藤井 紀恵

Abstract Background Ethylenediamine tetraacetate/glycine acid (EGA) and chloroquine diphosphate (CDP) are used in transfusion testing to dissociate IgG antibodies from red blood cells (RBCs). However, the ability of these reagents to dissociate IgM antibodies sensitized to RBCs has not been comprehensively elucidated. We investigated whether EGA and CDP could dissociate cold‐reactive antibodies from RBCs and their effect on RBCs after dissociation treatment. Study Design and Methods Cold‐reactive antibody‐sensitized RBC samples were prepared by mixing group A RBCs and group B plasma and treated with EGA, CDP, and dithiothreitol (DTT). Before and after the dissociation treatment, changes in the agglutination of these RBCs were assessed using the test tube method. Flow cytometric analysis was used to confirm the nature of antibodies bound to RBCs. Additionally, RBC morphology was evaluated using scanning electron microscopy. This study utilized off‐label use of EGA and CDP. Results Flow cytometric analysis showed that antibodies sensitized to RBCs were mainly IgM antibodies. After antibody dissociation, agglutination disappeared in the EGA‐treated samples to the same degree as in the DTT‐treated samples. However, IgM antibodies remained in the CDP‐treated samples. Regarding RBC morphology, RBC surface appeared coarser in both EGA‐ and CDP‐treated samples, and RBC area was significantly smaller in the CDP‐treated samples than in the EGA‐treated samples. Discussion EGA could dissociate cold‐reactive antibodies, whereas CDP had a higher residual antibody content. This difference in dissociation ability appears to correlate with the antibody pH of the dissociation reagent. EGA treatment may be useful in cases of sensitization by high‐titer cold‐reactive antibodies.

Abstract Exposure to ionizing radiation can induce harmful biological effects on the human body, particularly in cases of high-dose γ-irradiation affecting the gastrointestinal tract, bone marrow, skin and lung. Such exposures lead to lethal outcomes as individuals experience a breakdown in their immune system’s ability to defend against pathogens, predisposing them to sepsis-induced multiple organ failures. Mesenchymal stromal/stem cells (MSCs) possess diverse biological characteristics, including immunomodulation, anti-inflammation and tissue regeneration. Off-the-shelf culture-expanded human bone marrow- or adipose tissue-derived MSCs are clinically available to treat graft-versus-host disease following hematopoietic cell transplantation and perianal fistulas in Crohn’s disease in Japan. While preclinical studies showcase encouraging outcomes in radiation-induced injuries, the effectiveness of MSC transplantation in addressing acute radiation syndrome affecting organs in irradiated individuals is limited. Recent studies have highlighted MSC-releasing extracellular vesicles as nanoparticle substances responsible for outlining the mechanism of action and have identified various components, including proteins and microRNA, that serve as functional molecules. MSC-releasing extracellular vesicle-based therapy emerges as a promising avenue, offering a potential solution to the challenges posed by radiation-induced injuries. However, further investigation is required, especially regarding whether MSC-releasing extracellular vesicles have regenerative effects on tissue-resident stem cells. These unresolved issues represent key aspects that need to be addressed to optimize the therapeutic potential of cell-based and extracellular vesicle-based MSC therapies for interventions in the context of radiation-induced injuries.

Abstract Cytopenia is a well-documented complication in the treatment of hematological malignancies with lenalidomide and pomalidomide. Although prior studies have highlighted direct effects on hematopoietic cells to explain this adverse effect, the involvement of hematopoietic-supportive stroma remains less understood. This study examined the effects of lenalidomide/pomalidomide on the expansion and differentiation of human CD34⁺ hematopoietic stem/progenitor cells (HSPCs) in vitro, in co-culture with human bone-marrow mesenchymal stromal/stem cells (MSCs). Our findings indicate that lenalidomide/pomalidomide increases the population of immature CD34⁺CD38⁻ cells while decreasing the number of mature CD34⁺CD38⁺ cells, suggesting a mechanism that inhibits early HSPC maturation. This effect persisted across myeloid, megakaryocytic, and erythroid lineages, with MSCs playing a key role in preserving immature progenitors and inhibiting their differentiation. Furthermore, in myeloid differentiation assays augmented by granulocyte-colony stimulating factor, lenalidomide/pomalidomide not only enhanced the presence of CD34⁺ cells with mature myeloid markers such as CD11b but also reduced the populations lacking CD34 yet positive for these markers, irrespective of MSC presence. Thus, while MSCs support the presence of these immature cell populations, they simultaneously inhibit their maturation. This finding provides novel mechanistic insights into lenalidomide- and pomalidomide-induced cytopenia, and could guide therapeutic strategies for its mitigation.

The development of human mesenchymal stromal/stem cell (MSC)-based therapy has focused on exploring biological nanoparticles secreted from MSCs. There is emerging evidence that the immunomodulatory and regenerative effects of MSCs can be recapitulated by extracellular vesicles released from MSCs (MSC-EVs). Off-the-shelf allogeneic human MSC products are clinically available to treat acute graft-versus-host disease (GVHD), but real-world data have revealed the limitations of these products as well as their feasibility, safety, and efficacy. MSC-EVs may have advantages over parental MSCs as drugs because of their distinguished biodistribution and importantly dose-dependent therapeutic effects. Recent research has shed light on the role of microRNAs in the mode-of-action of MSC-EVs. A group of specific microRNAs alone or in combination with membrane proteins, membrane lipids, and soluble factors present in MSC-EVs play key roles in the regulation of GVHD. In this concise review, we review the regulation of T cell-mediated adaptive immunity and antigen-presenting cell-mediated innate immunity by MSC-EVs and the direct regenerative effects on damaged cells in association with the immunopathology of GVHD.

INTRODUCTION: Quantitative measurement of anti-A/-B antibody titers is important during ABO-incompatible living kidney transplantation (ABOi-LKT). METHODS: We conducted a multi-institutional study to measure the antibody titers using the automated column agglutination technique (auto-CAT) and tube test (TT) method in ABOi-LKT recipients. Statistical analysis was performed to evaluate the two methods. RESULTS: We examined 111 samples from 35 ABOi-LKT recipients at four institutions. The correlation coefficient of the two methods was >0.9; the concordance rate and clinically acceptable concordance rate for the IgG titers were 60.4% and 88.3%, respectively. Perioperative status did not influence the statistical significance. Parallel changes were observed in the IgG antibody titers measured using the auto-CAT or TT technique by desensitizing therapy in time-course monitoring. CONCLUSION: Auto-CAT is comparable with the TT technique and is feasible for IgG anti-A/B antibody titration in ABOi-LKT recipients.

SARS-CoV-2 infection has been reported to be associated with a positive direct antiglobulin test (DAT). In this study, an analysis of 40 consecutive coronavirus disease 2019 (COVID-19) cases from December 2020 to September 2021 in Japan revealed that patients of 70 years and over were predisposed to a positive DAT. DAT positivity was related to a decrease in the hemoglobin level. Anemia in DAT-positive COVID-19 patients was attributed to hemolysis, which was corroborated by high reticulocyte counts and an increase in the red blood cell distribution width. Human leukocyte antigen (HLA)-DRB1*12:01 and DRB1*12:02 were exclusively found in DAT-positive COVID-19 patients. In silico assays for the Spike protein of SARS-CoV-2 predicted several common core peptides that met the criteria for a B cell epitope and strong binding to both HLA-DRB1*12:01 and DRB1*12:02. Among these peptides, the amino acids sequence TSNFR, which is found within the S1 subunit of SARS-CoV-2 Spike protein, is shared by human blood group antigen Rhesus (Rh) CE polypeptides. In vitro analysis showed that the expression of HLA-DR in CD4+ T cells and CD8+ T cells from a DAT-positive patient was increased after pulsation with TSNFR-sequence-containing peptides. In summary, positive DAT is related to enhanced anemia and to HLA-DR12 in the Japanese population. A peptide sequence within SARS-CoV-2 Spike protein may act as an epitope for IgG binding to RBCs in DAT-positive COVID-19 patients.

A 43-year-old Japanese male, who had undergone open liver surgery for tumor resection, presented with decreased hemoglobin levels on Day 13 post-emergency-release transfusion of 16 units of Fy(a +) red blood cells. As the anemia was accompanied by increased lactate dehydrogenase, indirect bilirubin, and reticulocytes, as well as decreased haptoglobin, it was attributed to hemolysis. In the diagnostic workup for hemolytic reaction, the direct antiglobulin test result for IgG was positive and the antibody dissociated from the patient's peripheral red blood cells was identified as anti-Fya (titer, 4). The hemolytic reaction was transient (approximately 10 days), of moderate severity, and did not result in any obvious organ damage. However, a single compatible red blood cell transfusion of 2 units was required on Day 17 after the causative transfusion. Notably, HLA typing revealed that the patient carried the HLA-DRB1*04:03 allele, which has been implicated in immunogenicity and induction of anti-Fya response in Caucasian populations. In summary, this is the first documented case of definitive anti-Fya-mediated delayed hemolytic transfusion reaction associated with HLA-DRB1*04:03 in the Japanese population.

ヒト間葉系幹細胞(間葉系間質細胞/human mesenchymal stromal/stem cell:MSC)は制御性免疫細胞である。間葉系幹細胞の造血細胞移植後GVHDにおける免疫制御メカニズムは複雑であり、さまざまな免疫細胞に対して液性因子や細胞接着を介して作用する。近年では間葉系幹細胞が放出する細胞外小胞(extracellular vesicle:EV)の寄与が重要視されている。ヒト間葉系幹細胞の骨髄以外の代替ソース開発が進められてきたが、最近ではiPS細胞作製技術を応用し製造された間葉系幹細胞製剤の臨床応用がはじまっており、間葉系幹細胞を応用した細胞治療のgame changerになるか注目される。(著者抄録)

In the original publication of the article, the Figs. 4 C, F and 5 B, C were published with unexpected appearance of dots.