RESUMO
BACKGROUND: Stored red blood cells (RBCs) release hemoglobin (Hb) that leads to oxidative damage, which may contribute to thrombosis in susceptible transfusion recipients. Oxidative stress stimulates the generation of a new class of lipid mediators called F2 -isoprostanes (F2 -IsoPs) and isofurans (IsoFs) that influence cellular behavior. This study investigated RBC-derived F2 -IsoPs and IsoFs during storage and their influence on human platelets (PLTs). STUDY DESIGN AND METHODS: F2 -IsoP and IsoF levels in RBC supernatants were measured by mass spectrometry during storage and after washing. The effects of stored supernatants, cell-free Hb, or a key F2 -IsoP, 8-iso-prostaglandin F2α (PGF2α ), on PLT function were examined in vitro. RESULTS: F2 -IsoPs, IsoFs, and Hb accumulated in stored RBC supernatants. Prestorage leukoreduction reduced supernatant F2 -IsoPs and IsoFs levels, which increased again over storage time. Stored RBC supernatants and 8-iso-PGF2α induced PLT activation marker CD62P (P-selectin) expression and prothrombotic thromboxane A2 release. Cell-free Hb did not alter PLT mediator release, but did inhibit PLT spreading. Poststorage RBC washing reduced F2 -IsoP and IsoF levels up to 24 hours. CONCLUSIONS: F2 -IsoPs and IsoFs are produced by stored RBCs and induce adverse effects on PLT function in vitro, supporting a potential novel role for bioactive lipids in adverse transfusion outcomes. F2 -IsoP and IsoF levels could be useful biomarkers for determining the suitability of blood components for transfusion. A novel finding is that cell-free Hb inhibits PLT spreading and could adversely influence wound healing. Poststorage RBC washing minimizes harmful lipid mediators, and its use could potentially reduce transfusion complications.
Assuntos
Plaquetas/metabolismo , Eritrócitos/metabolismo , Furanos/metabolismo , Isoprostanos/metabolismo , Dinoprosta/análogos & derivados , Dinoprosta/metabolismo , F2-Isoprostanos/metabolismo , Humanos , Imunoensaio , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: ABO-mismatched platelets (PLTs) are commonly transfused despite reported complications. We hypothesized that because PLTs possess A and B antigens on their surface, ABO-mismatched transfused or recipient PLTs could become activated and/or dysfunctional after exposure to anti-A or -B in the transfused or recipient plasma. We present here in vitro modeling data on the functional effects of exposure of PLTs to ABO antibodies. STUDY DESIGN AND METHODS: PLT functions of normal PLTs of all ABO types were assessed before and after incubation with normal saline, ABO-identical plasma samples, or O plasma samples with varying titers of anti-A and anti-B (anti-A/B). Assays used for this assessment include PLT aggregation, clot kinetics, thrombin generation, PLT cytoskeletal function, and mediator release. RESULTS: Exposure of antigen-bearing PLTs to O plasma with moderate to high titers of anti-A/B significantly inhibits aggregation, prolongs PFA-100 epinephrine closure time, disrupts clot formation kinetics, accelerates thrombin generation, reduces total thrombin production, alters PLT cytoskeletal function, and influences proinflammatory and prothrombotic mediator release. CONCLUSIONS: Our findings demonstrate a wide range of effects that anti-A/B have on PLT function, clot formation, thrombin generation, PLT cytoskeletal function, and mediator release. These data provide potential explanations for clinical observations of increased red blood cell utilization in trauma and surgical patients receiving ABO-nonidentical blood products. Impaired hemostasis caused by anti-A/B interacting with A and B antigens on PLTs, soluble proteins, and perhaps even endothelial cells is a potential contributing factor to hemorrhage in patients receiving larger volumes of ABO-nonidentical transfusions.
Assuntos
Sistema ABO de Grupos Sanguíneos/imunologia , Anticorpos/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Adulto , Coagulação Sanguínea/fisiologia , Tipagem e Reações Cruzadas Sanguíneas , Plaquetas/imunologia , Feminino , Humanos , Técnicas In Vitro , Cinética , Masculino , Pessoa de Meia-Idade , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/fisiologia , Fatores de Tempo , TitulometriaRESUMO
Fibrosis can occur in any human tissue when the normal wound healing response is amplified. Such amplification results in fibroblast proliferation, myofibroblast differentiation, and excessive extracellular matrix deposition. Occurrence of these sequelae in organs such as the eye or lung can result in severe consequences to health. Unfortunately, medical treatment of fibrosis is limited by a lack of safe and effective therapies. These therapies may be developed by identifying agents that inhibit critical steps in fibrotic progression; one such step is myofibroblast differentiation triggered by transforming growth factor-ß1 (TGFß1). In this study, we demonstrate that TGFß1-induced myofibroblast differentiation is blocked in human fibroblasts by a candidate endogenous aryl hydrocarbon receptor (AhR) ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Our data show that ITE disrupts TGFß1 signaling by inhibiting the nuclear translocation of Smad2/3/4. Although ITE functions as an AhR agonist, and biologically persistent AhR agonists, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, cause severe toxic effects, ITE exhibits no toxicity. Interestingly, ITE effectively inhibits TGFß1-driven myofibroblast differentiation in AhR(-/-) fibroblasts: Its ability to inhibit TGFß1 signaling is AhR independent. As supported by the results of this study, the small molecule ITE inhibits myofibroblast differentiation and may be useful clinically as an antiscarring agent.
Assuntos
Indóis/farmacologia , Receptores de Hidrocarboneto Arílico/química , Tiazóis/farmacologia , Hidrocarboneto de Aril Hidroxilases/metabolismo , Diferenciação Celular , Citocromo P-450 CYP1B1 , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Humanos , Ligantes , Miofibroblastos/citologia , Órbita/citologia , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , CicatrizaçãoRESUMO
OBJECTIVE: To investigate the presence and role of NF-kappaB proteins in megakaryocytes and platelets. The nuclear factor-kappaB (NF-kappaB) transcription factor family is well known for its role in eliciting inflammation and promoting cell survival. We discovered that human megakaryocytes and platelets express the majority of NF-kappaB family members, including the regulatory inhibitor-kappaB (I-kappaB) and I-kappa kinase (IKK) molecules. METHODS AND RESULTS: Anucleate platelets exposed to NF-kappaB inhibitors demonstrated impaired fundamental functions involved in repairing vascular injury and thrombus formation. Specifically, NF-kappaB inhibition diminished lamellapodia formation, decreased clot retraction times, and reduced thrombus stability. Moreover, inhibition of I-kappaB-alpha phosphorylation (BAY-11-7082) reverted fully spread platelets back to a spheroid morphology. Addition of recombinant IKK-beta or I-kappaB-alpha protein to BAY inhibitor-treated platelets partially restored platelet spreading in I-kappaB-alpha inhibited platelets, and addition of active IKK-beta increased endogenous I-kappaB-alpha phosphorylation levels. CONCLUSIONS: These novel findings support a crucial and nonclassical role for the NF-kappaB family in modulating platelet function and reveal that platelets are sensitive to NF-kappaB inhibitors. As NF-kappaB inhibitors are being developed as antiinflammatory and anticancer agents, they may have unintended effects on platelets. On the basis of these data, NF-kappaB is also identified as a new target to dampen unwanted platelet activation.
Assuntos
Plaquetas/metabolismo , Megacariócitos/metabolismo , NF-kappa B/metabolismo , Adulto , Idoso , Plaquetas/efeitos dos fármacos , Plaquetas/patologia , Adesão Celular/efeitos dos fármacos , Diferenciação Celular , Linhagem Celular Tumoral , Forma Celular/efeitos dos fármacos , Feminino , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Humanos , Leucemia Megacarioblástica Aguda/metabolismo , Leucemia Megacarioblástica Aguda/patologia , Masculino , Megacariócitos/efeitos dos fármacos , Megacariócitos/patologia , Pessoa de Meia-Idade , NF-kappa B/antagonistas & inibidores , Subunidade p50 de NF-kappa B/metabolismo , Nitrilas/farmacologia , Sulfonas/farmacologia , Fator de Transcrição RelA/metabolismoRESUMO
Herpes simplex virus type-1 (HSV-1) amplicon vectors are being explored for a wide range of potential applications, including vaccine delivery and immunotherapy of cancer. While extensive effort has been directed towards the improvement of the amplicon "payload" in these vectors, relatively little attention has been paid to the effect of the packaging HSV-1 strains on the biological properties of co-packaged amplicon vectors. We therefore compared the biological properties of amplicon stocks prepared using a panel of primary HSV-1 isolates, a molecularly cloned strain used to package helper-free amplicons (designated here as F5), and two laboratory isolates (KOS and strain 17, which is the parent of the F5 clone). This analysis revealed considerable inter-strain variability in the ability of amplicon stocks packaged by different primary HSV-1 isolates to efficiently transduce established cell lines and primary human dendritic cells (DC). Amplicons packaged by both the F5 molecularly cloned virus and its laboratory-adapted parent (strain 17) were very inefficient at transducing DC, when compared to amplicons packaged by KOS or by several of the primary virus isolates. These finding have important implications for the future development of improved amplicon-based vaccine delivery systems and suggest that DC tropism may be an instrinsic property of some HSV-1 strains, independent of passage history or molecular cloning.
Assuntos
Células Dendríticas/virologia , Vetores Genéticos , Herpesvirus Humano 1/fisiologia , Transfecção/métodos , Animais , Linhagem Celular , Chlorocebus aethiops , Células Dendríticas/citologia , Humanos , Células Vero , Montagem de VírusRESUMO
Given the participation of amyloid beta (Abeta) in Alzheimer's disease (AD) pathogenesis the derivation of experimental therapeutics to prevent Abeta fibrillogenesis and/or enhance removal of parenchymal amyloid deposits represent viable disease-modifying approaches. Active Abeta-based immunotherapies have shown promise in mouse AD models, but application in human trials was accompanied by moderate brain inflammation in a subset of patients. Immune-shaping vaccine platforms may mitigate adverse effects. Herein, we describe the use of herpes simplex virus (HSV)-derived amplicons to elicit distinctive immune responses against Abeta. Two vaccine vectors were constructed: one expressing Abeta1-42 alone (HSVAbeta), and a second expressing Abeta1-42 fused with the molecular adjuvant tetanus toxin Fragment C (HSVAbeta/TtxFC). Peripheral administration of these vaccines augmented humoral responses to Abeta and reduced CNS Abeta deposition in Tg2576 AD mice. Interestingly and unexpectedly, HSVAbeta vaccination was uniquely toxic and incited the expression of pro-inflammatory molecule transcripts within the hippocampi of Tg2576 mice, suggesting that this paradigm may serve as a relevant model to study Abeta vaccine-elicited CNS inflammatory syndromes.
Assuntos
Doença de Alzheimer/imunologia , Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/uso terapêutico , Amiloidose/prevenção & controle , Imunoterapia Ativa/métodos , Simplexvirus/genética , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/biossíntese , Peptídeos beta-Amiloides/imunologia , Peptídeos beta-Amiloides/metabolismo , Análise de Variância , Animais , Antígenos/imunologia , Contagem de Células/métodos , Linhagem Celular , Cricetinae , Diagnóstico por Imagem , Modelos Animais de Doenças , Relação Dose-Resposta Imunológica , Ensaio de Imunoadsorção Enzimática/métodos , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Vetores Genéticos/uso terapêutico , Hipocampo/metabolismo , Humanos , Isotipos de Imunoglobulinas/biossíntese , Isotipos de Imunoglobulinas/uso terapêutico , Imuno-Histoquímica/métodos , Interferons/classificação , Interferons/metabolismo , Interleucina-6/metabolismo , Antígeno de Macrófago 1/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Transgênicos , Microglia/metabolismo , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/uso terapêutico , Placa Amiloide/metabolismo , Placa Amiloide/patologia , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Linfócitos T/metabolismo , Toxina Tetânica/imunologia , Toxina Tetânica/uso terapêutico , Fatores de TempoRESUMO
Human platelets play a key role in hemostasis and thrombosis and have recently emerged as key regulators of inflammation. Platelets stored for transfusion produce pro-thrombotic and pro-inflammatory mediators implicated in adverse transfusion reactions. Correspondingly, these mediators are central players in pathological conditions including cardiovascular disease, the major cause of death in diabetics. In view of this, a mass spectrometry based proteomics study was performed on platelets collected from healthy and type-2 diabetics stored for transfusion. Strikingly, our innovative and sensitive proteomic approach identified 122 proteins that were either up- or down-regulated in type-2 diabetics relative to nondiabetic controls and 117 proteins whose abundances changed during a 5-day storage period. Notably, our studies are the first to characterize the proteome of platelets from diabetics before and after storage for transfusion. These identified differences allow us to formulate new hypotheses and experimentation to improve clinical outcomes by targeting "high risk platelets" that render platelet transfusion less effective or even unsafe.
Assuntos
Plaquetas/metabolismo , Preservação de Sangue/métodos , Diabetes Mellitus Tipo 2/sangue , Proteoma/análise , Proteômica/métodos , Adulto , Idoso , Bancos de Sangue , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Integrina alfa2beta1/sangue , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Transfusão de Plaquetas , Proteoma/classificação , Fatores de Tempo , Adulto JovemRESUMO
The herpes simplex virus (HSV)-derived amplicon vector has evolved into a promising gene transfer platform for widespread DNA delivery in gene replacement strategies and vaccine development given its ease of molecular manipulation, large transgene capacity, and transduction efficiencies of numerous cell types in vivo. The recent development of helper virus-free packaging methodologies bodes well for this vector system in its eventual implementation as a clinically viable therapeutic modality. For realization of clinical application, efforts have been made to enhance yields and quality of helper-free amplicon stocks. Hexamethylene bisacetamide (HMBA), a hybrid polar compound that exhibits stimulatory activity of HSV-1 immediate-early gene expression, has been employed as a standard reagent in helper virus-free packaging given its purported mode of action on virus gene expression kinetics. Unexpectedly, we have found that HMBA exhibits no titer-enhancing activity; in contrast, the compound enhances the proportion of amplicon virions that are non-expressive. Omission of HMBA during vector packaging led to a marked reduction in the ratios of vector genome-transducing to transgene-expressing virions. This effect was neither packaging-cell-specific nor amplicon-promoter-dependent. Analysis of resultant vector stocks indicated amplicon genome replication/concatenation was unaffected, but the level of particle-associated ICP0 was reduced in stocks packaged in the presence of HMBA. Inclusion of a co-transfected, ICP0-expressing plasmid into the packaging process led to significant rescue of amplicon expression titers, indicating that regulation of ICP0 concentrations is critical for maintenance of the amplicon genome expressive state.
Assuntos
Acetamidas/farmacologia , Vírus Auxiliares/efeitos dos fármacos , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/genética , Proteínas Imediatamente Precoces/antagonistas & inibidores , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Animais , Estruturas do Núcleo Celular/efeitos dos fármacos , Estruturas do Núcleo Celular/metabolismo , Chlorocebus aethiops , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Vírus Auxiliares/genética , Vírus Auxiliares/fisiologia , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Camundongos , Células NIH 3T3 , Regiões Promotoras Genéticas/genética , Transcrição Gênica/efeitos dos fármacos , Transfecção , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Células Vero , Vírion/efeitos dos fármacos , Montagem de Vírus/efeitos dos fármacosRESUMO
Chronic lymphocytic leukemia (CLL) is a B lymphocyte malignancy that remains a largely incurable disease. CLL B cells possess the ability to process and present tumor antigens but lack expression of costimulatory molecules, rendering them inefficient effectors of T-cell activation. We previously demonstrated that helper virus-free preparations of herpes simplex virus (HSV) amplicon vectors encoding CD40L efficiently transduce CLL B cells and render them capable of eliciting specific anti-tumor T-cell responses. LIGHT (TNFSF14), a member of the tumor necrosis factor (TNF) superfamily, efficiently activates both T cells and antigen-presenting cells (APCs). We employed an HSV amplicon vector expressing human LIGHT (hf-HSV-LIGHT) to transduce CLL B cells and compared the immunomodulatory function and T-cell activation induced by hf-HSV-LIGHT transduction to that observed with a CD40L-expressing HSV amplicon (hf-HSV-CD40L). hf-HSV-LIGHT transduction induced expression of endogenous B7.1, B7.2, and ICAM.1 on CLL cells, albeit to a lesser degree than that observed in response to transduction with hf-HSV-CD40L. hf-HSV-LIGHT enhanced the antigen-presenting capacity of CLL B cells, as measured by induction of T-cell proliferation in an allogeneic mixed lymphocyte tumor reaction. Finally, hf-HSV-LIGHT-transduced CLL B cells successfully stimulated the outgrowth of autologous cytotoxic T-lymphocytes in vitro. In aggregate, these data suggest that hf-HSV-LIGHT transduction may be useful for induction of immune responses to CLL and other B-cell lymphoid malignancies.