Effects of Randomized Treatment With Icosapent Ethyl and a Mineral Oil Comparator on Interleukin-1ß, Interleukin-6, C-Reactive Protein, Oxidized Low-Density Lipoprotein Cholesterol, Homocysteine, Lipoprotein(a), and Lipoprotein-Associated Phospholipase A2: A REDUCE-IT Biomarker Substudy.

BACKGROUND: REDUCE-IT (Reduction of Cardiovascular Events With Icosapent Ethyl-Intervention Trial) reported a 25% relative risk reduction in major adverse cardiovascular events with use of icosapent ethyl compared with pharmaceutical grade mineral oil. The mechanisms underlying this benefit remain uncertain. We explored whether treatment allocation in REDUCE-IT might affect a series of biomarkers in pathways known to associate with atherosclerosis risk. METHODS: Serum levels of interleukin-1ß, interleukin-6, high-sensitivity C-reactive protein, oxidized low-density lipoprotein cholesterol, homocysteine, lipoprotein(a), and lipoprotein-associated phospholipase A2 (Lp-PLA2) were measured at baseline, at 12 months, at 24 months, and at the end-of-study visit among REDUCE-IT participants with triglyceride levels ≥135 mg/dL and <500 mg/dL who were randomly allocated to treatment with either 4 grams daily of icosapent ethyl or mineral oil used as a comparator. RESULTS: At baseline, median levels of each biomarker were similar in the 2 treatment groups. The levels of biomarkers associated with atherosclerosis increased over time among those allocated to mineral oil treatment; in this group at 12 months, the median percent increases from baseline were 1.5% for homocysteine, 2.2% for lipoprotein(a), 10.9% for oxidized low-density lipoprotein cholesterol, 16.2% for interleukin-6, 18.5% for lipoprotein-associated phospholipase A2, 21.9% for high-sensitivity C-reactive protein, and 28.9% for interleukin-1ß (all P values <0.001), with similar changes at 24 months. In the icosapent ethyl group, there were minimal changes in these biomarkers at 12 and 24 months. As such, at study conclusion, between-group treatment differences largely reflected increases in the mineral oil group with median percent differences of 2.4% for lipoprotein(a), 3.0% for homocysteine, 4.2% for oxidized low-density lipoprotein cholesterol, 19.8% for interleukin-6, 26.2% for Lp-PLA2, 38.5% for high-sensitivity C-reactive protein, and 48.7% for interleukin-1ß (all P values ≤0.007). These data are consistent with previous REDUCE-IT results in which the median percent change for low-density lipoprotein cholesterol at 12 months was -1.2% among those allocated to icosapent ethyl and 10.9% among those allocated to the mineral oil comparator. CONCLUSIONS: Among participants in REDUCE-IT, allocation to icosapent ethyl had minimal effects on a series of biomarkers associated with atherosclerotic disease, whereas levels increased among those allocated to mineral oil. The effect of these findings on interpretation of the overall risk reductions in clinical events observed within REDUCE-IT is uncertain. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT01492361.

Atherogenicity of low-density lipoproteins after switching from a protease inhibitor to dolutegravir: a substudy of the NEAT022 study.

BACKGROUND: The aim of this study was to investigate whether switching from a ritonavir-boosted PI-based regimen to a dolutegravir-based regimen improved the atherogenic properties of LDL particles in patients with HIV. METHODS: This was a substudy of the NEAT022 study (ClinicalTrials.gov NCT02098837). Adults with HIV with a Framingham score >10% or aged >50 years and being treated with a stable boosted PI-based regimen were randomized to either switch to dolutegravir or continue with boosted PI. At baseline and Week 48, we assessed atherogenic LDL properties: LDL particle size and phenotype (A, intermediate, B), oxidized LDL (ox-LDL) and lipoprotein-associated phospholipase A2 (Lp-PLA2) activity. RESULTS: Eighty-six participants (dolutegravir 44; PI 42) were included. Participants had a median (IQR) age of 54 (51-57) years and 79.1% were male. In the dolutegravir arm, after 48 weeks, we observed: (1) an increase in LDL size [median 1.65 Š(IQR -0.60 to 4.20); P = 0.007], correlated with the decrease in triglyceride concentration [Spearman correlation = -0.352 (P = 0.001)], with a corresponding decrease of subjects with atherogenic LDL phenotype B (36.4% to 20.5%; P = 0.039); (2) a decrease in Lp-PLA2 activity [median 1.39 µmol/min/mL (IQR -2.3 to 0.54); P = 0.002]; and (3) a decrease in ox-LDL [median 14 U/L (IQR -102 to 13); P = 0.006]. In the PI arm, none of these favourable lipid modifications was observed. CONCLUSIONS: Forty-eight weeks after switching from a PI-based to a dolutegravir-based regimen, patients with Framingham score >10% or aged >50 years showed improvement of several atherogenic lipid features, including LDL particle phenotype, ox-LDL and Lp-PLA2.

Re-Evaluating Biologic Pharmacotherapies that Target the Host Response during Sepsis.

Multiple organ dysfunction syndrome (MODS) caused by the systemic inflammatory response during sepsis is responsible for millions of deaths worldwide each year, and despite broad consensus concerning its pathophysiology, no specific or effective therapies exist. Recent efforts to treat and/or prevent MODS have included a variety of biologics, recombinant proteins targeting various components of the host response to the infection (e.g., inflammation, coagulation, etc.) Improvements in molecular biology and pharmaceutical engineering have enabled a wide range of utility for biologics to target various aspects of the systemic inflammatory response. The majority of clinical trials to date have failed to show clinical benefit, but some have demonstrated promising results in certain patient populations. In this review we summarize the underlying rationale and outcome of major clinical trials where biologics have been tested as a pharmacotherapy for MODS in sepsis. A brief description of the study design and overall outcome for each of the major trials are presented. Emphasis is placed on discussing targets and/or trials where promising results were observed. Post hoc analyses of trials where therapy demonstrated harm or additional risk to certain patient subgroups are highlighted, and details are provided about specific trials where more stringent inclusion/exclusion criteria are warranted.

Inhibiting PLA2G7 reverses the immunosuppressive function of intratumoral macrophages and augments immunotherapy response in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is an exceptionally immunosuppressive malignancy characterized by limited treatment options and a dismal prognosis. Macrophages constitute the primary and heterogeneous immune cell population within the HCC microenvironment. Our objective is to identify distinct subsets of macrophages implicated in the progression of HCC and their resistance to immunotherapy. METHODS: Intratumoral macrophage-specific marker genes were identified via single-cell RNA sequencing analyses. The clinical relevance of phospholipase A2 Group VII (PLA2G7), a pivotal enzyme in phospholipid metabolism, was assessed in patients with HCC through immunohistochemistry and immunofluorescence. Flow cytometry and an in vitro co-culture system were used to elucidate the specific role of PLA2G7 in macrophages. Orthotopic and subcutaneous HCC mouse models were employed to evaluate the potential of the PLA2G7 inhibitor in complementing immune checkpoint blockade (ICB) therapy. RESULTS: Single-cell RNA sequencing analyses disclosed predominant PLA2G7 expression in intratumoral macrophages within the HCC microenvironment. The macrophage-specific PLA2G7 was significantly correlated with poorer prognosis and immunotherapy resistance in patients with HCC. PLA2G7high macrophages represent a highly immunosuppressive subset and impede CD8 T-cell activation. Pharmacological inhibition of PLA2G7 by darapladib improved the therapeutic efficacy of anti-programmed cell death protein 1 antibodies in the HCC mouse models. CONCLUSIONS: Macrophage-specific PLA2G7 serves as a novel biomarker capable of prognosticating immunotherapy responsiveness and inhibiting PLA2G7 has the potential to enhance the efficacy of ICB therapy for HCC.

Biological Properties and Clinical Significance of Lipoprotein-Associated Phospholipase A2 in Ischemic Stroke.

Ischemic stroke, which occurs following blockage of the blood supply to the brain, is a leading cause of death worldwide. Its main cause is atherosclerosis, a disease of the arteries characterized by the deposition of plaques of fatty material on the inner artery walls. Multiple proteins involved in the inflammation response have been identified as diagnosing biomarkers of ischemic stroke. One of these is lipoprotein-associated phospholipase A2 (Lp-PLA2), an enzyme that can hydrolyze circulating oxidized phospholipids, generating proinflammatory lysophosphatidylcholine and promoting the development of atherosclerosis. In the last two decades, a number of studies have revealed that both the concentration and the activity of Lp-PLA2 are independent biomarkers of ischemic stroke. The US Food and Drug Administration (FDA) has approved two tests to determine Lp-PLA2 mass and activity for predicting stroke. In this review, we summarize the biological properties of Lp-PLA2, the detection sensitivity and limitations of Lp-PLA2 measurement, the clinical significance and association of Lp-PLA2 in ischemic stroke, and the prospects of therapeutic inhibition of Lp-PLA2 as an intervention and treatment.

Evaluation of recombinant platelet-activating factor acetylhydrolase for reducing the incidence and severity of post-ERCP acute pancreatitis.

BACKGROUND: Pancreatitis is the most common major complication of diagnostic and therapeutic ERCP. Platelet-activating factor (PAF) has been implicated in the pathophysiologic events associated with acute pancreatitis. Animal and human studies suggested that recombinant PAF acetylhydrolase (rPAF-AH) might ameliorate the severity of acute pancreatitis. OBJECTIVE: Our purpose was to determine whether prophylactic rPAF-AH administration reduces the frequency or severity of post-ERCP pancreatitis in high-risk patients. DESIGN: Randomized, multicenter, double-blind, placebo-controlled study. INTERVENTIONS: Patients received rPAF-AH at a dose of either 1 or 5 mg/kg or placebo. Patients were administered a single intravenous infusion over 10 minutes of study drug or placebo <1 hour before ERCP. MAIN OUTCOME MEASUREMENTS: Standardized criteria were used to diagnose and grade the severity of post-ERCP pancreatitis. Adverse events were prospectively recorded. RESULTS: A total of 600 patients were enrolled. There were no statistically significant differences among the treatment groups with respect to patient demographics, ERCP indications, and patient and procedure risk factors for post-ERCP pancreatitis with the following exceptions: the rPAF-AH 5 mg/kg group had significantly fewer patients younger than 40 years old and scheduled to undergo a therapeutic ERCP involving the pancreatic sphincter or duct. Post-ERCP pancreatitis occurred in 17.5%, 15.9%, and 19.6% of patients receiving rPAF-AH (1 mg/kg), rPAF-AH (5 mg/kg), and placebo, respectively (P = .59 for rPAF-AH 1 mg/kg vs placebo and P = .337 for rPAF-AH 5 mg/kg vs placebo). There was no statistically significant difference between the groups with regard to the severity of pancreatitis, frequency of amylase/lipase elevation more than 3 times normal, or abdominal pain. CONCLUSIONS: There was no apparent benefit of rPAF-AH treatment compared with placebo in reducing the incidence of post-ERCP pancreatitis in subjects at increased risk.

Plasma PAF-acetylhydrolase: an unfulfilled promise?

Plasma Platelet-activating-Factor (PAF)-acetylhydrolase (PAF-AH also named lipoprotein-PLA(2) or PLA(2)G7 gene) is secreted by macrophages, it degrades PAF and oxidation products of phosphatidylcholine produced upon LDL oxidation and/or oxidative stress, and thus is considered as a potentially anti-inflammatory enzyme. Cloning of PAF-AH has sustained tremendous promises towards the use of PAF-AH recombinant protein in clinical situations. The reason for that stems from the numerous animal models of inflammation, atherosclerosis or sepsis, where raising the levels of circulating PAF-AH either through recombinant protein infusion or through the adenoviral gene transfer showed to be beneficial. Unfortunately, neither in human asthma nor in sepsis the recombinant PAF-AH showed sufficient efficacy. One of the most challenging questions nowadays is as to whether PAF-AH is pro- or anti-atherogenic in humans, as PAF-AH may possess a dual pro- and anti-inflammatory role, depending on the concentration and the availability of potential substrates. It is equally possible that the plasma level of PAF-AH is a diagnostic marker of ongoing atherosclerosis.

Clinical aspects of plasma platelet-activating factor-acetylhydrolase.

Plasma platelet-activating factor (PAF)-acetylhydrolase (PAF-AH), which is characterized by tight association with plasma lipoproteins, degrades not only PAF but also phospholipids with oxidatively modified short fatty acyl chain esterified at the sn-2 position. Production and accumulation of these phospholipids are associated with the onset of inflammatory diseases and preventive role of this enzyme has been evidenced by many recent studies including prevalence of the genetic deficiency of the enzyme in the patients and therapeutic effects of treatment with recombinant protein or gene transfer. With respect to the atherosclerosis, however, it is not fully cleared whether this enzyme plays an anti-atherogenic role or pro-atherogenic role because plasma PAF-AH also might produce lysophosphatidylcholine (LysoPC) and oxidatively modified nonesterified fatty acids with potent pro-inflammatory and pro-atherogenic bioactivities. These dual roles of plasma PAF-AH might be regulated by the altered distribution of the enzyme between low density lipoprotein (LDL) and high density lipoprotein (HDL) particles because HDL-associated enzymes are considered to contribute to the protection of LDL from oxidative modification. This review focuses on the recent findings which address the role of this enzyme in the human diseases especially including asthma, septic shock and atherosclerosis.

Neuroprotective role of transgenic PAF-acetylhydrolase II in mouse models of focal cerebral ischemia.

BACKGROUND AND PURPOSE: Platelet-activating factor (PAF) and oxidized unsaturated free fatty acids have been postulated to aggravate neuronal damage in the postischemic brain. Type II PAF-acetylhydrolase (PAF-AH II) not only terminates signals by PAF by its PAF-hydrolyzing activity but also protects cells against oxidative stress. We examined whether PAF-AH II can rescue cerebral neurons against ischemic insults. METHODS: Transgenic mice overexpressing human PAF-AH II in neurons were generated and enzyme expressions were examined biochemically and histochemically. The mice were subjected to 60 minutes of transient middle cerebral artery occlusion followed by reperfusion for 24 hours. The infarction and apoptosis were estimated by TTC staining and fluorescence TUNEL staining, respectively. RESULTS: Overexpression of PAF-AH II was found in brains of transgenic mice by Western blot and enzymatic activity analyses. In immunohistochemistry, human PAF-AH II expression was found throughout the central nervous system, especially in neurons of neocortex, hippocampus, and basal ganglia. The neurological deficit scores, cerebral edema index, and relative infarction volume were all significantly (P<0.05) lower in transgenic mice (1.30+/-0.72, 1.12+/-0.04, and 14.0+/-7.7%, respectively) than in wild-type mice (2.56+/-0.93, 1.23+/-0.12, and 31.9+/-9.7%, respectively). Percentages of apoptotic cells were also significantly (P<0.001) lower in transgenic mice (cortex, 5.2+/-3.3%; hippocampus, 3.4+/-7.0%) than in wild-type mice (cortex, 41.1+/-16.9%; hippocampus, 58.9+/-15.3%). CONCLUSIONS: These results indicate that PAF-AH II exerts strong neuroprotective effects against ischemic injury and suggest a possibility for clinical use of this enzyme in cerebral ischemia.

The human asparaginase enzyme (ASPG) inhibits growth in leukemic cells.

The human protein ASPG is an enzyme with a putative antitumor activity. We generated in bacteria and then purified a recombinant GST-ASPG protein that we used to characterize the biochemical and cytotoxic properties of the human ASPG. We demonstrated that ASPG possesses asparaginase and PAF acetylhydrolase activities that depend on a critical threonine residue at position 19. Consistently, ASPG but not its T19A mutant showed cytotoxic activity in K562, NALM-6 and MOLT-4 leukemic cell lines but not in normal cells. Regarding the mechanism of action of ASPG, it was able to induce a significant apoptotic death in K562 cells. Taken together our data suggest that ASPG, combining different enzymatic activities, should be considered a promising anti-cancer agent for inhibiting the growth of leukemia cells.

Neuroprotection by platelet-activating factor acetylhydrolase in a mouse model of transient cerebral ischemia.

Neuronal damage after transient cerebral ischemia is exacerbated by signaling pathways involving activated platelet-activating factor (PAF) and ameliorated by PAF-acetylhydrolase (PAF-AH); but whether cerebral neurons can be rescued by human recombinant PAF-AH (rPAF-AH) remains unknown. Adult male mice underwent a 60 min middle cerebral artery occlusion (MCAO) and reperfusion for 24h. Then, the mice received intravenous tail injections with different drugs. Neurological behavioral function was evaluated by Bederson's test, and cerebral infarction volume was assessed with tetrazolium chloride (TTC) staining. mRNA and protein expression levels of matrix metalloproteinase-2 (MMP-2, collagenase-1), MMP-9 (gelatinase-B), and vascular endothelial growth factor (VEGF) were determined by quantitative real-time PCR (RT-PCR) and western blot analysis, respectively. Compared with the vehicle group, rPAF-AH significantly improved sensorimotor function (42%, P=0.0001). The volume of non-infarcted brain tissue was increased by the rPAF-AH treatment (16.3±4.6% vs. 46.0±10.3%, respectively). rPAF-AH significantly reduced mRNA and protein levels of MMP-2 and MMP-9, but increased the mRNA (P<0.001) and protein levels (P<0.01) of VEGF. These results demonstrate that rPAF-AH provides neuroprotection against ischemic injury. Neuroprotection might be induced not only by decrease in MMP-2 and MMP-9 expression, but also by increased VEGF expression.

Platelet-activating factor inactivator (rPAF-AH) enhances liver's recovery after paracetamol intoxication.

Platelet-activating factor (PAF) is a potent endogenous phospholipid modulator of diverse biological activities, including inflammation. The aim of this study was to investigate the effects of PAF inactivator, recombinant PAF acetylhydrolase (rPAF-AH) on post-paracetamol treatment functional outcome of the liver in the rat. Fifty male Wistar rats were divided into two groups: the control group received by gastric tube a toxic dose of paracetamol (3.5 g/kg body weight) and the rPAF-AH-treated group received the same dose of paracetamol followed by a dose of rPAF-AH (10 mg/kg body weight) intraperitoneally. The animals were sacrificed at 8, 16, 24, 32, and 40 hr after paracetamol treatment. APAP was found to cause acute hepatic injury, evident by alterations of biochemical (serum enzymes: ALT, AST, and ALP) and liver histopathological (degree of inflammation and apoptosis) indexes, which was followed by liver regeneration evident by three independent indexes ([(3)H]thymidine incorporation into hepatic DNA, liver thymidine kinase activity, and hepatocyte mitotic index). Hepatic levels of malondialdehyde (MDA) and serum cholesterol/HDL cholesterol fraction were also measured as parameters of oxidant-antioxidant balance. The positive effects of rPAF-AH were expressed by (1) a reduction of oxidative stress, (2) a large decrease in hepatic injury, and (3) a reduction of regenerating activity. These results suggest that PAF plays an important role in paracetamol-induced liver injury and regeneration. Furthermore, PAF inactivator enhances liver's recovery and attenuates the severity of experimental liver injury, providing important means of improving liver function following paracetamol intoxication.

Recombinant human platelet-activating factor acetylhydrolase for treatment of severe sepsis: results of a phase III, multicenter, randomized, double-blind, placebo-controlled, clinical trial.

OBJECTIVE: Platelet-activating factor (PAF) and structurally-related oxidized phospholipids are proinflammatory mediators in systemic inflammatory states such as severe sepsis. The enzyme platelet-activating factor acetylhydrolase (PAF-AH) rapidly degrades PAF and oxidized phospholipids into inactive metabolites. Reduced PAF-AH activity has been observed in patients with severe sepsis and may contribute to their systemic inflammatory response and organ dysfunction. A previous clinical trial with recombinant human PAF-AH (rPAF-AH, Pafase) suggested that this treatment may decrease 28-day all-cause mortality in patients with severe sepsis. The current study was undertaken to confirm this result. DESIGN: A prospective, randomized, double-blind, placebo-controlled, multicenter, international trial. SETTING: One hundred forty-six intensive care units from nine countries. PATIENTS: Approximately 2,522 patients were planned to be enrolled < or =12 hrs after the onset of severe sepsis. Eligible patients were randomized to receive either rPAF-AH 1.0 mg/kg or placebo administered intravenously once daily for five consecutive days. MEASUREMENTS AND MAIN RESULTS: The study was terminated based on the recommendation of an independent data and safety monitoring committee after the second of three planned interim analyses, and the enrollment of 1,425 patients. rPAF-AH treatment was well tolerated among the 1,261 patients included in the interim analysis (643 rPAF-AH and 618 placebo), but did not decrease 28-day all-cause mortality compared with placebo (25% for rPAF-AH vs. 24% for placebo; relative risk, 1.03; 95% confidence interval, 0.85-1.25; p =.80). There were no statistically significant differences between treatment groups in any of the secondary efficacy end points. The overall incidence of adverse events was similar among rPAF-AH and placebo-treated patients, and no rPAF-AH-treated patients developed antibodies to PAF-AH. CONCLUSIONS: rPAF-AH was well tolerated and not antigenic, but did not decrease 28-day all-cause mortality in patients with severe sepsis.

Beneficial effects of recombinant platelet-activating factor acetylhydrolase and BN 52021 on bacterial translocation in cerulein-induced pancreatitis.

BACKGROUND: Bacterial translocation (BT) has been suggested to be responsible for the high incidence of infections occurring after acute pancreatitis (AP). The aim of this study was to investigate the effects of the platelet-activating factor (PAF) inactivator, recombinant PAF-acetylhydrolase (rPAF-AH), and the PAF receptor antagonist, BN 52021, in AP. METHODS: Forty-eight male Wistar rats were divided into 4 groups: the sham group received saline intraperitoneally every hour for 6 h; the control group received cerulein 50 g/kg i.p. every hour for 6 h; the rPAF-AH group received AP plus rPAF-AH (5 mg/kg i.v. bolus), and the BN52021 group received AP plus BN 52021 (5 mg/kg i.v. bolus). The animals were sacrificed 12 h after the first cerulein injection. RESULTS: Supramaximal cerulein stimulation induced an increase in serum pancreatic enzymes, interleukin (IL)-6, pancreatic edema, and produced histologic evidence of AP. Compared with the control group, the addition of PAF receptor antagonists had a significant effect on serum pancreatic enzymes, pancreatic edema, and the histologic score of the pancreatitis. AP caused significant increases in BT in mesenteric lymph nodes (MLNs), pancreas, liver, spleen and blood. Compared with the control group, both rPAF-AH and BN 52021 decreased BT in the pancreas and blood. In addition, rPAF-AH decreased BT in the MLNs. We also found that PAF receptor antagonists suppressed the elevation in IL-6 levels. CONCLUSION: PAF antagonists attenuated the severity of experimental AP and reduced pancreatitis-induced BT to distant sites.