RESUMO
BACKGROUND: Platelet transfusion is associated with logistical problems with the national storage guidelines of platelets. This results in decreased function in vivo as a result of the platelet storage lesion, and complications such as allergic or hemolytic reactions and thrombosis. We evaluated a new, freshly prepared platelet modified lysate (PML) product designed to be more procoagulant than fresh and stored platelets. METHODS: Fresh platelets were concentrated, sonicated, and centrifuged to produce PML. Samples of both washed and unwashed PML were evaluated for particle size, concentration, and activity, and then tested for clot kinetics and thrombin generation. PML samples were also stored at various temperatures for durations up to 6 months and evaluated for clot kinetics and thrombin generation throughout. RESULTS: PML showed significantly higher concentration of platelet microparticles, increased procoagulant properties, and increased thrombin generation as compared to fresh and stored platelets. In addition, PML maintained its clot kinetics over a 6-month storage period with variable storage conditions. CONCLUSIONS: The newly proposed PML product is more procoagulant, stable, and has additional potential applications than currently available platelet products. Further studies will be performed to assess its functions in vivo and to assess thrombotic potential.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/química , Micropartículas Derivadas de Células/química , Coagulantes , Coagulantes/química , Coagulantes/farmacologia , Avaliação Pré-Clínica de Medicamentos , Humanos , Transfusão de PlaquetasRESUMO
Human lung fibroblasts (HLFs) act as innate immune sentinel cells that amplify the inflammatory response to injurious stimuli. Here, we use targeted lipidomics to explore the hypothesis that HLFs also play an active role in the resolution of inflammation. We detected cyclooxygenase-2 (COX-2)-dependent production of both proinflammatory and proresolving prostaglandins (PGs) in conditioned culture medium from HLFs treated with a proinflammatory stimulus, IL-1ß. Among the proresolving PGs in the HLF lipidome were several known ligands for peroxisome proliferator-activated receptor-γ (PPARγ), a transcription factor whose activation in the lung yields potent anti-inflammatory, antifibrotic, and proresolving effects. Next, we used a cell-based luciferase reporter to confirm the ability of HLF supernatants to activate PPARγ, demonstrating, for the first time, that primary HLFs activated with proinflammatory IL-1ß or cigarette smoke extract produce functional PPARγ ligands; this phenomenon is temporally regulated, COX-2- and lipocalin-type PGD synthase-dependent, and enhanced by arachidonic acid supplementation. Finally, we used luciferase reporter assays to show that several of the PGs in the lipidome of activated HLFs independently activate PPARγ and/or inhibit NFκB. These results indicate that HLFs, as immune sentinels, regulate both proinflammatory and proresolving responses to injurious stimuli. This novel endogenous resolution pathway represents a new therapeutic target for globally important inflammatory diseases such as chronic obstructive pulmonary disease.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Fibroblastos/metabolismo , Pulmão/citologia , PPAR gama/metabolismo , Ácidos Araquidônicos/farmacologia , Meios de Cultivo Condicionados/farmacologia , Dinoprostona/metabolismo , Eicosanoides/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Células HEK293 , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-1beta/farmacologia , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Oxirredutases Intramoleculares/metabolismo , Ligantes , Lipocalinas/metabolismo , Masculino , NF-kappa B/metabolismo , Prostaglandina-E Sintases , Fumar , Regulação para Cima/efeitos dos fármacosRESUMO
Chronic obstructive pulmonary disease is characterized, in part, by chronic inflammation that persists even after smoking cessation, suggesting that a failure to resolve inflammation plays an important role in the pathogenesis of the disease. It is widely recognized that the resolution of inflammation is an active process, governed by specialized proresolving lipid mediators, including lipoxins, resolvins, maresins, and protectins. Here, we report that proresolving signaling and metabolic pathways are disrupted in lung tissue from patients with chronic obstructive pulmonary disease, suggesting that supplementation with proresolving lipid mediators might reduce the development of emphysema by controlling chronic inflammation. Groups of mice were exposed long-term to cigarette smoke and treated with the proresolving mediator resolvin D1. Resolvin D1 was associated with a reduced development of cigarette smoke-induced emphysema and airspace enlargement, with concurrent reductions in inflammation, oxidative stress, and cell death. Interestingly, resolvin D1 did not promote the differentiation of M2 macrophages and did not promote tissue fibrosis. Taken together, our results suggest that cigarette smoking disrupts endogenous proresolving pathways and that supplementation with specialized proresolving lipid mediators is an important therapeutic strategy in chronic lung disease, especially if endogenous specialized proresolving lipid mediator signaling is impaired.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Ácidos Docosa-Hexaenoicos/uso terapêutico , Pneumonia/prevenção & controle , Enfisema Pulmonar/prevenção & controle , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Apoptose/efeitos dos fármacos , Doença Crônica , Ácidos Docosa-Hexaenoicos/metabolismo , Ácidos Docosa-Hexaenoicos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Humanos , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/patologia , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Estresse Oxidativo/efeitos dos fármacos , Pneumonia/metabolismo , Pneumonia/patologia , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/metabolismo , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/patologia , Transdução de Sinais/fisiologia , Fumar/efeitos adversosRESUMO
Influenza viruses remain a critical global health concern. More efficacious vaccines are needed to protect against influenza virus, yet few adjuvants are approved for routine use. Specialized proresolving mediators (SPMs) are powerful endogenous bioactive regulators of inflammation, with great clinical translational properties. In this study, we investigated the ability of the SPM 17-HDHA to enhance the adaptive immune response using an OVA immunization model and a preclinical influenza vaccination mouse model. Our findings revealed that mice immunized with OVA plus 17-HDHA or with H1N1-derived HA protein plus 17-HDHA increased Ag-specific Ab titers. 17-HDHA increased the number of Ab-secreting cells in vitro and the number of HA-specific Ab-secreting cells present in the bone marrow. Importantly, the 17-HDHA-mediated increased Ab production was more protective against live pH1N1 influenza infection in mice. To our knowledge, this is the first report on the biological effects of ω-3-derived SPMs on the humoral immune response. These findings illustrate a previously unknown biological link between proresolution signals and the adaptive immune system. Furthermore, this work has important implications for the understanding of B cell biology, as well as the development of new potential vaccine adjuvants.
Assuntos
Ácidos Docosa-Hexaenoicos/farmacologia , Imunidade Humoral/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/imunologia , Infecções por Orthomyxoviridae/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Anticorpos Antivirais/imunologia , Formação de Anticorpos/efeitos dos fármacos , Formação de Anticorpos/imunologia , Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/efeitos dos fármacos , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Masculino , Camundongos , Infecções por Orthomyxoviridae/virologia , Plasmócitos/citologia , Plasmócitos/efeitos dos fármacos , Plasmócitos/imunologia , Plasmócitos/metabolismoRESUMO
Many diabetics are insensitive to aspirin's platelet anti-aggregation effects. The influence of co-administration of aspirin and fish oil (FO) on plasma lysophospholipids in subjects with diabetes is poorly characterized. Thirty adults with type 2 diabetes mellitus were treated with aspirin (81mg/day) for seven days, then with FO (4g/day) for 28 days, then in combination for another seven days. Lysophospholipids and platelet measures were determined after acute (4h) and chronic (7 days) ingestion of aspirin, FO, or both in combination. FO ingestion reduced all lysophosphatidic acid (LPA) concentrations, while EPA (20:5n-3) and DHA (22:6n-3) lysophosphatidylcholine (LPC) concentrations significantly increased after FO alone and in combination with aspirin. In vitro arachidonic acid-induced platelet aggregation was most strongly correlated with palmitoleic (16:1) and oleic (18:1) LPA and LPC concentrations at all time points. The ingestion of these agents may reduce cardiovascular disease risk in diabetic adults, with a disrupted lipid milieu, via lysolipid mediated mechanisms.
Assuntos
Aspirina/farmacologia , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/fisiopatologia , Óleos de Peixe/farmacologia , Lisofosfatidilcolinas/sangue , Lisofosfolipídeos/sangue , Agregação Plaquetária/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Ingestão de Alimentos/fisiologia , Comportamento Alimentar , Feminino , Peixes , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Corneal scarring remains a major cause of blindness world-wide, with limited treatment options, all of which have side-effects. Here, we tested the hypothesis that topical application of Rosiglitazone, a Thiazolidinedione and ligand of peroxisome proliferator activated receptor gamma (PPARγ), can effectively block scar formation in a cat model of corneal damage. Adult cats underwent bilateral epithelial debridement followed by excimer laser ablation of the central corneal stroma to a depth of ~160 µm as a means of experimentally inducing a reproducible wound. Eyes were then left untreated, or received 50 µl of either 10 µM Rosiglitazone in DMSO/Celluvisc, DMSO/Celluvisc vehicle or Celluvisc vehicle twice daily for 2 weeks. Cellular aspects of corneal wound healing were evaluated with in vivo confocal imaging and post-mortem immunohistochemistry for alpha smooth muscle actin (αSMA). Impacts of the wound and treatments on optical quality were assessed using wavefront sensing and optical coherence tomography at 2, 4, 8 and 12 weeks post-operatively. In parallel, cat corneal fibroblasts were cultured to assess the effects of Rosiglitazone on TGFß-induced αSMA expression. Topical application of Rosiglitazone to cat eyes after injury decreased αSMA expression and haze, as well as the induction of lower-order and residual, higher-order wavefront aberrations compared to vehicle-treated eyes. Rosiglitazone also inhibited TGFß-induced αSMA expression in cultured corneal fibroblasts. In conclusion, Rosiglitazone effectively controlled corneal fibrosis in vivo and in vitro, while restoring corneal thickness and optics. Its topical application may represent an effective, new avenue for the prevention of corneal scarring with distinct advantages for pathologically thin corneas.
Assuntos
Cicatriz/prevenção & controle , Córnea/efeitos dos fármacos , Tiazolidinedionas/administração & dosagem , Actinas/metabolismo , Administração Tópica , Animais , Gatos , Diferenciação Celular , Células Cultivadas , Córnea/patologia , Aberrações de Frente de Onda da Córnea/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/fisiologia , PPAR gama/agonistas , Rosiglitazona , Fator de Crescimento Transformador beta/fisiologia , Resultado do Tratamento , Cicatrização/efeitos dos fármacosRESUMO
Protective humoral immune responses critically depend on the optimal differentiation of B cells into Ab-secreting cells. Because of the important role of Abs in fighting infections and in successful vaccination, it is imperative to identify mediators that control B cell differentiation. Activation of B cells through TLR9 by CpG-DNA induces plasma cell differentiation and Ab production. Herein, we examined the role of the peroxisome proliferator-activated receptor (PPAR)gamma/RXRalpha pathway on human B cell differentiation. We demonstrated that activated B cells up-regulate their expression of PPARgamma. We also show that nanomolar levels of natural (15-deoxy-Delta(12,14)-prostaglandin J(2)) or synthetic (rosiglitazone) PPARgamma ligands enhanced B cell proliferation and significantly stimulated plasma cell differentiation and Ab production. Moreover, the addition of GW9662, a specific PPARgamma antagonist, abolished these effects. Retinoid X receptor (RXR) is the binding partner for PPARgamma and is required to produce an active transcriptional complex. The simultaneous addition of nanomolar concentrations of the RXRalpha ligand (9-cis-retinoic acid) and PPARgamma ligands to CpG-activated B cells resulted in additive effects on B cell proliferation, plasma cell differentiation, and Ab production. Furthermore, PPARgamma ligands alone or combined with 9-cis-retinoic acid enhanced CpG-induced expression of Cox-2 and the plasma cell transcription factor BLIMP-1. Induction of these important regulators of B cell differentiation provides a possible mechanism for the B cell-enhancing effects of PPARgamma ligands. These new findings indicate that low doses of PPARgamma/RXRalpha ligands could be used as a new type of adjuvant to stimulate Ab production.
Assuntos
Formação de Anticorpos/imunologia , Linfócitos B/citologia , Diferenciação Celular/imunologia , Ativação Linfocitária/imunologia , PPAR gama/imunologia , Linfócitos B/imunologia , Western Blotting , Proliferação de Células , Ciclo-Oxigenase 2/biossíntese , Ciclo-Oxigenase 2/imunologia , Citometria de Fluxo , Expressão Gênica/imunologia , Regulação da Expressão Gênica/imunologia , Humanos , Ligantes , PPAR gama/biossíntese , Fator 1 de Ligação ao Domínio I Regulador Positivo , Proteínas Repressoras/biossíntese , Proteínas Repressoras/imunologia , Receptor X Retinoide alfa/biossíntese , Receptor X Retinoide alfa/imunologia , Transfecção , Regulação para CimaRESUMO
Prostaglandins are potent lipid molecules that affect key aspects of immunity. The original view of prostaglandins was that they were simply immunoinhibitory. This review focuses on recent findings concerning prostaglandin E2 (PGE2) and the PGD2 metabolite 15-deoxy-Delta(12,14)-PGJ2, and their divergent roles in immune regulation. We will highlight how these two seminal prostaglandins regulate immunity and inflammation, and play an emerging role in cancer progression. Understanding the diverse activities of these prostaglandins is crucial for the development of new therapies aimed at immune modulation.