RESUMO
AIM: To evaluate the 6-year outcome of root canal treatment irrigated with 0.5% or 3% sodium hypochlorite (NaOCl). METHODOLOGY: The baseline trial was designed as a quasi-randomized clinical trial. Patients referred for root canal treatment to an endodontic specialist clinic were recruited to the study (n = 298). The concentration of NaOCl was allocated quasi-randomized to 271 subjects (0.5% [n = 139], 3% [n = 132]). Bacterial sampling was performed immediately before root canal filling. Samples were cultured and evaluated as growth or no growth. Patients were invited to a clinical and radiological follow-up >5 years postoperatively. The clinical outcome measurements were tooth survival, cumulative incidence of endodontic retreatments, patients' assessment of pain, clinical findings and radiological signs of apical periodontitis (AP). RESULTS: Tooth survival was 85.6% in the 0.5% NaOCl group and 81.1% in the 3% NaOCl group (p = .45). There was no record of retreatment in 94.4% in the 0.5% NaOCl group and in 92.2% in the 3% NaOCl group (p = .76). The percentage of asymptomatic cases were 87.8% in the 0.5% group and 85.3% in the 3% NaOCl group (p = .81). Absence of clinical signs of AP was seen in 86.6% in the 0.5% NaOCl group and in 83.6% in the 3% NaOCl group (p = .80). Absence of radiological signs of AP was seen in 74.0% in the 0.5% NaOCl group and 64.1% in the 3% NaOCl group (p = .20). Subjects with positive culture before root filling reported subjective pain with a significantly higher frequency as compared to negative-culture subjects (p = .014). CONCLUSIONS: The use of 0.5% or 3% NaOCl for irrigation during root canal treatment resulted in similar clinical outcomes 5-7 years postoperatively. Persisting bacteria immediately before root filling may predict future episodes of subjective pain.
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Candida albicans belongs to our commensal mucosal flora and in immune-competent individuals in the absence of epithelial damage, this fungus is well tolerated and controlled by our immune defense. However, C. albicans is an opportunistic microorganism that can cause different forms of infections, ranging from superficial to life-threatening systemic infections. C. albicans is polymorphic and switches between different phenotypes (e.g. from yeast form to hyphal form). C. albicans hyphae are invasive and can grow into tissues to eventually reach circulation. During fungal infections, neutrophils in particular play a critical role for the defense, but how neutrophils are directed toward the invasive forms of fungi is less well understood. We set out to investigate possible neutrophil chemoattractants released by C. albicans into culture supernatants. We found that cell-free culture supernatants from the hyphal form of C. albicans induced both neutrophil chemotaxis and concomitant intracellular calcium transients. Size separation and hydrophobic sorting of supernatants indicated small hydrophilic factors as responsible for the activity. Further analysis showed that the culture supernatants contained high levels of short-chain fatty acids with higher levels from hyphae as compared to yeast. Short-chain fatty acids are known neutrophil chemoattractants acting via the neutrophil free fatty acid receptor 2. In line with this, the calcium signaling in neutrophils induced by hyphae culture supernatants was blocked by a free fatty acid receptor 2 antagonist and potently increased in the presence of a positive allosteric modulator. Our data imply that short-chain fatty acids may act as a recruitment signal whereby neutrophils can detect C. albicans hyphae.
Assuntos
Candida albicans , Neutrófilos , Humanos , Ácidos Graxos não Esterificados/análise , Hifas/química , Hifas/genética , Quimiotaxia , Ácidos Graxos Voláteis/análise , Fatores QuimiotáticosRESUMO
Pseudomonas aeruginosa is a Gram-negative bacterium and an opportunistic pathogen ubiquitously present throughout nature. LecB, a fucose-, and mannose-binding lectin, is a prominent virulence factor of P. aeruginosa, which can be expressed on the bacterial surface but also be secreted. However, the LecB interaction with human immune cells remains to be characterized. Neutrophils comprise the first line of defense against infections and their production of reactive oxygen species (ROS) and release of extracellular traps (NETs) are critical antimicrobial mechanisms. When profiling the neutrophil glycome we found several glycoconjugates on granule and plasma membranes that could potentially act as LecB receptors. In line with this, we here show that soluble LecB can activate primed neutrophils to produce high levels of intracellular ROS (icROS), an effect that was inhibited by methyl fucoside. On the other hand, soluble LecB inhibits P. aeruginosa-induced icROS production. In support of that, during phagocytosis of wild-type and LecB-deficient P. aeruginosa, bacteria with LecB induced less icROS production as compared with bacteria lacking the lectin. Hence, LecB can either induce or inhibit icROS production in neutrophils depending on the circumstances, demonstrating a novel and potential role for LecB as an immunomodulator of neutrophil functional responses.
Assuntos
Armadilhas Extracelulares , Neutrófilos , Humanos , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/metabolismo , Espécies Reativas de Oxigênio/metabolismo , LectinasRESUMO
We describe a female patient suffering from severe chronic non-bacterial osteomyelitis (CNO) with systemic inflammation and advanced malnutrition and complete deficiency of myeloperoxidase (MPO). CNO is a rare autoinflammatory bone disorder associated with dysregulation of the innate immune system. MPO deficiency is a genetic disorder with partial or complete absence of the phagocyte peroxidase MPO. MPO deficiency has no established clinical phenotype but reports indicate increased susceptibility to infection and chronic inflammation. The patient's symptoms began at 10 years of age with pain in the thighs, systemic inflammation and malnutrition. She was diagnosed with CNO at 14 years of age. Treatment with nonsteroidal anti-inflammatory drugs, corticosteroids, bisphosphonates or IL1-receptor antagonists (anakinra) did not relieve the symptoms. However, the patient responded instantly and recovered from her clinical symptoms when treated with TNFα blockade (adalimumab). Three years after treatment initiation adalimumab was withdrawn, resulting in rapid symptom recurrence. When reintroducing adalimumab, the patient promptly responded and went into remission. In addition to clinical and laboratory profiles, neutrophil functions (reactive oxygen species, ROS; neutrophil extracellular traps, NETs; degranulation; apoptosis; elastase activity) were investigated both in a highly inflammatory state (without treatment) and in remission (on treatment). At diagnosis, neither IL1ß, IL6, nor TNFα was significantly elevated in serum, but since TNFα blockade terminated the inflammatory symptoms, the disease was likely TNFα-driven. All neutrophil parameters were normal both during treatment and treatment withdrawal, except for MPO-dependent intracellular ROS- and NET formation. The role of total MPO deficiency for disease etiology and severity is discussed.
Assuntos
Desnutrição , Osteomielite , Feminino , Humanos , Adalimumab/uso terapêutico , Inflamação , Osteomielite/diagnóstico , Osteomielite/tratamento farmacológico , Espécies Reativas de Oxigênio , Fator de Necrose Tumoral alfa , Criança , AdolescenteRESUMO
Papillon-Lefèvre Syndrome (PLS) is an autosomal recessive monogenic disease caused by loss-of-function mutations in the CTSC gene, thus preventing the synthesis of the protease Cathepsin C (CTSC) in a proteolytically active form. CTSC is responsible for the activation of the pro-forms of the neutrophil serine proteases (NSPs; Elastase, Proteinase 3 and Cathepsin G), suggesting its involvement in a variety of neutrophil functions. In PLS neutrophils, the lack of CTSC protease activity leads to inactivity of the NSPs. Clinically, PLS is characterized by an early, typically pre-pubertal, onset of severe periodontal pathology and palmoplantar hyperkeratosis. However, PLS is not considered an immune deficiency as patients do not typically suffer from recurrent and severe (bacterial and fungal) infections. In this study we investigated an unusual CTSC mutation in two siblings with PLS, a 503A>G substitution in exon 4 of the CTSC gene, expected to result in an amino acid replacement from tyrosine to cysteine at position 168 of the CTSC protein. Both patients bearing this mutation presented with pronounced periodontal pathology. The characteristics and functions of neutrophils from patients homozygous for the 503A>G CTSC mutation were compared to another previously described PLS mutation (755A>T), and a small cohort of healthy volunteers. Neutrophil lysates from patients with the 503A>G substitution lacked CTSC protein and did not display any CTSC or NSP activity, yet neutrophil counts, morphology, priming, chemotaxis, radical production, and regulation of apoptosis were without any overt signs of alteration. However, NET formation upon PMA-stimulation was found to be severely depressed, but not abolished, in PLS neutrophils.
Assuntos
Catepsina C/genética , Armadilhas Extracelulares/metabolismo , Neutrófilos/patologia , Doença de Papillon-Lefevre/genética , Serina Proteases/metabolismo , Adulto , Apoptose , Catepsina C/metabolismo , Citometria de Fluxo , Humanos , Mutação com Perda de Função/genética , Pessoa de Meia-Idade , Doença de Papillon-Lefevre/enzimologia , Doença de Papillon-Lefevre/patologia , Espécies Reativas de Oxigênio/metabolismo , Análise de Sequência de DNARESUMO
Fusobacterium nucleatum is a gram-negative and anaerobic oral commensal that is implicated in inflammatory conditions of the tooth-supporting structures, that is, periodontal diseases. One of the main characteristics of these conditions is an accumulation of neutrophil granulocytes in the gingival pockets where bacteria reside. Neutrophils are recruited to tissue-residing microbes by gradients of bacteria derived chemoattractants, and the cellular migration over the pocket epithelium into the gingival pocket is likely governed by chemoattractants released by the amino acid fermenting anaerobes typically colonising this site. However, the chemoattractants released by F. nucleatum and other oral anaerobes have long been unidentified. In the present study, we show that the major chemoattractants released during the growth of F. nucleatum are short chain fatty acids (SCFAs), primarily acetate and butyrate. These SCFAs, that are released at high levels as end-products of the metabolism of F. nucleatum, trigger chemotaxis of human neutrophils, as well as cytosolic Ca2+ signals, via free fatty acid receptor 2 (FFAR2). This finding establishes the SCFA-FFAR2 interaction as an important mechanism in the recruitment of neutrophils to the periodontal pocket, but could also be of importance in the pathogenesis of other medical conditions involving colonisation/infection of F. nucleatum.
Assuntos
Fusobacterium nucleatum , Neutrófilos , Fatores Quimiotáticos , Ácidos Graxos não Esterificados , Ácidos Graxos Voláteis , HumanosRESUMO
In recent years, the concept of distinct subpopulations of human neutrophils has attracted much attention. One bona fide subset marker, exclusively expressed by a proportion of circulating neutrophils in a given individual, and therefore dividing neutrophils in two distinct subpopulations, is the glycoprotein CD177. CD177 is expressed on the plasma and granule membranes of 0-100% of circulating neutrophils depending on the donor. Several in vitro studies have linked CD177 to neutrophil transmigration, yet very few have looked at the role of CD177 for tissue recruitment in vivo. We investigate whether the CD177+ and CD177- neutrophil subsets differ in their propensity to migrate to both aseptic- and microbe-triggered inflamed human tissues. Microbe-triggered neutrophil migration was evaluated in samples of gingival crevicular fluid (GCF) from patients with periodontitis, whereas neutrophil migration to aseptic inflammation was evaluated in synovial fluid from patients with inflammatory arthritis, as well as in exudate from experimental skin chambers applied on healthy donors. We found that the proportion of CD177+ neutrophils was significantly higher in GCF from patients with periodontitis, as compared to blood from the same individuals. Such accumulation of CD177+ neutrophils was not seen in the two models of aseptic inflammation. Moreover, the proportion of CD177+ neutrophils in circulation was significantly higher in the periodontitis patient group, as compared to healthy donors. Our data indicate that the CD177+ neutrophil subset is preferentially recruited to the gingival crevice of periodontitis patients, and may imply that this subtype is of particular importance for situations of microbe-driven inflammation.
Assuntos
Líquido do Sulco Gengival/citologia , Isoantígenos/metabolismo , Neutrófilos/metabolismo , Periodontite/imunologia , Periodontite/patologia , Receptores de Superfície Celular/metabolismo , Artrite/imunologia , Artrite/patologia , Morte Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Fatores Quimiotáticos/farmacologia , Proteínas Ligadas por GPI/sangue , Proteínas Ligadas por GPI/metabolismo , Líquido do Sulco Gengival/efeitos dos fármacos , Humanos , Inflamação/imunologia , Inflamação/patologia , Isoantígenos/sangue , Modelos Biológicos , Neutrófilos/efeitos dos fármacos , Periodontite/sangue , Periodontite/microbiologia , Receptores de Superfície Celular/sangue , Líquido Sinovial/efeitos dos fármacos , Líquido Sinovial/metabolismo , Doadores de TecidosRESUMO
Porphyromonas gingivalis (P. gingivalis) is a gram-negative bacterium and an important etiologic agent of periodontitis. P. gingivalis releases outer membrane vesicles containing lipopolysaccharides (LPS), which can penetrate periodontal tissues. Once in the periodontal tissues and in contact with immune cells, it may participate in the destructive innate host response associated with the disease. The exact mechanism of P. gingivalis LPS in the disease process is not clear, but it is known to affect a variety of immune responses. OBJECTIVES: To investigate how LPS from P. gingivalis affect neutrophil extracellular trap (NET) formation, cell death and production of cytokines from human neutrophils and peripheral mononuclear blood mononuclear cells (PBMCs). MATERIALS AND METHODS: Isolated neutrophils and PBMCs were cultured with LPS from P. gingivalis or Escherichia coli (E. coli) (control). The NET formation was measured using Sytox green stain. Cell death of neutrophils and PBMCs was analyzed using flow cytometry or Sytox green stain. Cytokine production was measured using enzyme-linked immunosorbent assay (ELISA) kit or Bio-Plex assay. RESULTS: Exposure to LPS from P. gingivalis and E. coli caused significantly lower cell death in neutrophils. NETs were formed after exposure to the two different LPS. In PBMCs, exposure to P. gingivalis and E. coli LPS caused increased levels of IL-1ß and IL-6 compared to unstimulated controls. Increased cell death in PBMCs after exposure to LPS from E. coli in comparison to LPS from P. gingivalis and unstimulated controls was also observed. CONCLUSIONS: LPS from P. gingivalis has the ability to affect both human neutrophils and PBMCs with regard to cytokine production, cell death and production of NETs. LPS from P. gingivalis could be involved in the pathogenesis of periodontitis, and our results may contribute information regarding possible markers for diagnosis and targets for treatment of periodontal disease.
Assuntos
Porphyromonas gingivalis , Citocinas , Escherichia coli , Humanos , Lipopolissacarídeos/toxicidade , PeriodontiteRESUMO
OBJECTIVES: The prevalent usage of methacrylates in modern dentistry demands good knowledge of their biological impacts. While there have been several studies demonstrating the effects of different methacrylic monomers on mononuclear white blood cells, very little is known about the effects caused by these monomers on neutrophilic granulocytes. The objective of this study was to add novel knowledge about how neutrophils are affected by exposure to triethylene glycol dimethacrylate (TEGDMA), urethane dimethacrylate (UDMA), and bisphenol A glycol dimethacrylate (Bis-GMA) alone or in combinations. MATERIALS AND METHODS: Isolated neutrophils were cultured in the presence or absence of methacrylates. The IL-8 release was measured using a DuoSet ELISA development kit. Apoptosis and necrosis were analyzed using flow cytometry. The formation of neutrophil extracellular traps (NETs) was investigated using Sytox green DNA staining combined with microscopically examination of released DNA and myeloperoxidase (MPO). RESULTS: The release of IL-8 was significantly increased after exposure to TEGDMA, Bis-GMA, UDMA, or TEGDMA in combination with Bis-GMA or UDMA compared to the unstimulated controls. Exposure to TEGDMA, UDMA, and Bis-GMA for 24 hr separately or in combination did not affect apoptosis or necrosis of the exposed neutrophils. NET structures were formed by neutrophils after exposure to the different combinations of the methacrylates. CONCLUSION: The combination of TEGDMA and Bis-GMA had a synergistic proinflammatory effect on neutrophils by increasing the release of IL-8 and the formation of NET structures. The changes in the normal functions of neutrophils caused by methacrylate exposure may lead to altered inflammatory response and relate to previously reported adverse immune reactions caused by these substances.
Assuntos
Compostos Benzidrílicos/farmacologia , Bis-Fenol A-Glicidil Metacrilato/farmacologia , Citocinas/metabolismo , Teste de Materiais/métodos , Metacrilatos/farmacologia , Neutrófilos/efeitos dos fármacos , Polietilenoglicóis/farmacologia , Ácidos Polimetacrílicos/farmacologia , Poliuretanos/farmacologia , Resinas Compostas , Humanos , Técnicas In Vitro , Neutrófilos/metabolismoRESUMO
Gout is an inflammatory disease caused by monosodium urate (MSU) crystals. The role of neutrophils in gout is less clear, although several studies have shown neutrophil extracellular trap (NET) formation in acutely inflamed joints of gout patients. MSU crystals are known to induce the production of reactive oxygen species (ROS) and NET formation in neutrophils isolated from blood, but there is inconclusive knowledge on the localization of ROS production as well as whether the ROS are required for NET formation. In this report we demonstrate that MSU crystals activate human neutrophils to produce ROS exclusively in intracellular compartments. Additionally, in vivo transmigrated neutrophils derived from experimental skin chambers displayed markedly increased ROS production as compared to resting blood neutrophils. We also confirmed that MSU stimulation potently induced NET formation, but this response was not primed in in vivo transmigrated neutrophils. In line with this we found that MSU-triggered NET formation was independent of ROS production and proceeded normally in neutrophils from patients with dysfunctional respiratory burst (chronic granulomatous disease (CGD) and complete myeloperoxidase (MPO) deficiency). Our data indicate that in vivo transmigrated neutrophils are markedly primed for oxidative responses to MSU crystals and that MSU triggered NET formation is independent of ROS production.
Assuntos
Gota/metabolismo , Neutrófilos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Adulto , Idoso , Células Cultivadas , Armadilhas Extracelulares/efeitos dos fármacos , Armadilhas Extracelulares/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , Neutrófilos/efeitos dos fármacos , Neutrófilos/fisiologia , Peroxidase/metabolismo , Migração Transendotelial e Transepitelial , Ácido Úrico/metabolismo , Ácido Úrico/farmacologiaRESUMO
Neutrophil migration from blood to tissue-residing microbes is governed by a series of chemoattractant gradients of both endogenous and microbial origin. Periodontal disease is characterized by neutrophil accumulation in the gingival pocket, recruited by the subgingival biofilm consisting mainly of gram-negative, anaerobic and proteolytic species such as Porphyromonas gingivalis. The fact that neutrophils are the dominating cell type in the gingival pocket suggests that neutrophil-specific chemoattractants are released by subgingival bacteria, but characterization of chemoattractants released by subgingival biofilm species remains incomplete. In the present study we characterized small (< 3 kDa) soluble chemoattractants released by growing P. gingivalis, and show that these are selective for neutrophils. Most neutrophil chemoattractant receptors are expressed also by mononuclear phagocytes, the free fatty acid receptor 2 (FFAR2) being an exception. In agreement with the selective neutrophil recruitment, the chemotactic activity found in P. gingivalis supernatants was mediated in part by a mixture of short chain fatty acids (SCFAs) that are recognized by FFAR2, and other leukocytes (including monocytes) did not respond to SCFA stimulation. Although SCFAs, produced by bacterial fermentation of dietary fiber in the gut, has previously been shown to utilize FFAR2, our data demonstrate that the pronounced proteolytic metabolism employed by P. gingivalis (and likely also other subgingival biofilm bacteria associated with periodontal diseases) may result in the generation of SCFAs that attract neutrophils to the gingival pocket. This finding highlights the interaction between SCFAs and FFAR2 in the context of P. gingivalis colonization during periodontal disease, but may also have implications for other inflammatory pathologies involving proteolytic bacteria.
Assuntos
Neutrófilos , Porphyromonas gingivalis , Fatores Quimiotáticos , Ácidos Graxos Voláteis , Interleucina-8RESUMO
Formyl peptide receptor (FPR)-desensitized neutrophils display increased production/release of superoxide (O2-) when activated by platelet-activating factor (PAF), a priming of the response achieved through a unique receptor crosstalk mechanism. The aim of this study was to determine the effect of an inhibitor selective for small, heterotrimeric G proteins belonging to the Gαq subclass on that receptor crosstalk. We show that signals generated by FPRs and the PAF receptor (PAFR) induce activation of the neutrophil O2-, producing NADPH-oxidase, and that response was sensitive to Gαq inhibition in cells activated by PAF, but no inhibition was obtained in cells activated by FPR agonists. Signaling in naive neutrophils is terminated fairly rapidly, and the receptors become homologously desensitized. The downstream sensitivity to Gαq inhibition in desensitized cells displaying increased production/release of O2- through the PAFR receptor crosstalk mechanism also comprised the reactivation of the FPRs, and the activation signals were redirected from the PAFR to the desensitized/reactivated FPRs. The Gαq-dependent activation signals generated by the PAFRs activate the Gαi-coupled FPRs, a receptor crosstalk that represents a novel pathway by which G protein-coupled receptors can be regulated and signaling can be turned on and off.
Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/imunologia , Neutrófilos/imunologia , Glicoproteínas da Membrana de Plaquetas/imunologia , Receptores de Formil Peptídeo/imunologia , Receptores Acoplados a Proteínas G/imunologia , Transdução de Sinais/imunologia , Humanos , NADPH Oxidases/imunologia , Fator de Ativação de Plaquetas/imunologia , Superóxidos/imunologiaRESUMO
Neutrophils have the ability to capture and kill microbes extracellularly through the formation of neutrophil extracellular traps (NETs). These are DNA and protein structures that neutrophils release extracellularly and are believed to function as a defense mechanism against microbes. The classic NET formation process, triggered by, e.g., bacteria, fungi, or by direct stimulation of protein kinase C through phorbol myristate acetate, is an active process that takes several hours and relies on the production of reactive oxygen species (ROS) that are further modified by myeloperoxidase (MPO). We show here that NET-like structures can also be formed by neutrophils after interaction with phenol-soluble modulin α (PSMα) that are cytotoxic membrane-disturbing peptides, secreted from community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). The PSMα-induced NETs contained the typical protein markers and were able to capture microbes. The PSMα-induced NET structures were disintegrated upon prolonged exposure to DNase-positive S. aureus but not on exposure to DNase-negative Candida albicans. Opposed to classic NETosis, PSMα-triggered NET formation occurred very rapidly, independently of ROS or MPO, and was also manifest at 4°C. These data indicate that rapid NETs release may result from cytotoxic membrane disturbance by PSMα peptides, a process that may be of importance for CA-MRSA virulence.