An update on the taxonomy and functional properties of the probiotic Enterococcus faecium SF68.

Enterococcus faecium SF68 (SF68) is a well-known probiotic with a long history of safe use. Recent changes in the taxonomy of enterococci have shown that a novel species, Enterococcus lactis, is closely related with E. faecium and occurs together with other enterococci in a phylogenetically well-defined E. faecium species group. The close phylogenetic relationship between the species E. faecium and E. lactis prompted a closer investigation into the taxonomic status of E. faecium SF68. Using phylogenomics and ANI, the taxonomic analysis in this study showed that probiotic E. faecium SF68, when compared to other E. faecium and E. lactis type and reference strains, could be re-classified as belonging to the species E. lactis. Further investigations into the functional properties of SF68 showed that it is potentially capable of bacteriocin production, as a bacteriocin gene cluster encoding the leaderless bacteriocin EntK1 together with putative Lactococcus lactis bacteriocins LsbA, and LsbB-like putative immunity peptide (LmrB) were found located in an operon on plasmid pF9. However, bacteriocin expression was not studied. Competitive exclusion experiments in co-culture over 7 days at 37 °C showed that the probiotic SF68 could inhibit the growth of specific E. faecium and Listeria monocytogenes strains, while showing little or no inhibitory activity towards an entero-invasive Escherichia coli and a Salmonella Typhimurium strain, respectively. In cell culture experiments with colon carcinoma HT29 cells, the probiotic SF68 was also able to strain-specifically inhibit adhesion and/or invasion of enterococcal and L. monocytogenes strains, while such adhesion and invasion inhibition effects were less pronounced for E. coli and Salmonella strains. This study therefore provides novel data on the taxonomy and functional properties of SF68, which can be reclassified as Enterococcus lactis SF68, thereby enhancing the understanding of its probiotic nature.

Outcome of radiotherapy for invasive bladder cancer in older patients - not candidates for or decline cystectomy.

PURPOSE: Retrospective study to assess the role of radiotherapy (RT) in bladder cancer (BC) in patients who decline cystectomy or for whom it was contraindicated, regarding BC related events. MATERIALS AND METHODS: A cross-sectional study, of patients with BC treated with RT, with or without chemotherapy was carried out, during the period March 2005 to March 2017, excluding patients who performed cystectomy. BC-related events were defined as haematuria, episodes requiring intravesical irrigations or transurethral resection or admission in Urology Department caused by symptoms related to BC. RESULTS: Fifty-eight patients were included, 44 men and 14 women, whose median age at diagnosis was 78.3 years. Nine patients were treated with a RT dose below 50Gy (20-45Gy), in relation with non-resectable disease, nodal involvement, presence of metastasis or poor performance status. Forty-nine patients were treated with a RT dose above 50Gy (maximum 60.4Gy). The median follow-up time was 24 months, with BC-specific survival at 1/5 years of 87.5%/0.0% and 83.1%/33.9% in the group treated with RT dose below 50Gy and above 50Gy, respectively. Results revealed that the number of events decreased significantly after RT for both groups. For the two groups, the median of events before RT was 2.0, decreasing for 1.0 after RT (RT dose<50Gy: P=0.011; RT dose≥50Gy: P=0.026). CONCLUSION: This therapeutic approach can have an important role in older patients with bladder cancer, in terms of reducing disease related events. Symptom control was significantly achieved in both therapeutic groups.

Oral Vaccination with Hepatitis E Virus Capsid Protein and Immunobiotic Bacterium-Like Particles Induce Intestinal and Systemic Immunity in Mice.

The hepatitis E virus (HEV) genotype 3 (GT3) is an emergent pathogen in industrialized countries. It is transmitted zoonotically and may lead to chronic hepatitis in immunocompromised individuals. We evaluated if the major antigen of HEV, the capsid protein, can be used in combination with immunobiotic bacterium-like particles (IBLP) for oral vaccination in a mouse model. We have cloned and expressed the RGS-His5-tagged HEV GT3 capsid protein (ORF2) in E. coli and purified it by NiNTA. IBLP were obtained from two immunobiotic Lactobacillus rhamnosus strains acid- and heat-treated. ORF2 and the IBLP were orally administered to Balb/c mice. After three oral immunizations (14-day intervals), blood, intestinal fluid, Peyer´s patches, and spleen samples were drawn. IgA- and IgG-specific antibodies were determined by ELISA. Mononuclear cell populations from Peyer's patches and spleen were analyzed by flow cytometry, and the cytokine profiles were determined by ELISA to study cellular immunity. Orally administered recombinant ORF2 and IBLP from two L. rhamnosus strains (CRL1505 and IBL027) induced both antigen-specific humoral and cellular immune responses in mice. IBLP027 was more effective in inducing specific secretory IgA in the gut. IFN-γ, TNF-α, and IL-4 were produced by Peyer's plaques lymphocytes stimulated with ORF2 ex vivo suggesting a mixed Th1/Th2-type adaptive immune response in immunized mice. Oral vaccines are not invasive, do not need to be administered by specialized personal, and elicit both systemic and local immune responses at the port of entry. Here, we present an experimental oral vaccine for HEV GT3, which could be further developed for human and/or veterinary use.

Influence of exposure parameters on the detection of simulated root fractures in the presence of various intracanal materials.

AIM: To investigate how the exposure parameters used when producing CBCT scans affect diagnostic accuracy when detecting simulated vertical root fractures and how various filling materials affect image quality. METHODOLOGY: A total of 160 extracted single-rooted human teeth were divided into four groups based on the materials within the root canals: unrestored, gutta-percha, metallic post and fibreglass post. Half of the sample of each group was selected for the induction of vertical root fractures. Each tooth was placed in an empty socket of a dry skull which was scanned on a CBCT unit (9000 3D scanner; Kodak Dental Systems, Carestream Health, Rochester, NY, USA) using the following exposure parameters: 74 kV/12 mA; 74 kV/10 mA; 74 kV/8 mA; 74 kV/6.3 mA; 70 kV/12 mA; 70 kV/10 mA; 70 kV/8 mA; 70 kV/6.3 mA. Two observers assessed all images using a 5-point confidence scale for fracture detection and a 4-point score for the presence of artefacts. Sensitivity, specificity, accuracy and area under ROC curve were compared by two-way anova and Tukey's test. Artefact formation was evaluated by descriptive statistics. RESULTS: There were no significant differences in the sensitivity (P = 0.370), specificity (P = 0.660), accuracy (P = 0.084) and area under the ROC curve (P = 0.674) values amongst the various exposure parameters within the same group. There were some significant differences when the groups were compared for each intracanal material. The sensitivity and accuracy of the group containing metallic posts were significantly lower than the unrestored and fibreglass post groups (P ≤ 0.017). CONCLUSION: The variations in exposure parameters did not interfere with the diagnosis of vertical root fractures, independent of the root canal restorative status. Metallic posts were associated with greater artefact formation and compromised the diagnostic performance. It is possible to decrease the kVp/mA settings to reduce the probability of biological effects due to radiation, without losing diagnostic accuracy.

Comparative effect of a new mouthrinse containing chlorhexidine, triclosan and zinc on volatile sulphur compounds: a randomized, crossover, double-blind study.

AIM: The aims of this study were to compare the volatile sulphur compounds (VSC)-reducing effect of two commercial mouthrinses using a morning bad breath model and to assess the role of mechanical plaque control (MPC) when performed previously to mouthrinse use. PATIENTS AND METHODS: Eleven volunteers with good oral health were enrolled in a double-blind, randomized, six-step crossover design study with a 7-day washout period. Two commercial mouthrinses were tested using a saline solution (NaCl 0.9%) as a negative control: one mouthrinse contained 0.05% chlorhexidine, 0.05% cetylpyridinium chloride and 0.14% zinc lactate (CHX-CPC-Zn), while the other contained 0.05% chlorhexidine, 0.15% triclosan and 0.18% zinc pidolate (CHX-triclosan-Zn). A portable sulphide monitor (Halimeter(®) ) was used for VSC quantification. Measurements were made at baseline, and 1, 3 and 5 h after rinsing. Significant differences were detected by analysis of variance. RESULTS: No significant differences between groups were detected at baseline. We were unable to demonstrate a significant influence of mechanical plaque control on the reduction of VSC levels when performed before mouthrinse use (P = 0.631). Both mouthrinses effectively lowered VSC levels in all test intervals (P < 0.05). No statistically significant differences were found between mouthrinses in any of the test intervals (P = 0.629, 0.069 and 0.598 at 1, 3 and 5 h). CONCLUSION: This study demonstrated that CHX-CPC-Zn and CHX-triclosan-Zn have significant and similar effects in reducing VSC levels, which persist for at least 5 h. Such effects were independent of previous MPC, which failed to improve on the results of mouthrinse use alone.

Expression and function of fibroblast growth factor 10 and its receptor, fibroblast growth factor receptor 2B, in bovine follicles.

Some fibroblast growth factors (FGFs) affect ovarian follicle cell growth and/or differentiation. Whereas many FGFs activate several FGF receptors, FGF7 and FGF10 primarily activate only one, FGFR2B. As FGF7 is produced by bovine theca cells and acts on granulosa cells, we tested the hypothesis that FGF10 may also play a role in folliculogenesis in cattle. Reverse transcription-polymerase chain reaction demonstrated the presence of FGF10 mRNA in the oocytes and theca cells of the antral follicles, as well as in the preantral follicles. FGF10 protein was detected by immunohistochemistry in the oocytes of the preantral and antral follicles, and in the granulosa and theca cells of the antral follicles. FGF10 expression in theca cells changed during follicle development; mRNA abundance decreased with increasing follicular estradiol concentration in healthy follicles, and was lowest in highly atretic follicles. Culturing of granulosa cells in serum-free medium revealed FSH regulation of FGF10 receptor expression. The addition of FGF10 to cultured granulosa cells decreased the level of estradiol but did not alter cell proliferation. These data support a role for FGF10 in signaling to granulosa cells from theca cells and/or the oocyte.

Expression of LH receptor mRNA splice variants in bovine granulosa cells: changes with follicle size and regulation by FSH in vitro.

In cattle, most evidence suggests that granulosa cells express LH receptors (LHR) after (or as) the follicle becomes dominant, however there is some suggestion that granulosa cells from smaller pre-dominant follicles may express several LHR mRNA splice variants. The objective of this study was to measure LHR expression in bovine follicles of defined size and steroidogenic ability, and in granulosa cells from small follicles (<6 mm diameter) undergoing differentiation in vitro. Semiquantitative RT-PCR demonstrated that LHR mRNA was undetectable in granulosa cells of follicles <7 mm diameter (nondominant follicles), and increased with follicle diameter in follicles >7 mm diameter. Splice variants with deletions of exon 10 and part of exon 11 were detected as previously described, and we detected a novel splice variant with a deletion of exon 3. Cultured granulosa cells contained LHR mRNA, but with significantly greater amounts of variants with deletions of exon 10 and/or exon 11 compared with cells from dominant follicles. FSH increased the abundance of some but not all LHR mRNA splice variants in cultured granulosa cells. The addition of LH to cultured cells did not increase progesterone secretion, despite the presence of LHR mRNA. Collectively, these data suggest that granulosa cells do not acquire functional LHR until follicle dominance occurs.

Scanning electron microscopy of the choroid plexus of the lateral ventricle of the horse.

The present study examined the ultrastructure of the choroid plexus of the lateral ventricle of the horse. The material was fixed in 2.5% glutaraldehyde in 0.1 m sodium phosphate buffer, pH 7.3, processed and analysed by scanning electron microscopy. The choroid plexus was characterized by regions with a predominance of villi, which resembled finger-like projections or bunches of grapes, and others where straight and uniform folds predominated. Epithelial cells projected into the ventricle and large amounts of cilia and microvilli were observed on their surface. The choroid glomus corresponded to a dilatation of the choroid plexus and was characterized by blood vessels of different calibres surrounded by connective tissue.

Expression of fibroblast growth factor-8 and regulation of cognate receptors, fibroblast growth factor receptor-3c and -4, in bovine antral follicles.

Paracrine cell signaling is believed to be important for ovarian follicle development, and a role for some members of the fibroblast growth factor (FGF) family has been suggested. In the present study, we tested the hypothesis that FGF-8 and its cognate receptors (FGFR3c and FGFR4) are expressed in bovine antral follicles. RT-PCR was used to analyze bovine Fgf8, Fgfr3c and Fgfr4 mRNA levels in oocytes, and granulosa and theca cells. Fgf8 expression was detected in oocytes and in granulosa and theca cells; this expression pattern differs from that reported in rodents. Granulosa and theca cells, but not oocytes, expressed Fgfr3c, and expression in granulosa cells increased significantly with follicle estradiol content, a major indicator of follicle health. Fgfr4 expression was restricted to theca cells in the follicle, and decreased significantly with increasing follicle size. To investigate the potential regulation of Fgfr3c expression in the bovine granulosa, cells were cultured in serum-free medium with FSH or IGF-I; gene expression was upregulated by FSH but not by IGF-I. The FSH-responsive and developmentally regulated patterns of Fgfr3c mRNA expression suggest that this receptor is a potential mediator of paracrine signaling to granulosa cells during antral follicle growth in cattle.