A quantitative phenytoin GC-MS method and it's validation for samples from human ex situ brain microdialysis, blood and saliva using solid-phase extraction

This study describes the development and validation of a gas chromatography-mass spectrometry (GC-MS) method to identify and quantitate phenytoin in brain microdialysate, saliva and blood from human samples. A solid-phase extraction (SPE) was performed with a nonpolar C8-SCX column. The eluate was evaporated with nitrogen (50°C) and derivatized with trimethylsulfonium hydroxide before GC-MS analysis. As the internal standard, 5-(p-methylphenyl)-5-phenylhydantoin was used. The MS was run in scan mode and the identification was made with three ion fragment masses. All peaks were identified with MassLib. Spiked phenytoin samples showed recovery after SPE of ≥94%. The calibration curve (phenytoin 50 to 1,200 ng/mL, n = 6, at six concentration levels) showed good linearity and correlation (r² > 0.998). The limit of detection was 15 ng/mL; the limit of quantification was 50 ng/mL. Dried extracted samples were stable within a 15% deviation range for ≥4 weeks at room temperature. The method met International Organization for Standardization standards and was able to detect and quantify phenytoin in different biological matrices and patient samples. The GC-MS method with SPE is specific, sensitive, robust and well reproducible, and is therefore an appropriate candidate for the pharmacokinetic assessment of phenytoin concentrations in different human biological samples.

Influence of physical activity and acute exercise on cognitive performance and saliva testosterone in preadolescent school children

We investigated whether the chronic physical activity participation had an impact on the acute effects of a short bout of 12 min of intensive physical activity on cognitive performance and testosterone concentration in primary school students (n = 42, mean age = 9.69, SD = .44; experimental group (EG), n = 27; control group (CG), n = 15). Furthermore, we looked for associations between testosterone concentration and cognitive performance. After the intervention, participants of the EG showed better cognitive performances as compared to the CG. We further observed a significant group (EG, CG) test (pre, post) activity level (high, low) interaction. Post hoc pairwise comparisons revealed that after acute physical activity the testosterone concentration was diminished only in habitually low active children. The results indicate that intensive physical activity only attenuates the reactivity of the hypothalamic-pituitary-gonadal axis in habitually low active preadolescents, but had a beneficial effect on cognitive performance for all participants independent of their physical activity level and testosterone.

The effect of acute exercise and psychosocial stress on fine motor skills and testosterone concentration in the saliva of high school students

Little is known about the influence of different stressors on fine motor skills, the concentration of testosterone (T), and their interaction in adolescents. Therefore, 62 high school students aged 14–15 years were randomly assigned to two experimental groups (exercise, psychosocial stress) and a control group. Exercise stress was induced at 65–75% of the maximum heart rate by running for 15 minutes (n = 24). Psychosocial stress was generated by an intelligence test (HAWIK- IV), which was uncontrollable and characterized by social-evaluative-threat to the students (n=21). The control group followed was part of a regular school lesson with the same duration (n = 28). Saliva was collected after a normal school lesson (pre-test) as well as after the intervention/control period (post-test) and was analyzed for testosterone. Fine motor skills were assessed pre- and post-intervention using a manual dexterity test (Flower Trail) from the Movement Assessment Battery for Children-2. A repeated measure ANCOVA including gender as a covariate revealed a significant group by test interaction, indicating an increase in manual dexterity only for the psychosocial stress group. Correlation analysis of all students shows that the change of testosterone from pre- to post-test was directly linked (r = 2.31, p = .01) to the changes in manual dexterity performance. Participants showing high increases in testosterone from pre- to post-test made fewer mistakes in the fine motor skills task. Findings suggest that manual dexterity increases when psychosocial stress is induced and that improvement of manual dexterity performance corresponds with the increase of testosterone.

The basel cocktail for simultaneous phenotyping of human cytochrome P450 isoforms in plasma, saliva and dried blood spots.

BACKGROUND AND OBJECTIVE Phenotyping cocktails use a combination of cytochrome P450 (CYP)-specific probe drugs to simultaneously assess the activity of different CYP isoforms. To improve the clinical applicability of CYP phenotyping, the main objectives of this study were to develop a new cocktail based on probe drugs that are widely used in clinical practice and to test whether alternative sampling methods such as collection of dried blood spots (DBS) or saliva could be used to simplify the sampling process. METHODS In a randomized crossover study, a new combination of commercially available probe drugs (the Basel cocktail) was tested for simultaneous phenotyping of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4. Sixteen subjects received low doses of caffeine, efavirenz, losartan, omeprazole, metoprolol and midazolam in different combinations. All subjects were genotyped, and full pharmacokinetic profiles of the probe drugs and their main metabolites were determined in plasma, dried blood spots and saliva samples. RESULTS The Basel cocktail was well tolerated, and bioequivalence tests showed no evidence of mutual interactions between the probe drugs. In plasma, single timepoint metabolic ratios at 2 h (for CYP2C19 and CYP3A4) or at 8 h (for the other isoforms) after dosing showed high correlations with corresponding area under the concentration-time curve (AUC) ratios (AUC0-24h parent/AUC0-24h metabolite) and are proposed as simple phenotyping metrics. Metabolic ratios in dried blood spots (for CYP1A2 and CYP2C19) or in saliva samples (for CYP1A2) were comparable to plasma ratios and offer the option of minimally invasive or non-invasive phenotyping of these isoforms. CONCLUSIONS This new combination of phenotyping probe drugs can be used without mutual interactions. The proposed sampling timepoints have the potential to facilitate clinical application of phenotyping but require further validation in conditions of altered CYP activity. The use of DBS or saliva samples seems feasible for phenotyping of the selected CYP isoforms.

Survey of selected tick-borne diseases in dogs in Finland

BACKGROUND: Due to climate changes during the last decades, ticks have progressively spread into higher latitudes in northern Europe. Although some tick borne diseases are known to be endemic in Finland, to date there is limited information with regard to the prevalence of these infections in companion animals. We determined the antibody and DNA prevalence of the following organisms in randomly selected client-owned and clinically healthy hunting dogs living in Finland: Ehrlichia canis (Ec), Anaplasma phagocytophilum (Ap), Borrelia burgdorferi (Bb) and Bartonella. METHODS: Anti-Ap, -Bb and -Ec antibodies were determined in 340 Finnish pet dogs and 50 healthy hunting dogs using the 4DX Snap(R)Test (IDEXX Laboratories). In addition, PCRs for the detection of Ap and Bartonella DNA were performed. Univariate and multivariate logistic regression analyses were used to identify risk factors associated with seropositivity to a vector borne agent. RESULTS: The overall seroprevalence was highest for Ap (5.3%), followed by Bb (2.9%), and Ec (0.3%). Seropositivities to Ap and Bb were significantly higher in the Aland Islands (p <0.001), with prevalence of Ap and Bb antibodies of 45 and 20%, respectively. In healthy hunting dogs, seropositivity rates of 4% (2/50) and 2% (1/50) were recorded for Ap and Bb, respectively. One client-owned dog and one hunting dog, both healthy, were infected with Ap as determined by PCR, while being seronegative. For Bartonella spp., none of the dogs tested was positive by PCR. CONCLUSIONS: This study represents the first data of seroprevalence to tick borne diseases in the Finnish dog population. Our results indicate that dogs in Finland are exposed to vector borne diseases, with Ap being the most seroprevalent of the diseases tested, followed by Bb. Almost 50% of dogs living in Aland Islands were Ap seropositive. This finding suggests the possibility of a high incidence of Ap infection in humans in this region. Knowing the distribution of seroprevalence in dogs may help predict the pattern of a tick borne disease and may aid in diagnostic and prevention efforts.

A tick-borne encephalitis model in infant rats infected with langat virus

Tick-borne encephalitis virus (TBEV) is the causative agent of human TBE, a severe infection that can cause long-lasting neurologic sequelae. Langat virus (LGTV), which is closely related to TBEV, has a low virulence for human hosts and has been used as a live vaccine against TBEV. Tick-borne encephalitis by natural infection of LGTV in humans has not been described, but one of 18,500 LGTV vaccinees developed encephalitis. The pathogenetic mechanisms of TBEV are poorly understood and, currently, no effective therapy is available. We developed an infant rat model of TBE using LGTV as infective agent. Infant Wistar rats were inoculated intracisternally with 10 focus-forming units of LGTV and assessed for clinical disease and neuropathologic findings at Days 2, 4, 7, and 9 after infection. Infection with LGTV led to gait disturbance, hypokinesia, and reduced weight gain or weight loss. Cerebrospinal fluid concentrations of RANTES, interferon-γ, interferon-β, interleukin-6, and monocyte chemotactic protein-1 were increased in infected animals. The brains of animals with LGTV encephalitis exhibited characteristic perivascular inflammatory cuffs and glial nodules; immunohistochemistry documented the presence of LGTV in the thalamus, hippocampus, midbrain, frontal pole, and cerebellum. Thus, LGTV meningoencephalitis in infant rats mimics important clinical and histopathologic features of human TBE. This new model provides a tool to investigate disease mechanisms and to evaluate new therapeutic strategies against encephalitogenic flaviviruses.

Saliva suppresses osteoclastogenesis in murine bone marrow cultures.

Saliva can reach mineralized surfaces in the oral cavity; however, the relationship between saliva and bone resorption is unclear. Herein, we examined whether saliva affects the process of osteoclastogenesis in vitro. We used murine bone marrow cultures to study osteoclast formation. The addition of fresh sterile saliva eliminated the formation of multinucleated cells that stained positive for tartrate-resistant acid phosphatase (TRAP). In line with the histochemical staining, saliva substantially reduced gene expression of cathepsin K, calcitonin receptor, and TRAP. Addition of saliva led to considerably decreased gene expression of receptor activator of nuclear factor kappa-B (RANK) and, to a lesser extent, that of c-fms. The respective master regulators of osteoclastogenesis (c-fos and NFATc1) and the downstream cell fusion genes (DC-STAMP and Atp6v0d2) showed decreased expression after the addition of saliva. Among the costimulatory molecules for osteoclastogenesis, only OSCAR showed decreased expression. In contrast, CD40, CD80, and CD86-all costimulatory molecules of phagocytic cells-were increasingly expressed with saliva. The phagocytic capacity of the cells was confirmed by latex bead ingestion. Based on these in vitro results, it can be concluded that saliva suppresses osteoclastogenesis and leads to the development of a phagocytic cell phenotype.

MRI findings in a case of canine tick born meningoencephalomyelitis

Tick borne encephalitis virus (TBE) is an endemic infectious agent in northeastern Switzerland causing mainly meningoencephalomyelitis in dogs. We report a canine case of tick born meningoencephalomyelitis resulting in flaccid tetraplegia and, subsequently, fatal respiratory failure. Magnetic resonance imaging (MRI) demonstrated intra-axial bilateral, symmetric, and hyperintense lesions in T2-weighted and Fluid Attenuated Inversion Recovery (FLAIR) sequences affecting thalamus, basal nuclei, cerebral white matter and ventral horns of the caudal cervical spine. These radiological findings overlap those described during flavivirus encephalitis affecting human beings. These lesions in MRI and diffusion weighted images correlated with areas of vasogenic edema detected histopathologically. In endemic regions, clinicians should be aware that bilateral, symmetrical hyperintense thalamic lesions in T2WI can be suggestive of flavivirus infection in dogs with encephalitis

Differential inflammatory response of dental pulp explants and fibroblasts to saliva

Abstract AIM: To investigate the inflammatory response of dental pulp fibroblasts and the respective explants to whole saliva. METHODOLOGY: Explants from human and porcine dental pulp tissue and isolated dental pulp fibroblasts were used to investigate the inflammatory response to sterile saliva. Cytokine and chemokine expression was assessed by RT-PCR. Western blot analysis and pharmacologic inhibitors were used to determine the involvement of signalling pathways. RESULTS: Dental pulp explants of human and porcine origin exposed to human saliva exhibited no major changes of IL-6 and IL-8 mRNA expression (P > 0.05). In contrast, isolated porcine and human dental pulp fibroblasts, when stimulated with human saliva, exhibited a vastly increased expression of IL-6 and IL-8 mRNA (P < 0.05). In pulp fibroblasts, saliva also increased the expression of other cytokines and chemokines via activation of NFkappaB, ERK and p38 signalling. Notably, a significantly reduced inflammatory response was elicited when pulp fibroblasts were transiently exposed to saliva. CONCLUSIONS: Saliva has a potential impact on inflammation of dental pulp fibroblasts in vitro but not when cells are embedded in the intrinsic extracellular matrix of the explant tissue.

Salivary Pellicle vs Whole Saliva: The Response of Oral Fibroblasts Based on a Genome-Wide Microarray.

PURPOSE Whole saliva comprises components of the salivary pellicle that spontaneously forms on surfaces of implants and teeth. However, there are no studies that functionally link the salivary pellicle with a possible change in gene expression. MATERIALS AND METHODS This study examined the genetic response of oral fibroblasts exposed to the salivary pellicle and whole saliva. Oral fibroblasts were seeded onto a salivary pellicle and the respective untreated surface. Oral fibroblasts were also exposed to freshly harvested sterile-filtered whole saliva. A genome-wide microarray of oral fibroblasts was performed, followed by gene ontology screening with DAVID functional annotation clustering, KEGG pathway analysis, and the STRING functional protein association network. RESULTS Exposure of oral fibroblasts to saliva caused 61 genes to be differentially expressed (P < .05). Gene ontology screening assigned the respective genes into 262 biologic processes, 3 cellular components, 13 molecular functions, and 7 pathways. Most remarkable was the enrichment in the inflammatory response. None of the genes regulated by whole saliva was significantly changed when cells were placed onto a salivary pellicle. CONCLUSION The salivary pellicle per se does not provoke a significant inflammatory response of oral fibroblasts in vitro, whereas sterile-filtered whole saliva does produce a strong inflammatory response.

Sterile-filtered saliva is a strong inducer of IL-6 and IL-8 in oral fibroblasts.

OBJECTIVES Saliva has been implicated to support oral wound healing, a process that requires a transient inflammatory reaction. However, definitive proof that saliva can provoke an inflammatory response remained elusive. MATERIALS AND METHODS We investigated the ability of freshly harvested and sterile-filtered saliva to cause an inflammatory response of oral fibroblasts and epithelial cells. The expression of cytokines and chemokines was assessed by microarray, RT-PCR, immunoassays, and Luminex technology. The involvement of signaling pathways was determined by Western blot analysis and pharmacologic inhibitors. RESULTS We report that sterile-filtered whole saliva was a potent inducer of IL-6 and IL-8 in fibroblasts from the gingiva, the palate, and the periodontal ligament, but not of oral epithelial cells. This strong inflammatory response requires nuclear factor-kappa B and mitogen-activated protein kinase signaling. The pro-inflammatory capacity is heat stable and has a molecular weight of <40 kDa. Genome-wide microarrays and Luminex technology further revealed that saliva substantially increased expression of other inflammatory genes and various chemokines. To preclude that the observed pro-inflammatory activity is the result of oral bacteria, sterile-filtered parotid saliva, collected under almost aseptic conditions, was used and also increased IL-6 and IL-8 expression in gingiva fibroblasts. The inflammatory response was, furthermore, independent of MYD88, an adapter protein of the Toll-like receptor signaling pathway. CONCLUSIONS We conclude that saliva can provoke a robust inflammatory response in oral fibroblasts involving the classical nuclear factor-kappa B and mitogen-activated protein kinase signaling pathway. CLINICAL RELEVANCE Since fibroblasts but not epithelial cells show a strong inflammatory response, saliva may support the innate immunity of defect sites exposing the oral connective tissue.

Prevalence estimation of tick-borne encephalitis virus (TBEV) antibodies in dogs from Finland using novel dog anti-TBEV IgG MAb-capture and IgG immunofluorescence assay based on recombinant TBEV subviral particles

Tick-borne encephalitis (TBE) is one of the most dangerous human neurological infections occurring in Europe and Northern parts of Asia with thousands of cases and millions vaccinated against it. The risk of TBE might be assessed through analyses of the samples taken from wildlife or from animals which are in close contact with humans. Dogs have been shown to be a good sentinel species for these studies. Serological assays for diagnosis of TBE in dogs are mainly based on purified and inactivated TBEV antigens. Here we describe novel dog anti-TBEV IgG monoclonal antibody (MAb)-capture assay which is based on TBEV prME subviral particles expressed in mammalian cells from Semliki Forest virus (SFV) replicon as well as IgG immunofluorescence assay (IFA) which is based on Vero E6 cells transfected with the same SFV replicon. We further demonstrate their use in a small-scale TBEV seroprevalence study of dogs representing different regions of Finland. Altogether, 148 dog serum samples were tested by novel assays and results were compared to those obtained with a commercial IgG enzyme immunoassay (EIA), hemagglutination inhibition test and IgG IFA with TBEV infected cells. Compared to reference tests, the sensitivities of the developed assays were 90-100% and the specificities of the two assays were 100%. Analysis of the dog serum samples showed a seroprevalence of 40% on Åland Islands and 6% on Southwestern archipelago of Finland. In conclusion, a specific and sensitive EIA and IFA for the detection of IgG antibodies in canine sera were developed. Based on these assays the seroprevalence of IgG antibodies in dogs from different regions of Finland was assessed and was shown to parallel the known human disease burden as the Southwestern archipelago and Åland Islands in particular had considerable dog TBEV antibody prevalence and represent areas with high risk of TBE for humans.

Two years survival rate of Class II ART restorations in primary molars using two ways to avoid saliva contamination

AIM To compare the survival rates of Class II Atraumatic Restorative Treatment (ART) restorations placed in primary molars using cotton rolls or rubber dam as isolation methods. METHODS A total of 232 children, 6-7 years old, both genders, were selected having one primary molar with proximal dentine lesion. The children were randomly assigned into two groups: control group with Class II ART restoration made using cotton rolls and experimental group using rubber dam. The restorations were evaluated by eight calibrated evaluators (Kappa > 0.8) after 6, 12, 18 and 24 months. RESULTS A total of 48 (20.7%) children were considered dropout, after 24 months. The cumulative survival rate after 6, 12, 18 and 24 months was 61.4%, 39.0%, 29.1% and 18.0%, respectively for the control group, and 64.1%, 55.1%, 40.1% and 32.1%, respectively for the rubber dam group. The log rank test for censored data showed no statistical significant difference between the groups (P = 0.07). The univariate Cox Regression showed no statistical significant difference after adjusting for independent variables (P > 0.05). CONCLUSION Both groups had similar survival rates, and after 2 years, the use of rubber dam does not increase the success of Class II ART restorations significantly.