卒業研究
Clinical usefulness of flow cytometry analysis using whole blood assay to analyze the complex formation of activated platelets/monocytes or granulocytes
Abstract
In recent years, the number of patients with arterial thromboembolism such as ischemic heart disease and cerebrovascular disorder is increasing in Japan due to the Westernization of lifestyle. Therefore, laboratory tests that accurately captures platelet activation that occurs in the early stages of arterial thrombus formation are extremely important. However, a laboratory test method that can quantify the in vivo platelet activation state with high reproducibility has not yet been established. Recently, we newly established a flow cytometry analysis using whole blood assay to analyze the complex formation of activated platelets/monocytes or granulocytes. The purpose of this study was to investigate whether the established flow cytometry analysis was useful for evaluating the pathophysiology and therapeutic effects of thrombosis on prospective clinical studies in the field of emergency medicine. We measured the positive rate of activated platelets/monocytes or granulocytes complexes in various patients such as Cerebral infarction, myocardial infarction, deep vein thrombosis, sepsis, head injury, and fracture. The positive rate of activated platelets/monocytes complexes was significantly higher in patients with cerebral infarction, myocardial infarction, deep vein thrombosis, and head injury than in healthy control. On the other hand, the positive rate of activated platelets/granulocytes complexes was obviously increased only in septic patients with DIC than in patients with other cases. In addition, when we compared the positive rate of activated platelets/monocytes complexes in patients with cerebral infarction before and after treatment, we confirmed that the formation of activated platelets/monocytes complexes was significantly reduced after treatment of cerebral infarction compared to before treatment. Furthermore, we confirmed the possibility in basic experiments that a platelet-activating factor exists in the plasma of patients with acute cerebral infarction. The present study suggested that flow cytometry analysis using whole blood assay to analyze the complex formation of activated platelets/monocytes were useful not only for evaluating the pathophysiology of arterial and venous thrombosis but also for determining the effectiveness of treatment.
Establishment of new ELISA for screening test of antiphospholipid antibodies
Abstract
Anti-phospholipid antibodies (aPLs) are frequently associated with arterial and/or venous thrombotic complications and recurrent fetal loss in patients with systemic lupus erythematosus (SLE). It is now generally accepted that aPLs do not bind directly to the negatively charged phospholipid itself but rather to complexes of the phospholipid and phospholipid-binding proteins and that the most common antigenic targets are β2-glycoprotein I (β2GPI) and prothrombin (PT). Therefore, multiple aPLs to β2GPI and PT must be measured by enzyme-linked immunosorbent assay (ELISA) to accurately diagnose antiphospholipid syndrome (APS). The purpose of this study was to establish a new ELISA that could detect more aPLs as the first screening test for the diagnosis of APS. As a first experiment, we have established 4 types home-made ELISAs by combining two immobilized phospholipids (cardiolipin: CL, phosphatidylserine: PS) and three epitope-providing proteins (β2GPI, PT, and adult bovine plasma: ABP). Then, we compared the aPLs detection sensitivities of the 4 types home-made ELISAs using eight plasmas from SLE patients. Based on the results of the basic study, we used aCL/ABP-ELISA and aCL/ABS-ELISA in the present study. We measured the levels of each aPLs using 2 home-made ELISAs and 10 commercial ELISA kits in 143 plasma samples including APS patients (n=66) and healthy subjects (n=77). We performed the receiver operating characteristic (ROC) curve analysis on the absorbance level in mAU detected by aCL/ABP and aCL/ABS ELISA in 143 plasma samples, and we set cut-off values for two home-made ELISAs. The positive rate of aPLs in the APS patient group was higher with two home-made ELISAs compared to other commercially available ELISA kits. ROC curve analysis showed that the accuracy of predicting APS based on the test results of aCL/ABS-ELISA was 0.73 in terms of the area under the ROC curve (AUC). In contrast, the test results of aCL/ABP-ELISA raised the accuracy (AUC) of predicting APS to 0.91.In summary, patients with APS frequently possess various types of aPLs such as aCL, aβ2GPI, aCL/β2GPI, and aPS/PT. The occurrence of recurrent thrombotic and obstetric complications in APS apparently depends on variable combinations of these types of aPLs. Therefore, it is important to detect many types of aPLs by ELISA in the screening test for APS diagnosis.
Analysis of binding ability to peripheral blood cells by anti-phospholipid antibodies
Abstract
Introduction:Anti-phospholipid antibodies (aPLs) are a heterogeneous group of autoantibodies that are frequently found in the plasma of patients with systemic lupus erythematosus (SLE). The presence of aPLs is associated with arterial and/or venous thrombotic complications in SLE patients. However, the pathological mechanism leading to arterial and/or venous thrombotic complications in these patients remain unclear. From our previous studies, we presumed that aPLs causes persistently high tissue factor (TF) expression and inflammatory cytokine production by interacting with peripheral blood monocytes and lymphocytes. In this study, we investigated whether IgG-aPLs purified from APS patient plasma bind to the cell surface of monocytes, lymphocytes and granulocytes in the peripheral blood of healthy individuals. We also examined monocyte surface receptors recognized by IgG-aPLs.
Materials and methods:Using flow cytometry analysis, we measured the binding activity of IgG-aPLs to monocytes, lymphocytes, and granulocytes in peripheral blood cells from six healthy control volunteers. In addition, we examined whether aPLs in the plasma of eight representative SLE patients bind to the CD14 and CD36 antigens immobilized on ELISA wells.
Results:Flow cytometry analysis revealed that IgG-aPLs bound to the surface of peripheral blood cells (92% of monocyte fraction, 99.8% of granulocyte fraction, and 16.3% of lymphocyte fraction). On the other hands, non-APS IgG showed little binding ability to peripheral blood cells. Furthermore, when we examined the binding activity of aPLs to CD14 and CD36 antigens immobilized on ELISA wells, we confirmed that the significant binding activity to only CD36 antigen, but not to CD14, in 3 out of 8 SLE patients' plasma.
Conclusion:These results suggest that IgG-aPLs cause persistently high TF expression and inflammatory cytokine production by directly bind to monocytes, which may be an important mechanism in the pathogenesis of arterial and venous thrombosis peculiar to APS patients.
Clinical significant of anti-phosphatidylserine/prothrombin antibodies.
Abstract
Anti-phospholipid antibodies (aPLs) are a heterogeneous group of autoantibodies that appear in a variety of autoimmune diseases, particularly systemic lupus erythematosus (SLE). The presence of aPLs is associated with clinical events such as arterial and/or venous thrombosis and recurrent fetal loss. Anti-phospholipid syndrome (APS) is currently diagnosed by both clinical findings (recurrent arterial and/or venous thrombosis and obstetric complications) and laboratory evidence of persistent aPLs. Detection of anti-cardiolipin (aCL) and anti-β2-glycoprotein I (aβ2GPI) by enzyme-linked immunosorbent assay (ELISA) and detection of lupus anticoagulant (LA) activity by phospholipid-dependent coagulation assays have been standardized for the diagnosis of APS. Recently, ELISA for anti-phosphatidylserine/prothrombin (aPS/PT) that recognizes epitope on prothrombin (PT) molecule bound to phosphatidylserine (PS) which is the main phospholipid of cell membrane, has been developed. To clarify the clinical significance of aPS/PT detected by ELISA, we examined the concentrations of aCL/β2GPI and aPS/PT in 178 patients with SLE comprising 28 cases with arterial thrombosis, 38 cases with venous thrombosis, 12 cases with pregnancy complications. The present study showed that both aCL/β2GPI and aPS/PT were significant risk factors for arterial and/or venous thrombosis in patients with SLE. However, ROC curve analysis showed that the accuracy of predicting arterial and venous thrombosis based on the test results of aCL/β2GPI -ELISA were 0.65 and 0.74 in terms of the area under the ROC curve (AUC). In contrast, the test results of aPS/PT-ELISA raised the AUC of predicting arterial and venous thrombosis to 0.92 and 0.86. It is important to note that the AUC for the presence of aPS/PT was much higher than that for the presence of aCL/β2GPI.Therefore, it is suggested that aPS/PT-ELISA is a very important marker in the diagnosis of APS. Furthermore, we verified the correlation between homemade ELISA and two commercially available ELISA kits in 72 patients with collagen disease (28 cases with APS and 44 cases without APS) and 69 healthy subjects. We found that the concentration of antibody measured by each ELISA greatly varied depending on the kit. However, we confirmed that the positive or negative judgment almost agreed by setting the clinical cutoff value for each ELISA. In summary, the positive results of aCL/β2GPI-ELISA and aPS/PT-ELISA are very important markers in the diagnosis of APS. The present study showed that the aPS/PT-ELISA correlates best with both arterial and venous thrombosis in patients with SLE.
Establishment of biomarkers useful for diagnosis of chronic fatigue syndrome - Verification of the oxidative stress index and the mononuclear cell surface antigen analysis -
Abstract
According to the guidelines of the United States Centers for Disease Control and Prevention, Chronic Fatigue syndrome (CFS) is a disorder diagnosed in the presence of at least 6 months of disabling, unexplained mental and physical fatigue accompanied by other physical and psychological symptoms. Psychological disorders such as depression, viral infections, autoimmune disease, and prolonged stresses have all been considered as potential causes behind CFS, although the mechanisms by which these conditions lead to the symptoms of CFS are still unclear. Furthermore, CFS is a highly heterogeneous and partly subjective illness, and no standard laboratory test is currently available for reliable diagnosis of CFS. The purpose of this study was to establish biomarkers useful for diagnosis of CFS. We measured the levels of oxidative stress index (OSI) in 15 healthy subjects and 28 patients with severe CFS whose brain neuroinflammation was confirmed by the PET / MRI imaging tests. The present study confirmed that the OSI levels were significantly higher in sera of the CFS patients with neuroinflammation than in those of the healthy volunteers. Our results provide the possibility that the evaluation of oxidative stress is a useful biomarker for the objective diagnosis of CFS. On the other hand, we confirmed that the proportion of B cells were significantly reduced in CFS patients compared with healthy subjects in lymphocyte subset analysis. Furthermore, the surface antigen analysis of monocytes revealed that the proportion of CD14+ / CD16- monocytes significantly decreased and the proportion of CD14+ / CD16+monocytes were significantly increased in CFS patient group as compared with healthy subjects. It is a very interesting finding that CD14+/ CD16+ monocytes associated with inflammatory diseases and autoimmune diseases increase in patients with CFS with intracerebral neuroinflammation. In addition, there was a significant correlation between the level of OSI and the proportion of inflammatory monocytes in CFS patients. These findings suggested that chronic inflammation caused by oxidative stress might be involved in the pathogenesis of CFS.
Establishment of clinical laboratory tests useful for determining the severity of chronic fatigue syndrome patient
Abstract
Chronic fatigue syndrome (CFS) are characterized by severe fatigue and a variety of non-specific symptoms such as fever, headache, muscle pain, sleep disorder. They are a disorder diagnosed in the presence of at least 6 months of disabling, unexplained mental and physical fatigue accompanied by other physical and psychological symptoms. Psychological disorders such as depression, viral infections, autoimmune diseases, and prolonged stresses have all been considered as potential causes behind CSF, although the mechanisms by which these conditions lead to the symptoms of CFS are still unclear. In addition, since CFS is a very heterogeneous and partially subjective disease, clinical laboratory tests that reflect the pathogenesis of CFS have not yet been established. The purpose of this study was to establish the clinical laboratory tests that accurately reflects the severity of CFS patients. In this study, we classified 36 CFS patients into 6 stages of severity according to the performance status (PS) level and examined whether there were clinical laboratory tests reflecting the severity of CFS patients. Test values indicating symptoms of anemia and infection were found in patients with severe CFS, but no clinical test items correlated with severity of CFS patients were found in this study. Recent studies suggested that oxidative stress and autonomic dysfunction may be related to brain neuroinflammation in CFS patients. Therefore, we investigated whether oxidative stress index (OSI), autonomic function test (LF / HF ratio), and monocyte surface antigen analysis reflect the severity of CFS patients. We analyzed heart rate variability by acceleration pulse waveform in 36 CFS patients and found that patient with relative sympathetic nerve hyperfunction by rise of the LF/HF ratio were existed in 28 (77.8%) of these CFS patients. Moreover, when 36 CFS patients were divided into 6 categories based on the performance status levels, the OSI levels and LF/HF ratios were increased significantly in relation to the performance status levels. On the other hand, the increase of CD14+ / CD16+ monocytes by monocyte surface antigen analysis was not related to the severity of CFS patients. These results suggest that the evaluation of oxidation stress index and relative sympathetic nerve hyperfunction reflects not only the diagnostic evaluation of fatigue states, but also the etiology of fatigue state.
Fundamental study for elucidation of pathophysiological mechanisms in antiphospholipid syndrome.
Abstract
Antiphospholipid syndrome is an autoimmune disease characterized by persistent of autoantibodies called antiphospholipid antibodies and arterial and/or venous thrombosis and recurrent fetal loss. The aim of our study was to elucidate the precise mechanism responsible for arterial or venous thrombosis in patients with APS. In previous studies, we found that the oxidation stress indexes were significantly higher in sera of the APS patients than in those of the healthy volunteers. Furthermore, we reported that oxidative stress and anti-phospholipid antibodies promote chemokine and cytokine production and tissue factor expression of mononuclear cells and vascular endothelial cells. In this study, we co-cultured human aortic endothelial cells (HAEC) and peripheral blood mononuclear cells (PBMC) to create a model close to in vivo environment. We conducted the fundamental studies to clarify the interaction between HAEC and PBMC using co-culture models. When HAEC and/or PBMC were treated by hydrogen peroxide or anti-phospholipid antibodies, we found that there was a difference in VCAM-1 expression of vascular endothelial cells and TNF-α production from PBMC between single culture model and co-culture model. However, we confirmed that the expression levels of VCAM-1 varies depending on the experiment day even under the same culture conditions. These results suggested that the further fundamental studies are necessary to investigate the effect of antiphospholipid antibodies and/or oxidative stress on adhesion factor expression in the co-culture model of endothelial cells and mononuclear cells.
The effect of oxidative stress on pathogenesis of disseminated intravascular coagulation syndrome (DIC)
Abstract
Disseminated intravascular coagulation (DIC) is an acquired syndrome characterized by systemic activation of blood coagulation occurring in the presence of various serious diseases, resulting in microvascular thrombosis in various organs. DIC exhibits extensive hemorrhagic symptoms or organ disorders due to an imbalance in blood coagulation and fibrinolysis. DIC, often associated with acute myelogenous leukemia (AML), characterizes both tissue-type plasminogen activator-induced fibrin/fibrinogen degradation and consumption coagulopathy, causing severe bleeding disorder. On the other hand, DIC associated with sepsis characterizes inhibition of fibrinolysis by plasminogen activator inhibitor-1 promotes microvascular thrombosis followed by multiple organ dysfunction syndrome. In all cases of DIC, one critical mediator of blood coagulation activation has been shown to be the release of a transmembrane glycoprotein called tissue factor (TF). However, clinical tests useful for predicting the development and progression of DIC have not yet been clarified. Recently, we revealed that the relative oxidation stress index might make a useful clinical marker for the evaluation of malignant grades and clinical stages of non-Hodgkin lymphoma. The main objective of this study was to clarify the role of the oxidation stress on the pathogenesis of DIC in patients with acute leukemia. We measured both oxidation and anti-oxidation activities simultaneously in sera from 40 patients with AML, 20 with chronic myelogenous leukemia (CML), and 665 healthy volunteers. To obtain a parameter representing an overall shift toward the oxidative stress, the oxidation stress index (OSI) was calculated by the following formula: OSI = (d-ROMs / BMP) × 8.85. We found that the OSI level was significantly higher in sera of AML patients than in those of CML and healthy volunteers. When 40 AML patients were subclassified into 5 diagnostic categories according to FAB classification, the OSI levels were obviously higher in the patients with M2 and M3 than in those with M1, M4, M5, and CML. In addition, the present study confirmed that the OSI levels were significantly higher in AML patients with DIC than in AML patients without DIC. Furthermore, we investigated the effect of oxidative stress on the expression of monocyte TF, which is the pathogenesis of DIC, and we found that the treatment with H2O2 led to increase in the expression of monocyte TF. These results suggest that a high oxidative stress condition promotes the expression of TF, which may contribute to the pathogenesis of DIC in patients with AML. Therefore, the evaluation of oxidative stress is a useful clinical test to predict the development and progression of DIC in patients with AML.
Reactivation of proliferation in human aortic endothelial cells by oxidative stress
Abstract
Antiphospholipid syndrome is a systemic autoimmune thromboembolic disease characterized by arterial and/or venous thromboembolic complications and recurrent fetal loss associated with laboratory evidence of persistent antiphospholipid antibodies. We found many APS patients were in hyper oxidative stress and that the incidence of arterial thrombo-embolism was extremely high in those patients. Therefore, we focused on the possibility that vascular endothelial cells disorder due to oxidative stress related to thrombosis in APS patients and examined effects of oxidative stress on human aortic endthelial cells (HAEC).In this study, we confirmed the phenomenon that cells undergoing oxidative stress stimulation caused proliferation rapidly when certain conditions were met. We confirmed the phenomenon that HAEC proliferated rapidly when stimulated by oxidative stress in certain condition. As a result, we were able to confirm the reactivation of HAEC proliferation and fluctuation of cell cycle due to oxidative stress. However, it was difficult to stably reproduce the phenomenon. Therefore, we were not able to clarify the mechanism. It is necessary to further study the conditions under which HAEC proliferation reactivation occurs, establish protocols and clarify the mechanism.
Evaluation of severity in chronic fatigue syndrome
Abstract
According to the guidelines of the United States Centers for Disease Control and Prevention, Chronic fatigue syndrome (CFS) is a disorder diagnosed in the presence of at least 6 months of disabling, unexplained mental and physical fatigue accompanied by other physical and psychological symptoms. Psychological disorders such as depression, viral infections, autoimmune diseases, and prolonged stresses have all been considered as potential causes behind CSF, although the mechanisms by which these conditions lead to the symptoms of CFS are still unclear. Furthermore, CFS is a highly heterogeneous and partly subjective illness, and no standard laboratory test is currently available for reliable diagnosis of CFS. The objective of this study was to establish a new biomarker for CSF. Since it is hypothesized that brain inflammation is involved in the pathophysiology of CFS, we used [11C]-(R)-PK11195 and PET to investigate the existence of neuroinflammation in CFS patients. Nine CFS patients and 10 healthy controls underwent [11C]-(R)-PK11195 PET and completed questionnaires about fatigue, fatigue sensation, cognitive impairments, pain, and depression. To measure the density of translocator protein, nondisplaceable binding potential (BPND) values were determined using linear graphical analysis with the cerebellum as a reference region. The BPND values in the cingulate cortex, hippocampus, amygdala, thalamus, midbrain, and pons were significantly higher in CFS patients than in healthy controls. In CFS patients, the BPND values in the amygdala, thalamus, and midbrain positively correlated with cognitive impairment score, the BPND values in the cingulate cortex and thalamus positively correlated with pain score, and the BPND value in the hippocampus positively correlated with depression score. These results suggest that the neuroinflammation is present in widespread brain areas in CFS patients and was associated with the severity of neuropsychologic symptoms. In addition, we measured both oxidation and anti-oxidation activities simultaneously in sera from 9 CFS patients with neuroinflammation and 25 CFS patients without neuroinflammations, the former by d-ROMs test and the latter by BAP test using a JCA-BM1650 automated analyzer. To obtain a parameter representing an overall shift toward the oxidative stress, the oxidation stress index (OSI) was calculated by the following formula: OSI = (d-ROMs / BMP) × 8.85. The present study confirmed that the OSI levels were significantly higher in CFS patients with neuroinflammation than in CFS patients without neuroinflammation. Our results provide the possibility that a high oxidative stress condition causes the neuroinflammation, which is contribute to the pathogenesis of CFS. Therefore, the evaluation of oxidative stress is a useful clinical test for the objective diagnosis and the evaluation of disease severity in CFS.
Endothelial cells injured by antiphospholipid antibodies contribute to pathogenesis of thromboembolic complications in antiphospholipid syndrome.
Abstract
Anti-phospholipid antibodies (aPLs) are a heterogeneous group of autoantibodies that appear in a variety of autoimmune diseases, particularly systemic lupus erythematosus. It is now generally accepted that aPLs do not bind primarily to the negatively charged phospholipid itself but rather to complexes of the phospholipid and phospholipid-binding proteins. The most common and best characterized aPLs are anti-cardiolipin/β2-glycoprotein I antibodies (aCL/β2GPI) and anti-phosphatidylserine/prothrombin antibodies (aPS/PT). Several clinical studies have established that the presence of aCL/β2GPI and aPS/PT is associated with clinical events such as arterial and/or venous thromboembolic complications and obstetric complications.However, the precise mechanism responsible for thromboembolic complications in these patients remains unclear. The main objective of this study was to clarify the role of aPLs in the pathogenesis of arterial thromboembolic complications in patients with antiphospholipid syndrome (APS). Therefore, several experiments were conducted to clarify the effect of aPLs on arterial endothelial cells. Human aorta endothelial cells (HAEC) were treated by aPLs (APS patients-derived IgG or anti-phosphatidylserine/prothrombinmonoclonal antibody (aPS/PT) IgM) with or without inhibitors of signaling molecule for 2 hours. Then, mRNA expression levels of tissue factor (TF), MCP-1 and IL-8 in HAEC were measured. The treatment with some APS patients-derived IgG or aPS/PT IgM caused significant increase of mRNA expression levels of TF, MCP-1 and IL-8. And increase of these mRNA expressions was suppressed by inhibitors of p38 MAPK and JNK. These results suggest that aPLs induce chemokine production of endothelial cells via p38 MAPK pathway and JNK pathway, and adhesion of monocytes and neutrophils on endothelial cells, which may contribute to development of thrombotic events of APS patients.
Establishment of new ELISA system for the laboratory diagnosis of the anti-phospholipid syndrome.
Abstract
Anti-phospholipid antibodies (aPLs) are a heterogeneous group of autoantibodies that can be detected by enzyme-linked immunosorbent assays (ELISAs) as anti-cardiolipin antibodies (aCL), anti-cardiolipin/β2-glycoprotein I (aCL/β2-GPI), anti-phosphatidylserine antibodies (aPS), and anti-phosphatidylserine/prothrombin antibodies (aPS/PT). These aPLs are frequently found in the plasma of patients with systemic lupus erythematosus (SLE) and have been reported to be associated with clinical events such as arterial and/or venous thrombosis and recurrent fetal loss. Although several aPL-ELISAs were established for the diagnosis of APS, the clinical importance of these ELISAs has not yet been determined. For the purpose of clarifying the clinical significance of aPLs detected by ELISA, we examined the presence of aCL, aCL/β2-GPI, aPS, and aPS/PT in SLE patients with with arterial thrombosis (32 cases), with venous thrombosis (42 cases), with recurrent fetal loss (24 cases), and patients without complications (98cases). The present study showed that the positive results of aCL/β2-GPI-ELISA and aPS/PT-ELISA could serve as markers of arterial and/or venous thrombosis in patients with SLE, whereas aCL-ELISA and aPS-ELISA are less reliable as markers of these complications. Furthermore, we newly developed an ELISA for detection of aAPC. They were measured in 80 healthy volunteers and 185 patients with SLE: arterial thrombosis (32 cases), venous thrombosis (62 cases), and no thrombotic complications (91 cases). The median aAPC concentration was significantly higher in the SLE patients with arterial thrombosis and venous thrombosis than in those SLE patients without thrombotic complications. ROC analysis revealed that the presence of aAPC was most closely associated with episodes of venous thrombosis.In summary, SLE patients frequently possess various type of aPLs such as aCL/β2GPI, aPS/PT, and aAPC. The occurrence of recurrent thrombotic complications in SLE apparently depends on variable combinations of these type of aPLs. Therefore, in the differential diagnosis of APS, it is essential to analyze the spectrum of aPL types by use of the ELISA.
Antiphospholipid antibodies promote platelet activation, which may contribute to the risk of cerebral infarction in patients with SLE
Abstract
Antiphospholipid syndrome (APS), which often complicates systemic lupus erythematosus (SLE), is a systemic autoimmune thromboembolic disease characterized by arterial and/or venous thromboembolic complications and recurrent fetal loss associated with laboratory evidence of persistent antiphospholipid antibodies (aPLs). Cerebral infarction is the most common arterial thromboembolic complication in APS patients. However, the precise mechanism responsible for cerebral infarction in patients with APS remains unclear. The main objective of this study was to clarify the role of aPLs in the pathogenesis of cerebral infarction in patients with SLE. We examined the levels of anti-cardiolipin/β2-glycoprotein I antibodies (aCL/β2GPI) and anti-phosphatidylserine/prothrombin antibodies (aPS/PT) in 80 patients with SLE including 24 patients with cerebral infarction. SLE patients were divided into four groups according to the results of ELISA : group A (n=35), aCL/β2GPI(+)・aPS/PT(+); group B (n=21), aCL/β2GPI(+)・aPS/PT(-); group C (n=10), aCL/β2GPI(-)・aPS/PT(+); group D (n=14), aCL/β2GPI(-)・aPS/PT(-). The prevalence of cerebral infarction was obviously higher in the patients who had both aCL/β2GPI and aPS/PT than in the other patients having aCL/β2GPI or aPS/PT alone or neither of them. Furthermore, we studied the in vitro effects of aCL/β2GPI and aPS/PT on the enhancement of the platelet activation and the complex formation of platelets and monocytes/granulocytes. We purified IgG fractions from SLE patients' plasma (7 cases of aCL/β2GPI(+)・aPS/PT(+), 3 cases of aCL/β2GPI(+)・aPS/PT(-),3 cases of aCL/β2GPI(-)・aPS/PT(+), and 4 cases of aCL/β2GPI(-)・aPS/PT(-) by use of Protein G Sepharose. Peripheral blood samples from normal healthy volunteers were incubated for 60 min at 37℃, we studied their in vitro effects on the enhancement of platelet activation. The platelet activation and the complex formation of platelets and monocytes/ granulocytes were assessed by measurement of surface expression of CD62p in CD41-positive platelets, CD14-positive monocytes, or CD16-positive granulocyte by the flow cytometry analysis using whole blood assay. The CD62P antigen is known as platelet activation-dependent granule-external membrane protein or granule membrane protein, is a member of the selectin family of adhesion molecules and mediates the adhesion of activated platelets to neutrophils and monocytes in hemostasis. The purified IgG containing both aCL/β2GPI and aPS/PT caused significant enhancement of the platelet activation and the complex formation of platelets and monocytes/granulocytes. However, the purified IgG containing either aCL/β2GPI or aPS/PT had no enhancing effects on them. These results indicate that aCL/β2GPI and aPS/PT may cooperate to promote platelet activation, which may contribute to the risk of cerebral infarction in patients with SLE.
Improvement of chronic fatigue states by antioxidant supplement "BAP mineral C120"
Abstract
Fatigue is a frequent condition experienced by healthy individuals. It is well known that the accurate assessment of fatigue states in each subject is quite difficult, because the level of fatigue depends on subjective feelings. Chronic fatigue syndrome (CFS) is characterized by intense feeling of fatigue and a variety of non-specific symptoms. According to the guidelines of Ministry of Health, Labour and Welfare, CFS is a disorder diagnosed in the presence of at least 6 months of disabling, unexplained mental and physical fatigue accompanied by other physical and psychological symptoms. CFS is a highly heterogeneous and mostly subjective illness, and no standard laboratory test is currently available for reliable diagnosis of CFS. Therefore, lots of people suffering from severe chronic fatigue are not receiving appropriate medical care. Recently, we revealed that evaluation of both oxidative stress (Reactive Oxygen Metabolites-derived compounds: d-ROMs) and antioxidation activities (Biological Antioxidant Potential: BAP) was useful not only in objectively assessing the level of fatigue states, but also the etiology of CFS. Particularly important evidence is that the decrease in antioxidation activities (BAP) was strongly related to the development of chronic fatigue states. In the present study, we examined the effect of the anti-oxidant supplement "BAP mineral C120" on physiological fatigue and chronic fatigue states. We measured d-ROMs and BAP simultaneously in sera from 10 female volunteers with physiological fatigue condition and 8 patients with chronic fatigue syndrome (CFS). The oxidation stress index (OSI) was calculated by the following formula: OSI = (d-ROMs / BMP) × 8.85 (a coefficient for standardization to set the mean of healthy individuals to 1.0). After 2-week oral administration of "BAP mineral C120" to the volunteers in physiological fatigue states, the levels of d-ROMs decreased significantly and the BAP levels obviously increased compared to the levels before starting the supplement. The oxidation stress index (OSI) of the volunteers were also obviously improved by oral administrations of BAP mineral C120. However, when the supplement was discontinued 2-weeks later, the OSI of the volunteers returned to the previous levels. In 8 patients with CFS, the levels of d-ROMs and BAP were not improved significantly even after 4-weeks administration of BAP mineral C120. However, the OSI levels improved obviously after the treatment. These results suggest that the enhancement of antioxidation activities by oral administration of "BAP mineral C120" is effective in improving the chronic fatigue states.
Endothelial cells injured by oxidative stress and antiphospholipid antibodies contribute to the pathogenesis of thromboembolic complications in patients with antiphospholipid syndrome
Abstract
Background: Antiphospholipid syndrome (APS) is a systemic autoimmune thromboembolic disease characterized by arterial and/or venous thromboembolic complications and recurrent fetal loss associated with laboratory evidence of persistent antiphospholipid antibodies. Especially, arterial and venous thromboembolism is a major cause of mortality in patients with APS. Recently, we found that oxidative stress level was significantly higher in sera of the APS patients than in those of the healthy subjects, and then we expected that APS patients were easy to cause thrombosis in the presence of high oxidative stress and antiphospholipid antibodies. In present study, we investigated the effects of oxidative stress and antiphospholipid antibody in the pathogenesis of venous thromboembolism in APS patients.
Methods: Human saphenous vein endothelial cells (HSaVECs) were treated 2 hours by H2O2 (100µM) or anti-phosphatidylserine/prothrombin monoclonal antibody (aPS/PT [231D], 5µg/ml) with or without prothrombin (0.5µg/ml). Then we measured mRNA expression levels (MCP-1, IL-8, tissue factor [TF] and eNOS) in HSaVECs.
Results: The treatment with H2O2 led to increase in the expression of TF, MCP-1 and IL-8 mRNA in HSaVECs. On the other hand, the expression of eNOS mRNA was decreased. And MCP-1 and IL-8 mRNA expression levels were also increased by aPS/PT stimulation.
Conclusions: High oxidative stress condition and antiphospholipid antibodies may induce expression of TF and secretion of chemokines and suppress NO secretion in venous endothelial cells. The results of these phenomena, endothelial cells are damaged and thrombotic events have come to occur easily. In APS patients, these chronical damage of endothelial cells by high oxidative stress and antiphospholipid antibodies may contribute to the pathogenesis of thromboembolism.
Inhibitory effect of anti-phospholipid antibodies on the anticoagulant activity of activated protein C
Abstract
Background: The activated protein C (APC) pathway is one of the important systems to suppress thrombus formation and inflammation. Protein C is activated on endothelial cells by thrombin bound to thrombomodulin. APC exerts its anticoagulant function by proteolytic cleavage of the procoagulant protein factors Va and factor VIIIa. The resistance to APC (APC-R) is defined as a decreased anticoagulant response to the APC pathway. Hereditary APC-R caused by the factor V Leiden mutation is strongly associated with an increased risk of venous thromboembolic events. Acquired APC-R, a phenotypic APC-R that occurs in the absence of the factor V Leiden mutation, has been reported in patients with systemic lupus erythematosus (SLE). However, the precise mechanism by which acquired APC-R is generated in SLE patients remains unclear. The objective of this study was to clarify the roles of anti-phospholipid antibodies (aPLs) in the pathogenesis of acquired APC-R in patients with SLE.
Methods and Results: APC-R assay was performed by use of a commercial kit, Coatest Activated Protein C Resistance, in plasma samples from 38 normal healthy volunteers and 97 patients with SLE, and 26 (37%) of SLE patients showed the positive results. Factor V Leiden was examined in 26 SLE patients with APC-R, and all of them tested negative for the factor V Leiden mutation. Therefore, all 26 SLE patients with APC-R were considered to have acquired APC-R. Next, we developed a new laboratory method to measure APC-sensitivity ratio with an activated partial thromboplastin time (APTT)-based assay using KC-4 coagulometer. APTT was measured in the presence and absence of APC, and the APC-sensitivity ratio were expressed as the ratio of APTT in the presence and absence of APC (APTT with APC/APTT without APC). The APC sensitivity ratio of less than 2.40, which was the mean-2SD in normal controls, was decided as the normal cut-off value in the APC-R study. We measured the APC-sensitivity ratio in plasma samples from 28 normal healthy volunteers and 20 patients with antiphospholipid syndrome (APS), and found that all of the APS patients' plasma caused a decrease of the APC-sensitivity ratio. Therefore, we studied the in vitro effects of IgG fractions (aPLs(-)-IgG, n=4; aPLs(+)-IgG, n-4) on APC-sensitivity ratio, and we confirmed that the purified IgG containing aPLs hampered significantly the anticoagulant activity of APC.
Conclusions: The present study showed that acquired APC-R is most strongly attributable to functional interference of the APC pathway by aPLs, which contribute to risk of thromboembolic complications.
A novel ELISA system for detection of autoantibodies against the activated protein C (APC) for differential diagnosis of anti-phospholipid syndrome (APS)
Abstract
Anti-phospholipid antibodies (aPLs) are a heterogeneous group of autoantibodies that appear in a variety of autoimmune diseases, particularly systemic lupus erythematosus (SLE). The presence of aPLs is associated with clinical events such as arterial and/or venous thromboembolic complications and recurrent fetal loss. Antiphospholipid syndrome (APS) is currently diagnosed by both clinical findings and laboratory evidence of persistent aPLs. At present, the most common aPLs detected by enzyme-linked immunosorbent assay (ELISA) are anti-cardiolipin (aCL) and anti-β2-glycoprotein I (aβ2GPI). These antibodies were reported to be involved in the pathogenesis of arterial thromboembolic complications in patients with SLE. However, the type of antibodies associated with the pathogenesis of venous thrombosis remains unclear. Recently, the abnormality in the activated protein C pathway has been reported in SLE patients with venous thromboembolic events. However, the precise mechanism by which the abnormality in the activated protein C pathway is generated in SLE patients remains unclear. We hypothesized the SLE patients have autoantibodies against the activated protein C (anti-APC), and we newly developed an ELISA for detection of anti-APC. Anti-APC were measured in plasma samples from 80 normal healthy volunteers and 185 patients with SLE. The SLE patients were divided into three groups according to their complications: arterial thrombosis (32 cases), venous thrombosis (62 cases), and no thrombotic complications (91 cases). The median anti-APC concentration was significantly higher in the SLE patients with arterial thrombosis and venous thrombosis than in those SLE patients without thrombotic complications. The upper 98 percentile points of anti-APC concentration in the 80 normal controls were set as the cut-off point. A result was regarded as positive when the absorbance exceeded each cut-off value. Anti-APC was detected in 88.7 % of the SLE patients with venous thrombosis. Furthermore, ROC analysis revealed that the presence of anti-APC was most closely associated with episodes of venous thrombosis. The present study showed that the abnormality in the activated protein C pathway might reflect functional interference of the activated protein C anticoagulant system by anti-APC, which may represent an important mechanism responsible for the development of venous thrombosis in patients with SLE.
Activated platelet assay by flow cytometry analysis using whole blood assay
Abstract
Background: Antiphospholipid syndrome (APS), which often complicates systemic lupus erythematosus (SLE), is a systemic autoimmune thromboembolic disease characterized by arterial and/or venous thromboembolic complications and recurrent fetal loss associated with laboratory evidence of persistent antiphospholipid antibodies (aPLs). Cerebral vascular disorder (CVD) such as cerebral infarction and transient ischemic attack is the most common arterial thromboembolic complication in APS patients. However, the precise mechanism responsible for cerebral infarction in patients with APS remains unclear. The main objective of this study was to clarify the role of aPLs in the pathogenesis of cerebral infarction in patients with SLE.
Methods: The activation of platelets was assessed by measurement of surface expression of CD62p in CD41-positive platelets by the flow cytometry analysis using whole blood assay. The CD41 antigen is a platelet-specific membrane glycoprotein IIb, and so FITC-conjugated anti-CD41 was used to measure the number of platelets. The CD62p antigen is known as platelet activation-dependent granule-external membrane protein, so PE-conjugated anti-CD62p was used to measure the number of activated platelets. Because the SLE patients' plasma might contain some components other than aPLs that might affect platelet activation, we purified IgG fractions from SLE patients' plasma (1 cases of aPLs-negative and 2 cases of aPLs-positive) by use of Protein G Sepharose. Peripheral blood samples from normal healthy volunteers were incubated for 10 min at 37℃ with aPLs-negative IgG-I or aPLs-positive IgG-I/II. After preparation of each IgG fraction (aPLs-negative IgG-I, aPLs-positive IgG-I, and aPLs-positive IgG-II), we studied their in vitro effects on the enhancement of platelet activation.
Results: When the peripheral blood from 6 normal healthy volunteers were pretreated with aPLs-negative IgG(I), the percentage of activated platelet increased to 2.3 ± 0.5 %, but the values were always below 5.0%. In contrast, the treatment of them with aPLs-positive IgG caused an apparent increase in the percentage of activated platelet (IgG-I, 18.0 ± 11.9 %, n=6; IgG-II, 12.8 ± 9.1 %, n=6). Furthermore, aPLs-positive IgG-I and IgG-II significantly enhanced platelet activation in a concentration-dependent manner.
Conclusions: We newly developed a flow cytometry analysis using whole blood assay, and confirmed that aPLs cause the activation of platelets directly. The enhancement of platelet activation by aPLs may be involved, at least partially, in the pathogenesis of cerebral infarction in patients with SLE.