Dataset Information

Novel Assay To Characterize Neutrophil Responses to Oral Biofilms.

ABSTRACT: Neutrophils, the most numerous leukocytes, play an important role in maintaining oral health through interactions with oral microbial biofilms. Both neutrophil hyperactivity and the bacterial subversion of neutrophil responses can cause inflammation-mediated tissue damage like that seen in periodontal disease. We describe here an assay that assesses neutrophil activation responses to monospecies biofilm bacteria in vitro based on the surface expression of cluster of differentiation (CD) markers associated with various neutrophil functions. Most of what we know about neutrophil responses to bacteria is based on in vitro assays that use planktonic bacteria and isolated/preactivated neutrophils, which makes interpretation of the neutrophil responses to bacteria a challenge. An understanding of how neutrophils differentially interact with and respond to commensal and pathogenic oral bacteria is necessary in order to further understand the neutrophil's role in maintaining oral health and the pathogenesis of periodontal disease. In this study, a flow cytometry-based in vitro assay was developed to characterize neutrophil activation states based on CD marker expressions in response to oral monospecies bacterial biofilms. Using this approach, changes in CD marker expressions in response to specific prominent oral commensal and pathogenic bacteria were assayed. Several functional assays, including assays for phagocytosis, production of reactive oxygen species, activation of the transcription factor Nrf2, neutrophil extracellular trap formation, and myeloperoxidase release, were also performed to correlate neutrophil function with CD marker expression. Our results demonstrate that neutrophils display bacterial species-specific responses. This assay can be used to characterize how specific biofilms alter specific neutrophil pathways associated with their activation.

SUBMITTER: Oveisi M

PROVIDER: S-EPMC6346121 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Novel Assay To Characterize Neutrophil Responses to Oral Biofilms.

Oveisi Morvarid M Shifman Harold H Fine Noah N Sun Chunxiang C Glogauer Naomi N Senadheera Dilani D Glogauer Michael M

Infection and immunity 20190124 2

Neutrophils, the most numerous leukocytes, play an important role in maintaining oral health through interactions with oral microbial biofilms. Both neutrophil hyperactivity and the bacterial subversion of neutrophil responses can cause inflammation-mediated tissue damage like that seen in periodontal disease. We describe here an assay that assesses neutrophil activation responses to monospecies biofilm bacteria <i>in vitro</i> based on the surface expression of cluster of differentiation (CD) m ...[more]

PMID: 30455195

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Project description:UnlabelledDenitrifying fungi produce nitrous oxide (N2O), a potent greenhouse gas, as they generally lack the ability to convert N2O to dinitrogen. Contrary to the case for bacterial denitrifiers, the prevalence and diversity of denitrifying fungi found in the environment are not well characterized. In this study, denitrifying fungi were isolated from various soil ecosystems, and novel PCR primers targeting the P450nor gene, encoding the enzyme responsible for the conversion of nitric oxide to N2O, were developed, validated, and used to study the diversity of cultivable fungal denitrifiers. This PCR assay was also used to detect P450nor genes directly from environmental soil samples. Fungal denitrification capabilities were further validated using an N2O gas detection assay and a PCR assay targeting the nirK gene. A collection of 492 facultative anaerobic fungi was isolated from 15 soil ecosystems and taxonomically identified by sequencing the internal transcribed spacer sequence. Twenty-seven fungal denitrifiers belonging to 10 genera had the P450nor and the nirK genes and produced N2O from nitrite. N2O production is reported in strains not commonly known as denitrifiers, such as Byssochlamys nivea, Volutella ciliata, Chloridium spp., and Trichocladium spp. The prevalence of fungal denitrifiers did not follow a soil ecosystem distribution; however, a higher diversity was observed in compost and agricultural soils. The phylogenetic trees constructed using partial P450nor and nirK gene sequences revealed that both genes clustered taxonomically closely related strains together.ImportanceA PCR assay targeting the P450nor gene involved in fungal denitrification was developed and validated. The newly developed P450nor primers were used on fungal DNA extracted from a collection of fungi isolated from various soil environments and on DNA directly extracted from soil. The results indicated that approximatively 25% of all isolated fungi possessed this gene and were able to convert nitrite to N2O. All soil samples from which denitrifying fungi were isolated also tested positive for the presence of P450nor The P450nor gene detection assay was reliable in detecting a large diversity of fungal denitrifiers. Due to the lack of homology existing between P450nor and bacterial denitrification genes, it is expected that this assay will become a tool of choice for studying fungal denitrifiers.

Dataset Information

Novel Assay To Characterize Neutrophil Responses to Oral Biofilms.

Publications

Novel Assay To Characterize Neutrophil Responses to Oral Biofilms.

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