Effect of ethylenediaminetetraacetic acid irrigation on immune-inflammatory response in teeth submitted to regenerative endodontic therapy.

AIM: To analyse longitudinally the immune-inflammatory response in teeth of mice that underwent a regenerative protocol with or without the use of ethylenediaminetetraacetic acid (EDTA) to irrigate the root canal system. METHODOLOGY: First maxillary molars of mice were devitalized using size 10 and 15 files. Teeth were divided into the following groups: Empty - the canals were left empty; Blood Clot (BC) - the canals were filled with a blood clot; and EDTA + Blood - the canals were irrigated with 0.06 mL of 17% EDTA for 1 min and filled with a blood clot. Access cavities were restored with Coltosol® . Animals were sacrificed at 7, 14 or 21 days after the operative procedures, and teeth were collected. RNA was extracted, mRNA expression of the cytokines IGF, NGF, IL-1α, IL-10, TGF and VEGF was assessed using real-time PCR, and the anova Kruskal-Wallis test was used. RESULTS: IL-1 mRNA expression was significantly higher in the EDTA + BC group than in the Empty and BC groups at the 7th and 14th days of evaluation (P < 0.05). IL-10 mRNA expression was similar across the three groups at all time periods. TGF-ß mRNA expression in the EDTA + BC group was significantly higher on the 7th and 21st days than on the 14th (P < 0.05); at day 21, TGF-ß mRNA expression was similar between the BC and EDTA + BC groups but significantly higher than in the Empty group (P < 0.05). IGF mRNA expression was significantly higher in the EDTA + BC group than in the other groups at all time periods. VEGF mRNA expression remained unchanged throughout the experimental period in all groups (P > 0.05). NGF mRNA expression was similar amongst all groups at the 7th and 21st days (P > 0.05). At the 14th day, however, there was a significant increase in NGF mRNA expression in the EDTA + Blood group (P < 0.05) when compared with the expression in the other groups. CONCLUSION: EDTA promoted increased expression of factors that have the potential to improve the outcome of regenerative endodontic treatment.

Immunological profile of teeth with inflammatory periapical disease from chronic liver disease patients.

AIM: To evaluate the mRNA expression levels of the cytokines interferon-γ, tumour necrosis factor-α, interleukin (IL)-1ß, IL-10, IL-6, VEGF, and AGT and the chemokine CCL2/MCP-1 in periapical interstitial fluid associated with root canal infections before and after the reduction of the bacterial load using a cleaning procedure. METHODOLOGY: The case group included 11 patients with chronic liver disease, and the control group included 11 healthy patients. Clinical samples were taken from teeth with pulp necrosis. After cleaning and drying the canal, three paper points were introduced into the root canal and passed through the root apex (2 mm) into the periapical tissues for 1 min. The samples were collected immediately after root canal cleaning and 7 days later to characterize those gene expression levels using real-time PCR. The data were subjected to the Shapiro-Wilk and the Wilcoxon tests. RESULTS: In the control group, significantly increased expression of the pro-inflammatory cytokines IFN-γ and TNF-α was observed in teeth with restrained bacterial loads (day 7) (P < 0.05). Similarly, increased TNF-α expression was found on day 7 in the liver group (P < 0.05). No differences were observed in the expression levels of the IL-1ß, IL-10 and, IL-6, MCP-1/CCL-2 and VEGF between the first collection (day 0) and second collection (day 7), over time in either group. CONCLUSION: Chronic liver disease patients exhibited sufficient immunologic ability showing relatively similar expression levels of cytokines, chemokines and angiogenic factors in periapical samples compared with the responses from no-chronic liver disease patients. The outcomes of this study suggest that liver impairment did not compromise the periapical immune response.

Technical note: PaGELL v.1.5: A flexible parametric program for the Bayesian analysis of longevity data within the context of animal breeding.

This technical note presents the program PaGELL v.1.5 (Parametric Genetic Evaluation of Lifespan in Livestock), a flexible software program to analyze (right-censored) longevity data in livestock populations, with a special emphasis on the genetic evaluation of the breeding stock. This software relies on a parametric generalization of the proportional hazard model; more specifically, the baseline hazard function follows a Weibull process and flexibility is gained by including an additional time-dependent effect with the number of change points defined by the user. The program can accommodate 3 different sources of variation (i.e., systematic, permanent environmental, and additive genetic effects) and both fixed and time-dependent patterns (only for systematic and permanent environmental effects). Analyses are performed within a Bayesian context by sampling from the joint posterior distribution of the model, and model fit can be easily determined by the calculation of the deviance information criterion. Although this software has already been used on field data sets, its performance has been double-checked on simulated data set, and results are presented in this technical note. PaGELL v.1.5 was written in Fortran 95 language and, after compiling with the GNU Fortran Compiler v.4.7 and later, it has been tested in Windows, Linux, and MacOS operating systems (both 32- and 64-bit platforms). This program is available at http://www.casellas.info/files/pageII.zip.

Western diet in the perinatal period promotes dysautonomia in the offspring of adult rats.

The present study investigated the impact of a western diet during gestation and lactation on the anthropometry, serum biochemical, blood pressure and cardiovascular autonomic control on the offspring. Male Wistar rats were divided into two groups according to their mother's diet received: control group (C: 18% calories of lipids) and westernized group (W: 32% calories of lipids). After weaning both groups received standard diet. On the 60th day of life, blood samples were collected for the analysis of fasting glucose and lipidogram. Cardiovascular parameters were measured on the same period. Autonomic nervous system modulation was evaluated by spectrum analysis of heart rate (HR) and systolic arterial pressure (SAP). The W increased glycemia (123±2 v. 155±2 mg/dl), low-density lipoprotein (15±1 v. 31±2 mg/dl), triglycerides (49±1 v. 85±2 mg/dl), total cholesterol (75±2 v. 86±2 mg/dl), and decreased high-density lipoprotein (50±4 v. 38±3 mg/dl), as well as increased body mass (209±4 v. 229±6 g) than C. Furthermore, the W showed higher SAP (130±4 v. 157±2 mmHg), HR (357±10 v. 428±14 bpm), sympathetic modulation to vessels (2.3±0.56 v. 6±0.84 mmHg2) and LF/HF ratio (0.15±0.01 v. 0.7±0.2) than C. These findings suggest that a western diet during pregnancy and lactation leads to overweight associated with autonomic misbalance and hypertension in adulthood.

Immunological profile of periapical endodontic infections from HIV- and HIV+ patients.

AIM: To evaluate CD4(+) CD28(+) and CD8(+) T-cell genes and the gene expression of IFN-γ, TNF-α, IL-1-ß, IL-17A, IL-10, CCL-2/MCP-1, CCL-4, CCL-5 (RANTES), CXCR4, CCR5 and RANKL from cells in the periapical interstitial fluid from root canal infections in healthy patients (HIV-) and HIV-positive individuals (HIV+). METHODOLOGY: Subjects included 20 HIV- and 23 HIV+ patients referred to the School of Dentistry at the Universidade Federal de Minas Gerais (Belo Horizonte, MG, Brazil). Almost all HIV+ patients were undergoing highly active antiretroviral therapy (HAART). Clinical samples were taken from teeth with pulp necrosis, and no patients had acute periapical symptoms at the time of the appointments. After cleaning and drying, 3 paper points were introduced into the root canal, passing passively through the root apex (2 mm) into the periapical tissues for 1 min. The samples were collected immediately after root canal cleaning and 7 days later (restrained root canal bacterial load) to characterize those gene expressions using real-time PCR. RESULTS: Significantly higher levels of CD4(+) CD28(+) and CD8(+) T cells in teeth with restrained bacterial loads (second collection) compared with the first collection were observed in both HIV- and HIV+ samples. In HIV- patients, an increase in IL-10 and CXCR4 expression was demonstrated as well as a decrease in pro-inflammatory cytokines such as RANKL, IFN-γ, IL1-ß and CCL5. However, in HIV+ patients an increase in cytokines IFN-γ, IL-1-ß, TNF-α and IL-17A, and chemokines CCL-2, CXCR4 and CCR5 were observed. The chemokine CCL-5 was not detected in HIV+ individuals. CONCLUSIONS: These findings suggest that after reducing the root canal bacterial load in HIV- individuals an anti-inflammatory response is generated whilst in HIV+ patients a pro-inflammatory response is sustained in the periapical area.

Influence of population and exercise protocol characteristics on hemodynamic determinants of post-aerobic exercise hypotension.

Due to differences in study populations and protocols, the hemodynamic determinants of post-aerobic exercise hypotension (PAEH) are controversial. This review analyzed the factors that might influence PAEH hemodynamic determinants, through a search on PubMed using the following key words: "postexercise" or "post-exercise" combined with "hypotension", "blood pressure", "cardiac output", and "peripheral vascular resistance", and "aerobic exercise" combined only with "blood pressure". Forty-seven studies were selected, and the following characteristics were analyzed: age, gender, training status, body mass index status, blood pressure status, exercise intensity, duration and mode (continuous or interval), time of day, and recovery position. Data analysis showed that 1) most postexercise hypotension cases are due to a reduction in systemic vascular resistance; 2) age, body mass index, and blood pressure status influence postexercise hemodynamics, favoring cardiac output decrease in elderly, overweight, and hypertensive subjects; 3) gender and training status do not have an isolated influence; 4) exercise duration, intensity, and mode also do not affect postexercise hemodynamics; 5) time of day might have an influence, but more data are needed; and 6) recovery in the supine position facilitates systemic vascular resistance decrease. In conclusion, many factors may influence postexercise hypotension hemodynamics, and future studies should directly address these specific influences because different combinations may explain the observed variability in postexercise hemodynamic studies.

Influence of population and exercise protocol characteristics on hemodynamic determinants of post-aerobic exercise hypotension

Due to differences in study populations and protocols, the hemodynamic determinants of post-aerobic exercise hypotension (PAEH) are controversial. This review analyzed the factors that might influence PAEH hemodynamic determinants, through a search on PubMed using the following key words: “postexercise” or “post-exercise” combined with “hypotension”, “blood pressure”, “cardiac output”, and “peripheral vascular resistance”, and “aerobic exercise” combined only with “blood pressure”. Forty-seven studies were selected, and the following characteristics were analyzed: age, gender, training status, body mass index status, blood pressure status, exercise intensity, duration and mode (continuous or interval), time of day, and recovery position. Data analysis showed that 1) most postexercise hypotension cases are due to a reduction in systemic vascular resistance; 2) age, body mass index, and blood pressure status influence postexercise hemodynamics, favoring cardiac output decrease in elderly, overweight, and hypertensive subjects; 3) gender and training status do not have an isolated influence; 4) exercise duration, intensity, and mode also do not affect postexercise hemodynamics; 5) time of day might have an influence, but more data are needed; and 6) recovery in the supine position facilitates systemic vascular resistance decrease. In conclusion, many factors may influence postexercise hypotension hemodynamics, and future studies should directly address these specific influences because different combinations may explain the observed variability in postexercise hemodynamic studies.

Genetic evaluation of Alpine goats using different milk control intervals.

The objective of this study was to compare the results of genetic evaluations by using different milk control intervals to reduce the cost of milk yield controls without harming the quality of genetic evaluation of the animals. We analyzed test day milk yield data from the Goat Sector of Universidade Federal de Viçosa. After editing and checking for errors in the database, there were 20,710 records of test day milk yield for the 667 first lactations of Alpine goats, constituting the complete file, with 7-day control intervals. Information on specific weeks was excluded from the complete file to create files with data on control intervals of 15, 21, and 28 days. The RENPED program was used to recode the pedigree and data files and correct pedigree errors; the WOMBAT program was used for genetic evaluations of the 4 files. The following comparison criteria of analysis results were used: logarithm of the function of the restricted maximum likelihood, length of the analyses in seconds, Pearson and Spearman correlations, and common elimination percentage among the areas below the regression curve of the genetic values of the animals. Overall, it is recommended that a 7-day interval among milk controls should be used in breeding programs and farms with a high technical level. Intervals of 14 and 21 days can achieve satisfactory results combined with a lower data collection cost for farms with an average-to-low technical level, less effective size, and genetic variability that depend on external genetic material for genetic improvement.

Microbiologic profile of endodontic infections from HIV- and HIV+ patients using multiple-displacement amplification and checkerboard DNA-DNA hybridization.

OBJECTIVE: To compare the microbiota of endodontic infections in necrotic pulp from HIV-negative and HIV-positive subjects. MATERIALS AND METHODS: Root canal samples from necrotic pulp were collected from 40 HIV- and 20 HIV+ subjects. Pulps were amplified using multiple displacement amplification (MDA). Then, checkerboard DNA-DNA hybridization was employed to assess the levels of 107 microbial taxa. The percentage of DNA probe count and the percentage of teeth colonized by each test species were investigated. Significant differences between groups regarding proportions of taxa and prevalence of the test species were sought using the Mann-Whitney test and the Chi-square analysis, respectively. RESULTS: The most prevalent taxa detected were Dialister pneumosintes, Stenotrophomonas maltophilia, Streptococcus sobrinus, Corynebacterium diphteriae, and Helicobacter pylori among HIV- subjects and D. pneumosintes, Prevotella tannerae, Porphyromonas gingivalis, Parvimonas micra, Prevotella nigrescens, and Corynebacterium diphtheriae among HIV+ individuals. D. pneumosintes, C. diphtheria, and C. albicans were the most abundant species in the HIV- group, whereas the predominant taxa in HIV+ samples were P. tannerae, D. pneumosintes and Olsenella uli. P. tannerae, O. uli, Veilonella dispar, Bacteroides fragilis, and Actinomyces meyeri were significantly more abundant in HIV+ samples. CONCLUSIONS: There were significant differences in the prevalence and proportions of specific microbial taxa between HIV- and HIV+ individuals. The root canal microbiota may represent a reservoir of important oral and medical pathogens, mainly in HIV+ individuals.

Cytokine expression in response to root canal infection in gnotobiotic mice.

AIM: To examine cytokine expression profiles during periapical lesion development in response to synergetic human pathogens in a gnotobiotic mouse model. METHODOLOGY: Human strains of Fusobacterium nucleatum and Peptostreptococcus prevotii were inoculated into the root canals of germ-free mice in either mono- or bi-association. Animals were killed 7 and 14 days after infection, and periapical tissues were collected. mRNA expression of the cytokines IFN-γ, TNF-α, Receptor activator of nuclear factor kappa-B ligand (RANKL), IL-10, IL-4 and transforming growth factor ß (TGF-ß) was assessed using real-time PCR. Levene's test was used to assess the equality of variance of the data, whereas a t-test for independent samples was used to evaluate the significance of the differences between groups (P < 0.05). RESULTS: The mRNA expression of IFN-γ and TNF-α was up-regulated by F. nucleatum during the acute (day 7) and chronic phase (day 14) of periapical lesion development. However, in bi-infection the expression of IFN-γ and TNF-α were effectively absent at both time-points. RANKL mRNA expression was down-regulated during dual infection at the chronic phase. As IL-4 expression was similar at both time-points, IL-4 does not appear to be involved in the periapical response to these bacterial strains. IL-10 was up-regulated during the chronic phase by mono-infection with either F. nucleatum or P. prevotii. Dual infection increased TGF-ß mRNA expression on day 7, which paralleled the decrease in IFN-γ and TNF-α mRNA levels at the same time-point. F. nucleatum increased TGF-ß mRNA expression during the chronic phase. CONCLUSION: Cytokine profiles depend on the nature of the bacterial challenge. Both TGF-ß and IL-10 appeared to be regulating the proinflammatory cytokine responses at both time-points of the periapical immune response.

Microbiota of deciduous endodontic infections analysed by MDA and Checkerboard DNA-DNA hybridization.

AIMS: To evaluate the microbiota of endodontic infections in deciduous teeth by Checkerboard DNA-DNA hybridization after uniform amplification of DNA in samples by multiple displacement amplification (MDA). METHODOLOGY: Forty samples from the root canal system of deciduous teeth exhibiting pulp necrosis with or without radiographically detectable periradicular/interradicular bone resorption were collected and 32 were analysed, with three individuals contributing two samples; these were MDA-amplified and analysed by Checkerboard DNA-DNA hybridization for levels of 83 bacterial taxa. Two outcome measures were used: the percentage of teeth colonized by each species and the mean proportion of each bacterial taxon present across all samples. RESULTS: The mean amount of DNA in the samples prior to amplification was 5.2 (±4.7) ng and 6.1 (±2.3) µg after MDA. The mean number of species detected per sample was 19 (±4) (range: 3-66) to the nearest whole number. The most prevalent taxa were Prevotella intermedia (96.9%), Neisseria mucosa (65.6%), Prevotella nigrescens (56.2%) and Tannerella forsythia (56.2%). Aggregatibacter (Haemophilus) aphrophilus and Helicobacter pylori were not detected. P. intermedia (10%), Prevotella tannerae (7%) and Prevotella nigrescens (4.3%) presented the highest mean proportions of the target species averaged across the positive samples. CONCLUSION: Root canals of infected deciduous teeth had a diverse bacterial population. Prevotella sp. were commonly found with P. intermedia, Prevotella tannerae and Prevotella nigrescens amongst the most prominent species detected.

Flow cytometry for diepoxybutane test analysis.

The main feature of Fanconi anemia (FA) is the high sensitivity of the cells to the clastogenic agent, diepoxybutane (DEB). Thus, differential diagnosis of this syndrome can be made by cytogenetic analysis; adding DEB to lymphocytes in culture (DEB test) increases the number of chromosome breaks. Fanconi anemia cells have an abnormal cell cycle, with an increased frequency of cells arrested at G2. In order to determine if flow cytometry can be utilized for FA diagnosis, we cultivated lymphocytes with DEB and analyzed them for G2 accumulation. Lymphocytes cultivated for 72 h were labeled with CD3 antibody and propidium iodide for analysis of the cells in the G2 phase. Cultures of lymphocytes from two FA patients who were diagnosed by the DEB test and six control individuals with a negative DEB test had 55.26% (SD +/- 6.97) and 2.81% (SD +/- 0.22) cells arrested at G2, respectively. We conclude that flow-cytometry analysis of cells exposed to DEB can be useful for FA diagnosis.

Use of multiple-displacement amplification and checkerboard DNA-DNA hybridization to examine the microbiota of endodontic infections.

Multiple-displacement amplification (MDA) has been used to uniformly amplify bacterial genomes present in small samples, providing abundant targets for molecular analysis. The purpose of this investigation was to combine MDA and checkerboard DNA-DNA hybridization to examine the microbiota of endodontic infections. Sixty-six samples were collected from teeth with endodontic infections. Nonamplified and amplified samples were analyzed by checkerboard DNA-DNA hybridization for levels and proportions of 77 bacterial taxa. Counts, percentages of DNA probe counts, and percentages of teeth colonized for each species in amplified and nonamplified samples were computed. Significance of differences for each species between amplified and nonamplified samples was sought with Wilcoxon signed-rank test and adjusted for multiple comparisons. The amount of DNA in the samples ranged from 6.80 (+/- 5.2) ng before to 6.26 (+/- 1.73) mug after MDA. Seventy of the 77 DNA probes hybridized with one or more of the nonamplified samples. All probes hybridized with at least one sample after amplification. Most commonly detected species at levels of >10(4) in both amplified and nonamplified samples were Prevotella tannerae and Acinetobacter baumannii at frequencies between 89 and 100% of samples. The mean number of species at counts of >10(4) in amplified samples was 51.2 +/- 2.2 and in nonamplified samples was 14.5 +/- 1.7. The endodontic microbiota was far more complex than previously shown, although microbial profiles at teeth with or without periradicular lesions did not differ significantly. Species commonly detected in endodontic samples included P. tannerae, Prevotella oris, and A. baumannii.

Protective effect of an aqueous extract of Harpagophytum procumbens upon Escherichia coli strains submitted to the lethal action of stannous chloride.

Regardless of its lethal effects upon Escherichia coli (E. coli) cultures trough the production of free radicals (FR), stannous chloride (SnCl2) remains to be the most used reducing agent on the production of technetium-99m radiopharmaceuticals, to obtain images on nuclear medicine. Moreover, authors have reported that vegetal extracts are able to protect Escherichia coli cultures against the cytotoxicity of this agent. Harpagophytum procumbens, also known as Devil's Claw, is a plant used in folk medicine, as an analgesic and anti-inflammatory in cases of joint and back pain, on the treatment of degenerative rheumatoid arthritis, osteoarthritis, kidney inflammation and heart diseases. The presence of this extract reduced the lesive effects of SnCl2 upon E. coli AB1157 (proficient in DNA repair), BW9091 (deficient in the xthA gene) and BH110 (deficient in the xthA, nfo and fpg genes) cultures, and the deficient strains (BW9091 e BH110) were more sensible to this SnCl2 action than the proficient one. The substances in the extract could be acting as: (i) chelator of the stannous ions, avoiding the generation of FR, (ii) FR scavenger, protecting the cells against the oxidation, and/or (iii) an oxidant compound acting upon the stannous ions, reducing the SnCl2 cytotoxicity.

Guava extract (Psidium guajava) alters the labelling of blood constituents with technetium-99m.

Psidium guajava (guava) leaf is a phytotherapic used in folk medicine to treat gastrointestinal and respiratory disturbances and is used as anti-inflammatory medicine. In nuclear medicine, blood constituents (BC) are labelled with technetium-99m ((99m)Tc) and used to image procedures. However, data have demonstrated that synthetic or natural drugs could modify the labelling of BC with (99m)Tc. The aim of this work was to evaluate the effects of aqueous extract of guava leaves on the labelling of BC with (99m)Tc. Blood samples of Wistar rats were incubated with different concentrations of guava extract and labelled with (99m)Tc after the percentage of incorporated radioactivity (%ATI) in BC was determined. The results suggest that aqueous guava extract could present antioxidant action and/or alters the membrane structures involved in ion transport into cells, thus decreasing the radiolabelling of BC with (99m)Tc. The data showed significant (P<0.05) alteration of ATI in BC from blood incubated with guava extract.

Citotoxic effects of stannous salts and the action of Mytenus ilicifolia, Bacchris genistelloides and Cymbopogon citratus aqueous extracts

In nuclear medicine, stannous, as stannous chloride (SnCl2) and stannous fluoride (SnF2), are used as reducing agents to obtain radiopharmaceuticals labeled with technetium-99m. In the literature, the SnCl2 action was studied and it seems to be mediated through free radicals (FR) production in a Fenton-like reaction. In this work it was evaluated: (i) the in vitro SnF2 effects in different concentrations using pBCKS plasmid deoxyribonucleid acid (DNA); (ii) the SnF, effects in different Escherichia coli (E.coli) cultures, proficient or deficient in DNA repair genes, treated simultaneously with FR scavengers; and (iii) the biological effects of Maytenus ilicifolia, Baccharis genistelloides and Cymbopogon citratus aqueous extracts on the SnCL2 action in E.coli culture. The SnF2 treatment induced plasmidd DNA damages (single and double DNA strand breaks), in a dose-dependent manner. Citotoxicity mediated by SNf2 was observed and the simultaneous tratment with FR scavengers has increased the cell survival, suggesting the participation of FR on the SnF2-deleterious effects. The vegetal extrracts prottected the E.coli cells agains the SnCl2 effects. The components of the extracts could be interacting with SnCl2 blocking its participation in the FR formation.