Hematopoietic Recovery Research Articles

Upregulation of SIRT1 Contributes to dmPGE2-dependent Radioprotection of Hematopoietic Stem Cells.

Exposure to potentially lethal high-dose ionizing radiation results in bone marrow suppression, known as the hematopoietic acute radiation syndrome (H-ARS), which can lead to pancytopenia and possible death from hemorrhage or infection. Medical countermeasures to protect from or mitigate the effects of radiation exposure are an ongoing medical need. We recently reported that 16,16 dimethyl prostaglandin E2 (dmPGE2) given prior to lethal irradiation protects hematopoietic stem (HSCs) and progenitor (HPCs) cells and accelerates hematopoietic recovery by attenuating mitochondrial compromise, DNA damage, apoptosis, and senescence. However, molecular mechanisms responsible for the radioprotective effects of dmPGE2 on HSCs are not well understood. In this report, we identify a crucial role for the NAD+-dependent histone deacetylase Sirtuin 1 (Sirt1) downstream of PKA and CREB in dmPGE2-dependent radioprotection of hematopoietic cells. We found that dmPGE2 increases Sirt1 expression and activity in hematopoietic cells including HSCs and pharmacologic and genetic suppression of Sirt1 attenuates the radioprotective effects of dmPGE2 on HSC and HPC function and its ability to reduce DNA damage, apoptosis, and senescence and stimulate autophagy in HSCs. DmPGE2-mediated enhancement of Sirt1 activity in irradiated mice is accompanied by epigenetic downregulation of p53 activation and inhibition of H3K9 and H4K16 acetylation at the promoters of the genes involved in DNA repair, apoptosis, and autophagy, including p53, Ku70, Ku80, LC3b, ATG7, and NF-κB. These studies expand our understanding of intracellular events that are induced by IR but prevented/attenuated by dmPGE2 and suggest that modulation of Sirt1 activity may facilitate hematopoietic recovery following hematopoietic stress. Graphical Abstract.

ATP citrate lyase controls hematopoietic stem cell fate and supports bone marrow regeneration.

In order to support bone marrow regeneration after myeloablation, hematopoietic stem cells (HSCs) actively divide to provide both stem and progenitor cells. However, the mechanisms regulating HSC function and cell fate choice during hematopoietic recovery remain unclear. We herein provide novel insights into HSC regulation during regeneration by focusing on mitochondrial metabolism and ATP citrate lyase (ACLY). After 5-fluorouracil-induced myeloablation, HSCs highly expressing endothelial protein C receptor (EPCRhigh ) were enriched within the stem cell fraction at the expense of more proliferative EPCRLow HSCs. These EPCRHigh HSCs were initially more primitive than EPCRLow HSCs and enabled stem cell expansion by enhancing histone acetylation, due to increased activity of ACLY in the early phase of hematopoietic regeneration. In the late phase of recovery, HSCs enhanced differentiation potential by increasing the accessibility of cis-regulatory elements in progenitor cell-related genes, such as CD48. In conditions of reduced mitochondrial metabolism and ACLY activity, these HSCs maintained stem cell phenotypes, while ACLY-dependent histone acetylation promoted differentiation into CD48+ progenitor cells. Collectively, these results indicate that the dynamic control of ACLY-dependent metabolism and epigenetic alterations is essential for HSC regulation during hematopoietic regeneration.

VCAM1 confers innate immune tolerance on haematopoietic and leukaemic stem cells.

Haematopoietic stem cells (HSCs) home to the bone marrow via, in part, interactions with vascular cell adhesion molecule-1 (VCAM1)1-3. Once in the bone marrow, HSCs are vetted by perivascular phagocytes to ensure their self-integrity. Here we show that VCAM1 is also expressed on healthy HSCs and upregulated on leukaemic stem cells (LSCs), where it serves as a quality-control checkpoint for entry into bone marrow by providing 'don't-eat-me' stamping in the context of major histocompatibility complex class-I (MHC-I) presentation. Although haplotype-mismatched HSCs can engraft, Vcam1 deletion, in the setting of haplotype mismatch, leads to impaired haematopoietic recovery due to HSC clearance by mononuclear phagocytes. Mechanistically, VCAM1 'don't-eat-me' activity is regulated by β2-microglobulin MHC presentation on HSCs and paired Ig-like receptor-B (PIR-B) on phagocytes. VCAM1 is also used by cancer cells to escape immune detection as its expression is upregulated in multiple cancers, including acute myeloid leukaemia (AML), where high expression associates with poor prognosis. In AML, VCAM1 promotes disease progression, whereas VCAM1 inhibition or deletion reduces leukaemia burden and extends survival. These results suggest that VCAM1 engagement regulates a critical immune-checkpoint gate in the bone marrow, and offers an alternative strategy to eliminate cancer cells via modulation of the innate immune tolerance.

Higher efficacy of oral etoposide for mobilization of peripheral blood stem cells in patients with multiple myeloma

Abstract This study compares the efficacy, toxicity, hematopoietic recovery, and cost of stem-cell mobilization using intermediate-dose cyclophosphamide (IDCy) plus granulocyte colony-stimulating factor (G-CSF) compared with etoposide (VP-16) plus pegylated granulocyte colony-stimulating factor (PEG-rhG-CSF) in multiple myeloma (MM) patients. Two hundred forty-four consecutive patients undergoing mobilization with IDCy (3-3.5 g/m 2 ) plus G-CSF (n = 155) were compared with patients receiving VP-16 plus PEG-rhG-CSF (n = 89), including oral etoposide (n = 65) and intravenous etoposide (n = 24). Compared with IDCy, VP-16 use was associated with significantly higher median peak peripheral blood CD34 + cell count (8.20 [range: 1.84-84] × 106/kg vs 4.58 [range: 0.1-27.9] × 106/kg, P = .000), and ideal CD34 + cell yield of more than 6 × 106/kg (56.8% vs 35.1%, P = .001), notably with a higher efficacy in oral VP-16 use compared with IDCy use (CD 34 + cell counts: median peak peripheral blood 5.87 vs 4.58 × 106/kg and ≥6 × 106/kg [48.4% vs 35.1%]). The median number of apheresis courses was reduced from two in the IDCy group to one in the VP-16 group (P = .000). IDCy use was associated with significantly more frequent episodes of neutropenia (70.2% vs 35.2%; P = .000), intravenous antibiotic use (13.2% vs 11.4%; P = .672), and hospitalization (P = .000). The recoveries of neutrophils and platelets after autologous stem-cell transplantation were significantly faster in the VP-16 group compared with the IDCy group (P = .000). Our data indicate robust stem-cell mobilization in MM patients with VP-16 delivered either orally or intravenously. When compared with intravenous VP-16, oral VP-16 mobilization was associated with significantly more convenient, lower average total costs, and especially decreased the risk of hospital visits and exposure.

Venetoclax-based combinations for acute myeloid leukemia: optimizing their use in Latin-America

ABSTRACT Objectives: Venetoclax combinations are a new standard for patients with acute myeloid leukemia (AML). We aimed to evaluate the safety and efficacy of these combinations in a period of accelerated approval in Latin-America. Methods: This observational study evaluated adults with acute myeloid leukemia who received venetoclax-based therapy in 11 public or private centers in Mexico and Peru for both newly diagnosed or relapsed and refractory AML. Results: Fifty patients were included; 28 with newly diagnosed (ND) AML and 22 with relapsed/refractory (RR) disease. ND patients were older (64 vs. 40 years; p < 0.001) with a lower functional capacity (ECOG ≥2 64.3% vs 9%; p < 0.001). Venetoclax was frequently combined with azacytidine (60%) and prophylactic azoles (82%) with a median maximum dose of 200 mg (range, 100–600 mg). Hematologic toxicities were common. Complete response rates including patients with incomplete hematopoietic recovery were 78.6% in ND and 45.5% in RR patients, with a median overall survival of 9.6 (95% CI 3.7–15.5) and 8 months (95% CI 4.8–11.2). Discussion: Our study showed a preferred use of venetoclax plus azacytidine over cyatrabine. Patients in the first-line setting were similar to those in the landmark studies, while most patients with relapsed disease had received prior intensive therapies. Responses were favorable, with a median survival in agreement to other reports, albeit shorter than that observed in the randomized phase-3 trials. Conclusion: Venetoclax-based therapy in AML was effective despite dose reductions and prophylactic antifungals in two middle-income countries outside of a clinical trial setting.

Long-term follow-up of haploidentical transplantation in relapsed/refractory severe aplastic anemia: a multicenter prospective study

In recent decades, haploidentical stem cell transplantation (haplo-SCT) to treat severe aplastic anemia (SAA) has achieved remarkable progress. However, long-term results are still lacking. We conducted a multicenter prospective study involving SAA patients who underwent haplo-SCT as salvage therapy. Long-term outcomes were assessed, mainly focusing on survival and quality of life (QoL). Longitudinal QoL was prospectively evaluated during pretransplantation and at 3 and 5 years posttransplantation using the SF-36 scale in adults and the PedsQL 4.0 scale in children. A total of 287 SAA patients were enrolled, and the median follow-up was 4.56 years (range, 3.01–9.05 years) among surviving patients. During the long-term follow-up, 268 of 275 evaluable patients (97.5%) obtained sustained full donor chimerism, and 93.4% had complete hematopoietic recovery. The estimated overall survival and failure-free survival for the whole cohort at 9 years were 85.4% ± 2.1% and 84.0% ± 2.2%, respectively. Age (≥18 years) and a poorer performance status (ECOG >1) were identified as risk factors for survival outcomes. For QoL recovery after haplo-SCT, we found that QoL progressively improved from pretransplantation to the 3-year and 5-year time points with statistical significance. The occurrence of chronic graft versus host disease was a risk factor predicting poorer QoL scores in both the child and adult cohorts. At the last follow-up, 74.0% of children and 72.9% of adults returned to normal school or work. These inspiring long-term outcomes suggest that salvage transplantation with haploidentical donors can be routine practice for SAA patients without human leukocyte antigen (HLA)-matched donors.

Prevalence and variation of CHIP in patients with aggressive lymphomas undergoing CD19-directed CAR T-cell treatment

AbstractInflammation plays an important role in chimeric antigen receptor (CAR) T-cell therapy, especially in the pathophysiology of cytokine-release syndrome (CRS) and immune effector cell–associated neurotoxicity syndrome (ICANS). Clonal hematopoiesis of indetermined potential (CHIP) has also been associated with chronic inflammation. The relevance of CHIP in the context of CAR T-cell treatment is widely unknown. We evaluated the prevalence of CHIP, using a targeted deep sequencing approach, in a cohort of patients with relapsed/refractory (r/r) B-cell non-Hodgkin lymphoma before and after CAR T-cell treatment. The aim was to define the prevalence and variation of CHIP over time and to assess the influence on clinical inflammation syndromes (CRS/ICANS), cytopenia, and outcome. Overall, 32 patients were included. CHIP was found in 11 of 32 patients (34%) before CAR T-cell therapy. CHIP progression was commonly detected in the later course. Patients with CHIP showed a comparable response rate to CAR T-cell treatment but had an improved overall survival (not reached vs 265 days, P = .003). No significant difference was observed in terms of the occurrence and severity of CRS/ICANS, therapeutic use of tocilizumab and glucocorticosteroids, paraclinical markers of inflammation (with the exception of ferritin), or dynamics of hematopoietic recovery. CHIP is commonly observed in patients undergoing CD19-directed CAR T-cell therapy and is not associated with an inferior outcome.

Single-center experience using anakinra for steroid-refractory immune effector cell-associated neurotoxicity syndrome (ICANS).

In addition to remarkable antitumor activity, chimeric antigen receptor (CAR) T-cell therapy is associated with acute toxicities such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Current treatment guidelines for CRS and ICANS include use of tocilizumab, a monoclonal antibody that blocks the interleukin (IL)-6 receptor, and corticosteroids. In patients with refractory CRS, use of several other agents as third-line therapy (including siltuximab, ruxolitinib, anakinra, dasatinib, and cyclophosphamide) has been reported on an anecdotal basis. At our institution, anakinra has become the standard treatment for the management of steroid-refractory ICANS with or without CRS, based on recent animal data demonstrating the role of IL-1 in the pathogenesis of ICANS/CRS. Here, we retrospectively analyzed clinical and laboratory parameters, including serum cytokines, in 14 patients at our center treated with anakinra for steroid-refractory ICANS with or without CRS after standard treatment with tisagenlecleucel (Kymriah) or axicabtagene ciloleucel (Yescarta) CD19-targeting CAR T. We observed statistically significant and rapid reductions in fever, inflammatory cytokines, and biomarkers associated with ICANS/CRS after anakinra treatment. With three daily subcutaneous doses, anakinra did not have a clear, clinically dramatic effect on neurotoxicity, and its use did not result in rapid tapering of corticosteroids; although neutropenia and thrombocytopenia were common at the time of anakinra dosing, there were no clear delays in hematopoietic recovery or infections that were directly attributable to anakinra. Anakinra may be useful adjunct to steroids and tocilizumab in the management of CRS and/or steroid-refractory ICANs resulting from CAR T-cell therapies, but prospective studies are needed to determine its efficacy in these settings.

Development of severe aplastic anemia during steroid therapy for autoimmune hemolytic anemia

A 50-year-old male patient was admitted for close monitoring of anemia (hemoglobin level, 5.0 g/dl). Autoimmune hemolytic anemia (AIHA) of warm type was diagnosed based on the elevated reticulocyte and bone marrow erythroblast counts, elevated indirect bilirubin level, serum haptoglobin level below the detection limit, and positive direct Coombs test result. The patient responded to prednisolone 60 mg/day (1.0 mg/kg); however, pancytopenia was observed during gradual dose tapering and maintenance therapy. The bone marrow showed remarkable hypoplastic findings, and magnetic resonance imaging scans of the thoracolumbar spinal cord showed an overgrowth of the adipose tissue. Thus, the patient was diagnosed with aplastic anemia (AA) stage 4. He was successfully treated with a combination of immunosuppressive therapy (anti-thymocyte globulin +cyclosporine), which allowed him to reduce his dependence on transfusions. However, the direct Coombs test result remained positive even after hematopoietic recovery. Aplastic anemia following AIHA treatment is extremely rare and has not been reported previously.

Development of low-cost ex vivo hematopoietic stem cell expansion

Hematopoietic stem cells (HSC) have self-renewal as well as multilineage differentiation capacity and maintain hematopoiesis throughout life. HSC transplantation (HSCT) is performed as a curative therapy for hematopoietic malignancies and nonmalignant hematopoietic disorders. Furthermore, bone marrow, mobilized peripheral blood, and cord blood are available sources for HSCT. HLA compatibility is the most critical factor for a successful HSCT. The HSC number in a graft is also invaluable for engraftment. Moreover, it is challenging to obtain an abundant number of HSC for patients with obesity, particularly, in cord blood. HSC ex vivo expansion is an appropriate solution for this problem. Extrinsic factors to expand and maintain HSCs, such as cytokines are identified from analysis of HSCs and their niche. Thus, HSC ex vivo expansion is improved by adding them in culture medium; however, it is still difficult for therapeutic applications. Recently, several small molecular compounds have been reported to facilitate ex vivo expansion of HSC. Clinical trials that transplant ex vivo expanded cord blood have been already expanded, and some trials demonstrate reduction of time to hematopoietic recovery. Thus, we anticipate that ex vivo expanded cord blood transplantation will be applied widely in the future.

Association between haploidentical hematopoietic stem cell transplantation combined with an umbilical cord blood unit and graft-versus-host disease in pediatric patients with acquired severe aplastic anemia.

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) based on granulocyte colony-stimulating factor plus anti-thymocyte regimens ('Beijing Protocol') provides a salvage treatment for patients of acquired severe aplastic anemia (SAA) in China. However, graft-versus-host disease (GVHD) is a major impediment of haplo-HSCT due to human leukocyte antigen disparity. Recently, haplo-HSCT combined with umbilical cord blood (UCB) (haplo-cord HSCT) is performed in clinical trials to potentially reduce the risk of severe GVHD. Nevertheless, studies comparing GVHD in pediatric patients receiving haplo and haplo-cord HSCT for SAA are limited. The objective of this study was to investigate the impact of UCB co-infusion on GVHD in pediatric patients receiving haplo-HSCT for SAA. We conducted a retrospective study of 91 consecutive SAA children undergoing haploidentical transplantation based on the 'Beijing Protocol' with or without co-infusion of UCB in our center. All patients received uniform non-myeloablative conditioning and GVHD prophylaxis. We compared baseline characteristics and transplant outcomes between the haplo (n = 35) and haplo-cord (n = 56) recipients. All 91 patients achieved hematopoietic recovery from haploidentical donors, with a higher incidence of peri-engraftment syndrome observed with the haplo-cord group as compared with the haplo group (75.0% versus 48.6%, p = 0.029). Notably, the haplo-cord group showed a lower incidence of II-IV acute GVHD (aGVHD) than the haplo group (16.1% versus 42.9%, p = 0.002). Observed incidences of chronic GVHD (cGVHD) and moderate to severe cGVHD in the haplo-cord group were also lower than that in the haplo group (25.6% versus 51.3%, p = 0.019; 16.2% versus 41.3%, p = 0.016, respectively). Haplo-cord HSCT was identified as the only factor associated with a lower incidence of II-IV aGVHD and cGVHD in multivariate analysis. However, no differences were observed between the two groups for infections and survival outcomes. Our data indicated that co-infusion of UCB in 'Beijing Protocol'-based haplo-HSCT may be effective for reducing the risk of severe GVHD in SAA children.

Current Limitations and Perspectives of Chimeric Antigen Receptor-T-Cells in Acute Myeloid Leukemia.

Simple SummaryAcute myeloid leukemia (AML) is the most frequent type of acute leukemia in adults. Allogeneic hematopoietic cell transplantation (allo-HCT) has been the only potentially curative treatment for the majority of patients. The ability of chimeric antigen receptor (CAR)-modified T-cell therapy directed against the CD19 antigen to induce durable remissions in patients with acute lymphoblastic leukemia (ALL) has provided optimism that this novel treatment paradigm can be extrapolated to AML. In this review, we provide an overview of candidate target antigens for CAR-T-cells in AML, an update on recent progress in preclinical and clinical development of investigational CAR-T-cell products, and discuss challenges for the clinical implementation of CAR-T-cell therapy in AML.Adoptive transfer of gene-engineered chimeric antigen receptor (CAR)-T-cells has emerged as a powerful immunotherapy for combating hematologic cancers. Several target antigens that are prevalently expressed on AML cells have undergone evaluation in preclinical CAR-T-cell testing. Attributes of an ‘ideal’ target antigen for CAR-T-cell therapy in AML include high-level expression on leukemic blasts and leukemic stem cells (LSCs), and absence on healthy tissues, normal hematopoietic stem and progenitor cells (HSPCs). In contrast to other blood cancer types, where CAR-T therapies are being similarly studied, only a rather small number of AML patients has received CAR-T-cell treatment in clinical trials, resulting in limited clinical experience for this therapeutic approach in AML. For curative AML treatment, abrogation of bulk blasts and LSCs is mandatory with the need for hematopoietic recovery after CAR-T administration. Herein, we provide a critical review of the current pipeline of candidate target antigens and corresponding CAR-T-cell products in AML, assess challenges for clinical translation and implementation in routine clinical practice, as well as perspectives for overcoming them.

Graft-versus-host disease after liver transplantation is associated with bone marrow failure, hemophagocytosis, and DNMT3A mutations

Graft-versus-host disease after liver transplantation (LT-GVHD) is rare, frequently fatal, and associated with bone marrow failure (BMF), cytopenias, and hyperferritinemia. Given hyperferritinemia and cytopenias are present in hemophagocytic lymphohistiocytosis (HLH), and somatic mutations in hematopoietic cells are associated with hyperinflammatory responses (clonal hematopoiesis of indeterminate potential, CHIP), we identified the frequency of hemophagocytosis and CHIP mutations in LT-GVHD. We reviewed bone marrow aspirates and biopsies, quantified blood/marrow chimerism, and performed next-generation sequencing (NGS) with a targeted panel of genes relevant to myeloid malignancies, CHIP, and BMF. In all, 12 marrows were reviewed from 9 LT-GVHD patients. In all, 10 aspirates were evaluable for hemophagocytosis; 7 had adequate DNA for NGS. NGS was also performed on marrow from an LT cohort (n=6) without GVHD. Nine of 10 aspirates in LT-GVHD patients showed increased hemophagocytosis. Five (71%) of 7 with LT-GVHD had DNMT3A mutations; only 1 of 6 in the non-GVHD LT cohort demonstrated DNMT3A mutation (p=.04). Only 1 LT-GVHD patient survived. BMF with HLH features was associated with poor hematopoietic recovery, and DNMT3A mutations were over-represented, in LT-GVHD patients. Identification of HLH features may guide prognosis and therapeutics. Further studies are needed to clarify the origin and impact of CHIP mutations on the hyperinflammatory state.

LNK (SH2B3) inhibition expands healthy and Fanconi anemia human hematopoietic stem and progenitor cells

AbstractHematopoietic stem cell transplantation (HSCT) remains the only curative treatment for a variety of hematological diseases. Allogenic HSCT requires hematopoietic stem cells (HSCs) from matched donors and comes with cytotoxicity and mortality. Recent advances in genome modification of HSCs have demonstrated the possibility of using autologous HSCT-based gene therapy to alleviate hematologic symptoms in monogenic diseases, such as the inherited bone marrow failure (BMF) syndrome Fanconi anemia (FA). However, for FA and other BMF syndromes, insufficient HSC numbers with functional defects results in delayed hematopoietic recovery and increased risk of graft failure. We and others previously identified the adaptor protein LNK (SH2B3) as a critical negative regulator of murine HSC homeostasis. However, whether LNK controls human HSCs has not been studied. Here, we demonstrate that depletion of LNK via lentiviral expression of miR30-based short hairpin RNAs results in robust expansion of transplantable human HSCs that provided balanced multilineage reconstitution in primary and secondary mouse recipients. Importantly, LNK depletion enhances cytokine-mediated JAK/STAT activation in CD34+ hematopoietic stem and progenitor cells (HSPCs). Moreover, we demonstrate that LNK depletion expands primary HSPCs associated with FA. In xenotransplant, engraftment of FANCD2-depleted FA-like HSCs was markedly improved by LNK inhibition. Finally, targeting LNK in primary bone marrow HSPCs from FA patients enhanced their colony forming potential in vitro. Together, these results demonstrate the potential of targeting LNK to expand HSCs to improve HSCT and HSCT-based gene therapy.

TiRNA signaling via stress-regulated vesicle transfer in the hematopoietic niche.

Extracellular vesicles (EVs) transfer complex biologic material between cells. However, the role of this process invivo is poorly defined. Here, we demonstrate that osteoblastic cells in the bone marrow (BM) niche elaborate extracellular vesicles that are taken up by hematopoietic progenitor cells invivo. Genotoxic or infectious stress rapidly increased stromal-derived extracellular vesicle transfer to granulocyte-monocyte progenitors. The extracellular vesicles contained processed tRNAs (tiRNAs) known to modulateprotein translation. 5'-ti-Pro-CGG-1 was preferentially abundant in osteoblast-derived extracellular vesicles and, when transferred to granulocyte-monocyte progenitors, increased protein translation, cell proliferation, and myeloid differentiation. Upregulating EV transfer improved hematopoietic recovery from genotoxic injury and survival from fungal sepsis. Therefore, EV-mediated tiRNA transfer provides astress-modulated signaling axis in the BM niche distinct from conventional cytokine-driven stress responses.

Engraftment Effects after Intra-Bone Marrow versus Intravenous Allogeneic Stem Cell Transplantation in a Reduced-Intensity Conditioning Dog Leukocyte Antigen-Identical Canine Model

Following conventional i.v. hematopoietic stem cell transplantation (IV-HSCT), most of the hematopoietic stem cells get trapped in peripheral organs and do not reach the bone marrow niche. A promising approach to overcome this cell loss during the homing process seems to be the infusion of hematopoietic stem cells directly into the bone marrow cavity (intra-bone marrow [IBM]-HSCT). This study aimed to investigate the engraftment efficiency of IBM-HSCT compared with IV-HSCT following reduced-intensity conditioning in a canine HSCT model. Furthermore, the impact of 2 different graft infusion rates during IBM-HSCT on the engraftment was evaluated. Dogs received 4.5 Gy total body irradiation for conditioning at day -1 and 15mg/kg cyclosporin A twice daily at days -1 to +35 as immunosuppression. The IV-HSCT group (n=7) received unmodified bone marrow. The IBM-HSCT cohorts received buffy coat-enriched bone marrow that was applied into the humerus and femur simultaneously with an infusion time of either 10 minutes (IBM10; n=8) or 60 minutes (IBM60; n=7). Statistical analyses were performed using the Kruskal-Wallis test followed by the Mann-Whitney U test with Bonferroni correction for multiple comparisons. Statistical significance was declared at Bonferroni-adjusted P < .017. All dogs initially engrafted. One dog of the IBM10 cohort died at day +15 from infection. All 21 evaluable dogs developed a durable mixed donor chimerism over the course of 112 days. Engraftment kinetics did not differ significantly across the 3 groups. Leukocyte and platelet nadirs, as well as the durations of leukopenia and thrombocytopenia, were comparable in the 3 groups. Signs of toxicity for ingestion, body temperature, activity, and defecation did not show statistically significant differences among the 3 groups; only weight loss was greater in the IBM60 group compared with the IV group. IBM-HSCT following reduced-intensity conditioning resulted in an engraftment efficiency and hematopoietic recovery comparable to that seen with conventional IV-HSCT. In addition, modification of the graft infusion rate had no impact on engraftment and hematopoietic recovery in the canine IBM-HSCT model.

Salvage Cord Blood Transplantation Using a Short-term Reduced-intensity Conditioning Regimen for Graft Failure.

Objective Graft failure (GF) is a life-threatening complication of hematopoietic stem cell transplantation (HSCT). A standardized conditioning regimen and an appropriate graft source of salvage HSCT for GF have not yet been established. Some case series have shown good hematopoietic recoveries after salvage HSCT using a short-term reduced-intensity preparative regimen consisting of fludarabine (30-90 mg/m2), cyclophosphamide (2 g/m2), and total-body irradiation (2 Gy). However, the dose of fludarabine has varied in these reports based on the clinical condition of the patients, resulting in very limited experiences with each dose of fludarabine. Methods We retrospectively analyzed 10 patients who developed GF after allogeneic HSCT and underwent salvage cord blood transplantation (CBT) using the above-mentioned conditioning regimen with a fixed dose (90 mg/m2) of fludarabine. Results Eight patients (80.0%) achieved neutrophil engraftment within 30 days from salvage HSCT with a median of 21 (range, 17-23) days. The 1-year overall survival (OS) rate after the salvage HSCT was 50.0%, and the median OS was 281 (range, 23-1,638) days. Cumulative incidences of non-relapse mortality and relapse at 1 year were 50.0% and 10.0%, respectively. Conclusion CBT using this short-term reduced-intensity conditioning regimen may be a promising salvage therapy for GF.

Immune composition and its association with hematologic recovery after chemotherapeutic injury in acute myeloid leukemia

Chemotherapy-induced bone marrow (BM) injury is a significant cause of morbidity and mortality in acute myeloid leukemia (AML). Time to hematologic recovery after standard ("7+3") myeloablative chemotherapy can vary considerably among patients, but the factors that drive or predict BM recovery remain incompletely understood. Here, we assessed the composition of innate and adaptive immune subsets in the regenerating BM (day 17) after induction chemotherapy and related it to hematologic recovery in AML. T cells, and in particular the CD4 central memory (CD4CM) T-cell subset, were significantly enriched in the BM after chemotherapy, suggesting the relative chemoresistance of cells providing long-term memory for systemic pathogens. In contrast, B cells and other hematopoietic subsets were depleted. Higher frequencies of the CD4CM T-cell subset were associated with delayed hematopoietic recovery, whereas a high frequency of natural killer (NK) cells was related to faster recovery of neutrophil counts. The NK/CD4CM ratio in the BM after chemotherapy was significantly associated with the time to subsequent neutrophil recovery (Spearman's ρ=-0.723, p < 0.001, false discovery rate <0.01). The data provide novel insights into adaptive immune cell recovery after injury and identify the NK/CD4CM index as a putative predictor of hematopoietic recovery in AML.

Medical management of heavily exposed victims: an experience at the Tokaimura criticality accident

A criticality accident occurred at the uranium conversion plant in Tokaimura, Ibaraki Prefecture, Japan on 30 September 1999. When uranyl nitrate was overloaded to a critical mass level, uncontrolled fission reaction occurred. A procedure was carried out according to the JCO manual, although not an officially approved manual. Three workers were heavily exposed to neutrons and γ-rays produced by nuclear fission, and they subsequently developed acute radiation syndrome (ARS). The average doses to the whole body of the three workers were approximately 25, 9, and 3 GyEq (biologically equivalent dose of γ-exposure), respectively; dose distribution analysis later revealed extreme heterogeneity of these doses in two workers. They were triaged according to the predicted clinical needs. Two of these workers developed severe bone marrow failure and received haematopoietic stem cell transplantation: one with peripheral stem cell transplantation from his Human Leukocyte Antigen compatible sister and the other with umbilical cord blood transplantation. The graft was initially successful in both workers; autologous haematopoietic recovery was observed after donor/recipient mixed chimerism in one of them. Despite of all medical efforts available including haematopoietic stem cell transplantation, investigational drugs, skin graft, two workers died of multiple organ involvement and failure 83 and 211 days after the accident, respectively. Clinically as well as pathologically, the direct cause of death was deemed to be intractable gastrointestinal (GI) bleeding in one, and thoraco-abdominal compartment syndrome due to dermal fibrosis/sclerosis in the other. The third worker also developed bone marrow suppression but was treated with granulocyte colony-stimulating factor. He recovered without major complications and is now under periodical medical follow-up. These experiences suggest that treatment of bone marrow is not a limiting factor for saving the life of ARS victims severely exposed. Successful treatment of other organs such as lungs, skin, and GI tract is also essential. Furthermore, the whole-body dose may not always reflect the prognosis of ARS victims because of the nature of accidental exposure, heterogenous exposure.

Hematopoietic Stem Cell Regeneration through Paracrine Regulation of the Wnt5a-Prox1 Signaling Axis

The crosstalk between bone marrow (BM) microenvironment (niche) and hematopoietic stem cells (HSCs) is critical for HSC regeneration after injury. Here we show that deletion of the genes encoding the DNA repair-deficient syndrome Fanconi anemia (FA), Fanca and Fancc, in mice dampens HSC regeneration through both direct effects on HSCs and indirect effects on BM niche cells. Specifically, Fanca- or Fancc-deficiency compromises hematologic recovery and dampens HSC regeneration following irradiation. FA HSCs show persistent upregulation of the Wnt target Prox1, a homeobox transcription factor, in response to total body irradiation (TBI). Accordingly, lineage-specific deletion of Prox1 improves long-term repopulation of the irradiated FA HSCs. Forced expression of Prox1 in wild-type (WT) HSC mimics the defective repopulation phenotype of FA HSCs. By analyzing paracrine factors in Wnt signaling, we found that WT mice, but not FA mice, show significant induction by TBI of BM stromal Wnt5a protein, which is produced in LepR +CXCL12 + BM stromal cells. Wnt5a treatment of irradiated FA mice enhances hematopoietic recovery and HSC regeneration. Conversely, Wnt5a neutralization in co-cultured LepR + BM stromal cells inhibits HSC regeneration and hematopoietic recovery following TBI. Mechanistically, Wnt5a secreted by LepR +CXCL12 + BM stromal cells inhibits b-catenin accumulation, thereby repressing Prox1 transcription in irradiated HSPCs. The detrimental effect of deregulated Wnt5a-Prox1 signaling on HSC regeneration and hematopoietic recovery is also observed in aged mice. Irradiation induces upregulation of Prox1 in the HSCs of aged mice, and deletion of Prox1 in aged HSCs improves HSC regeneration and hematopoietic recovery after irradiation. Finally, treatment of aged mice with Wnt5a enhances hematopoietic repopulation. Collectively, these findings identify the novel paracrine Wnt5a-Prox1 signaling axis in regulating HSC regeneration under conditions of injury and aging. DisclosuresNo relevant conflicts of interest to declare.