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1.
Front Immunol ; 13: 970499, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36148237

RESUMEN

Unintended immunogenicity can affect the safety and efficacy of therapeutic proteins and peptides, so accurate assessments of immunogenicity risk can aid in the selection, development, and regulation of biologics. Product- and process- related impurities can act as adjuvants that activate the local or systemic innate immune response increasing the likelihood of product immunogenicity. Thus, assessing whether products have innate immune response modulating impurities (IIRMI) is a key component of immunogenicity risk assessments. Identifying trace levels of individual IIRMI can be difficult and testing individually for all potential impurities is not feasible. Therefore, to mitigate the risk, cell-based assays that use human blood cells or monocyte-macrophage reporter cell lines are being developed to detect minute quantities of impurities capable of eliciting innate immune activation. As these are cell-based assays, there is concern that excipients could blunt the cell responses, masking the presence of immunogenic IIRMI. Here, we explore the impact of frequently used excipients (non-ionic detergents, sugars, amino acids, bulking agents) on the sensitivity of reporter cell lines (THP-1- and RAW-Blue cells) and fresh human blood cells to detect purified TLR agonists as model IIRMI. We show that while excipients do not modulate the innate immune response elicited by TLR agonists in vivo, they can impact on the sensitivity of cell-based IIRMI assays. Reduced sensitivity to detect LPS, FSL-1, and other model IIRMI was also evident when testing 3 different recombinant drug products, product A (a representative mAb), B (a representative growth factor), C (a representative peptide), and their corresponding formulations. These results indicate that product formulations need to be considered when developing and validating cell-based assays for assessing clinically relevant levels of IIRMI in therapeutic proteins. Optimization of reporter cells, culture conditions and drug product concentration appear to be critical to minimize the impact of excipients and attain sensitive and reproducible assays.


Asunto(s)
Productos Biológicos , Excipientes , Adyuvantes Inmunológicos , Amino Azúcares , Detergentes , Excipientes/química , Humanos , Inmunidad Innata , Péptidos y Proteínas de Señalización Intercelular , Lipopolisacáridos , Péptidos
2.
Front Immunol ; 13: 919815, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35967447

RESUMEN

Since first reported in 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is rapidly acquiring mutations, particularly in the spike protein, that can modulate pathogenicity, transmission and antibody evasion leading to successive waves of COVID19 infections despite an unprecedented mass vaccination necessitating continuous adaptation of therapeutics. Small animal models can facilitate understanding host-pathogen interactions, target selection for therapeutic drugs, and vaccine development, but availability and cost of studies in BSL3 facilities hinder progress. To generate a BSL2-compatible in vivo system that specifically recapitulates spike protein mediated disease we used replication competent, GFP tagged, recombinant Vesicular Stomatitis Virus where the VSV glycoprotein was replaced by the SARS-CoV-2 spike protein (rVSV-SARS2-S). We show that infection requires hACE2 and challenge of neonatal but not adult, K18-hACE2 transgenic mice (hACE2tg) leads to productive infection of the lungs and brains. Although disease progression was faster in SARS-CoV-2 infected mice, infection with both viruses resulted in neuronal infection and encephalitis with increased expression of Interferon-stimulated Irf7, Bst2, Ifi294, as well as CxCL10, CCL5, CLC2, and LILRB4, and both models were uniformly lethal. Further, prophylactic treatment targeting the Spike protein (Receptor Binding Domain) with antibodies resulted in similar levels of protection from lethal infection against rVSV-SARS2-S and SARS-CoV-2 viruses. Strikingly, challenge of neonatal hACE2tg mice with SARS-CoV-2 Variants of Concern (SARS-CoV-2-α, -ß, ϒ, or Δ) or the corresponding rVSV-SARS2-S viruses (rVSV-SARS2-Spike-α, rVSV-SARS2-Spike-ß, rVSV-SARS2-Spike-ϒ or rVSV-SARS2-Spike-Δ) resulted in increased lethality, suggesting that the Spike protein plays a key role in determining the virulence of each variant. Thus, we propose that rVSV-SARS2-S virus can be used to understand the effect of changes to SARS-CoV-2 spike protein on infection and to evaluate existing or experimental therapeutics targeting spike protein of current or future VOC of SARS-CoV-2 under BSL-2 conditions.


Asunto(s)
COVID-19 , Glicoproteína de la Espiga del Coronavirus , Animales , Modelos Animales de Enfermedad , Humanos , Glicoproteínas de Membrana/metabolismo , Ratones , Receptores Inmunológicos , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/genética
3.
Pharm Res ; 38(11): 1961-1975, 2021 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-34845573

RESUMEN

PURPOSE: Polysorbate excipients are commonly used as surfactants to stabilize therapeutic proteins in formulations. Degradation of polysorbates could lead to particle formation and instability of the drug formulation. We investigated how the fatty acid composition of polysorbate 80 impacts the degradation profile, particle formation, and product stability under stress conditions. METHODS: Two polysorbate 80-containing therapeutic protein formulations were reformulated with either Polysorbate 80 NF synthesized from a fatty acid mixture that contains mainly oleic acid (≥58%) or a version of polysorbate 80 synthesized with high oleic acid (>98%). Stress conditions, including high temperature and esterase spiking, were applied and changes to both the polysorbate and the therapeutic protein product were investigated for stability, purity, innate immune response and biological activity. RESULTS: The addition of esterase and storage at 37°C led to significant hydrolysis of the polysorbate and increases in sub-visible particle formation for both polysorbates tested. The fatty acid composition of polysorbate 80 did not directly alter the stability profile of either therapeutic protein as measured by size exclusion chromatography, or significantly impact innate immune response or biological activity. However, formulations with Polysorbate 80 NF showed greater propensity for sub-visible particle formation under stress conditions. CONCLUSIONS: These results suggest that composition of fatty acids in polysorbate 80 may be a promoter for sub-visible particulate formation under the stress conditions tested but may not impact protein aggregation or biological activity.


Asunto(s)
Excipientes/química , Ácidos Grasos/química , Polisorbatos/química , Rituximab/química , Línea Celular Tumoral , Química Farmacéutica , Composición de Medicamentos/métodos , Humanos , Inmunidad Innata/efectos de los fármacos , Leucocitos Mononucleares , Estabilidad Proteica , Rituximab/farmacología , Rituximab/uso terapéutico
4.
PLoS Negl Trop Dis ; 14(2): e0008050, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-32109251

RESUMEN

Cutaneous leishmaniasis (CL) affects the lives of 0.7-1 million people every year causing lesions that take months to heal. These lesions can result in disfiguring scars with psychological, social and economic consequences. Antimonials are the first line of therapy for CL, however the treatment is lengthy and linked to significant toxicities; further, its efficacy is variable and resistant parasites are emerging. Shorter or lower dose antimonial treatment regimens, which would decrease the risk of adverse events and improve patient compliance, have shown reduced efficacy and further increase the risk emergence of antimonial-resistant strains. The progression of lesions in CL is partly determined by the immune response it elicits, and previous studies showed that administration of immunomodulatory type D CpG ODNs, magnifies the immune response to Leishmania and reduces lesion severity in nonhuman primates (NHP) challenged with Leishmania major or Leishmania amazonensis. Here we explored whether the addition of a single dose of immunomodulating CpG ODN D35 augments the efficacy of a short-course, low-dose pentavalent antimonial treatment regimen. Results show that macaques treated with D35 plus 5mg/kg sodium stibogluconate (SbV) for 10 days had smaller lesions and reduced time to re-epithelization after infection with Leishmania major. No toxicities were evident during the studies, even at doses of D35 10 times higher than those used in treatment. Critically, pentavalent antimonial treatment did not modify the ability of D35 to induce type I IFNs. The findings support the efficacy of D35 as adjuvant therapy for shorter, low dose pentavalent antimonial treatment.


Asunto(s)
Leishmaniasis Cutánea/tratamiento farmacológico , Oligodesoxirribonucleótidos/clasificación , Oligodesoxirribonucleótidos/uso terapéutico , Animales , Antimonio/administración & dosificación , Antimonio/farmacología , Línea Celular , Quimiocinas/genética , Quimiocinas/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Humanos , Leishmania major , Leishmaniasis Cutánea/parasitología , Leucocitos Mononucleares/efectos de los fármacos , Macaca fascicularis , Masculino , Oligodesoxirribonucleótidos/administración & dosificación
5.
Mol Nutr Food Res ; 61(8)2017 08.
Artículo en Inglés | MEDLINE | ID: mdl-28102587

RESUMEN

SCOPE: α-Cyclodextrin (α-CD), a cyclic polymer of glucose, has been shown to lower plasma cholesterol in animals and humans; however, its effect on atherosclerosis has not been previously described. METHODS AND RESULTS: apoE-knockout mice were fed either low-fat diet (LFD; 5.2% fat, w/w), or Western high fat diet (21.2% fat) containing either no additions (WD), 1.5% α-CD (WDA); 1.5% ß-CD (WDB); or 1.5% oligofructose-enriched inulin (WDI). Although plasma lipids were similar after 11 weeks on the WD vs. WDA diets, aortic atherosclerotic lesions were 65% less in mice on WDA compared to WD (P < 0.05), and similar to mice fed the LFD. No effect on atherosclerosis was observed for the other WD supplemented diets. By RNA-seq analysis of 16S rRNA, addition of α-CD to the WD resulted in significantly decreased cecal bacterial counts in genera Clostridium and Turicibacterium, and significantly increased Dehalobacteriaceae. At family level, Comamonadaceae significantly increased and Peptostreptococcaceae showed a negative trend. Several of these bacterial count changes correlated negatively with % atherosclerotic lesion and were associated with increased cecum weight and decreased plasma cholesterol levels. CONCLUSION: Addition of α-CD to the diet of apoE-knockout mice decreases atherosclerosis and is associated with changes in the gut flora.


Asunto(s)
Aterosclerosis/dietoterapia , Microbioma Gastrointestinal/efectos de los fármacos , Lípidos/sangre , alfa-Ciclodextrinas/farmacología , Animales , Aorta/efectos de los fármacos , Aorta/patología , Aterosclerosis/microbiología , Aterosclerosis/patología , Peso Corporal/efectos de los fármacos , Ciego/efectos de los fármacos , Ciego/microbiología , Dieta con Restricción de Grasas , Dieta Alta en Grasa/efectos adversos , Suplementos Dietéticos , Femenino , Microbioma Gastrointestinal/genética , Absorción Intestinal , Lípidos/farmacocinética , Ratones Noqueados para ApoE , alfa-Ciclodextrinas/metabolismo , beta-Ciclodextrinas/metabolismo , beta-Ciclodextrinas/farmacología
6.
Arthritis Rheumatol ; 69(1): 148-160, 2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-27429362

RESUMEN

OBJECTIVE: Dysregulation of innate and adaptive immune responses contributes to the pathogenesis of systemic lupus erythematosus (SLE) and its associated premature vascular damage. No drug to date targets both systemic inflammatory disease and the cardiovascular complications of SLE. Tofacitinib is a JAK inhibitor that blocks signaling downstream of multiple cytokines implicated in lupus pathogenesis. While clinical trials have shown that tofacitinib exhibits significant clinical efficacy in various autoimmune diseases, its role in SLE and the associated vascular pathology remains to be characterized. METHODS: MRL/lpr lupus-prone mice were administered tofacitinib or vehicle by gavage for 6 weeks (therapeutic arm) or 8 weeks (preventive arm). Nephritis, skin inflammation, serum levels of autoantibodies and cytokines, mononuclear cell phenotype and gene expression, neutrophil extracellular traps (NETs) release, endothelium-dependent vasorelaxation, and endothelial differentiation were compared in treated and untreated mice. RESULTS: Treatment with tofacitinib led to significant improvement in measures of disease activity, including nephritis, skin inflammation, and autoantibody production. In addition, tofacitinib treatment reduced serum levels of proinflammatory cytokines and interferon responses in splenocytes and kidney tissue. Tofacitinib also modulated the formation of NETs and significantly increased endothelium-dependent vasorelaxation and endothelial differentiation. The drug was effective in both preventive and therapeutic strategies. CONCLUSION: Tofacitinib modulates the innate and adaptive immune responses, ameliorates murine lupus, and improves vascular function. These results indicate that JAK inhibitors have the potential to be beneficial in SLE and its associated vascular damage.


Asunto(s)
Lupus Eritematoso Sistémico/complicaciones , Lupus Eritematoso Sistémico/tratamiento farmacológico , Piperidinas/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/uso terapéutico , Pirroles/uso terapéutico , Enfermedades Vasculares/tratamiento farmacológico , Enfermedades Vasculares/etiología , Animales , Femenino , Lupus Eritematoso Sistémico/inmunología , Ratones , Ratones Endogámicos MRL lpr , Enfermedades Vasculares/inmunología
7.
Immunology ; 149(3): 306-319, 2016 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-27329564

RESUMEN

Interleukin-1ß (IL-1ß), a potent pro-inflammatory cytokine, has been implicated in many diseases, including atherosclerosis. Activation of IL-1ß is controlled by a multi-protein complex, the inflammasome. The exact initiating event in atherosclerosis is unknown, but recent work has demonstrated that cholesterol crystals (CC) may promote atherosclerosis development by activation of the inflammasome. High-density lipoprotein (HDL) has consistently been shown to be anti-atherogenic and to have anti-inflammatory effects, but its mechanism of action is unclear. We demonstrate here that HDL is able to suppress IL-1ß secretion in response to cholesterol crystals in THP-1 cells and in human-monocyte-derived macrophages. HDL is able to blunt inflammatory monocyte cell recruitment in vivo following intraperitoneal CC injection in mice. HDL appears to modulate inflammasome activation in several ways. It reduces the loss of lysosomal membrane integrity following the phagocytosis of CC, but the major mechanism for the suppression of inflammasome activation by HDL is decreased expression of pro-IL-1ß and NLRP3, and reducing caspase-1 activation. In summary, we have described a novel anti-inflammatory effect of HDL, namely its ability to suppress inflammasome activation by CC by modulating the expression of several key components of the inflammasome.


Asunto(s)
Antiinflamatorios/uso terapéutico , Aterosclerosis/tratamiento farmacológico , Inflamasomas/metabolismo , Inflamación/tratamiento farmacológico , Interleucina-1beta/metabolismo , Lipoproteínas HDL/uso terapéutico , Macrófagos/efectos de los fármacos , Animales , Aterosclerosis/inmunología , Línea Celular , Colesterol/inmunología , Femenino , Humanos , Inflamación/inmunología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo
8.
PLoS One ; 11(2): e0150083, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26919698

RESUMEN

Human familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is characterized by low HDL, accumulation of an abnormal cholesterol-rich multilamellar particle called lipoprotein-X (LpX) in plasma, and renal disease. The aim of our study was to determine if LpX is nephrotoxic and to gain insight into the pathogenesis of FLD renal disease. We administered a synthetic LpX, nearly identical to endogenous LpX in its physical, chemical and biologic characteristics, to wild-type and Lcat-/- mice. Our in vitro and in vivo studies demonstrated an apoA-I and LCAT-dependent pathway for LpX conversion to HDL-like particles, which likely mediates normal plasma clearance of LpX. Plasma clearance of exogenous LpX was markedly delayed in Lcat-/- mice, which have low HDL, but only minimal amounts of endogenous LpX and do not spontaneously develop renal disease. Chronically administered exogenous LpX deposited in all renal glomerular cellular and matrical compartments of Lcat-/- mice, and induced proteinuria and nephrotoxic gene changes, as well as all of the hallmarks of FLD renal disease as assessed by histological, TEM, and SEM analyses. Extensive in vivo EM studies revealed LpX uptake by macropinocytosis into mouse glomerular endothelial cells, podocytes, and mesangial cells and delivery to lysosomes where it was degraded. Endocytosed LpX appeared to be degraded by both human podocyte and mesangial cell lysosomal PLA2 and induced podocyte secretion of pro-inflammatory IL-6 in vitro and renal Cxl10 expression in Lcat-/- mice. In conclusion, LpX is a nephrotoxic particle that in the absence of Lcat induces all of the histological and functional hallmarks of FLD and hence may serve as a biomarker for monitoring recombinant LCAT therapy. In addition, our studies suggest that LpX-induced loss of endothelial barrier function and release of cytokines by renal glomerular cells likely plays a role in the initiation and progression of FLD nephrosis.


Asunto(s)
Glomérulos Renales/efectos de los fármacos , Deficiencia de la Lecitina Colesterol Aciltransferasa/metabolismo , Lipoproteína X/toxicidad , Proteinuria/etiología , Animales , Apolipoproteína A-I/metabolismo , Células Cultivadas , Citoesqueleto/efectos de los fármacos , Citoesqueleto/ultraestructura , Células Endoteliales/metabolismo , Células Endoteliales/patología , Matriz Extracelular/metabolismo , Perfilación de la Expresión Génica , Membrana Basal Glomerular/efectos de los fármacos , Membrana Basal Glomerular/patología , Mesangio Glomerular/citología , Mesangio Glomerular/metabolismo , Mesangio Glomerular/patología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Interleucina-6/metabolismo , Glomérulos Renales/patología , Deficiencia de la Lecitina Colesterol Aciltransferasa/patología , Lipoproteína X/metabolismo , Lipoproteína X/farmacocinética , Lipoproteínas HDL/metabolismo , Lisosomas/metabolismo , Tasa de Depuración Metabólica , Ratones , Ratones Endogámicos C57BL , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Fosfolipasas A2/metabolismo , Pinocitosis , Podocitos/metabolismo , Podocitos/patología , Proteinuria/inducido químicamente , Proteinuria/genética , Proteinuria/patología
9.
J Lipid Res ; 56(7): 1282-95, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-25964513

RESUMEN

LCAT, a plasma enzyme that esterifies cholesterol, has been proposed to play an antiatherogenic role, but animal and epidemiologic studies have yielded conflicting results. To gain insight into LCAT and the role of free cholesterol (FC) in atherosclerosis, we examined the effect of LCAT over- and underexpression in diet-induced atherosclerosis in scavenger receptor class B member I-deficient [Scarab(-/-)] mice, which have a secondary defect in cholesterol esterification. Scarab(-/-)×LCAT-null [Lcat(-/-)] mice had a decrease in HDL-cholesterol and a high plasma ratio of FC/total cholesterol (TC) (0.88 ± 0.033) and a marked increase in VLDL-cholesterol (VLDL-C) on a high-fat diet. Scarab(-/-)×LCAT-transgenic (Tg) mice had lower levels of VLDL-C and a normal plasma FC/TC ratio (0.28 ± 0.005). Plasma from Scarab(-/-)×LCAT-Tg mice also showed an increase in cholesterol esterification during in vitro cholesterol efflux, but increased esterification did not appear to affect the overall rate of cholesterol efflux or hepatic uptake of cholesterol. Scarab(-/-)×LCAT-Tg mice also displayed a 51% decrease in aortic sinus atherosclerosis compared with Scarab(-/-) mice (P < 0.05). In summary, we demonstrate that increased cholesterol esterification by LCAT is atheroprotective, most likely through its ability to increase HDL levels and decrease pro-atherogenic apoB-containing lipoprotein particles.


Asunto(s)
Aterosclerosis/sangre , Aterosclerosis/enzimología , Antígenos CD36/deficiencia , Antígenos CD36/genética , Colesterol/metabolismo , Dieta Alta en Grasa/efectos adversos , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Animales , Aterosclerosis/etiología , Aterosclerosis/metabolismo , Transporte Biológico , Plaquetas/metabolismo , Plaquetas/patología , Colesterol/sangre , Recuento de Eritrocitos , Eritrocitos/metabolismo , Eritrocitos/patología , Esterificación , Femenino , Regulación Enzimológica de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Lipoproteínas VLDL/biosíntesis , Lipoproteínas VLDL/sangre , Lipoproteínas VLDL/química , Hígado/metabolismo , Ratones , Ratones Transgénicos , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Recuento de Plaquetas
10.
J Clin Lipidol ; 9(1): 81-90, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25670364

RESUMEN

BACKGROUND: Low high-density lipoprotein cholesterol (HDL-C) is a risk factor for coronary artery disease. Investigating mechanisms underlying acquired severe HDL deficiency in noncritically ill patients ("disappearing HDL syndrome") could provide new insights into HDL metabolism. OBJECTIVE: To determine the cause of low HDL-C in patients with severe acquired HDL deficiency. METHODS AND RESULTS: Patients with intravascular large B-cell lymphoma (n = 2), diffuse large B-cell lymphoma (n = 1), and autoimmune lymphoproliferative syndrome (n = 1) presenting with markedly decreased HDL-C, low low-density lipoprotein cholesterol (LDL-C), and elevated triglycerides were identified. The abnormal lipoprotein profile returned to normal after therapy in all 4 patients. All patients were found to have markedly elevated serum interleukin-10 (IL-10) levels that also normalized after therapy. In a cohort of autoimmune lymphoproliferative syndrome patients (n = 93), IL-10 showed a strong inverse correlation with HDL-C (R(2) = 0.3720, P < .0001). A direct causal role for increased serum IL-10 in inducing the observed changes in lipoproteins was established in a randomized, placebo-controlled clinical trial of recombinant human IL-10 in psoriatic arthritis patients (n = 18). Within a week of initiating subcutaneous recombinant human IL-10 injections, HDL-C precipitously decreased to near-undetectable levels. LDL-C also decreased by more than 50% (P < .0001) and triglycerides increased by approximately 2-fold (P < .005). All values returned to baseline after discontinuing IL-10 therapy. CONCLUSION: Increased IL-10 causes severe HDL-C deficiency, low LDL-C, and elevated triglycerides. IL-10 is thus a potent modulator of lipoprotein levels, a potential new biomarker for B-cell disorders, and a novel cause of disappearing HDL syndrome.


Asunto(s)
HDL-Colesterol/sangre , Dislipidemias/diagnóstico , Interleucina-10/sangre , Adulto , Artritis Psoriásica/tratamiento farmacológico , Síndrome Linfoproliferativo Autoinmune/sangre , Síndrome Linfoproliferativo Autoinmune/diagnóstico , Niño , LDL-Colesterol/sangre , Estudios de Cohortes , Dislipidemias/sangre , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Lactante , Interleucina-10/genética , Interleucina-10/uso terapéutico , Linfoma de Células B/diagnóstico , Linfoma de Células B Grandes Difuso/diagnóstico , Masculino , Persona de Mediana Edad , Efecto Placebo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapéutico , Resultado del Tratamiento , Triglicéridos/sangre , Receptor fas/genética
11.
Chembiochem ; 15(14): 2087-96, 2014 Sep 22.
Artículo en Inglés | MEDLINE | ID: mdl-25154602

RESUMEN

We report a synthetic route to BODIPY-cholesterol conjugates in which the key steps were Suzuki or Liebeskind-Srogl cross-coupling of cholesterol phenyl moieties with structurally diverse BODIPY scaffolds. All conjugates feature single-bonded and hydrophobic linkages between the fluorophore and sterol that are devoid of heteroatoms. Using HeLa cells, we show that these BODIPY-cholesterol analogues can be used simultaneously with the parent BODIPY-cholesterol for cell imaging and flow cytometry. The BODIPY-cholesterol analogues exhibit similar cellular localization in HeLa cells and show similar cholesterol efflux properties from THP-1 cells to HDL acceptors. These results demonstrate that the red-shifted BODIPY-cholesterol analogues behave in a manner similar to unlabeled cholesterol and are useful probes for simultaneous visualization of intracellular cholesterol pools and for monitoring cholesterol efflux from cells to extracellular acceptors.


Asunto(s)
Compuestos de Boro/análisis , Colesterol/análisis , Colorantes Fluorescentes/análisis , Colesterol/análogos & derivados , Citometría de Flujo , Células HeLa , Humanos , Imagen Óptica
12.
Arthritis Rheum ; 64(9): 2975-85, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22549550

RESUMEN

OBJECTIVE: Patients with systemic lupus erythematosus (SLE) have a notable increase in atherothrombotic cardiovascular disease (CVD) which is not explained by the Framingham risk equation. In vitro studies indicate that type I interferons (IFNs) may play prominent roles in increased CV risk in SLE. However, the in vivo relevance of these findings, with regard to the development of CVD, has not been characterized. This study was undertaken to examine the role of type I IFNs in endothelial dysfunction, aberrant vascular repair, and atherothrombosis in murine models of lupus and atherosclerosis. METHODS: Lupus-prone New Zealand mixed 2328 (NZM) mice and atherosclerosis-prone apolipoprotein E- knockout (apoE(-/-) ) mice were compared to mice lacking type I IFN receptor (INZM and apoE(-/-) IFNAR(-/-) mice, respectively) with regard to endothelial vasodilatory function, endothelial progenitor cell (EPC) function, in vivo neoangiogenesis, plaque development, and occlusive thrombosis. Similar experiments were performed using NZM and apoE(-/-) mice exposed to an IFNα-containing or empty adenovirus. RESULTS: Loss of type I IFN receptor signaling improved endothelium-dependent vasorelaxation, lipoprotein parameters, EPC numbers and function, and neoangiogenesis in lupus-prone mice, independent of disease activity or sex. Further, acute exposure to IFNα impaired endothelial vasorelaxation and EPC function in lupus-prone and non-lupus-prone mice. Decreased atherosclerosis severity and arterial inflammatory infiltrates and increased neoangiogenesis were observed in apoE(-/-) IFNAR(-/-) mice, compared to apoE(-/-) mice, while NZM and apoE(-/-) mice exposed to IFNα developed accelerated thrombosis and platelet activation. CONCLUSION: These results support the hypothesis that type I IFNs play key roles in the development of premature CVD in SLE and, potentially, in the general population, through pleiotropic deleterious effects on the vasculature.


Asunto(s)
Aterosclerosis/metabolismo , Interferón Tipo I/metabolismo , Lupus Eritematoso Sistémico/metabolismo , Placa Aterosclerótica/metabolismo , Trombosis/metabolismo , Cicatrización de Heridas/fisiología , Animales , Aterosclerosis/fisiopatología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Femenino , Lupus Eritematoso Sistémico/fisiopatología , Masculino , Ratones , Vasodilatación/fisiología
13.
J Immunol ; 187(11): 6143-56, 2011 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-22058412

RESUMEN

Systemic lupus erythematosus (SLE) is an autoimmune disease with heterogeneous manifestations including severe organ damage and vascular dysfunction leading to premature atherosclerosis. IFN-α has been proposed to have an important role in the development of lupus and lupus-related cardiovascular disease, partly by repression of IL-1 pathways leading to impairments in vascular repair induced by endothelial progenitor cells (EPCs) and circulating angiogenic cells (CACs). Counterintuitively, SLE patients also display transcriptional upregulation of the IL-1ß/IL-18 processing machinery, the inflammasome. To understand this dichotomy and its impact on SLE-related cardiovascular disease, we examined cultures of human and murine control or lupus EPC/CACs to determine the role of the inflammasome in endothelial differentiation. We show that caspase-1 inhibition improves dysfunctional SLE EPC/CAC differentiation into mature endothelial cells and blocks IFN-α-mediated repression of this differentiation, implicating inflammasome activation as a crucial downstream pathway leading to aberrant vasculogenesis. Furthermore, serum IL-18 levels are elevated in SLE and correlate with EPC/CAC dysfunction. Exogenous IL-18 inhibits endothelial differentiation in control EPC/CACs and neutralization of IL-18 in SLE EPC/CAC cultures restores their capacity to differentiate into mature endothelial cells, supporting a deleterious effect of IL-18 on vascular repair in vivo. Upregulation of the inflammasome machinery was operational in vivo, as evidenced by gene array analysis of lupus nephritis biopsies. Thus, the effects of IFN-α are complex and contribute to an elevated risk of cardiovascular disease by suppression of IL-1ß pathways and by upregulation of the inflammasome machinery and potentiation of IL-18 activation.


Asunto(s)
Células Endoteliales/inmunología , Inflamasomas/inmunología , Interferón-alfa/inmunología , Interleucina-18/inmunología , Lupus Eritematoso Sistémico/inmunología , Animales , Western Blotting , Diferenciación Celular/inmunología , Separación Celular , Células Endoteliales/metabolismo , Células Endoteliales/patología , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Interferón-alfa/metabolismo , Interleucina-18/metabolismo , Lupus Eritematoso Sistémico/metabolismo , Lupus Eritematoso Sistémico/patología , Ratones , Ratones Endogámicos MRL lpr , Análisis por Micromatrices , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/inmunología , Células Madre/metabolismo , Células Madre/patología
14.
J Immunol ; 185(7): 4457-69, 2010 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-20805419

RESUMEN

Systemic lupus erythematosus (SLE) is characterized by increased vascular risk due to premature atherosclerosis independent of traditional risk factors. We previously proposed that IFN-α plays a crucial role in premature vascular damage in SLE. IFN-α alters the balance between endothelial cell apoptosis and vascular repair mediated by endothelial progenitor cells (EPCs) and myeloid circulating angiogenic cells (CACs). In this study, we demonstrate that IFN-α promotes an antiangiogenic signature in SLE and control EPCs/CACs, characterized by transcriptional repression of IL-1α and ß, IL-1R1, and vascular endothelial growth factor A, and upregulation of IL-1R antagonist and the decoy receptor IL-1R2. IL-1ß promotes significant improvement in the functional capacity of lupus EPCs/CACs, therefore abrogating the deleterious effects of IFN-α. The beneficial effects from IL-1 are mediated, at least in part, by increases in EPC/CAC proliferation, by decreases in EPC/CAC apoptosis, and by preventing the skewing of CACs toward nonangiogenic pathways. IFN-α induces STAT2 and 6 phosphorylation in EPCs/CACs, and JAK inhibition abrogates the transcriptional antiangiogenic changes induced by IFN-α in these cells. Immunohistochemistry of renal biopsies from patients with lupus nephritis, but not anti-neutrophil cytoplasmic Ab-positive vasculitis, showed this pathway to be operational in vivo, with increased IL-1R antagonist, downregulation of vascular endothelial growth factor A, and glomerular and blood vessel decreased capillary density, compared with controls. Our study introduces a novel putative pathway by which type I IFNs may interfere with vascular repair in SLE through repression of IL-1-dependent pathways. This could promote atherosclerosis and loss of renal function in this disease.


Asunto(s)
Aterosclerosis/metabolismo , Interferón-alfa/metabolismo , Interleucina-1/metabolismo , Lupus Eritematoso Sistémico/metabolismo , Neovascularización Fisiológica , Adulto , Aterosclerosis/inmunología , Diferenciación Celular/fisiología , Células Endoteliales/metabolismo , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Proteína Antagonista del Receptor de Interleucina 1/biosíntesis , Interleucina-1/inmunología , Riñón/irrigación sanguínea , Riñón/metabolismo , Riñón/patología , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/patología , Nefritis Lúpica/metabolismo , Nefritis Lúpica/patología , Masculino , Microscopía Fluorescente , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores de Interleucina-1/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiología , Células Madre , Factor A de Crecimiento Endotelial Vascular/biosíntesis
15.
J Immunol ; 184(6): 3284-97, 2010 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-20164424

RESUMEN

Neutrophil-specific genes are abundant in PBMC microarrays from lupus patients because of the presence of low-density granulocytes (LDGs) in mononuclear cell fractions. The functionality and pathogenicity of these LDGs have not been characterized. We developed a technique to purify LDGs from lupus PBMCs and assessed their phenotype, function, and potential role in disease pathogenesis. LDGs, their autologous lupus neutrophils, and healthy control neutrophils were compared with regard to their microbicidal and phagocytic capacities, generation of reactive oxygen species, activation status, inflammatory cytokine profile, and type I IFN expression and signatures. The capacity of LDGs to kill endothelial cells and their antiangiogenic potential were also assessed. LDGs display an activated phenotype, secrete increased levels of type I IFNs, TNF-alpha, and IFN-gamma, but show impaired phagocytic potential. LDGs induce significant endothelial cell cytotoxicity and synthesize sufficient levels of type I IFNs to disrupt the capacity of endothelial progenitor cells to differentiate into mature endothelial cells. LDG depletion restores the functional capacity of endothelial progenitor cells. We conclude that lupus LDGs are proinflammatory and display pathogenic features, including the capacity to synthesize type I IFNs. They may play an important dual role in premature cardiovascular disease development in systemic lupus erythematosus by simultaneously mediating enhanced vascular damage and inhibiting vascular repair.


Asunto(s)
Endotelio Vascular/inmunología , Endotelio Vascular/patología , Mediadores de Inflamación/fisiología , Interferón Tipo I/biosíntesis , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/patología , Neutrófilos/inmunología , Neutrófilos/patología , Adulto , Separación Celular , Células Cultivadas , Endotelio Vascular/metabolismo , Femenino , Granulocitos/inmunología , Granulocitos/metabolismo , Granulocitos/patología , Humanos , Mediadores de Inflamación/metabolismo , Interferón Tipo I/fisiología , Interferón-alfa/biosíntesis , Interferón-alfa/fisiología , Recuento de Leucocitos , Lupus Eritematoso Sistémico/fisiopatología , Masculino , Células Progenitoras Mieloides/inmunología , Células Progenitoras Mieloides/metabolismo , Células Progenitoras Mieloides/patología , Neutrófilos/metabolismo
16.
J Immunol ; 183(4): 2729-40, 2009 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-19620300

RESUMEN

Individuals with systemic lupus erythematosus (SLE) have a striking increase in the risk of premature atherosclerosis, a complication preceded by significant subclinical vascular damage. A proposed mechanism leading to accelerated vascular disease in SLE is an imbalance between vascular damage and repair, as patients with this disease display significant abnormalities in phenotype and function of endothelial progenitor cells. In addition, individuals with SLE have a higher incidence of insulin resistance which may further contribute to the increased cardiovascular risk. This study examined the role of the peroxisome proliferator activated receptor gamma agonist pioglitazone in improving endothelial function, endothelial progenitor cell numbers and functional capacity, metabolic parameters, and disease activity in the lupus-prone murine model New Zealand Black/New Zealand White (NZB x NZW)F(1). Ten-week-old prenephritic female NZB/NZW F(1) mice were exposed to 10 or 25 mg/kg/day of oral pioglitazone or vehicle for 15 or 24 wk. Mice exposed to pioglitazone exhibited pronounced enhancement in endothelial-dependent vasorelaxation of thoracic aortas and in endothelial progenitor cell function, as assessed by the capacity of bone marrow-derived endothelial progenitor cells to differentiate into mature endothelial cells. Pioglitazone-treated mice showed improvement in insulin resistance, adipokine, and lipid profile. Kidneys from pioglitazone-treated mice showed significant decreases in immune complex deposition, renal inflammation, T cell glomerular infiltration, and intrarenal synthesis of TNF-alpha, IL-1beta, and VCAM-1. These results indicate that peroxisome proliferator-activated receptor gamma agonists could serve as important tools in the prevention of premature cardiovascular disease and organ damage in SLE.


Asunto(s)
Cardiomiopatías/metabolismo , Mediadores de Inflamación/fisiología , Riñón/patología , Lupus Eritematoso Sistémico/tratamiento farmacológico , Lupus Eritematoso Sistémico/patología , PPAR gamma/agonistas , PPAR gamma/fisiología , Tiazolidinedionas/farmacología , Animales , Cardiomiopatías/tratamiento farmacológico , Cardiomiopatías/patología , Modelos Animales de Enfermedad , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Endotelio Vascular/fisiología , Femenino , Mediadores de Inflamación/agonistas , Riñón/efectos de los fármacos , Riñón/fisiopatología , Lupus Eritematoso Sistémico/metabolismo , Lupus Eritematoso Sistémico/fisiopatología , Ratones , Ratones Endogámicos NZB , Pioglitazona , Factores de Riesgo , Células Madre/efectos de los fármacos , Células Madre/patología , Células Madre/fisiología
17.
J Immunol ; 180(5): 2989-98, 2008 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-18292521

RESUMEN

Fibroblast-like synoviocytes (FLS) and T cells can activate each other in vitro, and in vivo interactions between these cells may be important in rheumatoid arthritis (RA), yet FLS lack significant expression of CD28 ligands. We sought to identify molecules homologous to CD28 ligands that are strongly expressed by FLS, and documented strong B7-H3 expression on FLS and by fibroblasts of other tissues, which was unaffected by a variety of cytokines. Western blot analysis of FLS lysates showed predominant expression of the larger, four Ig-like domain isoform of B7-H3. Immunohistological sections of RA synovial tissue showed strong staining for B7-H3 on FLS. Cells expressing B7-H3 were distinct from but in close proximity to cells that expressed CD45, CD20, and CD3. Confocal microscopy of FLS and T cell cocultures showed localization of B7-H3 in the region of the T cell-FLS contact point, but distinct from the localization of T cell CD11a/CD18 (LFA-1) and FLS CD54 (ICAM-1). Reduction of B7-H3 expression on FLS by RNA interference affected interactions of FLS with resting T cells or cytokine-activated T cells. Resting T cells showed increased production of TNF-alpha, IFN-gamma, and IL-2, whereas cytokine-activated T cells showed reduced cytokine production relative to control. However, cytokine production by T cells activated through their TCR was not notably altered by knock down of B7-H3. These observations suggest that B7-H3 may be important for the interactions between FLS and T cells in RA, as well as other diseases, and the outcome of such interactions depends on the activation state of the T cell.


Asunto(s)
Antígenos CD/fisiología , Antígeno B7-1/fisiología , Comunicación Celular/inmunología , Fibroblastos/inmunología , Activación de Linfocitos/inmunología , Receptores Inmunológicos/fisiología , Membrana Sinovial/inmunología , Subgrupos de Linfocitos T/inmunología , Antígenos CD/genética , Antígenos CD/metabolismo , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Antígenos B7 , Células Cultivadas , Técnicas de Cocultivo , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Activación de Linfocitos/genética , Osteoartritis/inmunología , Osteoartritis/metabolismo , Osteoartritis/patología , Receptores Inmunológicos/deficiencia , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Membrana Sinovial/metabolismo , Membrana Sinovial/patología , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/patología
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