RESUMEN
Antiphospholipid syndrome (APS) is characterized by thrombus formation, poor pregnancy outcomes, and a proinflammatory response. H3K4me3-related monocytes activation are key regulators of APS pathogenesis. Therefore, H3K4me3 CUT&Tag and ATAC-seq are performed to examine the epigenetic profiles. The results indicate that the H3K4me3 signal and chromatin accessibility at the FOXJ2 promoter are enhanced in an in vitro monocyte model by stimulation with ß2GPI/anti-ß2GPI, which mimics APS, and decreases after OICR-9429 administration. Furthermore, FOXJ2 is highly expressed in patients with primary APS (PAPS) and is the highest in patients with triple-positive antiphospholipid antibodies (aPLs). Mechanistically, FOXJ2 directly binds to the SLAMF8 promoter and activates SLAMF8 transcription. SLAMF8 further interacts with TREM1 to stimulate TLR4/NF-κB signaling and prohibit autophagy. Knockdown of FOXJ2, SLAMF8, or TREM1 blocks TLR4/NF-κB and provokes autophagy, subsequently inhibiting the release of inflammatory and thrombotic indicators. A mouse model of vascular APS is established via ß2GPI intraperitoneal injection, and the results suggest that OICR-9429 administration attenuates the inflammatory response and thrombus formation by inactivating FOXJ2/SLAMF8/TREM1 signaling. These findings highlight the overexpression of H3K4me3-mediated FOXJ2 in APS, which consequently accelerates APS pathogenesis by triggering inflammation and thrombosis via boosting the SLAMF8/TREM1 axis. Therefore, OICR-9429 is a promising candidate drug for APS therapy.
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Modelos Animales de Enfermedad , Factores de Transcripción Forkhead , Inflamación , Monocitos , Trombosis , Animales , Femenino , Humanos , Ratones , Anticuerpos Antifosfolípidos/metabolismo , Síndrome Antifosfolípido/metabolismo , Síndrome Antifosfolípido/genética , beta 2 Glicoproteína I/metabolismo , beta 2 Glicoproteína I/genética , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/genética , Histonas/metabolismo , Histonas/genética , Inflamación/metabolismo , Inflamación/genética , Monocitos/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Trombosis/metabolismo , Trombosis/genéticaRESUMEN
Apolipoprotein H (APOH) downregulation can cause hepatic steatosis and gut microbiota dysbiosis. However, the mechanism by which APOH-regulated lipid metabolism contributes to metabolic dysfunction-associated steatotic liver disease (MASLD) remains undetermined. Herein, we aim to explore the regulatory effect of APOH, mediated through various pathways, on metabolic homeostasis and MASLD pathogenesis. We analyzed serum marker levels, liver histopathology, and cholesterol metabolism-related gene expression in global ApoH-/- C57BL/6 male mice. We used RNA sequencing and metabolomic techniques to investigate the association between liver metabolism and bacterial composition. Fifty-two differentially expressed genes were identified between ApoH-/- and WT mice. The mRNA levels of de novo lipogenesis genes were highly upregulated in ApoH-/- mice than in WT mice. Fatty acid, glycerophospholipid, sterol lipid, and triglyceride levels were elevated, while hyodeoxycholic acid levels were significantly reduced in the liver tissues of ApoH-/- mice than in those of WT mice. Microbial beta diversity was lower in ApoH-/- mice than in WT mice, and gut microbiota metabolic functions were activated in ApoH-/- mice. Moreover, ApoH transcripts were downregulated in patients with MASLD, and APOH-related differential genes were enriched in lipid metabolism. Open-source transcript-level data from human metabolic dysfunction-associated steatohepatitis livers reinforced a significant association between metabolic dysfunction-associated steatohepatitis and APOH downregulation. In conclusion, our studies demonstrated that APOH downregulation aggravates fatty liver and induces gut microbiota dysbiosis by dysregulating bile acids. Our findings offer a novel perspective on APOH-mediated lipid metabolic dysbiosis and provide a valuable framework for deciphering the role of APOH in fatty liver disease.
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Microbioma Gastrointestinal , Enfermedad del Hígado Graso no Alcohólico , Humanos , Masculino , Ratones , Animales , Metabolismo de los Lípidos/genética , beta 2 Glicoproteína I/genética , beta 2 Glicoproteína I/metabolismo , beta 2 Glicoproteína I/farmacología , Regulación hacia Abajo , Disbiosis/metabolismo , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Hígado/metabolismo , Ácidos Grasos/metabolismoRESUMEN
The expression of TTR and apolipoprotein H (APOH) genes and their relationship with prognosis in patients with colorectal cancer (CRC) metastasis by using bioinformatics analysis techniques are explored. The expression profiles of related genes in patients with CRC metastasis are retrieved from the Gene Expression Omnibus (GEO) database. The core genes transthyretin (TTR) and APOH are screened by constructing protein-protein interaction (PPI) network, and the corresponding patient data of 327 patients are extracted and included in the metastasis group. The TTR and APOH genes of 300 patients without CRC metastasis are screened and included in the control group. The relationship between the expression levels of TTR and APOH and the clinicopathological parameters of patients with CRC metastasis is analyzed. Kaplan-Meier survival curve is drawn to observe the influence of overexpression and low expression of TTR and APOH on the prognosis and survival of patients in the metastatic group. Receiver operating characteristic (ROC) curve is drawn to observe the prognostic efficacy of combined TTR and APOH detection in patients with CRC metastasis. The experimental results show that bioassay can confirm the close relationship between TTR, APOH, and patients with CRC metastasis. Regular detection of serum TTR and APOH expression can effectively assess the patient's condition and take measures to improve the prognosis of the patients.
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Neoplasias Colorrectales , Biología Computacional , Prealbúmina/metabolismo , Neoplasias Colorrectales/patología , Biología Computacional/métodos , Regulación Neoplásica de la Expresión Génica , Humanos , Prealbúmina/genética , Pronóstico , beta 2 Glicoproteína I/genéticaRESUMEN
OBJECTIVES: Tissue factor (TF) and Human apolipoprotein H (APOH) seem to be significantly associated with a clinical manifestation in systemic lupus erythematosus (SLE) patients with or without APS, mostly because of thrombotic events and coagulation processes. Additionally, according to recent studies, these two factors appear to be an important part of immune response and inflammation. METHODS: The objective of this study was to investigate three SNPs of APOH (rs4581, rs8178835 and rs818819) and three of TF (rs958587, rs3917615, rs1361600) in SLE patients and healthy subjects using TaqMan genotyping assay and their association with inflammatory cytokines level in serum and selected clinical parameters. RESULTS: Present study revealed that TF rs3917615 and rs958587 and APOH rs4581 possibly predispose to joint involvement in SLE. CONCLUSIONS: Analysed genetic variants of TF and APOH may have an impact on inflammatory processes and clinical relevance in SLE patients in the Caucasian population.
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Lupus Eritematoso Sistémico , Tromboplastina , beta 2 Glicoproteína I/genética , Estudios de Casos y Controles , Citocinas/genética , Humanos , Lupus Eritematoso Sistémico/genética , Polimorfismo de Nucleótido Simple , Tromboplastina/genéticaRESUMEN
OBJECTIVE: Lp(a) (lipoprotein[a]) is an independent risk factor for cardiovascular diseases and plasma levels are primarily determined by variation at the LPA locus. We performed a genome-wide association study in the UK Biobank to determine whether additional loci influence Lp(a) levels. Approach and Results: We included 293 274 White British individuals in the discovery analysis. Approximately 93 095 623 variants were tested for association with natural log-transformed Lp(a) levels using linear regression models adjusted for age, sex, genotype batch, and 20 principal components of genetic ancestry. After quality control, 131 independent variants were associated at genome-wide significance (P≤5×10-8). In addition to validating previous associations at LPA, APOE, and CETP, we identified a novel variant at the APOH locus, encoding ß2GPI (beta2-glycoprotein I). The APOH variant rs8178824 was associated with increased Lp(a) levels (ß [95% CI] [ln nmol/L], 0.064 [0.047-0.081]; P=2.8×10-13) and demonstrated a stronger effect after adjustment for variation at the LPA locus (ß [95% CI] [ln nmol/L], 0.089 [0.076-0.10]; P=3.8×10-42). This association was replicated in a meta-analysis of 5465 European-ancestry individuals from the Framingham Offspring Study and Multi-Ethnic Study of Atherosclerosis (ß [95% CI] [ln mg/dL], 0.16 [0.044-0.28]; P=0.0071). CONCLUSIONS: In a large-scale genome-wide association study of Lp(a) levels, we identified APOH as a novel locus for Lp(a) in individuals of European ancestry. Additional studies are needed to determine the precise role of ß2GPI in influencing Lp(a) levels as well as its potential as a therapeutic target.
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Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/genética , Sitios Genéticos , Variación Genética , Lipoproteína(a)/sangre , beta 2 Glicoproteína I/genética , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Enfermedades Cardiovasculares/epidemiología , Femenino , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Factores de Riesgo de Enfermedad Cardiaca , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Medición de RiesgoRESUMEN
BACKGROUND: Apolipoprotein H (APOH), also known as beta2-glycoprotein I (beta2-GPI), is an acute phase protein in hepatitis B virus (HBV) infection and binds to hepatitis B surface antigen (HBsAg) with high-affinity. APOH expression is upregulated by HBV and the large surface protein (LHBs), but also elevated in HBV-related hepatoma cells. Previous studies show that intracellular retention of HBsAg induces endoplasmic reticulum (ER) stress, a key driver of hepatocyte damage during chronic liver injury, but the mechanisms are unclear. We hypothesize that APOH mediates HBV-induced ER stress through increased retention of HBsAg. METHODS: VR-APOH-myc and VR-LHBs-flag plasmids were constructed by PCR using pcDNA3.1(-)-APOH or an HBV expression vector, respectively. APOH and ER stress markers were examined at protein and mRNA levels by Western Blot or RT-qPCR. HBsAg titer was assayed by ELISA. RNA-seq was performed to elucidate the transcriptional impact of APOH manipulation in HBV-producing cells (HepG2.2.15 cells). RESULTS: We found that HBV upregulates APOH expression in 293 T cells, and APOH overexpression subsequently inhibits secretion of HBsAg. Next, we show that LHBs overexpression in conjunction with APOH leads to ER stress in 293 T cells, as evidenced by production of the binding immunoglobulin protein (BiP) and C/EBP homologous protein (CHOP), as well as increased splicing of X-box binding protein 1 (XBP1). We further observed that loss of beta2-GPI reduced CHOP expression in HepG2.2.15 cells, while beta2-GPI overexpression enhanced CHOP production. CONCLUSION: The interaction of beta2-GPI and HBV initiates ER stress through driving intracellular retention of HBsAg and activates the UPR.
Asunto(s)
Estrés del Retículo Endoplásmico/genética , Retículo Endoplásmico/genética , Antígenos de Superficie de la Hepatitis B/genética , Virus de la Hepatitis B/genética , Interacciones Huésped-Patógeno/genética , beta 2 Glicoproteína I/genética , Retículo Endoplásmico/inmunología , Retículo Endoplásmico/virología , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/inmunología , Regulación de la Expresión Génica , Células HEK293 , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/inmunología , Células Hep G2 , Antígenos de Superficie de la Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Interacciones Huésped-Patógeno/inmunología , Humanos , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Transducción de Señal , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/inmunología , Transfección , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Proteína 1 de Unión a la X-Box/genética , Proteína 1 de Unión a la X-Box/inmunología , beta 2 Glicoproteína I/inmunologíaRESUMEN
AIMS: ß2-glycoprotein I/anti-ß2-glycoprotein I antibody complex (ß2/aß2) could promote oxLDL-induced endothelial inflammation through Toll-like receptor 4 (TLR4), therefore accelerates atherosclerosis in patients with anti-phospholipid syndrome (APS). However, effects of ß2/aß2 and TLR4 on oxLDL-induced CD36 activation in macrophages remain to be elucidated and are currently under investigation. MATERIALS AND METHODS: THP-1 macrophages with or without the pre-treatment of TAK-242, a TLR4 inhibitor, were treated with RPMI 1640, oxLDL, oxLDL+ß2/aß2 or oxLDL + LPS.CD36 expression and subsequent intracellular lipid accumulation, cholesterol-transportation-related proteins (ACAT1, ABCG1 and ABCA1) expression, inflammatory cytokines (IL-1ß, TNF-α and IL-6) secretion, focal adhesion kinases (FAK) activation and matrix metalloproteinases (MMP-2 and MMP-9) expression by these THP-1 macrophages were evaluated. Moreover, effects of TLR4 on oxLDL+ß2/aß2-induced peroxisome proliferators-activated receptor-γ (PPAR-γ) expression and CD36 translocation have also been observed. KEY FINDINGS: Compared with oxLDL-treated ones, CD36 expression, intracellular lipid accumulation and FAK activation were inhibited, whereas the levels of inflammatory cytokines and MMPs were upregulated in THP-1 macrophages treated with oxLDL+ß2/aß2 (p < 0.05). Moreover, observed differences between oxLDL-treated and oxLDL+ß2/aß2-treated THP-1 macrophages could be reversed by TAK-242 pre-treatment (p < 0.05). Furthermore, oxLDL+ß2/aß2 promoted PPAR-γ expression and CD36 cytoplasmic translocation in THP-1 macrophages, these effects could also be attenuated by TAK-242 (pâ¯<â¯0.05). SIGNIFICANCE: Through a TLR4 dependent manner, ß2/aß2 inhibited oxLDL-induced CD36 expression, lipid accumulation and FAK activation, while promoted inflammatory cytokines and MMPs expression in THP-1 macrophages, indicating the novel dual roles played by ß2/aß2 in APS-related atherosclerosis.
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Antígenos CD36/genética , Lipoproteínas LDL/farmacología , Macrófagos/efectos de los fármacos , beta 2 Glicoproteína I/antagonistas & inhibidores , beta 2 Glicoproteína I/genética , Proteínas Portadoras/metabolismo , Línea Celular , Colesterol/metabolismo , Citocinas/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Macrófagos/metabolismo , Metaloproteinasas de la Matriz/metabolismo , PPAR gamma/biosíntesis , Sulfonamidas/farmacología , Receptor Toll-Like 4/antagonistas & inhibidores , beta 2 Glicoproteína I/inmunologíaRESUMEN
We previously found that circulating ß2 -glycoprotein I inhibits human endothelial cell migration, proliferation, and angiogenesis by diverse mechanisms. In the present study, we investigated the antitumor activities of ß2 -glycoprotein I using structure-function analysis and mapped the critical region within the ß2 -glycoprotein I peptide sequence that mediates anticancer effects. We constructed recombinant cDNA and purified different ß2 -glycoprotein I polypeptide domains using a baculovirus expression system. We found that purified ß2 -glycoprotein I, as well as recombinant ß2 -glycoprotein I full-length (D12345), polypeptide domains I-IV (D1234), and polypeptide domain I (D1) significantly inhibited melanoma cell migration, proliferation and invasion. Western blot analyses were used to determine the dysregulated expression of proteins essential for intracellular signaling pathways in B16-F10 treated with ß2 -glycoprotein I and variant recombinant polypeptides. Using a melanoma mouse model, we found that D1 polypeptide showed stronger potency in suppressing tumor growth. Structural analysis showed that fragments A and B within domain I would be the critical regions responsible for antitumor activity. Annexin A2 was identified as the counterpart molecule for ß2 -glycoprotein I by immunofluorescence and coimmunoprecipitation assays. Interaction between specific amino acids of ß2 -glycoprotein I D1 and annexin A2 was later evaluated by the molecular docking approach. Moreover, five amino acid residues were selected from fragments A and B for functional evaluation using site-directed mutagenesis, and P11A, M42A, and I55P mutations were shown to disrupt the anti-melanoma cell migration ability of ß2 -glycoprotein I. This is the first study to show the therapeutic potential of ß2 -glycoprotein I D1 in the treatment of melanoma progression.
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Movimiento Celular/efectos de los fármacos , Melanoma Experimental/tratamiento farmacológico , Péptidos/farmacología , beta 2 Glicoproteína I/química , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Línea Celular Tumoral , Masculino , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Péptidos/química , Péptidos/metabolismo , Dominios Proteicos , Homología de Secuencia de Aminoácido , beta 2 Glicoproteína I/genética , beta 2 Glicoproteína I/metabolismoRESUMEN
The highly glycosylated ß-2-glycoprotein-1 (B2GP1), also called apolipoprotein H, is a 50â¯kDa human plasma protein with four or five N-glycosylation sites. Glycosylation of B2GP1 can impact auto antibody recognition leading to the development of antiphospholipid syndrome (APS), which can result in miscarriages or thrombosis. Next to its glycosylation different genetic variants are known to increase the risk of suffering from APS. Here we show that ESI-q/TOF-MS of intact B2GP1 can be used to analyze genetic variants and glycosylation simultaneously. After enrichment of B2GP1 from 16 different plasma samples and subsequent ESI-MS measurement of the intact protein, we detected five different SNPs in our samples either homozygous or heterozygous. The dominant glycan composition shows four biantennary, fully sialylated glycan structures, with a relative proportion of about 30%. We also detected compositions with one or two triantennary glycan structures in lower amounts and fucosylated species with one or two fucosyl residues. Two of our samples showed an unreported partially occupied fifth glycosylation site presumably arising from the presence of SNP variant S88N. Our method allows a fast determination of genetic variants and glycan compositions of human B2GP1 to be potentially used as diagnostic marker.
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Polimorfismo de Nucleótido Simple , Polisacáridos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , beta 2 Glicoproteína I/química , beta 2 Glicoproteína I/metabolismo , Síndrome Antifosfolípido/diagnóstico , Autoanticuerpos/metabolismo , Sitios de Unión , Glicosilación , Heterocigoto , Homocigoto , Humanos , Modelos Moleculares , Polisacáridos/química , Procesamiento Proteico-Postraduccional , beta 2 Glicoproteína I/sangre , beta 2 Glicoproteína I/genéticaRESUMEN
This study aimed to explore the mechanism of a novel mutation (p.Lys38Glu) in apolipoprotein H (APOH) gene causing hereditary beta2-glycoprotein I (ß2GPI) deficiency and thrombosis in a proband with thrombophilia. The plasma level of ß2GPI was measured by ELISA and Western blotting, and anti-ß2GPI antibody by ELISA. Lupus anticoagulant (LA) was assayed using the dilute Russell viper venom time. Deficiency of the major natural anticoagulants including protein C (PC), protein S (PS), antithrombin (AT) and thrombomodulin (TM) was excluded from the proband. A mutation analysis was performed by amplification and sequencing of the APOH gene. Wild type and mutant (c.112A>G) APOH expression plasmids were constructed and transfected into HEK293T cells. The results showed that the thrombin generation capacity of the proband was higher than that of the other family members. Missense mutation p.Lys38Glu in APOH gene and LA coexisted in the proband. The mutation led to ß2GPI deficiency and thrombosis by impairing the protein production and inhibiting the platelet aggregation. It was concluded that the recurrent thrombosis of the proband is associated with the coexistence of p.Lys38Glu mutation in APOH gene and LA in plasma.
Asunto(s)
Mutación/genética , Trombofilia/etiología , Trombofilia/genética , beta 2 Glicoproteína I/deficiencia , beta 2 Glicoproteína I/genética , Secuencia de Aminoácidos , Secuencia de Bases , Biología Computacional , Demografía , Familia , Femenino , Células HEK293 , Humanos , Masculino , Linaje , Agregación Plaquetaria , Unión Proteica , Proteínas Recombinantes/metabolismo , Resonancia por Plasmón de Superficie , Trombina/metabolismo , Trombofilia/sangre , Adulto Joven , beta 2 Glicoproteína I/sangre , beta 2 Glicoproteína I/químicaRESUMEN
The study aimed to evaluate the clinical significance of laboratory findings in patients with catastrophic antiphospholipid syndrome (CAPS) and to report the effects of a well-defined treatment protocol in 14 consecutive cases. Thirteen patients (12 presenting one and one presenting two episodes of CAPS) were consecutively treated and monitored between 1986 and 2017. Antiphospholipid antibody (aPL) characteristics of the patients were compared with those of 64 matched controls (45 antiphospholipid syndrome patients and 19 aPL carriers) who did not develop CAPS during the same mean follow-up period (12 years⯱â¯9.9 SD). Triple aPL positivity (IgG/IgM anticardiolipin + IgG/IgM anti-ß2Glycoprotein I + lupus anticoagulants) significantly prevailed in the CAPS patients with respect to the controls (p = 0.003). IgG anticardiolipin and IgG anti-ß2Glycoprotein I mean antibody titers of the CAPS patients were significantly higher than those of the controls (p = 0.0018 and p = 0.003, respectively). Triple therapy (anticoagulation + plasma exchange + steroids) was administered to all the CAPS cases except for one. Beginning in 2009, intravenous immunoglobulin infusion has also been included in the triple therapy protocol (six patients). All the patients recovered from CAPS; five showed renal failure and one a I-II class New York Heart Association (NYHA) dilated cardiomyopathy. Long-term outcomes of CAPS included a gradual worsening of renal failure in one patient who required hemodialysis 30 years after the acute episode. Renal function improved in the other four patients. The patient affected with dilated cardiomyopathy worsened to a II class NYHA over a five year period. Currently all the patients are alive. A specific antiphospholipid antibody profile could be considered a risk factor associated to CAPS. Early use of a defined treatment protocol based on triple therapy either or not associated with IVIG was associated with recovery in all CAPS patients.
Asunto(s)
Corticoesteroides/uso terapéutico , Anticoagulantes/uso terapéutico , Síndrome Antifosfolípido/terapia , Inmunoglobulinas Intravenosas/uso terapéutico , Intercambio Plasmático/métodos , Adolescente , Adulto , Anticuerpos Anticardiolipina/sangre , Anticuerpos Antifosfolípidos/sangre , Síndrome Antifosfolípido/diagnóstico , Síndrome Antifosfolípido/inmunología , Síndrome Antifosfolípido/patología , Estudios de Casos y Controles , Enfermedad Catastrófica , Femenino , Estudios de Seguimiento , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Inhibidor de Coagulación del Lupus/sangre , Masculino , Persona de Mediana Edad , Factores de Riesgo , Resultado del Tratamiento , beta 2 Glicoproteína I/antagonistas & inhibidores , beta 2 Glicoproteína I/genética , beta 2 Glicoproteína I/inmunologíaRESUMEN
DNA vaccines have recently emerged as a therapeutic agent for treating autoimmune diseases, such as multiple sclerosis. Antiphospholipid antibody syndrome (APS) is an autoimmune disease characterized by ß2-glycoprotein I (ß2-GPI)-targeting antiphospholipid antibodies (APAs) and vascular thrombosis or obstetrical complications. To examine the therapeutic potential of a ß2-GPI DNA vaccine, we administered a vaccine mixed with FK506 as an adjuvant to a mouse model of obstetric APS. First, the pCMV3-ß2-GPI DNA vaccine, which encodes the full-length human ß2-GPI gene, was constructed. Then, we administered the ß2-GPI DNA vaccine in 0.1 ml of saline, mixed with or without 100 µg of FK506, intramuscularly to the mice on days 28, 35 and 42. Blood titers of the anti-ß2-GPI antibody, platelet counts, activated partial thromboplastin times (aPTTs), and the percentage of fetal loss were measured. We also stimulated murine splenic T cells ex vivo with ß2-GPI and determined the T helper cell proportion and cytokine secretion. The administration of the ß2-GPI DNA vaccine mixed with FK506 reduced the blood IgG anti-ß2-GPI antibody titers and suppressed APS manifestations in mice. The combination also suppressed interferon-γ and interleukin (IL)-17A secretion but increased the Treg cell proportion and IL-10 secretion in murine splenic T cells following ex vivo stimulation with ß2-GPI. Our results demonstrated the therapeutic efficacy of a ß2-GPI DNA vaccine and FK506 as an adjuvant in a murine model of obstetric APS. Possible mechanisms include the inhibition of Th1 and Th17 responses and the up-regulation of Treg cells.
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Adyuvantes Farmacéuticos/administración & dosificación , Síndrome Antifosfolípido/prevención & control , Modelos Animales de Enfermedad , Tacrolimus/administración & dosificación , Vacunas de ADN/administración & dosificación , beta 2 Glicoproteína I/genética , Animales , Anticuerpos Antifosfolípidos/sangre , Anticuerpos Antifosfolípidos/inmunología , Síndrome Antifosfolípido/genética , Síndrome Antifosfolípido/inmunología , Síndrome Antifosfolípido/patología , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Proliferación Celular , Citocinas/metabolismo , Femenino , Inmunosupresores/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Embarazo , Linfocitos T Reguladores/inmunología , Vacunas de ADN/genéticaRESUMEN
Antiphospholipid antibody (aPL)mediated antiphospholipid syndrome (APS) is an autoimmune disease. Upon binding to aPL, the primary antigen of aPL, ß2glycoprotein I (ß2GP I), induces abnormal immune function, which further activates downstream signaling pathways in the cell and eventually leads to APS. The present study aimed to determine whether ß2GP I antigen and antiß2glycoprotein I antibody (aß2GP I), which belong to the aPL class of antibodies, may affect human chorionic epithelium cell (JEG3) proliferation, migration and invasion. Recombinant human (rh)ß2GP I protein was expressed using a prokaryotic expression system and aß2GP I antibody was purified from the blood serum of 10 patients with recurrent pregnancy loss. JEG3 cells were stimulated with rhß2GP I and aß2GP I separately or simultaneously, and serum immunoglobulin G of normal pregnant women was used as negative control. Using cell counting kit8, cell cycle and transwell assays in addition to EdU staining, it was determined that aß2GP I/rhß2GP I complex markedly increased JEG3 cell proliferation, migration and invasion. The results revealed that mRNA levels of inhibitor of nuclear factor (NF)κB kinase subunit (IKKß), myeloid differentiation primary response protein MyD88 (MyD88), NFκB and NFκB inhibitor α (IκBα), as well as the protein levels of MyD88, IκBα and phospho(p)IκBα in JEG3 cells increased following incubation with the aß2GP I/rhß2GP I complex. The observed upregulation of pIκBα protein suggested that IκBαmediated inhibition of NFκB was weakened. Furthermore, JEG3 cells were transfected with PGMLVNFκBLu vector. Luciferase activity in JEG3NFκBLuc1 and JEG3NFκBLuc2 cells was enhanced following treatment with aß2GP I/rhß2GP I complex. The present study demonstrated that aß2GP I/rhß2GP I complex activates NFκB through MyD88 signal transduction pathway, which further enhances JEG3 cell proliferation, migration and invasion.
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Anticuerpos Antifosfolípidos/inmunología , Complejo Antígeno-Anticuerpo/inmunología , Autoantígenos/inmunología , beta 2 Glicoproteína I/inmunología , Complejo Antígeno-Anticuerpo/metabolismo , Autoantígenos/genética , Autoantígenos/aislamiento & purificación , Ciclo Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Expresión Génica , Humanos , FN-kappa B/metabolismo , Unión Proteica , Proteínas Recombinantes , beta 2 Glicoproteína I/genética , beta 2 Glicoproteína I/aislamiento & purificaciónRESUMEN
The immune responses of males and females to bacterial infections display differences. The mechanisms that underlie this sexual dimorphism are multifactorial. Lipopolysaccharide (LPS) contributes to the pathogenesis of endotoxaemia. We have previously demonstrated that the plasma protein beta-2 glycoprotein-1 (ß2GPI) reduces LPS-induced inflammation in male mice. In the present study using a more robust infection model of septicaemia the role of ß2GPI is examined in both male and female wild type (WT) and ß2GPI deficient (ß2GPI-/-) mice challenged with Escherichia coli (E. coli) intravenously. ß2GPI deficiency led to an increase of E. coli colony forming units (CFU) in the circulation of both male and female mice. In male ß2GPI-/- mice this was associated with a worse clinical severity score. This difference was not observed between female ß2GPI-/- and female WT mice. Male WT mice had decreased levels of total and increased levels of free thiol ß2GPI following administration of LPS or E. coli. This pattern of sexual dimorphic response was also observed in our cohort of humans with sepsis. These findings support a role for ß2GPI in modulating the sex-specific susceptibility to gram-negative septicaemia.
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Endotoxemia/genética , Endotoxemia/inmunología , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/inmunología , Escherichia coli/inmunología , beta 2 Glicoproteína I/genética , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Endotoxemia/sangre , Endotoxemia/diagnóstico , Infecciones por Escherichia coli/sangre , Infecciones por Escherichia coli/diagnóstico , Femenino , Predisposición Genética a la Enfermedad , Humanos , Lipopolisacáridos/efectos adversos , Lipopolisacáridos/inmunología , Masculino , Ratones , Ratones Noqueados , Especificidad de Órganos , Sepsis/genética , Sepsis/inmunología , Índice de Severidad de la Enfermedad , Factores Sexuales , beta 2 Glicoproteína I/sangreRESUMEN
The first, obligatory replication phase of malaria parasite infections is characterized by rapid expansion and differentiation of single parasites in liver cells, resulting in the formation and release of thousands of invasive merozoites into the bloodstream. Hepatic Plasmodium development occurs inside a specialized membranous compartment termed the parasitophorous vacuole (PV). Here, we show that, during the parasite's hepatic replication, the C-terminal region of the parasitic PV membrane protein exported protein 1 (EXP-1) binds to host Apolipoprotein H (ApoH) and that this molecular interaction plays a pivotal role for successful Plasmodium liver-stage development. Expression of a truncated EXP-1 protein, missing the specific ApoH interaction site, or down-regulation of ApoH expression in either hepatic cells or mouse livers by RNA interference resulted in impaired intrahepatic development. Furthermore, infection of mice with sporozoites expressing a truncated version of EXP-1 resulted in both a significant reduction of liver burden and delayed blood-stage patency, leading to a disease outcome different from that generally induced by infection with wild-type parasites. This study identifies a host-parasite protein interaction during the hepatic stage of infection by Plasmodium parasites. The identification of such vital interactions may hold potential toward the development of novel malaria prevention strategies.
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Hígado/parasitología , Malaria/parasitología , Proteínas de la Membrana/metabolismo , Plasmodium berghei/fisiología , Proteínas Protozoarias/metabolismo , beta 2 Glicoproteína I/metabolismo , Animales , Animales Modificados Genéticamente , Sitios de Unión , Regulación hacia Abajo , Genes Protozoarios , Células HEK293 , Hepatocitos/parasitología , Humanos , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Plasmodium berghei/genética , Plasmodium berghei/crecimiento & desarrollo , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/genética , Eliminación de Secuencia , Esporozoítos/fisiología , Vacuolas/parasitología , beta 2 Glicoproteína I/antagonistas & inhibidores , beta 2 Glicoproteína I/genéticaRESUMEN
Homozygote genotype V247 of the ß2-glycoprotein-I (ß2GP-I) gene has been associated with anti-ß2GP-I and thrombosis in patients with primary anti-phospholipid syndrome APS (PAPS). However, the cellular immune response to ß2GP-I247 has been little studied. OBJECTIVE: To evaluate the immune cellular proliferation in response to native and non-native ß2GP-I247 valine/leucine phenotype from Mexican patients with PAPS. METHODS: We studied 10 patients with PAPS and 10 healthy control subjects (HC). The polymorphism at position 247 of the ß2GP-I gene was determined by PCR-RFLP and the corresponding ß2GP-I protein was subsequently purified from normal human plasma by affinity chromatography. PBMC purified from patients and controls were stimulated with ß2GP-I under native and in non native (reduced) conditions. We also determined the anti-ß2GP-I production in vitro by B cell clones (EBV) generated in cocultures experiments. Differential Scanning Calorimetry (DSC) was studied to determine the structural differences between the ß2GP-I247 valine/leucine isoforms. Cytokine profile (IL-2, IL-4, IL-6, TNFα, INFγ) was evaluated in culture supernatants. RESULTS: PAPS and healthy control PBMCs had a higher proliferative response when stimulated with ß2GP-I under reduced cultures conditions compared to non-denatured conditions. PBMCs response from PAPS patients was higher. We observed more cell proliferation in response to ß2GP-I247 valine/leucine or valine isoforms in non-native conditions. In contrast, this response was not significant against ß2GP-I247 leucine. These findings were T CD4+-dependent. Similar results were obtained with B cell clones derived from PAPS patients, which showed more pronounced proliferation in non native conditions and higher against ß2GP-I247 valine. No differences were found in anti-ß2GP-I production, but high levels of IL-6 in vitro were identified. The structural analysis of both ß2GP-I247 isoforms by DSC showed a major conformational change due to a single mutation in the ß2GP-I variants. CONCLUSIONS: PAPS PBMCs had a higher cellular response against ß2GP-I247 in non-native culture conditions preferentially to the ß2GP-I247 valine phenotype. This effect is T CD4+ dependent and appears to be driven by tertiary structural changes adopted by ß2GP-I247 polymorphism.
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Síndrome Antifosfolípido/genética , Linfocitos T CD4-Positivos/inmunología , beta 2 Glicoproteína I/genética , Adulto , Síndrome Antifosfolípido/inmunología , Células Clonales , Citocinas/metabolismo , Femenino , Genotipo , Humanos , Inmunidad Celular , Leucina , Masculino , México , Persona de Mediana Edad , Mutación/genética , Fenotipo , Polimorfismo Genético , ValinaRESUMEN
A significant amount of myocardial damage during a myocardial infarction (MI) occurs during the reperfusion stage, termed ischaemia/reperfusion (I/R) injury, and accounts for up to 50% of total infarcted tissue post-MI. During the reperfusion phase, a complex interplay of multiple pathways and mechanisms is activated, which ultimately leads to cell death, primarily through apoptosis. There is some evidence from a lupus mouse model that lupus IgG, specifically the antiphospholipid (aPL) antibody subset, is pathogenic in mesenteric I/R injury. Furthermore, it has previously been shown that the immunodominant epitope for the majority of circulating pathogenic aPLs resides in the N-terminal domain I (DI) of beta-2 glycoprotein I (ß2GPI). This study describes the enhanced pathogenic effect of purified IgG derived from patients with lupus and/or the antiphospholipid syndrome in a cardiomyocyte H/R in vitro model. Furthermore, we have demonstrated a pathogenic role for aPL containing samples, mediated via aPL-ß2GPI interactions, resulting in activation of the pro-apoptotic p38 MAPK pathway. This was shown to be inhibited using a recombinant human peptide of domain I of ß2GPI in the fluid phase, suggesting that the pathogenic anti-ß2GPI antibodies in this in vitro model target this domain.
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Anticuerpos Antifosfolípidos/genética , Infarto del Miocardio/genética , beta 2 Glicoproteína I/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Adulto , Animales , Anticuerpos Antifosfolípidos/metabolismo , Apoptosis/genética , Hipoxia de la Célula/genética , Modelos Animales de Enfermedad , Humanos , Inmunoglobulina G/inmunología , Masculino , Ratones , Persona de Mediana Edad , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Ratas , Daño por Reperfusión/genética , Daño por Reperfusión/fisiopatología , beta 2 Glicoproteína I/genéticaRESUMEN
BACKGROUND: Immunoglobulin (Ig)A anti-ß2-glycoprotein I (aB2GP1) antibodies are associated with thrombotic events, cardiovascular morbidity, and death in dialysis patients. About 30% of patients with chronic renal disease are positive for IgA aB2GP1; however, the origin of these antibodies is unknown. It has been speculated that dialysis membranes, age, or etiology of renal base disease are possible precipitating factors, although these factors do not appear to be the source of antibodies. B2GP1 is a protein of 326 amino acids grouped into five domains. Eight polymorphisms have been described; the most important are Val/Leu247, which appears to predispose aB2GP1 antibody production in patients with anti-phospholipid syndrome, and Trp/Ser316, which appears to have protective antibody production of aB2GP1. METHODS: DNA samples from 92 patients with renal failure on hemodialysis were randomly collected with a 1:1 ratio for the positivity for IgA aB2GP1. Forty-six samples were positive for IgA aB2GP1 (group 1) and 46 negative for IgA aB2GP1 (group 2). All samples were anonymized to study polymorphism Val/Leu247 and polymorphism Trp/Ser316. RESULTS: No significant differences were observed between those who were positive or negative for IgA aB2GP1 in patients with renal failure treated with hemodialysis and the polymorphism located in codons 247 and 316. CONCLUSIONS: The two groups of patients have the same prevalence in polymorphisms 247 and 316, and therefore there appears not to be a genetic predisposition in our population. New trigger factors must be studied.
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Autoanticuerpos/sangre , Inmunoglobulina A/sangre , Fallo Renal Crónico/sangre , Polimorfismo Genético , beta 2 Glicoproteína I/genética , Adulto , Autoanticuerpos/genética , Autoanticuerpos/inmunología , Codón , Femenino , Humanos , Inmunoglobulina A/genética , Inmunoglobulina A/inmunología , Fallo Renal Crónico/genética , Fallo Renal Crónico/inmunología , Masculino , Persona de Mediana Edad , Diálisis Renal , beta 2 Glicoproteína I/sangre , beta 2 Glicoproteína I/inmunologíaRESUMEN
BACKGROUND: Antiphospholipid antibodies (aPL) have been advocated as potential mediators of unexplained female infertility, but no evidence has yet been raised to support such an association. OBJECTIVES: To test the hypothesis that aPL might interfere with uterine decidualization, a gene expression study was performed on decidual stromal cells treated with different aPL preparations. METHODS: Decidual stromal cells were isolated from first-trimester deciduas obtained from two women undergoing elective abortion, and treated with: (i) a ß2GPI-dependent aPL monoclonal antibody (IS3); (ii) IS3 plus TIFI, a synthetic peptide mimicking PL-binding region of ß2GPI; and (iii) IgG from healthy subjects (NHS). Gene expression data were acquired using human HT-12 v3 beadchip arrays (Illumina). Differential expression analysis was performed by fitting a gene-wise linear model using the treatment group and decidual source as covariates. RESULTS: In the comparison of IS3 versus IgG NHS-treated decidual cells, gene ontology (GO) enrichment was expressed in terms relating to well-characterized aPL-mediated cellular effects: "inflammatory response," "immune response," "response to stress," "oxydoreductase activity," "metalloendopeptidase activity," and "cytokine/chemokine activity." As expected, almost all genes were up-regulated by IS3 treatment. The same GO categories appeared to be differentially expressed when IS3 treatment was compared to IS3 + TIFI, but with most genes being down-regulated. CONCLUSIONS: Given the inflammatory response evinced on gene expression analysis of decidual stromal cells treated with a ß2GPI -dependent aPL monoclonal antibody, it is feasible that aPL might interfere with uterine decidualization, affecting the early stages of implantation and ultimately resulting in female infertility.
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Anticuerpos Antifosfolípidos/genética , Anticuerpos Monoclonales/farmacología , Decidua , Estradiol/farmacología , Infertilidad Femenina , Acetato de Medroxiprogesterona/farmacología , Células del Estroma , beta 2 Glicoproteína I , Adulto , Células Cultivadas , Anticonceptivos Femeninos/farmacología , Decidua/inmunología , Decidua/patología , Regulación hacia Abajo , Estrógenos/farmacología , Femenino , Perfilación de la Expresión Génica , Humanos , Factores Inmunológicos/farmacología , Infertilidad Femenina/genética , Infertilidad Femenina/inmunología , Infertilidad Femenina/terapia , Embarazo , Células del Estroma/efectos de los fármacos , Células del Estroma/inmunología , Células del Estroma/patología , Resultado del Tratamiento , beta 2 Glicoproteína I/genética , beta 2 Glicoproteína I/inmunologíaRESUMEN
OBJECTIVE: To investigate the role of Toll-like receptor 2 (TLR2) in ß2-glycoprotein 1/anti-ß2-glycoprotein 1 (ß2GP1/anti-ß2GP1)-mediated tumor necrosis factor α (TNF-α) expression in mouse peritoneal macrophages. METHODS: The peritoneal macrophages from BALB/c mice were treated with ß2GP1/anti-ß2GP1 complex, agonist of TLR2 (Pam3CSK4) and agonist of TLR4 [lipopolysaccharide (LPS)], inhibitor of TLR2 [monoclonal IgG to mouse TLR2 (anti-mTLR2-IgG)] and inhibitor of TLR4 (TAK-242) in vitro. The mRNA level of TNF-α in the peritoneal macrophages was tested by real-time quantitative PCR, the protein expression of TNF-α was detected by Western blotting and immunofluorescence cytochemistry, and the expression of TLR2 on the surface of the peritoneal macrophages was assessed by flow cytometry. RESULTS: The mRNA and protein expression of TNF-α was significantly enhanced in mouse peritoneal macrophages treated with ß2GP1/anti-ß2GP1 complex, Pam3CSK4 and LPS. Anti-mTLR2-IgG could inhibit the effects of the above stimuli on TNF-α expression, but its effects were weaker than those of TAK-242. Meanwhile, the combination of anti-mTLR2-IgG and TAK-242 did not show much stronger inhibitory effects. Flow cytometry analysis showed that expression of TLR2 was enhanced by ß2GP1/anti-ß2GP1 complex, Pam3CSK4 and LPS in mouse peritoneal macrophages. However, anti-mTLR2-IgG, or TAK-242, or combination of both could not inhibit TLR2 expression in macrophages. CONCLUSION: Both TLR4 and TLR2 could increase the stimulating effect of ß2GP1/anti-ß2GP1 complex on the expression of TNF-α in mouse peritoneal macrophages.