Determination of diagnostic threshold in harmonization and comparison of clinical utility for five major antiphospholipid antibody assays used in Japan.

BACKGROUND: Anticardiolipin antibodies (aCL) and anti-ß2 -glycoprotein I antibodies (aß2 GPI) are essential in diagnosing antiphospholipid syndrome (APS) according to the international APS guideline. Five commercial assays for aCL and aß2 GPI are available in Japan, but their test results are quite discordant. For harmonization of diagnosing APS, upper reference limit (URL) and diagnostic accuracy of each assay were evaluated and compared by testing common sets of specimens across all assays. METHODS: We evaluated two manual and three automated assays for aCL and aß2 GPI of IgG- and IgM classes. 99%URL (the upper limit of reference interval: as per guideline) together with 97.5%URL were determined by testing sera from 198 to 400 well-defined healthy subjects. Both URLs were compared with the cutoff values, which were determined based on ROC analysis by testing 50 each of plasma specimens from patients with/without APS. Diagnostic accuracy was evaluated as area under curve (AUC) of the ROC curve. RESULTS: A variable degree of discrepancy between URLs and the cutoff values was observed, which was partly attributable to between-year assay variability. 97.5%URLs were set lower and closer to the cutoff values than 99%URLs. For all assays, diagnostic accuracies of both aß2 GPI-IgG and aCL-IgG were generally high (AUC: 0.84-0.93); whereas those for IgM-class assays were low (AUC: 0.57-0.67), implicating its utility is limited to rare IgG negative APS cases. CONCLUSION: To ensure harmonized APS diagnosis, the diagnostic thresholds of the five assays were evaluated by common procedures. Contrary to the guideline, 97.5%URL is rather recommended for diagnosing APS, which showed a closer match to the cutoff value.

Exploring the seasonal and regional features of cat-scratch disease on the basis of anti-Bartonella henselae IgM/IgG positive rates in Japan.

OBJECTIVES: This study aimed to explore the seasonal and regional features of cat-scratch disease (CSD) based on 15-years of test results for anti-Bartonella henselae IgG and IgM by immunofluorescence assay (IFA) performed as a laboratory specialized in diagnostic testing of CSD in Japan. A literature search was performed to put our findings in perspective. METHODS: A total of 956 sera from patients suspected of CSD were submitted to our laboratory from nationwide. Seasonal changes in the monthly positive rates of IgG/IgM antibodies and regional distribution of the test specimens were analyzed. RESULTS: The monthly positive rates of anti-B. henselae IFA of IgG and IgM were both significantly high between September and January and low between March and July. The seasonal pattern observed in this study was similar to the ones reported from US and France, which were analyzed from a clinical database (monthly incidence of CSD diagnosis) or from monthly positive rates of either B. henselae PCR or anti-B. henselae IFA. However, fluctuations in the IFA monthly positive rates in this study were more pronounced than other reports. Regarding regionality, the test specimens submitted to us for IFA were prominently more from southwestern areas than from northern/middle-northern areas of Japan. The distribution coincided well with the regional distribution of CSD case reports and with a known regional prevalence of Bartonella-species bacteremia among pet cats in Japan. CONCLUSION: These epidemiological features in Japan are of relevance in the clinical diagnoses of CSD.

aCL/ß2GPI and aPS/PT show synergic thrombogenic effects in suppressing anticoagulant activity of APC and stimulating tissue factor expression and TNF-α secretion by mononuclear cells.

INTRODUCTION: Patients with systemic lupus erythematosus (SLE) possessing anti-phospholipid antibodies (aPLs) are often complicated by thrombotic vascular events. aPLs commonly associated with the complications are anti-cardiolipin/ß2-glycoprotein I antibodies (aCL/ß2GPI) and anti-phosphatidylserine/prothrombin antibodies (aPS/PT). However, the pathological mechanisms leading to thrombosis remain unclear. We explored clinical features of SLE patients with aCL/ß2GPI and aPS/PT and investigated thrombogenic effects of their IgG fractions. MATERIALS AND METHODS: We enrolled 97 SLE patients and 38 healthy control volunteers and performed activated protein C (APC) resistance screening test using their plasma samples. To detect the direct effect of aPLs IgG on APC, we developed an APC sensitivity ratio assay. Effects of aPLs IgG on monocytes were studied by measuring the surface expression of tissue factor (TF) and excretion of TNF-α from peripheral blood mononuclear cell culture. RESULTS AND CONCLUSION: Thrombotic complications among SLE patients were closely associated with aCL/ß2GPI or aPS/PT, with higher prevalence in patients with both antibodies. Addition of aPLs(+)-IgG to the APC sensitivity ratio assay led to significant suppression of the anticoagulant activity of APC. The suppression was more pronounced in double-positive cases. TF expression on monocytes and concentration of TNF-α in culture medium were increased by aPLs, again more pronounced in double-positive cases. These results indicate that the effects of aCL/ß2GPI and aPS/PT are synergic both for APC anticoagulant activity and for production of TF and TNF-α from mononuclear cells. These modes of thrombogenic action of aPLs could be an important target for developing specific measures to prevent complications of SLE.

Probability of Soluble Tissue Factor Release Lead to the Elevation of D-dimer as a Biomarker for Traumatic Brain Injury.

d-dimer is a potential biomarker for the detection of traumatic brain injury (TBI). However, the mechanisms that trigger elevation of d-dimer in TBI remain unclear. The purpose of this study was to evaluate the reliability of d-dimer in blood as a biomarker for TBI and to determine the mechanisms involved in regulating its blood levels. Nine patients with moderate to severe isolated TBI (Glasgow Coma Scale [GCS] score 7-13) were admitted to our hospital from May 2013 to June 2014. Blood samples were collected from systemic arteries on arrival and at 1, 3, 5, and 7 days after injury. Blood levels of neuron specific enolase (NSE), d-dimer, and soluble tissue factor (sTF) were measured. NSE (33.4 ng/ml: normal <12.0 ng/ml) and d-dimer (56.1 µg/ml: normal <1.0 µg/ml) were elevated at admission and declined on day 1 after injury. At admission, there were significant correlations of d-dimer levels with NSE (R = 0.727, P = 0.026) and sTF (R = 0.803, P = 0.009) levels. The blood level of d-dimer accurately reflects the degree of brain tissue damage indicated by NSE levels. Our data suggest that release of sTF induced by brain tissue damage may activate the coagulation cascade, leading to elevation of d-dimer.

Novel enzyme immunoassay system for simultaneous detection of six subclasses of antiphospholipid antibodies for differential diagnosis of antiphospholipid syndrome.

: Antiphospholipid syndrome, which often complicates systemic lupus erythematosus (SLE), features high occurrence of arterial and/or venous thrombosis and recurrent fetal loss. However, which antibody subclass contributes to which clinical event remains uncertain. We newly developed an up-to-date enzyme immunoassay system using the AcuStar automated analyzer (Instrumentation Laboratory, Bedford, Massachusetts, USA) for parallel detection of six subclasses of antiphospholipid antibodies (aPLs): anticardiolipin antibodies (aCL) of IgG, IgM, and IgA and anti-ß2-glycoprotein I antibodies (aß2GPI) of IgG, IgM, and IgA. They were measured in 276 healthy volunteers and 138 patients with SLE: 45 with thromboembolic complications (29 arterial; 16 venous) and 93 without. Lupus anticoagulant activity in their plasma was measured according to the guidelines recommended by the Subcommittee on Lupus Anticoagulant/Phospholipid-Dependent Antibodies. aCL/ß2GPI was measured with a standard ELISA kit commonly used in Japan. The positive results of IgG aCL, IgA aCL, and IgG aß2GPI were closely associated with thromboembolic complications, whereas IgM aCL and IgM aß2GPI were not. receiver operating characteristic analysis revealed that the accuracy of predicting thromboembolic complications based on the composite test results of the former three antibodies were obviously higher than by each alone. Regarding agreement with the test results of lupus anticoagulant activity, IgG aß2GPI showed the closest match. Patients with SLE frequently possess various combinations of the six aPL subclasses, and this antibody spectrum is closely associated with thromboembolic events in these patients. This new automated enzyme immunoassay system allows simultaneous analysis of the profile of aPL subclasses for the differential diagnosis of antiphospholipid antibody syndrome in its early stage.

A potential biomarker for fatigue: Oxidative stress and anti-oxidative activity.

We sought to determine whether oxidative stress and anti-oxidative activity could act as biomarkers that discriminate patients with chronic fatigue syndrome (CFS) from healthy volunteers at acute and sub-acute fatigue and resting conditions. We calculated the oxidative stress index (OSI) from reactive oxygen metabolites-derived compounds (d-ROMs) and the biological antioxidant potential (BAP). We determined changes in d-ROMs, BAP, and OSI in acute and sub-acute fatigue in two healthy groups, and compared their values at rest between patients with CFS (diagnosed by Fukuda 1994 criteria) and another group of healthy controls. Following acute fatigue in healthy controls, d-ROMs and OSI increased, and BAP decreased. Although d-ROMs and OSI were significantly higher after sub-acute fatigue, BAP did not decrease. Resting condition yielded higher d-ROMs, higher OSI, and lower BAP in patients with CFS than in healthy volunteers, but lower d-ROMs and OSI when compared with sub-acute controls. BAP values did not significantly differ between patients with CFS and controls in the sub-acute condition. However, values were significantly higher than in the resting condition for controls. Thus, measured of oxidative stress (d-ROMS) and anti-oxidative activity (BAP) might be useful for discriminating acute, sub-acute, and resting fatigue in healthy people from patients with CFS, or for evaluating fatigue levels in healthy people.

A novel ELISA system for simultaneous detection of six subclasses of anti-phospholipid antibodies for prediction of thrombotic complications among SLE patients.

BACKGROUND: Anti-phospholipid antibodies (aPLs) are frequently associated with arterial and/or venous thromboembolic complications and recurrent fetal loss in patients with systemic lupus erythematosus (SLE). We recently reported that the clinical picture of SLE apparently depends on subclasses of aPLs in the patient's sera, but the contribution of each subclass remains uncertain. METHODS: We newly developed an ELISA system for simultaneous detection of six specific categories of aPLs: anti-cardiolipin (aCL), anti-ß2-glycoprotein I (aß2GPI), anti-cardiolipin/ß2-glycoprotein I (aCL/ß2GPI), anti-phosphatidylserine (aPS), anti-prothrombin (aPT), and anti-phosphatidylserine/prothrombin (aPS/PT). They were measured in 331 patients with SLE including 63 patients with arterial thromboembolic complications, 64 with venous thromboembolic complications, and 43 with recurrent fetal loss. Lupus anticoagulant (LA) activity in their plasma was measured according to the guidelines recommended by the Subcommittee on Lupus Anticoagulant/Phospholipid-Dependent Antibodies. RESULTS: Multivariate logistic analysis revealed that the concentration of aPS/PT was most closely associated with arterial thrombosis. In contrast, the concentration of aß2GPI was most closely related to venous thrombosis. Furthermore, both aCL/ß2GPI and aPS/PT were independently associated with episodes of recurrent fetal loss. Regarding the relation between APLs and LA activity, aPS/PT, followed by aß2GPI and aPT, showed the closest association with the presence of LA activity. CONCLUSIONS: Anti-phospholipid syndrome in patients with SLE can be classified by antigenic specificities of their aPLs as to their susceptibility to arterial and/or venous thromboembolic complications or obstetric complications.

Hypothermia reduces toll-like receptor 3-activated microglial interferon-ß and nitric oxide production.

Therapeutic hypothermia protects neurons after injury to the central nervous system (CNS). Microglia express toll-like receptors (TLRs) that play significant roles in the pathogenesis of sterile CNS injury. To elucidate the possible mechanisms involved in the neuroprotective effect of therapeutic hypothermia, we examined the effects of hypothermic culture on TLR3-activated microglial release of interferon (IFN)- ß and nitric oxide (NO), which are known to be associated with neuronal cell death. When rat or mouse microglia were cultured under conditions of hypothermia (33°C) and normothermia (37°C) with a TLR3 agonist, polyinosinic-polycytidylic acid, the production of IFN- ß and NO in TLR3-activated microglia at 48 h was decreased by hypothermia compared with that by normothermia. In addition, exposure to recombinant IFN- ß and sodium nitroprusside, an NO donor, caused death of rat neuronal pheochromocytoma PC12 cells in a concentration-dependent manner after 24 h. Taken together, these results suggest that the attenuation of microglial production of IFN- ß and NO by therapeutic hypothermia leads to the inhibition of neuronal cell death.

Temperature- and time-dependent changes in TLR2-activated microglial NF-κB activity and concentrations of inflammatory and anti-inflammatory factors.

PURPOSE: Therapeutic hypothermia protects neurons following injury to the central nervous system (CNS). Microglia express toll-like receptors (TLRs) that play significant roles in pathological processes in sterile CNS injury. We have examined the effects of culture temperature on the TLR2-activated microglial production of cytokines and nitric oxide (NO), which are known to be associated with CNS damage, and the possible involvement of nuclear factor-κB (NF-κB) activation underlying such effects. METHODS: Rat microglia were cultured with a selective TLR2 agonist, Pam(3)CSK(4), under hypothermic, normothermic, and hyperthermic conditions, and with Pam(3)CSK(4) in the presence of a NF-κB activation inhibitor at 37 °C. Cytokine and NO levels and NF-κB p65 activation were measured. RESULTS: The production of tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), and NO and the activation of NF-κB p65 were reduced by hypothermia, but augmented by hyperthermia at 3-6, 24-48, 48, and 0.5 h, post-treatment initiation, respectively. Pharmacological inhibition of NF-κB activation impaired the Pam(3)CSK(4)-induced TNF-α, IL-10, and NO production. CONCLUSIONS: In TLR2-activated microglia, hypothermia reduced, while hyperthermia increased, the early activation of NF-κB and the subsequent NF-κB-mediated production of TNF-α, IL-10, and NO in a time-dependent manner, suggesting that attenuation of these factors via suppression of NF-κB in microglia is one possible neuroprotective mechanism of therapeutic hypothermia. Moreover, temperature-dependent changes in microglial TNF-α production during the early phase and IL-10 and NO production during the late phase indicate that these factors might be useful as clinical markers to monitor hypothermia-related neuronal protection and hyperthermia-related neuronal injury.

Anti-phospholipid antibodies contribute to arteriosclerosis in patients with systemic lupus erythematosus through induction of tissue factor expression and cytokine production from peripheral blood mononuclear cells.

INTRODUCTION: In systemic lupus erythematosus (SLE) patients, the prevalence of arteriosclerosis obliterans (ASO) is high despite a lack of common risk factors for ASO. The main objective of this study was to investigate a possible direct role of anti-phospholipid antibodies (aPLs), which are frequently detected in SLE patients, in the pathogenesis of ASO. MATERIALS AND METHODS: We examined tissue factor (TF) expression on the monocyte surface by flow cytometric analysis in 89 SLE patients with or without ASO and/or aPLs and studied the in vitro effect of purified IgG fractions from plasma of SLE patients or normal healthy volunteers (aPLs(+) IgG, n=8; aPLs(-) IgG, n=6; Normal IgG, n=6) on the expression of TF and production of TNF-α and IL-1ß in healthy peripheral blood mononuclear cells (PBMCs) or isolated monocytes. RESULTS: We confirmed that high expression of monocyte TF was strongly associated with the prevalence of ASO and the presence of aPLs. Treatments of PBMCs with aPLs(-) IgG or normal IgG did not significantly increase expression of TF, TNF-α, and IL-1ß messenger RNA (mRNA) and the production of TNF-α and IL-1ß. However, stimulation of PBMCs with aPLs(+) IgG caused significant increase in expression of TF, TNF-α, and IL-1ß mRNA. Moreover, aPLs(+) IgG stimulated PBMCs and significantly enhanced the production of TNF-α and IL-1ß. CONCLUSION: These results suggest that IgG-aPLs cause persistently high TF expression and inflammatory cytokine production by interacting with peripheral blood monocytes and lymphocytes, which may be an important mechanism in the pathogenesis of ASO peculiar to SLE patients.

Temperature-related effects of adenosine triphosphate-activated microglia on pro-inflammatory factors.

BACKGROUND: Therapeutic hypothermia protects neurons after severe brain injury. Activated microglia produce several neurotoxic factors, such as pro-inflammatory cytokines and nitric oxide (NO), during neuron destruction. Hence, suppression of microglial release of these factors is thought to contribute partly to the neuroprotective effects of hypothermia. After brain insults, adenosine triphosphate (ATP) is released from injured cells and activates microglia. Here, we examined the acute effects of temperature on ATP-activated microglial production of inflammatory factors, and the possible involvement of p38 mitogen-activated protein kinase (p38) underlying such effects. METHODS: Microglia were cultured with ATP at 33, 37, and 39°C, or with ATP in the presence of a p38 inhibitor, SB203580, at 37°C. Cytokine and NO levels, and p38 activation were measured. RESULTS: Compared to 37°C, TNF-α was reduced at 33°C and augmented at 39°C for 1.5 h. IL-6 was reduced at 33°C for 6 h. NO was reduced at 33°C, but augmented at 39°C for 6 h. p38 was reduced at 33°C for 1 min. SB203580 inhibited ATP-induced TNF-α, IL-6, and NO production. CONCLUSION: Lowering temperature rapidly reduced p38 activation and the subsequent p38-regulated production of pro-inflammatory cytokines and NO in ATP-activated microglia, suggesting that attenuation of early phase inflammatory responses via suppression of p38 in microglia is one possible neuroprotective mechanism of therapeutic hypothermia. Temperature elevation increased TNF-α and NO production in these cells. These temperature-dependent changes imply that monitoring of TNF-α and NO in the cerebrospinal fluid during the early phase might be useful as biomarkers for responses to therapeutic hypothermia and hyperthermia.