Reaction Product Variability and Biological Activity of the Lactoperoxidase System Depending on Medium Ionic Strength and pH, and on Substrate Relative Concentration.

The potential of ions produced in water by the lactoperoxidase system against plant pests has shown promising results. We tested the bioactivity of ions produced by the lactoperoxidase oxidation of I- and SCN- in several buffers or in tap water and characterized the ions produced. In vitro biological activity was tested against Penicillium expansum, the causal agent of mold in fruits, and the major cause of patulin contamination of fruit juices and compotes. In buffers, the ionic concentration was increased 3-fold, and pathogen inhibition was obtained down to the 1:15 dilution. In tap water, the ionic concentration was weaker, and pathogen inhibition was obtained only down to the 1:3 dilution. Acidic buffer increased ion concentrations as compared to less acidic (pH 5.6 or 6.2) or neutral buffers, as do increased ionic strength. 13 C-labelled SCN- and MS showed that different ions were produced in water and in buffers. In specific conditions the ion solution turned yellow and a product was formed, probably diiodothiocyanate (I2 SCN- ), giving an intense signal at 49.7 ppm in 13 C-NMR. The formation of the signal was unambiguously favored in acidic media and disadvantaged or inhibited in neutral or basic conditions. It was enhanced at a specific SCN- : I- ratio of 1:4.5, but decreased when the ratio was 1:2, and was inhibited at ratio SCN- >I- . We demonstrated that the formation of the signal required the interaction between I2 and SCN- , and MS showed the presence of I2 SCN- .

Discrimination and evaluation of lactoferrin and delta-lactoferrin gene expression levels in cancer cells and under inflammatory stimuli using TaqMan real-time PCR.

The lactoferrin gene is known to be expressed either constitutively or under inducible conditions such as hormonal stimuli or inflammation. Its transcription from alternative promoters leads to two products, lactoferrin (Lf) and delta-lactoferrin (DeltaLf) mRNAs the expressions of which are altered during oncogenesis. The comparison of the two enhancer/promoter regions revealed that the two isoforms might be differentially trans-activated. Nevertheless, concomitant expression of both transcripts has been found in some normal tissues and in a subset of breast cancer cell lines and biopsies. Moreover, we found putative inflammatory response elements in both P1 and P2 promoter regions suggesting that both Lf and DeltaLf might be upregulated under inflammatory stimuli. Therefore, a duplex Taqman gene expression assay has been developed and used to profile mRNA expression of the Lf gene in the case of cancer and under inflammatory conditions. Discrimination between the two transcripts is achieved by using a primer pairs/probe set within exon 1beta for DeltaLf and a primer pairs/probe set within exon 1 and exon 2 for Lf. In this study, we confirmed that Lf/DeltaLf Taqman gene expression assay is a powerful tool to investigate the expression of both Lf and DeltaLf transcripts. We also showed that lymphocytes and leukocytes isolated from fresh human blood expressed an extremely high level of DeltaLf messengers. An extensive series of cancer cell lines has been studied confirming that both P1 and P2 promoter regions of the Lf gene are downregulated or silenced in the case of cancer. Furthermore, using stimulation by bacterial lipopolysaccharides (LPS), we showed that in MDA-MB-231 and HT-29 epithelial cells, Lf expression is strongly increased with a higher expression level in MDA-MB-231 whereas DeltaLf expression is not. These results suggest that the NF-kappaB/cRel response elements present in the P1 promoter region are functional whereas those present in the P2 promoter region are not and show that DeltaLf is not regulated in inflammatory conditions.

Ex vivo decontamination of yeast-colonized dentures by iodine-thiocyanate complexes.

INTRODUCTION: Under well-defined experimental conditions, and in the presence of hydrogen peroxide, lactoperoxidase produces stable iodine-thiocyanate complexes that have antimicrobial properties. A novel process was developed to short circuit the consumption of hydrogen peroxide by microbial catalases by producing iodine-thiocyanate complexes prior to contact with microorganisms, with the aim of being able to decontaminate the ex vivo dentures colonized by yeasts. MATERIALS AND METHODS: Teabags containing lactoperoxidase adsorbed on inert clay beads were immersed for 1 minute in phosphate buffer solution (0.1 M pH 7.4) containing 5.2 mM potassium iodide, 1.2 mM potassium thiocyanate, and 5.5 mM hydrogen peroxide. After removing the adsorbed lactoperoxidase, the stability and efficacy of iodine-thiocyanate complexes for Candida-colonized denture decontamination were verified. Investigations were performed in vitro on Candida albicans ATCC 10231 and on clinical isolates from 46 dentures. A Candida plate count was performed after a 24-hour incubation at 37°C on Sabouraud-chloramphenicol or CHROMagar solid media; then, the yeast growth was evaluated in Sabouraud broth by turbidimetry and biofilm biomass by crystal violet staining. RESULTS: In vitro tests demonstrated the effectiveness of the oxidant solution in sterilizing a suspension of 106Candida cells per milliliter after a 5-minute incubation. A single ex vivo immersion of contaminated dentures in a solution of iodine-thiocyanate complexes led to a decrease of at least 1 log unit in the number of colony-forming units in 58.3% of the tested dentures, while immersing in water alone had no effect on denture colonization (significant c2: p = 0.0006). CONCLUSION: These data suggest a promising new strategy for decontamination of dentures.

Denture contamination by yeasts in the elderly.

OBJECTIVES: The aim of this study was to investigate yeast carriage in healthy denture wearers by swabbing and to evaluate the effect of denture hygiene habits. MATERIALS AND METHODS: Denture wearers (n = 87) without evidence of denture stomatitis or any other oral disease were investigated by separately swabbing the fitting surface of the upper denture and the corresponding palatal mucosa in contact with the appliance. In a group of volunteers, a gel without any active compound was spread on the palatal side of the denture once in every morning for 2 weeks. RESULTS: Screening showed Candida colonisation of upper prosthesis in 75.9% of individuals. The most frequent species isolated were Candida albicans (77.9% of the positive cultures), Candida glabrata (44.1%) and Candida tropicalis (19.1%). Carriage of more than one yeast species was found in 48.5% of the contaminated dentures. There was a statistically significant association between denture contamination and palatal mucosa colonisation (chi-squared test: p < 0.0001). Repeated swabbings after 1 week as well as during a weekly follow-up for 1 month confirmed the denture contamination and its degree of severity. A daily gel application produced a yeast-count decrease to 10% of the initial value after 2 weeks (chi-squared test: p = 0.0134 and p = 0.2841 for prosthesis and palatal mucosa, respectively). CONCLUSION: This study documented the reliability of oral swabbing when investigating yeast carriage in healthy denture wearers. Moreover, just a diagnostic tool, sampling upper dentures for Candida could be the opportunity to verify the patient's compliance to hygiene advice.