Cigarette smoke modifies neutrophil chemotaxis, neutrophil extracellular trap formation and inflammatory response-related gene expression.

BACKGROUND AND OBJECTIVE: Cigarette smoking is a major risk factor for periodontitis, and smoking perturbs neutrophil reactive oxygen species production. This study tested the hypothesis that cigarette smoke extract (CSE) and its components/metabolites nicotine, cotinine and thiocyanate (SCN-), may influence neutrophil functions. MATERIAL AND METHODS: Chemotaxis was assessed in neutrophils pre-treated with CSE using real-time video microscopy. Neutrophil extracellular trap (NET) release in response to CSE, nicotine, cotinine, SCN- as well as to phorbol 12-myristate-13-acetate and hypochlorous acid following pre-treatment with CSE, nicotine, cotinine or SCN- was assessed using fluorescence-based assays. The impact of CSE and SCN- treatment on neutrophil respiratory burst- and inflammation-related gene expression (NFKBIE, DNAJB1, CXCL8, NCF1, NCF2, CYBB) was determined by real-time polymerase chain reaction. RESULTS: Both CSE and SCN- pre-treatment inhibited phorbol 12-myristate-13-acetate-stimulated NET release. Additionally, SCN- inhibited hypochlorous acid-stimulated NET formation, while SCN- alone stimulated NET release. Overall, neutrophils pre-treated with CSE exhibited reduced speed, velocity and directionality relative to untreated neutrophils. Although CSE and SCN- promoted DNAJB1 expression, increased redox-related gene expression was only detected in response to SCN-. CONCLUSION: These results suggest that CSE can alter ex vivo neutrophil activation by mechanisms independent of SCN- and nicotine, and SCN- may contribute to the perturbed innate immune responses observed in smokers.

CDCP1 cleavage is necessary for homodimerization-induced migration of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a highly aggressive and metastatic form of breast cancer that lacks the estrogen, progesterone and HER2 receptors and is resistant to targeted and hormone therapies. TNBCs express high levels of the transmembrane glycoprotein, complement C1r/C1s, Uegf, Bmp1 (CUB)-domain containing protein 1 (CDCP1), which has been correlated with the aggressiveness and poor prognosis of multiple carcinomas. Full-length CDCP1 (flCDCP1) can be proteolytically cleaved, resulting in a cleaved membrane-bound isoform (cCDCP1). CDCP1 is phosphorylated by Src family kinases in its full-length and cleaved states, which is important for its pro-metastatic signaling. We observed that cCDCP1, compared with flCDCP1, induced a dramatic increase in phosphorylation of the migration-associated proteins: PKCδ, ERK1/2 and p38 mitogen-activated protein kinase in HEK 293T. In addition, only cCDCP1 induced migration of HEK 293T cells and rescued migration of the TNBC cell lines expressing short hairpin RNA against CDCP1. Importantly, we found that only cCDCP1 is capable of dimerization, which can be blocked by expression of the extracellular portion of cCDCP1 (ECC), indicating that dimerization occurs through CDCP1's ectodomain. We found that ECC inhibited phosphorylation of PKCδ and migration of TNBC cells in two-dimensional culture. Furthermore, ECC decreased cell invasiveness, inhibited proliferation and stimulated apoptosis of TNBC cells in three-dimensional culture, indicating that the cCDCP1 dimer is an important contributor to TNBC aggressiveness. These studies have important implications for the development of a therapeutic to block CDCP1 activity and TNBC metastasis.

Hypochlorous acid regulates neutrophil extracellular trap release in humans.

Neutrophil extracellular traps (NETs) comprise extracellular chromatin and granule protein complexes that immobilize and kill bacteria. NET release represents a recently discovered, novel anti-microbial strategy regulated non-exclusively by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generation of reactive oxygen intermediates (ROIs), particularly hydrogen peroxide. This study aimed to characterize the role of ROIs in the process of NET release and to identify the dominant ROI trigger. We employed various enzymes, inhibitors and ROIs to record their effect fluorometrically on in vitro NET release by human peripheral blood neutrophils. Treatment with exogenous superoxide dismutase (SOD) supported the established link between hydrogen peroxide and NET production. However, treatment with myeloperoxidase inhibitors and direct addition of hypochlorous acid (HOCl; generated in situ from sodium hypochlorite) established that HOCl was a necessary and sufficient ROI for NET release. This was confirmed by the ability of HOCl to stimulate NET release in chronic granulomatous disease (CGD) patient neutrophils which, due to the lack of a functional NADPH oxidase, also lack the capacity for NET release in response to classical stimuli. Moreover, the exogenous addition of taurine, abundantly present within the neutrophil cytosol, abrogated NET production stimulated by phorbol myristate acetate (PMA) and HOCl, providing a novel mode of cytoprotection by taurine against oxidative stress by taurine.

Extracellular deoxyribonuclease production by periodontal bacteria.

BACKGROUND AND OBJECTIVE: Whilst certain bacteria have long been known to secrete extracellular deoxyribonuclease (DNase), the purpose in microbial physiology was unclear. Recently, however, this enzyme has been demonstrated to confer enhanced virulence, enabling bacteria to evade the host's immune defence of extruded DNA/chromatin filaments, termed neutrophil extracellular traps (NETs). As NETs have recently been identified in infected periodontal tissue, the aim of this study was to screen periodontal bacteria for extracellular DNase activity. MATERIAL AND METHODS: To determine whether DNase activity was membrane bound or secreted, 34 periodontal bacteria were cultured in broth and on agar plates. Pelleted bacteria and supernatants from broth cultures were analysed for their ability to degrade DNA, with relative activity levels determined using an agarose gel electrophoresis assay. Following culture on DNA-supplemented agar, expression was determined by the presence of a zone of hydrolysis and DNase activity related to colony size. RESULTS: Twenty-seven bacteria, including red and orange complex members Porphyromonas gingivalis, Tannerella forsythia, Fusobacterium nucleatum, Parvimonas micra, Prevotella intermedia, Streptococcus constellatus, Campylobacter rectus and Prevotella nigrescens, were observed to express extracellular DNase activity. Differences in DNase activity were noted, however, when bacteria were assayed in different culture states. Analysis of the activity of secreted DNase from bacterial broth cultures confirmed their ability to degrade NETs. CONCLUSION: The present study demonstrates, for the first time, that DNase activity is a relatively common property of bacteria associated with advanced periodontal disease. Further work is required to determine the importance of this bacterial DNase activity in the pathogenesis of periodontitis.

Fusobacterium nucleatum regulation of neutrophil transcription.

BACKGROUND AND OBJECTIVE: Abnormal neutrophil responses have been observed in periodontitis patients, including hyper-reactivity in terms of production of reactive oxygen species (ROS) following exposure to the key quorum-sensing plaque bacterium, Fusobacterium nucleatum. This study was designed to characterize the transcriptional response of neutrophils to F. nucleatum. MATERIAL AND METHODS: Peripheral blood neutrophils were exposed to F. nucleatum, and gene expression was analysed using high-throughput transcriptomics. RESULTS: Microarray technology demonstrated differential expression of 208 genes (163 increased and 43 decreased relative to control genes), which identified regulation of several ontological classes, including signal transduction (13%), transcription regulation (7%) and ROS response (14%). Individual gene expression analysis of selected transcripts, including CSF, CXCL3, FOS, HMOX1, HSP40, SOD2, NFKB2 and GP91, in individual and pooled RNA samples from control and F. nucleatum-exposed neutrophils corroborated microarray data. Analysis of ROS generation, combined with transcript analysis, in response to a panel of proinflammatory stimuli (F. nucleatum, Porphyromonas gingivalis, Escherichia coli lipopolysaccharide and opsonized Staphylococcus aureus) identified significant differences in ROS and transcript regulatory control. Further analyses of neutrophils from periodontitis patients and periodontally healthy control subjects stimulated with F. nucleatum indicated significant differential induction of several ROS response-related transcripts. CONCLUSION: These data demonstrate that neutrophils are transcriptionally active in response to the periodontal pathogen F. nucleatum and that these changes in gene expression are likely to affect neutrophil function. The differential response of neutrophils to a range of stimuli combined with data demonstrating differences between patient and control neutrophils indicate the importance of this cell and its interaction with the local tissue environment in the pathogenesis of periodontitis.

Neutrophil hyper-responsiveness in periodontitis.

Peripheral neutrophil hyper-responsiveness in chronic periodontitis leads to excessive reactive oxygen species (ROS) production. We aimed to determine whether neutrophil hyper-responsiveness was constitutive or reactive, and to discover the effect of non-surgical therapy. Peripheral blood neutrophils from patients (n = 19), before and 3 months after therapy, and matched control individuals were Fc gamma-receptor-stimulated with/without priming with P. gingivalis and F. nucleatum. Total and extracellular ROS were determined by luminol/isoluminol chemiluminescence. The high total ROS generation of patients' neutrophils compared with that of control individuals (P = 0.016) continued at a reduced level post-therapy (P = 0.059). Reduced activity post-therapy was also seen with priming. Unstimulated total ROS levels did not differ between patients and control individuals before or after therapy. However, the high unstimulated, extracellular ROS production by patients' neutrophils compared with control individuals (P < 0.05) continued post-therapy and was unaffected by priming. Therapy reduced Fc gamma-receptor-stimulated total ROS production, but not unstimulated extracellular radical release, suggesting that constitutive and reactive mechanisms underlie neutrophil hyper-responsiveness.

Differential activation of NF-kappaB and gene expression in oral epithelial cells by periodontal pathogens.

To investigate the molecular effects of the periodontopathogens Fusobacterium nucleatum (FN) and Porphyromonas gingivalis (PG) on the oral epithelium, the H400 oral epithelial cell line was cultured in the presence of non-viable bacteria. Following confirmation of the presence of transcripts for the bacterial pattern recognition receptors in H400 cells, Toll-like receptors -2, -4 and -9, and components of the NF-kappaB signalling pathway, immunocytochemical analyses were performed showing that NF-kappaB was activated within 1 h of exposure to both periodontopathogens. A significantly greater number of NF-kappaB nuclear translocations were apparent following H400 cell exposure to FN as compared with PG. Gene expression analyses indicated that transcripts known to be regulated by the NF-kappaB pathway, including cytokines/chemokines TNF-alpha, IL-1beta, IL-8, MCP-1/CCL2 and GM-CSF, were up-regulated following 4 and 24 h of exposure to both periodontopathogens. In addition, H400 periodontopathogen exposure resulted in differential regulation of transcripts for several cytokeratin gene family members. Consistent with the immunocytochemical data, microarray results indicated that FN induced a greater number of gene expression changes than PG following 24 h of exposure, 609 and 409 genes, respectively. Ninety-one genes were commonly differentially expressed by both periodontopathogens and represented biological processes commonly associated with periodontitis. Gene expression analyses by reserve transcriptase-polymerase chain reaction (RT-PCR) of molecules identified from the microarray data sets, including Heme oxygenase-1, lysyl oxidase, SOD2, CCL20 and calprotectin components, confirmed their differential expression profiles induced by the two periodontopathogens. FN and PG have clearly different molecular effects on oral epithelial cells, potentially highlighting the importance of the composition of the plaque biofilm in periodontitis pathogenesis.

Hyperactivity and reactivity of peripheral blood neutrophils in chronic periodontitis.

Some evidence exists that peripheral neutrophils from patients with chronic periodontitis generate higher levels of reactive oxygen species (ROS) after Fcgamma-receptor stimulation than those from healthy controls. We hypothesized that peripheral neutrophils in periodontitis also show both hyper-reactivity to plaque organisms and hyperactivity in terms of baseline, unstimulated generation and release of ROS. Peripheral neutrophils from chronic periodontitis patients and age/sex/smoking-matched healthy controls (18 pairs) were assayed for total ROS generation and extracellular ROS release, with and without stimulation (Fcgamma-receptor and Fusobacterium nucleatum), using luminol and isoluminol chemiluminescence. Assays were performed with and without priming with Escherichia coli lipopolysaccharide (LPS) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Phox gene expression (p22, p47, p67, gp91) was investigated using reverse transcription-polymerase chain reaction (RT-PCR). Neutrophils from patients produced higher mean levels of ROS in all assays. Total generation and extracellular release of ROS by patients' cells were significantly greater than those from controls after FcgammaR-stimulation, with (P = 0.023) and without (P < or = 0.023) priming with GM-CSF. Differences in unstimulated total ROS generation were not significant. By contrast, patients' cells demonstrated greater baseline, extracellular ROS release than those from controls (P = 0.004). This difference was maintained after priming with LPS (P = 0.028) but not GM-CSF (P = 0.217). Phox gene expression was similar in patient and control cells at baseline and stimulation with F. nucleatum (3 h) consistently reduced gp91(PHOX) transcripts. Our data demonstrate that peripheral neutrophils from periodontitis patients exhibit hyper-reactivity following stimulation (Fcgamma-receptor and F. nucleatum) and hyperactivity in terms of excess ROS release in the absence of exogenous stimulation. This hyperactive/-reactive neutrophil phenotype is not associated with elevated phox gene expression.

Platelet-derived growth factor (PDGF) isoform and PDGF receptor expression in drug-induced gingival overgrowth and hereditary gingival fibrosis.

OBJECTIVE: To investigate possible associations between platelet-derived growth factor (PDGF), PDGF receptor expression and macrophages in drug-induced and hereditary gingival overgrowth. MATERIALS AND METHODS: Tissues from patients with drug-induced gingival overgrowth (DIGO) (n = 10) and hereditary gingival fibrosis (n = 10) were studied and compared with 'control' gingiva (n = 10). Expression of PDGF and its alpha and beta receptors was investigated immunohistochemically and by RT-PCR. Macrophages were identified by immunostaining for CD68. RESULTS: PDGF isoforms and receptors were detected in most cells within all specimens. There were no differences in the numbers of macrophages, or fibroblasts expressing PDGF or receptors, between groups. The level of PDGF expression by fibroblasts, determined by absorbance measurements, was similar between groups for PDGF A. Significantly lower levels of total PDGF and the receptors were detected in drug-induced overgrowth compared to those in hereditary fibrosis (P < 0.004) and control specimens (P < 0.034). All specimens expressed mRNA for PDGF A, PDGF B and alpha and beta receptors. CONCLUSIONS: These data do not support a pivotal role for macrophage-derived PDGF B in the pathogenesis of DIGO. They suggest that fibroblasts in drug-induced lesions have a lowered capacity to produce, and respond to, PDGF, a property not shared by fibroblasts associated with hereditary fibrosis.

Levels of TGFbeta1 in gingival crevicular fluid during a 21-day experimental model of gingivitis.

OBJECTIVE: TGFbeta1 is a multifunctional growth factor with both pro- and anti-inflammatory properties. This study aimed to determine levels of TGFbeta1 in gingival crevicular fluid (GCF), serum and plasma in the early stages of gingival inflammation. DESIGN: A 21-day experimental model of gingivitis employing a split mouth design with a soft vinyl splint used to cover test teeth during brushing. SUBJECTS: Ten healthy volunteers (mean age 21 years; five males and five females). METHODS: GCF and blood (with and without EDTA) was collected on days 0, 7, 14 and 21. GCF volumes were measured on a precalibrated Periotron 8000TM. Clinical indices of gingival inflammation and plaque levels were obtained after GCF sampling. Normal brushing resumed after GCF collection on day 21 and final samples were collected on day 35. TGFbeta1 and alkaline phosphatase (ALP) levels were determined using enhanced chemiluminescent methods. RESULTS: Clinical indices and GCF volumes increased at test sites during the 21-day test period. Concentrations of TGFbeta1 and ALP in GCF (test and control), serum and plasma did not change throughout the study (P > 0.3). However, total amounts of TGFbeta1 (pg sample-1) and ALP (mu IU sample-1) in GCF increased at test sites and were significantly higher than baseline values at days 7, 14 and 21 (P < 0.04). Control sites showed no variation in TGFbeta1 or ALP levels throughout the study period (P > 0.35). All parameters at test sites returned to control levels at day 35 (P > 0.3). CONCLUSION: The data indicate that GCF TGFbeta1 levels increase early in plaque-induced inflammation. Whether the biological consequence of this site-specific increase is pro- or anti-inflammatory in nature remains to be elucidated.

TGF-beta isoforms and TGF-beta receptors in drug-induced and hereditary gingival overgrowth.

Drug therapy and hereditary factors are two of the main causes of gingival overgrowth (GO). Both of these forms of GO are associated with increased extracellular matrix production by fibroblasts. Transforming growth factor beta (TGF-beta) is an important mediator of wound healing and tissue regeneration, which stimulates fibroblasts to produce extracellular matrix materials. The aim of this immunohistochemical study was to determine whether there is any altered expression of TGF-beta isoforms or its receptors in tissue from patients with drug-induced GO (DIGO; n=10) and hereditary gingival fibromatosis (n=10) when compared to non-overgrowth tissue (n=10). Compared to control tissues, significantly more fibroblasts expressed TGF-beta1 in both DIGO and hereditary gingival fibromatosis tissues (P<0.03). Cells expressing TGF-beta2 were present at control levels in DIGO but were significantly reduced in hereditary gingival fibromatosis (P<0.02). By contrast, the number of TGF-beta3-positive cells was the same in overgrowth tissues and controls. However, because of differences in total fibroblast densities between groups, there was a proportional increase in TGF-beta3 as well as TGF-beta1 expressing cells within both overgrowth populations (P<0.0001). Furthermore, representation of the TGF-beta2-positive phenotype was reduced in hereditary gingival fibromatosis (P<0.01) but increased in DIGO (P<0.005) compared to controls. Absorbance measurements of the positive cell populations showed that the level of expression was significantly higher for TGF-beta1 in hereditary gingival fibromatosis (P<0.002) and significantly lower for TGF-beta3 in DIGO (P<0.03). No significant differences in the numbers of TGF-betaRI- or RII-positive cells were detected between overgrowth tissues and controls. However, there were increases in the proportion of receptor-positive cells in the total cell population analysed in overgrowth tissues (P<0.0001). These results indicate qualitative and quantitative differences in TGF-beta isoform and receptor expression by fibroblasts in gingival overgrowth that may contribute to disease pathogenesis.

Injury initiation and progression in the anterior cruciate ligament.

OBJECTIVE: To develop a theoretical model to identify mechanisms by which total and partial tears of the anterior cruciate ligament could occur. DESIGN: A sagittal-plane knee model was used to investigate anterior cruciate ligament injury due to excessive anterior tibial translation. The ligament was modelled as an ordered array of fibres linking femur and tibia. BACKGROUND: Despite years of research, the detailed biomechanics of anterior cruciate ligament injury is not well understood. METHODS: A "critical strain criterion" was used to identify the onset and progression of model ligament fibre disruption. The associated forces were also calculated. RESULTS: At low flexion angles (<20 degrees ), the posterior fibre of the model ligament failed first, and the tear progressed anteriorly through the ligament. At higher flexion angles, the anterior fibre failed first, and the tear progressed posteriorly. Near the flexion angle at which the progression of injury changed direction, all fibres failed at approximately the same anterior tibial translation. At all but very high flexion angles, the force supported by the injured ligament was maximum when initial fibre failure occurred; the force then decreased with increasing anterior tibial translation. CONCLUSIONS: Near (20 degrees ) flexion, all model anterior cruciate ligament fibres fail at approximately the same anterior tibial translation, implying that a partial ligament tear may be impossible in this flexion region. Relevance. This study provides insight into possible mechanisms of initiation and progression of anterior cruciate ligament injury. It suggests that a partial tear of the posterior half of the ligament may be difficult to detect clinically.

The female perspective: women's attitudes toward urogenital aging.

Different perspectives toward symptoms of urogenital aging exist among postmenopausal women, influenced by social and cultural factors, but one constant is that women rarely feel able to discuss urogenital problems freely--with their physicians, husbands, or friends. European women >55 years old were brought up to prize "modesty" and to consider urogenital symptoms as private matters that the woman must solve herself. Sexuality in older age groups is recognized as important, but many postmenopausal women are reluctant to complain of deteriorating sex lives. In spite of a different view of the menopause, similar reticence is found in Japan. Physicians need to be better equipped to improve communications between themselves and their postmenopausal patients.

A method of assessment during vocational training: report of a pilot study.

Problems arising from the present rate of failure of vocational trainees in the MRCGP examination are outlined; and the role of formative assessment during training in reducing this rate is considered. A study is described in which trainees in a number of centres were assessed by a method designed to measure specified cognitive abilities and areas of knowledge. The method, based on written papers, provides each candidate with a profile of performance and generates comparative standards. Reliability of marking, the distribution of candidate-scores within and between areas of assessment and techniques for monitoring the effectiveness of questions are reported. Use of the method by College as an educational service to trainees is considered, along with its potential as a Part I MRCGP examination.

Randomised double blind controlled clinical trial of intranasal budesonide in treatment of hay fever.

The effectiveness of budesonide, a new non-halogenated glucocorticoid administered by nasal inhaler, was evaluated in a double blind comparison with placebo in patients presenting with hay fever. Patients were supplied with antihistamine tablets and eye drops for use when they considered that their symptoms were inadequately controlled by their inhaler. Patients recorded the severity of their symptoms in a daily diary card and visited their general practitioner for assessment weekly for four weeks. All nasal symptoms of hay fever were appreciably reduced in the group taking budesonide and, although their eye symptoms were more severe than in the group taking placebo, they did not use appreciably more eye drops than the latter. The placebo group used appreciably more antihistamine tablets than those in the budesonide group. No patients were withdrawn from the budesonide group because of treatment failure or unacceptable side effects. The results suggest that budesonide is an acceptable and effective treatment for the nasal symptoms of hay fever.

Toxicity of nitro compounds fromLotus pedunculatus to grass grub (Costelytra zealandica) (Coleoptera: Scarabaeidae).

Crude extracts of root of the forage legumesLotus pedunculatus andCoronilla varia (crownvetch) were toxic when administered orally to 3rd instarCostelytra zealandica larvae. A group of 3-nitropropanoyl-D-glucopyranoses was isolated from active fractions of the crude extracts. These toxins, some of which were already known fromC. varia, have not previously been reported fromL. pedunculatus. The compounds were present in root tissue of this species at a concentration of about 1% dry weight. They include the triester karakin and the diesters coronarian and cibarian, all of which were toxic to larvae ofC. zealandica at levels which could account fully for the activity of the crude extracts.

The prediction of choice in infant feeding: a study of primiparae.

A random sample of 617 white Caucasian primiparae was identified from notifications made to the Leeds Area Health Authority over a 12-month period. Of these, 534 were interviewed regarding personal characteristics, events during pregnancy, and their experience of infant feeding. Significant associations between these factors and the mode of infant feeding initially used are described.From discriminant analysis of two half-populations, weightings were derived for each significantly associated variable, and discriminant scores were calculated for each participant. The distributions of these scores were similar in both half-populations, suggesting that the weightings were stable and thus have potential predictive importance. Further analysis showed that, from all the significant variables, the choice of two (maternal age-group at confinement and age at leaving school) correctly predicted 79 per cent of mothers who will choose to bottle-feed. The potential clinical yield of using these two factors is discussed, and a simple predictive tool for use in everyday practice is presented.

Prediction of duration of breast feeding in primiparas.

A random sample of 617 primiparas was identified from birth notifications over a 12 month period and 534 of these were interviewed four weeks after confinement. Those breast feeding at the time of interview were contacted again at four months and those still breast feeding then were contacted at six and a half months. Duration of breast feeding was found to be significantly associated with five interassociated personal characteristics of the mother and with specific aspects of her knowledge and attitudes regarding breast feeding. In hospital the timing of the first breast feed and difficulties with subsequent feeds, were important indicators; while at home the use of additional formula feeds was associated with a reduced prevalence of breast feeding by 18 weeks. A combination of older maternal age at confinement and older age at leaving school showed a tenfold increase of prevalence rates in breast feeding at 16 weeks between groups of mothers. The use of these two factors alone may thus help doctors, midwives, and health visitors in assessing the risk of premature termination of breast feeding and in planning programmes of preventive care.