Evaluation of PCR primers to identify Porphyromonas endodontalis in apical periodontitis clinical samples.

We aimed to evaluate whether two commonly used PCR primers are effective to identify P. endodontalis and discriminate from other prevalent black-pigmented bacteria in apical periodontitis (AP). Endodontic canal samples from patients with asymptomatic AP (n = 20) were collected and cultured in anaerobiosis. Two primer sets to detect P. endodontalis were selected from the literature and first analyzed for their specificity in silico; and then tested on clinical isolates in vitro and finally, in apical exudates ex vivo. The identity of P. endodontalis was verified by PCR and Sanger sequencing with universal primers for bacterial V3-V6 regions 16S rDNA. Only one primer set showed specificity only for P. endodontalis clones in silico and also was specific for P. endodontalis in vitro and ex vivo.

Epigenetic regulation of TLR2-mediated periapical inflammation.

AIM: To determine the methylation pattern of TLR2 gene promoter and its association with the transcriptional regulation of periapical inflammatory and angiogenic responses in symptomatic and asymptomatic forms of apical periodontitis. METHODOLOGY: In this cross-sectional study, apical lesions were obtained from volunteers with asymptomatic apical periodontitis (AAP) (n = 17) and symptomatic apical periodontitis (SAP) (n = 17) scheduled for tooth extraction, and both total RNA and DNA were extracted. DNA was bisulfite-treated, a region of CpG island within the TLR2 gene was amplified by qPCR and the products were sequenced. Additionally, the mRNA expression of TLR2, TLR4, IL-6, IL-12, TNFalpha, IL-23, IL-10, TGFbeta, VEGFA and CDH5 was analysed by qPCR. The data were analysed with chi-square tests, Mann-Whitney or unpaired t-tests, and Spearman´s correlation; variable adjustments were performed using multiple linear regression (P < 0.05). RESULTS: TLR2 depicted a hypomethylated DNA profile at the CpG island in SAP when compared with AAP, along with upregulated expression of TLR2, with pro-inflammatory cytokines IL-6 and IL-23, and the angiogenesis marker CDH5 (P < 0.05). TLR2 methylation percentage negatively correlated with mRNA levels of IL-23 and CDH5 in apical periodontitis. Lower methylation frequencies of single CpG dinucleotides -8 and -10 localized in close proximity to nuclear factor κB (NFκB) binding within the TLR2 promoter were identified in SAP versus AAP (P < 0.05). Finally, unmethylated -10 and -8 single sites demonstrated up-regulation of IL-23, IL-10 and CDH5 transcripts compared to their methylated counterparts (P < 0.05). CONCLUSIONS: TLR2 gene promoter hypomethylation was linked to transcriptional activity of pro-inflammatory cytokines and angiogenic markers in exacerbated periapical inflammation. Moreover, unmethylated single sites in close proximity to NFκB binding were involved in active transcription of IL-23, IL-10 and CDH5.

Expression of Toll-like receptors 2 and 4 and its association with matrix metalloproteinases in symptomatic and asymptomatic apical periodontitis.

To determine Toll-like receptors (TLR)2 and TLR4 expression levels and associate them with matrix metalloproteinases (MMPs) in asymptomatic apical periodontitis (AAP), symptomatic apical periodontitis (SAP), and healthy controls. Apical tissue/lesion samples were obtained from chronic AAP (n = 35) and SAP (n = 29), and healthy periodontal ligament (HPL, n = 10) with indication of tooth extraction, respectively. mRNA expression levels of TLR2, TLR4, MMP-1, MMP-2, MMP-8, and MMP-13 were determined by real-time reverse-transcription polymerase chain reaction. The data were analyzed with Kruskal-Wallis and Dunn's pot hoc test (p < 0.05). The correlation coefficient was obtained using the Spearman correlation (p < 0.05). TLR2, MMP-1, MMP-2, and MMP-13 mRNA levels were the highest in SAP followed by AAP and controls (p < 0.05). TLR4 and MMP-8 were over expressed in AAP and SAP compared to HPL (p < 0.05). TLR2 positively correlated with TLR4, MMP-1, MMP-8, and MMP-13 in SAP (p < 0.05). TLR2 and TLR4 are overexpressed in apical lesions versus healthy periodontal ligament and correlate with collagenolytic MMPs. Particularly, TLR2 is overexpressed in SAP in association with MMP-1, MMP-8, and MMP-13. Our results suggest that the activation of TLR2 along with MMP overexpression might contribute to SAP clinical presentation and progression. TLRs, MMPs, and their interaction can explain the clinical presentations and evolution of apical periodontitis and might represent key targets for new diagnostic and treatment approaches.

Human periodontal ligament fibroblasts synthesize C-reactive protein and Th-related cytokines in response to interleukin (IL)-6 trans-signalling.

AIM: To characterize the potential of human periodontal ligament fibroblasts (HPLF) to synthesize CRP and Th-related cytokines in response to IL-6 in periodontal health and apical inflammation. METHODOLOGY: Primary HPLF stimulated with IL-6, soluble(s) IL-6 receptor (R) and controls were assayed for CRP, Th1, Th2, Th17 and Treg-related cytokines by quantitative real-time PCR and ELISA, respectively. IL-6R mRNA expression and its soluble protein levels were screened in HPLF cultures, and ex vivo samples of healthy periodontal ligaments (n = 5) and apical lesions (n = 13). Data were analysed with ANOVA or unpaired t-test. RESULTS: 0.5 ng mL-1 IL-6 plus 1 ng mL-1 of its soluble receptor (sIL-6R) for 24 h was effective in inducing CRP production. IL-6 alone had a mild dose-dependent effect; co-stimulation with sIL-6R significantly enhanced this effect, whereas it was completely abolished by the addition of IL-6R blocking antibody (P < 0.05). Similarly, higher mRNA expression and protein levels of Th1, Th17 and partially Treg-related cytokines were found for IL-6 combined with its soluble receptor versus the nonstimulated group and IL-6R antibody (P < 0.05). IL-6R mRNA expression was slightly induced by IL-6 compared to THP-1 cells, but sILR-6 protein could not be detected in HPLF. High sIL-6R levels were detected in apical lesions and were immunolocalized to mononuclear inflammatory cells and proliferating epithelium. CONCLUSION: IL-6 trans-signalling induced Th1 and Th17-related cytokines and represents an extra-hepatic mechanism for PCR synthesis in human periodontal ligament fibroblasts, contributing to explain the bone-destructive phenotype of apical lesions and eventually its systemic complications.

[D-repeat polymorphism in the ASPN gene in knee osteoarthritis in females in Torreón, Coahuila. Case-control study]. / Polimorfismo del D repetido en gen ASPN en osteoartritis de rodilla en mujeres de Torreón, Coahuila. Estudio de casos y controles.

BACKGROUND: Knee osteoarthritis (OA) is one of the most common and disabling disorders of the musculoskeletal system. It may affect any ethnic group and causes variable degrees of disability. Various risk factors have been associated with the development and progression of this condition, such as: age, genetic and occupational factors, trauma, menopause, diabetes mellitus, obesity, and gender, among others. Distinguishing these factors, whether individually or altogether, is important to prevent or diagnose and treat the disease early on. METHODS: A case-control study was conducted in 260 females in Torre6n, Coahuila, to analyze the relationship between primary knee osteoarthritis and the D-repeat polymorphism in the ASPN gene (asporin). 130 females with knee osteoarthritis and 130 healthy female controls were included. RESULTS: In this study, menopause and the D16 allele variant were found to be significant risk factors for knee osteoarthritis (p = 0.002, OR 2.656, CI 95% 1.412-4.998; p = 0.026, OR 2.418, CI 95% 1.111-5.263, respectively). The D12 variant was found to be a significant protective allele. CONCLUSIONS: As far as we know, this is the first case-control study in Mexican women that suggests that menopause and the D-repeat polymorphism in the ASPN gene are associated with knee OA.

Near-universal prevalence of Pneumocystis and associated increase in mucus in the lungs of infants with sudden unexpected death.

BACKGROUND: Pneumocystis without obvious accompanying pathology is occasionally reported in autopsied infant lungs. Its prevalence and significance are unknown. Interestingly, this mild infection induces a strong activation of mucus secretion-related genes in young immunocompetent rodents that has not been explored in infants. Excess mucus is induced by multiple airway offenders through nonspecific pathways and would explain a cofactor role of Pneumocystis in respiratory disease. We undertook characterization of the prevalence of Pneumocystis and associated mucus in infant lungs. METHODS: Samples from 128 infants (mean age, 101 days) who died suddenly and unexpectedly in Santiago during 1999-2004 were examined for Pneumocystis using nested polymerase chain reaction (nPCR) amplification of the P. jirovecii mtLSU ribosomal RNA gene and immunofluorescence microscopy (IF). Pneumocystis-negative infants 28 days and older and their age-closest positives were studied for MUC5AC expression and Pneumocystis burden by Western blot and quantitative PCR, respectively. RESULTS: Pneumocystis DNA was detected by nPCR in 105 of the 128 infants (82.0%) and Pneumocystis organisms were visualized by IF in 99 (94.3%) of the DNA-positive infants. The infection was commonest at 3-4 months with 40 of 41 (97.6%) infants of that age testing positive. MUC5AC was significantly increased in Pneumocystis-positive tissue specimens (P = .013). Death was unexplained in 113 (88.3%) infants; Pneumocystis was detected in 95 (84.0%) of them vs 10 of 15 (66.7%) with explained death (P = .28). CONCLUSIONS: A highly focal Pneumocystis infection associated to increased mucus expression is almost universally present in the lungs of infants dying unexpectedly in the community regardless of autopsy diagnosis.

Heterozygote excess through life history stages in Cestrum miradorense Francey (Solanaceae), an endemic shrub in a fragmented cloud forest habitat.

Comparisons of genetic diversity and population genetic structure among different life history stages provide important information on the effect of the different forces and micro-evolutionary processes that mould diversity and genetic structure after fragmentation. Here we assessed genetic diversity and population genetic structure using 32 allozymic loci in adults, seeds, seedlings and juveniles of eight populations of the micro-endemic shrub Cestrum miradorense in a highly fragmented cloud forest in central-eastern Mexico. We expected that due to its long history or rarity, this species may have endured the negative effects of fragmentation and would show moderate to high levels of genetic diversity. High genetic diversity (H(e) = 0.445 ± 0.03), heterozygote excess (F(IT) = -0.478 ± 0.034, F(IS) = -0.578 ± 0.023) and low population differentiation (F(ST) = 0.064 ± 0.011) were found. Seeds had higher genetic diversity (H(e) = 0.467 ± 0.05) than the later stages (overall mean for adults, seedlings and juveniles H(e) = 0.438 ± 0.08). High gene flow was observed despite the fact that the fragmentation process began more than 100 years ago. We conclude that the high genetic diversity was the result of natural selection, which favours heterozygote excess in all stages, coupled with a combination of a reproductive system and seed/pollen dispersal mechanisms that favour gene flow.

Estudo da rede de serviços de saude na região de fronteira Argentina, Brasil e Paraguai: 2001-2002

A área da fronteira entre a Argentina-Brasil-Paragua, constituída pelas cidade de Porto Iguaçu, Foz de Iguaçu(Foz) e Cidade do Leste(CDE), é um setor com um importante fluxo de pessoas entre os três países, o que tem impacto na demanda de atenção de saúde dos mesmos, em um grau que é necessário conhecer. Com esta finalidade foi contratada uma consultoria que, em primeiro lugar, permitisse elaborar um diagnóstico da situação dos sistemas de saúde nas três cidades fronteiriças, que permitisse estabelecer uma proposta tri-nacional para solução dos principais problemas de interesse comum. Para levar adiante o estudo aplicou-se a metodologia de análise de Rede Assistencial elaborada e aplicada no Chile pelos técnicos do Ministério da Saúde. Essa metodologia consistiu na elaboração e aplicação de um guia para coleta de informações, associadas à visita dos consultores aos três países. Não foi possível levantar a totalidade da informação solicitada, obtendo-se, contudo, um nível que permite realizar análises e elaborar propostas para a região. O estudo abrangeu as cidades Tríplice Fronteira

Riluzole decreases the abstinence syndrome and physical dependence in morphine-dependent mice.

The effects of the antiglutamatergic agent, riluzole, were examined on the antinociceptive action of morphine, on the induction of physical dependence, and on the expression of the abstinence syndrome to the opiate in mice. Morphine was administered as a single dose (200 mg/kg) of a slow-release preparation. Acute and chronic administration of riluzole decreased the analgesic response to morphine, the intensity of abstinence behavior (administered 30 min before a dose of naloxone), and the development of physical dependence (repeatedly administered during the period of chronic morphine treatment).