This study, developed within the Innovative Medicines Initiative Joint Undertaking project PRECISESADS framework, aimed at functionally characterize the monocyte subsets in RA patients, and analyze ...their involvement in the increased CV risk associated with RA.
The frequencies of monocyte subpopulations in the peripheral blood of 140 RA patients and 145 healthy donors (HDs) included in the PRECISESADS study were determined by flow cytometry. A second cohort of 50 RA patients and 30 HDs was included, of which CD14
and CD16
monocyte subpopulations were isolated using immuno-magnetic selection. Their transcriptomic profiles (mRNA and microRNA), proinflammatory patterns and activated pathways were evaluated and related to clinical features and CV risk. Mechanistic
analyses were further performed.
CD14
CD16
intermediate monocytes were extended in both cohorts of RA patients. Their increased frequency was associated with the positivity for autoantibodies, disease duration, inflammation, endothelial dysfunction and the presence of atheroma plaques, as well as with the CV risk score. CD14
and CD16
monocyte subsets showed distinctive and specific mRNA and microRNA profiles, along with specific intracellular signaling activation, indicating different functionalities. Moreover, that specific molecular profiles were interrelated and associated to atherosclerosis development and increased CV risk in RA patients.
, RA serum promoted differentiation of CD14
CD16
to CD14
CD16
monocytes. Co-culture with RA-isolated monocyte subsets induced differential activation of endothelial cells.
Our overall data suggest that the generation of inflammatory monocytes is associated to the autoimmune/inflammatory response that mediates RA. These monocyte subsets, -which display specific and distinctive molecular signatures- might promote endothelial dysfunction and in turn, the progression of atherosclerosis through a finely regulated process driving CVD development in RA.
Objective
To investigate the expression of protease‐activated receptors (PARs), their potential regulation by anticardiolipin antibodies (aCL), and their association with the expression of other ...molecules relevant to thrombosis in monocytes obtained from 62 patients with primary antiphospholipid syndrome (APS).
Methods
Monocytes were isolated from peripheral blood mononuclear cells by magnetic depletion of nonmonocytes. Expression of tissue factor (TF) and PARs 1–4 genes was measured by quantitative real‐time reverse transcription–polymerase chain reaction. Cell surface TF and PARs 1–4 expression was analyzed by flow cytometry. For in vitro studies, purified normal monocytes were incubated with purified APS patient IgG, normal human serum IgG, or lipopolysaccharide, in the presence or absence of specific monoclonal antibodies anti–PAR‐1 (ATAP2) or anti–PAR‐2 (SAM11) to test the effect of blocking the active site of PAR‐1 or PAR‐2.
Results
Analysis of both mRNA and protein for the 4 PARs revealed significantly increased expression of PAR‐2 as compared with the control groups. PAR‐1 was significantly overexpressed in APS patients with thrombosis and in the control patients with thrombosis but without APS. PAR‐3 expression was not significantly altered. PAR‐4 expression was absent in all groups analyzed. In addition, we demonstrated a correlation between the levels of PAR‐2 and the titers of IgG aCL, as well as parallel behavior of TF and PAR‐2 expression. In vitro, IgG from APS patients significantly increased monocyte expression of PAR‐1 and PAR‐2. Inhibition studies suggested that there was direct cross‐talk between TF and PAR‐2, such that inhibition of PAR‐2 prevented the aCL‐induced expression of TF.
Conclusion
These results provide the first demonstration of increased expression of PARs in monocytes from patients with APS. Thus, PAR antagonists might have therapeutic potential as antithrombotic agents in APS.
Antiphospholipid syndrome (APS) is a disorder characterized by the association of arterial or venous thrombosis and/or pregnancy morbidity with the presence of antiphospholipid antibodies ...(anticardiolipin antibodies, lupus anticoagulant antibodies, and/or anti–β2-glycoprotein I antibodies). Several studies have contributed to uncovering the basis of antiphospholipid antibody pathogenicity, including the targeted cellular components, affected systems, involved receptors, intracellular pathways used, and the effector molecules that are altered in the process. Therapy for thrombosis traditionally has been based on long-term oral anticoagulation; however, bleeding complications and recurrence despite high-intensity anticoagulation can occur. Based on all the data obtained, new potential therapeutic agents have been proposed. Statins have a variety of direct effects on gene expression and the function of cells of both the innate and adaptive immune systems, many of which are related to blockade of GTPase isoprenylation. In APS, statins have multiple profound effects on monocyte, lymphocyte, and endothelial cell activities, all of which may contribute to thrombosis prevention in APS patients. Nevertheless, larger randomized trials are needed to validate the role of statins in the treatment of this autoimmune disease.
Objective Aberrant activation of tyrosine kinase receptors is frequently observed in acute myelogenous leukemia (AML). Moreover, activating mutations of the fms-like tyrosine kinase 3 (FLT3) receptor ...can be found in approximately 30% of patients, thereby representing one of the most frequent single genetic alterations in AML. AEE788, a novel dual receptor tyrosine kinase inhibitor of endothelial growth factor and vascular endothelial growth factor (VEGF), is being studied in several solid tumors with remarkable success. It is not known, however, about the efficacy of this inhibitor in the treatment of AML. Therefore, we investigated the effect of AEE788 in the treatment of three human AML cell lines and seven AML patient samples. Materials and Methods Cell survival in THP-1, MOLM-13, and MV4-11 cell lines (the two last harboring the FLT3/internal tandem duplication mutation) and AML blasts incubated with 0.5 to 15 μM AEE788 were quantified. We also studied the activation of VEGF/VEGF receptors loop, FLT3, and their downstream effectors (Akt, extracellular signal-regulated kinase, signal transducers and activators of transcription 5, and nuclear factor−κB). Results Our data showed that AEE788 was a tyrosine kinase inhibitor of FLT3 activity and had antiproliferative and proapoptotic activity in AML-derived cell lines and AML blasts that presented phosphorylation of the FLT3 receptor. Consistently, in these cells AEE788 abrogated VEGF/VEGF receptors activation and the survival signaling pathways studied. Conclusion Taken together, the activity of AEE788 might represent a promising new option of targeting FLT3 for the treatment of AML.
Objective
To examine the prevalence of isolated IgA anti–β
2
‐glycoprotein I (anti‐β
2
GPI) positivity and the association of these antibodies, and a subgroup that bind specifically to domain IV/V of ...β
2
GPI, with clinical manifestations of the antiphospholipid syndrome (APS) in 3 patient groups and to evaluate the pathogenicity of IgA anti‐β
2
GPI in a mouse model of thrombosis.
Methods
Patients with systemic lupus erythematosus (SLE) from a multiethnic, multicenter cohort (LUpus in MInorities, NAture versus nurture LUMINA) (n = 558), patients with SLE from the Hopkins Lupus Cohort (n = 215), and serum samples referred to the Antiphospholipid Standardization Laboratory (APLS) (n = 5,098) were evaluated. IgA anti‐β
2
GPI titers and binding to domain IV/V of β
2
GPI were examined by enzyme‐linked immunosorbent assay (ELISA). CD1 mice were inoculated with purified IgA anti‐β
2
GPI antibodies, and surgical procedures and ELISAs were performed to evaluate thrombus development and tissue factor (TF) activity.
Results
A total of 198 patients were found to be positive for IgA anti‐β
2
GPI isotype, and 57 patients were positive exclusively for IgA anti‐β
2
GPI antibodies. Of these, 13 of 23 patients (56.5%) in the LUMINA cohort, 17 of 17 patients (100%) in the Hopkins cohort, and 10 of 17 patients (58.9%) referred to APLS had at least one APS‐related clinical manifestation. Fifty‐four percent of all the IgA anti‐β
2
GPI–positive serum samples reacted with domain IV/V of anti‐β
2
GPI, and 77% of those had clinical features of APS. Isolated IgA anti‐β
2
GPI positivity was associated with an increased risk of arterial thrombosis (
P
< 0.001), venous thrombosis (
P
= 0.015), and all thrombosis (
P
< 0.001). The association between isolated IgA anti‐β
2
GPI and arterial thrombosis (
P
= 0.0003) and all thrombosis (
P
= 0.0003) remained significant after adjusting for other risk factors for thrombosis. In vivo mouse studies demonstrated that IgA anti‐β
2
GPI antibodies induced significantly larger thrombi and higher TF levels compared to controls.
Conclusion
Isolated IgA anti‐β
2
GPI–positive titers may identify additional patients with clinical features of APS. Testing for these antibodies when other antiphospholipid tests are negative and APS is suspected is recommended. IgA anti‐β
2
GPI antibodies directed to domain IV/V of β
2
GPI represent an important subgroup of clinically relevant antiphospholipids.
Objective To examine the prevalence of isolated IgA anti-beta2-glycoprotein I (anti-beta2GPI) positivity and the association of these antibodies, and a subgroup that bind specifically to domain IV/V ...of beta2GPI, with clinical manifestations of the antiphospholipid syndrome (APS) in 3 patient groups and to evaluate the pathogenicity of IgA anti-beta2GPI in a mouse model of thrombosis. Methods Patients with systemic lupus erythematosus (SLE) from a multiethnic, multicenter cohort (LUpus in MInorities, NAture versus nurture LUMINA) (n = 558), patients with SLE from the Hopkins Lupus Cohort (n = 215), and serum samples referred to the Antiphospholipid Standardization Laboratory (APLS) (n = 5,098) were evaluated. IgA anti-beta2GPI titers and binding to domain IV/V of beta2GPI were examined by enzyme-linked immunosorbent assay (ELISA). CD1 mice were inoculated with purified IgA anti-beta2GPI antibodies, and surgical procedures and ELISAs were performed to evaluate thrombus development and tissue factor (TF) activity. Results A total of 198 patients were found to be positive for IgA anti-beta2GPI isotype, and 57 patients were positive exclusively for IgA anti-beta2GPI antibodies. Of these, 13 of 23 patients (56.5%) in the LUMINA cohort, 17 of 17 patients (100%) in the Hopkins cohort, and 10 of 17 patients (58.9%) referred to APLS had at least one APS-related clinical manifestation. Fifty-four percent of all the IgA anti-beta2GPI-positive serum samples reacted with domain IV/V of anti-beta2GPI, and 77% of those had clinical features of APS. Isolated IgA anti-beta2GPI positivity was associated with an increased risk of arterial thrombosis (P < 0.001), venous thrombosis (P = 0.015), and all thrombosis (P < 0.001). The association between isolated IgA anti-beta2GPI and arterial thrombosis (P = 0.0003) and all thrombosis (P = 0.0003) remained significant after adjusting for other risk factors for thrombosis. In vivo mouse studies demonstrated that IgA anti-beta2GPI antibodies induced significantly larger thrombi and higher TF levels compared to controls. Conclusion Isolated IgA anti-beta2GPI-positive titers may identify additional patients with clinical features of APS. Testing for these antibodies when other antiphospholipid tests are negative and APS is suspected is recommended. IgA anti-beta2GPI antibodies directed to domain IV/V of beta2GPI represent an important subgroup of clinically relevant antiphospholipids. PUBLICATION ABSTRACT
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