Oxygen tension modulates the cytokine response of oral epithelium to periodontal bacteria

Background: There is an inverse relationship between pocket depth and pocket oxygen tension with deep pockets being associated with anaerobic bacteria. However, little is known about how the host tissues respond to bacteria under differing oxygen tensions within the periodontal pocket. Aim: To investigate the effect of different oxygen tensions upon nuclear factor-kappa B (NF-?B) activation and the inflammatory cytokine response of oral epithelial cells when exposed to nine species of oral bacteria. Materials and Methods: H400 oral epithelial cells were equilibrated at 2%, 10% or 21% oxygen. Cells were stimulated with heat-killed oral bacteria at multiplicity of infection 10:1, Escherichia coli lipopolysaccharide (15 µg/ml) or vehicle control. Interleukin-8 (IL-8) and tumour necrosis factor-alpha (TNF-a) levels were measured by enzyme-linked immunosorbent assay and NF-?B activation was measured by reporter vector or by immunohistochemical analysis. Results: Tannerella forsythensis, Porphyromonas gingivalis and Prevotella intermedia elicited the greatest epithelial NF-?B activation and cytokine responses. An oxygen-tension-dependent trend in cytokine production was observed with the highest IL-8 and TNF-a production observed at 2% oxygen and lowest at 21% oxygen. Conclusions: These data demonstrate a greater pro-inflammatory host response and cell signalling response to bacteria present in more anaerobic conditions, and hypersensitivity of epithelial cells to pro-inflammatory stimuli at 2% oxygen, which may have implications for disease pathogenesis and/or therapy.

Occupational exposure to blood borne pathogens among healthcare workers and preventative strategies

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Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense against pathogens

Plant oxylipins are a large family of metabolites derived from polyunsaturated fatty acids. The characterization of mutants or transgenic plants affected in the biosynthesis or perception of oxylipins has recently emphasized the role of the so-called oxylipin pathway in plant defense against pests and pathogens. In this context, presumed functions of oxylipins include direct antimicrobial effect, stimulation of plant defense gene expression, and regulation of plant cell death. However, the precise contribution of individual oxylipins to plant defense remains essentially unknown. To get a better insight into the biological activities of oxylipins, in vitro growth inhibition assays were used to investigate the direct antimicrobial activities of 43 natural oxylipins against a set of 13 plant pathogenic microorganisms including bacteria, oomycetes, and fungi. This study showed unequivocally that most oxylipins are able to impair growth of some plant microbial pathogens, with only two out of 43 oxylipins being completely inactive against all the tested organisms, and 26 oxylipins showing inhibitory activity toward at least three different microbes. Six oxylipins strongly inhibited mycelial growth and spore germination of eukaryotic microbes, including compounds that had not previously been ascribed an antimicrobial activity such as 13-keto-9(Z),11(Z),15(Z)- octadecatrienoic acid and 12-oxo-10,15(Z)-phytodienoic acid. Interestingly this first large-scale comparative assessment of the antimicrobial effects of oxylipins reveals that regulators of plant defense responses are also the most active oxylipins against eukaryotic microorganisms, suggesting that such oxylipins might contribute to plant defense through their effects both on the plant and on pathogens, possibly through related mechanisms. © 2005 American Society of Plant Biologists.

Efeitos da azilsartana sobre a doença periodontal em um modelo experimental com ratos wistar

Periodontal diseases, highly prevalent disease in worldwide population, manifest primarily in two distinct entities: plaque-induced gingivitis and periodontitis. Periodontitis is a chronic inflammatory disease characterized of different levels of collagen, cementum, and alveolar bone destruction. Recent experimental studies demonstrated anti-inflammatory and antirreabsortive effect of antihypertensive agents of the angiotensin II receptor blockers class on periodontal disease. The aim of this study was to evaluate the effects of azilsartan (AZT), a potent inhibitor of the angiotensin II receptor which has minimal adverse effects on bone loss, inflammation, and the expression of matrix metallo proteinases (MMPs), receptor activator of nuclear factor kB ligand (RANKL), receptor activator of nuclear factor kB (RANK), osteoprotegerin (OPG), cyclooxygenase-2 (COX-2), and cathepsin K in periodontal tissue in a rat model of ligature-induced periodontitis. Male Wistar albino rats were randomly divided into 5 groups of 20 rats each: (1) nonligated, water; (2) ligated, water; (3) ligated, 1 mg/kg AZT; (4) ligated, 5 mg/kg AZT; and (5) ligated, 10 mg/kg AZT. All groups were treated with water or AZT for 10 days. Periodontal tissues were analyzed by morphometric exam, histopathology and immunohistochemical detection of MMP-2, MMP-9, COX-2, RANKL, RANK, OPG, and cathepsin K. Levels of IL-1b, IL-10, TNF-a, myeloperoxidase (MPO), and glutathione (GSH) were determined by ELISA. Treatment with 5 mg/kg AZT resulted in reduced MPO (p˂0.05) and IL-1b (p˂0.05) levels and increased in Il-10 levels (p˂0.05). It was observed a reduced expression of MMP-2, MMP-9, COX-2, RANK, RANKL, cathepsin K, and a increased expression of OPG in the animals subjected to experimental periodontitis and threated with AZT (5 mg/kg). Conclusions: These findings suggest an anti-inflammatory and anti-reabsortive effects of AZT on ligature-induced periodontitis in rats.

Análise da imuno expressão das proteínas IL-17, IL-23 E RORγt na patogenia da doença periodontal

Periodontal disease is a chronic inflammatory condition primarily caused by bacteria in dental biofilm, which interact with the host, thus determining the nature of the resulting disease. Despite the wide knowledge about the pathogenesis of these diseases, the exact composition of the T cell profile during the active phase of the disease (Th1, Th2 or Th17) remains unknown. This study aimed to evaluate by immunohistochemical expression, the presence of the markers (IL-17, IL-23 and RORγt), involved in Th17 response in clinically healthy gingiva cases (n = 32), biofilm-induced gingivitis (n = 30), chronic periodontitis (n = 32) and aggressive periodontitis (n = 25), in order to analyze if the expression and/or distribution of these molecules in lymphocytes and macrophages, present in the inflammatory infiltrate of periodontal tissue, influences the tissue destruction observed in these diseases. The morphological analysis of cases was performed which assessed the intensity of the inflammatory infiltrate in mild, moderate and intense. For each case, in the area with the most representative immunostaining, 5 fields were chosen and analyzed, both for the intensity of the inflammatory infiltrate as for the quantity of immunostained cells, based on predetermined scores: score 0 (absence of inflammatory infiltrate/immunostaining), score 1 (the infiltrate/immunostaining covered less than 25% of the field area), score 2 (the infiltrate/immunostaining occupied between 25 and 50%) and score 3 (infiltrate/immunostaining present in over 50% of the field area). From this, a median was generated representing each case. The intensity of the inflammatory infiltrate correlated with the disease progression, in other words, it was crescent from clinically healthy gingiva to aggressive periodontitis (P <0.001). It was detected the presence of IL-17, IL-23 and RORγt in most of the evaluated cases and the number of immunostained cells correlated with the intensity of the inflammatory infiltrate (P <0.001) and with the clinical parameters analyzed (P <0.001), showing a positive correlation, mainly moderate. Aggressive periodontitis showed a higher percentage of immunostaining for all markers in relation to other clinical conditions assessed, suggesting a possible association of these markers with the progression of this disease, in which the higher the loss of periodontal support, the greater the amount of inflammatory infiltrate and larger the number of immunostained cells.

Avaliação do efeito anti-inflamatório dos fármacos: atorvastatin, carvedilol, olmesartan e telmisartan, em modelo experimental da doença periodontal induzida em ratos

A periodontite é uma doença crônica inflamatória mediada por marcadores inflamatórios, tais como as citocinas: IL-1β, IL-10 e TNF-α, que provoca a destruição dos tecidos gengivais e osso alveolar, causando perda de inserção dentária e posterior perda dental. A perda óssea é causada pela ativação de prostaglandinas oriundas do ácido araquidônico, através da ação da enzima ciclooxigenase 2 (COX-2), promovendo a liberação de enzimas proteolíticas, as metaloproteinases de matriz, principalmente a MMP-2 e MMP-9, que promovem reabsorção óssea. Além disso, ocorre o desequilíbrio entre a ação de RANKL e OPG, havendo uma maior ativação de RANKL, e por consequência a maior ativação de osteoclastos e maior reabsorção óssea. Mediadores inflamatórios e espécies reativas de oxigênio (ROS) produzidos localmente possuem potencial para disseminar na corrente sanguínea e iniciar ou exacerbar doenças sistêmicas como as cardiovasculares. O tratamento atual da doença consiste em terapêutica local, mas a necessidade de estudos sobre fármacos de atuação sistêmica culminou nesta pesquisa, que realizou a avaliação dos fármacos: atorvastatin, carvedilol, olmesartan e telmisartan, quanto a sua ação anti-inflamatória sobre a doença periodontal induzida por ligadura em ratos Wistar. Os animais foram divididos em 5 grupos, para cada fármaco, separadamente: (NL) grupo não ligado, (L) grupo ligado sem tratamento, (1mg/Kg) grupo ligado que recebeu dose de 1mg/Kg de fármaco, (5 ou 6 mg/Kg) grupo ligado que recebeu dose de 5 ou 6 mg/Kg de fármaco, (10 mg/Kg) grupo ligado que recebeu dose de 10mg/Kg de fármaco. Foram realizadas avaliações: histopatológica, perda óssea alveolar, imuno-histoquímica (para COX-2, MMP-2, MMP-9, RANK-L, RANK e OPG), e ELISA (para mieloperoxidase, glutationa, malonaldeído e as citocinas: IL-1β, IL-10 e TNF-α). Os grupos tratados com olmesartana a 6 mg/Kg, e atorvastatin, carvedilol e telmisartan a 10mg/Kg, mostraram diminuição da perda óssea, redução de: MPO, MDA, IL-1β, TNF-α, MMP-2, MMP-9, COX-2, RANKL/RANK, e aumento na expressão da OPG e da IL-10.

Estudo Clínico Longitudinal Comparativo da Condição Periodontal de Pilares Diretos de Próteses Parciais Removíveis Dento- Suportada e Dento-Muco-Suportada

Objective: To perform a long-term clinical evaluation of the periodontium of removable parti al denture (RPD) wearers, comparing the direct pillar teeth of tooth-supported and toothtissue supported RPDs. Method: Fifty patients with mean age of 45 years were enrolled in the study. The individuals were examined by a single examiner at the moment of denture installation and after 3, 6, 9 and 12 months. In each exam, the following parameters were verified: gingival recession (GR), probing depth (PD), plaque index (PI), gingival index (GI) e amount kerati nized mucosa (KM). All patients received oral hygiene instructions and prophylaxis and, when necessary, scaling and root planing. An analysis from the confidence interval was done to evaluate the endpoints regarding the type of denture in the direct pillar group. Results: The tooth-tissue supported dentures showed significantly higher GR, GI and PI values, and significantly lower KM values. Over time, neither of the types of denture presented statistically significant difference from the initial to the final examination for the parameters GR, PD, KM and GI, while the PI was significant only for the tooth-supported dentures. Conclusion: Pillar teeth adjacent to free ends presented a less favorable periodontal conditi on than the pillar teeth adjacent to intercalated spaces. However, the use of RPD did not aggravate the initial condition, after a follow-up period of 12 months. The findings of the study indicate that, within 1 year, there were no significant differences between the direct pillars of the toothsupported and tooth-ti ssue supported dentures, and suggest the need of professional follow up for a longer period.

The use of a magnetic attachment in a removable partial denture of a patient with periodontal tissue loss

O uso dos magnetos em prótese parcial removível é uma alternativa viável para eliminar a estrutura metálica que pode interferir na estética sem perder retenção e estabilidade. Os magnetos podem ser recomendados para pacientes com perda de tecido periodontal, desde que eles diminuam a transmissão de forças ao dente remanescente. O objetivo deste relato de caso clínico foi apresentar uma alternativa de tratamento protético para dentes pilares comprometidos periodontalmente e descrever as vantagens e desvantagens do uso dos magnetos em prótese parcial removível sobre os pontos de vista funcionais, biológicos e estéticos.

Estudo Clínico Longitudinal Comparativo da Condição Periodontal de Pilares Diretos de Próteses Parciais Removíveis Dento- Suportada e Dento-Muco-Suportada

Objective: To perform a long-term clinical evaluation of the periodontium of removable parti al denture (RPD) wearers, comparing the direct pillar teeth of tooth-supported and toothtissue supported RPDs. Method: Fifty patients with mean age of 45 years were enrolled in the study. The individuals were examined by a single examiner at the moment of denture installation and after 3, 6, 9 and 12 months. In each exam, the following parameters were verified: gingival recession (GR), probing depth (PD), plaque index (PI), gingival index (GI) e amount kerati nized mucosa (KM). All patients received oral hygiene instructions and prophylaxis and, when necessary, scaling and root planing. An analysis from the confidence interval was done to evaluate the endpoints regarding the type of denture in the direct pillar group. Results: The tooth-tissue supported dentures showed significantly higher GR, GI and PI values, and significantly lower KM values. Over time, neither of the types of denture presented statistically significant difference from the initial to the final examination for the parameters GR, PD, KM and GI, while the PI was significant only for the tooth-supported dentures. Conclusion: Pillar teeth adjacent to free ends presented a less favorable periodontal conditi on than the pillar teeth adjacent to intercalated spaces. However, the use of RPD did not aggravate the initial condition, after a follow-up period of 12 months. The findings of the study indicate that, within 1 year, there were no significant differences between the direct pillars of the toothsupported and tooth-ti ssue supported dentures, and suggest the need of professional follow up for a longer period.

Estudo imunoistoquímico do CD34 e podoplanina na doença periodontal

Angiogenesis and lymphangiogenesis are changes that occur due to gingival inflammation caused by microorganisms present in the biofilm, as well as the migration of immune cells and secretion of mediators in the aggressed site. This study aimed to research angiogenesis and lymphangiogenesis in 90 specimens of clinically healthy, with gingivitis and chronic periodontitis gingival tissue biopsies. The histological sections were evaluated by hematoxylin and eosin and the immunohistochemical technique through immunostaining for CD34 and podoplanin. To evaluate the angiogenic and lymphangiogenic indexes we performed a microvessel counting technique. The results showed that there is a correlation between the indexes (p = 0.030), however, we observed that periodontitis showed less lymphatic vessels than clinically healthy gingival tissue (p = 0.016). Podoplanin showed positive staining in the basal layers of the epithelium, and we observed a relationship between immunostaining intensity and the intensity of inflammatory infiltrate, with more intense staining in the presence of severe inflammatory infiltrate (p = 0.033). For this study, we concluded that there are fewer blood vessels in periodontitis compared with clinically healthy gingiva. The signaling present in the inflammatory process and the actual role of gingival blood and lymphatic vasculature are not fully understood, with further studies on angiogenesis and lymphangiogenesis being suggested.

Estudo imunoistoquímico do CD34 e podoplanina na doença periodontal

Angiogenesis and lymphangiogenesis are changes that occur due to gingival inflammation caused by microorganisms present in the biofilm, as well as the migration of immune cells and secretion of mediators in the aggressed site. This study aimed to research angiogenesis and lymphangiogenesis in 90 specimens of clinically healthy, with gingivitis and chronic periodontitis gingival tissue biopsies. The histological sections were evaluated by hematoxylin and eosin and the immunohistochemical technique through immunostaining for CD34 and podoplanin. To evaluate the angiogenic and lymphangiogenic indexes we performed a microvessel counting technique. The results showed that there is a correlation between the indexes (p = 0.030), however, we observed that periodontitis showed less lymphatic vessels than clinically healthy gingival tissue (p = 0.016). Podoplanin showed positive staining in the basal layers of the epithelium, and we observed a relationship between immunostaining intensity and the intensity of inflammatory infiltrate, with more intense staining in the presence of severe inflammatory infiltrate (p = 0.033). For this study, we concluded that there are fewer blood vessels in periodontitis compared with clinically healthy gingiva. The signaling present in the inflammatory process and the actual role of gingival blood and lymphatic vasculature are not fully understood, with further studies on angiogenesis and lymphangiogenesis being suggested.

Resposta clínica periodontal e controle glicêmico em pacientes com e sem diabetes tipo II portadores de periodontite crônica ao tratamento periodontal “full mouth desinfection”

There is a bidirectional association between periodontal disease (PD) and diabetes mellitus, in which diabetes favors the development of PD and PD, if left untreated, can worsen the metabolic control of diabetes. Thus, periodontal disease should be treated to restore periodontal health and reduce the complications of diabetes. Therefore, the objective is assess the effect of full mouth periodontal therapy decontamination (Full Mouth Desinfection - FMD) in diabetic type II patients with chronic periodontitis during 12 months. Thirty-one patients in group one (G1) and 12 in group two (G2) were followed at baseline, 03, 06 09 and 12 months. There following clinical parameters were accessed: probing on bleeding (BOP), visible plaque index (PI), probing depth (PD), clinical attachment level (CAL) and gingival recession (GR). For diabetic patients, there were also made laboratory tests to evaluate blood parameters: fasting glucose and glycated hemoglobin. The results had been analyzed in two ways: all sites in the mouth and another with diseased sites. The Mann-Whitney, Friedman and Wilcoxon tests were used with 5% significance. Intergroup analysis of all sites it is clear that there was no significant difference over time concerning PD, BOP, PI, CAL and RG. However, when evaluating the diseased sites, we observed significant difference for CAL and PD, with higher values in G1. The intragroup analysis for all sites showed a statistically significant reduction at PD, PI and BOP in both groups. Intragroup analysis of periodontal affected sites showed a statistically significant reduction in PD, BOP and CAL in both groups. There was also a statistically significant increase in RG values. There was no significant change concerning glycated hemoglobin and fasting glucose in the G1. Therefore, it can be concluded that there were improvements in periodontal parameters over the 12 months of research, but without changes in glycemic levels of diabetic patients. Thus, periodontal therapy proved effective in maintaining oral health.

Inter-relação entre lesão cervical não cariosa e comprometimento periodontal: diagnóstico, tratamento e previsibilidade - estudo clínico prospectivo e laboratorial

The coexistence of gingival recession (GR) with root coverage indication and non-carious cervical lesions (LCNC) generates the need for a protocol that respects and promotes health of dental and periodontal tissues and allows treatment predictability. The main objectives of this theses were: (1) verify, through clinical evaluations, the connective tissue graft for root coverage on direct and indirect restorations made of ceramic resin; (2) analyze the influence of the battery level of the LED curing unit in the composite resin characteristics; (3) assess the influence of restorative materials, composite resin and ceramics, on the viability of gingival fibroblasts from primary culture. Nine patients with good oral hygiene and occlusal stability diagnosed with LCNCs the anterior teeth including premolars associated with gingival recession (class I and II of Miller) and only gingival recession were selected. After initial clinical examination, occlusal adjustment was performed and the patients had their teeth randomized allocated on direct composite resin restoration of LCNC, polishing and GR treatment with connective tissue graft and advanced coronally flap CR group (n = 15); and indirect ceramic restoration of the LCNC's and GR treatment (CTG+CAF) Group C (n = 15). The GR presented teeth with no clinically formed LCNCs cavity were treated using (CTG+CAF) being the control group (n = 15). Sorption and solubility tests, analysis of the degree of conversion and diametral tensile strength were performed in composite resin samples (n = 10) photoactivated by 100, 50 and 10% battery charge LED unit. The viability of fibroblasts on composite resin, ceramics and dentin disks (n = 3) was examined. Clinical follow-up was performed for three months. The data obtained at different stages were tabulated and subjected to analysis for detection of normal distribution and homogeneity. The results showed that: the LED unit with 10% battery affects the characteristics of the composite resin; restorative materials present biocompatibility with gingival fibroblasts; and the association of surgical and restorative treatment of teeth affected by NCCL and GR presents successful results at 3-month follow-up.

Rab32 restriction of intracellular bacterial pathogens

Experiments in the authors’ laboratory are supported by the Wellcome Trust (grant no. 109680/Z/15/Z), the Royal Society (grant no. RG150386), and Tenovus Scotland (grant no. G14/19) to SS. VSC is recipient of a European Union’s Horizon 2020 research and innovation programme Marie Sklodowska Curie Fellowship (grant no. 706040).

An investigation of the molecular interactions between statins and bacterial pathogens, and their combined impact on the human immune system

Statins are a class of drug that inhibits cholesterol biosynthesis, and are used to treat patients with high serum cholesterol levels. They exert this function by competitively binding to the enzyme 3-hydroxy-3-methylglutaryl-CoenzymeA reductase (HMGR), which catalyses the formation of mevalonate, a rate-limiting step in cholesterol biosynthesis. In addition, statins have what are called “pleiotropic effects”, which include the reduction of inflammation, immunomodulation, and antimicrobial effects. Statins can also improve survival of patients with sepsis and pneumonia. Cystic fibrosis (CF) is the most common recessive inherited disease in the Caucasian population, which is characterised by factors including, but not limited to, excessive lung inflammation and increased susceptibility to infection. Therefore, the overall objective of this study was to examine the effects of statins on CFassociated bacterial pathogens and the host response. In this work, the prevalence of HMGR was examined in respiratory pathogens, and several CF-associated pathogens were found to possess homologues of this enzyme. HMGR homology was analysed in Staphylococcus aureus, Burkholderia cenocepacia and Streptococcus pneumoniae, and the HMGR of B. cenocepacia was found to have significant conservation to that of Pseudomonas mevalonii, which is the most widely-characterised bacterial HMGR. However, in silico analysis revealed that, unlike S. aureus and S. pneumoniae, B. cenocepacia did not possess homologues of other mevalonate pathway proteins, and that the HMGR of B. cenocepacia appeared to be involved in an alternative metabolic pathway. The effect of simvastatin was subsequently tested on the growth and virulence of S. aureus, B. cenocepacia and S. pneumoniae. Simvastatin inhibited the growth of all 3 species in a dose-dependent manner. In addition, statin treatment also attenuated biofilm formation of all 3 species, and reduced in vitro motility of S. aureus. Interestingly, simvastatin also increased the potency of the aminoglycoside antibiotic gentamicin against B. cenocepacia. The impact of statins was subsequently tested on the predominant CF-associated pathogen Pseudomonas aeruginosa, which does not possess a HMGR homologue. Mevastatin, lovastatin and simvastatin did not influence the growth of this species. However, sub-inhibitory statin concentrations reduced the swarming motility and biofilm formation of P. aeruginosa. The influence of statins was also examined on Type 3 toxin secretion, quorum sensing and chemotaxis, and no statin effect was observed on any of these phenotypes. Statins did not appear to have a characteristic effect on the P. aeruginosa transcriptome. However, a mutant library screen revealed that the effect of statins on P. aeruginosa biofilm was mediated through the PvrR regulator and the Cup fimbrial biosynthesis genes. Furthermore, proteomic analysis demonstrated that 6 proteins were reproducibly induced by simvastatin in the P. aeruginosa swarming cells. The effect of statins on the regulation of the host-P. aeruginosa immune response was also investigated. Statin treatment increased expression of the pro-inflammatory cytokines IL-8 and CCL20 in lung epithelial cells, but did not attenuate P. aeruginosa-mediated inflammatory gene induction. In fact, simvastatin and P. aeruginosa caused a synergistic effect on CCL20 expression. The expression of the transcriptional regulators KLF2 and KLF6 was also increased by statins and P. aeruginosa, with the induction of KLF6 by simvastatin proving to be a novel effect. Interestingly, both statins and P. aeruginosa were capable of inducing alternative splicing of KLF6. P. aeruginosa was found to induce KLF6 alternative splicing by way of the type 3 secreted toxin ExoS. In addition, a mechanistic role was elucidated for KLF6 in the lung, as it was determined that statin-mediated induction of this protein was responsible for the induction of the host response genes CCL20 and iNOS. Moreover, statin treatment caused a slight increase in infection-related cytotoxicity, and increased bacterial adhesion to cells. Taken together, these data demonstrate that statins can reduce the virulence of CFassociated bacterial pathogens and alter host response effectors. Furthermore, novel statin effectors were identified in both bacterial and host cells.