Flavonifractor plautii Bacteremia With Generalized Peritonitis: A Case Report and Literature Review.

Flavonifractor plautii is an obligate anaerobic rod bacterium that is part of the human gut microbiota. We describe a case of bacteremia caused by F. plautii in a mildly immunocompromised patient with acute generalized peritonitis. The patient is an 83-year-old male, with a history of stage III hepatocellular carcinoma 11 months prior, stage I gastric cancer, and cerebral infarction three months prior. He visited the emergency room of our hospital with a chief complaint of right-sided abdominal pain. A partial resection of the colon was performed due to stenosis of the transverse colon. Due to increasing abdominal pain, the patient underwent surgery for acute generalized peritonitis on the 11th postoperative day. F. plautii was detected in blood cultures collected prior to surgery, and the patient was treated with piperacillin/tazobactam 2.25 g four times a day for 11 days. The patient resumed eating and was discharged with no recurrence. This species may also stain gram-negative, and caution should be exercised in reporting results due to the potential impact on initial antimicrobial therapy. Gram staining showed variation in the length of the bacterium, which is considered a characteristic of this species. Appropriate antimicrobial therapy for F. plautii has yet to be established, and further accumulation of cases is needed to understand the resistance mechanism and confirm the effectiveness of different antimicrobials.

BRD9 determines the cell fate of hematopoietic stem cells by regulating chromatin state.

ATP-dependent chromatin remodeling SWI/SNF complexes exist in three subcomplexes: canonical BAF (cBAF), polybromo BAF (PBAF), and a newly described non-canonical BAF (ncBAF). While cBAF and PBAF regulate fates of multiple cell types, roles for ncBAF in hematopoietic stem cells (HSCs) have not been investigated. Motivated by recent discovery of disrupted expression of BRD9, an essential component of ncBAF, in multiple cancers, including clonal hematopoietic disorders, we evaluate here the role of BRD9 in normal and malignant HSCs. BRD9 loss enhances chromatin accessibility, promoting myeloid lineage skewing while impairing B cell development. BRD9 significantly colocalizes with CTCF, whose chromatin recruitment is augmented by BRD9 loss, leading to altered chromatin state and expression of myeloid-related genes within intact topologically associating domains. These data uncover ncBAF as critical for cell fate specification in HSCs via three-dimensional regulation of gene expression and illuminate roles for ncBAF in normal and malignant hematopoiesis.

Heme-dependent induction of mitophagy program during differentiation of murine erythroid cells.

Although establishment and maintenance of mitochondria are essential for the production of massive amounts of heme in erythroblasts, mitochondria must be degraded upon terminal differentiation to red blood cells (RBCs), thus creating a biphasic regulatory process. Previously, we reported that iron deficiency in mice promotes mitochondrial retention in RBCs, suggesting that a proper amount of iron and/or heme is necessary for the degradation of mitochondria during erythroblast maturation. Because the transcription factor GATA1 regulates autophagy in erythroid cells, which involves mitochondrial clearance (mitophagy), we investigated the relationship between iron or heme and mitophagy by analyzing the expression of genes related to GATA1 and autophagy and the impact of iron or heme restriction on the amount of mitochondria. We found that heme promotes the expression of GATA1-regulated mitophagy-related genes and the induction of mitophagy. GATA1 might induce the expression of the autophagy-related genes Atg4d and Stk11 for mitophagy through a heme-dependent mechanism in murine erythroleukemia (MEL) cells and a genetic rescue system with G1E-ER-GATA1 erythroblast cells derived from Gata1-null murine embryonic stem cells. These results provide evidence for a biphasic mechanism in which mitochondria are essential for heme generation, and the heme generated during differentiation promotes mitophagy and mitochondrial disposal. This mechanism provides a molecular framework for understanding this fundamentally important cell biological process.

Exploring the mechanistic link between SF3B1 mutation and ring sideroblast formation in myelodysplastic syndrome.

Acquired sideroblastic anemia, characterized by bone marrow ring sideroblasts (RS), is predominantly associated with myelodysplastic syndrome (MDS). Although somatic mutations in splicing factor 3b subunit 1 (SF3B1), which is involved in the RNA splicing machinery, are frequently found in MDS-RS, the detailed mechanism contributing to RS formation is unknown. To explore the mechanism, we established human umbilical cord blood-derived erythroid progenitor-2 (HUDEP-2) cells stably expressing SF3B1K700E. SF3B1K700E expressing cells showed higher proportion of RS than the control cells along with erythroid differentiation, indicating the direct contribution of mutant SF3B1 expression in erythroblasts to RS formation. In SF3B1K700E expressing cells, ABCB7 and ALAS2, known causative genes for congenital sideroblastic anemia, were downregulated. Additionally, mis-splicing of ABCB7 was observed in SF3B1K700E expressing cells. ABCB7-knockdown HUDEP-2 cells revealed an increased frequency of RS formation along with erythroid differentiation, demonstrating the direct molecular link between ABCB7 defects and RS formation. ALAS2 protein levels were obviously decreased in ABCB7-knockdown cells, indicating decreased ALAS2 translation owing to impaired Fe-S cluster export by ABCB7 defects. Finally, RNA-seq analysis of MDS clinical samples demonstrated decreased expression of ABCB7 by the SF3B1 mutation. Our findings contribute to the elucidation of the complex mechanisms of RS formation in MDS-RS.

[Recent advances in the knowledge of sideroblastic anemia].

Sideroblastic anemias (SAs) are a group of heterogeneous congenital and acquired disorders characterized by anemia and presence of ring sideroblasts in the bone marrow. Congenital SA is a rare condition caused by mutations of genes involved in heme biosynthesis, iron-sulfur cluster biosynthesis, and mitochondrial protein synthesis. SAs can also occur following exposure to certain drugs or alcohol or caused by copper deficiency (secondary SA). SAs have been found to be associated with myelodysplastic syndrome (idiopathic SA), which strongly correlates with specific somatic mutations in SF3B1 (splicing factor 3b subunit 1), involved in the RNA splicing machinery. The recent widespread use of genome-editing technology and next-generation sequencing has led to a better understanding of the molecular pathophysiology of SAs. This review discusses the current understanding of the pathophysiology of SAs.

TM5614, an Inhibitor of Plasminogen Activator Inhibitor-1, Exerts an Antitumor Effect on Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is triggered by t(9;22)(q34;q11.2) translocation, leading to the formation of the BCR-ABL1 fusion gene. Although the development of BCR-ABL1 tyrosine kinase inhibitors (TKIs) has dramatically improved the prognosis of CML, the disease could often relapse, presumably because leukemic stem cell fraction of CML (CML-LSC) may reside in specific niches, and also acquire an ability to resist the cytotoxic agents. Recently a study indicated that pharmacological inhibition of plasminogen activator inhibitor-1 (PAI-1, also known as SERPINE1) would cause detachment of CML-LSCs from their niche by inducing maturation of membrane-type matrix metalloprotease-1 (MT1-MMP), leading to increased susceptibility of CML-LSCs against TKIs. However, the direct antitumor effect of PAI-1 inhibition in CML remains unclear. Because PAI-1 mRNA expression was lower in CML cell line (K562) than bone marrow mononuclear cells derived from CML patients, we established K562 cell clones stably expressing exogenous PAI-1 (K562/PAI-1). We found that TM5614 treatment significantly suppressed cell proliferation and induced apoptosis in K562/PAI-1 cells, accompanied by increased activity of Furin protease, which is a known target of PAI-1. Besides processing mature MT1-MMP, Furin is in charge of cleaving the NOTCH receptor to form a heterodimer before exporting it to the cell surface membrane. In K562/PAI-1 cells, TM5614 treatment increased NOTCH1 intracellular domain (NICD) protein expression as well as NOTCH1 target of HEY1 mRNA levels. Finally, forced expression of either Furin or NICD in K562/PAI-1 cells significantly inhibited cell proliferation and induced apoptosis. Collectively, PAI-1 inhibition may have an antitumor effect by modulating the Furin/NICD pathway.

Unrelated cord blood transplantation for adult-onset EBV-associated T-cell and NK-cell lymphoproliferative disorders.

Adult-onset EBV-associated T-cell and NK-cell lymphoproliferative disorders (EBV-T/NK-LPDs) often progress rapidly, and require allogeneic stem cell transplantation early in the course of treatment. Unrelated cord blood transplantation (UCBT) is a readily available option for patients without HLA-matched donors. We retrospectively analyzed the outcomes of 12 UCBT in adult patients with chronic active EBV infection (CAEBV, n = 8), EBV-positive hemophagocytic lymphohistiocytosis following primary EBV infection (n = 2), hydroa vacciniforme-like lymphoproliferative disorder (n = 1), and systemic EBV-positive T-cell lymphoma of childhood (STCLC, n = 1). The median age at transplantation was 31.5 years (range 19-58). At the median follow-up time for survivors, which was 6.3 years (range 0.3-11.3), 3-year overall survival (OS) rates in all patients and 8 CAEBV patients were 68.2% (95% CI 28.6-88.9) and 83.3% (95% CI 27.3-97.5), respectively. Graft failure occurred in 4 of 8 CAEBV patients, requiring a second UCBT to achieve neutrophil engraftment. The cumulative incidence of grade II-IV acute GVHD was 33.3% (95% CI 9.1-60.4%). The EBV-DNA load became undetectable or very low after UCBT in all cases. UCBT may be a promising treatment option for adult-onset EBV-T/NK-LPDs.

Salvage Cord Blood Transplantation for Sustained Remission of Acute Megakaryoblastic Leukemia That Relapsed Early after Myeloablative Transplantation.

Acute megakaryoblastic leukemia (AMKL) is a rare subtype of acute myeloid leukemia accompanied by an aggressive clinical course and dismal prognosis. We herein report a case of AMKL preceded by mediastinal germ cell tumor that relapsed early after allogeneic hematopoietic stem cell transplantation with myeloablative conditioning but was successfully treated using salvage cord blood transplantation (CBT) with reduced-intensity conditioning. Although several serious complications developed, sustained remission with a favorable general condition was ultimately achieved. Although an optimal therapeutic strategy remains to be established, the graft-versus-leukemia effect of CBT may be promising, even for the treatment of refractory AMKL.

Targeting stanniocalcin-1-expressing tumor cells elicits efficient antitumor effects in a mouse model of human lung cancer.

Lung cancer is the most common cause of cancer-related death in developed countries; therefore, the generation of effective targeted therapeutic regimens is essential. Recently, gene therapy approaches toward malignant cells have emerged as attractive molecular therapeutics. Previous studies have indicated that stanniocalcin-1 (STC-1), a hormone involved in calcium and phosphate homeostasis, positively regulates proliferation, apoptosis resistance, and glucose metabolism in lung cancer cell lines. In this study, we investigated if targeting STC-1 in tumor cells could be a promising strategy for lung cancer gene therapy. We confirmed that STC-1 levels in peripheral blood were higher in lung cancer patients than in healthy donors and that STC-1 expression was observed in five out of eight lung cancer cell lines. A vector expressing a suicide gene, uracil phosphoribosyltransferase (UPRT), under the control of the STC-1 promoter, was constructed (pPSTC-1 -UPRT) and transfected into three STC-1-positive cell lines, PC-9, A549, and H1299. When stably transfected, we observed significant cell growth inhibition using 5-fluorouracil (5-FU) treatment. Furthermore, growth of the STC-1-negative lung cancer cell line, LK-2 was significantly arrested when combined with STC-1-positive cells transfected with pPSTC-1 -UPRT. We believe that conferring cytotoxicity in STC-1-positive lung cancer cells using a suicide gene may be a useful therapeutic strategy for lung cancer.

Acquisition of monosomy 7 and a RUNX1 mutation in Pearson syndrome.

Pearson syndrome (PS) is a very rare and often fatal multisystem disease caused by deletions in mitochondrial DNA that result in sideroblastic anemia, vacuolization of marrow precursors, and pancreatic dysfunction. Spontaneous recovery from anemia is often observed within several years of diagnosis. We present the case of a 4-month-old male diagnosed with PS who experienced prolonged severe pancytopenia preceding the emergence of monosomy 7. Whole-exome sequencing identified two somatic mutations, including RUNX1 p.S100F that was previously reported as associated with myeloid malignancies. The molecular defects associated with PS may have the potential to progress to advanced myelodysplastic syndrome .

[Ring sideroblasts and iron metabolism].

Ring sideroblasts show abnormal mitochondrial iron accumulation, and their emergence in the bone marrow is a characteristic of sideroblastic anemias (SAs). SAs are a group of heterogeneous congenital and acquired disorders. Congenital SA is a rare disease caused by gene mutations involved in heme biosynthesis, iron-sulfur cluster biosynthesis, and mitochondrial protein synthesis. SAs can also occur after exposure to certain drugs or alcohol and due to copper deficiency (secondary SA). Furthermore, SAs are associated with myelodysplastic syndrome (idiopathic SA), strongly correlating with specific somatic mutations in splicing factor 3b subunit 1 (SF3B1), which is involved in the RNA splicing machinery. Recent reports have indicated that common defects in iron/heme metabolism underlie in the mechanisms of ring sideroblast formation in congenital and acquired SAs. Current understanding of SA pathophysiology, including the mechanisms of ring sideroblast formation, is discussed in this review.

Flow Cytometry-Based Photodynamic Diagnosis with 5-Aminolevulinic Acid for the Detection of Minimal Residual Disease in Multiple Myeloma.

Multiple myeloma is the cancer of plasma cells. Along with the development of new and effective therapies, improved outcomes in patients with multiple myeloma have increased the interest in minimal residual disease (MRD) monitoring. However, the considerable heterogeneity of immunophenotypic and molecular markers of myeloma cells has limited its clinical application. 5-Aminolevulinic acid (ALA) is a natural compound in the heme biosynthesis pathway. Following ALA treatment, tumor cells preferentially accumulate porphyrins because of the differential activities of aerobic glycolysis, known as Warburg effect. Among various porphyrins, protoporphyrine IX is a strong photosensitizer; thus, ALA-based photodynamic diagnosis has been widely used in various solid cancers. Here, the feasibility of flow cytometry-based photodynamic detection of MRD was tested in multiple myeloma. Among various human cell lines of hematological malignancies, including K562 erythroleukemia, Jurkat T-cell leukemia, Nalm6 pre-B cell leukemia, KG1a myeloid leukemia, and U937 monocytic leukemia, human myeloma cell line, KMS18, and OPM2 abundantly expressed ALA transporters, such as SLC36A1 and SLC15A2, and 1 mM ALA treatment for 24 h resulted in nearly 100% porphyrin fluorescence expression, which could be competitively inhibited by ALA transport with gamma-aminobutyric acid. Titration studies revealed that the lowest ALA concentration required to achieve nearly 100% porphyrin fluorescence in KMS18 cells was 0.25 mM, with an incubation period of 2 h. Under these conditions, incubation of primary peripheral blood mononuclear cells resulted in only 1.8 % of the cells exhibiting porphyrin fluorescence. Therefore, flow cytometry-based photodynamic diagnosis is a promising approach for detecting MRD in multiple myeloma.

Study of pathophysiology and molecular characterization of congenital anemia in India using targeted next-generation sequencing approach.

Most patients with anemia are diagnosed through clinical phenotype and basic laboratory testing. Nonetheless, in cases of rare congenital anemias, some patients remain undiagnosed despite undergoing an exhaustive workup. Genetic testing is complicated by the large number of genes that are involved in rare anemias, due to similarities in the clinical presentation. We sought to enhance the diagnosis of patients with congenital anemias by using targeted next-generation sequencing. The genetic diagnosis was performed by gene capture followed by next-generation sequencing of 76 genes known to cause anemia syndromes. Genetic diagnosis was achieved in 17 of 21 transfusion-dependent patients and undiagnosed by conventional workup. Four cases were diagnosed with red cell membrane protein defects, four patients were diagnosed with pyruvate kinase deficiency, one case of adenylate kinase deficiency, one case of glucose phosphate isomerase deficiency, one case of hereditary xerocytosis, three cases having combined membrane and enzyme defect, two cases with Diamond-Blackfan anemia (DBA) and 1 with CDA type II with 26 different mutations, of which 21 are novel. Earlier incorporation of this NGS method into the workup of patients with congenital anemia may improve patient care and enable genetic counselling.

[Sideroblastic anemia].

Sideroblastic anemia (SA) signifies a group of heterogeneous congenital and acquired disorders characterized by anemia and the presence of ring sideroblasts in the bone marrow. Congenital SA is a rare disease caused by mutations of genes involved in heme biosynthesis, iron-sulfur cluster biosynthesis, and mitochondrial protein synthesis. In addition, SA can occur after exposure to certain drugs or alcohol and because of copper deficiency (secondary SA). Of note, SA also correlates with myelodysplastic syndrome (idiopathic SA). Recent progress in the genome analysis technology has enabled the identification of novel causative genes for SA, elucidating the molecular pathophysiology of these disorders. Accordingly, the significance of genetic diagnosis for SA has been increasing. This review discusses the current understanding of genetic mutations involved in the pathophysiology of SA.

Generation and Molecular Characterization of Human Ring Sideroblasts: a Key Role of Ferrous Iron in Terminal Erythroid Differentiation and Ring Sideroblast Formation.

Ring sideroblasts are a hallmark of sideroblastic anemia, although little is known about their characteristics. Here, we first generated mutant mice by disrupting the GATA-1 binding motif at the intron 1 enhancer of the ALAS2 gene, a gene responsible for X-linked sideroblastic anemia (XLSA). Although heterozygous female mice showed an anemic phenotype, ring sideroblasts were not observed in their bone marrow. We next established human induced pluripotent stem cell-derived proerythroblast clones harboring the same ALAS2 gene mutation. Through coculture with sodium ferrous citrate, mutant clones differentiated into mature erythroblasts and became ring sideroblasts with upregulation of metal transporters (MFRN1, ZIP8, and DMT1), suggesting a key role for ferrous iron in erythroid differentiation. Interestingly, holo-transferrin (holo-Tf) did not induce erythroid differentiation as well as ring sideroblast formation, and mutant cells underwent apoptosis. Despite massive iron granule content, ring sideroblasts were less apoptotic than holo-Tf-treated undifferentiated cells. Microarray analysis revealed upregulation of antiapoptotic genes in ring sideroblasts, a profile partly shared with erythroblasts from a patient with XLSA. These results suggest that ring sideroblasts exert a reaction to avoid cell death by activating antiapoptotic programs. Our model may become an important tool to clarify the pathophysiology of sideroblastic anemia.

Umbilical Cord Blood Transplantation Using Reduced-Intensity Conditioning without Antithymocyte Globulin in Adult Patients with Severe Aplastic Anemia.

Umbilical cord blood transplantation (UCBT) is a possible option for patients with aplastic anemia (AA) without a related or unrelated HLA-matched donor, particularly if immunosuppressive therapy (IST) has failed or transplantation is urgently needed. However, a higher rate of graft failure after UCBT remains a major problem, and the optimal conditioning regimen for stable engraftment after UCBT has not been established. Here we investigated 6 adult patients with AA who underwent UCBT using a reduced-intensity conditioning (RIC) regimen comprising fludarabine 125 mg/m2, cyclophosphamide 120 mg/kg, and 4 Gy of total body irradiation (Flu/CY/TBI4Gy) without antithymocyte globulin (ATG). Five patients underwent UCBT after IST failure, and 1 patient underwent UCBT as a first-line treatment due to a fulminant clinical finding of a neutrophil count of 0, despite granulocyte colony-stimulating factor administration. Regarding graft-versus-host disease (GVHD) prophylaxis, 2 patients received tacrolimus plus short-term methotrexate and 4 patients received tacrolimus plus mycophenolate mofetil, and all patients achieved sustained engraftment of both neutrophils and platelets, at a median of 17.5 days (range, 14 to 37 days) and 38.5 days (range, 31 to 86 days), respectively, with complete donor chimerism confirmed in all patients at a median of 14 days (range, 14 to 32 days). Three patients developed grade II acute GVHD (aGVHD), but grade III/IV aGVHD was not observed, whereas 4 patients developed chronic GVHD involving only skin. At the time of this report, all 6 patients were alive without the need for blood transfusion, at a median follow-up of 16 months (range, 12 to 131 months). Although further study is needed, our findings suggest that conditioning with Flu/CY/TBI4Gy without ATG might allow stable engraftment in UCBT for adults with AA.

[Molecular pathophysiology of sideroblastic anemia].

Sideroblastic anemias (SAs) are heterogeneous congenital and acquired disorders characterized by anemia and the presence of ring sideroblasts in bone marrow. Congenital sideroblastic anemia (CSA) is a rare disease caused by mutations in genes that are involved in heme biosynthesis, iron-sulfur [Fe-S] cluster biosynthesis, and mitochondrial protein synthesis. The most common form of CSA is X-linked sideroblastic anemia; it occurs because of mutations in the erythroid-specific δ-aminolevulinate synthase gene (ALAS2), which is the first enzyme of the heme biosynthesis pathway in erythroid cells. Additionally, SAs can occur after exposure to certain drugs or alcohol and with copper deficiency (secondary SA) ; they are also detected in association with myelodysplastic syndrome (idiopathic SA). Among all types of SAs, idiopathic SA is the most common form. This review encompasses the current understanding of the molecular pathophysiology of SA.