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1.
Circulation ; 146(10): 724-739, 2022 09 06.
Artículo en Inglés | MEDLINE | ID: mdl-35899625

RESUMEN

BACKGROUND: Atherosclerotic cardiovascular disease is the main cause of mortality worldwide and is strongly influenced by circulating low-density lipoprotein (LDL) cholesterol levels. Only a few genes causally related to plasma LDL cholesterol levels have been identified so far, and only 1 gene, ANGPTL3, has been causally related to combined hypocholesterolemia. Here, our aim was to elucidate the genetic origin of an unexplained combined hypocholesterolemia inherited in 4 generations of a French family. METHODS: Using next-generation sequencing, we identified a novel dominant rare variant in the LIPC gene, encoding for hepatic lipase, which cosegregates with the phenotype. We characterized the impact of this LIPC-E97G variant on circulating lipid and lipoprotein levels in family members using nuclear magnetic resonance-based lipoprotein profiling and lipidomics. To uncover the mechanisms underlying the combined hypocholesterolemia, we used protein homology modeling, measured triglyceride lipase and phospholipase activities in cell culture, and studied the phenotype of APOE*3.Leiden.CETP mice after LIPC-E97G overexpression. RESULTS: Family members carrying the LIPC-E97G variant had very low circulating levels of LDL cholesterol and high-density lipoprotein cholesterol, LDL particle numbers, and phospholipids. The lysophospholipids/phospholipids ratio was increased in plasma of LIPC-E97G carriers, suggestive of an increased lipolytic activity on phospholipids. In vitro and in vivo studies confirmed that the LIPC-E97G variant specifically increases the phospholipase activity of hepatic lipase through modification of an evolutionarily conserved motif that determines substrate access to the hepatic lipase catalytic site. Mice overexpressing human LIPC-E97G recapitulated the combined hypocholesterolemic phenotype of the family and demonstrated that the increased phospholipase activity promotes catabolism of triglyceride-rich lipoproteins by different extrahepatic tissues but not the liver. CONCLUSIONS: We identified and characterized a novel rare variant in the LIPC gene in a family who presents with dominant familial combined hypocholesterolemia. This gain-of-function variant makes LIPC the second identified gene, after ANGPTL3, causally involved in familial combined hypocholesterolemia. Our mechanistic data highlight the critical role of hepatic lipase phospholipase activity in LDL cholesterol homeostasis and suggest a new LDL clearance mechanism.


Asunto(s)
Mutación con Ganancia de Función , Lipasa , Proteína 3 Similar a la Angiopoyetina , Proteínas Similares a la Angiopoyetina/genética , Animales , HDL-Colesterol , LDL-Colesterol , Humanos , Lipasa/genética , Lipoproteínas , Ratones , Fosfolipasas/genética
2.
J Lipid Res ; 62: 100096, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34280453

RESUMEN

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes lysosomal degradation of the LDL receptor and is a key regulator of cholesterol metabolism. After the liver, the small intestine is the second organ that highly expresses PCSK9. However, the small intestine's ability to secrete PCSK9 remains a matter of debate. While liver-specific PCSK9-deficient mice present no PCSK9 in systemic blood, human intestinal Caco-2 cells can actively secrete PCSK9. This raises the possibility for active intestinal secretion via the portal blood. Here, we aimed to determine whether enterocytes can secrete PCSK9 using in vitro, ex vivo, and in vivo approaches. We first observed that PCSK9 secretion from Caco-2 cells was biphasic and dependent on Caco-2 maturation status. Transcriptional analysis suggested that this transient reduction in PCSK9 secretion might be due to loss of SREBP2-mediated transcription of PCSK9. Consistently, PCSK9 secretion was not detected ex vivo in human or mouse intestinal biopsies mounted in Ussing chambers. Finally, direct comparison of systemic versus portal blood PCSK9 concentrations in WT or liver-specific PCSK9-deficient mice confirmed the inability of the small intestine to secrete PCSK9 into the portal compartment. Altogether, our data demonstrate that mature enterocytes do not secrete PCSK9 and reinforce the central role of the liver in the regulation of the concentration of circulating PCSK9 and consequently of cellular LDL receptors.


Asunto(s)
Proproteína Convertasa 9/metabolismo , Animales , Células CACO-2 , Diferenciación Celular , Células Cultivadas , Humanos , Intestino Delgado/citología , Intestino Delgado/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proproteína Convertasa 9/sangre , Proproteína Convertasa 9/deficiencia
3.
Arterioscler Thromb Vasc Biol ; 38(3): 592-598, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29284604

RESUMEN

OBJECTIVE: Evolocumab, a PCSK9 (proprotein convertase subtilisin kexin type 9)-neutralizing antibody, lowers low-density lipoprotein cholesterol (LDL-C) in homozygous familial hypercholesterolemic (HoFH) patients with reduced LDLR (low-density lipoprotein receptor) function. However, their individual responses are highly variable, even among carriers of identical LDLR genetic defects. We aimed to elucidate why HoFH patients variably respond to PCSK9 inhibition. APPROACH AND RESULTS: Lymphocytes were isolated from 22 HoFH patients enrolled in the TAUSSIG trial (Trial Assessing Long Term Use of PCSK9 Inhibition in Subjects With Genetic LDL Disorders). Ten patients were true homozygotes (FH1/FH1) and 5 identical compound heterozygotes (FH1/FH2). Lymphocytes were plated with or without mevastatin, recombinant PCSK9 (rPCSK9), or a PCSK9-neutralizing antibody. Cell surface LDLR expression was analyzed by flow cytometry. All HoFH lymphocytes had reduced cell surface LDLR expression compared with non-FH lymphocytes, for each treatment modality. Lymphocytes from FH1/FH2 patients (LDLR defective/negative) displayed the lowest LDLR expression levels followed by lymphocytes from FH1/FH1 patients (defective/defective). Mevastatin increased, whereas rPCSK9 reduced LDLR expression. The PCSK9-neutralizing antibody restored LDLR expression. Lymphocytes displaying higher LDLR expression levels were those isolated from patients presenting with lowest levels of LDL-C and apolipoprotein B, before and after 24 weeks of evolocumab treatment. These negative correlations remained significant in FH1/FH1 patients and appeared more pronounced when patients with apolipoprotein E3/E3 genotypes were analyzed separately. Significant positive correlations were found between the levels of LDLR expression and the percentage reduction in LDL-C on evolocumab treatment. CONCLUSIONS: Residual LDLR expression in HoFH is a major determinant of LDL-C levels and seems to drive their individual response to evolocumab.


Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Anticolesterolemiantes/uso terapéutico , Homocigoto , Hiperlipoproteinemia Tipo II/tratamiento farmacológico , Linfocitos/efectos de los fármacos , Mutación , Inhibidores de PCSK9 , Receptores de LDL/genética , Inhibidores de Serina Proteinasa/uso terapéutico , Adolescente , Adulto , Anticuerpos Monoclonales Humanizados , Apolipoproteína B-100/sangre , Células Cultivadas , LDL-Colesterol/sangre , Quimioterapia Combinada , Ezetimiba/uso terapéutico , Femenino , Predisposición Genética a la Enfermedad , Heterocigoto , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Hiperlipoproteinemia Tipo II/sangre , Hiperlipoproteinemia Tipo II/diagnóstico , Hiperlipoproteinemia Tipo II/genética , Lovastatina/análogos & derivados , Lovastatina/uso terapéutico , Linfocitos/enzimología , Masculino , Persona de Mediana Edad , Fenotipo , Receptores de LDL/metabolismo , Resultado del Tratamiento , Adulto Joven
4.
Clin Sci (Lond) ; 132(10): 1075-1083, 2018 05 31.
Artículo en Inglés | MEDLINE | ID: mdl-29724769

RESUMEN

Therapeutic antibodies targeting proprotein convertase subtilisin kexin type 9 (PCSK9) (e.g. alirocumab) lower low-density lipoprotein cholesterol (LDL-C) and lipoprotein (a) [Lp(a)] levels in clinical trials. We recently showed that PCSK9 enhances apolipoprotein(a) [apo(a)] secretion from primary human hepatocytes but does not affect Lp(a) cellular uptake. Here, we aimed to determine how PCSK9 neutralization modulates Lp(a) levels in vivoSix nonhuman primates (NHP) were treated with alirocumab or a control antibody (IgG1) in a crossover protocol. After the lowering of lipids reached steady state, NHP received an intravenous injection of [2H3]-leucine, and blood samples were collected sequentially over 48 h. Enrichment of apolipoproteins in [2H3]-leucine was assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Kinetic parameters were calculated using numerical models with the SAAMII software. Compared with IgG1, alirocumab significantly reduced total cholesterol (TC) (-28%), LDL-C (-67%), Lp(a) (-56%), apolipoprotein B100 (apoB100) (-53%), and apo(a) (-53%). Alirocumab significantly increased the fractional catabolic rate of apoB100 (+29%) but not that of apo(a). Conversely, alirocumab sharply and significantly reduced the production rate (PR) of apo(a) (-42%), but not significantly that of apoB100, compared with IgG1, respectively.In line with the observations made in human hepatocytes, the present kinetic study establishes that PCSK9 neutralization with alirocumab efficiently reduces circulating apoB100 and apo(a) levels by distinct mechanisms: apoB primarily by enhancing its catabolism and apo(a) primarily by lowering its production.


Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticolesterolemiantes/farmacología , Lipoproteína(a)/sangre , Inhibidores de PCSK9 , Animales , Anticuerpos Monoclonales Humanizados , Apoproteína(a)/biosíntesis , Colesterol/sangre , Estudios Cruzados , Femenino , Lípidos/sangre , Macaca fascicularis , Masculino
6.
Clin Sci (Lond) ; 131(4): 261-268, 2017 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-28108631

RESUMEN

Since 2012, clinical trials dedicated to proprotein convertase subtilisin kexin type 9 (PCSK9) inhibition with monoclonal antibodies (mAbs) have unambiguously demonstrated robust reductions not only in low-density lipoprotein (LDL) cholesterol (LDL-C) but also in lipoprotein (a) [Lp(a)] levels. The scientific literature published prior to those studies did not provide any evidence for a link between PCSK9 and Lp(a) metabolism. More recent investigations, either in vitro or in vivo, have attempted to unravel the mechanism(s) by which PCSK9 mAbs reduce circulating Lp(a) levels, with some showing a specific implication of the LDL receptor (LDLR) in Lp(a) clearance whereas others found no significant role for the LDLR in that process. This elusive pathway appears clearly distinct from that of the widely prescribed statins that also enhance LDLR function but do not lower circulating Lp (a) levels in humans. So how does PCSK9 inhibition with mAbs reduce Lp(a)? This still remains to be established.


Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Hiperlipidemias/tratamiento farmacológico , Lipoproteína(a)/sangre , Inhibidores de PCSK9 , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Humanos , Hiperlipidemias/sangre , Hipolipemiantes/farmacología , Hipolipemiantes/uso terapéutico , Proproteína Convertasa 9/inmunología , Receptores de LDL/fisiología , Proyectos de Investigación
7.
Arterioscler Thromb Vasc Biol ; 36(8): 1647-50, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27079874

RESUMEN

OBJECTIVE: Proprotein convertase subtilisin kexin type 9 (PCSK9) inhibitors lower low-density lipoprotein (LDL) cholesterol in the vast majority of patients with autosomal dominant familial hypercholesterolemia. Will PCSK9 inhibition with monoclonal antibodies, in particular alirocumab, be of therapeutic value for patients with autosomal recessive hypercholesterolemia (ARH)? APPROACH AND RESULTS: Primary lymphocytes were obtained from 28 genetically characterized ARH patients and 11 controls. ARH lymphocytes treated with mevastatin were incubated with increasing doses of recombinant PCSK9 with or without saturating concentrations of alirocumab. Cell surface LDL receptor expression measured by flow cytometry and confocal microscopy was higher in ARH than in control lymphocytes. PCSK9 significantly reduced LDL receptor expression in ARH lymphocytes albeit to a lower extent than in control lymphocytes (25% versus 76%, respectively), an effect reversed by alirocumab. Fluorescent LDL cellular uptake, also measured by flow cytometry, was reduced in ARH lymphocytes compared with control lymphocytes. PCSK9 significantly lowered LDL cellular uptake in ARH lymphocytes, on average by 18%, compared with a 46% reduction observed in control lymphocytes, an effect also reversed by alirocumab. Overall, the effects of recombinant PCSK9, and hence of alirocumab, on LDL receptor expression and function were significantly less pronounced in ARH than in control cells. CONCLUSIONS: PCSK9 inhibition with alirocumab on top of statin treatment has the potential to lower LDL cholesterol in some autosomal recessive hypercholesterolemia patients.


Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticolesterolemiantes/farmacología , Hipercolesterolemia/tratamiento farmacológico , Linfocitos/efectos de los fármacos , Inhibidores de PCSK9 , Inhibidores de Serina Proteinasa/farmacología , Proteínas Adaptadoras Transductoras de Señales/genética , Adolescente , Adulto , Anticuerpos Monoclonales Humanizados , Estudios de Casos y Controles , Células Cultivadas , LDL-Colesterol/metabolismo , Quimioterapia Combinada , Femenino , Predisposición Genética a la Enfermedad , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Hipercolesterolemia/diagnóstico , Hipercolesterolemia/enzimología , Hipercolesterolemia/genética , Lovastatina/análogos & derivados , Lovastatina/farmacología , Linfocitos/enzimología , Masculino , Persona de Mediana Edad , Mutación , Fenotipo , Proproteína Convertasa 9/metabolismo , Receptores de LDL/metabolismo , Adulto Joven , Hiperlipoproteinemia Tipo III
9.
J Immunol ; 190(9): 4868-76, 2013 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-23530148

RESUMEN

Human Vγ9Vδ2 T cells exert potent in vitro and in vivo antitumor activities, making them promising candidates for immunotherapy strategies. Recognition of tumor cells by Vγ9Vδ2 T cells requires engagement of the TCR and/or NK receptors. Recently, one of the novel NK receptors, the class I-restricted T cell-associated molecule (CRTAM), has been described to promote cytotoxic function of NK cells and to lead to IFN-γ secretion by CD8(+) T cells through interaction with its ligand, Necl-2. A better understanding of the role of CRTAM in Vγ9Vδ2 T cell functions is highly relevant to optimize innate-like T cell-based cancer immunotherapy. In this article, we report that CRTAM is transiently expressed on activated Vγ9Vδ2 T lymphocytes following TCR engagement. However, CRTAM-Necl-2 interaction does not modify the cytotoxic function or IFN-γ secretion of Vγ9Vδ2 T cells. The expression of CRTAM in activated Vγ9Vδ2 T cells is quickly downregulated following interaction with Necl-2 on tumor cells. Of interest, CRTAM is concurrently acquired at the cell surface of Necl-2(+) tumor cells through Vγ9Vδ2 T cell membrane capture. Finally, we highlight that coculture experiments with tumor cells expressing Necl-2 result in significant cell death of CRTAM(+) Vγ9Vδ2 T cells. CRTAM-mediated cell death is dependent on an autophagic process, but not on apoptosis or necroptosis, as attested by the expression of characteristic markers and blocking experiments with specific inhibitors. On the basis of these findings, we propose that Necl-2 on tumor cells represents a new tumor counterattack mechanism and a potential target to improve efficiency of γδ T cell-based immunotherapy.


Asunto(s)
Autofagia/inmunología , Moléculas de Adhesión Celular/metabolismo , Inmunoglobulinas/inmunología , Inmunoglobulinas/metabolismo , Neoplasias/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Linfocitos T/inmunología , Antígenos/inmunología , Molécula 1 de Adhesión Celular , Moléculas de Adhesión Celular/inmunología , Línea Celular Tumoral , Membrana Celular/inmunología , Membrana Celular/metabolismo , Citotoxicidad Inmunológica/inmunología , Regulación hacia Abajo/inmunología , Células HT29 , Células Hep G2 , Humanos , Inmunoterapia/métodos , Interferón gamma/inmunología , Interferón gamma/metabolismo , Células K562 , Neoplasias/metabolismo , Neoplasias/patología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Receptores de Células Asesinas Naturales/inmunología , Receptores de Células Asesinas Naturales/metabolismo , Linfocitos T/citología , Linfocitos T/metabolismo
10.
Med Sci (Paris) ; 30(5): 537-43, 2014 May.
Artículo en Francés | MEDLINE | ID: mdl-24939541

RESUMEN

Nectin and nectin-like (Necl) are cell adhesion molecules expressed in various tumors. They were alternatively reported as involved in tumor suppressor or oncogenic functions that led to their use as histological or serological cancer markers. Gene inactivation in lung carcinoma but overexpression in leukemia were reported for Necl-2. DNAM-1 and CRTAM are emerging NK receptors of immune cells that were described to interact with nectin and Necl. DNAM-1, constitutively expressed by CD8(+) T cells, NK or γδ T lymphocytes, is a ligand of Necl-5. It participates to tumor immunosurveillance promoting Necl-5 expressing tumor cell lysis. CRTAM, only expressed after lymphocyte activation, is a ligand of Necl-2. Engagement of CRTAM with Necl-2 has opposite effects depending on the type of lymphocyte. For NK or CD8(+) T cells, it promotes cytotoxicity and IFNγ secretion favoring immunosurveillance. By contrast, CRTAM/Necl-2 interaction triggers cell death of activated TVg9Vd2 γδ T cells favoring immune escape. Nectin and Necl-mediated interactions appear to be crucial for the delicate balance between tumor escape and antitumor response.


Asunto(s)
Antígenos de Diferenciación de Linfocitos T/inmunología , Moléculas de Adhesión Celular/inmunología , Inmunoglobulinas/inmunología , Escape del Tumor , Animales , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/inmunología , Humanos , Inmunidad Celular/genética , Nectinas
11.
J Transl Med ; 11: 147, 2013 Jun 13.
Artículo en Inglés | MEDLINE | ID: mdl-23763830

RESUMEN

Epithelial ovarian cancer (EOC) is a significant cause of cancer-related mortality in women, and there has been no substantial decrease in the death rates due to EOC in the last three decades. Thus, basic knowledge regarding ovarian tumor cell biology is urgently needed to allow the development of innovative treatments for EOC. Traditionally, EOC has not been considered an immunogenic tumor, but there is evidence of an immune response to EOC in patients. Clinical data demonstrate that an antitumor immune response and immune evasion mechanisms are correlated with a better and lower survival, respectively, providing evidence for the immunoediting hypothesis in EOC. This review focuses on the immune response and immune suppression in EOC. The immunological roles of chemotherapy and surgery in EOC are also described. Finally, we detail pilot data supporting the efficiency of immunotherapy in the treatment of EOC and the emerging concept that immunomodulation aimed at counteracting the immunosuppressive microenvironment must be associated with immunotherapy strategies.


Asunto(s)
Carcinoma/inmunología , Tolerancia Inmunológica , Neoplasias Glandulares y Epiteliales/inmunología , Neoplasias Ováricas/inmunología , Carcinoma/mortalidad , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunoterapia/métodos , Neoplasias Glandulares y Epiteliales/mortalidad , Neoplasias Ováricas/mortalidad , Resultado del Tratamiento
12.
Int J Cancer ; 131(4): E449-62, 2012 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-22095289

RESUMEN

Epithelial ovarian cancer (EOC) usually spreads into the peritoneal cavity, thereby providing an opportunity for intraperitoneal adoptive immunotherapy with Vγ9Vδ2 T lymphocytes, a T cell subpopulation endowed with high lytic properties against tumor cells. However, previous studies have reported that Vγ9Vδ2 T cells fail to expand from peripheral blood mononuclear cells in one-third of patients with cancer. Here, from a cohort of 37 patients with EOC, a multiple correspondence analysis identified three populations, one of which was not suitable for Vγ9Vδ2 T-cell adoptive therapy. Interestingly, the ineligible patients were identified based on the frequency of Vγ9Vδ2 T cells in their peripheral blood and the patients' age. The average time to tumor recurrence was also found to be significantly different between the three populations, suggesting that the innate immune response is involved in EOC prognosis. A dramatic decrease in the lytic properties of Vγ9Vδ2 T cells occurred following incubation with ascitic supernatant and was found to be associated with reduced perforin/granzyme degranulation. Prostaglandin E2, but not IL-6, IL-10, VEGF or TGF-ß, showed immunosuppressive effects in Vγ9Vδ2 T cells. Interestingly, our results emphasize that pretreating ovarian tumor cells with zoledronate partially reverses the immunosuppressive effects of ovarian cancer-associated ascites and restores a high level of lytic activity. These data sustain that optimal Vγ9Vδ2 T-cell adoptive immunotherapy previously requires counteracting the tumor immunosuppressive microenvironment. Altogether, our findings provide a rationale for clinically evaluating Vγ9Vδ2 T-cell adoptive immunotherapy with intraperitoneal carcinomatosis presensitization by zoledronate in patients with EOC.


Asunto(s)
Dinoprostona/fisiología , Difosfonatos/farmacología , Imidazoles/farmacología , Inmunoterapia , Neoplasias Ováricas/patología , Linfocitos T Citotóxicos/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Ascitis/fisiopatología , Línea Celular Tumoral , Femenino , Citometría de Flujo , Humanos , Persona de Mediana Edad , Neoplasias Ováricas/terapia , Ácido Zoledrónico
13.
STAR Protoc ; 3(4): 101680, 2022 12 16.
Artículo en Inglés | MEDLINE | ID: mdl-36115027

RESUMEN

This manuscript proposes an efficient and reproducible protocol for the generation of genetically modified human induced pluripotent stem cells (hiPSCs) by genome editing using CRISPR-Cas9 technology. Here, we describe the experimental strategy for generating knockout (KO) and knockin (KI) clonal populations of hiPSCs using single-cell sorting by flow cytometry. We efficiently achieved up to 15 kb deletions, molecular tag insertions, and single-nucleotide editing in hiPSCs. We emphasize the efficacy of this approach in terms of cell culture time. For complete details on the use and execution of this protocol, please refer to Canac et al. (2022) and Bray et al. (2022).


Asunto(s)
Edición Génica , Células Madre Pluripotentes Inducidas , Humanos , Edición Génica/métodos , Sistemas CRISPR-Cas , Células Clonales , Técnicas de Cultivo de Célula
14.
J Immunol ; 182(6): 3423-31, 2009 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-19265120

RESUMEN

Vgamma9Vdelta2 T lymphocytes are a major human gammadelta T cell subset that react against a wide array of tumor cells, through recognition of phosphorylated isoprenoid pathway metabolites called phosphoantigens. Immunotherapeutic protocols targeting Vgamma9Vdelta2 T cells have yielded promising, yet limited, signs of antitumor efficacy. To improve these approaches, we analyzed the effects on gammadelta T cells of IL-21, a cytokine known to enhance proliferation and effector functions of CD8(+) T cells and NK cells. IL-21 induced limited division of phosphoantigen-stimulated Vgamma9Vdelta2 T cells, but did not modulate their sustained expansion induced by exogenous IL-2. Vgamma9Vdelta2 T cells expanded in the presence of IL-21 and IL-2 showed enhanced antitumor cytolytic responses, associated with increased expression of CD56 and several lytic molecules, and increased tumor-induced degranulation capacity. IL-21 plus IL-2-expanded Vgamma9Vdelta2 T cells expressed higher levels of inhibitory receptors (e.g., ILT2 and NKG2A) and lower levels of the costimulatory molecule NKG2D. Importantly, these changes were rapidly and reversibly induced after short-term culture with IL-21. Finally, IL-21 irreversibly enhanced the proinflammatory Th1 polarization of expanded Vgamma9Vdelta2 T cells when added at the beginning of the culture. These data suggest a new role played by IL-21 in the cytotoxic and Th1 programming of precommitted Ag-stimulated gammadelta T cells. On a more applied standpoint, IL-21 could be combined to IL-2 to enhance gammadelta T cell-mediated antitumor responses, and thus represents a promising way to optimize immunotherapies targeting this cell subset.


Asunto(s)
Adyuvantes Inmunológicos/fisiología , Citotoxicidad Inmunológica , Inmunoterapia Adoptiva/métodos , Mediadores de Inflamación/fisiología , Interleucinas/fisiología , Receptores de Antígenos de Linfocitos T gamma-delta/fisiología , Células TH1/inmunología , Adenocarcinoma/inmunología , Adenocarcinoma/patología , Adenocarcinoma/prevención & control , Adulto , Linfoma de Burkitt/inmunología , Linfoma de Burkitt/patología , Linfoma de Burkitt/prevención & control , Diferenciación Celular/inmunología , Línea Celular Tumoral , Polaridad Celular/inmunología , Proliferación Celular , Humanos , Interleucina-2/fisiología , Neoplasias Renales/inmunología , Neoplasias Renales/patología , Neoplasias Renales/prevención & control , Activación de Linfocitos , Fosfoproteínas/fisiología , Receptores de Antígenos de Linfocitos T gamma-delta/biosíntesis , Proteínas Recombinantes/farmacología , Células TH1/patología , Células TH1/trasplante
15.
Atherosclerosis ; 326: 47-55, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-33933263

RESUMEN

BACKGROUND AND AIMS: Proprotein Convertase Subtilisin Kexin Type 9 (PCSK9) is an endogenous inhibitor of the LDL receptor (LDLR). Mendelian randomization studies suggest that PCSK9 deficiency increases diabetes risk, but the underlying mechanisms remain unknown. The aim of our study was to investigate whether PCSK9 or its inhibition may modulate beta cell function. METHODS: We assessed PCSK9 and insulin colocalization in human pancreatic sections by epifluorescent and confocal microscopy. We also investigated the expression and the function of PCSK9 in the human EndoC-ßH1 beta cell line, by ELISA and flow cytometry, respectively. PCSK9 was inhibited with Alirocumab or siRNA. LDLR expression and LDL uptake were assessed by flow cytometry. RESULTS: PCSK9 was expressed and secreted from beta cells isolated from human pancreas as well as from EndoC-ßH1 cells. PCSK9 secretion was enhanced by statin treatment. Recombinant PCSK9 decreased LDLR abundance at the surface of these cells, an effect abrogated by Alirocumab. Alirocumab as well as PCSK9 silencing increased LDLR expression at the surface of EndoC-ßH1 cells. Neither exogenous PCSK9, nor Alirocumab, nor PCSK9 silencing significantly altered glucose-stimulated insulin secretion (GSIS) from these cells. High-low density lipoproteins (LDL) concentrations decreased GSIS, but the addition of PCSK9 or its inhibition did not modulate this phenomenon. CONCLUSIONS: While PCSK9 regulates LDLR abundance in beta cells, inhibition of exogenous or endogenous PCSK9 does not appear to significantly impact insulin secretion. This is reassuring for the safety of PCSK9 inhibitors in terms of beta cell function.


Asunto(s)
Células Secretoras de Insulina , Proproteína Convertasa 9 , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas , Receptores de LDL , Subtilisinas
16.
Atherosclerosis ; 293: 49-56, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31835041

RESUMEN

BACKGROUND AND AIMS: PCSK9 is an endogenous inhibitor of the LDL receptor pathway. Recently, Mendelian randomization studies have raised a doubt about the diabetogenic risk of PCSK9 inhibitors. Here, we assessed the relationship between plasma PCSK9 levels and the risk of new onset diabetes (NOD). METHODS: Fasting plasma PCSK9 levels were measured at baseline by ELISA in subjects without lipid lowering treatment in IT-DIAB (n = 233 patients with prediabetes, follow-up 5 years) and ELSA-Brasil (n = 1751; 27.5% with prediabetes, follow-up 4 years) prospective cohorts. The primary outcome in both studies was the incidence of NOD. The association of NOD with plasma PCSK9 levels was studied using multivariable Cox models. RESULTS: Plasma PCSK9 levels were not significantly associated with NOD in IT-DIAB (HR (+1SD) 0.96, CI95% [0.76; 1.21]) and ELSA-Brasil (OR (+1SD) 1.13 [0.89; 1.42]). In ELSA-Brasil, a significant positive association between PCSK9 and worsening of glucose homeostasis, including the progression from normoglycemia to prediabetes, was found (OR (+1SD) 1.17 [1.04; 1.30], p = 0.0074). Plasma PCSK9 concentration was also positively associated with the change in fasting plasma glucose between the first and second visit in ELSA-Brasil (ß = 0.053, CI95% [0.006; 0.10], p = 0.026). Plasma PCSK9 levels positively correlated with total cholesterol in IT-DIAB and ELSA-Brasil, but not with glucose homeostasis parameters, except for a positive correlation with HOMA-IR in ELSA-Brasil. CONCLUSIONS: Plasma PCSK9 levels were not significantly associated with NOD risk in longitudinal analyses. These data suggest that inhibition of the PCSK9 extra-cellular pathway should not be deleterious for glucose homeostasis.


Asunto(s)
Diabetes Mellitus Tipo 2/sangre , Proproteína Convertasa 9/sangre , Biomarcadores/sangre , Brasil/epidemiología , Diabetes Mellitus Tipo 2/epidemiología , Progresión de la Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Estudios de Seguimiento , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Factores de Riesgo , Factores de Tiempo
17.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1044-1045: 24-29, 2017 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-28064066

RESUMEN

The combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and trypsin proteolysis is an effective tool for accurate quantitation of multiple proteins in a single run. However, expensive samples pre-treatment as immunoenrichment are often required to analyze low abundant proteins. Plasma proprotein convertase subtilisin/kexin type 9 (PCSK9), a circulating regulator of low-density lipoprotein metabolism, was studied as an example of a low abundant plasma protein. We investigated post-proteolysis solid-phase extraction (SPE) as an alternative strategy to improve its detection. After optimization of pretreatment, including denaturation, reduction, alkylation, tryptic digestion and selective SPE concentration, 91±7% of PCSK9 was recovered from human plasma samples and coefficients of variation were less than 13.2% with a lower limit of quantification of 37.5ng/ml. This LC-MS/MS method was compared with standard enzyme-linked immunosorbent assay in 30 human plasma samples with a broad range of PCSK9 concentrations. Both methods were significantly correlated (r=0.936, p<0.001) with less than 7% of the values out of the 95% confidence interval and similar concentrations were measured using either LC-MS/MS or ELISA methods (514.2±217.2 vs. 504.2±231.0ng/ml, respectively- p=NS). This method involving SPE is an effective measurement tool for low abundant plasma protein analysis that could be easily included in multiplexed assays.


Asunto(s)
Cromatografía Liquida/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Proproteína Convertasa 9/sangre , Espectrometría de Masas en Tándem/métodos , Adulto , Femenino , Humanos , Modelos Lineales , Masculino , Persona de Mediana Edad , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Extracción en Fase Sólida/métodos , Tripsina/metabolismo
18.
Atherosclerosis ; 254: 249-253, 2016 11.
Artículo en Inglés | MEDLINE | ID: mdl-27594539

RESUMEN

BACKGROUND AND AIMS: We aimed to assess whether elevated PCSK9 and lipoprotein (a) [Lp(a)] levels associate with coronary artery calcification (CAC), a good marker of atherosclerosis burden, in asymptomatic familial hypercholesterolemia. METHODS: We selected 161 molecularly defined FH patients treated with stable doses of statins for more than a year. CAC was measured using the Agatston method and quantified as categorical variable. Fasting plasma samples were collected and analyzed for lipids and lipoproteins. PCSK9 was measured by ELISA, Lp(a) and apolipoprotein (a) concentrations by inmunoturbidimetry and LC-MS/MS, respectively. RESULTS: Circulating PCSK9 levels were significantly reduced in patients without CAC (n = 63), compared to those with CAC (n = 99). Patients with the highest CAC scores (above 100) had the highest levels of circulating PCSK9 and Lp(a). In multivariable regression analyses, the main predictors for a positive CAC score was age and sex followed by circulating PCSK9 and Lp(a) levels. CONCLUSIONS: In statin treated asymptomatic FH patients, elevated PCSK9 and Lp(a) levels are independently associated with the presence and severity of CAC, a good predictor of coronary artery disease.


Asunto(s)
Calcinosis/sangre , Vasos Coronarios/patología , Hiperlipoproteinemia Tipo II/sangre , Lipoproteína(a)/sangre , Proproteína Convertasa 9/sangre , Adulto , Anciano , Calcio/sangre , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/genética , Vasos Coronarios/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Hiperlipoproteinemia Tipo II/genética , Masculino , Persona de Mediana Edad , Análisis Multivariante , Mutación , Fenotipo , Tomografía Computarizada por Rayos X
19.
JACC Basic Transl Sci ; 1(6): 419-427, 2016 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-29308438

RESUMEN

To elucidate how the proprotein convertase subtilisin kexin type 9 (PCSK9) inhibitor alirocumab modulates lipoprotein(a) [Lp(a)] plasma levels, the authors performed a series of Lp(a) uptake studies in primary human hepatocytes and dermal fibroblasts and measured Lp(a) secretion from human hepatocytes. They found that Lp(a) cellular uptake occurred in a low-density lipoprotein receptor-independent manner. Neither PCSK9 nor alirocumab altered Lp(a) internalization. By contrast, the secretion of apolipoprotein (a) from human hepatocytes was sharply increased by PCSK9, an effect that was reversed by alirocumab. They propose that PCSK9 does not significantly modulate Lp(a) catabolism, but rather enhances the secretion of Lp(a) from liver cells.

20.
PLoS One ; 8(5): e63322, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23717410

RESUMEN

Vγ9Vδ2 cells are cytotoxic T cells that are able to recognize epithelial ovarian carcinoma (EOC) cells. Therefore, Vγ9Vδ2 cell-based adoptive transfer is an attractive therapy for EOC. However, the inefficient ex vivo expansion after specific stimulation of Vγ9Vδ2 cells from some patients and the relationships between Vγ9Vδ2 cells and clinical course of EOC are issues that remain to be clarified. Herein, peripheral blood mononuclear cells (PBMCs) from 60 EOC patients were stimulated with bromohydrin pyrophosphate (BrHPP) or zoledronate, which are specific agonists of Vγ9Vδ2 cells. The compounds differed in their efficacies to induce ex vivo Vγ9Vδ2 PBMC expansion, but 16/60 samples remained inefficiently expanded with both stimuli. Interestingly, the Vγ9Vδ2 cells in these low-responding PBMCs displayed before expansion (ex vivo PBMCs) an altered production of the pro-inflammatory cytokines IFN-γ and TNF-α, a decreased naive fraction and a reduced frequency. No evidence of an involvement of CD4(+)CD25(+)Foxp3(+) regulatory cells was observed. Importantly, our data also demonstrate that a Vγ9Vδ2 cell frequency of 0.35% or less in EOC PBMCs could be used to predict low responses to both BrHPP and zoledronate. Moreover, our data highlight that such a deficiency is not correlated with advanced EOC stages but is associated with more refractory states to platinum-based chemotherapy and is an independent predictor of shorter disease-free survival after treatment. These results are the first to suggest a potential contribution of Vγ9Vδ2 cells to the anti-tumor effects of chemotherapeutic agents and they strengthen interest in strategies that might increase Vγ9Vδ2 cells in cancer patients.


Asunto(s)
Biomarcadores de Tumor/inmunología , Neoplasias Glandulares y Epiteliales/inmunología , Neoplasias Ováricas/inmunología , Linfocitos T Citotóxicos/fisiología , Anciano , Biomarcadores de Tumor/sangre , Complejo CD3/metabolismo , Carcinoma Epitelial de Ovario , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Terapia Combinada , Difosfatos/farmacología , Difosfonatos/farmacología , Supervivencia sin Enfermedad , Resistencia a Antineoplásicos , Humanos , Imidazoles/farmacología , Interferón gamma/metabolismo , Persona de Mediana Edad , Neoplasias Glandulares y Epiteliales/sangre , Neoplasias Glandulares y Epiteliales/mortalidad , Neoplasias Glandulares y Epiteliales/terapia , Neoplasias Ováricas/sangre , Neoplasias Ováricas/mortalidad , Neoplasias Ováricas/terapia , Pronóstico , Análisis de Supervivencia , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Reguladores/metabolismo , Resultado del Tratamiento , Factor de Necrosis Tumoral alfa/metabolismo , Ácido Zoledrónico
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