Cutaneous mastocytosis: A dermatological perspective.

Mastocytosis is a rare disease characterised by expansion and collection of clonal mast cells in various organs including the skin, bone marrow, spleen, lymph nodes and gastrointestinal tract. The prevalence of mastocytosis has been estimated to be one in 10 000, while the estimated incidence is one per 100 000 people per year. Cutaneous mastocytosis is classified into (i) maculopapular cutaneous mastocytosis, also known as urticaria pigmentosa; (ii) diffuse cutaneous mastocytosis; and (iii) mastocytoma of the skin. In adults, cutaneous lesions are usually associated with indolent systemic mastocytosis and have a chronic evolution. Paediatric patients, on the contrary, have often cutaneous manifestations without systemic involvement and usually experience a spontaneous regression. Diagnosis of cutaneous mastocytosis may be challenging due to the rarity of the disease and the overlap of cutaneous manifestations. This short review describes pathogenesis and clinical aspects of cutaneous mastocytosis with a focus on diagnosis and currently available therapies.

Metabolically unhealthy overweight individuals have high lysophosphatide levels, phospholipase activity, and oxidative stress.

BACKGROUND & AIMS: Metabolically unhealthy overweight (MUO) individuals and metabolically healthy overweight (MHO) individuals differ in biomarkers of atherogenesis. Metabolomic approaches enable studies of the metabolic variables underlying these differences. METHODS: We determined the metabolomes in plasma samples from 34 MUO and 34 MHO individuals matched for sex, age, and body mass index (BMI) to identify potential metabolic markers or pathways associated with atherogenic traits. RESULTS: This analysis revealed that the MUO group had significantly higher levels of glycolic acid, 6 lysophosphatidylethanolamines (lysoPEs), and 12 lysophosphatidylcholines (lysoPCs). Although the two groups had similar total body fat percentages and lean body masses, MUO individuals had larger visceral fat areas (VFAs). They also had greater circulating lipoprotein-associated phospholipase A2 (Lp-PLA2) activity and higher levels of oxidized low-density lipoprotein (ox-LDL) and urinary 8-epi-prostaglandin F2α (8-epi-PGF2α), reflecting higher risks for oxidative and lipid-related tissue damage. The following measures were positively correlated: VFA and ox-LDL; ox-LDL and Lp-PLA2 activity; and lysoPC, lysoPE, and 8-epi-PGF2α levels. Chronic plasma lysoPC increases were associated with atherogenic traits, higher levels of mean ox-LDL, 8-epi-PGF2α, Lp-PLA2, and visceral fat accumulation in MUO compared to MHO individuals. CONCLUSIONS: This panel of plasma metabolites distinguishes MUO from MHO individuals and will enable future research on fat dysregulation and obesity.

Study of serum paraoxonase and phospholipase activities in pregnant women in relation to birth weight.

AIM: Low birth weight is an important issue due to its dreadful consequences in future. Well prevailing over the world, this is important in view of developing countries. Low birth weight is associated with high neonatal and infant mortality, lower trajectory of growth during childhood and adolescence, increases risk of non-communicable diseases during adult life. Oxidative stress is a major player among the various etiologies. Paraoxonase1 is an important antioxidant defense. Phospholipase is required to release free fatty acids from phosphoglycerides utilized for fetal growth. We conducted the study to look for the predictive value of serum paraoxonase and phospholipase. METHODS: With binding to Helsinki declaration and approval from Institutional Ethical Committee, we have selected 100 pregnant ladies. Serum PON1 arylesterase (ARE), lactonase (LACT) and serum phospholipase (PL) activities are measured. We used SPSS 20.0 for linear and logistic regression models to assess the predictability of the ARE, LACT and PL for predicting the IUGR. RESULTS: R value increases in the order of maternal age, maternal weight, phospholipase, arylesterase, lactonase. Logistic regression analysis with different models and with birth weight as dependent factor, maternal age is flagged out as not significant. PL, LACT, ARE emerge out to be good predictors of IUGR. CONCLUSION: From this study we have concluded that PON1 LACT, ARE and serum PL, could be the newer markers IUGR in maternal serum. IUGR can be predicted beforehand by using PON1 LACT, PON1 ARE and serum phospholipase. This surely will help in timely diagnosis and treatment accordingly of possible adverse pregnancy outcome.

Determination of phospholipase activity of PAF acetylhydrolase.

This article presents a radiometric assay to determine the enzymatic activity of platelet-activating factor (PAF) acetylhydrolase (PAF-AH), also known as lipoprotein-associated phospholipase A2 and phospholipase A2 group 7A. The method is based on the release of radioactively labeled acetate from sn-2-labeled PAF and separation of substrate and product using reversed-phase column chromatography on octadecyl silica gel cartridges. The assay is fast, convenient, reproducible, sensitive, and inexpensive. The instrumentation required includes standard laboratory equipment and a liquid scintillation counter. The assay is also useful to determine the activity of intracellular PAF-AH (PAF-AH II), provided that a few modifications are included. The enzymatic activity determined using PAF as the substrate is a direct indication of the ability of plasma samples, purified preparations, and cellular and tissue lysates to hydrolyze short- and medium-chain phospholipids that may or may not harbor oxidized functionalities. In addition, the assay can be used to test the suitability of other phospholipids, including species containing oxidized, long-chain sn-2 fatty acyl groups, as PAF-AH substrates. This versatile assay can be used to accurately determine PAF-AH activity in biological samples and preliminarily assess affinity and efficiency of the hydrolysis of potential substrates present in complex mixtures.

Endothelial lipase activity predicts high-density lipoprotein catabolism in hemodialysis: novel phospholipase assay in postheparin human plasma.

OBJECTIVE: A novel phospholipase assay was used to measure for the first time the behavior of endothelial and hepatic phospholipase activities in postheparin human plasma of hemodialyzed patients and its relationship with atherogenic and antiatherogenic lipoprotein levels. METHODS AND RESULTS: Endothelial and hepatic phospholipase activity was assessed in a total SN1-specific phospholipase assay, using (1-decanoylthio-1-deoxy-2-decanoyl-sn-glycero-3-phosphoryl) ethylene glycol as the substrate. Hemodialyzed patients presented lower values of total and hepatic phospholipase activity than controls: 4.4 (1.9-9.0) versus 7.5 (3.6-18.0) and 2.6 (0.7-6.2) versus 6.6 (1.3-15.2) µmol of fatty acid released per milliliter of postheparin plasma per hour, respectively (P<0.001); however, endothelial lipase (EL) phospholipase activity was increased in patients: 1.7 (0.8-3.0) versus 1.1 (0.1-2.7) µmol of fatty acid released per milliliter of postheparin plasma per hour (P=0.008). EL was negatively associated with high-density lipoprotein (HDL)-cholesterol (r=-0.427; P=0.001), and apolipoprotein A-I levels, total phospholipase, and hepatic lipase activity were directly associated with low-density lipoprotein-cholesterol and apolipoprotein B. The association of EL and HDL-cholesterol remained significant when adjusting for waist circumference (ß=-0.26; P=0.05), and the effect of hepatic lipase on low-density lipoprotein-cholesterol continued after adjusting for age (ß=0.46; P= 0.001). CONCLUSIONS: Our results support the hypothesis that EL is the predominant enzyme responsible for lipolytic catabolism of HDLs in hemodialyzed patients and resolve the apparent paradox observed between low hepatic lipase activity and decreased HDL-cholesterol levels observed in these patients. In addition, the ability to assess total hepatic lipase and EL phospholipase activity in plasma will increase our knowledge of the mechanisms involved in controlling HDL levels and cardiovascular risk in hemodialyzed patients, as well as other populations with low levels of HDL-cholesterol.

From circulating biomarkers to genomics and imaging in the prediction of cardiovascular events in the general population.

Cardiovascular disease (CVD) is the leading cause of death and disability worldwide. In the last decades numerous markers have been considered and investigated for the prediction of CV events, but only a few of them resulted in improved global risk assessment beyond traditional risk factors when incorporated into coronary evaluation scores. Recent genetic studies have pointed out a few but consistent loci or genes which are independently associated with CV risk. The idea is fascinating that these genetic markers could lead to improved individual CV risk assessment and tailored pharmacological interventions. In this brief review we will not make a systematic review of all non-genetic and genetic markers of CV risk but we will try to make a brief overview of the most interesting ones with the aim to underline potential 'pros' and 'cons' of their implementation in clinical practice.

Patients with primary antiphospholipid syndrome and coronary microvascular dysfunction--a distinct clinical subset.

UNLABELLED: Morbidity of patients with cardiac syndrome X (CSX) is high. Impairment of microvascular endothelial function has been suggested to be a mechanism of the disease. The study was undertaken to assess some of the characteristics of patients with primary antiphospholipid syndrome (pAPS) and CSX. METHODS: We studied 36 patients with CSX, 14 patients having pAPS and 10 healthy controls. Patients evaluation included: clinical examination, 12-lead ECG, effort treadmill test (protocol Bruce modified), determination of plasma triglycerides, cholesterol, antiphospholipid antibodies (APLA). There were determined as markers of the inflammatory state: serum phospholipase (PL-A2) and peripheral neutrophils activity. RESULTS: Patients with pAPS presented normal values of serum cholesterol and triglycerides levels, normal PL-A2 activity, moderate superoxide anion generation. Patients without APLA presented hyperlipidemia, increased PL-A2 activity, increased superoxide anion generation. During the follow-up period we found a correlation between P1-A2 activity and ischemic episodes, but only in patients with CSX and pAPS there were registered cardiovascular events. CONCLUSION: Patients with SCX and pAPS represent a distinct clinical subset, being characterized by minimal inflammation, absence of usual risk factors for coronary heart disease, more severe prognosis related to recurrent thromboses and the need for early anticoagulant therapy.

Phospholipase and lysophospholipase activity of rat eosinophil leukocytes.

Previous studies have shown the high lysophospholipase activity of rat eosinophilic leukocytes and used this enzyme to measure the rise in eosinophilic population of peripheral tissues caused by parasitic infections. This report details the methods and results of an investigation showing the presence in the same cells of high phospholipase (PLA) activity. Unfractionated and metrizamide-purified peritoneal eosinophil preparations were assayed using a mixed micelle substrate (6/15 mM lecithin/Triton X-100) at experimentally determined pH (6.4) and ionic strength (I=0.2) optima: the attendant reaction products included free fatty acids and organic P in a 2/1 molar proportion with a correspondent loss in the initial phospholipid concentration. The organic P fragment was further characterized as GPC (glycerylphosphorylcholine) by quantitative precipitation and acid hydrolysis. Estimates of PLA activity averaged 5 micromol/h/10(6) unfractionated eosinophils and metrizamide-purified eosinophil preparations. Paired tests for PLA and LysoPLA on unfractionated and enriched cell preparations, cytosolic extracts, and chromatographic fractions yielded similar activity ratios, supporting the inference of a close association of the two activities which could also be confirmed for the major tissues of eosinophil production and distribution.

Improved high-performance liquid chromatographic method for the combined analysis of phospholipase, lipoxygenase and cyclooxygenase activities.

We report herein an improved method for the high-performance liquid chromatographic separation and analysis of eicosanoids formed during the stimulation of human platelets in vitro with collagen. Since the products of interest, excepting arachidonic acid, contain hydroxyl groups (one to several), our method involves the conversion of the hydroxyl groups into acetates (pyridine/acetic anhydride) after derivatization with anthryl diazomethane (ADAM) rendering the compounds with much decreased polarity for separation on a reversed-phase column. This procedure is superior to that involving ADAM esters only, i.e. with free hydroxyl groups, as it leads to the excellent separation of the desired compounds from each other and from extraneous peaks observed due to the ADAM reagent and sharpens the peak of thromboxane. We have successfully applied the method to investigate the formation of thromboxane B2 and 12-hydroxyheptadecatrienoic acid (HHT) (products of cyclooxygenase and thromboxane A2 synthase), 12-hydroxyeicosatetraenoic acid (12-HETE, a 12-lipoxygenase product) and arachidonic acid (AA, product of phospholipase A2) formed during the in vitro aggregation of human platelets induced by collagen. A correlation between the inhibition of aggregation by aspirin and thromboxane/HHT formation was observed. All four compounds can be chromatographed in a single run. We employed prostaglandin B1 (PGB1) as internal reference standard to quantify the products. The method is useful to investigate selectivity of drugs which may affect either or all of these enzyme pathways at the same time.

Similar turnover and shedding of the cellular prion protein in primary lymphoid and neuronal cells.

The cellular prion protein (PrP(C)) is essential for pathogenesis and transmission of prion diseases. Although prion replication in the brain is accompanied by neurodegeneration, prions multiply efficiently in the lymphoreticular system without any detectable pathology. We have used pulse-chase metabolic radiolabeling experiments to investigate the turnover and processing of PrP(C) in primary cell cultures derived from lymphoid and nervous tissues. Similar kinetics of PrP(C) degradation were observed in these tissues. This indicates that the differences between these two organs with respect to their capacity to replicate prions is not due to differences in the turnover of PrP(C). Substantial amounts of a soluble form of PrP that lacks the glycolipid anchor appeared in the medium of splenocytes and cerebellar granule cells. Soluble PrP was detected in murine and human serum, suggesting that it might be of physiological relevance.

Lysosomal phospholipase activity is decreased in mucolipidosis II and III fibroblasts.

Mucolipidosis (ML) II and III are rare autosomal recessively inherited diseases characterized by deficiency of multiple lysosomal enzymes and, as a result, a generalized storage of macromolecules in lysosomes of cells of mesenchymal origin. In ML II and ML III fibroblasts, most, but not all, newly synthesized lysosomal enzymes are secreted into the medium instead of being targeted correctly to lysosomes. Defects in the enzyme UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase underlie this effect. It is unknown how lysosomal phospholipases are targeted to the lysosomes of fibroblasts. In the present study lysosomal phospholipase activity was determined in delipidated fibroblast homogenates and plasma from ML II and ML III patients and controls using a [3H]choline-labeled phosphatidylcholine. After incubation, residual phosphatidylcholine and its labeled degradation products (lysophosphatidylcholine, glycerophosphorylcholine and choline phosphate) were quantified. We found that ML II and ML III fibroblasts are deficient in lysosomal phospholipase A and C activity. These enzymes were present in elevated amounts in plasma of ML II and ML III patients. These data indicate that phospholipases, like most other lysosomal enzymes in these diseases, are secreted into the blood instead of being targeted specifically to lysosomes. Thus, the mannose-6-phosphate receptor pathway is needed for proper delivery of lysosomal phospholipases to lysosomes. We also found that production of labeled choline phosphate was mainly due to the activity of acid sphingomyelinase instead of phospholipase C under the assay conditions used. Other active lipolytic enzymes were phospholipase A and lysophospholipase. No evidence for phospholipase D activity was found.

Occurrence of hemolytic activity in the skin secretion of the caecilian siphonops paulensis.

The skin secretion of the caecilian Siphonops paulensis (SpSS) induces a time-and dose-dependent hemolytic response on red blood cells (RBC). When RBC from various animals species were subjected to the action of SpSS, a range of sensitivities was evident, sheep erythrocytes being the most susceptible, human, mouse and rabbit having moderate susceptibility, cow, snake and toad erythrocytes being more resistant, while S. paulensis RBC were entirely resistant. The hemolytic activity of SpSS was inhibited at temperatures higher than 60 degrees C. Both trypsin- and chymotrypsin-treated SpSS were ineffective in inducing RBC lysis. The treatment of SpSS with sheep RBC ghosts reduced its activity. There is no phospholipase activity in the SpSS.

Alterations in individual molecular species of human platelet phospholipids during thrombin stimulation: electrospray ionization mass spectrometry-facilitated identification of the boundary conditions for the magnitude and selectivity of thrombin-induced platelet phospholipid hydrolysis.

Although the rapid thrombin-induced release of arachidonic acid in human platelets has been known for over 20 years, the amount of arachidonic acid mass mobilized and the source of the released arachidonic acid has remained a subject of intense controversy. Herein, we exploit the analytic power and sensitivity of electrospray ionization mass spectrometry to identify plasmenylethanolamines as the largest source of arachidonic acid mass released during thrombin stimulation and to demonstrate the presence of multiple novel molecular species of plasmenylethanolamines in human platelets. Specifically, 90 s after thrombin stimulation a total of 60.1 nmol of arachidonic acid-containing phospholipids/10(9) platelets was hydrolyzed which included the loss of 31.8 nmol/10(9) platelets from ethanolamine glycerophospholipids (hydrolysis of plasmenylethanolamines represented 63% of the mass lost from the ethanolamine glycerophospholipid pool) but only 10.9 nmol/10(9) platelets from choline glycerophospholipids. Human platelet phosphatidylserine and phosphatidylinositol pools contained similar amounts of arachidonic acid mass in resting platelets (approximately equal to 20 nmol/10(9) platelets), and each pool contributed 8.7 nmol/10(9) platelets after thrombin stimulation. From these results, a lower boundary for the rate of thrombin-induced arachidonic acid mobilization in human platelets can be set at > 60 nmol/10(9) platelets, thereby identifying specific kinetic characteristics and substrate selectivities of the phospholipase(s) activated during platelet stimulation. Collectively, these results underscore the importance of plasmenylethanolamines as the major storage depot of arachidonic acid in resting platelets and as the major source of arachidonic acid mobilized after thrombin stimulation of human platelets.

Hepatic lipase gene therapy in hepatic lipase-deficient mice. Adenovirus-mediated replacement of a lipolytic enzyme to the vascular endothelium.

Hepatic lipase (HL) is an endothelial-bound lipolytic enzyme which functions as a phospholipase as well as a triacylglycerol hydrolase and is necessary for the metabolism of IDL and HDL. To evaluate the feasibility of replacing an enzyme whose in vivo physiologic function depends on its localization on the vascular endothelium, we have infused recombinant replication-deficient adenovirus vectors expressing either human HL (HL-rAdV; n = 7) or luciferase cDNA (Lucif-rAdV; n = 4) into HL-deficient mice with pretreatment plasma cholesterol, phospholipid, and HDL cholesterol values of 176 +/- 9, 314 +/- 12, and 129 +/- 9, respectively. After infusion of HL-rAdV, HL could be detected in the postheparin plasma of HL-deficient mice by immunoblotting and postheparin plasma HL activities were 25,700 +/- 4,810 and 1,510 +/- 688 nmol/min/ml on days 5 and 15, respectively. Unlike the mouse HL, 97% of the newly synthesized human HL was heparin releasable, indicating that the human enzyme was virtually totally bound to the mouse vascular endothelium. Infusion of HL-rAdV in HL-deficient mice was associated with a 50-80% decrease in total cholesterol, triglyceride, phospholipids, cholesteryl ester, and HDL cholesterol (P < 0.001) as well as normalization of the plasma fast protein liquid chromatography lipoprotein profile by day 8. These studies demonstrate successful expression and delivery of a lipolytic enzyme to the vascular endothelium for ultimate correction of the HL gene defect in HL-deficient mice and indicate that recombinant adenovirus vectors may be useful in the replacement of endothelial-bound lipolytic enzymes in human lipolytic deficiency states.

Selective regulation of human neutrophil functions by the cell activation inhibitor CI-959.

The cell activation inhibitor CI-959 [5-methoxy-3-(1-methylethoxy)-N-1H-tetrazol-5-ylbenzo[ b]thiophene-2- carboxamide, monosodium salt] was evaluated for its effects on human neutrophil functions. CI-959 inhibited spontaneous migration and chemotaxis toward N-formyl-methionyl-L-leucyl-L-phenylalanine (fMLP) with 50% inhibition (IC50) values of 3.6 and 3.1 microM, respectively. CI-959 also inhibited superoxide anion generation in response to C5a, fMLP, serum-opsonized zymosan (SOZ), concanavalin A (Con A), and calcium ionophore A23187 with IC50 values of 2.5, 4.7, 14.5, 5.4, and 14.8 microM, respectively. In comparison, CI-959 inhibited myeloperoxidase microM, respectively. In comparison, CI-959 inhibited myeloperoxidase release in response to C5a, fMLP, SOZ, and Con A with IC50 values of 11.6, 16.1, 7.5, and < 1.0 microM, respectively, while inhibiting the response to A23187 by only 5.5% at 100 microM. At concentrations up to 100 microM, CI-959 had no effect on the respiratory burst or degranulation in response to L-alpha-1,2-dioctanoylglycerol (DiC8) or phorbol 12-myristate 13-acetate (PMA). In addition, the compound inhibited leukotriene B4 release stimulated by fMLP and SOZ (IC50 values 4.0 and 2.5 microM, respectively), while having less activity against the A23187-stimulated response (IC50 > 100 microM). These results demonstrate that CI-959 inhibits cellular responses to stimuli that mobilize intracellular calcium. For cellular responses to inophore-mediated calcium influx, only oxygen radical production was inhibited by CI-959. CI-959 was further evaluated for its effects on neutrophil stimulus-response coupling. At 100 microM, CI-959 had no effect on human neutrophil phospholipase C or protein kinase C. CI-959 inhibited fMLP-stimulated intracellular calcium mobilization and calcium influx with IC50 values of 16.7 and 3.1 microM, respectively, and exhibited less potent calmodulin antagonist activity (IC50 = 90.5 microM). These results indicate that CI-959 may exert its stimulus- and response-specific inhibitory effects on neutrophil functions, in part, through inhibition of calcium-regulated signalling mechanisms.

Phospholipase C activity in platelets from Bernard-Soulier syndrome patients.

The levels of glycoprotein (GP) Ib and GPV and phospholipase C activity were measured in platelets from three Bernard-Soulier syndrome patients. The patients' platelets had 46%, 46%, and 24% of control levels of GPIb alpha and 43%, trace, and 13% of control levels of GPV as determined by immunoblot analysis. Stimulation by thrombin, trypsin, the thromboxane analogue U46619, and the combination of U46619 and trypsin caused the formation of [32P]phosphatidic acid, an index of phospholipase C activity, in [32P]orthophosphate-prelabeled platelets. With all agonists, however, the formation of [32P]phosphatidic acid was markedly reduced in Bernard-Soulier syndrome platelets compared with control platelets. These data indicated a postreceptor defect in phospholipase C activation in Bernard-Soulier syndrome platelets and confirmed earlier observations of potential proteolytic and nonproteolytic mechanisms of platelet activation.

[Structure and function of blood platelets]. / Structure et fonctions des plaquettes sanguines.

Blood platelets are discoid cellular fragments without nucleus originating from megakaryocytes. Platelets are able to respond to a great variety of agonists which bind to specific receptors localized on the plasma membrane. This process takes place when blood vessels are cut. Platelets then change their shape, adhere to newly exposed subendothelial tissues, release the content of numerous secretory granules and aggregate together. During this process, a great numbers of biochemical reactions are triggered such as phospholipases activation, synthesis of mediators and protein phosphorylation. These events result from increased cytoplasmic free calcium originating through calcium channels from the extracellular medium and from internal stores. Involvement of blood platelets in cardiovascular diseases may result from an exaggeration of these mechanisms by risk factors and are also discussed.