Characterization, Quantification, and Visualization of Neutrophil Extracellular Traps.

Following the discovery of neutrophil extracellular traps (NETs) in 2004 by Brinkmann and colleagues, there has been extensive research into the role of NETs in a number of inflammatory diseases, including periodontitis. This chapter describes the current methods for the isolation of peripheral blood neutrophils as well as of oral neutrophils for subsequent NET experiments, including approaches to quantify and visualize NET production, the ability of NETs to entrap and kill bacteria, and the removal of NETs by nuclease-containing plasma.

Anti-bacterial antibodies in multiple myeloma patients at disease presentation, in response to therapy and in remission: implications for patient management.

Multiple myeloma (MM) is associated with increased risk of infection, but little is known regarding antibody levels against specific bacteria. We assessed levels of polyclonal immunoglobulin and antibacterial antibodies in patients recruited to the TEAMM trial, a randomised trial of antibiotic prophylaxis at the start of anti-myeloma treatment. Polyclonal IgG, IgA and IgM levels were below the reference range in 71%, 83% and 90% of 838 MM patients at diagnosis. Anti-vaccine targeted tetanus toxoid antibodies were protective in 95% of 193 healthy controls but only 41% of myeloma patients. In healthy controls, protective antibodies against 6/12 pneumococcal serotypes, haemophilus and meningococcus A were present in 67%, 41% and 56% compared to just 15%, 21% and 17% of myeloma patients. By 1 year, myeloma patients IgG levels had recovered for 57% of patients whilst the proportion with protective levels of IgG against thymus-dependent protein antigen tetanus toxoid had changed little. In contrast the proportions of patients with protective levels against thymus independent polysaccharide antigens pneumococcus, haemophilus and meningococcus had fallen from 15 to 7%, 21 to 0% and 17 to 11%. Findings highlight the need for strategies to protect patients against bacterial infections during therapy and vaccination programmes during remission.

Development and Application of High-Content Biological Screening for Modulators of NET Production.

Neutrophil extracellular traps (NETs) are DNA-based antimicrobial web-like structures whose release is predominantly mediated by reactive oxygen species (ROS); their purpose is to combat infections. However, unbalanced NET production and clearance is involved in tissue injury, circulation of auto-antibodies and development of several chronic diseases. Currently, there is lack of agreement regarding the high-throughput methods available for NET investigation. This study, therefore, aimed to develop and optimize a high-content analysis (HCA) approach, which can be applied for the assay of NET production and for the screening of compounds involved in the modulation of NET release. A suitable paraformaldehyde fixation protocol was established to enable HCA of neutrophils and NETs. Bespoke and in-built bioinformatics algorithms were validated by comparison with standard low-throughput approaches for application in HCA of NETs. Subsequently, the optimized protocol was applied to high-content screening (HCS) of a pharmaceutically derived compound library to identify modulators of NETosis. Of 56 compounds assessed, 8 were identified from HCS for further characterization of their effects on NET formation as being either inducers, inhibitors or biphasic modulators. The effects of these compounds on naïve neutrophils were evaluated by using specific assays for the induction of ROS and NET production, while their modulatory activity was validated in phorbol 12-myristate 13-acetate-stimulated neutrophils. Results indicated the involvement of glutathione reductase, Src family kinases, molecular-target-of-Rapamycin, and mitogen-activated-protein-kinase pathways in NET release. The compounds and pathways identified may provide targets for novel therapeutic approaches for treating NET-associated pathologies.

Inflammation and Regeneration in the Dentin-pulp Complex: Net Gain or Net Loss?

The balance between the immune/inflammatory and regenerative responses in the diseased pulp is central to the clinical outcome, and this response is unique within the body because of its tissue site. Cariogenic bacteria invade the dentin and pulp tissues, triggering molecular and cellular events dependent on the disease stage. At the early onset, odontoblasts respond to bacterial components in an attempt to protect the tooth's hard and soft tissues and limit disease progression. However, as disease advances, the odontoblasts die, and cells central to the pulp core, including resident immune cells, pulpal fibroblasts, endothelial cells, and stem cells, respond to the bacterial challenge via their expression of a range of pattern recognition receptors that identify pathogen-associated molecular patterns. Subsequently, recruitment and activation occurs of a range of immune cell types, including neutrophils, macrophages, and T and B cells, which are attracted to the diseased site by cytokine/chemokine chemotactic gradients initially generated by resident pulpal cells. Although these cells aim to disinfect the tooth, their extravasation, migration, and antibacterial activity (eg, release of reactive oxygen species [ROS]) along with the bacterial toxins cause pulp damage and impede tissue regeneration processes. Recently, a novel bacterial killing mechanism termed neutrophil extracellular traps (NETs) has also been described that uses ROS signaling and results in cellular DNA extrusion. The NETs are decorated with antimicrobial peptides (AMPs), and their interaction with bacteria results in microbial entrapment and death. Recent data show that NETs can be stimulated by bacteria associated with endodontic infections, and they may be present in inflamed pulp tissue. Interestingly, some bacteria associated with pulpal infections express deoxyribonuclease enzymes, which may enable their evasion of NETs. Furthermore, although NETs aim to localize and kill invading bacteria using AMPs and histones, limiting the spread of the infection, data also indicate that NETs can exacerbate inflammation and their components are cytotoxic. This review considers the potential role of NETs within pulpal infections and how these structures may influence the pulp's vitality and regenerative responses.

Characterization, Quantification, and Visualization of Neutrophil Extracellular Traps.

Following the discovery of neutrophil extracellular traps (NETs) in 2004 by Brinkmann and colleagues, there has been extensive research into the role of NETs in a number of inflammatory diseases, including periodontitis. This chapter describes the current methods for the isolation of peripheral blood neutrophils for subsequent NET experiments, including approaches to quantify and visualize NET production, the ability of NETs to entrap and kill bacteria, and the removal of NETs by nuclease-containing plasma.