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. STUDY DESIGN AND METHODS: To investigate the contribution of HLA Class II antibody and monocytes to the development of pulmonary edema in TRALI, we studied whether the permeability of human lung microvascular endothelial cells (HMVECs) could be enhanced by coculturing HMVECs with peripheral blood mononuclear cells (PBMNCs) in the presence of HLA Class II antibody-containing plasma, which was implicated in TRALI (anti-HLA-DR plasma). In addition, similar experiments were performed with human umbilical vein endothelial cells (HUVECs). The endothelial permeability to fluoresceinated dextran, which was added from the start of coculture, was measured. RESULTS: The coculture of HMVECs or HUVECs with PBMNCs in the presence of anti-HLA-DR plasma resulted in the increase of endothelial permeability in the corresponding antigen-antibody-dependent manner. CV-3988, a platelet-activating factor (PAF) receptor antagonist, almost completely suppressed the increase in endothelial permeability. Neutralizing antibodies to tumor necrosis factor (TNF)-α alone and simultaneous addition of the antibodies to TNF-α and interleukin (IL)-1ß to the coculture partially suppressed the permeability increase of HMVECs and HUVECs, respectively. CONCLUSIONS: HLA Class II antibody and monocytes in the corresponding antigen-antibody combination caused the enhancement of endothelial permeability. PAF, TNF-α, and/or IL-1ß might be involved in the endothelial permeability increase. HLA Class II antibody-initiated monocyte activation could lead to the development of pulmonary edema in TRALI.

Biocompatibility study of hemoglobin vesicles, cellular-type artificial oxygen carriers, with human umbilical cord hematopoietic stem/progenitor cells using an in vitro expansion system.

Hemoglobin vesicles (HbVs), liposomal oxygen carriers containing human hemoglobin, are candidates for development of a clinically useful transfusion alternative. Our previous in vivo animal studies of massive HbV dosages demonstrated the lack of any suppressive effect on hematopoiesis. Before starting clinical trials, we aimed to examine the details of the biocompatibility of HbVs with human hematopoietic stem/progenitor cells. We investigated the effect of HbVs at a concentration of up to 3 vol/vol (%) on expansion of human umbilical cord (CB) hematopoietic stem/progenitor cells, using a coculture system of human TERT-transfected bone marrow stromal cells and CD34+ cells in vitro. The exposure of CB CD34+ cells to HbVs up to 14 days suppressed the expansion of total cells and the CD34+ cells themselves, whereas the empty liposomes, that did not contain Hb, had modestly inhibitory effects on the expansion of these cells. As a result, the number of colonies obtained from the expanded CD34+ cells was inhibited by the exposure to HbVs. In contrast, exposure to HbVs for 3 days had no effect on the expansion of CD34+ cells and only slightly decreased the number of total cells. Our in vitro experimental condition does not fully recreate the physiological condition, and the effect of the direct contact of HbV would be magnified because of the absence of shielding by the vasculature and the lack of the reticuloendothelial system and blood stream. However, the present data raise some concern regarding hematopoiesis, and one has to pay attention to this in future human clinical trials.

Influence of hemoglobin vesicles, cellular-type artificial oxygen carriers, on human umbilical cord blood hematopoietic progenitor cells in vitro.

Hemoglobin vesicles (HbVs), liposomal oxygen carriers containing human hemoglobin, are candidates for development as clinically useful blood substitutes. Although HbVs are shown to distribute transiently into the bone marrow in animal models, the influence of HbVs on human hematopoietic stem/progenitor cells has not yet been studied. Therefore, we investigated the influence of HbVs at a concentration of up to 3 vol/vol % on the clonogenic activity (in semisolid culture) and proliferative activity (in liquid culture) of human hematopoietic progenitor cells derived from umbilical cord blood (CB) in vitro. Continuous exposure of CB mononuclear cells to HbVs tended to decrease the number and size of mature-committed colonies and most notably reduced the number of colonies of high-proliferative potential colony-forming cells (HPP-CFC). In contrast, exposure to HbVs for 20 h or 3 days, which is more relevant to the clinical setting, had no effect on the number of mature-committed colonies and only modestly decreased the number of HPP-CFC. Continuous exposure (10 days) to HbVs significantly suppressed the cellular proliferation and differentiation of both the erythroid and myeloid lineages in liquid culture. Again, short exposure (20 h or 3 days) did not affect these parameters. Thus, our results show that HbVs, under conditions relevant to the clinical setting, have no adverse effect on human CB hematopoietic progenitor activity in vitro.

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. STUDY DESIGN AND METHODS: We investigated whether inflammatory mediators released from monocytes upon stimulation by HLA Class II antibodies could increase endothelial permeability. Human endothelial cell monolayers were incubated with cell-free supernatants of peripheral blood mononuclear cells (PBMNCs) stimulated with HLA Class II antibody-containing plasma (anti-HLA-DR plasma), which has been implicated in severe nonhemolytic transfusion reactions. The permeability of endothelial cells to dextran was measured. RESULTS: The supernatants of PBMNCs stimulated with the anti-HLA-DR plasma in corresponding antigen-antibody combinations were able to increase endothelial permeability. At least 3 hours of exposure of PBMNCs to anti-HLA-DR plasma was required to produce a supernatant that could induce a significant increase in permeability. Simultaneous addition of tumor necrosis factor alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta) neutralizing antibodies to the activated PBMNC supernatant significantly reduced the increase in permeability. Treatment of the endothelial cells with an inhibitor of nuclear factor kappaB (NF-kappaB), but not inhibitors of apoptosis, significantly prevented the increase in permeability. CONCLUSION: Both TNF-alpha and IL-1 beta, generated from PBMNCs by anti-HLA-DR plasma in a corresponding antigen-antibody-dependent manner, led to an increase in endothelial permeability. The activation of monocytes by the HLA-DR antibodies and the resultant inflammatory mediators could contribute to the pathogenesis of rash, urticaria, angioedema, and pulmonary edema after transfusion.

Influence of O(2)-carrying plasma hemoprotein "albumin-heme" on complement system and platelet activation in vitro and physiological responses to exchange transfusion.

Recombinant human serum albumin (HSA) including the synthetic iron(II)-porphyrin (FeP), albumin-heme (HSA-FeP), is a unique O(2)-carrying plasma hemoprotein as a red blood cell substitute. We have investigated the possible influence of HSA-FeP on the complement system and platelet activation in vitro. The amounts of the serum complement titer CH(50) and terminal complement complex SC5b-9 of human blood serum, incubated with HSA-FeP (10, 20, and 40 vol %), were almost the same as those of the corresponding samples with HSA. The effect of HSA-FeP on the platelet reactivity has been demonstrated by conformational changes in the membrane glycoprotein IIb/IIIa and surface expression of an alpha-granule membrane protein P-selectin. Platelet activation in response to the ADP-stimulation was not influenced by the presence of HSA-FeP. It can be concluded that the albumin-heme solution does not facilitate the immunological reaction and platelet activation. Moreover, a 20% exchange transfusion with HSA-FeP into anesthetized rats has been performed to evaluate the circulation and blood parameters for 6 h. Time course changes in all parameters showed features identical to the control group (without infusion) and HSA group.

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 alphaIIbbeta3 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 beta-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 beta-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.

Life-threatening adverse reaction followed by thrombocytopenia after passive transfusion of fresh frozen plasma containing anti-CD36 (Nak) 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. CASE REPORT: A 67-year-old Japanese woman underwent brain surgery. On the 6th postoperative day, the patient experienced tightness of the chest and nausea after receiving a transfusion of fresh frozen plasma (FFP). When she manifested hypotension, the transfusion was discontinued. No cutaneous manifestation was observed. The patient's condition gradually improved soon after the administration of steroids. RESULTS: Her pretransfusion PLT count was 17.1 x 10(4) per microL. It decreased to 1.9 x 10(4) per microL 12 hours after transfusion and recovered to 15.4 x 10(4) per microL 8 days after transfusion. The donor of the FFP had a Type I CD36 deficiency. Flow cytometric analysis identified anti-CD36 isoantibody in the FFP. The cross-match between the patient's PLTs and the FFP was positive. The FFP induced the aggregation of PLTs derived from healthy adults. CONCLUSION: This is the first reported case of life-threatening adverse effects and thrombocytopenia caused by passively transfused anti-CD36 isoantibody. The possibility of passive infusion of this antibody should be considered in the evaluation of life-threatening transfusion reactions followed by thrombocytopenia.

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. STUDY DESIGN AND METHODS: To investigate the association of RANTES with NHTRs, the biologic activity of RANTES in the supernatant of stored PC at the intravascular concentration expected after PC transfusion was assessed by examining chemotaxis and histamine release in human basophils. In addition, the levels of RANTES in PCs involved in NHTRs were compared with those in PCs causing no transfusion reactions. RESULTS: The supernatant of PC diluted to contain 1 nM RANTES significantly increased the migration of and release of histamine from basophils. Neutralizing antibody to RANTES suppressed the PC-triggered migration, but not histamine release. The levels of RANTES in PCs involved in NHTRs after PC transfusion were comparable to those in PCs that did not cause any transfusion reactions. CONCLUSION: RANTES that accumulated in PCs during storage was biologically active in a basophil chemotaxis assay at the intravascular concentration expected after PC transfusion. However, the NHTRs after PC transfusion were not simply related to the RANTES level in PCs.

Thrombopoietin upregulates nucleolin mRNA and protein in thrombopoietin-dependent megakaryocytic cell line, UT-7/TPO.

Thrombopoietin (TPO) is a hematopoietic cytokine that regulates megakaryocytosis and thrombocytosis by binding to its receptor (c-Mpl). The signaling pathways downstream of c-Mpl include the Ras/Raf/MAP kinase and JAK/STAT pathway and are transduced into the regulation of immediate early-, early- and delayed-response genes. How these genes couple c-Mpl activation to the biochemical machinery of cell growth and cell cycle progression in hematopoietic cells is still unclear. UT-7/TPO is a recently characterized TPO-dependent cell line. Using RNA fingerprinting with arbitrarily primed PCR (RAP-PCR) to identify the TPO-regulated genes in this cell line, we found that the mRNA expression of nucleolin was upregulated in the UT-7/TPO cells in response to TPO. Concomitantly, the TPO-stimulated cells expressed an increased amount of full length nucleolin as determined by immunoblot analysis. The TPO-induced upregulation of nucleolin mRNA was not inhibited by the MEK1/2 inhibitor PD98059, suggesting that ERK/MAPK activation is not necessary for elevation of nucleolin gene expression in response to TPO in UT-7/TPO. Nucleolin is a multifunctional nucleolar protein thought to be involved in many cellular processes, including ribosome biogenesis, the processing of ribosomal RNA (rRNA), mRNA stability, transcriptional regulation, and cell proliferation. Thus, these results indicate that the upregulation of nucleolin mRNA and protein may be important for the TPO-induced effects of hematopoietic cells.

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. STUDY DESIGN AND METHODS: Cord blood CD34+ cells were cultured with thrombopoietin, stem cell factor, and flt3/flk2 ligand on a monolayer of human marrow primary stromal cells prepared using long-term culture medium or AB serum-based medium. After 2 weeks, clonogenic progenitor activity and SCID mouse-reconstituting cell activity were assayed. mRNA expression of cytokines and Notch ligand by stromal cells was also examined. RESULTS: There were no remarkable differences in expansion-supporting activity and mRNA expression between stromal cells established by the two methods. CONCLUSION: An ex vivo expansion system completely based on AB serum has been established.