Role of lipoprotein-associated phospholipase A2 in atherosclerosis and its potential as a therapeutic target.

Despite substantial progress in preventing adverse cardiovascular events with current therapeutic strategies, there remains an extensive residual risk of clinical events, particularly in high-risk patients. Because of the evidence implicating inflammation in the pathogenesis of atherosclerosis, identifying and targeting inflammatory pathways could help further reduce cardiovascular risk. There has been controversy regarding the role of lipoprotein-associated phospholipase A2 (Lp-PLA2) in atherosclerosis, partly because of the lack of simple animal models with a human-like pattern of Lp-PLA2 lipoprotein distribution. However, accumulating evidence from pathology, biology and epidemiology studies favors a pro-atherogenic rather than an atheroprotective role for the enzyme. In particular, Lp-PLA2 might play an important role in plaque vulnerability. As a result, additional studies are warranted to determine whether Lp-PLA2 inhibition improves plaque stability and ultimately clinical outcomes for high-risk patients.

Toll-like receptor ligands as adjuvants in allergen-specific immunotherapy.

BACKGROUND: Allergen-specific immunotherapy (SIT) leads to long-term amelioration of T-helper type 2 (Th2)-mediated allergic symptoms and is therefore recommended as a first line therapy for allergies. The major disadvantage of SIT is its low efficiency, requiring treatment over years. OBJECTIVE: In this study, we evaluated the potential of Toll-like receptor (TLR) ligands to facilitate Th1-type immune responses. METHODS: The immunogenicity and therapeutic potential of the major bee venom allergen phospholipase A2 (PLA2) combined with various TLR ligands were tested in mice and compared with immune responses induced by conventional aluminium-based preparations. RESULTS: Regarding total IgG against PLA2, TLR2/4-binding lipopolysaccharide and TLR3-binding polyriboinosinic polyribocytidylic (PolyI:C) were the superior adjuvants for prophylactic vaccination. However, TLR9-binding phosphorothioate-modified cytosine-guanosine-rich oligonucleotide (CpG), TLR-3-binding PolyI:C, and TLR2/6-binding peptidoglycan skewed the immune responses more towards IgG2a isotype and Th1 cytokines. Furthermore, in a therapeutic approach, CpG, PolyI:C and TLR7/8-binding 3M003 had immune modulating properties as they suppressed established IgE titres. CONCLUSION: The potential of TLR ligands to adjuvate the immunogenicity of bee venom PLA2 and to skew the Th1-Th2 balance proved very heterogeneous. With respect to SIT, CpG, PolyI:C, and 3M003 were very promising. Hence, TLR ligands should be considered as adjuvants or immune modulators in SIT in human as to improve its efficiency regarding the Th1-Th2 balance of the immune response with a likely effect on therapy duration.

Para-Bromophenacyl bromide alleviates airway hyperresponsiveness and modulates cytokines, IgE and eosinophil levels in ovalbumin-sensitized and -challenged mice.

Airway hyperresponsiveness, airway eosinophilia and increased IgE levels in serum are the important characteristic features of asthma. We evaluated the potential of para-Bromophenacyl bromide (PBPB), a known phospholipase A(2) inhibitor, on allergen-induced airway hyperresponsiveness in a mouse model. We sensitized and challenged mice with ovalbumin (OVA) to develop airway hyperresponsiveness as assessed by airway constriction and airway hyperreactivity (AHR) to methacholine (MCh) induced by allergen. The mice were orally treated with PBPB (0.1, 1 and 10 mg/kg) during or after OVA-sensitization and OVA-challenge to evaluate its protective or reversal effect on airway constriction and AHR to MCh. Determination of OVA-induced airway constriction and AHR to MCh were performed by measuring specific airway conductance (SGaw) using non-invasive dual-chamber whole body-plethysmography. We observed that PBPB (1 mg/kg) significantly reduced OVA-induced airway constriction and AHR to MCh (p<0.01). PBPB (1 mg/kg) treatment significantly inhibited PLA(2) activity in the BAL fluid. Cytokine analysis of the BAL fluid revealed that PBPB caused an increase in interferon-gamma (IFN-gamma) (p<0.02) and a decrease in interleukin-4 (IL-4) (p<0.05) and interleukin-5 (IL-5) (p<0.05) levels. The OVA-specific serum IgE levels (p<0.01) and the BAL eosinophils (p<0.001) were also reduced significantly. Thus, PBPB is capable of modulating allergen induced cytokine levels and serum IgE levels, and alleviating allergen induced airway hyperresponsiveness and eosinophils in mice. These data suggest that PBPB could be useful in the development of novel agents for the treatment of allergen induced airway hyperresponsiveness.

Immobilization of phospholipase A2 on porous glass and its application for lowering serum cholesterol concentration.

Phospholipase A2 (PLA2; EC 3.1.1.4) is a lipolytic enzyme that hydrolysis the ester bond in sn-2 position of phospholipids. In this work, the PLA2 from hog pancreas was covalently coupled to porous glass. The properties of free and immobilized enzyme were also investigated and compared. The optimum pH and temperature were found as 8.5 and 50 degrees C, respectively for both free and immobilized enzyme. The immobilized enzyme had good properties that potential for medical application is considerable. Its use in lowering plasma cholesterol concentrations in blood samples was also demonstrated.

Effects of recombinant platelet activating factor acetylhydrolase in obstructive jaundice.

BACKGROUND/AIMS: The aim of this study was to investigate effects of recombinant PAH (platelet activating factor acetylhydrolase), on tissue damage and on antioxidant response. METHODOLOGY: A total of 30 Wistar-Albino type rats were used in this PAH treatment, and the subjects were divided into 3 groups namely sham, ligation and PAH treatment groups, each containing 10 rats. In PAH treatment and ligation groups, laparotomy was made; common bile duct was ligated and incised. Following ligation, blood and liver tissue were taken. In the sham group, the common bile duct was turned but no other procedure was applied. The subjects in the ligation group were given intraperitoneal placebo on the 2nd to 6th days; and those in the PAH treatment group were applied PAH at a 5-mg/kg dose. Blood and liver tissue were taken on postoperative day 7. The parameters examined in the blood sample included, liver function tests, superoxide dismutase, glutathione peroxidase, tumor necrosis factor-alpha and interleukin-6. In liver tissue, histopathologic examination was made. RESULTS: Levels of AST, ALT, GGT, ALP, tumor necrosis factor-alpha and interleukin-6 were significantly higher than the levels in the sham group. These parameters, measured on the 7th day in the PAH treatment group were found to be significantly lower than the ligation group. Portal inflammation in the PAH given group was significantly lower than the ligation group. CONCLUSIONS: Administration of PAH in experimental jaundice has produced improvement in liver functions, significant reductions in serum GGT and ALP, tumor necrosis factor-alpha and interleukin-6, and in liver damage whereas it has brought about an increase in the levels of antioxidant enzymes.

Recombinant platelet-activating factor acetylhydrolase to prevent acute respiratory distress syndrome and mortality in severe sepsis: Phase IIb, multicenter, randomized, placebo-controlled, clinical trial.

OBJECTIVE: Platelet-activating factor (PAF) is a potent proinflammatory mediator implicated in the pathogenesis of both severe sepsis and acute respiratory distress syndrome. One of the regulatory pathways for PAF involves degradation to the inactive metabolite lyso-PAF by the enzyme PAF acetylhydrolase (PAF-AH). Because reduced concentrations of the natural form of PAF-AH have been reported in septic patients, the present study was conducted to determine whether treatment with recombinant human PAF-AH (rPAF-AH, Pafase) was safe when administered after the onset of severe sepsis and whether it decreases the prevalence of acute respiratory distress syndrome and 28-day all-cause mortality. DESIGN: A prospective, randomized, double-blind, placebo-controlled, multicenter trial. SETTING: Thirty-three medical and surgical intensive care units located in the United States. PATIENTS: A total of 127 patients with severe sepsis, but without established acute respiratory distress syndrome, were enrolled in the study. Randomization occurred within 12 hrs of the onset of severe sepsis. Patients then received 1.0 mg/kg rPAF-AH (n = 45), 5.0 mg/kg rPAF-AH (n = 39), or placebo (n = 43) administered intravenously, once daily, for five consecutive days. MEASUREMENTS AND MAIN RESULTS: Demographic and baseline clinical characteristics of the three treatment groups were similar, except for a significantly higher prevalence of respiratory tract infections as the cause of severe sepsis in patients treated with 1.0 mg/kg rPAF-AH. There were no treatment-related deaths, and the overall prevalence of adverse events was similar among rPAF-AH-treated and placebo-treated patients. There were no significant differences in the prevalence of acute respiratory distress syndrome among the three treatment groups. However, 28-day all-cause mortality was 21% in the 1.0 mg/kg rPAF-AH group, 28% in the 5.0 mg/kg rPAF-AH group, and 44% in the placebo group (overall chi-square p =.07; 1.0 mg/kg rPAF-AH vs. placebo, p =.03). A trend toward reduced multiple organ dysfunction also was observed in the 1.0 mg/kg rPAF-AH group compared with the placebo group (p =.11). CONCLUSION: The results from this study indicate that rPAF-AH was well tolerated and should be pursued as a potential new treatment to decrease mortality in patients with severe sepsis.

Inducing tolerance by intranasal administration of long peptides in naive and primed CBA/J mice.

To assess the capacity of a peptide-based immunotherapy to induce systemic tolerance via the nasal route, we designed three long overlapping peptides of 44-60 aa covering the entire sequence of phospholipase A2 (PLA2), a major bee venom allergen. Both prophylactic and therapeutic intranasal administrations of long peptides to PLA2-hypersensitive CBA/J mice induced specific T cell tolerance to the native allergen. In prophylactic conditions, this tolerance was marked by a suppression of subsequent specific IgE response, whereas the therapeutic approach in presensitized mice induced a more than 60% decrease in PLA2-specific IgE. This decline was associated with a shift in the cytokine response toward a Th1 profile, as demonstrated by decreased PLA2-specific IgG1 and enhanced IgG2a levels, and by a decline in the specific IL-4/IFN-gamma ratios. T cell transfer from long peptide-tolerized mice to naive animals abrogated the expected anti-PLA2 IgE and IgG1 Ab response, as well as specific T cell proliferation, but enhanced specific IgG2a response upon sensitization with PLA2. These events were strongly suggestive of a clonal anergy affecting more profoundly Th2 than the Th1 subsets. In conclusion, these results demonstrate that allergen-derived long peptides delivered via the nasal mucosa may offer an alternative to immunotherapy with native allergens without the inherent risk of systemic anaphylactic reactions. Moreover, long peptides, in contrast to immunotherapy strategies based on short peptides, have the advantage of covering all potential T cell epitopes, and may represent novel and safe tools for the therapy of allergic diseases.

Effect of recombinant human platelet-activating factor-acetylhydrolase on allergen-induced asthmatic responses.

Platelet-activating factor (PAF) is a potent lipid mediator associated with key features of asthma such as airway constriction, eosinophil infiltration, edema, and mucus accumulation. Regulation of PAF occurs primarily through degradation to biologically inactive lyso-PAF by cellular and secreted PAF-acetylhydrolase (PAF-AH). We evaluated the effect of human recombinant PAF-AH (rPAF-AH) on the dual phase asthmatic response in atopic subjects with mild asthma. Effects on induced sputum cell counts and differentials, eosinophilic cationic protein (ECP), and tryptase were evaluated. Enrolled subjects demonstrated a positive skin test and a dual asthmatic response to allergen inhalation challenge. Fourteen subjects received rPAF-AH (1 mg/kg) or placebo intravenously in a randomized, double blind, placebo-controlled, two-period crossover study. Treatment with rPAF-AH did not significantly reduce either the early- or late-asthmatic response. Sputum eosinophil cell counts were not affected by treatment, but there was a trend toward a reduction in sputum neutrophils. No significant change in sputum ECP and tryptase was observed between rPAF-AH and placebo. Thus, at the dose studied, the unique anti-PAF agent rPAF-AH demonstrated no significant effect on the allergen-induced dual-phase asthmatic response.

Effect of human plasma-type platelet-activating factor acetylhydrolase in two anaphylactic shock models.

The effect of human recombinant plasma-type platelet-activating factor (PAF) acetylhydrolase was examined in two murine models, PAF-induced death and active anaphylactic models. In the PAF-induced death model where mice were injected intravenously with 40 microg/kg of PAF, the administration of PAF acetylhydrolase reduced mortality in a dose-dependent manner, showing complete prevention of mortality at 1.0 mg/kg. Myeloperoxidase activity, the marker for neutrophils, was increased in the lung by PAF injection, and the PAF acetylhydrolase treatment significantly reversed the increase in myeloperoxidase activity. As in the PAF-induced model, PAF acetylhydrolase also decreased mortality in the active anaphylactic shock model where bovine serum albumin was injected intravenously to mice previously immunized with bovine serum albumin. The protective effect of PAF acetylhydrolase on mortality in this model was significant at 1.0 mg/kg. These results suggest that PAF is an important mediator in the lethality of systemic anaphylaxis, and that PAF acetylhydrolase may be beneficial for treatment of anaphylactic shock.

Vasoactive side effects of intravenous immunoglobulin preparations in a rat model and their treatment with recombinant platelet-activating factor acetylhydrolase.

Previously, we observed in a rat model that intravenous administration of intramuscular immunoglobulin preparations induced a long-lasting hypotension, which appeared to be associated with the presence of IgG polymers and dimers in the preparations, but unrelated to complement activation. We found evidence that this hypotensive response is mediated by platelet-activating factor (PAF) produced by macrophages. In this study, we compared the vasoactive effects of 16 intravenous immunoglobulin (IVIG) products from 10 different manufacturers, in anesthetized rats. Eight of the IVIG preparations showed no hypotensive effects (less than 15% decrease), whereas the other 8 had relatively strong effects (15%-50% decrease). The hypotensive effects correlated with the IgG dimer content of the preparations. Pretreatment of the rats with recombinant PAF acetylhydrolase completely prevented the hypotensive reaction on IVIG infusion, and administration after the onset of hypotension resulted in normalization of the blood pressure. We also observed PAF production on in vitro incubation of human neutrophils with IVIG, which could be blocked by anti-Fcgamma receptor antibodies. This indicates that induction of PAF generation may also occur in a human system. Our findings support the hypothesis that the clinical side effects of IVIG in patients may be caused by macrophage and neutrophil activation through interaction of IgG dimers with Fcgamma receptors. Because phagocyte activation may also lead to the release of other inflammatory mediators, recombinant PAF acetylhydrolase (rPAF-AH) provides a useful tool to determine whether PAF plays a role in the clinical side effects of IVIG. If so, rPAF-AH can be used for the treatment of those adverse reactions. (Blood. 2000;95:1856-1861)

Effect of recombinant platelet-activating factor acetylhydrolase on two models of experimental acute pancreatitis.

BACKGROUND & AIMS: Recent reports suggest that platelet-activating factor (PAF) plays a role in pancreatitis and pancreatitis-associated lung injury. In this study, the effects on these processes of termination of PAF action by recombinant PAF-acetylhydrolase (rPAF-AH) were investigated. METHODS: Rats were given rPAF-AH and then infused with a supramaximally stimulating dose of cerulein to induce mild pancreatitis. Opossums underwent biliopancreatic duct ligation to induce severe pancreatitis, and rPAF-AH administration was begun 2 days later. RESULTS: In mild, secretagogue-induced pancreatitis, rPAF-AH given before the cerulein reduced hyperamylasemia, acinar cell vacuolization, and pancreatic inflammation but did not alter pancreatic edema or pulmonary microvascular permeability. In severe, biliopancreatic duct ligation-induced pancreatitis, rPAF-AH delayed and reduced the extent of inflammation and acinar cell injury/necrosis and completely prevented lung injury even though the rPAF-AH administration was begun after the onset of pancreatitis. CONCLUSIONS: PAF plays an important role in the regulation of pancreatic injury but not pancreatic edema or increased pulmonary microvascular permeability in mild, secretagogue-induced pancreatitis. PAF plays a critical role in the regulation of progression of pancreatic injury and mediation of pancreatitis-associated lung injury in severe biliary pancreatitis. Amelioration of pancreatitis and prevention of pancreatitis-associated lung injury can be achieved with rPAF-AH even if treatment is begun after pancreatitis is established.

Successful immunotherapy with T-cell epitope peptides of bee venom phospholipase A2 induces specific T-cell anergy in patients allergic to bee venom.

BACKGROUND: Specific immunotherapy with honeybee venom (BV) is highly effective, but allergic side effects can occur during treatment. Immunotherapy with peptides containing major T-cell epitopes of the relevant allergen or allergens provides an alternative strategy without these problems. OBJECTIVE: The study investigates the immunologic mechanisms and clinical effects of immunotherapy with T-cell epitope peptides of the major BV allergen, the phospholipase A2 (PLA). METHODS: Five patients with IgE-mediated systemic allergic reactions to bee stings were treated with a mixture of three T-cell epitope peptides of PLA. Ten patients allergic to BV receiving whole BV immunotherapy served as control subjects. Increasing doses of the peptide mixture, up to a maintenance dose of 100 microg, were administered subcutaneously within 2 months. The patients were then challenged with PLA and 1 week later with a bee sting. The cellular and humoral immune response was measured in vitro. RESULTS: No allergic side effects were caused by the peptide immunotherapy, and all patients tolerated the challenge with PLA without systemic allergic symptoms. Two patients developed mild systemic allergic reactions after the bee sting challenge. After peptide immunotherapy, specific proliferative responses to PLA and the peptides in peripheral blood mononuclear cells were decreased in successfully treated patients. The production of TH2 and TH1 cytokines was inhibited, and B cells were not affected in their capacity to produce specific IgE and IgG4 antibodies. Their levels increased after allergen challenge in favor of IgG4. CONCLUSIONS: Immunotherapy of BV allergy with short T-cell peptides of PLA induces epitope-specific anergy in peripheral T cells and changes the specific isotype ratio in a fashion similar to that of conventional immunotherapy in successfully treated patients.

The central role of PAF in necrotizing enterocolitis development.

We have addressed two critical questions concerning NEC development. 1) Why is the neonatal intestine particularly susceptible to necrosis? and 2) Does PAF play a critical role in NEC development? We have found that intestinal tissue of the newborn has the highest specific activity for the acetyltransferase of the de novo pathway. It is suggested that the high capacity of this tissue to synthesize PAF may contribute to the fact that the necrosis of the newborn is more prevalent in this tissue. We have previously reported that dexamethasone lowers the activity of acetyl-CoA:lyso-PAF acetyltransferase in liver and spleen. This hormone also cause an increase in plasma PAF-acetylhydrolase activity and an increased secretion of PAF-acetylhydrolase by various macrophages. It would, therefore, appear that the beneficial effects of glucocorticoids on the prevention of NEC may be due to both increased inactivation of PAF as caused by the increase in PAF-acetylhydrolase as well as a decrease in PAF synthesis. We are presently investigating the effect of glucocorticoids on acetyl-CoA: alkyl-lyso-sn-glycero-3-phosphate acetyltransferase. The reported studies in which NEC was prevented by intravenous infusion of recombinant PAF-acetylhydrolase provides further documentation as to the importance of PAF in the development of NEC. The specific activity of PAF-acetylhydrolase required for protection by dexamethasone was similar. This finding would be suggestive of the fact that the mechanisms by which dexamethasone causes a complete protection against NEC may be mediated by increasing the plasma activity. Other mechanisms have been proposed such as facilitating the maturation of the small bowel. As discussed, other factors such as hypoxia, endotoxins, TNF alpha, and enternal feeding have been suggested to be contributing agents of NEC development. Many of these factors and procedures are known to increase in PAF. We have suggested a mechanism to explain the increase in PAF formation as caused LPS, TNF alpha, and interleukins being the inhibition of the secretion of PAF-AH by macrophages. Our previous reports on the mechanisms involve in the prevention of NEC by glucocorticoids and the reported findings that human recombinant PAF-acetylhydrolase can prevent NEC provide further support for a central role for PAF in NEC development. Furthermore, the presence of a high PAF biosynthetic activity in the neonatal intestine affords an explanation as to why this tissue is highly susceptible to this disease.

Highly efficient immobilization of phospholipase A2 and its biomedical applications.

A new method for the immobilization of phospholipase A2 (PLA2) has been developed to enhance the activity retention of immobilized PLA2. When PLA2 from the venom of Agkistrodon piscivorus piscivorus was pretreated with 4-nitro-3-octanoyl-oxybenzoic acid to acylate epsilon-amino groups of two lysines (Lys-7 and Lys-10) and the resulting acylated enzyme was covalently coupled onto carbonyldiimidazole-activated cross-linked agarose beads, the immobilized acylated enzyme showed high retention of activity toward various aggregated phospholipids. Toward densely packed phospholipid bilayers, such as large unilamellar vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, the immobilized acylated A. p. piscivorus PLA2 was 25-fold more active than the soluble A. p. piscivorus PLA2. The general applicability of our immobilization protocol was demonstrated by the high retention of activity achieved for the immobilized acylated PLA2 from the venom of Naja naja naja. In particular, full activity retention of the immobilized acylated A. p. piscivorus PLA2 toward phospholipids on the surface of human low density lipoproteins suggests its potential usefulness in a newly developed PLA2-based therapy for hypercholesterolemia.

Enzymatic modification of plasma low density lipoproteins in rabbits: a potential treatment for hypercholesterolemia.

Phospholipase A2 (EC 3.1.1.4) hydrolyzes certain phospholipids of low density lipoprotein (LDL). Plasma clearance of phospholipase A2-modified human LDL is up to 17 times faster than that of native human LDL in hypercholesterolemic rabbits. Modification of blood lipoproteins of hypercholesterolemic rabbits was performed by using an extracorporeal circuit containing immobilized phospholipase A2. After 90-min treatments, nearly 30% decreases in plasma cholesterol concentrations were observed. Erythrocyte, leukocyte, and platelet counts showed no net change after treatment. This technique does not require any fluid replacement or sorbent regeneration and offers a potential approach for lowering serum cholesterol and LDL levels.

Suppression of experimental autoimmune uveitis in guinea-pigs by inhibition of phospholipase A2.

Retinal S-antigen mixed with complete Freund's adjuvant was used to induce experimental autoimmune uveitis (EAU) in guinea-pigs. Guinea-pigs receiving no treatment, was compared with test animals which received topically and systemically administered KLM-583B, a phospholipase A2 (PLA2) inhibitor, or subcutaneous (sub. cut) and topical corticosteroid treatment, as well as a test group which received cyclosporin A suc. cut. The best clinical suppression of EAU was obtained in the group treated suc. cut with KLM-538B. Steroids also suppressed the inflammation in the eyes but was not as effective as KLM-583B or cyclosporine A. PLA 2 activity in the aqueous humour and the myeloperoxidase (MPO) levels measured from iris-ciliary body were significantly lower in the groups treated suc. cut. with KLM-583B or cyclosporin A. Guinea-pigs treated suc. cut. with KLM-583B and cyclosporin A had the lowest antiserum titres to retinal S-antigen.