RESUMO
OBJECTIVES: To evaluate the mechanical properties of root canal dentin treated with sodium hypochlorite (NaOCl) in combination with hydroxyethylidene diphosphonic acid (HEDP) or ethylenediaminetetraacetic acid (EDTA). METHODS: For testing fracture resistance, 45 single-rooted teeth were instrumented and irrigated with NaOCl/HEDP, NaOCl/EDTA, or distilled water. Fifteen untreated teeth served as control. After obturation, specimens from the experimental groups were thermocycled, dynamically-loaded, and then statically-loaded in a universal testing machine until failure. For flexural strength analysis, 15 teeth were instrumented and irrigated with NaOCl/HEDP or NaOCl/EDTA. Root segments were sectioned into dentin bars and tested for flexural strength using a universal testing machine. For microhardness evaluation, 20 teeth were instrumented and irrigated with NaOCl/HEDP or NaOCl/EDTA. Dentin disks from the coronal-third of each root segment were prepared, one before and one after irrigation, for microhardness testing with a Knoop hardness tester. RESULTS: The highest fracture resistance was recorded in the untreated group, and the lowest in the EDTA group. Although the HEDP group had higher fracture resistance than the EDTA group, the distilled water group demonstrated even greater fracture resistance than the HEDP group. Specimens treated with HEDP had significantly higher flexural strength and microhardness values when compared with those treated with EDTA. CONCLUSION: The fracture resistance, flexural strength, and microhardness of root canal dentin were higher when root canals were irrigated with NaOCl/HEDP, when compared with NaOCl/EDTA. CLINICAL SIGNIFICANCE: Irrigating root canals with NaOCl combined with HEDP significantly improves the mechanical integrity of root canal dentin compared to the use of NaOCl with EDTA.
Assuntos
Quelantes , Dentina , Ácido Edético , Dureza , Teste de Materiais , Irrigantes do Canal Radicular , Hipoclorito de Sódio , Dentina/efeitos dos fármacos , Hipoclorito de Sódio/farmacologia , Humanos , Ácido Edético/farmacologia , Irrigantes do Canal Radicular/farmacologia , Quelantes/farmacologia , Estresse Mecânico , Ácido Etidrônico/farmacologia , Cavidade Pulpar/efeitos dos fármacos , Resistência à Flexão , Análise do Estresse Dentário , Preparo de Canal Radicular/métodos , Fraturas dos Dentes/prevenção & controle , Raiz Dentária/efeitos dos fármacos , Maleabilidade , Temperatura , Obturação do Canal Radicular/métodosRESUMO
Cryptococcal meningitis is the most common cause of meningitis among HIV/AIDS patients in sub-Saharan Africa, and worldwide causes over 223,000 cases leading to more than 181,000 annual deaths. Usually, the fungus gets inhaled into the lungs where the initial interactions occur with pulmonary phagocytes such as dendritic cells and macrophages. Following phagocytosis, the pathogen can be killed or can replicate intracellularly. Previous studies in mice showed that different subsets of these innate immune cells can either be antifungal or permissive for intracellular fungal growth. Our studies tested phagocytic antigen-presenting cell (APC) subsets from the human lung against C. neoformans. Human bronchoalveolar lavage was processed for phagocytic APCs and incubated with C. neoformans for two hours to analyze the initial interactions and fate of the fungus, living or killed. Results showed all subsets (3 macrophage and 3 dendritic cell subsets) interacted with the fungus, and both living and killed morphologies were discernable within the subsets using imaging flow cytometry. Single cell RNA-seq identified several different clusters of cells which more closely related to interactions with C. neoformans and its protective capacity against the pathogen rather than discrete cellular subsets. Differential gene expression analyses identified several changes in the innate immune cell's transcriptome as it kills the fungus including increases of TNF-α (TNF) and the switch to using fatty acid metabolism by upregulation of the gene FABP4. Also, increases of TNF-α correlated to cryptococcal interactions and uptake. Together, these analyses implicated signaling networks that regulate expression of many different genes - both metabolic and immune - as certain clusters of cells mount a protective response and kill the pathogen. Future studies will examine these genes and networks to understand the exact mechanism(s) these phagocytic APC subsets use to kill C. neoformans in order to develop immunotherapeutic strategies to combat this deadly disease.
Assuntos
Criptococose , Cryptococcus neoformans , Humanos , Animais , Camundongos , Apresentação de Antígeno , Fator de Necrose Tumoral alfa , FagócitosRESUMO
Bacillus anthracis, the causative agent of inhalation anthrax, is a serious concern as a bioterrorism weapon. The vegetative form produces two exotoxins: Lethal toxin (LT) and edema toxin (ET). We recently characterized and compared six human airway and alveolar-resident phagocyte (AARP) subsets at the transcriptional and functional levels. In this study, we examined the effects of LT and ET on these subsets and human leukocytes. AARPs and leukocytes do not express high levels of the toxin receptors, tumor endothelium marker-8 (TEM8) and capillary morphogenesis protein-2 (CMG2). Less than 20% expressed surface TEM8, while less than 15% expressed CMG2. All cell types bound or internalized protective antigen, the common component of the two toxins, in a dose-dependent manner. Most protective antigen was likely internalized via macropinocytosis. Cells were not sensitive to LT-induced apoptosis or necrosis at concentrations up to 1000 ng/mL. However, toxin exposure inhibited B. anthracis spore internalization. This inhibition was driven primarily by ET in AARPs and LT in leukocytes. These results support a model of inhalation anthrax in which spores germinate and produce toxins. ET inhibits pathogen phagocytosis by AARPs, allowing alveolar escape. In late-stage disease, LT inhibits phagocytosis by leukocytes, allowing bacterial replication in the bloodstream.
Assuntos
Antígenos de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Leucócitos/efeitos dos fármacos , Macrófagos Alveolares/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Pinocitose/efeitos dos fármacos , Adolescente , Adulto , Idoso , Animais , Apoptose/efeitos dos fármacos , Bacillus anthracis/metabolismo , Relação Dose-Resposta a Droga , Feminino , Humanos , Leucócitos/metabolismo , Leucócitos/patologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patologia , Masculino , Camundongos , Proteínas dos Microfilamentos/metabolismo , Pessoa de Meia-Idade , Necrose , Células RAW 264.7 , Receptores de Superfície Celular/metabolismo , Receptores de Peptídeos/metabolismo , Esporos Bacterianos/metabolismo , Adulto JovemRESUMO
Rapid and accurate differentiation of Salmonella spp. causing enteric fever from nontyphoidal Salmonella is essential for clinical management of cases, laboratory risk management, and implementation of public health measures. Current methods used for confirmation of identification, including biochemistry and serotyping as well as whole-genome sequencing analyses, take several days. Here we report the development and evaluation of a real-time PCR assay that can be performed directly on crude DNA extracts from bacterial colonies for the rapid identification of typhoidal and nontyphoidal Salmonella.
Assuntos
Infecções por Salmonella/microbiologia , Salmonella typhi/classificação , Salmonella/classificação , DNA Bacteriano/genética , Genoma Bacteriano , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Infecções por Salmonella/diagnóstico , Salmonella enterica/classificação , Sensibilidade e Especificidade , Sorogrupo , Febre Tifoide/microbiologia , Sequenciamento Completo do GenomaRESUMO
The lung is the entry site for Bacillus anthracis in inhalation anthrax, the most deadly form of the disease. Spores must escape through the alveolar epithelial cell (AEC) barrier and migrate to regional lymph nodes, germinate and enter the circulatory system to cause disease. Several mechanisms to explain alveolar escape have been postulated, and all these tacitly involve the AEC barrier. In this study, we incorporate our primary human type I AEC model, microarray and gene enrichment analysis, qRT-PCR, multiplex ELISA, and neutrophil and monocyte chemotaxis assays to study the response of AEC to B. anthracis, (Sterne) spores at 4 and 24â¯h post-exposure. Spore exposure altered gene expression in AEC after 4 and 24â¯h and differentially expressed genes (±1.3 fold, pâ¯≤â¯0.05) included CCL4/MIP-1ß (4â¯h), CXCL8/IL-8 (4 and 24â¯h) and CXCL5/ENA-78 (24â¯h). Gene enrichment analysis revealed that pathways involving cytokine or chemokine activity, receptor binding, and innate immune responses to infection were prominent. Microarray results were confirmed by qRT-PCR and multiplex ELISA assays. Chemotaxis assays demonstrated that spores induced the release of biologically active neutrophil and monocyte chemokines, and that CXCL8/IL-8 was the major neutrophil chemokine. The small or sub-chemotactic doses of CXCL5/ENA-78, CXCL2/GROß and CCL20/MIP-3α may contribute to chemotaxis by priming effects. These data provide the first whole transcriptomic description of the human type I AEC initial response to B. anthracis spore exposure. Taken together, our findings contribute to an increased understanding of the role of AEC in the pathogenesis of inhalational anthrax.
Assuntos
Células Epiteliais Alveolares/microbiologia , Bacillus anthracis/patogenicidade , Quimiocinas/metabolismo , Perfilação da Expressão Gênica , Esporos Bacterianos/patogenicidade , Antraz/genética , Antraz/metabolismo , Quimiocina CCL20/genética , Quimiocina CCL20/metabolismo , Quimiocina CXCL5/genética , Quimiocina CXCL5/metabolismo , Quimiocinas/genética , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Monócitos/metabolismo , Monócitos/microbiologia , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Fator Plaquetário 4/genética , Fator Plaquetário 4/metabolismo , Infecções Respiratórias/genética , Infecções Respiratórias/metabolismo , Regulação para CimaRESUMO
Dendritic cells (DCs) and macrophages (MΦs) are antigen-presenting phagocytic cells found in many peripheral tissues of the human body, including the blood, lymph nodes, skin, and lung. They are vital to maintaining steady-state respiration in the human lung based on their ability to clear airways while also directing tolerogenic or inflammatory responses based on specific stimuli. Over the past three decades, studies have determined that there are multiple subsets of these two general cell types that exist in the airways and interstitium. Identifying these numerous subsets has proven challenging, especially with the unique microenvironments present in the lung. Cells found in the vasculature are not the same subsets found in the skin or the lung, as demonstrated by surface marker expression. By transcriptional profiling, these subsets show similarities but also major differences. Primary human lung cells and/ or tissues are difficult to acquire, particularly in a healthy condition. Additionally, surface marker screening and transcriptional profiling are continually identifying new DC and MΦ subsets. While the overall field is moving forward, we emphasize that more attention needs to focus on replicating the steady-state microenvironment of the lung to reveal the physiological functions of these subsets.
Assuntos
Células Dendríticas/citologia , Células Dendríticas/imunologia , Pulmão/citologia , Pulmão/imunologia , Macrófagos/citologia , Macrófagos/imunologia , Descanso , Animais , Humanos , Pulmão/patologiaRESUMO
The respiratory system is a complex network of many cell types, including subsets of macrophages and dendritic cells that work together to maintain steady-state respiration. Owing to limitations in acquiring cells from healthy human lung, these subsets remain poorly characterized transcriptionally and phenotypically. We set out to systematically identify these subsets in human airways by developing a schema of isolating large numbers of cells by whole-lung bronchoalveolar lavage. Six subsets of phagocytic APC (HLA-DR+) were consistently observed. Aside from alveolar macrophages, subsets of Langerin+, BDCA1-CD14+, BDCA1+CD14+, BDCA1+CD14-, and BDCA1-CD14- cells were identified. These subsets varied in their ability to internalize Escherichia coli, Staphylococcus aureus, and Bacillus anthracis particles. All subsets were more efficient at internalizing S. aureus and B. anthracis compared with E. coli Alveolar macrophages and CD14+ cells were overall more efficient at particle internalization compared with the four other populations. Subsets were further separated into two groups based on their inherent capacities to upregulate surface CD83, CD86, and CCR7 expression levels. Whole-genome transcriptional profiling revealed a clade of "true dendritic cells" consisting of Langerin+, BDCA1+CD14+, and BDCA1+CD14- cells. The dendritic cell clade was distinct from a macrophage/monocyte clade, as supported by higher mRNA expression levels of several dendritic cell-associated genes, including CD1, FLT3, CX3CR1, and CCR6 Each clade, and each member of both clades, was discerned by specific upregulated genes, which can serve as markers for future studies in healthy and diseased states.
Assuntos
Células Dendríticas/fisiologia , Pulmão/imunologia , Macrófagos Alveolares/fisiologia , Macrófagos/fisiologia , Adulto , Idoso , Antígenos CD/análise , Antígenos CD1/análise , Antígeno B7-2/análise , Células Dendríticas/classificação , Perfilação da Expressão Gênica , Glicoproteínas/análise , Humanos , Imunoglobulinas/análise , Receptores de Lipopolissacarídeos/análise , Pulmão/microbiologia , Macrófagos/classificação , Glicoproteínas de Membrana/análise , Pessoa de Meia-Idade , Antígeno CD83RESUMO
The lung is the entry site for Bacillus anthracis in inhalation anthrax, the most deadly form of the disease. Spores escape from the alveolus to regional lymph nodes, germinate and enter the circulatory system to cause disease. The roles of carrier cells and the effects of B. anthracis toxins in this process are unclear. We used a human lung organ culture model to measure spore uptake by antigen presenting cells (APC) and alveolar epithelial cells (AEC), spore partitioning between these cells, and the effects of B. anthracis lethal toxin and protective antigen. We repeated the study in a human A549 alveolar epithelial cell model. Most spores remained unassociated with cells, but the majority of cell-associated spores were in AEC, not in APC. Spore movement was not dependent on internalization, although the location of internalized spores changed in both cell types. Spores also internalized in a non-uniform pattern. Toxins affected neither transit of the spores nor the partitioning of spores into AEC and APC. Our results support a model of spore escape from the alveolus that involves spore clustering with transient passage through intact AEC. However, subsequent transport of spores by APC from the lung to the lymph nodes may occur.
Assuntos
Antraz/patologia , Antígenos de Bactérias/metabolismo , Bacillus anthracis/patogenicidade , Toxinas Bacterianas/metabolismo , Pulmão/microbiologia , Linfonodos/microbiologia , Movimento , Esporos Bacterianos/patogenicidade , Células Apresentadoras de Antígenos/microbiologia , Sangue/microbiologia , Linhagem Celular , Células Epiteliais/microbiologia , Humanos , Modelos Teóricos , Técnicas de Cultura de ÓrgãosRESUMO
Dendritic cells (DC) are generally categorized as a group of rare antigen presenting cells that are to the crucial development of immune responses to pathogens and also of tolerance to self-antigens. Therefore, having the ability to identify DC in specific tissues and to test their functional abilities in the steady state are scientific gaps needing attention. Research on primary human DC is lacking due to their rarity and the difficulty of obtaining tissue samples. However, recent findings have shown that several different DC subsets exist, and that these subsets vary both by markers expressed and functions depending on their specific microenvironment. After discriminating from other cell types, DC can be split into myeloid and plasmacytoid fractions. While plasmacytoid DC express definite markers, CD123 and BDCA-2, myeloid DC encompass several different subsets with overlapping markers expressed. Such markers include the blood DC antigens BDCA-1 and BDCA-3, along with Langerin, CD1a and CD14. Marker specificity is further reduced when accounting for microenvironmental differences, as observed in the blood, primary lymphoid tissues, skin and lungs. The mixed leukocyte reaction (MLR) has been used to measure the strength of antigen presentation by specific DC subsets. Surface markers and MLR require standardization to enable consistent identification of and comparisons between DC subsets. To alleviate these issues, researchers have begun comparing DC subsets at the transcriptional level. This has allowed degrees of relatedness to be determined between DC in different microenvironments, and should be a continued area of focus in years to come.
Assuntos
Células Dendríticas/citologia , Microambiente Celular , Humanos , Pulmão/citologia , Reconhecimento Automatizado de Padrão , Pele/citologiaRESUMO
The lung is the site of entry for Bacillus anthracis in inhalation anthrax, the deadliest form of the disease. Bacillus anthracis produces virulence toxins required for disease. Alveolar macrophages were considered the primary target of the Bacillus anthracis virulence factor lethal toxin because lethal toxin inhibits mouse macrophages through cleavage of MEK signaling pathway components, but we have reported that human alveolar macrophages are not a target of lethal toxin. Our current results suggest that, unlike human alveolar macrophages, the cells lining the respiratory units of the lung, alveolar epithelial cells, are a target of lethal toxin in humans. Alveolar epithelial cells expressed lethal toxin receptor protein, bound the protective antigen component of lethal toxin, and were subject to lethal-toxin-induced cleavage of multiple MEKs. These findings suggest that human alveolar epithelial cells are a target of Bacillus anthracis lethal toxin. Further, no reduction in alveolar epithelial cell viability was observed, but lethal toxin caused actin rearrangement and impaired desmosome formation, consistent with impaired barrier function as well as reduced surfactant production. Therefore, by compromising epithelial barrier function, lethal toxin may play a role in the pathogenesis of inhalation anthrax by facilitating the dissemination of Bacillus anthracis from the lung in early disease and promoting edema in late stages of the illness.