Sofosbuvir Suppresses the Genome Replication of DENV1 in Human Hepatic Huh7 Cells.

Dengue virus (DENV) causes dengue fever and dengue hemorrhagic fever, and DENV infection kills 20,000 people annually worldwide. Therefore, the development of anti-DENV drugs is urgently needed. Sofosbuvir (SOF) is an effective drug for HCV-related diseases, and its triphosphorylated metabolite inhibits viral RNA synthesis by the RNA-dependent RNA polymerase (RdRp) of HCV. (2'R)-2'-Deoxy-2'-fluoro-2'-methyluridine (FMeU) is the dephosphorylated metabolite produced from SOF. The effects of SOF and FMeU on DENV1 replication were analyzed using two DENV1 replicon-based methods that we previously established. First, a replicon-harboring cell assay showed that DENV1 replicon replication in human hepatic Huh7 cells was decreased by SOF but not by FMeU. Second, a transient replicon assay showed that DENV1 replicon replication in Huh7 cells was decreased by SOF; however, in hamster kidney BHK-21 cells, it was not suppressed by SOF. Additionally, the replicon replication in Huh7 and BHK-21 cells was not affected by FMeU. Moreover, we assessed the effects of SOF on infectious DENV1 production. SOF suppressed infectious DENV1 production in Huh7 cells but not in monkey kidney Vero cells. To examine the substrate recognition of the HCV and DENV1 RdRps, the complex conformation of SOF-containing DENV1 RdRp or HCV RdRp was predicted using AlphaFold 2. These results indicate that SOF may be used as a treatment for DENV1 infection.

[Immune pathophysiology of bone marrow failure].

Bone marrow (BM) failure is a condition characterized by peripheral pancytopenia due to decreased BM function. It includes conditions such as acquired aplastic anemia (AA), myelodysplastic syndrome (MDS), and paroxysmal nocturnal hemoglobinuria (PNH). AA is characterized by pancytopenia and BM hypoplasia, and is primarily caused by an autoimmune mechanism involving cytotoxic T cells that damage hematopoietic stem cells (HSCs). Recent genomic research has revealed that patients with AA often exhibit clonal hematopoiesis by HSCs with genetic alterations, such as PIGA, DNMT3A, ASXL1, BCOR/BCORL1, copy-number neutral LOH of chromosome 6p (6pLOH), and HLA class I allele mutations. The genomic landscape of AA is distinct from MDS and age-related clonal hematopoiesis. Most notably, the presence of PNH-type cells and HLA class I allele-lacking cells indicates the presence of HSCs that have escaped from autoimmunity. We recently identified a common nonsense mutation at codon19 (c.19C>T, p.R7X) in exon1 (Exon1mut) of different HLA-A and HLA-B alleles, and HLA-DR loss of hematopoietic stem progenitor cells in AA patients carrying HLA-DR15. These results provide important clues for understanding the immune pathophysiology of BM failure.

A longitudinal analysis of paroxysmal nocturnal haemoglobinuria-type cells in patients with bone marrow failure: Results of a prospective multi-centre study in Japan.

To determine the prevalence and clinical relevance of glycosylphosphatidylinositol-anchored protein-deficient (GPI[-]) cell populations (paroxysmal nocturnal haemoglobinuria [PNH]-type cells) in patients with acquired aplastic anaemia (AA) or myelodysplastic syndrome (MDS), we prospectively studied peripheral blood samples of 2402 patients (1075 with AA, 900 with MDS, 144 with PNH, and 283 with other anaemia) using a high-sensitivity flow cytometry assay in a nationwide multi-centre observational study. PNH-type cells were detected in 52.6% of AA and 13.7% of MDS patients. None of the 35 patients with refractory anaemia (RA) with ringed sideroblasts or the 86 patients with RA with excess of blasts carried PNH-type cells. Among the 317 patients possessing PNH-type granulocytes, the percentage of PNH-type granulocytes increased by ≥10% in 47 patients (14.8%), remained unchanged in 240 patients (75.7%), and decreased by ≥10% in 30 patients (9.5%) during 3 years of follow-up. PNH-type granulocyte expansion occurred more frequently (27.1%) in the 144 patients who originally carried PNH-type granulocytes ≥1% than in the 173 patients with PNH-type granulocytes <1% (4.6%). This study confirmed that PNH-type cells are undetectable in authentic clonal MDS patients, and the presence of ≥1% PNH-type granulocytes predicts a higher likelihood of PNH-type cell expansion than with <1% PNH-type granulocytes.

Hemophagocytic Lymphohistiocytosis With Elevated Cytokines Related to Macrophage Activation After Liver Transplantation for Autoimmune Hepatitis: A Case Report.

Hemophagocytic lymphohistiocytosis (HLH) is a rare but lethal complication of liver transplantation (LT). HLH is characterized by pathologic macrophage activation with hypercytokinemia, excessive inflammation, and tissue destruction, resulting in progressive organ dysfunction. HLH is also known as macrophage activation syndrome (MAS) when complicated by rheumatic or autoinflammatory diseases. Measuring several serum cytokines could be helpful in diagnosing HLH and MAS. Cytokines related to macrophage activation: neopterin, interleukin-18 (IL-18), and soluble tumor necrosis factor receptors (sTNF-R) I and II have not been assessed in patients with HLH complicated by LT. In this case, these cytokines were evaluated in the perioperative period of LT. The patient was a 24-year-old woman who underwent living-donor LT for acute worsening of autoimmune hepatitis. On postoperative day 12, the patient was diagnosed with HLH on the basis of the criteria. Plasma exchange, steroid pulse therapy, intravenous immunoglobulin and granulocyte-colony stimulating factor effectively inhibited progression to lethal HLH. When HLH occurred after LT, cytokine analysis showed that neopterin, IL-18, sTNFR-I, and II were elevated: cytokine storm. Of note, cytokine analysis on hospital admission also revealed elevated cytokine levels. Particularly, IL-18 levels were markedly elevated, suggesting that activation of the innate immune system was involved. These results revealed that a cytokine storm and macrophage activation developed before LT. Based on these findings, cytokine analysis related to macrophage activation may be useful for diagnosing and predicting HLH and MAS in patients with LT.

Clinical significance of the increased expression of the WT1 gene in peripheral blood of patients with acquired aplastic anemia.

To determine the significance of increased Wilms tumor 1 (WT1) gene expression in the peripheral blood of patients with acquired aplastic anemia (AA), we analyzed serial changes in WT1 mRNA copy number (WT1cn) in 63 patients with AA as well as in five patients with myelodysplastic syndromes (MDS) and seven patients with paroxysmal nocturnal hemoglobinuria (PNH). WT1cn was higher than the cut-off (≥50 copies/µg RNA) at the time of the first measurement in 41% of untreated (60-190 copies/µg RNA [median 130]) and 59% of treated (59-520 copies/µg RNA [median 150]) AA patients. Although WT1cns gradually increased in most AA patients during the 2-105 months follow-up period, they did not lead to clonal evolution except in three patients in whom the maximum change ratio of WT1cn (WT1cn-change max), defined as the ratio of WT1cn at the first examination to that of the maximum value, exceeded 20.0 and who developed MDS at 2, 46, and 105 months. Increased WT1 gene expression was enriched in granulocytes rather than in mononuclear cells in most WT1-positive AA patients and did not correlate with mutations of genes associated with myeloid malignancy. WT1cns were high at 690-5700 (median 2000) in MDS patients and remained high thereafter, while WT1cns in PNH patients (77-200; median 96) were similar to those in AA. Thus, moderate increases in WT1cns up to 600 are common in AA patients in stable remission. An increase in the WT1cn-change max over 20.0 may portend transformation from AA to MDS.

Hematopoietic stem progenitor cells with malignancy-related gene mutations in patients with acquired aplastic anemia are characterized by the increased expression of CXCR4.

The phenotypic changes in hematopoietic stem progenitor cells (HSPCs) with somatic mutations of malignancy-related genes in patients with acquired aplastic anemia (AA) are poorly understood. As our initial study showed increased CXCR4 expression on HLA allele-lacking (HLA[-]) HSPCs that solely support hematopoiesis in comparison to redundant HLA(+) HSPCs in AA patients, we screened the HSPCs of patients with various types of bone marrow (BM) failure to investigate their CXCR4 expression. In comparison to healthy individuals (n = 15, 12.3%-49.9%, median 43.2%), the median CXCR4+ cell percentages in the HSPCs of patients without somatic mutations were low: 29.3% (14.3%-37.3%) in the eight patients without HLA(-) granulocytes, 8.8% (4.1%-9.8%) in the five patients with HLA(-) cells accounting for >90% of granulocytes, and 7.8 (2.1%-8.7%) in the six patients with paroxysmal nocturnal hemoglobinuria. In contrast, the median percentage was much higher (78% [61.4%-88.7%]) in the five AA patients without HLA(-) granulocytes possessing somatic mutations (c-kit, t[8;21], monosomy 7 [one for each], ASXL1 [n = 2]), findings that were comparable to those (66.5%, 63.1%-88.9%) in the four patients with advanced myelodysplastic syndromes. The increased expression of CXCR4 may therefore reflect intrinsic abnormalities of HSPCs caused by somatic mutations that allow them to evade restriction by BM stromal cells.

[Clinical significance of detecting human leukocyte antigen class I allele-lacking leukocytes in aplastic anemia].

Human leukocyte antigen (HLA) class I allele-lacking [HLA (-)] leukocytes provide compelling evidence that cytotoxic T-lymphocytes are involved in the development of aplastic anemia (AA). However, the clinical significance and precise mechanisms underlying clonal hematopoiesis by HLA (-) hematopoietic stem progenitor cells remain unknown. In HLA (-) cells from patients with AA, we discovered a common nonsense mutation at codon19 (c.19C>T, p.R7X) in exon1 (Exon1mut) of different HLA-A and HLA-B alleles. Exon1 mutation detection using droplet digital polymerase chain reaction (ddPCR) is a powerful tool for diagnosing immune pathophysiology in patients with bone marrow failure. We also looked at the prognosis of 633 patients with AA, including 127 with HLA (-) leukocytes who had been followed up for a long time. In Japanese patients with AA, HLA (-) leukocytes and concomitant aberrant clones were not associated with clonal evolution to MDS/AML. In patients with AA and both marker (-) leukocytes, HLA (-) leukocytes may indicate a lower risk of developing secondary paroxysmal nocturnal hemoglobinuria (PNH). Detecting HLA (-) leukocytes is critical for managing patients with AA and assisting physicians in selecting appropriate therapy.

Somatic mutations and clonal expansions in paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic stem cell disorder caused by a mutation of the X-linked PIGA gene, resulting in a deficient expression of glycosylphosphatidylinositol (GPI)-anchored proteins. While large clonal expansions of GPI(-) cells cause hemolytic symptoms, tiny GPI(-) cell populations can be found in healthy individuals and remain miniscule throughout life. The slight expansion of PNH clones often occurs in patients with acquired aplastic anemia (AA), an autoimmune bone marrow (BM) failure caused by autoreactive cytotoxic T lymphocyte attack on hematopoietic stem and progenitor cells (HSPCs). The presence of PNH clones is thought to represent the immune pathophysiology of BM failure and be derived from GPI(-) HSPCs that evaded immune attack against HSPCs. However, which mechanisms underlie the selection of GPI(-) HSPCs as well as their overwhelming clonal expansion remains unclear. Ancestral or secondary somatic mutations in GPI(-) HSPCs contribute to the clonal expansion of the aberrant HSPCs in certain patients with PNH; however, it remains unclear whether such driver mutations are responsible for clonal expansion of all patients. Increased sensitivity to TGF-ß in GPI(-) HSPCs partly explains the predominance of GPI(-) erythrocytes in immune-mediated BM failure. CD4+ T cells specific to antigens presented by HLA-DR15 on HSPCs also contribute to the immune escape of GPI(-) HSPCs. Studying the evolution of HSPCs in AA and PNH will yield further information for understanding human autoimmunity and stem cell biology.

Anti-COX-2 autoantibody is a novel biomarker of immune aplastic anemia.

In immune aplastic anemia (IAA), severe pancytopenia results from the immune-mediated destruction of hematopoietic stem cells. Several autoantibodies have been reported, but no clinically applicable autoantibody tests are available for IAA. We screened autoantibodies using a microarray containing >9000 proteins and validated the findings in a large international cohort of IAA patients (n = 405) and controls (n = 815). We identified a novel autoantibody that binds to the C-terminal end of cyclooxygenase 2 (COX-2, aCOX-2 Ab). In total, 37% of all adult IAA patients tested positive for aCOX-2 Ab, while only 1.7% of the controls were aCOX-2 Ab positive. Sporadic non-IAA aCOX-2 Ab positive cases were observed among patients with related bone marrow failure diseases, multiple sclerosis, and type I diabetes, whereas no aCOX-2 Ab seropositivity was detected in the healthy controls, in patients with non-autoinflammatory diseases or rheumatoid arthritis. In IAA, anti-COX-2 Ab positivity correlated with age and the HLA-DRB1*15:01 genotype. 83% of the >40 years old IAA patients with HLA-DRB1*15:01 were anti-COX-2 Ab positive, indicating an excellent sensitivity in this group. aCOX-2 Ab positive IAA patients also presented lower platelet counts. Our results suggest that aCOX-2 Ab defines a distinct subgroup of IAA and may serve as a valuable disease biomarker.

Incidence of acquired pure red cell aplasia: a nationwide epidemiologic analysis with 2 registry databases in Japan.

Acquired pure red cell aplasia (PRCA) is a rare syndrome characterized by anemia with reticulocytopenia and a marked reduction in erythroid precursors. Given its rarity, the true incidence is largely unknown, and epidemiological data representing the general population, with a description of the full spectrum of etiologies, are scarce. An epidemiological study on PRCA in Japan conducted 30 years ago estimated the annual incidence as 0.3 per million. To update the data and investigate the incidence and demographics of PRCA, we conducted a nationwide epidemiological study using the Japanese Society of Hematology (JSH) Hematologic Disease Registry, a hematologic disease registration database managed by the JSH and the Diagnosis Procedure Combination (DPC) study data available at a website of the Ministry of Health, Labor, and Welfare (MHLW) of Japan. A total of 1055 patients with newly diagnosed acquired PRCA were identified between 2012 and 2019, and the average annual incidence was calculated at 1.06 (95% confidence interval [CI], 0.83-1.28) per million. The median age was 73 (range, 18-99) years. The female-to-male ratio was 1.5:1, and the female predominance was most prominent in the child-bearing age group. Sixty-nine percent of acquired PRCA was idiopathic. The incidence of PRCA was approximately 20% of that of aplastic anemia (AA) during the same period. Approximately 0.98 patients per million per year (95% CI, 0.89-1.07) required hospitalization for the treatment of PRCA. These results are expected to contribute to the discussion of resource allocation for PRCA in the aging population in many countries, including Japan.

Hemolytic paroxysmal nocturnal hemoglobinuria: 20 years of medical progress.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by intravascular hemolysis, thrombosis and bone marrow failure. Prior to the availability of specific therapy, PNH led to the death of around half of affected individuals, mainly through thrombotic complications, with a particular grim prognosis for patients presenting with classic PNH. The anti-C5 monoclonal antibody eculizumab has revolutionized treatment, controlling intravascular hemolysis and thrombosis occurrence, with improved long-term survival. However, eculizumab is infused on a lifelong 2 week basis and most of the patients are anemic, with some remaining transfusion-dependent. New anti-C5 agents reproduce the safety and efficacy of eculizumab, with improved patient convenience, while proximal complement inhibitors have been developed to address C3-mediated extravascular hemolysis, aiming to eventually improve hematological response. This review will describe the spectacular medical progress in PNH of the last 20 years, as well as the risks and benefits of a novel approach.

Frequent HLA-DR loss on hematopoietic stem progenitor cells in patients with cyclosporine-dependent aplastic anemia carrying HLA-DR15.

To determine whether antigen presentation by HLA-DR on hematopoietic stem progenitor cells (HSPCs) is involved in the development of acquired aplastic anemia (AA), we studied the HLA-DR expression on CD45dimCD34+CD38+ cells in the peripheral blood of 61 AA patients including 23 patients possessing HLA-class I allele-lacking (HLA-class I[-]) leukocytes. HLA-DR-lacking (DR[-]) cells accounted for 13.0-57.1% of the total HSPCs in seven (11.5%) patients with HLA-DR15 who did not possess HLA-class I(-) leukocytes. The incubation of sorted DR(-) HSPCs in the presence of IFN-γ for 72 h resulted in the full restoration of the DR expression. A comparison of the transcriptome profile between DR(-) and DR(+) HSPCs revealed the lower expression of immune response-related genes including co-stimulatory molecules (e.g., CD48, CD74, and CD86) in DR(-) cells, which was not evident in HLA-class I(-) HSPCs. DR(-) cells were exclusively detected in GPI(+) HSPCs in four patients whose HSPCs could be analyzed separately for GPI(+) and GPI(-) HSPCs. These findings suggest that CD4+ T cells specific to antigens presented by HLA-DR15 on HSPCs may contribute to the development of AA as well as the immune escape of GPI(-) HSPCs in a distinct way from CD8+ T cells recognizing HLA-class I-restricted antigens.

The GPI-anchored protein CD109 protects hematopoietic progenitor cells from undergoing erythroid differentiation induced by TGF-ß.

Although a glycosylphosphatidylinositol-anchored protein (GPI-AP) CD109 serves as a TGF-ß co-receptor and inhibits TGF-ß signaling in keratinocytes, the role of CD109 on hematopoietic stem progenitor cells (HSPCs) remains unknown. We studied the effect of CD109 knockout (KO) or knockdown (KD) on TF-1, a myeloid leukemia cell line that expresses CD109, and primary human HSPCs. CD109-KO or KD TF-1 cells underwent erythroid differentiation in the presence of TGF-ß. CD109 was more abundantly expressed in hematopoietic stem cells (HSCs) than in multipotent progenitors and HSPCs of human bone marrow (BM) and cord blood but was not detected in mouse HSCs. Erythroid differentiation was induced by TGF-ß to a greater extent in CD109-KD cord blood or iPS cell-derived megakaryocyte-erythrocyte progenitor cells (MEPs) than in wild-type MEPs. When we analyzed the phenotype of peripheral blood MEPs of patients with paroxysmal nocturnal hemoglobinuria who had both GPI(+) and GPI(-) CD34+ cells, the CD36 expression was more evident in CD109- MEPs than CD109+ MEPs. In summary, CD109 suppresses TGF-ß signaling in HSPCs, and the lack of CD109 may increase the sensitivity of PIGA-mutated HSPCs to TGF-ß, thus leading to the preferential commitment of erythroid progenitor cells to mature red blood cells in immune-mediated BM failure.

Pax5 mediates the transcriptional activation of the CD81 gene.

CD81 is an integral membrane protein of the tetraspanin family and forms complexes with a variety of other cell surface membrane proteins. CD81 is involved in cell migration and B cell activation. However, the mechanism of the transcriptional regulation of the CD81 gene remains unclear. Here, we revealed that CD81 transcriptional activation was required for binding of the transcription factor Pax5 at the Pax5-binding sequence (-54)GCGGGAC(-48) located upstream of the transcriptional start site (TSS) of the CD81 gene. The reporter assay showed that the DNA sequence between - 130 and - 39 bp upstream of the TSS of the CD81 gene had promoter activity for CD81 transcription. The DNA sequence between - 130 and - 39 bp upstream of TSS of CD81 harbors two potential Pax5-binding sequences (-87)GCGTGAG(-81) and (-54)GCGGGAC(-48). Reporter, electrophoresis mobility shift, and chromatin immunoprecipitation (ChIP) assays disclosed that Pax5 bound to the (-54)GCGGGAC(-48) in the promoter region of the CD81 gene in order to activate CD81 transcription. Pax5 overexpression increased the expression level of CD81 protein, while the Pax5-knockdown by shRNA decreased CD81 expression. Moreover, we found that the expression level of CD81 was positively correlated with Pax5 expression in human tumor cell lines. Because CD81 was reported to be involved in cell migration, we evaluated the effects of Pax5 overexpression by wound healing and transwell assays. The data showed that overexpression of either Pax5 or CD81 promoted the epithelial cell migration. Thus, our findings provide insights into the transcriptional mechanism of the CD81 gene through transcription factor Pax5.

Human hepatitis B virus-derived virus-like particle as a drug and DNA delivery carrier.

The controlled release of medications using nanoparticle-based drug delivery carriers is a promising method to increase the efficacy of pharmacotherapy and gene therapy. One critical issue that needs to be overcome with these drug delivery carriers is their target specificity. We focused on the cell tropism of a virus to solve this issue, i.e., we attempted to apply hepatitis B virus-like particle (HBV-VLP) as a novel hepatic cell-selective carrier for medication and DNA. To prepare HBV-VLP, 293T cells were transfected with expression plasmids carrying HBV envelope surface proteins, large envelope protein (L), and small envelope protein (S). After 72 h post-transfection, VLP-containing culture supernatants were harvested, and HBV-VLP was labeled with red fluorescent dye (DiI) and was purified by sucrose gradient ultracentrifugation. An anticancer drugs (geldanamycin or doxorubicin) and GFP-expressing plasmid DNA were incorporated into HBV-VLP, and medication- and plasmid DNA-loaded VLPs were prepared. We evaluated their delivery capabilities into hepatocytes, other organ-derived cells, and hepatocytes expressing sodium taurocholate cotransporting polypeptide (NTCP), which functions as the cellular receptor for HBV by binding to HBV L protein. HBV-VLP selectively delivered both anticancer drugs and plasmid DNA not into HepG2, Huh7, and other organ cells but into HepG2 cells expressing NTCP. In summary, we developed a novel delivery nanocarrier using HBV-VLP that could be used as a hepatitis selective drug- and DNA-carrier for cancer treatment and gene therapy.

Congestive heart failure associated with POEMS syndrome that was adequately distinguished from cardiac amyloidosis: a case report and literature review.

Congestive heart failure (CHF) is a common complication in patients with AL amyloidosis but is rare in another plasma cell dyscrasia, POEMS syndrome. A 52-year-old man developed POEMS syndrome with a solitary plasmacytoma complicated by CHF mimicking cardiac amyloidosis (CA). His neurological symptoms and CHF did not improve after radiotherapy (50 Gy) targeting the plasmacytoma. Based on typical findings of noninvasive examinations such as elevated serum NT-proBNP (12,631 pg/mL), a pseudo-infarct pattern on electrocardiography, interventricular septal thickening with a granular sparkling appearance and an apical sparing pattern of longitudinal strain on echocardiography, and late gadolinium enhancement of the left ventricular wall on cardiac magnetic resonance imaging (MRI), severe CA ineligible for autologous peripheral blood stem cell transplantation (auto-PBSCT) was strongly suspected. However, myocardial biopsy failed to reveal amyloid deposits, and CHF markedly improved after only one cycle of chemotherapy with melphalan and dexamethasone. Accordingly, CA was denied as the etiology of his heart failure, and the patient was finally diagnosed with POEMS syndrome. As a result, high-dose melphalan followed by auto-PBSCT improved his neurological symptoms. Careful evaluation is therefore needed to appropriately treat patients with POEMS syndrome complicated by CHF, even when the results of non-invasive examinations are typical for AL amyloidosis.

The effectiveness of immunosuppressive therapy in patients with aplastic anaemia secondary to chemoradiotherapy for cancers.

The outcome of immunosuppressive therapy (IST) and prognosis in patients with aplastic anaemia (AA) secondary to chemotherapy or radiotherapy for cancers remains unknown. A total of 43 of 2559 patients with AA referred to our hospital had previously received chemoradiotherapy for various types of solid tumours (n = 25) or haematological malignancies (n = 18). Their cancer status was complete remission (CR) in 27, non-CR in 13, and unknown in three. Small populations of glycosylphosphatidylinositol-anchored protein-deficient [GPI(-)] granulocytes were detected in 16 patients (37·2%). Of 18 patients who were treated with IST, 50% improved regardless of the presence of GPI(-) cells. The overall survival (OS) rate was significantly higher in patients with a history of solid tumours patients than in those of haematological malignancies (median OS, 87 vs. 11 months, P = 0·0003), and in patients treated with IST than in those of untreated patients (median OS, 115 vs. 20 months, P = 0·028). Cancer aggravation occurred in two of four patients who were treated with IST while in non-CR of their original cancers. Progression to myelodysplastic syndromes was observed in two patients not possessing GPI(-) cells. IST should thus be considered for patients with AA secondary to chemoradiotherapy for cancers, particularly when their original solid tumours are in CR.