Shinobu Wakamoto

Bone marrow stromal cells prepared using AB serum and bFGF for hematopoietic stem cells expansion

BACKGROUND: An ex vivo culture system was previously established for stem cell expansion using human marrow stromal cells and serum‐free medium. However, the stromal cells were prepared using long‐term culture medium containing horse serum and FCS, which may transmit infectious diseases of xenogeneic origin. In this study, therefore, a method was established to prepare stromal cells using an AB serum‐based medium. In the case that serum from a transplant recipient or PBPC donor is available, additional infectious diseases would not be transmitted.

Biologic activity of RANTES in apheresis PLT concentrates and its involvement in nonhemolytic transfusion reactions

BACKGROUND: RANTES, one of the PLT‐derived biologic response modifiers, accumulates in PLT concentrates (PCs) during storage and may play a causative role in nonhemolytic transfusion reactions (NHTRs) after PC transfusion.

Effects of filtration and gamma radiation on the accumulation of RANTES and transforming growth factor-β1 in apheresis platelet concentrates during storage

BACKGROUND: Platelet‐derived biologic response modifiers (BRMs) including RANTES and transforming growth factor (TGF)‐β1 accumulate in platelet components during storage because of platelet activation, and they may play a causative role in nonhemolytic febrile transfusion reactions. The majority of PCs with high unit values are provided by single donor apheresis in Japan.

Lipopolysaccharide‐triggered desensitization of TNF‐α mRNA expression involves lack of phosphorylation of IκBα in a murine macrophage‐like cell line, P388D1

Activation of nuclear factor κB (NF‐κB) is thought to be required for cytokine production by lipopolysaccharide (LPS)‐responsive cells. Here, we investigated the contribution of NF‐κB in preventing LPS‐induced transcription of the tumor necrosis factor α (TNF‐α) gene in a murine macrophage cell line, P388D1, when tolerance was induced in the cells with a short exposure to a higher dose of LPS. Electrophoretic mobility shift assays with the κB elements of the murine TNF‐α promoter and enhancer revealed that nuclear mobilization of heterodimers of p65/p50, c‐rel/p50 and p65/c‐rel, and homodimers of p65 was markedly reduced in LPS‐tolerant cells, whereas that of p50 homodimers was only slightly increased. Western blot analysis showed that the phosphorylation of Ser32 on IκBα and its transient degradation did not occur in LPS‐tolerant cells. These results thus suggest that desensitization of TNF‐α gene expression in this LPS‐tolerant state is closely associated with down‐regulation of transactivating NF‐κB and may involve a defect in the LPS‐induced IκBα kinase pathway.

Life-threatening adverse reaction followed by thrombocytopenia after passive transfusion of fresh frozen plasma containing anti-CD36 (Naka) isoantibody

BACKGROUND: Anti‐CD36 isoantibody in blood recipients is reported to cause refractoriness to platelet (PLT) transfusions and posttransfusion purpura–like syndrome. There are few reports, however, about the effects of passively transfused blood products containing this isoantibody on recipients.

Effects of hemoglobin vesicles on resting and agonist-stimulated human platelets in vitro

Hemoglobin vesicles (HbV) are artificial oxygen carriers that encapsulate a concentrated hemoglobin (Hb) solution with a phospholipid bilayer membrane. The oxygen transporting ability of HbV in vivo has been demonstrated by the transfusion of HbV into hemorrhagic shock rodent models. However, the compatibility of HbV with human blood cells must be evaluated. Preincubation of platelets with concentrations of 20% or 40% HbV had no effect on the binding of PAC-1, a monoclonal antibody that detects activation-dependent conformational changes in αIIbβ3 on platelets, or the surface expression of CD62P in whole blood. ADP-induced increases in PAC-1 binding were significantly enhanced by exposing the platelets to concentrations of either 20% or 40% HbV, whereas the ADP-induced increases in CD62P expression were not affected by HbV treatment at either concentration. Preincubation of platelet-rich plasma (PRP) with HbV minimally reduced the spontaneous release of TXB2 and RANTES, but did not significantly affect the formation of TXB2 or the release of RANTES and β-TG in platelets stimulated with ADP. Similarly, preincubation of PRP with HbV minimally reduced the spontaneous release of RANTES but did not significantly affect the formation of TXB2 or the release of RANTES and β-TG in platelets stimulated with collagen, although collagen-induced serotonin release tended to decrease with HbV pretreatment. These data suggest that the exposure of human platelets to high concentrations of HbV (up to 40%) in vitro did not cause platelet activation and did not adversely affect the formation and secretion of prothrombotic substances or proinflammatory substances triggered by platelet agonists, although one of the earliest events in ADP-induced platelet activation was slightly potentiated by HbV pretreatment at the doses tested. Taken together, these results imply that HbV, at concentrations of up to 40%, do not have any aberrant interactions with either unstimulated or agonist-induced platelets.

Effects of poly(ethyleneglycol)-modified hemoglobin vesicles on agonist-induced platelet aggregation and RANTES release in vitro.

We studied the effects of hemoglobin-vesicles modified with PEG (PEG-HbV), a type of liposome-encapsulated hemoglobin (LEH), on human platelet functions in vitro. The effect of a low concentration of PEG-HbV (Hb; 5.8 mg/dl) was assessed by examining an agonist-induced aggregation response, and that of relatively high concentrations of PEG-HbV (Hb; 0.29, 1 and 2 g/dl) by measuring the release of RANTES (Regulated upon activation, normal T-cell expressed and presumably secreted) from platelets, which is regarded as a marker of platelet activation. The preincubation of platelets with PEG-HbV at 5.8 mg/dl of Hb did not affect platelet aggregation induced by collagen, thrombin and ristocetin. The pretreatment of platelet-rich plasma (PRP) with PEG-HbV at concentrations up to 2 g/dl of Hb had no aberrant effects on the collagen-induced RANTES release. Furthermore, the collagen-induced release of RANTES from PRP was not affected by longer incubation with PEG-HbV at 2 g/dl of Hb. The basal levels of RANTES from PRP were unchanged in the presence of PEG-HbV. These results suggest that PEG-HbV, at the concentrations studied, have no aberrant effects on platelet functions in the presence of plasma.

Heterogeneity of platelet responsiveness to anti-CD36 in plasma associated with adverse transfusion reactions

Background and Objectives  Antibodies to CD36 (anti‐CD36) are clinically important. As some platelet immunoglobulins produced by transfusion or pregnancy have been shown to induce platelet activation and to play roles in non‐haemolytic transfusion reactions (NHTRs), we investigated the in vitro response of platelets to plasma containing anti‐CD36.

Enhancement of endothelial permeability by coculture with peripheral blood mononuclear cells in the presence of HLA Class II antibody that was associated with transfusion-related acute lung injury.

BACKGROUND: HLA Class II antibody–initiated activation of monocytes possessing the corresponding antigen is thought to participate in the pathogenesis of transfusion‐related acute lung injury (TRALI). Pulmonary edema, a hallmark of TRALI, is caused by increasing vascular permeability.

Endothelial permeability is increased by the supernatant of peripheral blood mononuclear cells stimulated with HLA Class II antibody

BACKGROUND: The generation of inflammatory mediators from monocytes activated by HLA Class II antibodies is thought to play important roles in the etiology of nonhemolytic transfusion reactions. Increased permeability of endothelial cells contributes to the pathogenesis of rash, urticaria, angioedema, and pulmonary edema, which are symptoms of transfusion reactions.