in cytokine production was observed with the highest IL-8 and TNF-a production observed at 2% oxygen and lowest at 21% oxygen. Conclusions: These data demonstrate a greater pro-inflammatory host response and cell signalling response to bacteria present in more anaerobic conditions, and hypersensitivity of epithelial cells to pro-inflammatory stimuli at 2% oxygen, which may have implications for disease pathogenesis and/or therapy.

Assessment of different formulations of oral Mycobacterium bovis Bacille Calmette-Guerin (BCG) vaccine in rodent models for immunogenicity and protection against aerosol challenge with M-bovis

Bovine tuberculosis (bTB) caused by infection with Mycobacterium bovis is causing considerable economic loss to farmers and Government in the United Kingdom as its incidence is increasing. Efforts to control bTB in the UK are hampered by the infection in Eurasian badgers (Metes metes) that represent a wildlife reservoir and source of recurrent M. bovis exposure to cattle. Vaccination of badgers with the human TB vaccine, M. bovis Bacille Calmette-Guerin (BCG), in oral bait represents a possible disease control tool and holds the best prospect for reaching badger populations over a wide geographical area. Using mouse and guinea pig models, we evaluated the immunogenicity and protective efficacy, respectively, of candidate badger oral vaccines based on formulation of BCG in lipid matrix, alginate beads, or a novel microcapsular hybrid of both lipid and alginate. Two different oral doses of BCG were evaluated in each formulation for their protective efficacy in guinea pigs, while a single dose was evaluated in mice. In mice, significant immune responses (based on lymphocyte proliferation and expression of IFN-gamma) were only seen with the lipid matrix and the lipid in alginate microcapsular formulation, corresponding to the isolation of viable BCG from alimentary tract lymph nodes. In guinea pigs, only BCG formulated in lipid matrix conferred protection to the spleen and lungs following aerosol route challenge with M. bovis. Protection was seen with delivery doses in the range 10(6)-10(7) CFU, although this was more consistent in the spleen at the higher dose. No protection in terms of organ CFU was seen with BCG administered in alginate beads or in lipid in alginate microcapsules, although 10(7) in the latter formulation conferred protection in terms of increasing body weight after challenge and a smaller lung to body weight ratio at necropsy. These results highlight the potential for lipid, rather than alginate, -based vaccine formulations as suitable delivery vehicles for an oral BCG vaccine in badgers.

Consideration of the efficacy of non-ionic vesicles in the targeted delivery of oral vaccines

The fundamentals of this research were to exploit non-ionic surfactant technology for delivery and administration of vaccine antigens across the oral route and to gain a better understanding of vaccine trafficking. Using a newly developed method for manufacture of non-ionic surfactant vesicles (niosomes and bilosomes) lower process temperatures were adopted thus reducing antigen exposure to potentially damaging conditions. Vesicles prepared by this method offered high protection to enzymatic degradation, with only ~10 % antigen loss measured when vesicles incorporating antigen were exposed to enzyme digestion. Interestingly, when formulated using this new production method, the addition of bile salt to the vesicles offered no advantage in terms of stability within simulated gastro-intestinal conditions. Considering their ability to deliver antigen to their target site, results demonstrated that incorporation of antigen within vesicles enhanced delivery and targeting of the antigen to the Peyer's Patch, again with niosomes and bilosomes offering similar efficiency. Delivery to both the Peyer's patches and mesentery lymphatics was shown to be dose dependent at lower concentrations, with saturation kinetics applying at higher concentrations. This demonstrates that in the formulation of vaccine delivery systems, the lipid/antigen dose ratio is not only a key factor in production cost, but is equally a key factor in the kinetics of delivery and targeting of a vaccine system. © 2013 Controlled Release Society.

Consideration of the efficacy of non-ionic vesicles in the targeted delivery of oral vaccines

The fundamentals of this research were to exploit non-ionic surfactant technology for delivery and administration of vaccine antigens across the oral route and to gain a better understanding of vaccine trafficking. Using a newly developed method for manufacture of non-ionic surfactant vesicles (niosomes and bilosomes) lower process temperatures were adopted thus reducing antigen exposure to potentially damaging conditions. Vesicles prepared by this method offered high protection to enzymatic degradation, with only ~10 % antigen loss measured when vesicles incorporating antigen were exposed to enzyme digestion. Interestingly, when formulated using this new production method, the addition of bile salt to the vesicles offered no advantage in terms of stability within simulated gastro-intestinal conditions. Considering their ability to deliver antigen to their target site, results demonstrated that incorporation of antigen within vesicles enhanced delivery and targeting of the antigen to the Peyer's Patch, again with niosomes and bilosomes offering similar efficiency. Delivery to both the Peyer's patches and mesentery lymphatics was shown to be dose dependent at lower concentrations, with saturation kinetics applying at higher concentrations. This demonstrates that in the formulation of vaccine delivery systems, the lipid/antigen dose ratio is not only a key factor in production cost, but is equally a key factor in the kinetics of delivery and targeting of a vaccine system. © 2013 Controlled Release Society.