Increased frequency of immunoglobulin (Ig)A-secreting cells following Toll-like receptor (TLR)-9 engagement in patients with Kawasaki disease.

Kawasaki disease (KD) is an acute vasculitis affecting mainly infants and children. Human B cells express Toll-like receptor (TLR)-9, whose natural ligands are unmethylated cytosine-guanine dinucleotide (CpG) motifs characteristic of bacterial DNA. The aim of this study was to clarify the pathogenesis of KD analysing the activation status of peripheral blood mononuclear cells (PBMC), focusing on B lymphocyte activation and functions. Ten patients and 10 age-matched healthy donors were recruited from the Bambino Gesù Hospital of Rome, Italy and enrolled into this study. We determined phenotype profile and immunoglobulin (Ig) production of PBMC from KD patients and age-matched controls. We found that the frequency of CD19(+) B lymphocytes and CD19(+) /CD86(+) activated B lymphocytes from KD patients during the acute phase before therapy was increased significantly. Moreover, B lymphocytes of acute-phase KD patients were more prone to CpG oligodeoxynucleotide (ODN) activation compared with the age-matched controls, as assessed by a significant increase of the number of IgA-secreting cells (SC). In the same patients we found a marked increase of IgM, IgG, interleukin (IL)-6 and tumour necrosis factor (TNF)-α production compared with the control group. In addition, in two convalescent KD patients, conventional treatment with intravenous immunoglobulin (IVIG) restored the normal frequency of CD19(+) B cells, the number of IgA-, IgM- and IgG-SC and the production of IL-6 and TNF-α. Our findings indicate that the percentages of peripheral B lymphocytes of acute-phase KD patients are increased and are prone to bacterial activation in terms of increased numbers of IgA-SC and increased production of IL-6 and TNF-α inflammatory cytokines. Thus, our data support the hypothesis of an infectious triggering in KD.

Trans-plasma membrane electron transport in human blood platelets.

The plasma membrane redox (PMR) system is important for cell metabolism and survival; it is also crucial for blood coagulation and thrombosis. This review will give an update on the PMR system, with a particular regard to platelets, and on the role of antioxidant vitamins belonging to this system.

Hydrogen peroxide release from human blood platelets.

The release of hydrogen peroxide from human blood platelets after stimulation with particulate membrane-perturbing agents has been determined by fluorescence using scopoletin as the detecting agent. Platelet suspensions containing less than 1 polymorphonuclear leukocyte/10(8) platelets showed a significant release of hydrogen peroxide (6.11 nmol/10(9) platelets per 20 min, S.D., 0.26, n = 9) after addition of zymosan or latex particles, compared to unstimulated platelets. The release of hydrogen peroxide was only observed when the scopoletin was added to the platelet suspensions during the stimulation. Any attempt to determine hydrogen peroxide release in the supernatant at the end of the incubation with zymosan or latex failed. A NADH-dependent production of hydrogen peroxide was observed by measuring the difference of oxygen uptake in the presence and absence of catalase (500 units), which was not inhibited by potassium cyanide (1 mM). By this method the NADH-dependent cyanide-insensitive peroxide production and release was 6.0 nmol/10(9) platelets per 20 min from resting platelets (S.D., 2, n = 6) vs 15 nmol/10(9) platelets per 20 min from stimulated platelets (S.D., 2, n = 6).

Stimulated platelets release factor(s) affecting the in vitro response of human polymorphonuclear cells.

The metabolic and functional responses of human polymorphonuclear cells (PMNs) to thrombin-activated platelet supernatants were studied. The incubation of PMNs with supernatants from stimulated platelets (SPS) caused a 50% decrease in both killing of Staphylococcus aureus and luminol-enhanced chemiluminescence (CL) by PMNs stimulated by opsonized-zymosan (OZ), Concanavalin A (Con A), or calcium ionophore A23187. The levels of PMN intracellular fluorescence measured by flow cytometry, using the fluorochrome dichlorofluorescein diacetate (DCF-DA), were considerably less in the presence of SPS than in resting platelet supernatants (RPS). No influence of platelet supernatant on O2 consumption and O2- generation by OZ-activated PMNs was observed. The incubation of PMNs with SPS caused a significant increase in the rate of chemotaxis and aggregation elicited by Con A, OZ, and phorbol myristate acetate (PMA). The supernatant from resting platelets did not show any of the above-reported effects. Platelets previously degranulated by thrombin were unable to inhibit CL when activated with agonists. Studies on the differential release of the granules by platelets showed that the CL-quenching activity paralleled the discharge of lysosomal content. The release of myeloperoxidase (MPO) from PMNs elicited by OZ was reduced in the presence of SPS. The platelet supernatant did not affect the MPO activity if PMNs were lysed with Triton X-100. The leakage of lactate dehydrogenase (LDH) from platelets was less than 3%, and no catalase or superoxide dismutase was released. This activity withstood lyophilization, but was destroyed by 10 min heating at 100 degrees C or by treatment with proteolytic enzymes.

A surface NAD-glycohydrolase of human platelets may influence their aggregation.

Human platelets may hydrolyze externally added NAD+ yielding ADPR and nicotinamide. The extent of hydrolysis is significantly higher when the platelets are stimulated. The presence of external NAD+ strongly inhibits the aggregation induced by every agonist used. It seems that adenosine or ADPR itself generated by NAD+ hydrolysis may be responsible for the inhibition of aggregation. Evidence is given that some of the NAD+ hydrolysis product is taken up by stimulated platelets.

Anandamide activates human platelets through a pathway independent of the arachidonate cascade.

Anandamide (arachidonoylethanolamide, AnNH) is shown to activate human platelets, a process which was not inhibited by acetylsalicylic acid (aspirin). Unlike AnNH, hydroperoxides generated thereof by lipoxygenase activity, and the congener (13-hydroxy)linoleoylethanolamide, were unable to activate platelets, though they counteracted AnNH-mediated stimulation. On the other hand, palmitoylethanolamide neither activated human platelets nor blocked the AnNH effects. AnNH inactivation by human platelets was afforded by a high-affinity transporter, which was activated by nitric oxide-donors up to 225% of the control. The internalized AnNH could thus be hydrolyzed by a fatty acid amide hydrolase (FAAH), characterized here for the first time.

Cytoskeleton alterations of erythrocytes from patients with Fanconi's anemia.

Fanconi's anemia (FA) is a very rare genetically heterogeneous disease which has been hypothesized to be defective in the detoxification of reactive oxygen species. In this work we report the results obtained by morphometric and biochemical analyses on the red blood cells (RBCs) from FA patients. With respect to RBCs from healthy donors the following changes have been detected: (i) a variety of ultrastructural alterations, mainly surface blebbing typical of acanthocytes and stomatocytes; (ii) a significant quantitative increase of these altered forms; (iii) modifications of spectrin cytoskeleton network; (iv) an altered redox balance, e.g. a decreased catalase activity and significant variations in the GSSG/GSH ratio. We hypothesize that remodeling of the redox state occurring in FA patients results in cytoskeleton-associated alterations of red blood cell integrity and function.

Hydrogen peroxide has a role in the aggregation of human platelets.

The aggregation of platelets induced by soluble and particulate stimuli is enhanced by the addition of minute amounts of H2O2. Externally added catalase strongly inhibits the aggregation induced by particulate stimuli and by phorbol myristate acetate (PMA). The addition of aminotriazole to stimulated platelets causes a significant inhibition of intracellular catalase. This indicates the formation of H2O2 inside the platelets during activation. No effects were observed when the platelets were stimulated by the ionophore A23187.

Oxygen consumption in platelets of newborn infants before and after stimulation by thrombin.

The authors compared the oxygen consumption in platelets from the umbilical cord blood of 36 healthy newborn infants with that of 27 adult subjects, before and after thrombin addition (1.67 U/ml). Oxygen consumption at rest was 6 mumol/10(9)/min in adult control platelets and 5.26 in newborn infants. The burst in oxygen consumption after thrombin addition was 26.30 mumol/10(9)/min in adults and 24.90 in infants. Dinitrophenol did not inhibit the burst of O2 consumption in platelets in 8 out of 10 newborn infants, while the same concentration caused a decrease in 9 out of 10 adult subjects. Deoxyglucose inhibited the burst in O2 consumption in newborn infant and adult platelets by about 50%. KCN at the concentration of 10(-4) M completely inhibited basal oxygen consumption but did not completely inhibit the burst after thrombin. At the concentration of 10(-3) M, it inhibited both basal O2 consumption and the burst in infants and adult subjects.

Labelling of blood platelets with stable tracers.

Half-time values of platelets labelled with stable rubidium are compared to those of platelets labelled with Cr51. Platelets labelled with stable rubidium are assayed by a very simple version of the X-Ray fluorescence equipment. The mean quantity of rubidium incorporated by the cells is of about some microgram Rb per ml blood. The in vitro half-time of human Rb labelled platelets stored at 22 degrees C is 41.2 +/- 3h compared with the value 44.8 +/- 3h for platelets labelled with Cr51, as deduced by six experiments. The in vivo half-time of rabbit platelets labelled with stable rubidium is 22 +/- 3h compared with the value 18 +/- 3h of platelets labelled with Cr51; ten experiments were carried out.

Cyanide insensitive oxidase in platelets of newborn infant.

The authors studied the behaviour of neonate platelet O2 consumption after the addition of pyridine nucleotide compared to adult controls. O2 consumption of neonate platelets after NADH addition was 103,2 millimicronmol O2/10(9)/hr (SE = 24,74) and in adult controls 188,8 millimicronmol O2/10(9)/hr (SE = 36,46). After the addition of NADPH O2 consumption was, respectively, 233,5 millimicronmol (SE = 46,29) and 218,3 millimicronmol (SE = 30,01).

The in vitro inhibitory effect on thrombin by 2,3-diphosphoglycerate.

Thrombin incubated with 2,3-diphosphoglycerate (150 nmol 2,3-DPG/1 NIH thrombin unit) lost up to 70% of its clotting activity, whereas the esterase activity remained unchanged. No fibrinopeptide release by thrombin was observed in the presence of 2,3-DPG. The fibrin polymerization was normal. By chromatography on Amberlite IRC-50, alpha-thrombin was eluted at pH 8.0. In presence of 2,3-DPG, alpha-thrombin was not eluted. Likely, 2,3-DPG can interfere with thrombin.

Involvement of oxygen radicals in cytarabine-induced apoptosis in human polymorphonuclear cells.

We investigated apoptosis in polymorphonuclear neutrophils (PMNs) induced by cytarabine (Ara-C). This drug increased apoptosis by 100% with respect to the controls after 3 hr of incubation. This increase was inhibited by N-acetyl-L-cysteine (NAC) or diphenyleneiodonium chloride (DPI). Ara-C alone caused an early increase (after a 30-min incubation) in intracellular oxidant generation (inhibitable by rotenone, fumonisin b1, and DPI) and in protein tyrosine phosphorylations (inhibitable by NAC). The drug also affected the observed reduction of dimethylthiazol diphenyltetrazolium bromide (MTT). No extracellular release of reactive oxygen species (ROS) was elicited by the addition of Ara-C, while the drug increased the release of ROS by N-formyl-leucyl-phenylalanine-(f-MLP) but not phorbol 12-myristate 13-acetate-stimulated PMNs. This phenomenon was abolished by the addition of genistein, whereas such an effect was not observed following the addition of 1-(5-isoquinolynilsulfonyl)-2-methylpiperazine (H7). Ara-C induced ROS release from PMNs in the presence of subthreshold concentrations of f-MLP (priming effect). These results indicate that intracellular ROS production from mitochondria promotes Ara-C-induced apoptosis. Ara-C primes plasma membranes by a mechanism involving protein tyrosine phosphorylations and may also contribute to ROS generation from the granules.

Surface reactions of a plasma-sprayed CaO-P2O5-SiO2-based glass with albumin, fibroblasts and granulocytes studied by XPS, fluorescence and chemiluminescence.

X-ray photoelectron spectroscopy (XPS) was used to define the chemical composition of the outermost surface layer and the surface modification of a plasma-coated phospho-silicate glass (identified as BVA) when immersed in K-phosphate buffer or in phosphate buffered human albumin solution. Its behavior was compared with that of a soda-lime-based glass (identified as BVH) treated in the same way. The surface % composition of plasma-sprayed glass was consistent with bulk composition. After incubation with buffer, a Ca-P-rich layer developed only on the surface of BVA glass. Human serum albumin was bound reversibly to both glasses maintaining its native state. However, the protein completely covered the BVA glass surface within 24 h, with the formation of a mixed albumin-Ca-P layer, while 4 days incubation was necessary for complete coverage of BVH glass surface. Murine fibroblasts seeded on plasma-coated BVA glass showed a proliferation pattern similar to that of control cells grown on Petri dish, while cells seeded on BVH had more restricted growth. A limited response was induced in polymorphonuclear granulocytes by both bulk glasses powder. In conclusion, the glass identified as BVA has the suitable characteristics of its surface layers to be considered biologically active from both a chemical and a cellular point of view.

Livedo vasculopathy vs small vessel cutaneous vasculitis: cytokine and platelet P-selectin studies.

OBJECTIVE: To assess the role of platelets and lymphocyte-related immunological mechanisms in livedo vasculopathy (LV) and cutaneous small vessel vasculitis (CSVV). Livedo vasculopathy is thought to be related to the thrombotic occlusion of small and medium-sized dermal vessels. Cutaneous small vessel vasculitis comprises a heterogeneous group of disorders in which the main pathogenetic events could be modulated by circulating cytokines. DESIGN: Case series study of 2 groups of patients affected respectively with LV and CSVV. SETTING: A large clinical and research institute for the study and treatment of cutaneous diseases. PATIENTS: Consecutive patients with clinically and histologically proved idiopathic LV (n = 8) and CSVV (n = 20) were studied and compared with healthy donors (n = 20). Patients with potentially correlated systemic diseases were excluded. MAIN OUTCOME MEASURES: Surface expression of platelet P-selectin and circulating level of interleukin (IL) 1beta, tumor necrosis factor alpha (TNF-alpha), IL-8, IL-2, and soluble IL-2 receptor. RESULTS: The IL-2 and soluble IL-2 receptor levels were significantly higher in serum samples from patients with both LV (1.24 +/- 0.46 IU/mL [mean +/- SD] vs 0.46 +/- 0.24 IU/mL, P<.001; 899 +/- 368 IU/mL vs 628 +/- 132 IU/mL, P<.02) and CSVV (0.91 +/- 0.57 IU/mL, P<.02; 1087 +/- 451 IU/mL, P<.001) than in those from the healthy controls. The serum levels of IL-1beta, TNF-alpha, and IL-8 were higher in patients with CSVV than in controls (7.53 +/- 6.7 pg/mL vs 4.58 +/- 2.72 pg/mL; 23.7 +/- 12.6 pg/mL vs 10.82 +/- 2.46 pg/mL, P<.001; 37.8 +/- 46 pg/mL vs 8.25 +/- 3.53 pg/mL, P<.02, respectively). No significant difference in the serum levels of IL-1beta (7.2 +/- 4.9 pg/mL), TNF-alpha (12.9 +/- 3.47 pg/mL), and IL-8 (5.9 +/- 4.13 pg/mL) was observed in patients with LV compared with controls. An increased expression of platelet P-selectin was also detected in patients with LV in comparison with controls and patients with CSVV. The mean +/- SD percentage of positive cells for P-selectin was 43% +/- 5% in the patients with LV, 5.1% +/- 2% in the controls (P<.001), and 5.3% +/- 2% in the patients with CSVV (P<.001). CONCLUSIONS: Taken together, these data demonstrate that different pathogenetic mechanisms are operative in LV and CSVV. In fact, platelet and lymphocyte activation is present in LV, whereas the levels of inflammatory mediators are in a normal range. In CSVV, the high serum levels of proinflammatory cytokines suggest that they are actively involved in the pathogenesis of cutaneous vasculitis.

Hydrogen peroxide is an intermediate in the platelet activation cascade triggered by collagen, but not by thrombin.

Human blood platelets produce oxidant species when stimulated by collagen and thrombin. The oxidative burst of platelets has been studied by cytofluorimetry taking advantage of the fluorogenic dye DCFH2-DA, which is taken up and deacetylated by platelets and then oxidized to the fluorescent derivative DCF. The oxidation of DCFH2 is induced by stimulation with collagen but not with thrombin and inhibited by external catalase. Catalase also inhibited the aggregation induced by collagen, but not that induced by thrombin. Aspirin and indomethacin inhibited the formation of the fluorochrome only when platelets were stimulated by thrombin. Externally added H2O2 increased the cytoplasmic calcium content as probed by the fluorescence of Indo-1. The present data suggest that collagen induces the production of H2O2, which in turn may stimulate the aggregation of platelets through a calcium mobilization. Instead the stimulation by thrombin does not require the intermediacy of H2O2.

The effect of 2,3-diphosphoglycerate on oxygen consumption burst in thrombin-stimulated platelets.

2,3-Diphosphoglycerate (2,3-DPG) modifies platelet function; it diminishes aggregation and the release reaction. The hypothesis that this occurs through a modification of the intracellular level of cyclic AMP or through an alteration in the synthesis of prostaglandins has been proposed. Since the release reaction occurs simultaneously with a burst in the consumption of oxygen, the authors have studied the effect of 2,3-DPG on oxygen consumption after the addition of thrombin with or without the addition of substances which modify platelet metabolism (aspirin, theophylline, glucagon, etc.). It was observed that 2,3-DPG diminishes oxygen consumption induced by thrombin. This mechanism alters the platelet membrane function.

Effect of amlodipine on exercise-induced platelet activation in patients affected by chronic stable angina.

BACKGROUND: Literature concerning exercise-induced platelet activation in chronic stable angina is somewhat confusing. The reason lies in the type of exercise as well as in methodological problems. A powerful, recently introduced procedure to detect platelet activation is flow cytometry. Platelet response to activating factors is mediated by calcium uptake; however, calcium antagonist effect on platelet activity is still unclear. HYPOTHESIS: The study was undertaken to investigate exercise-induced platelet activation before and after treatment with amlodipine in chronic stable angina. METHODS: Twenty patients with chronic stable angina were entered into the study. Each subject underwent a symptom-limited cycloergometer stress test following a washout period of 2 weeks. Blood samples were collected before and immediately after exercise. All subjects were then randomized into two groups of 10 patients each, with Group 1 and Group 2 taking amlodipine 10 mg/day, and placebo for 4 weeks, respectively. They subsequently underwent a second exercise stress test, and blood samples were obtained before and immediately after exercise. Flow-cytometric evaluation of platelet activity was performed in order to recognize GMP-140 expression on platelet membrane. RESULTS: Strenuous exercise induced a significant increase in platelet activation in all subjects prior to therapy. No significant differences were observed in platelet activity at rest between Groups 1 and 2, whereas a significant decrease in exercise-induced platelet activation was demonstrated in Group 1 compared with Group 2. CONCLUSION: Our data provide evidence of the favorable effect of amlodipine on exercise-induced platelet activation in patients affected by chronic stable angina.

Congenital disorders sharing oxidative stress and cancer proneness as phenotypic hallmarks: prospects for joint research in pharmacology.

In spite of very distinct genotypic assets, a number of congenital conditions include oxidative stress as a phenotypic hallmark. These disorders include Fanconi's anaemia, ataxia telangiectasia, xeroderma pigmentosum and Bloom's syndrome, as well as two frequent congenital conditions: Down's syndrome and cystic fibrosis. Cancer proneness is a clinical feature shared by these disorders, while other manifestations include early ageing, neurological symptoms or congenital malformations. The onset of oxidative stress has been related to excess formation, or defective detoxification, of reactive oxygen species (ROS). This can arise from either the abnormal expression or inducibility of ROS-detoxifying enzymes, or by defective absorption of nutrient antioxidants. Resulting oxidative injury has been characterized through: (i) DNA, protein or lipid oxidative damage; (ii) excess ROS formation (in vitro and ex vivo); (iii) sensitivity to oxygen-related toxicity; (iv) improvement of cellular defects by either hypoxia or antioxidants; and (v) circumstantial evidence for in vivo oxidative stress (as e.g. clastogenic factors). Investigations conducted so far have been confined to individual disorders. Comparative studies of selected indicators for oxidative stress could provide further insights into the pathogenesis of each individual condition. Such a unified approach may have wide-ranging consequences for studies of ageing and cancer.

Gender disparity in pediatric diseases.

Sex/gender differences in terms of incidence, prevalence, age at onset and severity have been documented for several complex adulthood diseases. However, several pediatric diseases also displayed a gender disparity. Unfortunately, epidemiologic studies investigating gender disparity in pediatric age show dissimilar results often depending on the spatial and temporal issues, to considerable regional environmental variations, to social conditions or to infectious agent virulence. Anyway, studies over time showed that gender disparity in childhood mortality and morbidity may be narrow in some pathological conditions whereas in other severe diseases, e.g. sepsis, some cancers and some immune disorders, the disproportion was found as significant. In this work we briefly review literature data dealing with sex/gender differences in morbidity and mortality observed during the pediatric age. In particular, communicable and non-communicable diseases, including cancer, have been considered. The possible mechanisms underlining these differences, e.g. hormonal and epigenetic, are also discussed. The analysis of literature available as concerns pediatric age seems to underline that gender differences start very early in human beings and that hormones as well as gene expression in XX and XY cells can play a role. A reappraisal of the gender issue in pediatric research could thus be pivotal: it might contribute to the improvement of diagnostic and therapeutic strategies as well as to the improvement of the appropriateness of the cures.