Mitsuhiro Fujihara

Reduction in adverse reactions to platelets by the removal of plasma supernatant and resuspension in a new additive solution (M-sol).

BACKGROUND: Leukodepletion reduces but does not eliminate adverse reactions to platelet concentrate (PC). As an alternative strategy, plasma reduction or washing of platelets should be considered. However, the efficacy of this strategy is still unclear.

Storage of platelets in a novel additive solution (M-sol), which is prepared by mixing solutions approved for clinical use that are not especially for platelet storage

BACKGROUND: To reduce adverse reactions due to platelet (PLT) transfusion, medical solutions on the market, such as saline and ACD‐A, are used to replace the plasma of PLT concentrates in Japan; however, they are not strongly preservative. Here, an attempt was made to develop a novel additive solution (M‐sol) having the ability to preserve PLTs stably, with only approved solutions for clinical use.

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.

Virus Trap in Human Serum Albumin Nanotube

Infectious hepatitis B virus (HBV), namely Dane particles (DPs), consists of a core nucleocapsid including genome DNA covered with an envelope of hepatitis B surface antigen (HBsAg). We report the synthesis, structure, and HBV-trapping capability of multilayered protein nanotubes having an anti-HBsAg antibody (HBsAb) layer as an internal wall. The nanotubes were prepared using an alternating layer-by-layer assembly of human serum albumin (HSA) and oppositely charged poly-L-arginine (PLA) into a nanoporous polycarbonate (PC) membrane (pore size, 400 nm), followed by depositions of poly-L-glutamic acid (PLG) and HBsAb. Subsequent dissolution of the PC template yielded (PLA/HSA)(2)PLA/PLG/HBsAb nanotubes (AbNTs). The SEM measurements revealed the formation of uniform hollow cylinders with a 414 ± 16 nm outer diameter and 59 ± 4 nm wall thickness. In an aqueous medium, the swelled nanotubes captured noninfectious spherical small particles of HBsAg (SPs); the binding constant was 3.5 × 10(7) M(-1). Surprisingly, the amount of genome DNA in the HBV solution (HBsAg-positive plasma or DP-rich solution) decreased dramatically after incubation with the AbNTs (-3.9 log order), which implies that the infectious DPs were completely entrapped into the one-dimensional pore space of the AbNTs.

Possible contribution of apoptosis-inducing factor (AIF) and reactive oxygen species (ROS) to UVB-induced caspase-independent cell death in the T cell line Jurkat

We analyzed the mechanism of UVB‐induced cell death using the Jurkat T cell line. Apoptosis was assessed by measuring phosphatidylserine (PS) externalization, caspase activity, the decrease in mitochondrial membrane potential (ΔΨm), nucleosomal DNA fragmentation, and morphological changes such as chromatin condensation. The mitochondrio‐nuclear translocation of apoptosis‐inducing factor (AIF) was evaluated by confocal laser microscopy. The cell death pattern of UVB‐irradiated cells was similar to the Fas‐induced cell death pattern. However, zVAD‐fmk inhibited the nucleosomal fragmentation of DNA but not the externalization of PS, decrease in ΔΨm, or mitochondrio‐nuclear translocation of AIF. N‐acetyl L‐cysteine significantly inhibited the translocation of AIF induced by UVB. These results suggested that caspase‐dependent and ‐independent pathways were involved in UVB‐induced cell death in Jurkat cells, and the mitochondrio‐nuclear translocation of AIF was associated with the latter pathway. In addition, reactive oxygen species generated by UVB might be involved in inducing the mitochondrio‐nuclear translocation of AIF.

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.

Culture system for extensive production of CD19+IgM+ cells by human cord blood CD34+ progenitors.

We established a co-culture system with a monolayer of the murine bone marrow (BM) stroma cell line, MS-5, in which human cord blood CD34+ cells differentiated to CD19+ cells. The addition of stem cell factor (SCF) and granulocyte colony- stimulating factor (G-CSF) highly enhanced the production of CD19+ cells. The expansion of the cell numbers was over 103-fold. Furthermore, a significant proportion (<45%) of the cells expressed surface igm (sigm) after 5 weeks of co-culture. cd34+CD19− cells also showed a similar development of CD19+ cells and CD19+sIgM+ cells. Filter separation of MS-5 cells and CD34+ cells did not inhibit the growth of CD19+ cells. However, when further purified CD34+CD19−CD13− CD33− cells were cultured in the presence of MS-5 cells with or without a separation filter, CD19+ cells did not appear in the non-contact setting. This result suggested that the highly purified CD34+CD19−CD13−CD33− progenitors require the cell–cell contact for the development of CD19+ cells, whereas other CD34+ fractions contain progenitors that do not require the contact. This co-culture system should be useful for the study of early human B-lymphopoiesis.

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.

Effects of hemoglobin vesicles, a liposomal artificial oxygen carrier, on hematological responses, complement and anaphylactic reactions in rats

Hemoglobin vesicle (HbV), a liposomal oxygen carrier containing human hemoglobin, was intravenously infused into rats. After the infusion of saline, the HbV or empty vesicle (EV), numbers of red cells, leukocytes and platelets in peripheral blood were unchanged during the observation period of one week in addition to each time point among three groups. However, the lymphocyte ratio transiently decreased and the granulocyte ratio increased in the HbV and EV groups at 6 h after the infusion. Those changes returned to the initial value one day after the infusion and those were maintained for the subsequent observation period. No dramatic change was seen in the ratio of CD4+/CD8+ T cells. A transient decrease of the complement titer was observed three days after the infusion of HbV and EV, although the consumption of complement titer was not detected in rat serum by mixing HbV or EV in vitro, indicating that the transient decrease of complement titer in vivo was not due to the consumption of complement due to the interaction with HbV or EV. Multiple infusions of HbV caused the decrease of complement titer only after the first infusion and no allergic reaction was observed. No anaphylactic shock was observed in rats administered with EV several times, while ovalbumin (OVA) sensitized rats died with symptoms of respiratory distress after the second OVA administration. These results indicate that HbV could be administered without serious clinical symptoms or adverse reactions.