Yasuo Miura

Abstract This study investigated the anti-tumor effects of andrographolide, a diterpene lactone derived from Andrographis paniculata , on T-cell acute lymphoblastic leukemia (T-ALL) cells. Andrographolide induced dose-dependent cytotoxicity and morphological changes in the T-ALL cell line Jurkat cells, including cell shrinkage and chromatin condensation. Mechanistically, andrographolide triggers apoptosis through reactive oxygen species (ROS) generation, mitochondrial membrane depolarization, and cytochrome c release. These effects were reversed by the ROS inhibitor N-acetyl-L-cysteine (NAC), indicating that andrographolide induces apoptosis through a ROS-dependent apoptotic pathway. In contrast, NAC treatment did not reverse cytarabine- and vincristine-induced apoptosis or the ROS-dependent apoptotic pathway in Jurkat cells. Intriguingly, andrographolide also induced ferroptosis, as evidenced by increased expression of the ferroptosis marker fatty acid-CoA ligase 4 and ultrastructural changes such as reduced mitochondrial area and disappearance of cristae. These effects were likewise reversed by NAC, further implicating ROS in the ferroptotic process. In MOLT-4 cells, where andrographolide suppressed viability, increased Annexin V positivity and ROS levels, and upregulated FACL4 expression in a NAC-sensitive manner. Unlike cytarabine and vincristine, andrographolide did not significantly alter cell cycle distribution. In conclusion, andrographolide induces both apoptosis and ferroptosis in T-ALL cells via ROS-dependent mechanisms that are distinct from those of conventional chemotherapeutic agents. These dual actions position andrographolide as a candidate for standalone or combination therapy in T-ALL.

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 Background Donor-specific antibodies (DSAs) targeting human leukocyte antigens (HLAs) substantially reduce the longevity of transplanted organs. Desensitization of DSA-positive renal transplant recipients is achieved through intravenous administration of immunoglobulin (IVIg). However, the presence and detectability of anti-HLA antibodies in IVIg preparations following administration are not fully understood. We aimed to assess whether immunoglobulin preparations contain anti-HLA antibodies that can be detected as passive antibodies when administered into the body. Methods We evaluated 3 immunoglobulin preparations from different pharmaceutical companies, using anti-HLA class I and II antibody specificity tests and immunocomplex capture fluorescence analysis (ICFA). Results Direct testing for anti-HLA antibodies resulted in high background errors, particularly for Venoglobulin. Diluting Venoglobulin to physiological concentrations revealed the presence of anti-HLA class I antibodies; however, no common alleles were found between the specificity identification test and ICFA. For Glovenin and Venilon, anti-HLA class I and II antibodies were detected; however, variability was observed across different test reagent lots. Moreover, dilution of the globulin formulation revealed a prozone phenomenon. Conclusion The administration of IVIg complicates the accurate detection of anti-HLA antibodies, underscoring the need for careful interpretation of test results post-IVIg administration.

Extracellular vesicles (EVs) serve as an intrinsic system for delivering functional molecules within our body, playing significant roles in diverse physiological phenomena and diseases. Both native and engineered EVs are currently the subject of extensive research as promising therapeutics and drug delivery systems, primarily due to their remarkable attributes, such as targeting capabilities, biocompatibility, and low immunogenicity and mutagenicity. Nevertheless, their clinical application is still a long way off owing to multiple limitations. In this context, the Science Board of the Pharmaceuticals and Medical Devices Agency (PMDA) of Japan has conducted a comprehensive assessment to identify the current issues related to the quality and safety of EV-based therapeutic products. Furthermore, we have presented several examples of the state-of-the-art methodologies employed in EV manufacturing, along with guidelines for critical processes, such as production, purification, characterization, quality evaluation and control, safety assessment, and clinical development and evaluation of EV-based therapeutics. These endeavors aim to facilitate the clinical application of EVs and pave the way for their transformative impact in healthcare.

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 treatment of human immunodeficiency virus (HIV-1) has evolved since the establishment of combination antiretroviral therapy (ART) in the 1990s, providing HIV-infected individuals with approaches that suppress viral replication, prevent acquired immunodeficiency syndrome (AIDS) throughout their lifetime with continuous therapy, and halt HIV transmission. However, despite the success of these regimens, the global HIV epidemic persists, prompting a comprehensive exploration of potential strategies for an HIV cure. Here, we offer a consolidated overview of cell-based therapies for HIV-1, focusing on CAR-T cell approaches, gene editing, and immune modulation. Persistent challenges, including CAR-T cell susceptibility to HIV infection, stability, and viral reservoir control, underscore the need for continued research. This review synthesizes current knowledge, highlighting the potential of cellular therapies to address persistent challenges in the pursuit of an HIV cure.

Abstract 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.

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.

BACKGROUND: The occurrence of transfusion-transmitted hepatitis B virus (HBV) infection has fallen dramatically due to continuous improvements in pre-transfusion laboratory testing. However, the characteristics of transfusion-transmitted HBV infection caused by individual donor nucleic acid amplification test (ID-NAT)-negative blood products are unclear. CASE PRESENTATION: A 76-year-old woman with acute myeloid leukemia was diagnosed with transfusion-transmitted HBV infection after receiving apheresis platelets derived from an ID-NAT-negative blood donation. This case was diagnosed definitively as transfusion-mediated because complete nucleotide homology of a 1556 bp region of the HBV Pol/preS1-preS2-S genes and a 23 bp region of the HBV core promoter/precore between the donor and recipient strains was confirmed by PCR-directed sequencing. The case is uncommon with respect to the unexpectedly prolonged HBV-DNA incubation period of nearly 5 months after transfusion (previously, the longest period observed since the recent implementation of ID-NAT pre-transfusion laboratory testing in Japan was 84 days). Slow-replicating HBV genotype A2 may contribute to the prolonged incubation period; also, the quantity of apheresis platelets delivered in a large volume of plasma, and/or the immune response of the recipient suffering from a hematological neoplasm, may have contributed to establishment of HBV infection in the recipient. This was supported by analysis of three previously documented cases of transfusion-transmitted HBV infection by blood products derived from ID-NAT-negative donations in Japan. CONCLUSION: Continuous monitoring of HBV infection for longer periods (>3 months) may be required after transfusion of blood components from an ID-NAT-negative HBV window donation.