Kamon Shirakawa

Elevated circulating levels and cardiac secretion of soluble fas ligand in patients with congestive heart failure

The circulating levels of soluble Fas ligand was increased in patients with advanced congestive heart failure. This study also indicates that the failing heart may contribute to the increased concentration of soluble Fas ligand in patients with congestive heart failure.

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

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 β‐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. GLIA 38:303–312, 2002.

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.

Presepsin (sCD14-ST): development and evaluation of one-step ELISA with a new standard that is similar to the form of presepsin in septic patients

The current international consensus guidelines for the management of sepsis and septic shock emphasize the need to establish a rapid method to diagnose sepsis (1). Currently, procalcitonin (PCT) is used as a marker to diagnose sepsis or severe sepsis (2). However, a meta-analysis of PCT showed that this marker has poor diagnostic utility for differentiating sepsis from other non-infectious causes. Thus, better biomarkers of sepsis are needed (3). As a result, it is still a challenge to rapidly and accurately diagnose sepsis and severe sepsis. We previously identified a sepsis-specific novel form of CD14, the soluble CD14 subtype (sCD14-ST: renamed presepsin). To measure presepsin specifically, we developed a two-step sandwich enzyme-linked immunosorbent assay (ELISA), and demonstrated that presepsin was elevated specifically in septic patients (4). Receiver operating characteristic (ROC) curve analysis of presepsin revealed that presepsin is a more specific and sensitive marker for sepsis compared with conventional markers of sepsis, including interleukin-6 (IL-6) and PCT (5). Although the first generation two-step ELISA was sufficient to evaluate presepsin as a diagnostic marker for sepsis, this assay was not convenient and lacked the rapidity and precision that is required for routine presepsin tests in intensive care units. In this letter, we describe the development and evaluation of a one-step ELISA that uses recombinant presepsin as a new standard. The two-step ELISA measured plasma presepsin levels within 4 h, and concentrations were extrapolated based on recombinant CD14 (S286C) antigen (approx. 40 kDa) as the standard. However, an analysis of native presepsin in septic patients revealed that this protein consists of the N-terminal 13 kDa fragment of the CD14 protein wwhen examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditionsx (6). Based on these data, we prepared recombinant human pre-

Expression of FasL and Fas Protein and Their Soluble Form in Patients with Hypersensitivity Pneumonitis

Background: Hypersensitivity pneumonitis (HP) is characterized by a lymphocytic alveolitis and loosely formed granulomas in lung biopsy specimens. HP improves or disappears altogether after cessation of antigen exposure. The Fas-Fas ligand (FasL) system is one of the representative systems of apoptosis-signaling receptor molecules, and is involved in various inflammatory diseases. We hypothesized that the Fas-FasL system may be associated with this disorder. Methods: We examined the expression of FasL and Fas proteins in lung tissues from patients with HP using immunohistochemistry. We also measured the soluble form of FasL (sFasL) and sFas levels in serum and bronchoalveolar lavage fluid (BALF) from patients with HP using enzyme-linked immunosorbent assay (ELISA). Furthermore, we also measured the cytotoxic activity of BALF sFasL in vitro. Results: FasL was detected in infiltrating mononuclear cells, and Fas was detected in infiltrating mononuclear cells, alveolar macrophages, and epithelioid cells in HP, whereas FasL was not detected and Fas was detected in few alveolar macrophages in controls. The levels of sFasL and sFas in BALF, but not in serum, were significantly increased in HP compared with controls. BALF of HP that included high levels of sFasL had no cytotoxic activity for bronchiolar epithelial cells in vitro. Conclusions: In HP, there is an upregulation of FasL and Fas in lung tissues. Since there is no incidence of apoptosis and no cytotoxic activity for lung epithelial cells in BALF from patients with HP, the increased levels of BALF sFasL and sFas may reflect the activation and sequestration of inflammatory cells rather than apoptosis.

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.

Increased levels of soluble Fas ligand in CSF of rapidly progressive HTLV-1-associated myelopathy/tropical spastic paraparesis patients

The interaction of Fas ligand (FasL) with Fas-bearing cells induces apoptosis and contributes to the negative regulation of peripheral T-cell responses. Membrane-bound FasL is cleaved by a matrix metalloproteinase-like enzyme and converted to a soluble form (sFasL). Recent studies suggest that such sFasL can cause systemic tissue damage. Here we report that serum and CSF levels of soluble FasL (sFasL) are markedly higher in three active phase patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). All of these patients showed higher sFasL levels in CSF than in serum. Although the HTLV-1 proviral load of patients showed no correlation with serum or with CSF sFasL, CSF sFasL levels of 14 HAM/TSP patients correlated with the anti-HTLV-1 antibody titer and neopterin concentration in CSF. These results indicate that sFasL mediated mechanisms may contribute to the inflammatory process and subsequent spinal tissue damage seen in HAM/TSP patients.

The new sepsis marker, sCD14-ST, induction mechanism in the rabbit sepsis models

Soluble CD14 subtype (sCD14-ST) is a fragment of CD14 and is markedly increased in sepsis patients. We developed a new immunoassay to detect sCD14-ST and evaluated the efficacy of this marker for diagnosis of sepsis. For developing the strategies of sCD14-ST as a sepsis diagnostic marker, the induction mechanism must be known.

Properties of soluble glycoprotein VI, a potential platelet activation biomarker

Abstract 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.