Avatrombopag as alternative therapy for severe aplastic anemia patients who are intolerant or unresponsive to eltrombopag.

Introduction: Eltrombopag (EPAG), a thrombopoietin receptor agonist, was approved for the treatment of severe aplastic anemia (SAA) combined with immunosuppressive therapy (IST). However, EPAG contains a typical biphenyl structure, which causes liver function damage. Methods: Twenty patients with SAA who were intolerant or refractory to EPAG were enrolled in a multicenter prospective registry of the Chinese Eastern Collaboration Group of Anemia (ChiCTR2100045895) from October 2020 to June 2023. Results: Eight patients who were ineffective to EPAG, six with kidney impairment, and nine with abnormal liver function (two with concomitant liver and kidney impairment) were converted to avatrombopag (AVA) therapy with the median duration of AVA treatment was 6 (3-24) months. 17 cases (85%) achieved trilineage hematological response (HR): complete remission (CR) in 3 cases (15%), good partial remission (GPR) in 4 cases (20%), partial remission (PR) in 10 cases (50%), and no response (NR) in 3 cases (15%). The median time to response was 1.7 (0.5-6.9) months, with 16 cases (94%) achieving response within six months and 17 cases (100%) within 12 months. 9 cases (50%) achieved transfusion independence. AVA converted treatment was associated with higher neutrophil counts (0.8×109/L vs 2.2×109/L, p=0.0003), platelet counts (11×109/L vs 39×109/L, p=0.0008), hemoglobin count (59g/L vs 98g/L, p=0.0002), red cell count (1.06×1012/L vs 2.97×1012/L, p=0.001), and absolute reticulocyte count (31.99 ×109/L vs 67.05×109/L p=0.0004) were all significantly elevated compared with the pre-treatment level. After the conversion to AVA therapy, liver and kidney function indexes were maintained within the normal range, no AVA related grade 2 or higher adverse events occurred, and no thrombotic events occurred. Conclusion: The conversion to AVA was an optimal choice for patients with SAA who were EPAG intolerant or refractory. Clinical trial registration: http://www.chictr.org.cn/showproj.html?proj=125480, identifier ChiCTR2100045895.

Refractory pure red cell aplasia associated with T-cell large granular lymphocyte leukemia treated by ruxolitinib.

Acquired pure red cell aplasia (PRCA) is a rare syndrome characterized by normocytic normochromic anemia with severe reticulocytopenia and absence of erythroid precursors in the bone marrow. For refractory PRCA patients, the low response rate and high toxicity of alternative therapies pose a great challenge. T-cell large granular lymphocyte (T-LGL) leukemia is one of the most common conditions in secondary PRCA and also the most difficult form to manage with an inferior treatment response to other secondary PRCA forms. T-LGL leukemia exhibits sustained activation of the intracellular JAK-STAT signaling pathway. We herein report a case of PRCA associated with T-LGL leukemia that had been refractory to multiple lines of therapies and was successfully treated by ruxolitinib. The patient achieved complete remission and tolerated ruxolitinib well without occurrence of neutropenia or thrombocytopenia. This preliminary finding favors ruxolitinib as a potential salvage therapy for refractory PRCA associated with T-LGL leukemia.

Severe hemolytic exacerbations of Chinese PNH patients infected SARS-CoV-2 Omicron.

INTRODUCTION: Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by hemolytic anemia, bone marrow failure, thrombophilia. COVID-19, caused by a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with many variants including Omicron. METHODS: This study collected demographic and clinical data of 20 PNH patients with SARS-CoV-2 Omicron infection. RESULTS: They all were with high disease activity, and LDH level exceeded any documented since the diagnosis of PNH, and those reported in the literature for previously stable treatment with complement inhibitors. D-dimer level elevated in 10 patients. 2 patients developed mild pulmonary artery hypertension. Glomerular filtration rate declined in 5 patients. 1 patient developed acute renal failure and underwent hemodialysis. Anemia and hemolysis were improved in 5 patients treated with eculizumab. CONCLUSIONS: Hemolytic exacerbation of PNH with COVID-19 is severe and eculizumab may be an effective treatment.

Interlukin-4 weakens resistance to stress injury and megakaryocytic differentiation of hematopoietic stem cells by inhibiting Psmd13 expression.

Thrombocytopenia is a major and fatal complication in patients with acute myeloid leukemia (AML), which results from disrupted megakaryopoiesis by leukemic niche and blasts. Our previous research revealed that elevated interleukin-4 (IL-4) in AML bone marrow had adverse impact on multiple stages throughout megakaryopoiesis including hematopoietic stem cells (HSCs), but the specific mechanism remains unknown. In the present study, we performed single-cell transcriptome analysis and discovered activated oxidative stress pathway and apoptosis pathway in IL-4Rαhigh versus IL-4Rαlow HSCs. IL-4 stimulation in vitro led to apoptosis of HSCs and down-regulation of megakaryocyte-associated transcription factors. Functional assays displayed higher susceptibility of IL-4Rαhigh HSCs to tunicamycin and irradiation-induced apoptosis, demonstrating their vulnerability to endoplasmic reticulum (ER) stress injury. To clarify the downstream signaling of IL-4, we analyzed the transcriptomes of HSCs from AML bone marrow and found a remarkable down-regulation of the proteasome component Psmd13, whose expression was required for megakaryocytic-erythroid development but could be inhibited by IL-4 in vitro. We knocked down Psmd13 by shRNA in HSCs, and found their repopulating capacity and megakaryocytic differentiation were severely compromised, with increased apoptosis in vivo. In summary, our study uncovered a previous unrecognized regulatory role of IL-4-Psmd13 signaling in anti-stress and megakaryocytic differentiation capability of HSCs.

Transfusion-dependent non-severe aplastic anemia: characteristics and outcomes in the clinic.

Transfusion-dependent non-severe aplastic anemia (TD-NSAA) is a rare condition of bone marrow failure that can persist for a long time or develop into severe aplastic anemia (SAA). Little is known about the clinical and laboratory characteristics, and disease prognosis and outcomes in TD-NSAA patients. The clinical and laboratory data of 124 consecutive TD-NSAA patients in the Chinese Eastern Collaboration Group of Anemia from December 2013 and January 2017 were analyzed retrospectively. In 124 TD-NSAA patients, the median age was 32 years (range: 3-80) and the median disease course was 38 months (range: 3-363). Common complications were iron overload (53/101, 52.5%), liver and kidney dysfunction (42/124, 33.9%), diabetes mellitus/impaired glucose tolerance (24/124, 19.4%), and severe infection (29 cases, 23.4%). 58% of patients (57/124) developed severe aplastic anemia with a median progression time of 24 months (range: 3-216). Patients with absolute neutrophil count (ANC) <0.5×109/L, severe infection, or iron overload had a higher probability of progression to SAA (P=0.022, P=0.025, P=0.001). Patients receiving antithymocyte globulin (ATG) plus Cyclosporin A (CsA) had a higher overall response rate compared to those receiving CsA alone (56.7% vs 19.3%, P < 0.001). The addition of ATG was the favorable factor for efficacy (P=0.003). Fourteen patients developed secondary clonal hematologic disease: eleven patients with paroxysmal nocturnal hemoglobinuria, two patients with myelodysplastic syndromes, and one patient with acute myeloid leukemia, respectively. Ten patients (8.1%) died with a median follow-up of 12 months (range: 3- 36 months). Patients with TD-NSAA usually have a prolonged course of disease, and are prone to be complicated with important organ damage and disease progression to SAA. Intensive immunosuppressive therapy based on ATG might be an appropriate approach for TD-NSAA. Clinical trial registration: http://www.chictr.org.cn/edit.aspx?pid=125480&htm=4, identifier ChiCTR2100045895.

Prolonged use of eltrombopag in patients with severe aplastic anemia in the real world.

Eltrombopag (EPAG) can improve the efficacy of immunosuppressive therapy (IST) consisting of antithymocyte immunoglobulin (ATG) and cyclosporin in severe aplastic anemia (SAA) patients. This study explored whether patients with SAA could benefit from continuous usage of EPAG beyond 6 months.Seventy-four treatment-naive Chinese patients with SAA were administrated with rabbit ATG-based IST plus EPAG for 6 months. Patients not achieving complete remission (CR) at 6 months were treated with EPAG for another 6 months.At 1, 3, 6 and 12 months after IST, the cumulative response rates were 31%, 61%, 82% and 90%, and the cumulative CR rates were 0, 14%, 27% and 45%, respectively. The cumulative effect curve showed that 93% and 53% of all remission and CR occurred within 6 months, while 98% and 83% of all remission and CR occurred within 12 months. Thirty-seven percent of patients (11 of 30) with partial remission (PR) at 6 months continuously exposed to EPAG improved to CR within 3 (1-5) months of the extended median time. Six patients failing at 6 months continued to use EPAG. Three patients showed improved responses with an extended median time of 6 (1-6) months. The 2-year event-free survival (EFS) was better in those continuing with EPAG (89% vs. 49%, P = 0.006) for patients with PR or non-remission at 6 months.Continuous administration with EPAG could improve the hematologic response and EFS in patients without achieving CR at 6 months.This trial has been registered at the Chinese Clinical Trial Registry (ChiCTR2100045895).

Processes of nitrogen removal from rainwater runoff in bioretention filters modified with ceramsite and activated carbon.

Conventional bioretention filters lack satisfactory performance in nitrogen removal. In this study, we used a mixture of cultivated soil and river sand as the bioretention filter to remove nitrogen pollutants from simulated rainwater runoff. To improve its permeability and nitrogen removal performance, both activated carbon and ceramsite were used as additives. The nitrogen removal processes and its mass accumulation in the modified bioretention filters were studied. The contribution of adsorption and biotransformation processes, together with the effects of percolate rate on nitrogen removal performance was explored. The results showed that an activated carbon layer in the bioretention filters could obviously improve nitrogen removal efficiencies, but its location made no significant difference in nitrogen removal performance. Bioretention filters modified with 20% of ceramsite could achieve the optimal percolate rate and nitrogen removal efficiencies. At given conditions, the average removal efficiencies of ammonium nitrogen (NH3-N), nitrate-nitrogen (NO3-N), and total nitrogen (TN) by the modified bioretention filter reached 80.27%, 41.48%, and 59.45%, respectively. During the leaching processes, organic nitrogen originated in the filter materials can be mineralised into NH3-N, then be denitrified and completely removed in the anaerobic environment under flooding conditions. Biotransformation in the modified bioretention filters caused a reduction of NH3-N removal efficiency by 15.41% and an increase of NO3-N removal efficiency by 31.03%. The modified bioretention filter can withstand a long-term operation. Compared with NO3-N and TN, the pollutant of NH3-N in rainwater runoff is not easy to form a mass accumulation in the modified bioretention filter.Highlights The modified bioretention filter showed high percolation rate and nitrogen removal.Hydraulic residence time is a critical design parameter to achieve nitrogen removal.NH3-N is not easy to form a mass accumulation in the filler media as NO3-N.Biodegradation increased NO3-N removal efficiency by 31.03% at given conditions.

Recent Advancements in Poor Graft Function Following Hematopoietic Stem Cell Transplantation.

Poor graft function (PGF) is a life-threatening complication that occurs after transplantation and has a poor prognosis. With the rapid development of haploidentical hematopoietic stem cell transplantation, the pathogenesis of PGF has become an important issue. Studies of the pathogenesis of PGF have resulted in some success in CD34+-selected stem cell boosting. Mesenchymal stem cells, N-acetyl-l-cysteine, and eltrombopag have also been investigated as therapeutic strategies for PGF. However, predicting and preventing PGF remains challenging. Here, we propose that the seed, soil, and insect theories of aplastic anemia also apply to PGF; CD34+ cells are compared to seeds; the bone marrow microenvironment to soil; and virus infection, iron overload, and donor-specific anti-human leukocyte antigen antibodies to insects. From this perspective, we summarize the available information on the common risk factors of PGF, focusing on its potential mechanism. In addition, the safety and efficacy of new strategies for treating PGF are discussed to provide a foundation for preventing and treating this complex clinical problem.

Modeling the sources and retention of phosphorus nutrient in a coastal river system in China using SWAT.

The Soil Water Assessment Tool (SWAT) was used for exploring the sources and retention dynamics of phosphorus nutrient in the river system of the Yong River Basin, China. The performance of the SWAT model was assessed. The retention dynamics of phosphorus nutrient in the river continuum and the factors contributing to those patterns were studied. The results showed that an average of 1828 tons of TP entered the river network of the Yong River Basin annually and in-stream processes trapped 1161 tons yr-1 of TP in the watercourse, which accounted for 63.5% of the annual TP inputs. The TP retention rates in the river network ranged from 3.08 to 63.43 mg m-2 day-1. An average of 666.9 tons of TP was delivered from the estuary to the East China Sea annually. The unit area riverine exports of TP ranged from 102.21 to 244.00 kg km-2 yr-1. The river network is a net sink for TP and is going through a phosphorus accumulation phase. The results confirm that the river system has a considerable phosphorus retention capacity that is highly variable on a spatiotemporal scale. Because of the cumulative effect of continued phosphorus removal along the entire flow path, the retention fractions of phosphorus removed from all streams at the basin scale is considerably higher than that of an individual river portion. The variations of hydrological regimes, water surface area, unit area inputs of phosphorus, and the concentrations of suspended sediments have a great influence on phosphorus retention.

[Analysis of Rainwater Runoff Pollution Characteristics of Various Typical Underlying Surfaces in Ningbo].

The pollution of rainwater runoff in urban areas can cause nutrient enrichment and eutrophication in receiving waters. To explore the pollution characteristics of rainwater runoff in Ningbo, eight sampling campaigns were carried out during rainfall events from 2009 to 2019. Samples of rainwater runoff were collected from underlying surfaces of roofs, squares, grassland, main roads, and the roads in commercial streets and residential districts. The concentrations of runoff pollutants, their sources and correlations, and first flush effects were studied using frequency statistical analysis and correlation analysis. The average event mean concentrations of the chemical oxygen demand (COD), ammonia nitrogen, total nitrogen, total phosphorus, and total suspended solids (TSS) in the rainwater runoff in Ningbo were in the ranges of 23.88-102.31, 0.40-1.69, 3.41-8.71, 0.09-0.50, and 37.6-323.4 mg·L-1, respectively. Apart from the square surfaces, the COD and total nitrogen pollution of the underlying surfaces was severe. The ammonia nitrogen concentrations from the roof, commercial street, and residential district surfaces were significantly higher (P<0.05) than those from the square, grassland, and main road surfaces. The concentrations of total phosphorus from the commercial street, main road, and grassland surfaces were significantly higher (P<0.05) than those of the other underlying surfaces. The correlations of TSS with COD, ammonia nitrogen, and total phosphorus showed that the pollutants and TSS have the same original sources in roof, square, main road, and commercial street runoff, while ammonia nitrogen and total nitrogen have the same original sources in runoff from grassland and residential areas. Under the meteorological conditions of light and moderate rain, the first flush effects of ammonia nitrogen in the runoff of roofs and grassland were observed clearly, whereas this was not the same for the pollutants of total nitrogen and total phosphorus in the residential area, main road, and square runoff.

A Single-Cell Immunofluorescence Method for the Division Patterns Research of Mouse Bone Marrow-Derived Hematopoietic Stem Cells.

How hematopoietic stem cells (HSCs) maintain the balance of self-renewal and differentiation could be partially ascribed to asymmetric and symmetric division patterns. However, a simple and effective method to detect stem cell division patterns is lacking. In this study, we introduce a strategy to describe stem cells division patterns with high spatial resolution at the single-cell level. We show that the fate determinant, Numb, exhibits low expression levels in HSCs that increase upon the initiation of differentiation. Using this single-cell immunofluorescence technique, we found that HSCs mainly undergo symmetric self-renewal in the presence of only stem cell factor, but with the addition of trombopoietin this division pattern is transformed into a symmetric commitment dominant mode in vitro. In addition, our study indicated that the division pattern cannot be defined by cell size or the nuclear/cytoplasm ratio. These findings collectively demonstrate that this single-cell immunofluorescence technique provides a new biological strategy in stem cell division research, and can be more widely applied given its flexibility, easy operability, and inexpensiveness.

[Role of Vascular Niche of Bone Marrow in the Development of MLL-AF9 Induced Acute Myclaid Leukemia].

OBJECTIVE: To investigate the role of bone marrow vascular niche in the development of MLL-AF9 acute myeloid leukemia (AML). METHODS: Transplantation experiments were performed to establish non-radiated MLL-AF9 AML model; the half-bone immunofluorescence staining and tile-scan imaging of two-photon confocal microscopy were used to obtain the data of 3 main bone marrow niche cells; flow cytometry analysis was performed to characterize leukemia cells in different anatomical sites. RESULTS: In the early stage of MLL-AF9 AML, the proportion of leukemia cells in the metaphysis of the femur was significantly higher than that in the diaphysis. The detection of apoptosis and proliferation rate of leukemia cells showed that the percentage of leukemia cells in metaphysis significantly decreased, and the proliferation (S/G2/M phase) was also significantly more active. These different features of leukemia cells may relate with different bone marrow microenvironment. The image data of 3 major components of bone marrow niche (endothelial cells, endosteum, megakaryocytes) showed different distribution of blood vessels in metaphysis and diaphysis. Furtherly comparing the spatial distance between leukemia cells and endothelial cells, endosteum, megakaryocytes indicated that leukemia cells are closer to the blood vessels, suggesting the important role of blood vessels in the development of leukemia. Glucose uptake assays and intracellular ROS detection showed that the supportive role of blood vessels for leukemia cells did not related with nutrient metabolism pathway. CONCLUSION: The vascular niche plays an important role in the development of leukemia, and does not relate with the transport of nutrients and the elimination of metabolic waste, instead, which may relate with perivascular cytokines or other vascular functions.

Bone marrow endothelial cell-derived interleukin-4 contributes to thrombocytopenia in acute myeloid leukemia.

Normal hematopoiesis can be disrupted by the leukemic bone marrow microenvironment, which leads to cytopenia-associated symptoms including anemia, hemorrhage and infection. Thrombocytopenia is a major and sometimes fatal complication in patients with acute leukemia. However, the mechanisms underlying defective thrombopoiesis in leukemia have not been fully elucidated. In the steady state, platelets are continuously produced by megakaryocytes. Using an MLL-AF9-induced acute myeloid leukemia mouse model, we demonstrated a preserved number and proportion of megakaryocyte-primed hematopoietic stem cell subsets, but weakened megakaryocytic differentiation via both canonical and non-canonical routes. This primarily accounted for the dramatic reduction of megakaryocytic progenitors observed in acute myeloid leukemia bone marrow and a severe disruption of the maturation of megakaryocytes. Additionally, we discovered overproduction of interleukin-4 from bone marrow endothelial cells in acute myeloid leukemia and observed inhibitory effects of interleukin-4 throughout the process of megakaryopoiesis in vivo Furthermore, we observed that inhibition of interleukin-4 in combination with induction chemotherapy not only promoted recovery of platelet counts, but also prolonged the duration of remission in our acute myeloid leukemia mouse model. Our study elucidates a new link between interleukin-4 signaling and defective megakaryopoiesis in acute myeloid leukemia bone marrow, thereby offering a potential therapeutic target in acute myeloid leukemia.

Megakaryocyte-derived excessive transforming growth factor ß1 inhibits proliferation of normal hematopoietic stem cells in acute myeloid leukemia.

Impaired production of healthy hematopoietic cells from residual hematopoietic stem cells (HSCs) leads to high mortality in acute myeloid leukemia (AML). Previous studies have identified p21 and Egr3 as intrinsic factors responsible for the growth arrest and differentiation blockade of normal HSCs in leukemia; however, the related extrinsic factors remain unknown. In this study, we found that transforming growth factor ß (TGFß) signaling was upregulated in HSCs from bone marrow of mice with MLL-AF9-induced acute myeloid leukemia (AML) because of excessive production of TGFß1, especially from megakaryocytes, and overactivation of latent TGFß1 protein. We also found that SMAD3, a signal transducer of TGFß1, directly bound to Egr3 and upregulated its expression to arrest proliferation of HSCs. Our study provides evidence for targeting TGFß1 in AML to rectify normal hematopoiesis defects in clinical practice.

Chasing the precursor of functional hematopoietic stem cells at the single cell levels in mouse embryos.

BACKGROUND: Adult hematopoietic stem cells (HSCs), the ideal system for regenerative research, were isolated at single cell levels decades ago, whereas studies on embryonic HSCs are much more difficult. METHODS: Zhou et al identified a new pre-HSC cell surface marker, CD201, by which they isolated pre-HSCs at single cell levels for further analyses. RESULTS: The novel expression pattern of HSC development is revealed, including the fundamental role of mammalian targets of rapamycin (mTOR) signaling pathway in HSCs emergence, and the repopulation potential of S/G2/M phase pre-HSCs. CONCLUSIONS: Deeper understandings of the cellular origin and developmental regulatory network of HSCs are essential to develop new strategies of generating HSCs in vitro for clinical application.

NK cells play a significant role in immunosurveillance at the early stage of MLL-AF9 acute myeloid leukemia via CD226/CD155 interactions.

Acute myeloid leukemia (AML) is an aggressive hematological malignancy, and the mechanism underlying immune system involvement in leukemia development is unclear. In the present study, we utilized a myeloid/lymphoid or mixed-lineage leukemia; translocated to, 3 (MLLT3/MLL-AF9)-induced AML mouse model with or without exposure to irradiation. We found that the leukemia cells could survive and expand in hosts with intact immune systems, whereas leukemia progression was accelerated in mice with impaired immune systems. Moreover, the leukemia cells escaped from host immunosurveillance via editing their immunogenicity, including the up-regulation of an inhibitory antigen (i.e., CD47) and the down-regulation of active antigens (i.e., CD86, CD54, retinoic acid early transcript (RAE), histocompatibility 2, D region locus b (H2-Db) and H2-Dd). Natural killer (NK) cells were activated in the early phase of AML progression, whereas T cells were stimulated in the late phase. Furthermore, NK cell depletion showed that NK cells were necessary for the elimination of leukemia cells in our AML mouse model. Notably, CD155/CD226 primarily mediated the interaction between NK cells and leukemia cells and contributed to the antitumor effects of NK cells during the early phase of AML. Clinical data from patients with diverse hematological malignancies showed that CD155 expression was decreased in hematological malignancies. Taken together, our results demonstrate that NK cells play a pivotal role in immunosurveillance against leukemia cells during the early stage of AML primarily through the CD226/CD155 interaction; however, NK cells are not sufficient to eliminate leukemia cells.