Up-regulated PLA2G10 in cancer impairs T cell infiltration to dampen immunity.

T cells are often absent from human cancer tissues during both spontaneously induced immunity and therapeutic immunotherapy, even in the presence of a functional T cell-recruiting chemokine system, suggesting the existence of T cell exclusion mechanisms that impair infiltration. Using a genome-wide in vitro screening platform, we identified a role for phospholipase A2 group 10 (PLA2G10) protein in T cell exclusion. PLA2G10 up-regulation is widespread in human cancers and is associated with poor T cell infiltration in tumor tissues. PLA2G10 overexpression in immunogenic mouse tumors excluded T cells from infiltration, resulting in resistance to anti-PD-1 immunotherapy. PLA2G10 can hydrolyze phospholipids into small lipid metabolites, thus inhibiting chemokine-mediated T cell mobility. Ablation of PLA2G10's enzymatic activity enhanced T cell infiltration and sensitized PLA2G10-overexpressing tumors to immunotherapies. Our study implicates a role for PLA2G10 in T cell exclusion from tumors and suggests a potential target for cancer immunotherapy.

Double positivity to bee and wasp venom: improved diagnostic procedure by recombinant allergen-based IgE testing and basophil activation test including data about cross-reactive carbohydrate determinants.

BACKGROUND: Specific IgE (sIgE) antibodies to both bee and wasp venom can be due to a sensitivity to both insect venoms or due to cross-reactive carbohydrate determinants (CCDs). OBJECTIVE: Investigating whether a basophil activation test (BAT) with both venoms as well as with bromelain and horseradish peroxidase (HRP) or recombinant allergen-based IgE testing can improve the diagnostic procedure. METHODS: Twenty-two Hymenoptera-venom allergic patients with sIgE antibodies to both bee and wasp venom were studied. sIgE antibodies to MUXF3 CCD, bromelain, HRP, rApi m 1, and rVes v 5 were determined, and a BAT (Flow2 CAST) with venom extracts, bromelain, and HRP was performed. Further recombinant allergen-based IgE testing was done by using an ELISA, if required. The reactivity of basophils was calculated from the insect venom concentration at half-maximum stimulation. RESULTS: Double positivity/double negativity/single positivity to rApi m 1 and rVes v 5 was seen in 12/1/9 patients. Further recombinant allergen-based IgE testing in the last ones revealed positive results to the other venom in all cases except one. BAT was double positive/double negative/single positive in 6/2/14 patients. Four patients with negative results in sIgE antibodies to CCDs had positive results in BAT. BAT with bromelain/HRP showed a sensitivity of 50%/81% and a specificity of 91%/90%. CONCLUSION: Component-resolved IgE testing elucidates the pattern of double positivity, showing a majority of true double sensitizations independent of CCD sensitization. BAT seems to add more information about the culprit insect even if the true clinical relevance of BAT is not completely determined because of ethical limitations on diagnostic sting challenges. BAT with HRP is a good method to determine sensitivity to CCDs.

Intracellular phospholipase A2 expression and location in human macrophages: influence of synthetic material surface chemistry.

Phospholipase A(2) (PLA(2)) enzymes participate in a potent inflammatory pathway through the liberation of arachidonic acid upon hydrolysis of membrane glycerophospholipids. The presence of implanted polycarbonate-urethane (PCNU) materials, used in several medical applications, has the ability to influence inflammatory responses of human macrophages that are recruited to a tissue-material interface; however, the specific inflammatory pathways that are activated upon macrophage attachment to PCNU are largely unknown. Previous studies suggested the participation of PLA(2) pathways in material degradation with the use of chemical inhibitors, such as aristolochic acid (ARIST), however not accurately defining the specific PLA(2) enzymes involved. The current study aimed to establish specific groups of PLA(2) involved in the macrophage foreign body response to PCNU. ARIST was assessed for specific effects on secretory PLA(2) (sPLA(2)) protein expression and non-specific effects on key proteins, beta-actin and monocyte-specific esterase, implicated in the macrophage attack on PCNU materials. Macrophage attachment to PCNU materials induced increased intracellular expression of cytosolic PLA(2) (cPLA(2)), but not sPLA(2), relative to tissue culture polystyrene (TCPS) as detected by immunoblot analysis, demonstrating an early and delayed stimulation during the time course of increased cPLA(2) protein expression. Laser scanning confocal microscopy images indicated a change in location of cPLA(2) in macrophages adherent to PCNU surfaces compared to TCPS. This study has illustrated changes in macrophage cPLA(2) expression in response to cell-attachment to PCNU surfaces, demonstrating that the macrophage foreign body response to biomaterials induces a potent inflammatory pathway, which may lead to tissue damage near the site of material implantation.

Immune regulatory functions of human beta-defensin-2 in odontoblast-like cells.

AIM: To investigate the effects of human beta-defensins on the expression of genes involved in the host immune response of the dental pulp. METHODOLOGY: Human odontoblast-like cells were cultured in Dulbecco's modified Eagle's medium. Cells were stimulated by recombinant human beta-defensins (rhBDs) up to 4 h. RNA was extracted followed by cDNA synthesis (oligo-(dT)-primer). Samples were analysed by real-time polymerase chain reaction (PCR) technology. Genes of interest were: human beta-defensin-1, -2, interleukin (IL)-6, IL-8, tumour necrosis factor-alpha, cyclooxygenase-2, leukotriene-A4-hydrolase, cytosolic phospholipase-A-2 (cPLA(2)), and dentine sialophosphoprotein. Gene expression of beta-actin served as internal standard for normalizing real-time PCR data. Two-way anova and the paired t-test were applied for comparison of the gene expression. RESULTS: In odontoblast-like cells rhBD-2 stimulation led to a down-regulation of the gene expression of hBD-1 (P < 0.05), whilst the mRNA expression of IL-6 (P < 0.05), IL-8 (P < 0.05) and cPLA(2) was increased in response to rhBD-2. CONCLUSION: The results of the present study suggest immune regulatory functions of human beta-defensin-2 in odontoblast-like cells.

Autoantibody titers against OxLDL are correlated with Achilles tendon thickness in patients with familial hypercholesterolemia.

Achilles tendon xanthomas are associated with increased cardiovascular risk in patients with familial hypercholesterolemia (FH). Oxidized low density lipoprotein (OxLDL), the antibodies against OxLDL, and the LDL-associated phospholipase A(2) (Lp-PLA(2)) may play important roles in atherogenesis. We investigated the possible association between plasma levels of OxLDL, Lp-PLA(2) activity, and autoantibody titers against various types of mildly OxLDL with Achilles tendon thickness (ATT). ATT was determined by sonography in 80 unrelated heterozygous FH patients. Three different types of mildly OxLDL were prepared: OxLDL(L), OxLDL(P), and OxLDL(D), at the end of the lag, propagation, and decomposition phases of oxidation, respectively. Similar types of OxLDL were also prepared after inactivation of the LDL-associated Lp-PLA(2). These types were denoted OxLDL(-)(L), OxLDL(-)(P), and OxLDL(-)(D). FH patients exhibited significantly higher plasma OxLDL levels and serum IgG titers against OxLDL(P) and OxLDL(D) compared with 40 normolipidemic apparently healthy controls. ATT values were positively correlated with autoantibody titers against OxLDL(P) and OxLDL(D); however, in multiple regression analysis, ATT was independently associated only with the autoantibody titers against OxLDL(D). We conclude that the IgG autoantibody titers against OxLDL(D) but not OxLDL or Lp-PLA(2) may play an important role in the pathogenesis of Achilles tendon xanthomas in FH patients.

Induction of interleukin-10 and suppressor of cytokine signalling-3 gene expression following peptide immunotherapy.

BACKGROUND: Allergen-derived (T cell epitope) peptides may be safer for immunotherapy than native allergen, as they do not cross-link immunoglobulin (Ig)E. However, HLA polymorphism results in multiple potential epitopes. Synthetic peptides of phospholipase (PL) A(2) were selected for a peptide vaccine, on the basis of binding affinity for commonly expressed HLA-DR molecules. OBJECTIVE: To evaluate treatment with an HLA-DR-based PLA(2) peptide vaccine in subjects with mild honeybee allergy in an open, controlled study. METHODS: Twelve volunteers with allergy to bee venom received nine intradermal injections of PLA(2) peptides, with six untreated subjects serving as controls. Outcome was assessed by the size of the late-phase cutaneous reaction to allergen, peripheral blood mononuclear cell (PBMC) proliferation, cytokine release, and expression of genes associated with immune regulation. RESULTS: Subjects receiving peptides showed a decrease in the magnitude of the late-phase cutaneous reaction to bee venom compared with controls (P=0.03). The proliferation of venom-stimulated PBMCs decreased in treated subjects compared with controls (P=0.01). Peptide treatment reduced the production of IL-13 by PLA(2)-stimulated PBMCs (P<0.01) and IFN-gamma (P<0.01), and increased the production of IL-10 (P=0.02). Transcription of the suppressor of cytokine signalling (Socs)3 gene was significantly increased following therapy. A transient, but modest, increase in allergen-specific IgG was also observed. CONCLUSION: HLA-DR-based T cell epitopes modify surrogate markers associated with successful immunotherapy and induction of immune regulation, supporting the concept that this form of treatment may be efficacious in human allergic disease.

Inhibition of platelet-activating factor biosynthesis by adenosine and histamine in human neutrophils: involvement of cPLA2alpha and reversal by lyso-PAF.

Leukotrienes (LT) and platelet-activating factor (PAF) are important lipid mediators of inflammation. We and others reported previously that autacoids such as adenosine, histamine, prostaglandin E2, and beta-adrenergic agents inhibit LT biosynthesis in activated human polymorphonuclear leukocytes (PMN). In this study, we demonstrate that CGS-21680 (a selective agonist of the adenosine A2A receptor) and histamine also potently inhibit PAF biosynthesis in agonist [formyl Met-Leu-Phe (fMLP)]- and thapsigargin-activated human PMN. The observed inhibitions of PAF biosynthesis were reversed effectively by exogenous 1-O-alkyl-lyso-sn-glyceryl-3-phosphocholine (lyso-PAF), suggesting that these effects of CGS-21680 and histamine implicate the blockade of cytosolic phospholipase A2alpha (cPLA2alpha) activity and lyso-PAF release and that the acetyl-coenzyme A/lyso-PAF acetyl transferase is not inhibited by the autacoids. Accordingly, the cPLA2alpha inhibitor pyrrophenone completely blocked PAF formation, and lyso-PAF similarly prevented this effect of pyrrophenone. The inhibitory effects of CGS-21680 and histamine on PAF biosynthesis were prevented by the protein kinase A inhibitor H-89, supporting roles for the Gs -coupled receptors A2A and H2, respectively, and cyclic adenosine monophosphate in the inhibitory mechanism. The fMLP-induced phosphorylations of p38 and extracellular signal-regulated kinase 1/2 were not altered significantly by the CGS-21680, indicating that inhibition of these kinases is not involved in the inhibitory effect of the adenosine A2A receptor ligand on LT and PAF biosynthesis. These data further emphasize the multiple and potent inhibitory effects of adenosine and histamine on leukocyte functions, in particular, on the biosynthesis of two classes of important lipid mediators and their putative regulatory roles in immune processes in health and diseases.

Impaired NADPH oxidase activity in Rac2-deficient murine neutrophils does not result from defective translocation of p47phox and p67phox and can be rescued by exogenous arachidonic acid.

Rac2 is a hematopoietic-specific Rho-GTPase that plays a stimulus-specific role in regulating reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and other functional responses in neutrophils. In this study, rac2-/- neutrophils were shown to have significantly decreased NADPH oxidase activity and actin remodeling in response to exogenous arachidonic acid (AA), as previously observed for phorbol 12-myristate 13-acetate (PMA) or formyl-Met-Leu-Phe (fMLP) as agonists. PMA-, fMLP-, or AA-induced translocation of p47phox and p67phox to the plasma membrane was not impaired in rac2-/- neutrophils. Combined stimulation of rac2-/- neutrophils with exogenous AA and PMA had a synergistic effect on NADPH oxidase activity, and superoxide production increased to a level that was at least as high as wild-type cells and had no effect on fMLP-elicited enzyme activity. Membrane translocation of p47phox and p67phox as well as Rac1 activation was not increased further by combined PMA and AA stimulation. Inhibitor studies were consistent with important roles for phorbol ester-activated protein kinase C (PKC) isoforms and an atypical isoform, PKCzeta, in superoxide production by wild-type and rac2-/- neutrophils stimulated with AA and PMA. In addition, PMA-stimulated release of AA and cytoplasmic phospholipase A2 expression in rac2-/- neutrophils were similar to wild-type, suggesting that deficient AA production by PMA-stimulated rac2-/- neutrophils does not explain the effect of exogenous AA on oxidase activity. Although not required for translocation of p47phox and p67phox, Rac2 is necessary for optimal activity of the assembled oxidase complex, an effect that can be replaced by exogenous AA, which may act directly or via an exogenous AA-induced mediator.

Rabbit IgG antibodies against Phospholipase A2 from Crotalus durissus terrificus neutralize the lethal activity of the venom

Crotalus durissus terrificus (C.d.t.) (South American rattlesnake) venom possesses myotoxic and neurotoxic activities, both of which are also expressed by crotoxin, the principal toxin of this venom. Crotoxin contains a basic phospholipase A2 (PLA2) and a non toxic acidic protein, crotapotin. We have produced and investigated the ability of IgG antibodies raised in rabbits against PLA2 to neutralize the lethality of the whole venom. PLA2 was isolated by gel filtration chromatography (Sephadex G-75). Specific antibodies were obtained by subcutaneous and intramuscular inoculation of PLA2 (700 µg) with Freund adjuvant. Groups of six mice (20 + 2 g) were inoculated with 0.5 ml i.p. of C. d. t. venom (4 µg) or a mixture of venom that had been preincubated with the desired volume of IgG antibodies. Mortality, recorded 24 and 48 h after inoculation, showed that IgG anti-PLA2 were more effective than anticrotalic serum in neutralizing the lethal activity. These results demonstrate that it could be possible to obtain an anti-venom made by specific antibodies with a high level of protection against the lethal component of C.d.t. venom, and/or the inclusion of these antibodies as a supplement in heterologous anti-venoms

El veneno de Crotalus durissus terrificus (C.d.t.) (Cascabel de Sud América) posee actividad miotóxica y neurotóxica, actividades que también exhibe el complejo crotoxina, principal componente tóxico de este veneno. El complejo crotoxina está constituido por una fosfolipasa A2 básica (PLA2) y una proteína acídica no tóxica, el crotapotín. En este trabajo se estudió la capacidad neutralizante de anticuerpos IgG anti-PLA2 sobre la letalidad inducida por el veneno entero. El antígeno PLA2, fue aislado por cromatografía de filtración en gel (Sephadex G-75). Se inocularon conejos machos por vía subcutánea e intramuscular, con 700 µg de PLA2 y adyuvante para la obtención de anticuerpos específicos. La capacidad neutralizante del antisuero se analizó en ratones por inoculación con diluciones de veneno entero preincubado con un volumen adecuado de anticuerpos IgG anti-PLA2. Se inocularon ratones controles con 0.5 ml i.p. de veneno (4 µg.ml-1). El número de muertes fue contabilizado a las 24 y 48 h posteriores a la inoculación, demostrándose que la capacidad neutralizante de los anticuerpos IgG anti-PLA2 fue superior a la obtenida con el antiveneno crotálico. Los resultados obtenidos demuestran la potencial aplicación de antivenenos constituidos por anticuerpos específicos contra PLA2, y/o la inclusión de estos anticuerpos como suplementos en antivenenos polivalentes

Peptide-based immunotherapy: a novel strategy for allergic disease.

The T-cell component of the antigen-specific immune response is the target of various novel interventions to modify chronic immunologic disorders, such as allergic diseases. Recent clinical trials have evaluated the safety and efficacy of therapeutic vaccines consisting of short, synthetic, allergen-derived peptides, corresponding to T-cell epitopes from the eliciting antigen. The main advantage of such an approach is the reduction in systemic, immunoglobulin E-mediated adverse events compared with existing whole allergen immunotherapy, often referred to as 'allergy shots'. T-cell peptide epitopes, although capable of inducing immunologic tolerance, are short linear structures that have reduced ability to cross-link mast cell- and basophil-bound immunoglobulin E. The precise mechanism of tolerance induction remains incompletely defined. However, recent data indicate that peptide therapy induces/expands a population of antigen-specific regulatory T-cells. A novel form of treatment combining efficacy with a substantially decreased occurrence of adverse events is likely to have a major impact on the management and prevalence of allergic diseases. Furthermore, the principles of epitope-specific therapy hold promise for the development of therapeutic vaccines for the treatment of autoimmune diseases.

TNF-alpha induces the late-phase airway hyperresponsiveness and airway inflammation through cytosolic phospholipase A(2) activation.

BACKGROUND: Late-phase airway hyperresponsiveness (AHR) in asthma is considered the event leading to persistent inflammation in the lungs, but the molecular mechanisms involved in this process are poorly understood. OBJECTIVE: To examine the role of TNF-alpha in the development of a late AHR and airway inflammation in asthma. METHODS: We established a murine model of asthma with not only biphasic AHR to methacholine but also airway eosinophilia. The effect of TNF-alpha blockade was determined by using anti-TNF-alpha antibody and TNF-alpha knockout mice. Cytosolic phospholipase A(2) (cPLA(2)) mRNA expression and activity were assessed by using RT-PCR and 1-stearoyl-2-[1-(14)C] arachidonyl-sn-glycero-3-phosphocholine as the substrate, respectively. RESULTS: TNF-alpha blockade resulted in significant inhibition of the late AHR without affecting the early AHR, and reduction in airway eosinophilia and inflammation. cPLA(2) activity was increased in asthmatic lungs in a TNF-alpha-dependent way, and cPLA(2) inhibitor blocked late AHR and airway eosinophilia. TNF-alpha also stimulated the synthesis of cPLA(2) metabolites such as leukotriene B(4) and platelet-activating factor in the airway. Specific inhibitors of cPLA(2) metabolites inhibited the late AHR and airway eosinophilia. CONCLUSIONS: TNF-alpha is the proximal key cytokine capable of developing late-phase AHR and subsequent airway inflammation through expression/activation of cPLA(2).

Phospholipase A2 pathway association with macrophage-mediated polycarbonate-urethane biodegradation.

Activation of the phospholipase A2 (PLA2) pathway is a key cell signaling event in the inflammatory response. The PLA2 family consists of a group of enzymes that hydrolyze membrane phospholipids, resulting in the liberation of arachidonic acid (AA), a precursor to pro-inflammatory molecules. Given the well-documented activating role of biomaterials in the inflammatory response to medical implants, the present study investigated the link between PLA2 and polycarbonate-based polyurethane (PCNU) biodegradation, and the effect that material surface had on PLA2 activation in the U937 cell line. PCNUs were synthesized with poly(1,6-hexyl 1,2-ethyl carbonate)diol, 1,4-butanediol and one of two diisocyanates (hexane 1,6-diisocyanate or 4,4'-methylene bisphenyl diisocyanate) in varying stoichiometries and incubated with adherent U937 cells. PLA2 inhibiting agents resulted in significantly decreased PCNU biodegradation (p < 0.05). Moreover, when activation of PLA2 was assessed (3H-AA release), significantly more 3H-AA was released from PCNU-adherent U937 cells than polystyrene-adherent U937 cells (p < 0.05) which was significantly decreased in the presence of PLA2 inhibitors. The pattern of inhibition of U937 cell-mediated biodegradation and 3H-AA release that was modulated by PCNU surface differences, suggests a role for secretory PLA2 along with cytosolic PLA2. Understanding PCNU activation of intracellular pathways, such as PLA2, will allow the design of materials optimized for their intended use.

Activation of cytokine production by secreted phospholipase A2 in human lung macrophages expressing the M-type receptor.

Secreted phospholipases A(2) (sPLA(2)) are enzymes released in plasma and extracellular fluids during inflammatory diseases. Because human group IB and X sPLA(2)s are expressed in the lung, we examined their effects on primary human lung macrophages (HLM). Both sPLA(2)s induced TNF-alpha and IL-6 release in a concentration-dependent manner by increasing their mRNA expression. This effect was independent of their enzymatic activity because 1) the capacity of sPLA(2)s to mobilize arachidonic acid from HLM was unrelated to their ability to induce cytokine production; and 2) two catalytically inactive isoforms of group IB sPLA(2) (bromophenacyl bromide-inactivated human sPLA(2) and the H48Q mutant of the porcine sPLA(2)) were as effective as the catalytically active sPLA(2)s in inducing cytokine production. HLM expressed the M-type receptor for sPLA(2)s at both mRNA and protein levels, as determined by RT-PCR, immunoblotting, immunoprecipitation, and flow cytometry. Me-indoxam, which decreases sPLA(2) activity as well as binding to the M-type receptor, suppressed sPLA(2)-induced cytokine production. Incubation of HLM with the sPLA(2)s was associated with phosphorylation of ERK1/2, and a specific inhibitor of this pathway, PD98059, significantly reduced the production of IL-6 elicited by sPLA(2)s. In conclusion, two distinct sPLA(2)s produced in the human lung stimulate cytokine production by HLM via a mechanism that is independent of their enzymatic activity and involves activation of the ERK1/2 pathway. HLM express the M-type receptor, but its involvement in eliciting cytokine production deserves further investigation.

Medicinal application of long synthetic peptide technology.

This review covers the latest developments of long synthetic peptide technology for the rapid identification and development of malaria vaccine candidates and immunological modulators. A brief description of the two most common solid-phase synthetic procedures, together with the latest advances in optimisation of peptide chain assembly and analytical instrumentation, is given, with special attention to non-specialists. Several examples of vaccine candidates developed in the authors' or their collaborators' laboratories are also provided.

Impaired secretion of interleukin-4 and interleukin-13 by allergen-specific T cells correlates with defective nuclear expression of NF-AT2 and jun B: relevance to immunotherapy.

BACKGROUND: Allergen immunotherapy (IT) is a successful treatment associated with decreased Th2 cytokine production by allergen-specific T cells. We have previously demonstrated (Faith et al., J Immunol 1997; 159:53-57) that inhibition of Th2 cytokine production in vitro correlates with impaired tyrosine kinase activity through the TCR. The transcription factor complex, nuclear factor of activated T cells (NF-AT), which regulates Th2 cytokine production is controlled by the activity of tyrosine kinases. OBJECTIVE: To address whether decreased Th2 cytokine production by allergen-specific CD4+ T cells following IT is correlated with altered translocation and nuclear expression of the NF-AT family member, NF-AT2, and the activator protein 1 (AP1) component of NF-AT, jun B. METHODS: T cell lines specific for insect venom phospholipase A2 (PLA) were derived from patients prior to and during conventional venom IT. Nuclear expressions of NF-AT and jun B were assessed following stimulation through the TCR. Th1 and Th2 cytokine and IL-10 production by insect venom-specific T cells were also determined. Results were compared with a well-established model system in which anergy was induced in cloned, allergen-specific Th2 cells. RESULTS: Impaired translocation and decreased expression of NF-AT2 and jun B were detected in PLA-specific T cell lines derived from bee venom-allergic individuals following 16 weeks treatment compared to pre-treatment. These results correlated with significantly reduced production of IL-4 and IL-13 and significantly increased production of IFN-gamma and IL-10 by PLA-specific T cells. Impaired IL-4 and IL-13 production also correlated with defective nuclear expression of NF-AT2/jun B in cloned, anergic allergen-specific Th2 cells. CONCLUSION: These results suggested that optimal production of IL-4 and IL-13 by allergen-specific T cells is dependent on the nuclear expression of NF-AT2 and jun B. Thus, specific inhibition of NF-AT2/jun B might be an option in novel and improved forms of allergen IT.

Crotapotin induced modification of T lymphocyte proliferative response through interference with PGE2 synthesis.

The immunosuppressive property has been demonstrated for the venom of the Crotalus durissus terrificus. Using a simple, novel method for obtaining crotapotin and phospholipase A2 isoforms from venom, it was possible to demonstrate that the addition of crotapotin to cultures of isolated lymphocytes resulted in a significant inhibition of the cellular proliferative response to Concanavalin A. This reduction in blastogenic response of lymphocytes is accompanied by a significant increase in the production of PGE2 by macrophages. This effect on the innate immune response suggests that this compound may modify the subsequent adaptative immune response.

Platelet-activating factor acetylhydrolase is increased in lung lavage fluid from patients with acute respiratory distress syndrome.

OBJECTIVE: Platelet-activating factor (PAF) is a proinflammatory phospholipid that may contribute to inflammation in the acute respiratory distress syndrome (ARDS). PAF acetylhydrolase (PAF-AH) degrades PAF and regulates its biological activity. We characterized PAF-AH in bronchoalveolar lavage fluid from ARDS patients (n = 33, 22 survivors), patients at risk for ARDS (n = 6), and healthy controls (n = 6). DESIGN: Bronchoalveolar lavage was performed during acute (<96 hrs from onset), plateau (6 to 12 days), and late (> or = 14 days) phases of ARDS. PATIENTS: Intubated patients with ARDS or a risk factor for ARDS. MEASUREMENTS AND MAIN RESULTS: In ARDS, total bronchoalveolar lavage PAF-AH activity was markedly increased in the acute phase (87 +/- 89 mU/mL, n = 33) and then decreased in the plateau (23 +/- 14 mU/mL, n = 10) and late phases (19 +/- 14 mU/mL, n = 7) (p = .003). Total bronchoalveolar lavage PAF-AH activity during the acute phase of ARDS was also increased as compared with patients at risk for ARDS (16 +/- 13 mU/mL, n = 6) and healthy controls (3 +/- 3 mU/mL, n = 6) (p < .001). In contrast, plasma PAF-AH activities were the same in controls (3215 +/- 858 mU/mL, n = 6), in patients at risk for ARDS (3606 +/- 1607 mU/mL, n = 6), and during the acute phase of ARDS (3098 +/- 2395 mU/mL, n = 33) (p = .18). PAF-AH mRNA was present in alveolar macrophages in the acute phase of ARDS (five of six) and in at-risk patients (two of three) but not in healthy controls. CONCLUSIONS: PAF-AH activity is increased in bronchoalveolar lavage fluid from patients with ARDS. Likely sources include leakage of plasma PAF-AH into alveoli or release of PAF-AH from injured cells; however, the presence of PAF-AH mRNA in alveolar macrophages suggests that PAF-AH may be actively synthesized in the lungs of patients with ARDS. PAF-AH activity in the lungs of ARDS patients may regulate inflammation caused by PAF and related oxidized phospholipids generated in the inflammatory response.

Macrophage-expressed group IIA secretory phospholipase A2 increases atherosclerotic lesion formation in LDL receptor-deficient mice.

OBJECTIVE: Transgenic mice expressing human group IIA secretory phospholipase A(2) (group IIA sPLA(2)) spontaneously develop atherosclerotic lesions. The mechanism for this proatherogenic effect is likely multifactorial, because HDL-cholesterol is significantly lower and LDL/VLDL cholesterol is slightly higher in transgenic mice compared with nontransgenic littermates. In the present study, we show for the first time that elicited peritoneal macrophages from transgenic mice express human group IIA sPLA(2). This study tested whether macrophage-expressed sPLA(2) contributes to atherogenesis. METHODS AND RESULTS: Bone marrow cells from either sPLA(2) transgenic mice or control C57BL/6 mice were transplanted into LDL receptor-deficient mice. After hematopoietic engraftment, animals were fed a diet enriched with saturated fat and cholesterol for 12 weeks. Despite a lack of effect on serum lipoprotein concentrations, the presence of bone marrow-derived cells expressing human group IIA sPLA(2) resulted in a significant increase in the extent of atherosclerosis in the aortic arch (12.8+/-1.4% versus 7.4+/-0.9%; P<0.005) and aortic sinus (0.3+/-0.03 mm(2) versus 0.2+/-0.04 mm(2); P<0.05). CONCLUSIONS: Group IIA sPLA(2) can contribute to atherosclerotic lesion development through a mechanism that is independent of systemic lipoprotein metabolism.

A molecular assembly system that renders antigens of choice highly repetitive for induction of protective B cell responses.

Virus like particles (VLPs) are known to induce potent B cell responses in the absence of adjuvants. Moreover, epitope-specific antibody responses may be induced by VLPs that contain peptides inserted in their immunodominant regions. However, due to steric problems, the size of the peptides capable of being incorporated into VLPs while still permitting capsid assembly, is rather limited. While peptides genetically fused to either the N- or C-terminus of VLPs present fewer assembly problems, the immune responses obtained against such epitopes are often limited, most likely because the epitopes are not optimally exposed. In addition, such particles may be less stable in vivo. Here, we show that peptides and proteins engineered to contain a free cys can be chemically coupled to VLPs formed from the hepatitis B core antigen (HBcAg) containing a lys in the immuno-dominant region. By using this approach steric hindrance of capsid assembly is abrogated. Peptides or protein coupled to VLPs in an oriented fashion are shown to induce strong and protective B cell responses even against self-epitopes in the absence of adjuvants. This molecular assembly system may be used to induce strong B cell responses against most antigens.