Background/Purpose: The mammalian target of rapamycin (mTOR) is a component of MTOR complex-1 that, when activated by upstream molecule protein kinase B (AKT) and another cell signaling pathway mitogen activated protein kinase p38 (p38/MAPK), is responsible for protective autophagy, cell proliferation and growth, and protein synthesis. These independent signaling pathways cross-talk and regulate each other. The mTOR pathway is responsible for the endothelial proliferation leading to chronic renal vascular lesions in antiphospholipid antibody (aPL)-positive patients. Firstly, our team developed a flow cytometry-based assay that assesses the basal and responsive mTOR phosphorylation in peripheral blood monocytes of patients in relation to the activation of AKT and p38/MAPK . Secondly, based on this assay, we evaluated three antiphospholipid syndrome (APS) patients.

Methods: We assayed unstimulated and lipopolysaccharide (LPS)-stimulated whole blood, 30 minutes at 37C, for peripheral blood classical monocyte (HLA-DR⁺⁺CD14⁺CD16^–), phosphorylated mTOR/AKT, and p38/MAPK analysis. Unstimulated and stimulated levels of phosphorylated mTOR/AKT and p38/MAPK were reported as percent positives and median fluorescence intensities, and compared against each other. Three APS patients, identified according to the Updated Sapporo Classification Criteria, were assessed with the assay. The mean results were compared with three age- and gender-matched healthy volunteers by the Mann-Whitney U test.

Results: All patients were male (mean age: 51 ± 18.5) with no other systemic autoimmune diseases. Patient #1 had history of arterial thrombosis, autoimmune hemolytic anemia, thrombocytopenia, white matter changes, aPL nephropathy, and livedo reticularis (on aspirin [ASA], azathioprine, and low dose prednisone). Patient #2 had arterial thrombosis, transient ischemic attacks, and microthrombotic APS with peripheral cyanosis, diffuse alveolar hemorrhage, and livedoid vasculopathy (on ASA, hydroxychloroquine, atorvastatin, and low-molecular-weight-heparin). Patient #3 had microthrombotic APS with livedoid vasculopathy (on mycophenolate mofetil and warfarin). All three patients had high mTOR levels both at baseline and after stimulation when compared to healthy controls (p: 0.02 and p: 0.007, respectively), which was independent of AKT and p38/MAPK activation (p: 0.02 and p: 0.03, respectively) (Table).

Conclusion: We developed a flow cytometry-based assay that assesses the activation of mTOR and related proteins; our preliminary analysis of three APS patients demonstrates that mTOR pathway is activated independent of AKT and p38/MAPK. Further clinical studies will determine the clinical utility of this assay.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/flow-cytometric-assessment-of-the-mammalian-target-of-rapamycin-pathway-using-antiphospholipid-syndrome-as-a-disease-model/