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Current antifibrinolytic agents reduce blood loss by inhibiting plasmin active sites (e.g., aprotinin) or by preventing plasminogen/tissue plasminogen activator (tPA) binding to fibrin clots (e.g., ε-aminocaproic acid and tranexamic acid); however, they have adverse side effects. Here, we expressed 60-residue (NH2NAE IEKCOOH) Kunitz domain1 (KD1) mutants of human tissue factor pathway inhibitor type-2 that inhibit plasmin as well as plasminogen activation. A single (KD1-L17R-KCOOH) and a double mutant (KD1-Y11T/L17R- KCOOH) were expressed in Escherichia coli as His-tagged constructs, each with enterokinase cleavage sites. KD1-Y11T/L17R-KCOOH was also expressed in Pichia pastoris. KD1-Y11T/L17R-KCOOH inhibited plasmin comparably to aprotinin and bound to the kringle domains of plasminogen/plasmin and tPA with Kd of ~50 nM and ~35 nM, respectively. Importantly, compared to aprotinin, KD1-L17R-KCOOH and KD1-Y11T/L17R-KCOOH did not inhibit kallikrein. Moreover, the antifibrinolytic potential of KD1-Y11T/L17R-KCOOH was better than that of KD1-L17R-KCOOH and similar to that of aprotinin in plasma clot-lysis assays. In thromboelastography experiments, KD1-Y11T/L17R-KCOOH was shown to inhibit fibrinolysis in a dose dependent manner and was comparable to aprotinin at a higher concentration. Further, KD1-Y11T/L17R-KCOOH did not induce cytotoxicity in primary human endothelial cells or fibroblasts. We conclude that KD1-Y11T/L17R-KCOOH is comparable to aprotinin, the most potent known inhibitor of plasmin and can be produced in large amounts using Pichia.
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UNLABELLED: Adeno-associated virus 2 (AAV2) and adenovirus 5 (Ad5) are promising gene therapy vectors. Both display liver tropism and are currently thought to enter hepatocytes in vivo through cell surface heparan sulfate proteoglycans (HSPGs). To test directly this hypothesis, we created mice that lack Ext1, an enzyme required for heparan sulfate biosynthesis, in hepatocytes. Ext1(HEP) mutant mice exhibit an 8-fold reduction of heparan sulfate in primary hepatocytes and a 5-fold reduction of heparan sulfate in whole liver tissue. Conditional hepatocyte Ext1 gene deletion greatly reduced AAV2 liver transduction following intravenous injection. Ad5 transduction requires blood coagulation factor X (FX); FX binds to the Ad5 capsid hexon protein and bridges the virus to HSPGs on the cell surface. Ad5.FX transduction was abrogated in primary hepatocytes from Ext1(HEP) mice. However, in contrast to the case with AAV2, Ad5 transduction was not significantly reduced in the livers of Ext1(HEP) mice. FX remained essential for Ad5 transduction in vivo in Ext1(HEP) mice. We conclude that while AAV2 requires HSPGs for entry into mouse hepatocytes, HSPGs are dispensable for Ad5 hepatocyte transduction in vivo. This study reopens the question of how adenovirus enters cells in vivo. IMPORTANCE: Our understanding of how viruses enter cells, and how they can be used as therapeutic vectors to manage disease, begins with identification of the cell surface receptors to which viruses bind and which mediate viral entry. Both adeno-associated virus 2 and adenovirus 5 are currently thought to enter hepatocytes in vivo through heparan sulfate proteoglycans (HSPGs). However, direct evidence for these conclusions is lacking. Experiments presented herein, in which hepatic heparan sulfate synthesis was genetically abolished, demonstrated that HSPGs are not likely to function as hepatocyte Ad5 receptors in vivo. The data also demonstrate that HSPGs are required for hepatocyte transduction by AAV2. These results reopen the question of the identity of the Ad5 receptor in vivo and emphasize the necessity of demonstrating the nature of the receptor by genetic means, both for understanding Ad5 entry into cells in vivo and for optimization of Ad5 vectors as therapeutic agents.
Asunto(s)
Adenoviridae/genética , Dependovirus/genética , Heparitina Sulfato/metabolismo , Hepatocitos/virología , Hígado/virología , Receptores Virales/metabolismo , Transducción Genética , Animales , Células Cultivadas , Femenino , Vectores Genéticos , Hepatocitos/química , Hígado/química , Masculino , RatonesRESUMEN
Cyclooxygenase-2 (COX-2) is an inducible enzyme that drives inflammation and is the therapeutic target for widely used nonsteroidal antiinflammatory drugs (NSAIDs). However, COX-2 is also constitutively expressed, in the absence of overt inflammation, with a specific tissue distribution that includes the kidney, gastrointestinal tract, brain, and thymus. Constitutive COX-2 expression is therapeutically important because NSAIDs cause cardiovascular and renal side effects in otherwise healthy individuals. These side effects are now of major concern globally. However, the pathways driving constitutive COX-2 expression remain poorly understood. Here we show that in the kidney and other sites, constitutive COX-2 expression is a sterile response, independent of commensal microorganisms and not associated with activity of the inflammatory transcription factor NF-κB. Instead, COX-2 expression in the kidney but not other regions colocalized with nuclear factor of activated T cells (NFAT) transcription factor activity and was sensitive to inhibition of calcineurin-dependent NFAT activation. However, calcineurin/NFAT regulation did not contribute to constitutive expression elsewhere or to inflammatory COX-2 induction at any site. These data address the mechanisms driving constitutive COX-2 and suggest that by targeting transcription it may be possible to develop antiinflammatory therapies that spare the constitutive expression necessary for normal homeostatic functions, including those important to the cardiovascular-renal system.
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Ciclooxigenasa 2/genética , FN-kappa B/metabolismo , Factores de Transcripción NFATC/genética , Transducción de Señal , Transcripción Genética , Animales , Ciclooxigenasa 2/metabolismo , Ciclosporina/farmacología , Citocinas/metabolismo , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Vida Libre de Gérmenes , Riñón/efectos de los fármacos , Riñón/metabolismo , Lipopolisacáridos/farmacología , Luciferasas/metabolismo , Masculino , Ratones Endogámicos C57BL , Factores de Transcripción NFATC/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Distribución Tisular/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
Tissue factor pathway inhibitor-2 (TFPI-2) is a homologue of TFPI-1 and contains three Kunitz-type domains and a basic C terminus region. The N-terminal domain of TFPI-2 is the only inhibitory domain, and it inhibits plasma kallikrein, factor XIa, and plasmin. However, plasma TFPI-2 levels are negligible (≤20 pM) in the context of influencing clotting or fibrinolysis. Here, we report that platelets contain significant amounts of TFPI-2 derived from megakaryocytes. We employed RT-PCR, Western blotting, immunohistochemistry, and confocal microscopy to determine that platelets, MEG-01 megakaryoblastic cells, and bone marrow megakaryocytes contain TFPI-2. ELISA data reveal that TFPI-2 binds factor V (FV) and partially B-domain-deleted FV (FV-1033) with K(d) ~9 nM and binds FVa with K(d) ~100 nM. Steady state analysis of surface plasmon resonance data reveal that TFPI-2 and TFPI-1 bind FV-1033 with K(d) ~36-48 nM and bind FVa with K(d) ~252-456 nM. Further, TFPI-1 (but not TFPI-1161) competes with TFPI-2 in binding to FV. These data indicate that the C-terminal basic region of TFPI-2 is similar to that of TFPI-1 and plays a role in binding to the FV B-domain acidic region. Using pull-down assays and Western blots, we show that TFPI-2 is associated with platelet FV/FVa. TFPI-2 (~7 nM) in plasma of women at the onset of labor is also, in part, associated with FV. Importantly, TFPI-2 in platelets and in plasma of pregnant women inhibits FXIa and tissue-type plasminogen activator-induced clot fibrinolysis. In conclusion, TFPI-2 in platelets from normal or pregnant subjects and in plasma from pregnant women binds FV/Va and regulates intrinsic coagulation and fibrinolysis.
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Plaquetas/citología , Fibrinólisis/fisiología , Glicoproteínas/metabolismo , Lipoproteínas/metabolismo , Megacariocitos/citología , Glicoproteína IIb de Membrana Plaquetaria/metabolismo , Coagulación Sanguínea , Plaquetas/enzimología , Células de la Médula Ósea/citología , Femenino , Sangre Fetal/enzimología , Regulación de la Expresión Génica , Glicoproteínas/genética , Hemostasis , Humanos , Ligandos , Lipoproteínas/genética , Embarazo , Inhibidores de Proteasas/química , Unión Proteica , Estructura Terciaria de Proteína , Resonancia por Plasmón de SuperficieRESUMEN
Prostaglandin-endoperoxide synthase 2 (PTGS2), also known as cyclooxygenase 2 (COX-2), plays a critical role in many normal physiological functions and modulates a variety of pathological conditions. The ability to turn endogenous COX-2 on and off in a reversible fashion, at specific times and in specific cell types, would be a powerful tool in determining its role in many contexts. To achieve this goal, we took advantage of a recently developed RNA interference system in mice. An shRNA targeting the Cox2 mRNA 3'untranslated region was inserted into a microRNA expression cassette, under the control of a tetracycline response element (TRE) promoter. Transgenic mice containing the COX-2-shRNA were crossed with mice encoding a CAG promoter-driven reverse tetracycline transactivator, which activates the TRE promoter in the presence of tetracycline/doxycycline. To facilitate testing the system, we generated a knockin reporter mouse in which the firefly luciferase gene replaces the Cox2 coding region. Cox2 promoter activation in cultured cells from triple transgenic mice containing the luciferase allele, the shRNA and the transactivator transgene resulted in robust luciferase and COX-2 expression that was reversibly down-regulated by doxycycline administration. In vivo, using a skin inflammation-model, both luciferase and COX-2 expression were inhibited over 80% in mice that received doxycycline in their diet, leading to a significant reduction of infiltrating leukocytes. In summary, using inducible RNA interference to target COX-2 expression, we demonstrate potent, reversible Cox2 gene silencing in vivo. This system should provide a valuable tool to analyze cell type-specific roles for COX-2.
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Ciclooxigenasa 2 , Regulación Enzimológica de la Expresión Génica/genética , Interferencia de ARN , ARN Interferente Pequeño , Elementos de Respuesta , Animales , Línea Celular , Ciclooxigenasa 2/biosíntesis , Ciclooxigenasa 2/genética , Técnicas de Sustitución del Gen , Ratones , Ratones Transgénicos , ARN Interferente Pequeño/biosíntesis , ARN Interferente Pequeño/genéticaRESUMEN
CD20 is a tetraspanning membrane protein expressed on B lymphocytes. CD20 deficiency in both mice and humans has recently been shown to have deleterious effects on Ab responses to T-independent Ags; however, no effect on T-dependent immunity has been reported. In this study, we used a Cd20â»/â» mouse line to evaluate Ab responses to adeno-associated virus and SRBCs. The neutralizing Ab response to adeno-associated virus was significantly reduced by CD20 deficiency; both primary (IgM) and secondary (IgG1 and IgG2b) responses to SRBC were also reduced in Cd20â»/â» mice, and this was associated with a reduction in the number of germinal center B cells. A successful humoral response requires the integration of intracellular signaling networks that critically rely on calcium mobilization. In this article, we confirm that BCR-mediated calcium mobilization is reduced in Cd20â»/â» murine B cells after BCR stimulation in vitro, and further show that the reduction is due to an effect on calcium influx rather than calcium release from intracellular stores. Calcium-dependent upregulation of CD69 was impaired in CD20-deficient B cells, as was upregulation of CD86. Altogether, this study demonstrates a role for CD20 in B cell activation and T-dependent humoral immunity.
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Antígenos CD20/inmunología , Linfocitos B/inmunología , Inmunidad Humoral/inmunología , Linfocitos T/inmunología , Animales , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Inmunohistoquímica , Activación de Linfocitos/inmunología , Ratones , Ratones NoqueadosRESUMEN
Cyclooxygenase 2 (COX)-2 is induced by bacterial and viral infections and has complex, poorly understood roles in anti-pathogen immunity. Here, we use a knock-in luciferase reporter model to image Cox2 expression across a range of tissues in mice following treatment with the either the prototypical bacterial pathogen-associated molecular pattern (PAMP), LPS, which activates Toll-like receptor (TLR)4, or with poly(I:C), a viral PAMP, which activates TLR3. LPS induced Cox2 expression in all tissues examined. In contrast, poly(I:C) elicited a milder response, limited to a subset of tissues. A panel of cytokines and interferons was measured in plasma of wild-type, Cox1(-/-) and Cox2(-/-) mice treated with LPS, poly(I:C), MALP2 (TLR2/6), Pam3CSK4 (TLR2/1), R-848 (TLR7/8) or CpG ODN (TLR9), to establish whether/how each COX isoform modulates specific PAMP/TLR responses. Only LPS induced notable loss of condition in mice (inactivity, hunching, piloerection). However, all TLR agonists produced cytokine responses, many of which were modulated in specific fashions by Cox1 or Cox2 gene deletion. Notably we observed opposing effects of Cox2 gene deletion on the responses to the bacterial PAMP, LPS, and the viral PAMP, poly(I:C), consistent with the differing abilities of the PAMPs to induce Cox2 expression. Cox2 gene deletion limited the plasma IL-1ß and interferon-γ responses and hypothermia produced by LPS. In contrast, in response to poly(I:C), Cox2(-/-) mice exhibited enhanced plasma interferon (IFNα,ß,γ,λ) and related cytokine responses (IP-10, IL-12). These observations suggest that a COX-2 selective inhibitor, given early in infection, may enhance and/or prolong endogenous interferon responses, and thereby increase anti-viral immunity.
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Ciclooxigenasa 2/metabolismo , Citocinas/metabolismo , Regulación de la Expresión Génica , Interferones/metabolismo , Receptores Toll-Like/metabolismo , Animales , Bacterias/metabolismo , Quimiocina CXCL10/metabolismo , Ciclooxigenasa 2/genética , Femenino , Eliminación de Gen , Técnicas de Sustitución del Gen , Interleucina-12/metabolismo , Interleucina-1beta/metabolismo , Lipopolisacáridos/metabolismo , Luciferasas , Masculino , Ratones , Ratones Endogámicos C57BL , Poli I-C/metabolismo , Virus/metabolismoRESUMEN
There are two schools of thought regarding the cyclooxygenase (COX) isoform active in the vasculature. Using urinary prostacyclin markers some groups have proposed that vascular COX-2 drives prostacyclin release. In contrast, we and others have found that COX-1, not COX-2, is responsible for vascular prostacyclin production. Our experiments have relied on immunoassays to detect the prostacyclin breakdown product, 6-keto-PGF1α and antibodies to detect COX-2 protein. Whilst these are standard approaches, used by many laboratories, antibody-based techniques are inherently indirect and have been criticized as limiting the conclusions that can be drawn. To address this question, we measured production of prostanoids, including 6-keto-PGF1α, by isolated vessels and in the circulation in vivo using liquid chromatography tandem mass spectrometry and found values essentially identical to those obtained by immunoassay. In addition, we determined expression from the Cox2 gene using a knockin reporter mouse in which luciferase activity reflects Cox2 gene expression. Using this we confirm the aorta to be essentially devoid of Cox2 driven expression. In contrast, thymus, renal medulla, and regions of the brain and gut expressed substantial levels of luciferase activity, which correlated well with COX-2-dependent prostanoid production. These data are consistent with the conclusion that COX-1 drives vascular prostacyclin release and puts the sparse expression of Cox2 in the vasculature in the context of the rest of the body. In doing so, we have identified the thymus, gut, brain and other tissues as target organs for consideration in developing a new understanding of how COX-2 protects the cardiovascular system.
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Vasos Sanguíneos/metabolismo , Ciclooxigenasa 1/genética , Ciclooxigenasa 2/genética , Epoprostenol/metabolismo , Transcriptoma , 6-Cetoprostaglandina F1 alfa/metabolismo , Animales , Aorta/metabolismo , Ciclooxigenasa 1/metabolismo , Ciclooxigenasa 2/metabolismo , Femenino , Masculino , Ratones , Ratones Noqueados , Especificidad de Órganos/genética , Prostaglandinas/metabolismo , Espectrometría de Masas en TándemRESUMEN
Serum coagulation factor X (FX) is proposed to play a major role in adenovirus tropism, promoting transduction by bridging the virus to cell-surface heparan sulfate proteoglycans (HSPGs). Both murine FX and human FX increased transduction by Ad.CMVfLuc, an adenovirus vector, in murine hepatocyte-like cells and human hepatocarcinoma cells. In contrast, only hFX increased transduction of several non-hepatic cancer cell lines and Chinese hamster ovary (CHO) cells. Not only was mFX unable to promote transduction in these cells, it competitively blocked hFX-enhanced transduction. Competition and HSPG digestion experiments suggested mFX- and hFX-enhanced transduction in hepatocyte-derived cells, and hFX-enhanced transduction in epithelial cancer cells were dependent on HSPGs. Ad·hFX-mediated transduction of CHO mutants unable to produce HSPGs was also curtailed. Hepatocyte-derived cells expressed substantially more HSPGs than the cancer cell lines. Dose-response curves and heparin-Sepharose binding suggested Ad·hFX has greater affinity for HSPGs than does Ad·mFX. In coagulation factor-depleted mice hFX also had enhanced ability, compared with mFX, to reconstitute hepatic adenovirus transduction. The results suggest that differences in Ad·hFX and Ad·mFX affinity to HSPGs may result in differences in their ability to enhance adenovirus transduction of many cells. These findings may have implications for murine models of adenovirus vector targeting.
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Adenoviridae/fisiología , Factor X/farmacología , Heparitina Sulfato/metabolismo , Proteoglicanos/metabolismo , Transducción Genética/métodos , Tropismo Viral/efectos de los fármacos , Animales , Células CHO , Cricetinae , Cricetulus , Factor X/química , Vectores Genéticos/metabolismo , Células Hep G2 , Heparitina Sulfato/química , Humanos , Ratones , Mutación , Especificidad de Órganos , Proteoglicanos/química , Especificidad de la Especie , Tropismo Viral/fisiologíaRESUMEN
Recombinant adenovirus serotype 5 (Ad5) vectors have been studied extensively in preclinical gene therapy models and in a range of clinical trials. However, innate immune responses to adenovirus vectors limit effectiveness of Ad5 based therapies. Moreover, extensive pre-existing Ad5 immunity in human populations will likely limit the clinical utility of adenovirus vectors, unless methods to circumvent neutralizing antibodies that bind virus and block target cell transduction can be developed. Furthermore, memory T cell and humoral responses to Ad5 are associated with increased toxicity, raising safety concerns for therapeutic adenovirus vectors in immunized hosts. Most preclinical studies have been performed in naïve animals; although pre-existing immunity is among the greatest hurdles for adenovirus therapies, it is also one of the most neglected experimentally. Here we summarize findings using adenovirus vectors in naïve animals, in Ad-immunized animals and in clinical trials, and review strategies proposed to overcome innate immune responses and pre-existing immunity.
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Adenoviridae/genética , Terapia Genética/métodos , Inmunidad Innata , Animales , Anticuerpos Neutralizantes/química , Encéfalo/metabolismo , Cápside/metabolismo , Ensayos Clínicos como Asunto , Hepatocitos/citología , Humanos , Memoria Inmunológica , Macrófagos del Hígado/citología , Ratones , Linfocitos T/citología , VacunaciónRESUMEN
Adenovirus is a nonenveloped dsDNA virus that activates intracellular innate immune pathways. In vivo, adenovirus-immunized mice displayed an enhanced innate immune response and diminished virus-mediated gene delivery following challenge with the adenovirus vector AdLacZ suggesting that antiviral Abs modulate viral interactions with innate immune cells. Under naive serum conditions in vitro, adenovirus binding and internalization in macrophages and the subsequent activation of innate immune mechanisms were inefficient. In contrast to the neutralizing effect observed in nonhematopoietic cells, adenovirus infection in the presence of antiviral Abs significantly increased FcR-dependent viral internalization in macrophages. In direct correlation with the increased viral internalization, antiviral Abs amplified the innate immune response to adenovirus as determined by the expression of NF-kappaB-dependent genes, type I IFNs, and caspase-dependent IL-1beta maturation. Immune serum amplified TLR9-independent type I IFN expression and enhanced NLRP3-dependent IL-1beta maturation in response to adenovirus, confirming that antiviral Abs specifically amplify intracellular innate pathways. In the presence of Abs, confocal microscopy demonstrated increased targeting of adenovirus to LAMP1-positive phagolysosomes in macrophages but not epithelial cells. These data show that antiviral Abs subvert natural viral tropism and target the adenovirus to phagolysosomes and the intracellular innate immune system in macrophages. Furthermore, these results illustrate a cross-talk where the adaptive immune system positively regulates the innate immune system and the antiviral state.
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Adenoviridae/inmunología , Anticuerpos Antivirales/inmunología , Inmunidad Innata/inmunología , Fagosomas/inmunología , Infecciones por Adenoviridae/inmunología , Animales , Macrófagos/inmunología , Macrófagos/virología , Ratones , Fagocitosis , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunologíaRESUMEN
The innate immune system recognizes nucleic acids during infection and tissue damage. Whereas viral RNA is detected by endosomal toll-like receptors (TLR3, TLR7, TLR8) and cytoplasmic RIG-I and MDA5, endosomal TLR9 and cytoplasmic DAI bind DNA, resulting in the activation of nuclear factor-kappaB and interferon regulatory factor transcription factors. However, viruses also trigger pro-inflammatory responses, which remain poorly defined. Here we show that internalized adenoviral DNA induces maturation of pro-interleukin-1beta in macrophages, which is dependent on NALP3 and ASC, components of the innate cytosolic molecular complex termed the inflammasome. Correspondingly, NALP3- and ASC-deficient mice display reduced innate inflammatory responses to adenovirus particles. Inflammasome activation also occurs as a result of transfected cytosolic bacterial, viral and mammalian (host) DNA, but in this case sensing is dependent on ASC but not NALP3. The DNA-sensing pro-inflammatory pathway functions independently of TLRs and interferon regulatory factors. Thus, in addition to viral and bacterial components or danger signals in general, inflammasomes sense potentially dangerous cytoplasmic DNA, strengthening their central role in innate immunity.
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Proteínas Portadoras/inmunología , Proteínas del Citoesqueleto/inmunología , Citosol/metabolismo , Citosol/virología , ADN/inmunología , Inmunidad Innata/inmunología , Inflamación/inmunología , Adenoviridae/genética , Adenoviridae/inmunología , Adenoviridae/fisiología , Animales , Proteínas Reguladoras de la Apoptosis , Proteínas Adaptadoras de Señalización CARD , Proteínas Portadoras/genética , Línea Celular , Proteínas del Citoesqueleto/deficiencia , Proteínas del Citoesqueleto/genética , Citosol/microbiología , ADN Viral/inmunología , Humanos , Inflamación/virología , Interleucina-1beta/inmunología , Interleucina-1beta/metabolismo , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR , Procesamiento Proteico-PostraduccionalRESUMEN
Adeno-associated virus (AAV) vectors are associated with relatively mild host immune responses in vivo. Although AAV induces very weak innate immune responses, neutralizing antibodies against the vector capsid and transgene still occur. To understand further the basis of the antiviral immune response to AAV vectors, studies were performed to characterize AAV interactions with macrophages. Primary mouse macrophages and human THP-1 cells transduced in vitro using an AAV serotype 2 (AAV2) vector encoding green fluorescent protein did not result in measurable transgene expression. An assessment of internalized vector genomes showed that AAV2 vector uptake was enhanced in the presence of normal but not heat-inactivated or C3-depleted mouse/human serum. Enhanced uptake in the presence of serum coincided with increased macrophage activation as determined by the expression of NF-kappaB-dependent genes such as macrophage inflammatory protein 2 (MIP-2), interleukin-1beta (IL-1beta), IL-8, and MIP-1beta. AAV vector serotypes 1 and 8 also activated human and mouse macrophages in a serum-dependent manner. Immunoprecipitation studies demonstrated the binding of iC3b complement protein to the AAV2 capsid in human serum. AAV2 did not activate the alternative pathway of the complement cascade and lacked cofactor activity for factor I-mediated degradation of C3b to iC3b. Instead, our results suggest that the AAV capsid also binds complement regulatory protein factor H. In vivo, complement receptor 1/2- and C3-deficient mice displayed impaired humoral immunity against AAV2 vectors, with a delay in antibody development and significantly lower neutralizing antibody titers. These results show that the complement system is an essential component of the host immune response to AAV.
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Proteínas del Sistema Complemento/fisiología , Dependovirus/inmunología , Vectores Genéticos/inmunología , Animales , Anticuerpos Antivirales/biosíntesis , Línea Celular , Dependovirus/genética , Expresión Génica , Humanos , Inmunoprecipitación , Activación de Macrófagos , Macrófagos/inmunología , Macrófagos/virología , Ratones , Ratones Endogámicos C57BLRESUMEN
Neutrophils are effectors of the innate immune response to adenovirus vectors. Following the systemic administration of Cy2-labeled AdLuc in mice, flow cytometry and PCR analysis of liver leukocytes revealed that 25% of recruited neutrophils interacted with adenovirus vectors. In vitro, flow cytometry of human neutrophils incubated with Cy2-labeled AdLuc also demonstrated a significant interaction with adenovirus vectors. Fluorescence and electron microscopy confirmed vector internalization by neutrophils. The AdLuc-neutrophil interaction reduced vector transduction efficiency by more than 50% in coincubation assays in epithelium-derived cells. Adenovirus vector uptake by neutrophils occurred independently of coxsackievirus adenovirus receptor (CAR) and capsid RGD motifs, since neutrophils do not express CAR and uptake of the RGD-deleted vector AdL.PB* was similar to that of AdLuc. Furthermore, both AdLuc and AdL.PB* activated neutrophils and induced similar degrees of L-selectin shedding. Neutrophil uptake of AdLuc was dependent on the presence of complement and antibodies, since the interaction between AdLuc and neutrophils was significantly reduced when they were incubated in immunoglobulin G-depleted or heat-inactivated human serum. Blocking of complement receptor 1 (CD35) but not complement receptor 3 (CD11b/CD18) significantly reduced neutrophil uptake of AdLuc. Blocking of Fc gammaRI (CD64), Fc gammaRII (CD32), and Fc gammaRIII (CD16) individually or together also reduced neutrophil uptake of AdLuc, although less than blocking of CD35 alone. Combined CR1 and Fc receptor blockade synergistically inhibited neutrophil-AdLuc interactions close to baseline. These results demonstrate opsonin-dependent adenovirus vector interactions with neutrophils and their corresponding receptors.
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Adenoviridae/fisiología , Neutrófilos/virología , Receptores de Complemento/fisiología , Receptores Fc/fisiología , Citometría de Flujo , Vectores Genéticos , HumanosRESUMEN
One of the biggest challenges in optimizing viral vectors for gene therapy relates to the immune response of the host. Adeno-associated virus (AAV) vectors are associated with low immunogenicity and toxicity, resulting in vector persistence and long-term transgene expression. The inability of AAV vectors to efficiently transduce or activate antigen presenting cells (APCs) may account for their decreased immunogenicity. AAV mediated gene therapy however, leads to the development of antibodies against the vector capsid. Anti-AAV antibodies have neutralizing effects that decrease the efficiency of in vivo gene therapy and can prevent vector re-administration. Furthermore, recent studies have shown that AAV vectors can elicit both cellular and humoral immune responses against the transgene product. Both cell-mediated response and humoral response to the delivered gene depend on a number of variables; including the nature of the transgene, the promoter used, the route and site of administration, vector dose and host factors. The response of the host to the vector, in terms of antigen-specific immunity, will play a substantial role in clinical outcome. It is therefore important to understand both, why AAV vectors are able to escape immunity and the circumstances and mechanisms that lead to the induction of immune responses. This review will summarize innate and adaptive immune responses to AAV vectors, discuss possible mechanisms and outline strategies, such as capsid modifications, use of alternative serotypes, or immunosuppression, which have been used to circumvent them.
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Dependovirus/genética , Dependovirus/inmunología , Vectores Genéticos/inmunología , Animales , Animales Modificados Genéticamente , Células Presentadoras de Antígenos/inmunología , Humanos , Inmunidad Innata , Células Asesinas Naturales/inmunologíaRESUMEN
Helper-dependent adenovirus (HD-Ad) vectors with all adenoviral genes deleted mediate very long-term expression of therapeutic transgenes in a variety of animal models of disease. These vectors are associated with reduced toxicity and improved safety relative to traditional early region 1 deletion first-generation Ad (FG-Ad) vectors. Many studies have clearly demonstrated that FG-Ad vectors induce innate and adaptive immune responses in vivo; however, a comprehensive analysis of host immune responses to HD-Ad vectors has not yet been performed. In DBA/2 mice, intravenous injection of HD-Ad vectors encoding LacZ (HD-AdLacZ) or a murine secreted alkaline phosphatase (HD-AdSEAP) induced an early expression of inflammatory cytokine and chemokine genes in the liver, including interferon-inducible protein 10, macrophage inflammatory protein 2, and tumor necrosis factor alpha, and were expressed in a pattern similar to that induced by FG-Ad vectors encoding AdSEAP. Like AdSEAP, and consistent with the pattern of cellular gene expression, HD-AdLacZ and HD-AdSEAP induced the recruitment of CD11b-positive leukocytes to the transduced liver within hours of administration. AdSEAP also induced a second phase of liver inflammation, consisting of inflammatory gene expression and CD3-positive lymphocytic infiltrates 7 days posttransduction. In contrast, beyond 24 h no infiltrates or expression of inflammatory genes was detected in the livers of mice receiving HD-AdSEAP. Despite the lack of liver inflammation at 7 days, Ad-specific cytotoxic T lymphocytes could be detected in mice receiving HD-AdSEAP. This lack of liver inflammation was not due to reduced transduction since levels of transgene expression and the amounts of vector DNA in the liver were equivalent in mice receiving HD-AdSEAP and AdSEAP. These results demonstrate that HD-Ad vectors induce intact innate but attenuated adaptive immune responses in vivo.
Asunto(s)
Adenoviridae/inmunología , Virus Defectuosos/inmunología , Vectores Genéticos/inmunología , Animales , Inmunidad Innata , Ratones , Ratones Endogámicos DBA , Transducción Genética , TransgenesRESUMEN
Adenovirus (Ad) vectors can produce inflammatory responses at high doses. Intravenous administration of an Ad vector expressing green fluorescent protein (AdGFP) to naive mice induced a biphasic pattern of liver cytokine/chemokine gene expression over 7 days. Tumor necrosis factor alpha (TNF-alpha), macrophage inflammatory protein 2 (MIP-2), and interferon gamma-inducible protein 10 (IP-10) genes were upregulated, with two distinct peaks of mRNA expression occurring at 6 hr and 5 days. The administration of transcription-defective AdGFP particles induced the early but not the late peak of chemokine/cytokine gene expression, confirming that Ad vector-induced inflammation is capsid dependent in the early phase and transcription dependent in the late phase. To determine the role of adenoviral capsid motifs in the early phase, capsid-modified Ad vectors were employed. The intravenous administration of the RGD-deleted Ad vector AdL.PB*, the fiber mutant AdL.F*, or the double mutant AdL.F*PB* induced similar levels of cytokine/chemokine expression compared with the wild-type vector AdLuc. Kupffer cell blockade significantly reduced liver TNF-alpha, MIP-2, and IP-10 gene expression and liver inflammation after the administration of AdL.PB* or AdL.F*PB*. Fluorescence microscopy of AdLuc- and AdL.PB*-transduced liver at 1 hr revealed localization of Ad vectors to liver sinusoids in Kupffer cell-depleted mice. AdL.PB* induced less E-selectin and VCAM-1 gene expression in liver, confirming reduced endothelial activation in mice receiving RGD-deleted Ad vectors. In vitro studies of endothelial cells demonstrated reduced transduction and endothelial activation by AdL.PB* compared with AdLuc. These results demonstrate that adenovirus capsid RGD motifs are required for efficient transduction and endothelial cell activation. Altering vector tropism represents a feasible strategy to modulate the innate response to Ad vectors in nontargeted tissues.
Asunto(s)
Adenoviridae/genética , Adenoviridae/inmunología , Vectores Genéticos/inmunología , Hígado/inmunología , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Cápside/inmunología , Cápside/metabolismo , Movimiento Celular , Quimiocinas/biosíntesis , Citocinas/biosíntesis , Virus Defectuosos/genética , Endotelio Vascular/metabolismo , Vectores Genéticos/análisis , Vectores Genéticos/toxicidad , Humanos , Macrófagos del Hígado/inmunología , Hígado/anatomía & histología , Hígado/virología , Ratones , Ratones Endogámicos DBA , Neutrófilos/inmunología , Oligopéptidos/genética , Linfocitos T Citotóxicos/inmunología , Transcripción Genética , Transducción GenéticaRESUMEN
The use of adenovirus vectors for human gene therapy is limited by potent inflammatory responses that result in significant morbidity. In kidney-derived epithelial cells (REC), activation of extracellular signal-regulated kinase 1/2 (ERK) and p38 kinase (p38) pathways occurred within 20 min of transduction with the serotype 5 adenovirus vector AdCMV beta gal. Inhibition of ERK and p38 with U0126 and SB203580, respectively, reduced the expression of IP-10 mRNA following transduction with AdCMV beta gal. To determine the role of the coxsackievirus-adenovirus receptor (CAR) or alpha(v) integrins in the activation of ERK and p38 and the expression of IP-10, REC cells were transduced with the fiber-modified and RGD-deleted adenovirus vectors AdL.F(RAEK-HA) and AdL.PB(HA), respectively. Compared with the wild-type capsid vector Ad5Luc, transduction with AdL.F(RAEK-HA) and AdL.PB(HA) resulted in reduced ERK-p38 activation and less IP-10 mRNA expression. The decreased IP-10 expression induced by the tropism-modified vectors was due to diminished transduction, since increasing multiplicity of infection resulted in increased IP-10 expression. Inhibition of adenovirus penetration with bafilomycin A1 or ammonium chloride attenuated the activation of ERK-p38 and IP-10 mRNA expression following infection, suggesting that endosomal escape was required to trigger these pathways. In vivo, direct inhibition of ERK and p38 signaling pathways inhibited adenovirus vector-induced IP-10 expression in mouse liver 1 h following transduction. These results demonstrate the importance of signaling via ERK and p38 in the early host response to adenovirus vectors and will permit the development of novel strategies to improve the safety and efficacy of these agents in human gene therapy.