Full automation of total metabolic tumor volume from FDG-PET/CT in DLBCL for baseline risk assessments.

BACKGROUND: Current radiological assessments of 18fluorodeoxyglucose-positron emission tomography (FDG-PET) imaging data in diffuse large B-cell lymphoma (DLBCL) can be time consuming, do not yield real-time information regarding disease burden and organ involvement, and hinder the use of FDG-PET to potentially limit the reliance on invasive procedures (e.g. bone marrow biopsy) for risk assessment. METHODS: Our aim is to enable real-time assessment of imaging-based risk factors at a large scale and we propose a fully automatic artificial intelligence (AI)-based tool to rapidly extract FDG-PET imaging metrics in DLBCL. On availability of a scan, in combination with clinical data, our approach generates clinically informative risk scores with minimal resource requirements. Overall, 1268 patients with previously untreated DLBCL from the phase III GOYA trial (NCT01287741) were included in the analysis (training: n = 846; hold-out: n = 422). RESULTS: Our AI-based model comprising imaging and clinical variables yielded a tangible prognostic improvement compared to clinical models without imaging metrics. We observed a risk increase for progression-free survival (PFS) with hazard ratios [HR] of 1.87 (95% CI: 1.31-2.67) vs 1.38 (95% CI: 0.98-1.96) (C-index: 0.59 vs 0.55), and a risk increase for overall survival (OS) (HR: 2.16 (95% CI: 1.37-3.40) vs 1.40 (95% CI: 0.90-2.17); C-index: 0.59 vs 0.55). The combined model defined a high-risk population with 35% and 42% increased odds of a 4-year PFS and OS event, respectively, versus the International Prognostic Index components alone. The method also identified a subpopulation with a 2-year Central Nervous System (CNS)-relapse probability of 17.1%. CONCLUSION: Our tool enables an enhanced risk stratification compared with IPI, and the results indicate that imaging can be used to improve the prediction of central nervous system relapse in DLBCL. These findings support integration of clinically informative AI-generated imaging metrics into clinical workflows to improve identification of high-risk DLBCL patients. TRIAL REGISTRATION: Registered clinicaltrials.gov number: NCT01287741.

Lipoprotein modifications by gingipains of Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: Several studies have shown an association between periodontitis and cardiovascular disease (CVD). Atherosclerosis is the major cause of CVD, and a key event in the development of atherosclerosis is accumulation of lipoproteins within the arterial wall. Bacteria are the primary etiologic agents in periodontitis and Porphyromonas gingivalis is the major pathogen in the disease. Several studies support a role of modified low-density lipoprotein (LDL) in atherogenesis; however, the pathogenic stimuli that induce the changes and the mechanisms by which this occur are unknown. This study aims to identify alterations in plasma lipoproteins induced by the periodontopathic species of bacterium, P. gingivalis, in vitro. MATERIAL AND METHODS: Plasma lipoproteins were isolated from whole blood treated with wild-type and gingipain-mutant (lacking either the Rgp- or Kgp gingipains) P. gingivalis by density/gradient-ultracentrifugation and were studied using 2-dimensional gel electrophoresis followed by matrix-assisted laser desorption/ionization mass spectrometry. Porphyromonas gingivalis-induced lipid peroxidation and antioxidant levels were measured by thiobarbituric acid-reactive substances and antioxidant assay kits, respectively, and lumiaggregometry was used for measurement of reactive oxygen species (ROS) and aggregation. RESULTS: Porphyromonas gingivalis exerted substantial proteolytic effects on the lipoproteins. The Rgp gingipains were responsible for producing 2 apoE fragments, as well as 2 apoB-100 fragments, in LDL, and the Kgp gingipain produced an unidentified fragment in high-density lipoproteins. Porphyromonas gingivalis and its different gingipain variants induced ROS and consumed antioxidants. Both the Rgp and Kgp gingipains were involved in inducing lipid peroxidation. CONCLUSION: Porphyromonas gingivalis has the potential to change the expression of lipoproteins in blood, which may represent a crucial link between periodontitis and CVD.

Magnetic resonance enterography is feasible and reliable in multicenter clinical trials in patients with Crohn's disease, and may help select subjects with active inflammation.

BACKGROUND: Reliable tools for patient selection are critical for clinical drug trials. AIM: To evaluate a consensus-based, standardised magnetic resonance enterography (MRE) protocol for selecting patients for inclusion in Crohn's disease (CD) multicenter clinical trials. METHODS: This study recruited 20 patients [Crohn's Disease Activity Index (CDAI) scores: <150 (n = 8); 150-220 (n = 4); 220-450 (n = 8)], to undergo ileocolonoscopy and two MREs (with and without colonic contrast) within a 14-day period. Procedures were scored centrally using, Magnetic Resonance Index of Activity (MaRIA), and both Crohn's Disease Endoscopic Index of Severity (CDEIS) and Simplified Endoscopic Score (SES-CD). RESULTS: 37 MREs were acquired. Both MREs were evaluable in 16 patients for calculation of test-retest and inter-reader reliability scores. The MaRIA scores for the terminal ileum had excellent test-retest and inter-reader reliability, with correlations >0.9. The proximal ileum showed strong within-reader agreement (0.90-0.96), and fair between-reader agreement (0.59-0.72). MRE procedures were tolerable. MaRIA scores correlated with CDEIS and SES-CD (0.63 and 0.71), but not with CDAI (0.34). MRE identified 3 patients with intra-abdominal complications, who would otherwise have been included in clinical trials. Furthermore, both MRE and ileocolonoscopy identified active bowel wall inflammation in 2 patients with CDAI <150, and none in 1 patient with CDAI > 220. Data quality was good/excellent in 85% of scans, and fair or better in 96%. CONCLUSIONS: Magnetic resonance enterography of high-quality and reproducibility was feasible in a global multi- centre setting, with evidence for improved selectivity over CDAI and ileocolonoscopy in identifying appropriate CD patients for inclusion in therapeutic intervention trials.

The role of phagocytosis, oxidative burst and neutrophil extracellular traps in the interaction between neutrophils and the periodontal pathogen Porphyromonas gingivalis.

Neutrophils are regarded as the sentinel cells of innate immunity and are found in abundance within the gingival crevice. Discovery of neutrophil extracellular traps (NETs) within the gingival pockets prompted us to probe the nature of the interactions of neutrophils with the prominent periopathogen Porphyromonas gingivalis. Some of the noted virulence factors of this Gram-negative anaerobe are gingipains: arginine gingipains (RgpA/B) and lysine gingipain (Kgp). The aim of this study was to evaluate the role of gingipains in phagocytosis, formation of reactive oxygen species, NETs and CXCL8 modulation by using wild-type strains and isogenic gingipain mutants. Confocal imaging showed that gingipain mutants K1A (Kgp) and E8 (RgpA/B) induced extracellular traps in neutrophils, whereas ATCC33277 and W50 were phagocytosed. The viability of both ATCC33277 and W50 dwindled as the result of phagocytosis and could be salvaged by cytochalasin D, and the bacteria released high levels of lipopolysaccharide in the culture supernatant. Porphyromonas gingivalis induced reactive oxygen species and CXCL8 with the most prominent effect being that of the wild-type strain ATCC33277, whereas the other wild-type strain W50 was less effective. Quantitative real-time polymerase chain reaction revealed a significant CXCL8 expression by E8. All the tested P. gingivalis strains increased cytosolic free calcium. In conclusion, phagocytosis is the primary neutrophil response to P. gingivalis, although NETs could play an accessory role in infection control. Although gingipains do not seem to directly regulate phagocytosis, NETs or oxidative burst in neutrophils, their proteolytic properties could modulate the subsequent outcomes such as nutrition acquisition and survival by the bacteria.

The role of Porphyromonas gingivalis gingipains in platelet activation and innate immune modulation.

Platelets are considered to have important functions in inflammatory processes and as actors in the innate immunity. Several studies have shown associations between cardiovascular disease and periodontitis, where the oral anaerobic pathogen Porphyromonas gingivalis has a prominent role in modulating the immune response. Porphyromonas gingivalis has been found in atherosclerotic plaques, indicating spreading of the pathogen via the circulation, with an ability to interact with and activate platelets via e.g. Toll-like receptors (TLR) and protease-activated receptors. We aimed to evaluate how the cysteine proteases, gingipains, of P. gingivalis affect platelets in terms of activation and chemokine secretion, and to further investigate the mechanisms of platelet-bacteria interaction. This study shows that primary features of platelet activation, i.e. changes in intracellular free calcium and aggregation, are affected by P. gingivalis and that arg-gingipains are of great importance for the ability of the bacterium to activate platelets. The P. gingivalis induced a release of the chemokine RANTES, however, to a much lower extent compared with the TLR2/1-agonist Pam3 CSK4 , which evoked a time-dependent release of the chemokine. Interestingly, the TLR2/1-evoked response was abolished by a following addition of viable P. gingivalis wild-types and gingipain mutants, showing that both Rgp and Kgp cleave the secreted chemokine. We also demonstrate that Pam3 CSK4 -stimulated platelets release migration inhibitory factor and plasminogen activator inhibitor-1, and that also these responses were antagonized by P. gingivalis. These results supports immune-modulatory activities of P. gingivalis and further clarify platelets as active players in innate immunity and in sensing bacterial infections, and as target cells in inflammatory reactions induced by P. gingivalis infection.

Suppression of inflammatory responses of human gingival fibroblasts by gingipains from Porphyromonas gingivalis.

The interaction between human gingival fibroblasts (HGFs) and Porphyromonas gingivalis plays an important role in the development and progression of periodontitis. Porphyromonas gingivalis possesses several virulence factors, including cysteine proteases, the arginine-specific (Rgp) and lysine-specific (Kgp) gingipains. Studying the mechanisms that P. gingivalis, and its derived virulence, use to propagate and interact with host cells will increase the understanding of the development and progression of periodontitis. In this study, we aimed to elucidate how P. gingivalis influences the inflammatory events in HGFs regarding transforming growth factor-ß1 (TGF-ß1 ), CXCL8, secretory leucocyte protease inhibitor (SLPI), c-Jun and indoleamine 2,3-dioxygenase (IDO). HGFs were inoculated for 6 and 24 h with the wild-type strains ATCC 33277 and W50, two gingipain-mutants of W50 and heat-killed ATCC 33277. The P. gingivalis regulated CXCL8 and TGF-ß1 in HGFs, and the kgp mutant gave significantly higher immune response with increased CXCL8 (P < 0.001) and low levels of TGF-ß1 . We show that HGFs express and secrete SLPI, which was significantly suppressed by P. gingivalis (P < 0.05). This suggests that by antagonizing SLPI, P. gingivalis contributes to the tissue destruction associated with periodontitis. Furthermore, we found that P. gingivalis inhibits the expression of the antimicrobial IDO, as well as upregulating c-Jun (P < 0.05). In conclusion, P. gingivalis both triggers and suppresses the immune response in HGFs. Consequently, we suggest that the pathogenic effects of P. gingivalis, and especially the activity of the gingipains on the inflammatory and immune response of HGFs, are crucial in periodontitis.

The periodontal pathogen Porphyromonas gingivalis sensitises human blood platelets to epinephrine.

Recent studies indicate connections between periodontitis and atherothrombosis, and the periodontal pathogen Porphyromonas gingivalis has been found within atherosclerotic lesions. P. gingivalis-derived proteases, designated gingipains activate human platelets, probably through a "thrombin-like" activity on protease-activated receptors (PARs). However, the potential interplay between P. gingivalis and other physiological platelet activators has not been investigated. The aim of this study was to elucidate consequences and mechanisms in the interaction between P. gingivalis and the stress hormone epinephrine. By measuring changes in light transmission through platelet suspensions, we found that P. gingivalis provoked aggregation, whereas epinephrine alone never had any effect. Intriguingly, pre-treatment of platelets with a low, sub-threshold number of P. gingivalis (i.e. a density that did not directly provoke platelet aggregation) resulted in a marked aggregation response when epinephrine was added. This synergistic action was not inhibited by the cyclooxygenas inhibitor aspirin. Furthermore, fura-2-measurements revealed that epinephrine caused an intracellular Ca(2+) mobilization in P. gingivalis pre-treated platelets, whereas epinephrine alone had no effect. Inhibition of the arg-specific gingipains, but not the lys-specific gingipains, abolished the aggregation and the Ca(2+) response provoked by epinephrine. Similar results were achieved by separate blockage of platelet alpha(2)-adrenergic receptors and PARs. In conclusion, the present study shows that a sub-threshold number of P. gingivalis sensitizes platelets to epinephrine. We suggest that P. gingivalis-derived arg-specific gingipains activates a small number of PARs on the surface of the platelets. This leads to an unexpected Ca(2+) mobilization and a marked aggregation response when epinephrine subsequently binds to the alpha(2)-adrenergic receptor. The present results are consistent with a direct connection between periodontitis and stress, and describe a novel mechanism that may contribute to pathological platelet activation.

Correlation between rises in Chlamydia pneumoniae-specific antibodies, platelet activation and lipid peroxidation after percutaneous coronary intervention.

We recently showed that Chlamydia pneumoniae activates platelets in vitro, with an associated oxidation of low-density lipoproteins. The aim of this study was to investigate whether C. pneumoniae is released during percutaneous coronary intervention (PCI) and, thereby, causes platelet activation and lipid peroxidation. Seventy-three patients undergoing coronary angiography and following PCI or coronary artery bypass graft (CABG) and 57 controls were included in the study. C. pneumoniae antibodies, serotonin and lipid peroxidation were measured before and 24 h, 1 month and 6 months after angiography. The results show that serum C. pneumoniae IgA concentrations were significantly higher in patients than in the controls. Furthermore, in 38% of the C. pneumoniae IgG positive patients, the C. pneumoniae IgG concentration increased 1 month after PCI. The levels of C. pneumoniae IgG antibodies 1 month after PCI correlated with plasma-lipid peroxidation (r = 0.91, P < 0.0001) and platelet-derived serotonin (r = 0.62, P = 0.02). There was no elevation in the total serum IgG 1 month after PCI. In conclusion, the present results suggest that PCI treatment of coronary stenosis releases C. pneumoniae from the atherosclerotic lesions, which leads to platelet activation and lipid peroxidation.

Beta(2)-Adrenergic activation increases glycogen synthesis in L6 skeletal muscle cells through a signalling pathway independent of cyclic AMP.

AIMS/HYPOTHESIS: In skeletal muscle, the storage of glycogen by insulin is regulated by glycogen synthase, which is regulated by glycogen synthase kinase 3 (GSK3). Here we examined whether adrenergic receptor activation, which can increase glucose uptake, regulates glycogen synthesis in L6 skeletal muscle cells. METHODS: We used L6 cells and measured glycogen synthesis (as incorporation of D: -[U-(14)C]glucose into glycogen) and GSK3 phosphorylation following adrenergic activation. RESULTS: Insulin (negative logarithm of median effective concentration [pEC(50)] 8.2 +/- 0.3) and the beta-adrenergic agonist isoprenaline (pEC(50) 7.5 +/- 0.3) induced a twofold increase in glycogen synthesis in a concentration-dependent manner. The alpha(1)-adrenergic agonist cirazoline and alpha(2)-adrenergic agonist clonidine had no effect. Both insulin and isoprenaline phosphorylated GSK3. The beta-adrenergic effect on glycogen synthesis is mediated by beta(2)-adrenoceptors and not beta(1)-/beta(3)-adrenoceptors, and was not mimicked by 8-bromo-cyclic AMP or cholera toxin, and also was insensitive to pertussis toxin, indicating no involvement of cyclic AMP or inhibitory G-protein (G(i)) signalling in the beta(2)-adrenergic effect on glycogen synthesis. 12-O-tetra-decanoylphorbol-13-acetate (TPA) increased glycogen synthesis 2.5-fold and phosphorylated GSK3 fourfold. Inhibition of protein kinase C (PKC) isoforms with 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrollo(3,4-c)-carbazole (Gö6976; inhibits conventional and novel PKCs) or 2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl)maleimide (Gö6983; inhibits conventional, novel and atypical PKCs) inhibited the stimulatory TPA effect, but did not significantly inhibit glycogen synthesis mediated by insulin or isoprenaline. Inhibition of phosphatidylinositol 3-kinase (PI3K) with wortmannin inhibited the effects of insulin and isoprenaline on glycogen synthesis. CONCLUSIONS/INTERPRETATION: These results demonstrate that in L6 skeletal muscle cells adrenergic stimulation through beta(2)-adrenoceptors, but not involving cyclic AMP or G(i), activates a PI3K pathway that stimulates glycogen synthesis through GSK3.

Beta-adrenoceptors, but not alpha-adrenoceptors, stimulate AMP-activated protein kinase in brown adipocytes independently of uncoupling protein-1.

AIMS/HYPOTHESIS: Brown adipocytes provide a potentially important model system for understanding AMP-activated protein kinase (AMPK) regulation, where adrenergic stimulation leads to mitochondrial uncoupling through uncoupling protein-1 (UCP1) activity. AMPK is a sensor of energy homeostasis and has been implicated in glucose and lipid metabolism in several insulin-sensitive tissues. The aim of this study was to characterise the potential role of AMPK in adrenergically mediated glucose uptake and to find out whether UCP1 is involved in the adrenergic activation of AMPK. METHODS: We used primary brown adipocytes differentiated in culture and measured AMPK phosphorylation and glucose uptake following adrenergic activation. RESULTS: Treatment of adipocytes with noradrenaline (norepinephrine) caused phosphorylation of AMPK via beta-adrenoceptors and not alpha(1)- or alpha(2)-adrenoceptors. This effect was not beta(3)-adrenoceptor specific, since responses remained intact in adipocytes from beta(3)-adrenoceptor knock-out mice. These effects were also mimicked by forskolin and cAMP analogues. Treatment of cells with adenine 8-beta-D-arabinofuranoside, an AMPK inhibitor, partially blocked beta-adrenoceptor-mediated increases in glucose uptake. Brown adipocytes are characterised by the production of UCP1, which can uncouple the mitochondria. Using adipocytes from Ucp1(+/+) and Ucp1(-/-) mice, we showed that noradrenaline-mediated phosphorylation of AMPK does not require the presence or activity of UCP1. CONCLUSIONS/INTERPRETATION: These results suggest a pathway where increases in cAMP mediated by beta-adrenoceptors leads to activation of AMPK in brown adipocytes, which contributes in part to beta-adrenoceptor-mediated increases in glucose uptake, an effect independent of the presence or function of UCP1.

Differential adrenergic regulation of the gene expression of the beta-adrenoceptor subtypes beta1, beta2 and beta3 in brown adipocytes.

In brown adipocytes, fundamental cellular processes (cell proliferation, differentiation and apoptosis) are regulated by adrenergic stimulation, notably through beta-adrenergic receptors. The presence of all three beta-receptor subtypes has been demonstrated in brown adipose tissue. Due to the significance of the action of these receptors and indications that the subtypes govern different processes, the adrenergic regulation of the expression of the beta(1)-(,) beta(2)- and beta(3)-adrenoceptor genes was examined in murine brown-fat primary cell cultures. Moderate levels of beta(1)-receptor mRNA, absence of beta(2)-receptor mRNA and high levels of beta(3)-receptor mRNA were observed in mature brown adipocytes (day 6 in culture). Noradrenaline (norepinephrine) addition led to diametrically opposite effects on beta(1)- (markedly enhanced expression) and beta(3)-gene expression (full cessation of expression, as previously shown). beta(2)-Gene expression was induced by noradrenaline, but only transiently (<1 h). The apparent affinities (EC(50)) of noradrenaline were clearly different (7 nM for the beta(1)-gene and

Acute effects of nicotine infusion on platelets in nicotine users with normal and impaired renal function.

The role of platelets in cardiovascular disease associated with smoking is becoming more established, but the effects of nicotine on platelets are unclear. Nicotine therapy is used for smoking cessation in both health and disease. Consequently, the effects of nicotine on platelets are of particular significance in disorders such as renal disease, which is associated with defective platelet function, increased cardiovascular morbidity, and altered nicotine metabolism. Thus, the aim of the present study was to investigate the acute effects of nicotine infusion (NI) on platelets in seven healthy subjects (HS) and seven patients with renal failure (RF). All subjects were nicotine users and had refrained from using nicotine for 36 h before NI. Blood was collected before, immediately after, and 2 h after NI. The plasma concentrations of nicotine and its main metabolite cotinine were determined by gas chromatography. Platelet responsiveness was assessed by aggregometry and flow cytometry in whole blood (P-selectin surface expression, fibrinogen- and von Willebrand factor-binding), P-selectin expression in isolated platelets, and immunoassays of platelet release (beta-thromboglobulin, platelet factor 4, and soluble P-selectin) and nitric oxide (NO) products. The plasma levels of cotinine, but not nicotine, were significantly higher in RF compared to HS at all time points. In both groups, collagen-induced platelet aggregation was restrained immediately after NI, when the plasma concentration of nicotine was maximal, and was restored after 2 h. Two hours after NI, activation-dependent P-selectin surface expression in isolated platelets increased in both groups. This increased platelet responsiveness occurred simultaneously with a significant increase of plasma cotinine and a decrease of NO products. Thus, the present study suggests that nicotine, directly or through some secondary mechanism or metabolite, only slightly potentiates some of the platelet responses. Renal failure appears not to influence the effects of nicotine on platelets.

beta1 to beta3 switch in control of cyclic adenosine monophosphate during brown adipocyte development explains distinct beta-adrenoceptor subtype mediation of proliferation and differentiation.

To explain the distinctive pharmacological profiles observed for adrenergic stimulation of cell proliferation (beta1) and cell differentiation (beta3), the adrenergic control of cAMP accumulation was investigated during brown adipocyte development. In preadipocytes, norepinephrine (NE) increased cAMP levels but the beta3-agonists BRL-37344 and CGP-12177 did not; in contrast, when the cells had differentiated into mature brown adipocytes, a large cAMP response to the beta3-agonists had emerged and was now double that to NE (although the affinity of NE had increased 10-fold). Beta1-messenger RNA (mRNA) levels were high in both pre- and mature brown adipocytes; beta3-mRNA did not appear until maturation but then abruptly. Although beta1-receptors remained detectable by [3H]CGP-12177 binding in the mature brown adipocytes, the cAMP response to NE (based on propranolol inhibitory potency) switched from beta1 to beta3. Even the established beta1-agonist dobutamine acted through beta3-receptors in the mature brown adipocytes. The increases in cAMP levels could adequately explain the increased cell proliferation in NE-stimulated preadipocytes and the NE-induced UCP1 gene expression in mature brown adipocytes. The distinctive adrenergic profiles for stimulation of proliferation and of differentiation were thus not due to the existence of additional pathways but to a switch in the type of beta-receptor mediating the NE response, coordinated with an alteration in the nuclear response to increased cAMP levels. The study implies that full recruitment of brown adipose tissue cannot be induced by exclusive beta3-stimulation.

Activation of the granule pool of the NADPH oxidase accelerates apoptosis in human neutrophils.

Oxidative stress induces apoptosis in many types of cells, including human neutrophils. Our objective was to determine whether reactive oxygen species (ROS) produced by activated neutrophils are associated with accelerated apoptosis. Exposing neutrophils to ionomycin or phorbol myristate acetate (PMA) induced intracellular H2O2 production and rapid onset of apoptosis, measured as condensed chromatin, cellular shrinkage, and DNA fragmentation. Neutrophils activated with formyl-methionyl-leucyl-phenylalanine (fMLP) generated mainly extracellular H2O2 and did not undergo apoptosis. Exogenously added H2O2, together with the catalase blocker sodium azide, induced apoptosis to the same extent and with similar kinetics as PMA and ionomycin. Adenosine inhibited ionomycin-induced intracellular H2O2 production and apoptosis. Neither PMA nor ionomycin caused apoptosis in dimethyl sulfoxide-differentiated HL-60 cells, which are incapable of intracellular H2O2 production, whereas H2O2 induced apoptosis more efficiently in these cells than in neutrophils. We propose that activated neutrophils use intracellularly formed H2O2 to commit suicide.

Modulation of the chemotactic peptide- and immunoglobulin G-triggered respiratory burst in human neutrophils by exogenous and endogenous adenosine.

The effects of exogenous and endogenous adenosine on the production of oxygen metabolites in neutrophils triggered by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) or immunoglobulin G (IgG)-opsonized yeast particles, were investigated. By using luminol-enhanced chemiluminescence, we found that adenosine A1 receptor activation did not affect, whereas adenosine A receptor activation, through a mechanism involving the cyclic AMP (cAMP)-protein kinase A signalling pathway, both inhibited the fMLP- and IgG-triggered respiratory burst. The adenosine-induced inhibition was however more pronounced after exposure to fMLP than to IgG-yeast. Stimulation with fMLP caused an extracellular accumulation of endogenous adenosine, which indicates that this event is a negative-feedback mechanism preventing an uncontrolled activation of chemoattractant-stimulated neutrophils. On the contrary, exposure of neutrophils to IgG-yeast did not appear to accumulate extracellular adenosine, probably due to increased adenosine deaminase activity during phagocytosis. In conclusion, this work accentuates the importance of adenosine, both exogenously applied and endogenously formed, as an inflammatory agent modulating the respiratory burst during the different phases in neutrophil activation.

Immunological findings in blood and bronchoalveolar lavage fluid in chronic bronchitis patients with recurrent infectious exacerbations.

Bronchial infections are common in smokers and seem to be related to the presence of chronic bronchitis (CB). Why only some smokers develop repeated bronchial infections is not known. The aim of this study was to screen for immunological changes associated with disease in patients with CB and recurrent infectious exacerbations compared to asymptomatic smokers. Sixteen smokers with stable CB and recurrent infectious exacerbations, and 18 asymptomatic smokers, all without any immunomodulating treatment, underwent bronchoscopy and bronchoalveolar lavage (BAL). Smoking history and current smoking status were comparable. Serum levels of immunoglobulin (Ig)A, IgM, IgG and IgG subclasses were measured. Blood and BAL lymphocyte phenotypes and proliferative responses of peripheral blood mononuclear cells (PBMCs) to various stimulators were analysed. Unstimulated and tetanus toxoid-stimulated production of cytokines in PBMC cultures was measured. Natural killer (NK-) cell activity was analysed. A significantly (p<0.05) lower level of IgG3 was found in the CB group, and a significantly (p<0.01) higher proliferative response of PBMCs was found in the CB group after stimulation with diphtheria toxoid. Detectable levels of interleukin (IL)-6, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma, but not of IL-2, IL-4 or transforming growth factor-beta2, were found in supernatants from cultured cells in both study groups. Stimulated TNF-alpha production was significantly (p<0.05) higher in the CB group. NK-cell activity did not differ significantly between the study groups. There were no major differences between the groups in lymphocyte subpopulations in blood or BAL. In conclusion, no major alterations in the analysed indices of cell-mediated and humoral immunity were found in patients with chronic bronchitis prone to recurrent infectious exacerbations when compared with asymptomatic smoking controls.

Adenosine inhibits actin dynamics in human neutrophils: evidence for the involvement of cAMP.

The mechanisms by which adenosine regulates the inflammatory reaction are poorly characterized. In this study, we investigated the effects of adenosine on neutrophil actin polymerization elicited by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) or IgG-opsonized yeast particles. We used bodipy-phallacidin staining in combination with flow cytometry and found that adenosine markedly reduced actin polymerization triggered by IgG-yeast, whereas the effect on the fMLP-response was less pronounced. Similar or even more pronounced effects were obtained with the adenosine A2 receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), suggesting an A2 receptor-mediated mechanism. The following observations indicate that the A2 receptor-induced effects involve the cAMP-protein kinase A (PKA) signaling pathway: (1) a combination of NECA and the cAMP-specific phosphodiesterase (PDE) inhibitor Ro 20-1724 raised the cAMP content in both unstimulated and stimulated neutrophils and also further inhibited the actin dynamics; (2) the PKA inhibitor H89 reversed the inhibitory effects of NECA on the actin dynamics; (3) Ro 20-1724, isoproterenol and dibutyryl cAMP (DBcAMP) reduced actin polymerization in almost the same way as NECA did. NECA together with Ro 20-1724 impaired the fMLP-induced shape changes and cortical accumulation of actin filaments. In contrast, H89 potentiated the fMLP-induced formation of a submembranous ring of actin filaments. Neutrophils phagocytosing yeast particles in the presence of NECA and Ro 20-1724 were predominantly round in shape, and their ability to extend actin-rich pseudopods around the prey was reduced. These effects were partly antagonized by H89. In correlation with the effects on actin polymerization, NECA more effectively diminished IgG-induced upregulation of the beta2 integrin CD11b/CD18 than such upregulation induced by fMLP. The inhibitory effects of A2-receptor activation on actin dynamics and beta2 integrin expression in neutrophils exposed to IgG-yeast were also associated with a cAMP-dependent reduction of the phagocytic capacity. In conclusion, we show that adenosine inhibits actin dynamics and shape changes in neutrophils via a cAMP-dependent pathway. This finding further characterizes the mechanisms by which adenosine functions as an important modulator of the inflammatory response.

Beta 3-adrenoceptor in guinea pig brown and white adipocytes: low expression and lack of function.

In the guinea pig, cold acclimation induced a conversion of unilocular to multilocular adipocytes in interscapular (IS) and retroperitoneal (RP) fat depots but not in the epididymal (EP) fat pad. The conversion was associated with an increase in mitochondriogenesis and the appearance of the uncoupling protein. The maximal lipolytic responses to norepinephrine and dibutyryl adenosine 3',5'-cyclic monophosphate were decreased in IS cells, unchanged in RP cells, and increased in EP cells, suggesting a site-specific regulation of lipolysis at the postreceptor level. beta 3-Adrenergic agonists were not lipolytic regardless of the depot and the thermal environment of the animal. These agents did not inhibit glucose transport and lipogenesis, as was previously reported for rodents. Cloning and sequencing of the guinea pig beta 3-adrenoceptor gene revealed a slightly higher amino acid sequence similarity with the human than with the rodent beta 3-adrenoceptors. beta 3-Adrenoceptor transcripts were present at a very low level in guinea pig adipocytes, and mRNA levels did not increase to a significant extent after cold acclimation. The guinea pig thus differs from rodents by an absence of beta 3-adrenergic effects and by low beta 3-adrenoceptor expression in brown and white adipose tissues.

Sulfatide-induced L-selectin activation generates intracellular oxygen radicals in human neutrophils: modulation by extracellular adenosine.

The sulfated form of galactocerebrosides (sulfatides) have recently been established as ligands for L-selectin. In this study we show that exposure of human neutrophils to sulfatides induces a transient generation of oxygen radicals, revealed by the luminol-enhanced chemiluminescence (CL) technique. The CL response was mainly located intracellularly, and was dependent on sulfation of the galactose ring, since non-sulfated galactocerebrosides had no effect. Sulfatides also dramatically amplified the CL response triggered by the chemotactic peptide formylmethionyl-leucyl-phenylalanine (fMLP). This effect was primarily due to an increased (up to 10-fold) intracellular generation of oxygen metabolites. Removal or blocking of L-selectin with chymotrypsin and monoclonal antibodies, respectively, markedly reduced the effects of sulfatides. Furthermore, sulfatides amplified the CL response triggered by ionomycin, whereas the response induced by phorbol-12-myristate-13-acetate was slightly reduced. The tyrosine kinase inhibitor, genistein, markedly inhibited the oxygen radical production induced by sulfatides, and totally abolished the potentiating effects of sulfatides in fMLP- and ionomycin-stimulated neutrophils. Sulfatides also triggered a transient rise in the intracellular free calcium concentration, [Ca2+]i. Consequently, L-selectin activation through sulfatides appear to affect oxidase activity through a Ca(2+)-dependent pathway involving tyrosine phosphorylation. Adenosine is an anti-inflammatory agent predominately released from the vascular endothelium which might suppress an inappropriate activation of the oxidase during L-selectin-mediated rolling of neutrophils. Indeed, we found that adenosine inhibited the oxidative burst induced by sulfatides, mainly by attenuating the intracellular generation of oxygen radicals. However, 10-100 times higher concentration of exogenous adenosine was required to inhibit the CL response induced by sulfatides to the same extent as the adenosine-mediated inhibition of the fMLP-induced response. This difference in sensitivity to adenosine could be explained by various expression of extracellular adenosine deaminase (ADA), since we found that the ADA-inhibitor erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) markedly reduced the oxygen radical production caused by sulfatides and almost totally abolished the potentiating effects of sulfatides on the fMLP-induced respiratory burst. In contrary, EHNA only slightly reduced the fMLP-triggered CL response. We suggest that the initial activation of L-selectin prepare the neutrophil for an effective microbicidal activity in the extravascular space. This process might be dependent on a L-selectin-mediated increase in the expression and activity of ADA, which locally reduces the extracellular level of adenosine.

Synergistic inhibition of thrombin-induced platelet aggregation by the novel nitric oxide-donor GEA 3175 and adenosine.

1. The influence of the novel nitric oxide-donor GEA 3175 on thrombin- and ionomycin-stimulated human platelets was investigated. The effect of GEA 3175 was compared with that of adenosine, an activator of platelet adenylyl cyclase. 2. GEA 3175 inhibited thrombin-induced secretion of ATP but did not affect aggregation; similar results were obtained with adenosine. 3. Thrombin-stimulated rises in the cytosolic free Ca2+ concentration, [Ca2+]i, were dose-dependently inhibited by GEA 3175 and adenosine. GEA 3175 and adenosine maximally reduced the initial rise in [Ca2+]i by 41% and 35%, respectively. 4. Simultaneous exposure to GEA 3175 and adenosine nearly abolished both the functional responses (i.e. aggregation and degranulation) and the rises in [Ca2+]i in thrombin-stimulated platelets. 5. Aggregation and increases in [Ca2+]i triggered in platelets by the Ca(2+)-ionophore ionomycin were only marginally affected by a combination of GEA 3175 and adenosine. 6. GEA 3175 potently increased the guanosine 3':5'-cyclic monophosphate (cyclic GMP) content in platelets but did not affect adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels. Adenosine did not increase either the cyclic AMP or the cyclic GMP levels in platelets. However, adenosine and GEA 3175 combined significantly elevated the platelet cyclic AMP content. 7. The results show that simultaneous exposure to GEA 3175 and adenosine promotes potent anti-aggregatory properties in platelets in vitro. The findings suggest that blockage of the cytosolic Ca(2+)-signal, which is probably mediated by an amplified cyclic nucleotide response, is an important event during the synergistic inhibition of thrombin-induced aggregation.