Membrane skeleton hyperstability due to a novel alternatively spliced 4.1R can account for ellipsoidal camelid red cells with decreased deformability.

The red blood cells (RBCs) of vertebrates have evolved into two basic shapes, with nucleated nonmammalian RBCs having a biconvex ellipsoidal shape and anuclear mammalian RBCs having a biconcave disk shape. In contrast, camelid RBCs are flat ellipsoids with reduced membrane deformability, suggesting altered membrane skeletal organization. However, the mechanisms responsible for their elliptocytic shape and reduced deformability have not been determined. We here showed that in alpaca RBCs, protein 4.1R, a major component of the membrane skeleton, contains an alternatively spliced exon 14-derived cassette (e14) not observed in the highly conserved 80 kDa 4.1R of other highly deformable biconcave mammalian RBCs. The inclusion of this exon, along with the preceding unordered proline- and glutamic acid-rich peptide (PE), results in a larger and unique 90 kDa camelid 4.1R. Human 4.1R containing e14 and PE, but not PE alone, showed markedly increased ability to form a spectrin-actin-4.1R ternary complex in viscosity assays. A similar facilitated ternary complex was formed by human 4.1R possessing a duplication of the spectrin-actin-binding domain, one of the mutations known to cause human hereditary elliptocytosis. The e14- and PE-containing mutant also exhibited an increased binding affinity to ß-spectrin compared with WT 4.1R. Taken together, these findings indicate that 4.1R protein with the e14 cassette results in the formation and maintenance of a hyperstable membrane skeleton, resulting in rigid red ellipsoidal cells in camelid species, and suggest that membrane structure is evolutionarily regulated by alternative splicing of exons in the 4.1R gene.

Genetic Risk Stratification of Primary Open-Angle Glaucoma in Japanese Individuals.

PURPOSE: To assess the impact of genetic risk estimation for primary open-angle glaucoma (POAG) in Japanese individuals. DESIGN: Cross-sectional analysis. PARTICIPANTS: Genetic risk scores (GRSs) were constructed based on a genome-wide association study (GWAS) of POAG in Japanese people. A total of 3625 Japanese individuals, including 1191 patients and 2434 controls (Japanese Tohoku), were used for the model selection. We also evaluated the discriminative accuracy of constructed GRSs in a dataset comprising 1034 patients and 1147 controls (the Japan Glaucoma Society Omics Group [JGS-OG] and the Genomic Research Committee of the Japanese Ophthalmological Society [GRC-JOS]) and 1900 participants from a population-based study (Hisayama Study). METHODS: We evaluated 2 types of GRSs: polygenic risk scores using the pruning and thresholding procedure and a GRS using variants associated with POAG in the GWAS of the International Glaucoma Genetics Consortium (IGGC). We selected the model with the highest areas under the receiver operating characteristic curve (AUC). In the population-based study, we evaluated the correlations between GRS and ocular measurements. MAIN OUTCOME MEASURE: Proportion of patients with POAG after stratification according to the GRS. RESULTS: We found that a GRS using 98 variants, which showed genome-wide significance in the IGGC, showed the best discriminative accuracy (AUC, 0.65). In the Japanese Tohoku, the proportion of patients with POAG in the top 10% individuals was significantly higher than that in the lowest 10% (odds ratio [OR], 6.15; 95% confidence interval [CI], 4.35-8.71). In the JGS-OG and GRC-JOS, we confirmed similar impact of POAG GRS (AUC, 0.64; OR [top vs. bottom decile], 5.81; 95% CI, 3.79-9.01). In the population-based study, POAG prevalence was significantly higher in the top 20% individuals of the GRS compared with the bottom 20% (9.2% vs. 5.0%). However, the discriminative accuracy was low (AUC, 0.56). The POAG GRS was correlated positively with intraocular pressure (r = 0.08: P = 4.0 × 10-4) and vertical cup-to-disc ratio (r = 0.11; P = 4.0 × 10-6). CONCLUSIONS: The GRS showed moderate discriminative accuracy for POAG in the Japanese population. However, risk stratification in the general population showed relatively weak discriminative performance. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Prognostic value of the "dynamic" R2-ISS in patients with multiple myeloma undergoing anti-CD38 antibody-based triplet therapies.

To retrospectively analyze whether the second revision of the international staging system (R2-ISS) influenced prognosis at treatment initiation in patients with multiple myeloma (MM) receiving anti-CD38 antibody-based triplet treatments. High-risk chromosomal abnormalities were examined from diagnosis to treatment initiation and considered positive if detected once. R2-ISS was recalculated at the initiation of treatment and defined as "dynamic R2-ISS." Data from 150 patients who underwent the defined treatments were analyzed. The median progression-free survival (PFS) was 19.5 months, and the median overall survival (OS) was 36.5 months. Dynamic R2-ISS significantly stratified prognoses for both PFS and OS. The median PFS for patients with dynamic R2-ISS IV was 3.3 months, and the median OS was 11.7 months, indicating extremely poor outcomes. Although the Revised International Staging System (R-ISS) calculated at the initiation of treatment significantly stratified treatment outcomes, the patients classified as R-ISS could be further stratified by R2-ISS to provide better prognostic information. Dynamic R2-ISS showed potential as a prognostic tool in patients with MM who are treated with anti-CD38 antibody-based triplet therapies.

Adherence to and clinical utility of "quality indicators" for Staphylococcus aureus bacteremia: a retrospective, multicenter study.

BACKGROUND: We aimed to improve the prognosis, treatment, and management of Staphylococcus aureus bacteremia (SAB) by evaluating the association between adherence to quality indicators (QIs) and clinical outcomes in patients with their clinical outcomes. METHODS: We retrospectively collected clinical and microbiological data on hospitalized patients with SAB from 14 hospitals (three with > 600, two with 401-600, five with 201-400, and four with ≤ 200 beds) in Japan from January to December 2022. The SAB management quality was evaluated using the SAB-QI score (ranging from 0 to 13 points), which consists of 13 QIs (grouped into five categories) based on previous literature. RESULTS: Of the 4,448 positive blood culture episodes, 289 patients with SAB (6.5%) were enrolled. The SAB-QI scores ranged from 3 to 13, with a median score of 9 points. The SAB-QI score was highest in middle-sized hospitals with 401-600 beds. Adherence to each of the four QI categories (blood culture, echocardiography, source control, and antibiotic treatment) was significantly higher in survived cases than in fatal cases. Kaplan-Meier curves with log-rank tests demonstrated that higher adherence to SAB-QIs indicated a better prognosis. Logistic regression analysis revealed that age, methicillin resistance, multiple comorbidities (≥ 2), and low SAB-QI score were significantly associated with 30-day mortality in patients with SAB. CONCLUSIONS: Our study highlights that greater adherence to the SAB-QIs correlates with improved patient outcomes. Management of patients with SAB should follow these recommended indicators to maintain the quality of care, especially for patients with poor prognosticators.

Cytomegalovirus Reactivation during Elotuzumab Therapy in Patients with Multiple Myeloma.

INTRODUCTION: Some treatments are associated with cytomegalovirus (CMV) reactivation (CMVRA) in patients with multiple myeloma (MM). However, no reports exist on the association between elotuzumab and CMVRA. Therefore, we assessed the incidence of CMVRA in patients with MM who received elotuzumab therapy. METHODS: The medical records of 85 patients who underwent elotuzumab therapy were included in the retrospective analysis for CMV positivity. RESULTS: Thirty patients were tested for CMV antigenemia during elotuzumab therapy, and 16 were positive for CMV antigenemia; the cumulative incidence rate of CMVRA 6 months after elotuzumab initiation was 18.4%. The history of allogeneic stem cell transplantation (allo-HSCT) was significantly more common in the CMVRA group (31.2%) than that of the group without CMVRA (8.7%). However, even among patients who did not undergo allo-HSCT, the cumulative incidence rate of CMVRA at 6 months was 15.1%. During CMVRA, the symptoms included fever in 8 cases, while retinitis was observed in 1 case. Five patients required antiviral therapy and CMV antigenemia resolved in all but 1 case. CONCLUSION: Although the patient population was heterogeneous, CMVRA cannot be underestimated during elotuzumab therapy, and evaluation of CMVRA, especially in symptomatic cases, is clinically important.

New Dihydropyridine Derivative Attenuates NF-κB Activation via Suppression of Calcium Influx in a Mouse BV-2 Microglial Cell Line.

Activated microglia contribute to many neuroinflammatory diseases in the central nervous system. In this study, we attempted to identify an anti-inflammatory compound that could suppress microglial activation. We performed high-throughput screening with a chemical library developed at our institute. We performed a luciferase assay of nuclear factor-kappa B (NF-κB) reporter stable HT22 cells and identified a compound that was confirmed to inhibit the anti-inflammatory response in BV2 microglial cells. The selected dihydropyridine derivative can suppress the expression response of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor (TNF), as well as NF-κB phosphorylation and nuclear translocation, and reduce the intracellular calcium level. Thus, our identified compound has a potential role in suppressing microglial activation and may contribute to the development of a new therapeutic molecule against neuroinflammatory diseases.

The relationship between equol production status and normal tension glaucoma.

PURPOSE: Equol is metabolized by intestinal bacteria from soy isoflavones and is chemically similar to estrogen. Dietary habits, such as consumption of soy products, influence equol production. A relationship between glaucoma and estrogen has been identified; here, we investigated the relationship between equol production status and glaucoma in Japan. METHODS: We recruited 68 normal-tension glaucoma (NTG) patients (male to female ratio 26:42, average age 63.0 ± 7.6 years) and 31 controls (male to female ratio 13:18, average age 66.0 ± 6.3 years) from our hospital. All women included were postmenopausal. Urinary equol concentration was quantified with the ELISA method. MD was calculated based on the Humphrey visual field. The association between MD and equol was analyzed with Spearman's rank correlation coefficient. The Mann-Whitney U test was used to compare the equol-producing (> 1 µM) and non-producing (< 1 µM) subjects. We also investigated the association between equol and glaucoma with a logistic regression analysis. RESULTS: There was a significant association between equol and MD (r = 0.36, P < 0.01) in the NTG patients. Glaucoma, represented by MD, was significantly milder in the equol-producing subjects than the non-equol producing subjects (P = 0.03). A multivariate analysis revealed the independent contributions of equol, cpRNFLT, and IOP to MD (P = 0.03, P = 0.04, and P < 0.01, respectively). CONCLUSION: Our results suggest that equol, acting through estrogen receptor-mediated neuroprotective effects, might be involved in suppressing the progression of NTG. This result also adds to evidence that glaucoma may be influenced by lifestyle.

[Retrospective analysis of allogeneic hematopoietic stem cell transplantation for multiple myeloma after myeloablative conditioning with 8 Gy of total body irradiation].

Allogeneic hematopoietic stem cell transplantation (allo-SCT) is a curative treatment option for multiple myeloma (MM), but few patients are eligible due to its high risk of treatment-related toxicity and relapse. Here, we report the feasibility and efficacy of allo-SCT after myeloablative conditioning with 8 Gy of total body irradiation (TBI) for reducing relapse of MM. We retrospectively analyzed data from 30 consecutive patients who received allo-SCT for MM after 8 Gy of TBI at Japanese Red Cross Medical Center between 2012 and 2021. Median age at allo-SCT was 47 (range 31-61) years. Stem-cell sources were peripheral blood from an HLA-matched related donor (MRD, n=5), bone marrow from an HLA-matched unrelated donor (MUD, n=5), bone marrow from an HLA-mismatched unrelated donor (MMUD, n=13), and cord blood (n=7). All patients received conditioning with 8 Gy of TBI combined with Flu/Mel (n=28) or others (n=2). Five-year PFS and 5-year OS were 36.7% and 46.2%, respectively. Sixteen patients died during the observation period (12 of primary disease and 4 of treatment-related toxicity). Patients with VGPR or better before allo-SCT had significantly better PFS (p=0.009) and OS (p=0.01) than others. Patients who received MMUD cells tended to have better PFS than those with other cell sources. Our report showed that allo-SCT for MM after 8 Gy of TBI is feasible, and the better PFS of MMUD suggests graft-versus-myeloma effects.

The GPR84 molecule is a mediator of a subpopulation of retinal microglia that promote TNF/IL-1α expression via the rho-ROCK pathway after optic nerve injury.

Resident microglia are important to maintain homeostasis in the central nervous system, which includes the retina. The retinal microglia become activated in numerous pathological conditions, but the molecular signatures of these changes are poorly understood. Here, using an approach based on FACS and RNA-seq, we show that microglial gene expression patterns gradually change during RGC degeneration induced by optic nerve injury. Most importantly, we found that the microglial cells strongly expressed Tnf and Il1α, both of which are known to induce neurotoxic reactive astrocytes, and were characterized by Gpr84high -expressing cells in a particular subpopulation. Moreover, ripasudil, a Rho kinase inhibitor, significantly blunted Gpr84 expression and cytokine induction in vitro and in vivo. Finally, GPR84-deficient mice prevented RGC loss in optic nerve-injured retina. These results reveal that Rho kinase-mediated GPR84 alteration strongly contribute to microglial activation and promote neurotoxicity, suggesting that Rho-ROCK and GPR84 signaling may be potential therapeutic targets to prevent the neurotoxic microglial phenotype induced by optic nerve damage, such as occurs in traumatic optic neuropathy and glaucoma.

ΔNp63 Regulates Radioresistance in Human Head and Neck Squamous Carcinoma Cells.

Radiation therapy is commonly used to treat head and neck squamous cell carcinoma (HNSCC); however, recurrence results from the development of radioresistant cancer cells. Therefore, it is necessary to identify the underlying mechanisms of radioresistance in HNSCC. Previously, we showed that the inhibition of karyopherin-ß1 (KPNB1), a factor in the nuclear transport system, enhances radiation-induced cytotoxicity, specifically in HNSCC cells, and decreases the localization of SCC-specific transcription factor ΔNp63. This suggests that ΔNp63 may be a KPNB1-carrying nucleoprotein that regulates radioresistance in HNSCC. Here, we determined whether ΔNp63 is involved in the radioresistance of HNSCC cells. Cell survival was measured by a colony formation assay. Apoptosis was assessed by annexin V staining and cleaved caspase-3 expression. The results indicate that ΔNp63 knockdown decreased the survival of irradiated HNSCC cells, increased radiation-induced annexin V+ cells, and cleaved caspase-3 expression. These results show that ΔNp63 is involved in the radioresistance of HNSCC cells. We further investigated which specific karyopherin-α (KPNA) molecules, partners of KPNB1 for nuclear transport, are involved in nuclear ΔNp63 expression. The analysis of nuclear ΔNp63 protein expression suggests that KPNA1 is involved in nuclear ΔNp63 expression. Taken together, our results suggest that ΔNp63 is a KPNB1-carrying nucleoprotein that regulates radioresistance in HNSCC.

Changes in glial cells and neurotrophic factors due to rotenone-induced oxidative stress in Nrf2 knockout mice.

Glaucoma is one of the most common causes of blindness worldwide. It is thought to be a multifactorial disease with underlying mechanisms that include mitochondrial dysfunction and oxidative stress. Here, we used NF-E2 related factor 2 (Nrf2) knockout (KO) mice, which are vulnerable to oxidative stress, to examine a neuroprotective effect against oxidative stress due to rotenone, a mitochondrial complex I inhibitor. Wild-type (WT) and Nrf2 KO mice received an oral solution of rotenone for 30 days. We then extracted the retinas and performed immunohistochemistry and quantitative RT-PCR. We also prepared a primary Müller cell culture of samples from each mouse, added 30 µM rotenone, and then measured cell viability, cytotoxicity and CellRox absorbance. We also examined gene expression. We found a significant increase in the number of 8-OHdG-positive retinal ganglion cells (RGCs) after rotenone administration in both the WT and Nrf2 KO mice. There was no difference in the number of RNA-binding protein with multiple splicing (RBPMS)-positive RGCs in the WT and Nrf2 KO mice, but Nrf2 KO mice that were given rotenone had significantly less retinal gene expression of RBPMS than Nrf2 KO mice given a control. Moreover, there was significantly higher mRNA gene expression of vimentin and glial fibrillary acidic protein (GFAP) in Nrf2 KO mice that received rotenone than WT mice that received rotenone. A statistical analysis of the in vitro experiment showed that cell viability was lower, cytotoxicity was higher, and oxidative stress was higher in the Müller cells of the Nrf2 KO mice than the WT mice. Finally, brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) were significantly higher in the Müller cells of the Nrf2 KO mice than the WT mice. These findings suggest that in Nrf2 KO mice under oxidative stress caused by rotenone, temporary neurotrophic factors are secreted from the Müller cells, conferring neuroprotection in these cells.

Real-world clinical outcomes in patients with relapsed and refractory multiple myeloma receiving VTD-PACE treatment in the era of monoclonal antibodies.

Bortezomib (Velcade), thalidomide, dexamethasone, platinum (cisplatin), adriamycin (doxorubicin), cyclophosphamide, and etoposide (VTD-PACE) are commonly used as salvage treatment for patients with relapsed/refractory multiple myeloma (RRMM). However, its outcomes in the era of monoclonal antibodies remain unclear. Therefore, this retrospective cohort study assessed the clinical outcomes of 60 patients with RRMM (median four prior treatment lines) administered VTD-PACE. The median follow-up period was 11.1 months, during which they received a median of two cycles of VTD-PACE. The overall response rate (ORR) was 66.7%; ORRs of 53.1 and 82.1% were noted in patients with ≥ 4 and ≤ 3 prior lines (P = 0.027), respectively. The median overall survival (OS) was 17 months, with a median progression-free survival (PFS) of 9.8 months. Using the 3-month time point after VTD-PACE treatment as a landmark, 54 patients were still alive. Landmark analysis was conducted for PFS and OS of patients who received or did not receive HSCT or CART after VTD-PACE treatment. Patients who underwent subsequent hematopoietic stem cell transplantation (HSCT) or chimeric antigen receptor T-cell therapy (CART) following VTD-PACE showed a trend of longer PFS and OS than those who did not undergo subsequent HSCT or CART. The median OS in patients with and without renal dysfunction was 10.7 months and 21.5 months, respectively (P = 0.0091). Therefore, VTD-PACE is useful as a bridging therapy for HSCT or CART, as a response can be expected regardless of organ damage, disease risk, or history of anti-CD38 antibody use.

Real-world clinical outcomes in patients with multiple myeloma treated with isatuximab after daratumumab treatment.

Isatuximab and daratumumab are anti-CD38 monoclonal antibodies used to treat refractory multiple myeloma. Isatuximab is often used after unsuccessful daratumumab treatment; however, the clinical benefits of receiving isatuximab after daratumumab treatment have not been fully evaluated. Therefore, this retrospective cohort study assessed the clinical outcomes of 39 patients with multiple myeloma who were administered isatuximab after daratumumab. The median follow-up period was 8.7 months (range 0.1-25.0 months). The overall response rate was 46.2% (18 patients). The 1-year overall survival was 53.9%, with a median progression-free survival of 5.6 months. The median progression-free survival in patients with high and normal lactate dehydrogenase levels was 4.5 and 9.6 months, respectively (P = 0.004). The median progression-free survival in patients with and without triple-class refractory disease was 5.1 months and not reached, respectively (P = 0.001). The median overall survival in patients with high and normal lactate dehydrogenase levels was not reached and 9.3 months, respectively (P = 0.001). The median overall survival in patients with and without triple-class refractory disease was 9.9 months and not reached, respectively (P = 0.038). Our findings provide insight into the optimal use and timing of anti-CD38 antibody therapy.

Novel partial deletions, frameshift and missense mutations of CSF1R in patents with CSF1R-related leukoencephalopathy.

BACKGROUND AND PURPOSE: Colony-stimulating factor 1 receptor (CSF1R)-related leukoencephalopathy is an adult-onset leukoencephalopathy caused by mutations in CSF1R. The present study aimed to explore the broader genetic spectrum of CSF1R-related leukoencephalopathy in association with clinical and imaging features. METHODS: Mutational analysis of CSF1R was performed for 100 consecutive patients with adult-onset leukoencephalopathy. Sequence and copy number variation (CNV) analyses of CSF1R were performed. The genomic ranges of the deletions were determined by long-read sequencing. Ligand-dependent autophosphorylation of CSF1R was examined in cells expressing the CSF1R mutants identified in this study. RESULTS: CSF1R mutations were identified in 15 patients, accounting for 15% of the adult-onset leukoencephalopathy cases. Seven novel and five previously reported CSF1R mutations were identified. The novel mutations, including three missense and one in-frame 3 bp deletion, were located in the tyrosine kinase domain (TKD) of CSF1R. Functional assays revealed that none of the novel mutations in the TKD showed autophosphorylation of CSF1R. Two partial deletions of CSF1R were identified that resulted in lack of the C-terminal region, including the distal TKD, in two patients. Various clinical features including cognitive impairment, psychiatric symptoms and gait disturbance were observed. Various degrees of the white matter lesions and corpus callosum abnormalities on magnetic resonance imaging and characteristic calcifications on computed tomography were observed as imaging features. CONCLUSIONS: Our results highlight the importance of examining the CNV of CSF1R even when Sanger or exome sequencing reveals no CSF1R mutations. Genetic examination of sequences and CNV analyses of CSF1R are recommended for an accurate diagnosis of CSF1R-related leukoencephalopathy.

Metabolic and pathologic profiles of human LSS deficiency recapitulated in mice.

Skin lesions, cataracts, and congenital anomalies have been frequently associated with inherited deficiencies in enzymes that synthesize cholesterol. Lanosterol synthase (LSS) converts (S)-2,3-epoxysqualene to lanosterol in the cholesterol biosynthesis pathway. Biallelic mutations in LSS have been reported in families with congenital cataracts and, very recently, have been reported in cases of hypotrichosis. However, it remains to be clarified whether these phenotypes are caused by LSS enzymatic deficiencies in each tissue, and disruption of LSS enzymatic activity in vivo has not yet been validated. We identified two patients with novel biallelic LSS mutations who exhibited congenital hypotrichosis and midline anomalies but did not have cataracts. We showed that the blockade of the LSS enzyme reaction occurred in the patients by measuring the (S)-2,3-epoxysqualene/lanosterol ratio in the forehead sebum, which would be a good biomarker for the diagnosis of LSS deficiency. Epidermis-specific Lss knockout mice showed neonatal lethality due to dehydration, indicating that LSS could be involved in skin barrier integrity. Tamoxifen-induced knockout of Lss in the epidermis caused hypotrichosis in adult mice. Lens-specific Lss knockout mice had cataracts. These results confirmed that LSS deficiency causes hypotrichosis and cataracts due to loss-of-function mutations in LSS in each tissue. These mouse models will lead to the elucidation of the pathophysiological mechanisms associated with disrupted LSS and to the development of therapeutic treatments for LSS deficiency.

[Acute myeloid leukemia with t (8;21) translocation diagnosed at 21 weeks of gestation resulting in full-term delivery without chemotherapy].

A 28-year-old female was diagnosed with acute myeloid leukemia (AML) due to t (8;21) (q22;q22.1); RUNX1-RUNX1T1 at 21 weeks of gestation. Because no adverse prognostic genetic mutations were discovered, we decided to continue the pregnancy without chemotherapy for as long as possible. After careful monitoring with blood tests every two weeks, the disease did not progress until full-term, and a cesarean section was performed at 39 weeks of gestation. About two months after delivery, blasts in the peripheral blood increased to 46.5%, and myeloblasts in the bone marrow increased to 21.2%. The patient received idarubicin and cytarabine induction therapy, followed by three cycles of high-dose cytarabine consolidation therapy, and complete remission was maintained. Here we report a rare case who could avoid chemotherapy until full-term labor without progression of AML.

[A retrospective analysis of 124 patients with multiple myeloma who received up-front autologous hematopoietic cell transplantation following triplet induction therapy].

The IFM/DFCI group reported that VRD induction followed by up-front autologous peripheral blood stem cell transplantation (ASCT) and maintenance therapy led to median PFS of 50 months, which established up-front ASCT as the standard of care even in the era of novel agents. We conducted a retrospective analysis on outcomes of patients who received triplet induction therapy followed by up-front ASCT at our institution. A total of 124 patients received ASCT between November 2016 and December 2021 at Japanese Red Cross Medical Center. Patient characteristics, treatment response before and after ASCT, and PFS and OS were retrospectively analyzed. VRD-based induction therapy was used for 94%. Among 118 evaluable patients, 116 (98%) received either consolidation and/or maintenance therapy. Best responses were ≥CR 77% and ≥VGPR 94%, respectively. Sixty-eight out of 104 patients achieved MRD-negativity by multiparameter FCM (<10-5). Five-year estimated PFS and OS were 54.7% and 80.2%, respectively. Age ≥65, high-risk cytogenetic abnormalities, and

Cholesterol-binding protein TSPO2 coordinates maturation and proliferation of terminally differentiating erythroblasts.

TSPO2 (translocator protein 2) is a transmembrane protein specifically expressed in late erythroblasts and has been postulated to mediate intracellular redistribution of cholesterol. We identified TSPO2 as the causative gene for the HK (high-K+) trait with immature red cell phenotypes in dogs and investigated the effects of the TSPO2 defects on erythropoiesis in HK dogs with the TSPO2 mutation and Tspo2 knockout (Tspo2-/-) mouse models. Bone marrow-derived erythroblasts from HK dogs showed increased binucleated and apoptotic cells at various stages of maturation and shed large nuclei with incomplete condensation when cultured in the presence of erythropoietin, indicating impaired maturation and cytokinesis. The canine TSPO2 induces cholesterol accumulation in the endoplasmic reticulum and could thereby regulate cholesterol availability by changing intracellular cholesterol distribution in erythroblasts. Tspo2-/- mice consistently showed impaired cytokinesis with increased binucleated erythroblasts, resulting in compensated anemia, and their red cell membranes had increased Na,K-ATPase, resembling the HK phenotype in dogs. Tspo2-deficient mouse embryonic stem cell-derived erythroid progenitor (MEDEP) cells exhibited similar morphological defects associated with a cell-cycle arrest at the G2/M phase, resulting in decreased cell proliferation and had a depletion in intracellular unesterified and esterified cholesterol. When the terminal maturation was induced, Tspo2-/- MEDEP cells showed delays in hemoglobinization; maturation-associated phenotypic changes in CD44, CD71, and TER119 expression; and cell-cycle progression. Taken together, these findings imply that TSPO2 is essential for coordination of maturation and proliferation of erythroblasts during normal erythropoiesis.

CHOP deletion and anti-neuroinflammation treatment with hesperidin synergistically attenuate NMDA retinal injury in mice.

Glaucoma is a leading cause of blindness worldwide and is characterized by degeneration associated with the death of retinal ganglion cells (RGCs). It is believed that glaucoma is a group of heterogeneous diseases with multifactorial pathomechanisms. Here, we investigate whether anti-inflammation treatment with an ER stress blockade can selectively promote neuroprotection against NMDA injury in the RGCs. Retinal excitotoxicity was induced with an intravitreal NMDA injection. Microglial activation and neuroinflammation were evaluated with Iba1 immunostaining and cytokine gene expression. A stable HT22 cell line transfected with an NF-kB reporter was used to assess NF-kB activity after hesperidin treatment. CHOP-deficient mice were used as a model of ER stress blockade. Retinal cell death was evaluated with a TUNEL assay. As results, in the NMDA injury group, Iba1-positive microglia increased 6 h after NMDA injection. Also at 6 h, pro-inflammatory cytokines and chemokine increased, including TNFα, IL-1b, IL-6 and MCP-1. In addition, the MCP-1 promoter-driven EGFP signal, which we previously identified as a stress signal in injured RGCs, also increased; hesperidin treatment suppressed this inflammatory response and reduced stressed RGCs. In CHOP-deficient mice that received an NMDA injection, the gene expression of pro-inflammatory cytokines, chemokines, markers of active microglia, and inflammatory regulators was greater than in WT mice. In WT mice, hesperidin treatment partially prevented retinal cell death after NMDA injury; this neuroprotective effect was enhanced in CHOP-deficient mice. These findings demonstrate that ER stress blockade is not enough by itself to prevent RGC loss due to neuroinflammation in the retina, but it has a synergistic neuroprotective effect after NMDA injury when combined with an anti-inflammatory treatment based on hesperidin.

Minimal residual disease detection in multiple myeloma: comparison between BML single-tube 10-color multiparameter flow cytometry and EuroFlow multiparameter flow cytometry.

Minimal residual disease (MRD)-negative status in multiple myeloma (MM) is associated with favorable outcomes. Although EuroFlow next-generation flow (NGF) is a global standard for MRD detection, its operating cost is high. Therefore, it is desirable to develop a less expensive method with equivalent sensitivity to that of EuroFlow-NGF. In this study, we compared the analytical ability of our BML 10-color multiparameter flow cytometry (MFC) to that of EuroFlow-NGF. Bone marrow samples collected from 51 patients with MM were subjected to MRD detection using BML 10-color-MFC and EuroFlow-NGF. Our antibody panel consisted of CD38 multiepitope, CD138, CD45, CD56, CD19, CD27, CD81, CD117, cytoplasmic immunoglobulin (cIg) κ, and cIgλ in a single tube. The median percentages of total plasma cells, as per 10-color-MFC and EuroFlow-NGF, were 0.2148% and 0.2200%, respectively, with a good correlation between the methods (r = 0.950). The median percentages of myeloma cells determined via 10-color-MFC and EuroFlow-NGF were 0.0012% and 0.0007%, respectively, with a strong correlation (r = 0.954). Our 10-color-MFC demonstrated high sensitivity to detect MRD; the results showed a good correlation with those obtained using EuroFlow-NGF. Therefore, our cost-effective single-tube MFC (approximately 100 USD/sample) is a promising alternative method for the detection of MRD in patients with MM.