土井 洋輝

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.

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.

BACKGROUND: Activated partial thromboplastin time (APTT) is susceptible to reagent composition. This study aimed to investigate a large number of specimens and determine the cause of discrepancies. METHOD: This study included 18,994 subjects who underwent coagulation tests at our hospital from May 2020 to December 2020. Measuring reagents included HemosIL SynthASil APTT (APTT-SS, Instrumentation Laboratory) and Coagpia APTT-N (APTT-N, Sekisui Medical). RESULTS: A total of 451 patients demonstrated APTT-N of >39 seconds and an APTT-N/SS ratio of >1.3. A C-reactive protein (CRP) level of ≥1.4 mg/L demonstrated a significant positive correlation, with a higher APTT-N/SS indicating higher CRP levels. All 28 subjects receiving no anticoagulants and who had remaining specimens underwent a cross-mixing test (CMT). Of them, 17 were suspected for lupus anticoagulant (LA) by both the waveform shape and the index of circulating anticoagulant (ICA) value, 6 by the ICA value, and 5 were difficult to determine. CONCLUSION: This study revealed that the APTT-N prolongation correlated with CRP degree and the transient involvement of LA in CMT results due to CRP. This study indicated various reactivities depending on the assay reagents used. Further testing is warranted if LA is suspected, considering the patient's background.

OBJECTIVES: We evaluated the applicability of a machine learning-based low-density lipoprotein-cholesterol (LDL-C) estimation method and the influence of the characteristics of the training datasets. METHODS: Three training datasets were chosen from training datasets: health check-up participants at the Resource Center for Health Science (N = 2664), clinical patients at Gifu University Hospital (N = 7409), and clinical patients at Fujita Health University Hospital (N = 14,842). Nine different machine learning models were constructed through hyperparameter tuning and 10-fold cross-validation. Another test dataset of another 3711 clinical patients at Fujita Health University Hospital was selected as the test set used for comparing and validating the model against the Friedewald formula and the Martin method. RESULTS: The coefficients of determination of the models trained on the health check-up dataset produced coefficients of determination that were equal to or inferior to those of the Martin method. In contrast, the coefficients of determination of several models trained on clinical patients exceeded those of the Martin method. The means of the differences and the convergences to the direct method were higher for the models trained on the clinical patients' dataset than for those trained on the health check-up participants' dataset. The models trained on the latter dataset tended to overestimate the 2019 ESC/EAS Guideline for LDL-cholesterol classification. CONCLUSION: Although machine learning models provide valuable method for LDL-C estimates, they should be trained on datasets with matched characteristics. The versatility of machine learning methods is another important consideration.

OBJECTIVES: Agaritine (AGT) is a hydrazine-containing compound derived from the mushroom Agaricus blazei Murill. We previously reported the antitumor effect of AGT on hematological tumor cell lines and suggested that AGT induces apoptosis in U937 cells via caspase activation. However, the antitumor mechanism of AGT has not been fully understood. METHODS: Four hematological tumor cell lines (K562, HL60, THP-1, H929) were used in this study. The cells were incubated in the presence of 50 μM AGT for 24 h and analyzed for cell viability, annexin V positivity, caspase-3/7 activity, mitochondrial membrane depolarization, cell cycle, DNA fragmentation, and the expression of mitochondrial membrane-associated proteins (Bax and cytochrome c). RESULTS: In HL60, K562, and H929 cells, AGT reduced cell viability and increased annexin V- and dead cell-positive rates; however, it did not affect THP-1 cells. In K562 and HL60 cells, caspase-3/7 activity, mitochondrial membrane depolarization, and expression of mitochondrial membrane proteins, Bax and cytochrome c, were all increased by AGT. Cell cycle analysis showed that only K562 exhibited an increase in the proportion of cells in G2/M phase after the addition of AGT. DNA fragmentation was also observed after the addition of AGT. CONCLUSIONS: These results indicate that AGT induces apoptosis in K562 and HL60 cells, like U937 reported previously, but showed no effect on THP-1 cells. It was suggested that AGT-induced apoptosis involves the expression of Bax and cytochrome c via mitochondrial membrane depolarization.

OBJECTIVES: Agaritine (AGT) is a hydrazine-containing compound derived from the mushroom Agaricus blazei Murill. We previously reported the antitumor effect of AGT on hematological tumor cell lines and suggested that AGT induces apoptosis in U937 cells via caspase activation. However, the antitumor mechanism of AGT has not been fully understood. METHODS: Four hematological tumor cell lines (K562, HL60, THP-1, H929) were used in this study. The cells were incubated in the presence of 50 μM AGT for 24 h and analyzed for cell viability, annexin V positivity, caspase-3/7 activity, mitochondrial membrane depolarization, cell cycle, DNA fragmentation, and the expression of mitochondrial membrane-associated proteins (Bax and cytochrome c). RESULTS: In HL60, K562, and H929 cells, AGT reduced cell viability and increased annexin V- and dead cell-positive rates; however, it did not affect THP-1 cells. In K562 and HL60 cells, caspase-3/7 activity, mitochondrial membrane depolarization, and expression of mitochondrial membrane proteins, Bax and cytochrome c, were all increased by AGT. Cell cycle analysis showed that only K562 exhibited an increase in the proportion of cells in G2/M phase after the addition of AGT. DNA fragmentation was also observed after the addition of AGT. CONCLUSIONS: These results indicate that AGT induces apoptosis in K562 and HL60 cells, like U937 reported previously, but showed no effect on THP-1 cells. It was suggested that AGT-induced apoptosis involves the expression of Bax and cytochrome c via mitochondrial membrane depolarization.

<ns3:p><ns3:bold>Background</ns3:bold>: Andrographolide (Andro) is a diterpenoid component of the plant <ns3:italic>Andrographis paniculata</ns3:italic> that is known for its anti-tumor activity against a variety of cancer cells.  </ns3:p><ns3:p> <ns3:bold>Methods</ns3:bold>: We studied the effects of Andro on the viability of the human leukemia monocytic cell line THP-1 and the human multiple myeloma cell line H929. Andro was compared with cytosine arabinoside (Ara-C) and vincristine (VCR), which are well-established therapeutics against hematopoietic tumors. The importance of reactive oxygen species (ROS) production for the toxicity of each agent was investigated by using an inhibitor of ROS production, N-acetyl-L-cysteine (NAC).   </ns3:p><ns3:p> <ns3:bold>Results</ns3:bold>:  Andro reduced the viability of THP-1 and H929 in a concentration-dependent manner. H929 viability was highly susceptible to Andro, although only slightly susceptible to Ara-C. The agents Andro, Ara-C, and VCR each induced apoptosis, as shown by cellular shrinkage, DNA fragmentation, and increases in annexin V-binding, caspase-3/7 activity, ROS production, and mitochondrial membrane depolarization. Whereas Ara-C and VCR increased the percentages of cells in the G0/G1 and G2/M phases, respectively, Andro showed little or no detectable effect on cell cycle progression. The apoptotic activities of Andro were largely suppressed by NAC, an inhibitor of ROS production, whereas NAC hardly affected the apoptotic activities of Ara-C and VCR. </ns3:p><ns3:p> <ns3:bold>Conclusions</ns3:bold>: Andro induces ROS-dependent apoptosis in monocytic leukemia THP-1 and multiple myeloma H929 cells, underlining its potential as a therapeutic agent for treating hematopoietic tumors. The high toxicity for H929 cells, by a mechanism that is different from that of Ara-C and VCR, is encouraging for further studies on the use of Andro against multiple myeloma.</ns3:p>

Background: Andrographolide (Andro) is a diterpenoid component of the plant Andrographis paniculata that is known for its anti-tumor activity against a variety of cancer cells.   Methods: We studied the effects of Andro on the viability of the human leukemia monocytic cell line THP-1 and the human multiple myeloma cell line H929. Andro was compared with cytosine arabinoside (Ara-C) and vincristine (VCR), which are well-established therapeutics against hematopoietic tumors. The importance of reactive oxygen species (ROS) production for the toxicity of each agent was investigated by using an inhibitor of ROS production, N-acetyl-L-cysteine (NAC).    Results:  Andro reduced the viability of THP-1 and H929 in a concentration-dependent manner. H929 viability was highly susceptible to Andro, although only slightly susceptible to Ara-C. The agents Andro, Ara-C, and VCR each induced apoptosis, as shown by cellular shrinkage, DNA fragmentation, and increases in annexin V-binding, caspase-3/7 activity, ROS production, and mitochondrial membrane depolarization. Whereas Ara-C and VCR increased the percentages of cells in the G0/G1 and G2/M phases, respectively, Andro showed little or no detectable effect on cell cycle progression. The apoptotic activities of Andro were largely suppressed by NAC, an inhibitor of ROS production, whereas NAC hardly affected the apoptotic activities of Ara-C and VCR.  Conclusions: Andro induces ROS-dependent apoptosis in monocytic leukemia THP-1 and multiple myeloma H929 cells, underlining its potential as a therapeutic agent for treating hematopoietic tumors. The high toxicity for H929 cells, by a mechanism that is different from that of Ara-C and VCR, is encouraging for further studies on the use of Andro against multiple myeloma.

Atellica IM 1300を用いた心筋バイオマーカー測定の基本性能評価を行った。患者検体血清を用いて、Low、Middle、Highの3濃度のpool血清を作製し、20回同時測定を行ったところ、トロポニンIはCV 1.7〜5.5、ミオグロビンはCV 1.2〜2.1、CK-MBはCV 1.3〜6.9であり、すべての項目において同時再現性は良好であった。日差再現性もトロポニンIはCV 2.6〜6.0、ミオグロビンはCV 2.8〜4.3、CK-MBはCV 2.2〜4.5であった。また、トロポニンI:〜25000.00pg/mL、ミオグロビン:〜1000.00ng/mL、CK-MB:〜300.00ng/mLまで希釈直線性が認められた。現行機器との相関については、ケミルミADVIA Centaur XPTとの良好な相関関係が認められた。さらに、トロポニンIに関して定量限界を確認したところ、2.206pg/mLという値が得られ、心筋梗塞の診断に用いることができる高感度測定試薬であると考えられた。検出限界は0.81ng/mLを示していた。

Conscious of the potential bioactivity of fluorine, an investigation was conducted using various fluorine-containing diaryliodonium salts in order to study and compare their biological activity against human lymphoma U937 cells. Most of the compounds tested are well-known reagents for fluoro-functionalized arylation reactions in synthetic organic chemistry, but their biological properties are not fully understood. Herein, after initially investigating 18 fluoro-functionalized reagents, we discovered that the ortho-fluoro-functionalized diaryliodonium salt reagents showed remarkable cytotoxicity in vitro. These results led us to synthesize more compounds, previously unknown sterically demanding diaryliodonium salts having a pentafluorosulfanyl (SF5) functional group at the ortho-position, that is, unsymmetrical ortho-SF5 phenylaryl-λ3-iodonium salts. Newly synthesized mesityl(2-(pentafluoro-λ6-sulfanyl)phenyl)iodonium exhibited the greatest potency in vitro against U937 cells. Evaluation of the cytotoxicity of selected phenylaryl-λ3-iodonium salts against AGLCL (a normal human B cell line) was also examined.