Elevated ratio of C-type lectin-like receptor 2 level and platelet count (C2PAC) aids in the diagnosis of post-operative venous thromboembolism in IDH-wildtype gliomas.

INTRODUCTION: Podoplanin (PDPN) is known to induce platelet aggregation via interacting with the C-type lectin-like receptor-2 on platelets and is involved in postoperative venous thromboembolism (VTE) formation. In this study, we investigate the correlation between soluble C-type lectin-like receptor (sCLEC-2) levels and PDPN expression in patients with high grade gliomas and the relationship between sCLEC-2 levels and the occurrence of VTE. MATERIALS AND METHODS: Forty-four patients harboring high grade gliomas, treated surgically at the Department of Neurosurgery, Niigata University from April 2018 to August 2020, were included. Patients with high grade gliomas were divided into isocitrate dehydrogenase (IDH)- wildtype and mutant groups, and the presence or absence of VTE and the intensity of PDPN by immunohistochemistry were confirmed. Platelet counts, as well as plasma sCLEC-2 and PDPN were measured in these patients. Furthermore, the levels of sCLEC-2 concentration were divided by the platelet count (C2PAC index) for comparison. RESULTS: IDH-wildtype glioma patients highly expressed PDPN (P < 0.001) compared to IDH-mutant glioma patients. In total, 9 (20.5 %) patients were diagnosed with VTE during the follow-up period, of which 8 patients harbored IDH-wildtype gliomas, and one patient an IDH-mutant glioma. Mean sCLEC-2 levels and C2PAC index in patients with IDH-wildtype gliomas were significantly higher than that of low or no PDPN expression group, which included patients with IDH-mutant gliomas (P = 0.0004, P = 0.0002). In patients with IDH-wildtype gliomas, the C2PAC index in patients with VTE was significantly higher than in patients without VTE (P = 0.0492). The optimal cutoff point of C2PAC for predicting VTE in IDH-wildtype glioma patients was 3.7 with a sensitivity of 87.5 % and specificity of 51.9 %. CONCLUSION: Platelet activation is strongly involved in the development of VTE in patients with IDH-wildtype high grade gliomas, and C2PAC index is a potential marker to detect VTE formation after surgery.

Development of a newly immunoassay specific for mouse presepsin (sCD14-ST).

Presepsin (sCD14-ST) is used as a marker for sepsis diagnosis. The production mechanism of presepsin is unique in that it is produced through phagocytosis of microorganisms. However, some studies have demonstrated that non-infected patients had increased presepsin levels and that presepsin is related to the risk or severity of diseases. This study was designed to describe a sensitive sandwich enzyme-linked immunosorbent assay for mouse presepsin developed to investigate the association of presepsin with diseases. Polyclonal antibodies were generated from peptide-immunized rabbit antiserum. Mouse presepsin standard was prepared using the recombinant method as an Fc-fusion protein. The linear detection range of the method was 4.7-300 pg/mL with a detection limit of 1.4 pg/mL. The assay detected mouse presepsin where mouse soluble CD14 (sCD14) was digested by cathepsin D proteinase and the cross-reactivity of sCD14 was not observed. The normal levels of mouse presepsin and sCD14 were compared; 65.9 ± 21.4 pg/mL and 43.2 ± 7.2 ng/mL were determined, respectively. Moreover, the levels of presepsin and sCD14 were compared with a lipopolysaccharide (LPS)-injected sepsis mouse model. The newly developed analytical method had high specificity to presepsin and is an efficient tool for studying the association between presepsin and diseases.

Antibody-mediated soluble CD14 stabilization prevents agitation-induced increases in presepsin levels in blood component specimens.

Presepsin is a 13-kDa N-terminal glycoprotein of CD14. Previously, agitation-induced increases in presepsin levels have been reported; however, the mechanism remains poorly understood. In this study, we aimed to reveal the mechanism of presepsin increase. The agitated plasma or serum was separated using gel exclusion chromatography and analyzed by ELISA. The effect of an anti-CD14 antibody (F1024-1-3) was examined. We observed elevated presepsin levels in the agitated plasma and aggregated soluble CD14 (sCD14). However, treatment with F1024-1-3 before agitation prevented the aggregation and the increase in presepsin levels. Depletion of aggregated sCD14 decreased the presepsin levels. Our findings indicate that agitation induces the aggregation of sCD14 and triggers an increase in presepsin. Anti-CD14 antibody prevents an increases in presepsin.

Anti-human CD14 monoclonal antibody improves survival following sepsis induced by endotoxin, but not following polymicrobial infection.

Cluster of differentiation 14 (CD14), a pattern recognition receptor expressed on myeloid cells and a critical component of the innate immune system, mediates local and systemic host responses to gram-negative bacterial products, including lipopolysaccharide (LPS). Therefore, CD14 is an attractive target for development of sepsis therapies, and several monoclonal anti-CD14 antibodies have been reported. In this study, we prepared an anti-human CD14 monoclonal antibody, F1024-1-3, which suppressed LPS-induced upregulation of pro-inflammatory cytokines and an adhesion molecule in human peripheral mononuclear cells and human vascular endothelial cells. Half-maximal inhibitory concentrations in these assays ranged from 0.1 to 1µg/ml. In rabbits, intravenous administration (3mg/kg) as well as in vitro exposure of F1024-1-3 suppressed LPS-induced cytokine production in whole blood. In endotoxemia models generated by three sequential injections of LPS, intravenous administration of F1024-1-3 at 0.3-3mg/kg sharply reduced pro-inflammatory responses in a dose-dependent manner and moderately attenuated pro-coagulant responses; at 1mg/kg, the protein protected rabbits from lethality even when administered 2h after the initial LPS injection. However, F1024-1-3 (10mg/kg) given 2h post-surgery did not prevent death of rabbits in a cecal ligation and puncture model. Thus, suppression of CD14-mediated activation of leukocytes and endothelial cells alone may not be clinically efficacious for the treatment of severe sepsis and septic shock.

Establishment of immunoassay for platelet-derived soluble glycoprotein VI, a novel platelet marker.

Soluble Glycoprotein VI (GPVI) is an attractive marker for disorders marked by platelet activation, such as thrombotic microangiopathy, myocardial infarction, and stroke. Several groups have already developed an immunoassay for soluble GPVI; however, there are several discrepancies between the groups' assays. In this study, we prepared the two types of recombinant soluble GPVI, the monomeric form GPVI (GPVI-His) and the dimeric form of GPVI (GPVI-Fc), moreover, we generated four anti-GPVI antibodies, F1232-7-1 (7S1), F1232-10-2 (10S2), F1232-19-1 (19D1), and F1232-21-1 (21D1). The former 2 antibodies (7S1 and 10S2) had a high affinity for both GPVI-His and GPVI-Fc, while the latter 2 antibodies (19D1 and 21D1) showed a high affinity for GPVI-Fc but low affinity for GPVI-His. All of the antibodies comparably recognized surface GPVI on resting platelets. Furthermore, we established two immunoassays for soluble GPVI, 7S1/10S2-HRP and 19D1/21D1-HRP (capture antibody/detection antibody). 7S1/10S2-HRP showed equivalent reactivity with GPVI-His and GPVI-Fc, whereas 19D1/21D1-HRP had high affinity for GPVI-Fc but low reactivity with GPVI-His. In terms of reactivity with platelet-derived soluble GPVI, 7S1/10S2-HRP demonstrated sensitive detection whereas 19D1/21D1-HRP was nonreactive. Taken together, 7S1/10S2-HRP is a suitable candidate for a reliable soluble GPVI immunoassay as it has a high affinity for monomeric GPVI.

Properties of soluble glycoprotein VI, a potential platelet activation biomarker.

Glycoprotein VI (GPVI) plays a critical role in the platelet response to collagen. Clinical studies suggest that the plasma level of soluble GPVI (sGPVI) is a highly specific and useful platelet activation marker. However, many properties of sGPVI have not been fully characterized, such as its sensitivity in detecting platelet activation and its elimination rate from the blood. In this study we established a sandwich enzyme-linked immunosorbent assay for human sGPVI, which cross-reacts to cynomolgus monkey sGPVI, and evaluated the time course of sGPVI production in a cynomolgus monkey model of lipopolysaccharide (LPS)-induced thrombocytopenia. The sGPVI levels in this model were dramatically elevated and returned to baseline by 24 hours after LPS injection, the change was more pronounced than the existing platelet activation biomarker, soluble P-selectin (sP-selectin) levels. The elimination half-life of recombinant human sGPVI was about 2.5 hours following intravenous administration to monkeys. These results suggest that plasma sGPVI closely reflects platelet activation in the bloodstream and has a short half-life. sGPVI would be a useful biomarker for disorders marked by platelet activation and for monitoring anti-platelet therapy.

A novel antiplatelet antibody therapy that induces cAMP-dependent endocytosis of the GPVI/Fc receptor gamma-chain complex.

Platelet adhesion to vascular subendothelium, mediated in part by interactions between collagen and glycoprotein VI (GPVI) complexed with Fc receptor gamma-chain, is crucial for thrombus formation. Antiplatelet therapy benefits patients with various thrombotic and ischemic diseases, but the safety and efficacy of existing treatments are limited. Recent data suggest GPVI as a promising target for a novel antiplatelet therapy, for example, GPVI-specific Abs that deplete GPVI from the surface of platelets. Here, we characterized GPVI-specific auto-Abs (YA-Abs) from the first reported patient with ongoing platelet GPVI deficiency caused by the YA-Abs. To obtain experimentally useful human GPVI-specific mAbs with characteristics similar to YA-Abs, we generated human GPVI-specific mouse mAbs and selected 2 representative mAbs, mF1201 and mF1232, whose binding to GPVI was inhibited by YA-Abs. In vitro, mF1201, but not mF1232, induced human platelet activation and GPVI shedding, and mF1232 inhibited collagen-induced human platelet aggregation. Administration of mF1201 and mF1232 to monkeys caused GPVI immunodepletion with and without both significant thrombocytopenia and GPVI shedding, respectively. When a human/mouse chimeric form of mF1232 (cF1232) was labeled with a fluorescent endocytosis probe and administered to monkeys, fluorescence increased in circulating platelets and surface GPVI was lost. Loss of platelet surface GPVI mediated by cF1232 was successfully reproduced in vitro in the presence of a cAMP-elevating agent. Thus, we have characterized cAMP-dependent endocytosis of GPVI mediated by a human GPVI-specific mAb as what we believe to be a novel antiplatelet therapy.

Evaluation of a newly identified soluble CD14 subtype as a marker for sepsis.

CD14, a high-affinity receptor for lipopolysaccharide (LPS), is a glycoprotein expressed on the surface membranes of monocytes/macrophages. We have identified a previously unknown form of soluble CD14, named soluble CD14 subtype (sCD14-ST), that is increased in patients with sepsis. To measure sCD14-ST concentrations in plasma, we prepared anti-sCD14-ST antibodies and developed an enzyme immunoassay (EIA) for this soluble form of CD14. With this assay, quantitative measurements are available within 4 h, and we compared the levels of sCD14-ST in plasma from normal subjects (healthy controls), patients with systemic inflammatory response syndrome (SIRS), and sepsis patients. The level of sCD14-ST in subjects with sepsis was much higher than the levels in subjects with SIRS and the healthy controls. Additionally, when a subject's sCD14-ST level was used as a diagnostic marker for sepsis, the area under the receiver operating characteristic (ROC) curve was 0.817, thereby demonstrating that elevated sCD14-ST levels were a better marker for sepsis than the other molecular markers we tested. sCD14-ST levels also correlated with procalcitonin (PCT) levels and with sequential organ failure assessment (SOFA) scores. Finally, changes in sCD14-ST concentration correlated with the severity of sepsis. Taken together, these results indicate that sCD14-ST is a useful marker for the rapid diagnosis of sepsis and for monitoring the severity of the disease.

Role of meltrin {alpha} (ADAM12) in obesity induced by high- fat diet.

Meltrin alpha is a member of the metalloprotease-disintegrin (ADAM) family. In this paper we demonstrate that meltrin alpha is involved in the development of white adipose tissue. Compared with wild-type mice, meltrin alpha(-/-) mice displayed moderate resistance to weight gain induced by a high-fat diet, mainly because of an impaired increase in the number of adipocytes. There was no obvious difference in adipocyte size between wild-type and meltrin alpha(-/-) mice, suggesting normal maturation of adipocytes of the latter under a high-fat diet. Embryonic fibroblasts and stromal-vascular cells lacking meltrin alpha exhibited impaired cell proliferation upon adipogenic stimulation, which was accompanied by moderate defects in adipose differentiation. Addition of culture medium conditioned with wild-type cells in an early phase of adipose differentiation did not restore the defects in the meltrin alpha(-/-) cells. These results uncover the involvement of meltrin alpha in the development of obesity and in adipogenic cell proliferation.

The expression of Fas and Fas ligand, and the effects of interferon in chronic liver diseases with hepatitis C virus.

In viral hepatitis, binding of Fas ligand (FasL) with Fas expressed on the surfaces of infected hepatocytes induces apoptosis, removing hepatitis virus along with infected hepatocytes. We measured serum concentrations of soluble Fas (sFas) and FasL (sFasL), expression of membrane-bound FasL, and expression of FasL-mRNA in patients with chronic hepatitis C without cirrhosis (CH-C) and chronic hepatitis C with liver cirrhosis (LC-C). In CH-C, sFasL concentrations were lower and FasL-mRNA expression was significantly less than in volunteers. In LC-C, sFas concentrations were significantly greater than in healthy volunteers, while sFasL, membrane-bound FasL expression, and FasL-mRNA expression did not show significant differences. We also examined these variables over 24 h following the first interferon (IFN) treatment in patients with CH-C. Serum concentrations of sFas and sFasL, and FasL-mRNA expression increased markedly beyond amounts present before IFN injection until 12 h after IFN injection. However, membrane-bound FasL expression decreased until 6 h, followed by an increase until 24 h. Our findings suggest that the ratio of membrane-bound FasL to sFasL may be regulated to remove virally infected cells in CH-C. In addition, apoptosis mediated by the Fas/FasL system may be influenced by IFN injection for treatment of CH-C.

Platelet-derived growth factor-b expression induced after rat peripheral nerve injuries.

Schwann cells are crucially important for peripheral nerve regeneration. These cells synthesize several factors that are supposed to enhance axonal regeneration when injured. Platelet-derived growth factor (PDGF) B-chain and its beta-receptor are expressed in Schwann cells in both normal peripheral nerves and culture. To elucidate the role of PDGF-B in peripheral nerve regeneration, we investigated its expression in cut or crush-injured rat sciatic nerves for up to 28 days. Northern blotting identified substantial increase of PDGF B-chain transcripts in injured nerves. Immunohistochemistry demonstrated that protein products of the transcripts were augmented at the distal tip of swollen axons in proximal nerve segments and in regenerating axons. Soon after both types of injury, considerable amounts of PDGF-B accumulated in numerous Schwann cells in distal segments of both models. With restoration of the axon-Schwann cell relationship in the crush model, levels of PDGF-B tended to decrease, eventually returning to normal. In the cut model in which the relationship cannot be restored, the PDGF-B was depleted to a very low level. The spatiotemporal correlation between PDGF-B and cell proliferation was very close throughout the study. These results differed strikingly from those of our previous study of rat optic nerve transection, in which PDGF-B was expressed only in a few recruited macrophages and glial cells. Augmented PDGF-B expression after sciatic nerve injury might contribute to peripheral nerve regeneration because PDGF-B is a mitogen and survival factor for Schwann cells and because it has trophic activity on neurons.

Plasma and urine levels of urinary trypsin inhibitor in patients with acute and fulminant hepatitis.

BACKGROUND AND AIM: Urinary trypsin inhibitor (UTI) is synthesized by hepatocytes and excreted into urine. Plasma and urine UTI levels have been measured to evaluate whether these levels may be useful markers in various pathological conditions. However, there has been no study on plasma and urine UTI levels in patients with acute liver diseases. The aim of the present study was to evaluate plasma and urine UTI levels and their relationship with the severity of hepatic damage in patients with acute liver diseases. METHODS: Plasma and urine UTI levels were measured by newly developed enzyme-linked immunosorbent assay in 15 patients with acute hepatitis (AH), 12 patients with acute severe hepatitis (ASH) and 10 patients with fulminant hepatitis (FH), as assessed on admission. The serial changes in plasma and urine UTI were also observed in some patients with AH and ASH. RESULTS: Plasma UTI levels (U/mL, median [25-75th percentile]) were: 11.0, (9.5-16.1) in patients with AH; 7.8 (5.6-11.5) in those with ASH; 6.5 (4.0-9.5) in patients with FH; and 9.7 (7.3-11.0) in normal controls. Plasma UTI levels in patients with FH were significantly lower than in those with AH. Plasma UTI levels showed significant positive correlations with the levels of prothrombin time (PT), hepaplastin test, antithrombin III, alpha2-plasmin inhibitor, plasminogen (Plg) and fibrinogen. After the recovery of liver dysfunction, increased plasma UTI levels in patients with AH were decreased, whereas previously decreased plasma UTI levels in patients with ASH were increased. Urine UTI levels were significantly increased in patients with AH compared with those of normal controls. In patients with ASH and FH, urine UTI levels were increased but not significantly. Urine UTI levels significantly positively correlated with PT and Plg. After the recovery of liver dysfunction, previously increased urine UTI levels in patients with AH were decreased. The correlation between plasma UTI and urine UTI levels was not significant. CONCLUSIONS: The findings of the present study suggested that the levels of plasma and urine UTI changed in patients with AH and were closely related to the abnormalities of coagulo-fibrinolysis, including PT. Further studies are needed to clarify whether these levels may be useful markers to predict the prognosis of acute hepatitis.

Increased circulating levels of soluble Fas ligand are correlated with disease activity in patients with fibrosing lung diseases.

OBJECTIVE: The Fas-Fas ligand (FasL) pathway is one of the important apoptosis-signalling molecule systems. We previously determined that this pathway may be involved in the pathogenesis of fibrosing lung diseases. In the present study, we evaluated the clinical significance of the levels of soluble forms of Fas (sFas) and FasL (sFasL) in serum from patients with fibrosing lung diseases. METHODOLOGY: We measured sFas, sFasL, KL-6 (a measure of alveolar type II cell damage), surfactant protein D (SP-D), and surfactant protein A (SP-A) levels in serum from 35 patients with idiopathic pulmonary fibrosis (IPF), 17 patients with interstitial pneumonia associated with collagen vascular diseases (CVD-IP), and 13 normal healthy controls using enzyme-linked immunosorbent assays (ELISA). RESULTS: The serum levels of sFasL were significantly increased in patients with active IPF and CVD-IP, compared with those with inactive disease and controls. There was no significant difference in sFasL levels between patients with inactive disease and controls. Serum sFasL levels were significantly correlated with lactate dehydrogenase and KL-6 levels in IPF. The decrease in sFasL levels following corticosteroid therapy was not correlated with the clinical course of IPF. There was no significant difference in serum sFas levels between IPF or CVD-IP patients and controls. CONCLUSIONS: Although further studies need to be performed on a large number of patients with histologically proven IPF or CVD-IP, it would seem that serum sFasL levels may reflect the activity of IPF and CVD-IP.