Local neutrophil and eosinophil extracellular traps formation in pyodermatitis pyostomatitis vegetans.

Pyodermatitis pyostomatitis vegetans is a rare inflammatory condition, affecting the skin and/or mucous membrane. Some cases include both skin and mucous involvement, whereas others develop either skin or mucous lesions only. The typically affected areas are the scalp, face, trunk and extremities, including the flexural areas and umbilicus. Clinical features show erosive granulomatous plaques, keratotic plaques with overlying crusts and pustular lesions. Among mucous lesions, oral mucosa is most frequently involved, and gingival erythema, shallow erosions, cobblestone-like papules on the buccal mucosa or upper hard palate of the oral cavity are also observed. Some of the lesions assume a 'snail track' appearance. Although there are several similarities between pyodermatitis pyostomatitis vegetans and other diseases, that is pyoderma gangrenosum, pemphigus vegetans and pemphigoid vegetans, the histopathological features of pyodermatitis pyostomatitis vegetans are unique in that epidermal hyperplasia, focal acantholysis and dense inflammatory infiltrates with intraepidermal and subepidermal eosinophilic microabscesses are observed. Direct immunofluorescence findings are principally negative. Activated neutrophils are supposed to play an important role in the pathogenesis of pyodermatitis pyostomatitis vegetans. The expression of IL-36 and neutrophil extracellular traps (NETs) was observed in the lesional skin, and additionally, eosinophil extracellular traps (EETs) was detected in pyodermatitis pyostomatitis vegetans. A possible pathogenic role of NETs and EETs in the innate immunity and autoinflammatory aspects of pyodermatitis pyostomatitis vegetans was discussed.

Porphyromonas gingivalis promotes the progression of oral squamous cell carcinoma by activating the neutrophil chemotaxis in the tumour microenvironment.

BACKGROUND: We aimed to determine the significance of Porphyromonas gingivalis (P. gingivalis) in promoting tumour progression in the tumour microenvironment (TME) of oral squamous cell carcinoma (OSCC). METHODS: The Gene Expression Omnibus (GEO) was used to screen out the differentially expressed genes from the two datasets of GEO138206 and GSE87539. Immunohistochemistry and immunofluorescence analysis of samples, cell biological behaviour experiments, and tumour-bearing animal experiments were used to verify the results in vivo and in vitro. The mechanism was revealed at the molecular level, and rescue experiments were carried out by using inhibitors and lentiviruses. RESULTS: CXCL2 was selected by bioinformatics analysis and was found to be related to a poor prognosis in OSCC patients. Samples with P. gingivalis infection in the TME of OSCC had the strongest cell invasion and proliferation and the largest tumour volume in tumour-bearing animal experiments and exhibited JAK1/STAT3 signalling pathway activation and epithelial-mesenchymal transition (EMT). The expression of P. gingivalis, CXCL2 and TANs were independent risk factors for poor prognosis in OSCC patients. A CXCL2/CXCR2 signalling axis inhibitor significantly decreased the invasion and proliferation ability of cells and the tumour volume in mice. When lentivirus was used to block the CXCL2/CXCR2 signalling axis, the activity of the JAK1/STAT3 signalling pathway was decreased, and the phenotype of EMT was reversed. CONCLUSION: Porphyromonas gingivalis promotes OSCC progression by recruiting TANs via activation of the CXCL2/CXCR2 axis in the TME of OSCC.

Neutrophil N1 and N2 Subsets and Their Possible Association with Periodontitis: A Scoping Review.

Periodontitis is a chronic non-communicable disease caused by dysbiotic changes that affect the subgingival microbiota. During periodontitis, neutrophils play a central role in the initial recognition of bacteria, and their number increases with the appearance of the first signs of periodontal inflammation. Recent evidence has led to the proposition that neutrophils can also functionally polarize, determining selective activity patterns related to different diseases. Two well-defined neutrophil phenotypes have been described, the pro-inflammatory N1 subset and the suppressor N2 subset. To date, it has not been established whether these different neutrophil subtypes play a role in the pathogenesis of periodontitis. Thus, this scoping review aimed to determine whether there was evidence to suggest that the neutrophils present in periodontal tissues can be associated with certain phenotypes. The research question, population, concept, and context sought to identify original articles, in humans, that detected the presence of neutrophils in the periodontal tissues of people affected by periodontitis. Based on the search strategy, we found 3658 studies. After removing the papers with abstracts not related to the outcome measures and eligibility criteria, 16 articles were included for qualitative analysis. Several studies identified the presence of different neutrophil subsets, specifically, the naive, pro- and para-inflammatory, hyper-reactive and hyper-active, and high- and low-responder phenotypes. The existing evidence demonstrates the presence of pro-inflammatory, hyper-reactive and high-responder neutrophils in periodontal tissues affected with periodontitis. There is no evidence demonstrating the presence of the N1 or N2 phenotypes in periodontal tissues during periodontitis. However, the existence of pro-inflammatory phenotypes, which increase NETosis and degranulation, and increase the production of pro-inflammatory cytokines, could be suggestive of the N1 phenotypes.

Triggering NETosis via protease-activated receptor (PAR)-2 signaling as a mechanism of hijacking neutrophils function for pathogen benefits.

Neutrophil-derived networks of DNA-composed extracellular fibers covered with antimicrobial molecules, referred to as neutrophil extracellular traps (NETs), are recognized as a physiological microbicidal mechanism of innate immunity. The formation of NETs is also classified as a model of a cell death called NETosis. Despite intensive research on the NETs formation in response to pathogens, the role of specific bacteria-derived virulence factors in this process, although postulated, is still poorly understood. The aim of our study was to determine the role of gingipains, cysteine proteases responsible for the virulence of P. gingivalis, on the NETosis process induced by this major periodontopathogen. We showed that NETosis triggered by P. gingivalis is gingipain dependent since in the stark contrast to the wild-type strain (W83) the gingipain-null mutant strain only slightly induced the NETs formation. Furthermore, the direct effect of proteases on NETosis was documented using purified gingipains. Notably, the induction of NETosis was dependent on the catalytic activity of gingipains, since proteolytically inactive forms of enzymes showed reduced ability to trigger the NETs formation. Mechanistically, gingipain-induced NETosis was dependent on proteolytic activation of protease-activated receptor-2 (PAR-2). Intriguingly, both P. gingivalis and purified Arg-specific gingipains (Rgp) induced NETs that not only lacked bactericidal activity but instead stimulated the growth of bacteria species otherwise susceptible to killing in NETs. This protection was executed by proteolysis of bactericidal components of NETs. Taken together, gingipains play a dual role in NETosis: they are the potent direct inducers of NETs formation but in the same time, their activity prevents P. gingivalis entrapment and subsequent killing. This may explain a paradox that despite the massive accumulation of neutrophils and NETs formation in periodontal pockets periodontal pathogens and associated pathobionts thrive in this environment.

Immune and regulatory functions of neutrophils in inflammatory bone loss.

Although historically viewed as merely anti-microbial effectors in acute infection or injury, neutrophils are now appreciated to be functionally versatile with critical roles also in chronic inflammation. Periodontitis, a chronic inflammatory disease that destroys the tooth-supporting gums and bone, is particularly affected by alterations in neutrophil numbers or function, as revealed by observations in monogenic disorders and relevant mouse models. Besides being a significant debilitating disease and health burden in its own right, periodontitis is thus an attractive model to dissect uncharted neutrophil-associated (patho)physiological pathways. Here, we summarize recent evidence that neutrophils can contribute to inflammatory bone loss not only through the typical bystander injury dogma but intriguingly also through their absence from the affected tissue, where they normally perform important immunomodulatory functions. Moreover, we discuss recent advances in the interactions of neutrophils with the vascular endothelium and - upon extravasation - with bacteria, and how the dysregulation of these interactions leads to inflammatory tissue damage. Overall, neutrophils have both protective and destructive roles in periodontitis, as they are involved in both the maintenance of periodontal tissue homeostasis and the induction of inflammatory bone loss. This highlights the importance of developing approaches that promote or sustain a fine balance between homeostatic immunity and inflammatory pathology.

Neutrophil-Mediated Delivery of Dexamethasone Palmitate-Loaded Liposomes Decorated with a Sialic Acid Conjugate for Rheumatoid Arthritis Treatment.

PURPOSE: The aim of this research was to design dexamethasone palmitate (DP) loaded sialic acid modified liposomes, with the eventual goal of using peripheral blood neutrophils (PBNs) that carried drug-loaded liposomes to improve the therapeutic capacity for rheumatoid arthritis (RA). METHODS: A sialic acid - cholesterol conjugate (SA-CH) was synthesized and anchored on the surface of liposomal dexamethasone palmitate (DP-SAL). The physicochemical characteristics and in vitro cytotoxicity of liposomes were evaluated. Flow cytometry and confocal laser scanning microscopy were utilized to investigate the accumulation of liposomes in PBNs. The adjuvant-induced arthritis was adopted to investigate the targeting ability and anti-inflammatory effect of DP loaded liposomes. RESULTS: Both DP-CL and DP-SAL existed an average size less than 200 nm with remarkably high encapsulation efficiencies more than 90%. In vitro and in vivo experiments manifested SA-modified liposomes provided a reinforced accumulation of DP in PBNs. As well, DP-SAL displayed a greater degree of accumulation in the joints and a stronger anti-inflammatory effect in terms of RA suppression. CONCLUSIONS: SA-modified liposomal DP was a promising candidate for RA-targeting treatment through the neutrophil-mediated drug delivery system.

Aggregatibacter actinomycetemcomitans induces detachment and death of human gingival epithelial cells and fibroblasts via elastase release following leukotoxin-dependent neutrophil lysis.

Aggregatibacter actinomycetemcomitans is considered to be associated with periodontitis. Leukotoxin (LtxA), which destroys leukocytes in humans, is one of this bacterium's major virulence factors. Amounts of neutrophil elastase (NE), which is normally localized in the cytoplasm of neutrophils, are reportedly increased in the saliva of patients with periodontitis. However, the mechanism by which NE is released from human neutrophils and the role of NE in periodontitis is unclear. In the present study, it was hypothesized that LtxA induces NE release from human neutrophils, which subsequently causes the breakdown of periodontal tissues. LtxA-treatment did not induce significant cytotoxicity against human gingival epithelial cells (HGECs) or human gingival fibroblasts (HGFs). However, it did induce significant cytotoxicity against human neutrophils, leading to NE release. Furthermore, NE and the supernatant from LtxA-treated human neutrophils induced detachment and death of HGECs and HGFs, these effects being inhibited by administration of an NE inhibitor, sivelestat. The present results suggest that LtxA mediates human neutrophil lysis and induces the subsequent release of NE, which eventually results in detachment and death of HGECs and HGFs. Thus, LtxA-induced release of NE could cause breakdown of periodontal tissue and thereby exacerbate periodontitis.

The Alzheimer's disease-protective CD33 splice variant mediates adaptive loss of function via diversion to an intracellular pool.

The immunomodulatory receptor Siglec-3/CD33 influences risk for late-onset Alzheimer's disease (LOAD), an apparently human-specific post-reproductive disease. CD33 generates two splice variants: a full-length CD33M transcript produced primarily by the "LOAD-risk" allele and a shorter CD33m isoform lacking the sialic acid-binding domain produced primarily from the "LOAD-protective" allele. An SNP that modulates CD33 splicing to favor CD33m is associated with enhanced microglial activity. Individuals expressing more protective isoform accumulate less brain ß-amyloid and have a lower LOAD risk. How the CD33m isoform increases ß-amyloid clearance remains unknown. We report that the protection by the CD33m isoform may not be conferred by what it does but, rather, from what it cannot do. Analysis of blood neutrophils and monocytes and a microglial cell line revealed that unlike CD33M, the CD33m isoform does not localize to cell surfaces; instead, it accumulates in peroxisomes. Cell stimulation and activation did not mobilize CD33m to the surface. Thus, the CD33m isoform may neither interact directly with amyloid plaques nor engage in cell-surface signaling. Rather, production and localization of CD33m in peroxisomes is a way of diminishing the amount of CD33M and enhancing ß-amyloid clearance. We confirmed intracellular localization by generating a CD33m-specific monoclonal antibody. Of note, CD33 is the only Siglec with a peroxisome-targeting sequence, and this motif emerged by convergent evolution in toothed whales, the only other mammals with a prolonged post-reproductive lifespan. The CD33 allele that protects post-reproductive individuals from LOAD may have evolved by adaptive loss-of-function, an example of the less-is-more hypothesis.

Non-invasive Imaging of Sendai Virus Infection in Pharmacologically Immunocompromised Mice: NK and T Cells, but not Neutrophils, Promote Viral Clearance after Therapy with Cyclophosphamide and Dexamethasone.

In immunocompromised patients, parainfluenza virus (PIV) infections have an increased potential to spread to the lower respiratory tract (LRT), resulting in increased morbidity and mortality. Understanding the immunologic defects that facilitate viral spread to the LRT will help in developing better management protocols. In this study, we immunosuppressed mice with dexamethasone and/or cyclophosphamide then monitored the spread of viral infection into the LRT by using a noninvasive bioluminescence imaging system and a reporter Sendai virus (murine PIV type 1). Our results show that immunosuppression led to delayed viral clearance and increased viral loads in the lungs. After cessation of cyclophosphamide treatment, viral clearance occurred before the generation of Sendai-specific antibody responses and coincided with rebounds in neutrophils, T lymphocytes, and natural killer (NK) cells. Neutrophil suppression using anti-Ly6G antibody had no effect on infection clearance, NK-cell suppression using anti-NK antibody delayed clearance, and T-cell suppression using anti-CD3 antibody resulted in no clearance (chronic infection). Therapeutic use of hematopoietic growth factors G-CSF and GM-CSF had no effect on clearance of infection. In contrast, treatment with Sendai virus-specific polysera or a monoclonal antibody limited viral spread into the lungs and accelerated clearance. Overall, noninvasive bioluminescence was shown to be a useful tool to study respiratory viral progression, revealing roles for NK and T cells, but not neutrophils, in Sendai virus clearance after treatment with dexamethasone and cyclophosphamide. Virus-specific antibodies appear to have therapeutic potential.

Use of cilomilast-loaded phosphatiosomes to suppress neutrophilic inflammation for attenuating acute lung injury: the effect of nanovesicular surface charge.

BACKGROUND: Cilomilast is a phosphodiesterase 4 (PDE4) inhibitor for treating inflammatory lung diseases. This agent has a narrow therapeutic index with significant adverse effects on the nervous system. This study was conducted to entrap cilomilast into PEGylated phosphatidylcholine-rich niosomes (phosphatiosomes) to improve pulmonary delivery via the strong affinity to pulmonary surfactant film. Neutrophils were used as a cell model to test the anti-inflammatory activity of phosphatiosomes. In an in vivo approach, mice were given lipopolysaccharide to produce acute lung injury. The surface charge in phosphatiosomes that influenced the anti-inflammatory potency is discussed in this study. RESULTS: The average diameter of the phosphatiosomes was about 100 nm. The zeta potential of anionic and cationic nanovesicles was - 35 and 32 mV, respectively. Cilomilast in both its free and nanocapsulated forms inhibited superoxide anion production but not elastase release in activated neutrophils. Cationic phosphatiosomes mitigated calcium mobilization far more effectively than the free drug. In vivo biodistribution evaluated by organ imaging demonstrated a 2-fold ameliorated lung uptake after dye encapsulation into the phosphatiosomes. The lung/brain distribution ratio increased from 3 to 11 after nanocarrier loading. The intravenous nanocarriers deactivated the neutrophils in ALI, resulting in the elimination of hemorrhage and alveolar wall damage. Only cationic phosphatiosomes could significantly suppress IL-1ß and TNF-α in the inflamed lung tissue. CONCLUSIONS: These results suggest that phosphatiosomes should further be investigated as a potential nanocarrier for the treatment of pulmonary inflammation.

A Phase III Study of Balugrastim Versus Pegfilgrastim in Breast Cancer Patients Receiving Chemotherapy With Doxorubicin and Docetaxel.

OBJECTIVES: This study aimed to evaluate the efficacy and safety of once-per-cycle balugrastim versus pegfilgrastim for neutrophil support in breast cancer patients receiving myelosuppressive chemotherapy. METHODS: Breast cancer patients (n = 256) were randomized to 40 or 50 mg of subcutaneous balugrastim or 6 mg of pegfilgrastim ≈24 hours after chemotherapy (60 mg/m(2) doxorubicin and 75 mg/m(2) docetaxel, every 21 days for up to 4 cycles). The primary efficacy parameter was the duration of severe neutropenia (DSN) in cycle 1. Secondary parameters included DSN (cycles 2-4), absolute neutrophil count (ANC) nadir, febrile neutropenia rates, and time to ANC recovery (cycles 1-4). Safety, pharmacokinetics, and immunogenicity were assessed. RESULTS: Mean cycle 1 DSN was 1.0 day with 40 mg of balugrastim, 1.3 with 50 mg of balugrastim, and 1.2 with pegfilgrastim (upper limit of 95% confidence intervals for between-group DSN differences was <1.0 day for both balugrastim doses versus pegfilgrastim). Between-group efficacy parameters were comparable except for time to ANC recovery in cycle 1 (40 mg of balugrastim, 2.0 days; 50 mg of balugrastim, 2.1; pegfilgrastim, 2.6). Median terminal elimination half-life was ≈37 hours for 40 mg of balugrastim, ≈36 for 50 mg of balugrastim, and ≈45 for pegfilgrastim. Antibody response to balugrastim was low and transient, with no neutralizing effect. CONCLUSION: Once-per-cycle balugrastim is not inferior to pegfilgrastim in reducing cycle 1 DSN in breast cancer patients receiving chemotherapy; both drugs have comparable safety profiles. IMPLICATIONS FOR PRACTICE: This paper provides efficacy and safety data for a new, once-per-cycle granulocyte colony-stimulating factor, balugrastim, for the prevention of chemotherapy-induced neutropenia in patients with breast cancer receiving myelosuppressive chemotherapy. In this phase III trial, balugrastim was shown to be not inferior to pegfilgrastim in the duration of severe neutropenia in cycle 1 of doxorubicin/docetaxel chemotherapy, and the safety profiles of the two agents were similar. Once-per-cycle balugrastim is a safe and effective alternative to pegfilgrastim for hematopoietic support in patients with breast cancer receiving myelosuppressive chemotherapy associated with a greater than 20% risk of developing febrile neutropenia.

Polymorphonuclear neutrophils in periodontitis and their possible modulation as a therapeutic approach.

The main focus of this review is polymorphonuclear neutrophilic granulocytes. Polymorphonuclear neutrophils play a pivotal role in normal host resistance to subgingival dental-plaque biofilm. Both hyper- and hypo-responsiveness of the immune system toward the microbial challenge in periodontitis have been described. We review polymorphonuclear neutrophil physiology with emphasis on the role of neutrophil functions and dysfunctions in periodontitis. Text boxes are given at the end of each subsection, which present the current knowledge on neutrophil-modulating agents as a potential therapeutic approach in periodontitis.

Ethylmercury and Hg2+ induce the formation of neutrophil extracellular traps (NETs) by human neutrophil granulocytes.

Humans are exposed to different mercurial compounds from various sources, most frequently from dental fillings, preservatives in vaccines, or consumption of fish. Among other toxic effects, these substances interact with the immune system. In high doses, mercurials are immunosuppressive. However, lower doses of some mercurials stimulate the immune system, inducing different forms of autoimmunity, autoantibodies, and glomerulonephritis in rodents. Furthermore, some studies suggest a connection between mercury exposure and the occurrence of autoantibodies against nuclear components and granulocyte cytoplasmic proteins in humans. Still, the underlying mechanisms need to be clarified. The present study investigates the formation of neutrophil extracellular traps (NETs) in response to thimerosal and its metabolites ethyl mercury (EtHg), thiosalicylic acid, and mercuric ions (Hg(2+)). Only EtHg and Hg(2+) triggered NETosis. It was independent of PKC, ERK1/2, p38, and zinc signals and not affected by the NADPH oxidase inhibitor DPI. Instead, EtHg and Hg(2+) triggered NADPH oxidase-independent production of ROS, which are likely to be involved in mercurial-induced NET formation. This finding might help understanding the autoimmune potential of mercurial compounds. Some diseases, to which a connection with mercurials has been shown, such as Wegener's granulomatosis and systemic lupus erythematosus, are characterized by high prevalence of autoantibodies against neutrophil-specific auto-antigens. Externalization in the form of NETs may be a source for exposure to these self-antigens. In genetically susceptible individuals, this could be one step in the series of events leading to autoimmunity.

Risk Factors for Life-Threatening Complications of Maxillofacial Space Infection.

The purpose of this study was to review the clinical features of maxillofacial space infection (MSI), and to identify the potential risk factors predisposing to life-threatening complications. A retrospective review of the medical charts of patients with MSI treated at Peking University School and Hospital of Stomatology from August 2008 to September 2013 was conducted. A total of 127 patients [75 men (59.1%) and 52 women (40.9%); mean age, 45.39 ± 21.18 years, with a range of 1-85 years] formed the study cohort. The most common cause of MSI was odontogenic infection (57.5%). The most common space involved was the submandibular space. All patients were treated by antibiotics as well as surgical incision and drainage. Sixteen patients developed life-threatening complications, and the dominant complication was respiratory obstruction. Multivariate logistic regression analysis revealed the percentage of neutrophils (NEUT%) upon hospital admission ≥ 85.0% to be associated with life-threatening complications (P < 0.05). Even though adequate antibiotic therapy and incision and drainage of abscess were given, MSI patients with NEUT% upon hospital admission ≥ 85.0% carry a higher risk of life-threatening complications. In these patients, an aggressive treatment strategy is mandatory.

Total and Differential Leukocyte Counts in the Peripheral Blood of Patients with Generalised Aggressive Periodontitis.

PURPOSE: To compare the total and differential leukocyte counts in the peripheral blood of generalised aggressive periodontitis patients with that of periodontally healthy subjects in a central Indian population. MATERIALS AND METHODS: Seventy-five patients with generalised aggressive periodontitis and 63 periodontally healthy subjects were enrolled for the purpose of the study. All participants received a full-mouth periodontal examination in which probing depth and clinical attachment level were recorded. The haematological variables analysed included total leukocyte count, neutrophil count, lymphocyte count, monocyte count, neutrophil percentage, lymphocyte percentage, monocyte percentage and platelet count. RESULTS: The patient group showed a significantly higher total leukocyte count (7.62 ± 1.70 x 109 cells/l, p = 0.008), neutrophil count (5.06 ± 1.47x109 cells/l, p < 0.001) and neutrophil percentage (70.61 ± 8.73, p < 0.001), as well as a significantly lower lymphocyte count (1.82 ± 0.65 x 109 cells/l, p = 0.002) and lymphocyte percentage (26.55 ± 8.05, p < 0.001) compared to the control group. Logistic regression analyses showed significant associations between aggressive periodontitis and elevated total leukocyte (p = 0.012) and neutrophil counts (p = 0.001). CONCLUSION: The findings of the present study suggest that patients with generalised aggressive periodontitis might also demonstrate a systemic inflammatory response, as evidenced by increased leukocyte counts. This systemic inflammatory response observed in patients with generalised aggressive periodontitis may be associated with an increased risk for cardiovascular diseases.

[Cigarette smoking in different manners induces acute lung injury in rats].

Objective: To investigate the effects of cigarette smoking in different manners on acute lung injury in rats. Methods: The commercially available cigarettes with tar of 1,5, 11 mg were smoked in Canada depth smoking (health canada method, HCM) manner, and those with tar of 11 mg were also smoked in international standard (ISO) smoking manner. Rats were fixed and exposed to mainstream in a manner of nose-mouth exposure. After 28 days, the bronchoalveolar lavage fluids from left lung were collected for counting and classification of inflammatory cells and determination of pro-inflammatory cytokines IL-1ß and TNF-α. The right lungs were subjected to histological examination and determination of myeloperoxidase (MPO) and superoxide dismutase (SOD) activities and glutathione, reactive oxygen species (ROS) and malondialdehyde (MDA) levels. Results: In both HCM and ISO manners, the degree of lung injury was closely related to the tar content of cigarettes, and significant decrease in the body weight of rats was observed after smoking for one week. In a HCM manner, smoking with cigarette of 11 mg tar resulted in robust infiltration of macrophages, lymphocytes and neutrophils into lungs, significant increase in IL-1ß and TNF-α levels and MPO activities, and significant decrease in GSH levels and SOD activities and increase in ROS and MDA levels (all P<0.05). Smoking with cigarette of 5 mg tar led to moderate increase in IL-1ß and TNF-α levels, and MPO activities (all P<0.05), and moderate decrease in GSH levels and SOD activities and increase of ROS and MDA levels (all P<0.05). However, smoking with cigarette of 1 mg tar affected neither inflammatory cell infiltration nor IL-1ß and TNF-α levels. Conclusion: Cigarette smoking in nose-mouth exposure manner can induce acute lung injury in rats; and the degree of lung injury is closely related to the content of tar and other hazards in cigarettes.

Increased levels of neutrophil extracellular trap remnants in the circulation of patients with small vessel vasculitis, but an inverse correlation to anti-neutrophil cytoplasmic antibodies during remission.

OBJECTIVES: Neutrophil extracellular traps (NETs) have been visualized at the site of ANCA-associated vasculitis (AAV) lesions. Increased levels of NET remnants in the circulation have been reported in some AAV patients with active disease. The aim of the present study was to analyse NET remnants in a larger cohort of AAV patients with varying degrees of disease activity and to elucidate possible factors responsible for remnant variation. METHODS: Levels of NET remnants in the circulation of healthy controls (HCs; n = 31) and AAV patients (n = 93) were determined with ELISA. NET remnants were then correlated with ANCA levels, spontaneous and induced cell death (NETosis/necrosis) in vitro, neutrophil count and corticosteroid therapy. RESULTS: Patients with active disease showed higher levels of circulating NET remnants compared with patients in remission (P = 0.026) and HCs (P = 0.006). From patients sampled during both remission and active disease, we found increased levels during active disease (P = 0.0010). In remission, ANCA-negative patients had higher levels of NET remnants than ANCA-positive patients and a negative correlation was observed between NET remnants and PR3-ANCA (rs = -0.287, P = 0.048). NET remnants correlated with neutrophil count in HCs (rs = 0.503, P = 0.014) but not in patients during remission. Neutrophils from patients showed enhanced spontaneous cell death (P = 0.043). CONCLUSION: We found increased levels of circulating NET remnants in patients with active AAV. Furthermore, AAV patients exhibited an increased propensity for spontaneous cell death. NET remnant levels seem to be positively related to disease activity and neutrophil count, but inversely related to ANCA at least during remission.

Quantifying oral inflammatory load: oral neutrophil counts in periodontal health and disease.

BACKGROUND AND OBJECTIVE: Neutrophils are the primary white blood cells that are recruited to fight the initial phases of microbial infections. While healthy norms have been determined for circulating blood neutrophil counts in order to identify patients with suspected systemic infections, the levels of oral neutrophils (oPMNs) in oral health and in the presence of periodontal diseases have not been described. It is important to address this deficiency in our knowledge as neutrophils are the primary immune cell present in the crevicular fluid and oral environment and previous work has suggested that they may be good indicators of overall oral inflammation and periodontal disease severity. The objective of this study was to measure oPMN counts obtained in a standardized oral rinse from healthy patients and from those with chronic periodontal disease in order to determine if oPMN levels have clinical relevance as markers of periodontal inflammation. A parallel goal of this investigation was to introduce the concept of 'oral inflammatory load', which constitutes the inflammatory burden experienced by the body as a consequence of oral inflammatory disease. MATERIAL AND METHODS: Periodontal examinations of patients with a healthy periodontium and chronic periodontal disease were performed (n = 124). Two standardized consecutive saline rinses of 30 s each were collected before patient examination and instrumentation. Neutrophils were quantified in the rinse samples and correlated with the clinical parameters and periodontal diagnosis. RESULTS: Average oPMN counts were determined for healthy patients and for those with mild, moderate and severe chronic periodontal diseases. A statistically significant correlation was found between oPMN counts and deep periodontal probing, sites with bleeding on probing and overall severity of periodontal disease. CONCLUSIONS: oPMN counts obtained through a 30-s oral rinse are a good marker of oral inflammatory load and correlate with measures of periodontal disease severity.

Periodontitis mainly increases osteoclast formation via enhancing the differentiation of quiescent osteoclast precursors into osteoclasts.

BACKGROUND AND OBJECTIVES: Tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts are formed in sequential steps: proliferation and differentiation of hematopoietic progenitors into quiescent osteoclast precursors (QOPs), followed by fusion of QOPs. In this study, we investigated whether enhancement of osteoclast formation by periodontitis is derived from the stimulation of proliferation of hematopoietic progenitors or the differentiation of QOPs into osteoclasts. MATERIAL AND METHODS: Ligatures were placed around the first molars in the left mandibles of Fischer 344 inbred rats. The rats received drinking water containing bromodeoxyuridine (BrdU) (which can be incorporated into dividing nuclei) after ligation during the experimental period. The number of inflammatory cells in the distal area was counted. Alveolar bone loss was histologically estimated by measuring the distance from the cementoenamel junction to the alveolar bone crest in the distal area and determining the percentage of periodontal ligament area in the furcation. The number of osteoclasts and percentage of BrdU(+) nuclei in total osteoclasts nuclei were counted after TRAP and BrdU double labeling. RESULTS: The number of polymorphonuclear cells increased on day 1 and then rapidly decreased. The number of mononuclear cells increased in a time-dependent manner up to day 5 and remained the same until day 10. Alveolar bone loss of ligatured teeth increased in a time-dependent manner. The number of osteoclasts peaked on day 3 then gradually decreased. At peak, the percentage of BrdU(+) nuclei in total osteoclasts nuclei in the distal and furcation areas were 7.9% and 4.1%, respectively. CONCLUSION: These results indicate that most of the osteoclasts formed after periodontitis induction are derived from preformed QOPs, suggesting that enhancement of osteoclast formation by periodontitis might be mainly caused by stimulating the differentiation of QOPs into osteoclasts.

Cellular composition of long-standing gingivitis and periodontitis lesions.

BACKGROUND AND OBJECTIVE: Insufficient information on the cellular composition of long-standing gingivitis lesions without signs of attachment loss makes an understanding of differences in cellular composition between "destructive" and "nondestructive" periodontal lesions difficult. The aim of the current study was to analyze differences in cell characteristics between lesions representing long-standing gingivitis and severe periodontitis. MATERIAL AND METHODS: Two groups of patients were recruited. One group consisted of 36 patients, 33-67 years of age, with severe generalized periodontitis (periodontitis group). The second group consisted of 28 patients, 41-70 years of age, with overt signs of gingival inflammation but no attachment loss (gingivitis group). From each patient a gingival biopsy was obtained from one selected diseased site and prepared for immunohistochemical analysis. RESULTS: Periodontitis lesions were twice as large and contained significantly larger proportions, numbers and densities of cells positive for CD138 (plasma cells) and CD68 (macrophages) than did gingivitis lesions. The proportion of B cells that expressed the additional CD5 marker (B-1a cells) was significantly larger in periodontitis lesions than in gingivitis lesions. The densities of T cells and B cells did not differ between periodontitis lesions and gingivitis lesions. T cells were not the dominating cell type in gingivitis lesions, as B cells together with their subset plasma cells comprised a larger number and proportion than T cells. CONCLUSION: Periodontitis lesions at teeth with advanced attachment and bone loss exhibit quantitative and qualitative differences in relation to gingivitis lesions at teeth with no attachment and bone loss. It is suggested that the large number and high density of plasma cells are the hallmarks of advanced periodontitis lesions and the most conspicuous difference in relation to long-standing gingivitis lesions.