Levels Of Secretory Phospholipase A2 Research Articles

Secretory Phospholipase A2 as a Promising Biomarker for Predicting Acute Chest Syndrome in Children With Sickle Cell Disease: A Systematic Review and Meta-Analysis.

Acute chest syndrome (ACS) is a severe and potentially life-threatening complication of sickle cell disease (SCD). Early identification of patients at risk for ACS is crucial for timely intervention. There is a potential association between ACS and elevated levels of secretory phospholipase A2 (sPLA2), an enzyme involved in the breakdown of phospholipids. sPLA2 has emerged as a promising biomarker for predicting ACS. This systematic review and meta-analysis aimed to assess the diagnostic value of PLA2 in predicting ACS among children with SCD. A comprehensive search was conducted across multiple databases, including MEDLINE, Embase, Cochrane Library, PubMed, and Web of Science. Studies assessing the relationship between sPLA2 levels and ACS in SCD patients were included. Pooled sensitivity, specificity, likelihood ratios, and the area under the receiver operating characteristic curve (AUC) were calculated to assess sPLA2's diagnostic accuracy. There is a potential association between significant association between elevated sPLA2 levels and increased ACS risk in SCD patients. The pooled sensitivity of sPLA2 for predicting ACS was 0.766 (95% CI: 0.620-0.877), with a pooled specificity of 0.736 (95% CI: 0.680-0.787). The AUC of the summary receiver operating characteristic (SROC) curve was 0.84, indicating good discriminatory ability. sPLA2 emerges as a promising biomarker for predicting ACS in SCD patients, potentially guiding risk stratification and early intervention strategies to enhance patient outcomes. Nonetheless, further prospective studies are warranted to validate its clinical utility and standardize sPLA2 assay protocols.

Secretory Phospholipase A2 Levels Are High in Women with Sickle Cell Disease and Menstruation-Induced Vaso-Occlusive Crises

Menstruation-induced vaso-occlusive crisis (MIVOC) is a significant cause of morbidity in women with sickle cell disease (SCD). Secretory phospholipase A2 (sPLA2) is an inflammatory biomarker that is elevated in vaso-occlusive events such as acute chest syndrome (ACS), but its role in MIVOC is not previously studied. This study compared the serum level of sPLA2 among women with MIVOC and those without MIVOC. This is a comparative cross-sectional study. 354 women with SCD were screened for MIVOC using a structured questionnaire. sPLA2 levels were assayed using a standard ELISA while full blood counts were performed on an automated hematology analyzer. Data were analyzed using the SPSS software v26.0. Results were summarized as frequencies, percentages, and mean ± standard deviation. Variables were compared using the Student’s t-test and Pearson’s correlation. A p-value of <.05 was considered significant. The prevalence of MIVOC was 26.8%. Participants with MIVOC (n = 95) had significantly lower mean hemoglobin concentration (8.00 ± 2.03g/dL vs. 9.95 ± 4.15g/dL, p < .000), significantly higher mean platelets count (518.71 ± 84.58 × 109/L vs 322.21 ± 63.80 × 109/L, p < .000) and higher sPLA2 level (6.58 ± 1.94 IU vs 6.03 ± 0.42 IU, p = .008) compared to those without MIVOC (n = 95). Among participants with MIVOC, sPLA2 levels positively correlated with total white blood cell, absolute neutrophil, and lymphocyte counts. This study demonstrates that MIVOC is common among women with SCD and that the pathophysiology of MIVOC may have an inflammatory basis similar to that of ACS. The potential role of anti-inflammatory and antiplatelet agents in preventing and treating MIVOC may be explored.

Antihypertensive activity of oleanolic acid is mediated via downregulation of secretory phospholipase A2 and fatty acid synthase in spontaneously hypertensive rats.

Oleanolic acid (OA) is reported to possess antihypertensive activity via the regulation of lipid metabolism; however, the mechanisms underlying lipid regulation by OA are yet to be fully elucidated. The aim of the present study was to evaluate the mechanisms via which OA regulates lipid metabolism in spontaneously hypertensive rats (SHRs) via ultra-performance liquid chromatography-quadrupole/Orbitrap-mass spectrometry (MS)-based lipidomics analysis. SHRs were treated with OA (1.08 mg/kg) for 4 weeks. The liver tissues were excised, homogenized in dichloromethane and centrifuged, and subsequently the supernatant layer was collected and concentrated under vacuum to dryness. The dichloromethane extract was subjected to MS analysis and database searching, and comparison of standards was performed to identify potential biomarkers. Partial least squares-discriminant analysis performed on the liver lipidome revealed a total of 14 endogenous metabolites that were significantly changed in the SHR model group (SH group) compared with Wistar Kyoto rats [normal control (NC group)], including glycerophospholipids, sphingolipids and glycerides. Heatmaps revealed that the liver lipid profiles in the OA group were clustered more closely compared with those observed in the NC group, indicating that the antihypertensive effect of OA was mediated via regulation of liver lipid metabolites. It was observed that the protein levels of secretory phospholipase A2 (sPLA2) and fatty acid synthase (FAS) were increased in the SH group compared with the NC group. In addition, the levels of lysophosphatidylcholine and triglycerides in the liver were elevated, whereas the levels of low-density lipoprotein cholesterol and high-density lipoprotein cholesterol were reduced in the SH group. Upon treatment with OA, the mRNA and protein levels of PLA2 and FAS were observed to be downregulated. Collectively, the present study indicated that the antihypertensive activity of OA was mediated via downregulation of sPLA2 and FAS in SHRs, and that treatment with OA resulted in significant improvements in blood pressure and associated abnormalities in the lipid metabolites.

High-Throughput Lipidomic and Transcriptomic Analysis To Compare SP2/0, CHO, and HEK-293 Mammalian Cell Lines.

A combined lipidomics and transcriptomics analysis was performed on mouse myeloma SP2/0, Chinese hamster ovary (CHO), and human embryonic kidney (HEK) cells in order to compare widely used mammalian expression systems. Initial thin layer chromatography (TLC) analysis indicated that phosphatidylethanolamine (PE) and phosphatidylcholine (PC) were the major lipid components in all cell lines with lower amounts of sphingomyelin (SM) in SP2/0 compared to CHO and HEK, which was subsequently confirmed and expanded upon following mass spectrometry (MS) analysis. HEK contained 4-10-fold higher amounts of lyso phosphatidylethanolamine (LPE) and 2-4-fold higher amounts of lyso phosphatidylcholine (LPC) compared to SP2/0 and CHO cell lines. C18:1 followed by C16:1 were the main contributors to the difference in both LPE and LPC levels. Alternatively, the SP2/0 cell line exhibited 30-65-fold lower amounts of SM principally in the amount of 16:0. By mapping the transcriptomics data to KEGG pathways, we found expression levels of secretory phospholipase A2 (sPLA2), lysophospholipid acyltransferase (LPEAT), lysophosphatidylcholine acyltransferase (LPCAT), and lysophospholipase (LYPLA) can contribute to the differences in LPE and LPC. Sphingomyelin synthases (SMS) and sphingomyelin phosphodiesterase (SMase) enzymes may play roles in SM differences across the three cell lines. The results of this study provide insights that will aid the understanding of the physiological and secretory differences across recombinant protein production systems.

Secretory phospholipase A2 activity in blood serum: The challenge to sense

Excess levels of secretory phospholipase A2 (sPLA2) is known to contribute to several inflammatory diseases including vascular inflammation correlating with coronary events in coronary artery disease. Thus a method to monitor sPLA2 activity in blood serum is urgently needed. Such method is still a challenge since existing fluorescent probes do not allow to monitor sPLA2 activity directly in blood serum. Here we analyze and overcome barriers in sPLA2 sensing methodology and report a fluorescent probe and a kinetic model of its hydrolysis by sPLA2. New probe is designed with a fluorophore and a quencher not interfering binding to the enzyme. At the same time phospholipid matrix bearing the probe promotes efficient initial quenching of the fluorophore. Kinetic model of probe hydrolysis takes into account signal change due to the side processes. The probe and the kinetic model applied together prove the concept that the activity of sPLA can be measured directly in blood serum.

Association of Biomarkers of Lipid Modification with Functional and Morphological Indices of Coronary Stenosis Severity in Stable Coronary Artery Disease

Biomarkers of blood lipid modification and oxidative stress have been associated with increased cardiovascular morbidity. We sought to determine whether these biomarkers were related to functional indices of stenosis severity among patients with stable coronary artery disease. We studied 197 consecutive patients with stable coronary artery disease due to single vessel disease. Fractional flow reserve (FFR) ≤ 0.80 was assessed as index of a functionally significant lesion. Serum levels of secretory phospholipase A2 (sPLA2) activity, secretory phospholipase A2 type IIA (sPLA2-IIA), myeloperoxydase (MPO), lipoprotein-associated phospholipase A2 (Lp-PLA2), and oxidized low-density lipoprotein (OxLDL) were assessed using commercially available assays. Patients with FFR > 0.8 had higher sPLA2 activity, sPLA2 IIA, and OxLDL levels than patients with FFR ≤ 0.8 (21.25 [16.03-27.28] vs 25.85 [20.58-34.63] U/mL, p < 0.001, 2.0 [1.5-3.4] vs 2.6 [2.0-3.4] ng/mL, p < 0.01; and 53.0 [36.0-71.0] vs 64.5 [50-89.25], p < 0.001 respectively). Patients with FFR > 0.80 had similar Lp-PLA2 and MPO levels versus those with FFR ≤ 0.8. sPLA2 activity, sPLA2 IIA significantly increased area under the curve over baseline characteristics to predict FFR ≤ 0.8 (0.67 to 0.77 (95 % confidence interval [CI]: 0.69-0.85) p < 0.01 and 0.67 to 0.77 (95 % CI: 0.69-0.84) p < 0.01, respectively). Serum sPLA2 activity as well as sPLA2-IIA level is related to functional characteristics of coronary stenoses in patients with stable coronary artery disease.

Secretory Phospholipase A2 Is Associated with the Odds of Acute Coronary Syndromes through Elevation of Serum Amyloid-A Protein

In coronary heart disease (CHD), levels of secretory phospholipase A2 (sPLA2) are commonly increased. Serum amyloid-A (SAA) is increased in acute coronary syndromes (ACS) as well. It is needed to verify the hypotheses that sPLA2 is associated with the odds of ACS through elevation of SAA. We conducted a case-control study with 57 male patients with ACS and 30 controls matched by gender category. Levels of sPLA2, SAA, and myeloperoxidase (MPO) were measured by immunoreactive assay on the basis of a double-antibody sandwich technique. Levels of sPLA2, MPO, and SAA were significantly higher in patients than those in controls (11,359.0 ± 10,372.4 pg/mL vs. 1,320.5 ± 654.5 pg/mL, p = 0.00; 438.6 ± 310.7 ng/mL vs. 271.1 ± 176.8 ng/mL, p = 0.01; 10,995.2 ± 2,842.6 ng/mL vs. 3,861.7 ± 3,173.5 ng/mL, p = 0.00). There were significant correlations between age, visceral obesity, MPO, sPLA2, and SAA (r = 0.43; p = 0.00; r = 0.30; p = 0.00; r = 0.28; p = 0.00 and r = 0.53; p = 0.00). On multivariate logistic regression analyses, there were significant and independent associations between sPLA2 and SAA with odds of ACS [OR (95% CI) = 14.2 (2.1 to 98.6), p = 0.00; OR (95% CI) = 44.9 (6.9 to 328.4), p = 0.00]. Our findings suggest that sPLA2 may be associated with the odds of ACS compared with controls through increased inflammation, represented by elevated SAA.

The Acute-phase Response Is Not Predictive for the Development of Arthritis in Seropositive Arthralgia – A Prospective Cohort Study

To evaluate whether markers of the acute-phase response in patients presenting with arthralgia and positive anticitrullinated protein antibodies (ACPA) and/or immunoglobulin M rheumatoid factor (IgM-RF) could be predictive for the development of arthritis. In total, 137 ACPA- and/or IgM-RF-positive patients were included. Patients were followed annually for the development of arthritis, defined as presence of 1 or more swollen joints at clinical examination. High-sensitivity C-reactive protein (hsCRP), procalcitonin (PCT), secretory phospholipase A2 (SPLA2), tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), IL-12p70, IL-10, and interferon-γ (IFN-γ) were measured in baseline serum samples. Gene expression focusing on a predefined panel of genes coding for inflammatory molecules was measured by multiplex ligation-dependent probe amplification. Thirty-five patients (26%) developed arthritis within a median time of 11 months (interquartile range 3.7-18 mo). Circulating levels of cytokines, SPLA2, hsCRP, and PCT were not different between patients with progression to clinical arthritis and those without progression. However, a trend for IL-12p70, TNF-α, IL-10, IL-6, and SPLA2 was observed. No correlation between messenger RNA (mRNA) expression levels of inflammatory genes and progression to arthritis was found. Subgroup analysis of patients with early progression to arthritis showed higher levels of mRNA expression of poly(A)-specific ribonuclease and polycomb complex protein BMI-1 compared to patients without progression to arthritis. Although low-grade inflammation is present before onset of clinical arthritis in large cohorts and can be detected using consecutive measurements, a single measurement of acute-phase reactants seems to have limited value for prediction of development of arthritis in individual patients.

Increased phospholipase A2 and lyso-phosphatidylcholine levels are associated with surfactant dysfunction in lung contusion injury in mice

Surfactant dysfunction is an important pathologic disturbance in various forms of acute inflammatory lung injury. Previously we reported the presence of marked alterations in the composition and activity of pulmonary surfactant in bilateral lung contusions (LC) injury induced by blunt trauma in rats. This is extended here to a mouse model of unilateral LC with a focus on compositional and functional changes in surfactant associated with permeability injury and increases in activity of secretory phospholipase A2. Surfactant-associated gene expression was not altered in mice with unilateral LC injury on the basis of Affymetrix analysis. LC mice had significant permeability injury with increased albumin and total protein in bronchoalveolar lavage at 5, 24, 48, and 72 hours after insult compared with uninjured controls. The percent content of large surfactant aggregates was depleted at all postinjury times, and pulmonary pressure-volume (P-V) mechanics and compliance were abnormal during this period. Surfactant dysfunction was evaluated in 24 hours, when permeability injury and P-V changes were most prominent. At this time, activity levels of secretory phospholipase A2 were increased in bronchoalveolar lavage, and chromatographic analysis showed that large surfactant aggregates had decreased levels of phosphatidylcholine and increased levels of lyso-phosphatidylcholine. These changes were accompanied by severe detriments in large aggregate surface activity by pulsating bubble surfactometry. Large aggregates from LC mice at 24 hours had minimum surface tensions of only 12.6 ± 1.1 mN/m after prolonged bubble pulsation (20 min) compared with 0.7 ± 0.03 mN/m for uninjured controls. These results document important detriments in the composition and activity of pulmonary surfactant in LC injury in mice and suggest that active synthetic phospholipase-resistant exogenous surfactants may have utility in treating surfactant dysfunction in this clinically important condition.

Refining the value of secretory phospholipase A2 as a predictor of acute chest syndrome in sickle cell disease: results of a feasibility study (PROACTIVE)

Acute chest syndrome (ACS) is defined as fever, respiratory symptoms and a new pulmonary infiltrate in an individual with sickle cell disease (SCD). Nearly half of ACS episodes occur in SCD patients already hospitalized, potentially permitting pre-emptive therapy in high-risk patients. Simple transfusion of red blood cells may abort ACS if given to patients hospitalized for pain who develop fever and elevated levels of secretory phospholipase A2 (sPLA2). In a feasibility study (PROACTIVE; ClinicalTrials.gov NCT00951808), patients hospitalized for pain who developed fever and elevated sPLA2 were eligible for randomization to transfusion or observation; all others were enrolled in an observational arm. Of 237 enrolled, only 10 were randomized; one of the four to receive transfusion had delayed treatment. Of 233 subjects receiving standard care, 22 developed ACS. A threshold level of sPLA2 ≥ 48 ng/ml gave optimal sensitivity (73%), specificity (71%) and accuracy (71%), but a positive predictive value of only 24%. The predictive value of sPLA2 was improved in adults and patients with chest or back pain, lower haemoglobin concentration and higher white blood cell counts, and in those receiving less than two-thirds maintenance fluids. The hurdles identified in PROACTIVE should facilitate design of a larger, definitive, phase 3 randomized controlled trial.

Inpatient management of sickle cell pain: A ‘snapshot’ of current practice

The Sickle Cell Disease Clinical Research Network (SCDCRN) designed the PROACTIVE Feasibility Study (ClinicalTrials.gov NCT00951808) to determine whether elevated serum levels of secretory phospholipase A2 (sPLA2) during hospitalization for pain would permit preemptive therapy of sickle cell acute chest syndrome (ACS) by blood transfusion. While PROACTIVE was not designed to assess pain management and was terminated early due to inadequate patient accrual, collection of clinical data allowed a "snapshot" of current care by expert providers. Nearly half the patients admitted for pain were taking hydroxyurea; hydroxyurea did not affect length of stay. Providers commonly administered parenteral opioid analgesia, usually morphine or hydromorphone, to adults and children, generally by patient-controlled analgesia (PCA). Adult providers were more likely to prescribe hydromorphone and did so at substantially higher morphine equivalent doses than were given to adults receiving morphine; the latter received doses similar to children who received either medication. All subjects treated with PCA received higher daily doses of opioids than those treated by time-contingent dosing. Physicians often restricted intravenous fluids to less than a maintenance rate and underutilized incentive spirometry, which reduces ACS in patients hospitalized for pain.

A-002 (Varespladib), a phospholipase A2inhibitor, reduces atherosclerosis in guinea pigs

BackgroundThe association of elevated serum levels of secretory phospholipase A2 (sPLA2) in patients with cardiovascular disease and their presence in atherosclerotic lesions suggest the participation of sPLA2 enzymes in this disease. The presence of more advanced atherosclerotic lesions in mice that overexpress sPLA2 enzymes suggest their involvement in the atherosclerotic process. Therefore, the sPLA2 family of enzymes could provide reasonable targets for the prevention and treatment of atherosclerosis. Thus, A-002 (varespladib), an inhibitor of sPLA2enzymes, is proposed to modulate the development of atherosclerosis.MethodsTwenty-four guinea pigs were fed a high saturated fat, high cholesterol diet (0.25%) for twelve weeks. Animals were treated daily with A-002 (n = 12) or vehicle (10% aqueous acacia; n = 12) by oral gavage. After twelve weeks, animals were sacrificed and plasma, heart and aorta were collected. Plasma lipids were measured by enzymatic methods, lipoprotein particles size by nuclear magnetic resonance, aortic cytokines by a colorimetric method, and aortic sinus by histological analyses.ResultsPlasma total cholesterol, HDL cholesterol and triglycerides were not different among groups. However, the levels of inflammatory cytokines interleukin (IL)-10, IL-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were significantly reduced in the treatment group. This group also had a significant 27% reduction in cholesterol accumulation in aorta compared with placebo group. Morphological analysis of aortic sinus revealed that the group treated with A-002 reduced atherosclerotic lesions by 24%.ConclusionThe use of A-002 may have a beneficial effect in preventing diet-induced atherosclerosis in guinea pigs.

Aldosterone Promotes Inflammation and Endothelial Cell-Sickle Cell Adhesion: Potential for a New Therapeutic Target in Sickle Cell Disease

Sickle cell disease is fundamentally an inflammatory state, and endothelial activation and dysfunction have significant roles in the pathophysiology of this disease. In the last decade, research in the cardiovascular field has proven that the hormone aldosterone, canonically viewed as a regulator of renal electrolyte handling and blood pressure, also has direct, pro-inflammatory effects on the vascular endothelium that are independent of its classical effects. Excessive aldosterone is now known to cause microvascular damage, vascular inflammation, oxidative stress and endothelial dysfunction although the molecular mechanisms remain poorly understood (Brown, Hypertension 2008). In addition, aldosterone decreases endothelial cell production of nitric oxide and upregulates VCAM-1 and ICAM-1 production, leading to increased leukocyte-endothelial cell adhesion (Oberleithner, PNAS, 2007; Krug, Hypertension 2007). In animal models, aldosterone-mediated vascular injury in the brain, heart, and kidneys leads to stroke, myocardial injury, and renal damage (Marney, Clin Sci 2007). In addition, several large clinical trials have shown that aldosterone-antagonizing medications decrease mortality in patients with renal and heart failure, due in part to the blocking of the inflammatory vascular effects of this hormone (Pitt, N Engl J Med, 2003). Although the vascular effects of aldosterone are similar to those that occur in sickle cell disease, no published studies to date have investigated the possible interactions between aldosterone and sickle cell disease. Furthermore, the efficacy of aldosterone-antagonists as a potential therapy/prophylaxis for sickle cell complications has not been evaluated.We found that patients with Hemoglobin SS (n=21) have abnormally elevated aldosterone plasma levels, as measured with ELISA, that range from 1.5–40 times (median: 8.6 times) higher than normal levels, similar in range to those of patients with heart failure (Struthers, Eur J of Heart Failure 2004). In addition, aldosterone levels in sickle cell patients positively correlated with secretory phospholipase A2 levels (R=0.43, p<0.05), a known biomarker for predicting acute chest syndrome. To determine how aldosterone affects endothelialsickle cell adhesion, we exposed human umbilical vein endothelial cells (HUVECs) and sickle erythrocytes and leukocytes isolated from patient samples to varying physiologic concentrations (1.0–100 nM) of aldosterone ex vivo for 2 hours and then utilized static and dynamic flow adhesion assays. We found that aldosterone increases sickle erythrocyte (but not normal erythrocytes), neutrophil and mononuclear cell (monocytes + lymphocytes) adhesion to endothelial cells in a dose-dependent manner (compared to controls, p<0.05 for all concentrations between 1–10 nM, p<0.001 for all concentrations >10nM) in static conditions. Compared to controls, endothelial-sickle blood cell adhesion increased up to 100 times with aldosterone exposure. Similarly, under physiologic flow conditions (shear stress: 1 dyne/cm2), endothelial cell exposure to aldosterone increased capture of sickle erythrocytes and leukocytes in a dose dependent manner (compared to controls, p<0.05 for all concentrations >10 nM). Furthermore, measurements with atomic force microscopy (AFM), a highly sensitive tool used to measure and track cell adhesion and deformability at the single cell level, revealed that the adhesive force between single sickle cell erythrocytes and HUVECs increases over time with aldosterone exposure. With the addition of spironolactone, an aldosterone antagonist, all adhesive interactions decreased to near baseline levels/controls (p>0.3 for all comparisons with baseline levels/controls) as measured with static and dynamic flow adhesion assays and AFM.To investigate the underlying mechanisms of these phenomena, fluorescence imaging revealed increased reactive oxygen species production and expression of VCAM-1 and ICAM-1 in HUVECs exposed to aldosterone for only 2 hr when compared to controls. Aldosterone exposure did not affect sickle erythrocyte or leukocyte deformability as measured with ektacytometry and AFM, respectively. Taken together, these results suggest that aldosterone may play an important role in sickle cell vasculopathy and the high levels of this hormone may provide an effective therapeutic target for this disease.

EFFECTS OF PREDNISOLONE ON THE SYSTEMIC RELEASE OF MEDIATORS OF CELL-MEDIATED CYTOTOXICITY DURING HUMAN ENDOTOXEMIA

Corticosteroids are widely used for the suppression of cell-mediated cytoxicity. This process is mediated by natural killer cells and cytotoxic T lymphocytes, and their activation can be monitored by levels of the chemokines CXCL9 and CXCL10, the degranulation product granzymes A and B, and by levels of secretory phospholipase A2. The current study aimed to determine the effects of increasing doses of prednisolone on the release of these mediators in healthy humans exposed to LPS. Therefore, 32 healthy men received prednisolone orally at doses of 0, 3, 10, or 30 mg (n = 8 per group) at 2 h before intravenous injection of Escherichia coil LPS (4 ng/kg). Prednisolone dose-dependently attenuated the LPS-induced rises in the plasma concentrations of the chemokines CXCL9 and CXCL10, as well as of granzymes A and B levels. CXCL10 and granzyme B release were most sensitive to prednisolone, with a significant inhibition already achieved at the lowest prednisolone dose (3 mg). The levels of secretory phospholipase A2 were increased after LPS administration but were not significantly affected by prednisolone. This study demonstrates that prednisolone differentially inhibits the systemic release of mediators involved in cell-mediated cytotoxicity in humans in vivo.

Secretory Phospholipase A2 Levels in Patients with Sickle Cell Disease and Acute Chest Syndrome

In a multicenter study (eight centers), we determined secretory phospholipase A2 (sPLA2) levels in patients with sickle cell disease and acute chest syndrome (ACS). The diagnosis of ACS was made according to established criteria. The sPLA2 levels were determined in blood samples collected at baseline (time of diagnosis) and serially thereafter up to day 22–35 follow-up visits. Thirty-four of 43 (80%) patients with ACS had enzyme levels ≥1.00 AU at baseline. The enzyme levels decreased significantly on Days 2 through Days 25–35 after baseline. Nine of 43 (20%) patients had baseline sPLA2 values of <1.00 AU with six of them never exceeding 1.00 AU at any point in time during follow-up. The data indicate that the reliability of sPLA2 for predicting the development of ACS is not perfect (100%) as was previously reported but occurs in about 80% of the patients.

Serum Biomarkers for Identifying Acute Chest Syndrome Among Patients Who Have Sickle Cell Disease and Present to the Emergency Department

To compare the accuracy of biomarkers for identifying acute chest syndrome (ACS) in patients with sickle cell disease presenting to a pediatric emergency department (ED). We conducted a 13-month-long (2002-2003) cohort study with nested case-control in patients with sickle cell disease presenting to the pediatric ED with vaso-occlusive crises or fever in which we compared levels of secretory phospholipase A2 (sPLA2), endothelin-1, interleukin-6 (IL-6), and peripheral white blood cell count (WBC) in cases that were complicated by ACS and in control subjects with uncomplicated illnesses. For diagnosis, a test was considered to be accurate when the area under its receiver operator characteristic curve (AUC) was >0.70. Laboratory tests with AUC values > or =0.70 were entered into a binary recursive partitioning model for diagnosis. For the period of study, samples from 72 visits were obtained from 51 patients who presented with vaso-occlusive crises (range: 1-4 visits per patient; 15 were enrolled more than once). ACS complicated 19 of 72 visits (26%, 95% confidence interval: 17%-38%). At an AUC value of 0.79, only the sPLA2 test was accurate for diagnosing ACS. AUC values for peripheral WBC, endothelin-1, and IL-6 were 0.68, 0.51, and 0.52, respectively. Binary recursive partitioning retained only sPLA2 at a cutoff of 13.7 ng/mL to be accurate for diagnosis. This cutoff had a sensitivity of 74% (14 of 19), a specificity of 87% (46 of 53), a positive likelihood ratio of 5.6, and a negative likelihood ratio of 0.18. Secretory phospholipase A2 but not endothelin-1, IL-6, or WBC is an accurate test for identifying present or incipient ACS in young patients who present to the ED with sickle cell pain crises.

Secretory phospholipase A2 attacks erythrocytes with externalized phosphatidylserine

Introduction. Membrane phospholipids are distributed asymmetrically in normal cells, with phosphatidylserine (PS) confined to the inner monolayer. Externalization of PS is a marker for apoptosis and follows ischemia-reperfusion injury. Circulating levels of secretory phospholipase A2 (sPLA2) are elevated in sepsis, the acute respiratory distress syndrome, and following trauma, pointing at an active role of the enzyme in disease-associated tissue destruction. PLA2 hydrolyzes membrane phospholipids and is an early and rate-limiting step in the production of powerful inflammatory mediators. We hypothesized that (1) normal cells are not susceptible to sPLA2, but that (2) erythrocytes with externalized PS are hemolyzed by sPLA2 at clinically relevant concentrations, (3) binding the externalized PS would protect the cells from sPLA2-induced lysis, and also that (4) inhibition of PLA2 would protect cells with externalized PS from hemolysis. Methods. Erythrocytes were experimentally induced to expose PS. Normal erythrocytes and PS-exposing erythrocytes were exposed to sPLA2, and vulnerability was measured by flow cytometry and hemolysis. Annexin V, and its dimmer, DiAnnexin V, were tested as cytoprotective agents given their affinity for binding externalized PS. Finally, PX-18 was tested for its ability to inhibit enzymatic activity of sPLA2 and protect the PS-exposing cells. Results. Normal erythrocytes are not susceptible to sPLA2, however, sPLA2 hemolyzes PS-exposing erythrocytes at clinically relevant concentrations in a dose- and time-dependent fashion. Increase in hemolysis was inversely related to removal of PS-exposing cells, indicating that PLA2 targets externalized PS as a marker for cytotoxicity. Annexin V and Di-Annexin V effectively prevented PLA2-induced hemolysis at 7 nmol per 5 × 10 7 cells, confirming that PLA2 requires externalization and recognition of PS for cytotoxicity. At 10 μM, PX-18 reduced hemolysis of PS-exposing cells to baseline levels, indicating that sPLA2 inhibition also protects PS-exposing cells. Conclusion. Secretory PLA2 effectively hemolyzes PS-exposing erythrocytes. Cellular protection can be achieved both by binding PS and by inhibiting sPLA2 activity. These inhibitors may have therapeutic efficacy in inflammatory diseases.

Inflammatory mediators in dengue virus infection in children: interleukin-6 and its relation to C-reactive protein and secretory phospholipase A2.

To assess the potential role of interleukin-6 (IL-6) in the pathogenesis of dengue virus infection, levels of this cytokine were measured in children with dengue virus infection on admission to the hospital. As presumed surrogate markers of IL-6, C-reactive protein (CRP) and secretory phospholipase A2 (sPLA2) were measured. Three groups were studied: 33 apparently healthy children as negative controls, 11 children with bacterial infections as positive controls, and 186 children with serologically documented dengue virus infection. One-hundred and fifteen patients had dengue fever (DF) and 71 had dengue hemorrhagic fever (DHF). Compared with healthy controls, dengue shock syndrome (DSS) patients had significantly higher levels of IL-6 on admission (P < 0.05), comparable with those in positive controls. Dengue patients with shock had significantly higher levels of IL-6 than normotensive patients (P < 0.001) and higher levels of IL-6 were associated with a higher incidence of ascites. C-reactive protein concentrations in dengue patients and in healthy children were not different, but lower than in children with bacterial infections (P = 0.008). Secretory phospholipase A2 levels were higher in dengue patients than in apparently healthy children (P < or = 0.05) and similar to those in children with bacterial infection. Dengue shock syndrome patients had significantly higher sPLA2 concentrations than normotensive patients (P = 0.02). These data indicate that IL-6 and sPLA2 may have a pathogenetic role only in the most severe forms of dengue virus infection.

Secretory phospholipase A2 cleavage of intravasated bone marrow primes human neutrophils.

Recent clinical reports suggest that early femoral intramedullary rod (IMR) fixation in patients with multiple injuries increases the risk of adult respiratory distress syndrome (ARDS). We have shown that lipid-mediated neutrophil (PMN) priming and elevated circulating levels of secretory phospholipase A2 (sPLA2) within the first 24 hours after injury correlate with the development of ARDS. We thus hypothesized that circulating lipid products, generated by sPLA2 cleavage of intravasated bone marrow, prime PMNs for enhanced superoxide anion (O2-) production. Isolated PMNs from healthy volunteers were incubated for 5 minutes with buffer or sPLA2-lysed bone marrow (100 U/mL) collected from trauma patients. After formyl-methionyleucylphenylalanine (fMLP) activation, O2- production was quantified by the superoxide dismutase-inhibitable reduction of cytochrome c. Blood samples were also drawn from five injured patients before and 24 hours after femoral IMR fixation. PMNs were isolated and assessed for in vivo priming. PMNs incubated with sPLA2-lysed bone marrow were primed for more than 3.5 times greater fMLP-induced O2- production. Furthermore, in patients with femoral fractures, PMN O2- release in response to fMLP after IMR fixation was more than 2.5 times higher than before fixation. Collectively, the findings suggest that bone marrow released from acute fracture sites may become a lipid substrate for the elevated sPLA2 levels found in injured patients. The resultant priming of PMNs may thus render the injured patient at risk for ARDS. Although clearly hypothetical at present, we submit that these observations warrant further investigation because of their clinical implications.

Complement activation in patients with sepsis is in part mediated by C-reactive protein.

The involvement of C-reactive protein (CRP) in the activation of complement in patients with sepsis was investigated. In 104 patients with infections of varying severity, circulating levels of CRP-complement complexes, which are specific indicators for CRP-mediated complement activation, were assessed. Complement-CRP complexes were increased in almost all patients and correlated significantly with levels of C3a (r = .59; P < .001) and C-reactive protein (r = .76; P < .001). In addition, they correlated with levels of secretory phospholipase A2 (r = .59; P < .001). Levels of complement-CRP complexes in patients with a pneumococcal type of infection were similar to those in patients with other types of infections. Complement-CRP complexes were significantly higher in patients with shock (P = .01) and in patients who died (P = .03). These results demonstrate that part of the complement activation in patients with sepsis is independent from a direct interaction with microorganisms but rather results from an endogenous mechanism involving CRP.