Total Bone Marrow Research Articles

T Cells Trafficking to Bone Marrow Have Different Characteristics from Those to Other Organs in an Immune-Mediated Bone Marrow Failure Mouse Model

Background and objective T-cell-mediated destruction of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) is the pathophysiology of aplastic anemia (AA). Human AA has been modeled in mice by adaptation of historic “runt disease” in which infusion of lymph node (LN) cells into recipients mismatched at MHC or minor histocompatibility (minor-H) antigen loci produced BM failure with severe pancytopenia and marrow hypoplasia that highly resemble manifestations of human AA. These models have been used to test the plausibility of immune mechanisms inferred from the study of patients and patient cells, and also to serve as a platform for testing new treatments. While much has been learned from these models, the dynamics of T cell homing, expansion and localization in target tissues have not been well examined. Thus, we employed C.B10 AA model to characterize T cells trafficking to BM and other organs. Methods C.B10 mice received 5 Gy total body irradiation (TBI) and were then infused with B6-DsRed LN cells at 5×106 cells/mouse via tail vein injection. On days 3, 6, 9, and 12, mice were sacrificed, and BM, spleen, LNs, and thymus were collected to analyze the dynamic change and activation status of donor T cells in these organs by flow cytometry. On day 12, blood was collected from retro-orbital sinus for the analyses of hematologic parameters, while donor-derived (DsRed) T cells were sorted from BM, spleen and LNs into CD3+CD4+ and CD3+CD8+ fractions for RNA isolation and gene expression analyses by PCR array. Results Relative to control animals that received TBI without LN cell infusion, AA mice developed severe BM failure with dramatic decrease in white blood cells, platelets and total BM cells on day 12 after LN cell infusion. Frequencies of DsRed T cells trafficking to BM, LNs and spleen increased with time. Surprisingly there were very few DsRed T cells in BM on days 3 and 6, significantly lower than those in spleen or LNs. On day 12, DsRed T cells increased dramatically in BM to a level much higher than those in the spleen and LNs, suggesting that donor T cells homed primarily to spleen and LNs first, expanded and differentiated, and then trafficked to BM. Frequency of DsRed T cells in thymus was the lowest during the whole process, implying that thymus is more resistant to donor T cell infiltration, which may represent an immune privilege site (Figure 1A). On day 12, CD3+CD8+ and CD3+CD4+ T cells were essentially of donor (DsRed) origin in BM, spleen and LNs of AA mice (CD3+CD8+ at 99.12±0.29%, 92.07±0.90%, and 93.59±1.04%, and CD3+CD4+ at 98.38±0.20%, 82.87±1.81%, and 39.78±4.24%, respectively) except CD3+CD4+ T cells in LNs. Transcriptome analyses showed higher expression in Cd38, Ifng, Lag3, Csf1, Spp1, and Tnfsf13b in BM CD3+CD4+ and CD3+CD8+ T cells, but lower expression in Foxp3 and Ctla4 in BM CD3+CD4+ T cells than did those in spleen and LNs, indicating that BM T cells were at a state with stronger activation but weaker immunosuppression signaling . Meanwhile, we assessed donor T cell phenotypes using CD44 and CD62L markers, and found that almost all donor CD4+ or CD8+ T cells in BM were of effector memory T cell phenotype (EM: CD44+CD62L-) on days 6 , 9 and 12, while T cells in spleen and LNs were of EM, central memory (CM:CD44+CD62L+), and naïve (NA: CD44-CD62L+) phenotypes on days 6 and 9 before converting to EM phenotype on day 12 (Figure 1B), indicating that T cells in the BM were at a more terminal differentiation stage ready for the initiation of HSPC destruction. The expansion of EM T cell phenotype in BM failure mice reflects our recent observation in AA patients from which we found clonal expansion enriched in effector memory CD8+ T cells defined by CD28-CD57+ phenotype (Giudice et al. ASH abstract, 2016). Conclusions We demonstrate here that donor CD3+CD8+ and CD3+CD4+ T cells first home to host spleen and LNs for expansion and differentiation, and then traffick to host BM where they display strong signaling of activation with a terminal effector memory phenotype. The discovery of stage-specific T cell lodging pattern provides fresh insight for the development of new treatment for AA targeting T cell trafficking intervention. [Display omitted] DisclosuresYoung:Novartis/GSK to institute: Research Funding.

Absence of Interleukin-6 Protects Murine Bone Marrow Erythropoiesis Under Inflammation, in a Process Reversible By Iron Mediated Reactive Oxygen Species Upregulation

Inflammatory states seen in infections and chronic disorders are often characterized by a condition called anemia of Inflammation (AI). Using a mouse model of AI generated by a single injection of the heat killed pathogen Brucella abortus (BA), we have previously shown that mice lacking IL6 (IL6 -KO mice) exhibited protected erythropoiesis in the bone marrow (BM) and a faster recovery from anemia compared to controls. To study the mechanism of IL6 mediated improved erythropoiesis under AI, we investigated erythroid recovery in WT and IL6 -KO mice injected with BA. 72 hours following BA administration, both genotypes showed impaired BM erythropoiesis associated by a surge in inflammatory cytokines such as IFNγ and TNFα and a concurrent increase in mitochondrial ROS (reactive oxygen species), specifically superoxide (SO) in erythroid progenitors. Cytokines levels were normalized after 24 hours. However, during the second phase of erythroid recovery (10-14 days following BA treatment), mice showed a second surge of inflammatory cytokines. During this phase, IL6 -KO mice showed significantly improved BM erythropoiesis and normalization of ROS levels. We analyzed SO levels by Mitosox red, generic ROS production using the chloromethyl derivative of 2′,7′-dichlorofluorescin diacetate and total protein oxidation (by looking at total protein carbonylation) by Western blot. These results were in sharp contrast to WT animals which continued to show upregulated ROS, total protein oxidation and slackened erythroid recovery.Given the extensive association of iron overloading conditions and oxidative stress, we investigated the improved erythroid recovery within IL6 -KO mice in the context of increased iron absorption. We asked if and how BM erythropoiesis would change in IL6 -KO mice lacking Hepcidin (Hamp), a key iron regulatory hormone whose absence induces iron overload. In double knock out mice lacking both IL6 and Hamp (DKO), we observed an initial improved erythroid recovery in the BM, as indicated by increased number of total BM erythroid progenitors and reticulocytes, and improved RBC lifespan. However, all of these factors regressed after 10 days, with a simultaneous increase of cytokine production. The deterioration of erythropoiesis in the BM was accompanied by a concurrent increase in ROS. Of note, compared to non-treated mice, erythroid progenitors in the spleen of DKO-BA did not show increased ROS levels, and this was associated with an increased number of splenic erythroid progenitors and RBC. These results point to a potential role in the microenvironment in influencing erythropoiesis under inflammation. Furthermore, these results suggest that while lack of IL6 protects BM erythropoiesis, iron overload could antagonize it. In fact, iron overloaded IL6 -KO mice (with iron dextran) did show an impairment in BM erythroid recovery and upregulation of ROS, similarly to the DKO. Finally, we hypothesized that while there is a distinct positive correlation between upregulated ROS and weakened BM erythropoiesis, this comes into effect only in the context of inflammation. In other words, we speculated that some level of ROS upregulation is otherwise a physiological byproduct of erythropoietic stress associated with the onset of anemia. In line with this theory, we found that ROS levels were indeed upregulated in BM erythroid progenitors of phlebotomized animals. Our data suggests a model of erythroid recovery under AI wherein inflammation exacerbates ROS production triggered by stress erythropoiesis, and that this phenomenon exquisitely impairs BM erythropoiesis. This negative effect on BM erythropoiesis is mitigated by the absence of IL6, but the lack of IL6 mediated protective function is reversible under conditions of iron overload (Figure 1).Our results, in combination with the upregulation of TNFa, suggest the involvement of non-apoptotic ROS associated programmed cell death such as necroptosis or ferroptosis, which are under investigation. We also have preliminary data pointing to mechanism of increased erythroid iron uptake that could potentially contribute to oxidative stress under AI. With these additional studies, we hope to further characterize the mechanisms surrounding improved BM erythropoiesis in the absence of ROS and IL6. Our studies could potentially characterize cellular responses that can be targeted in conditions of inflammation for therapeutic benefits. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Characterizing Graft-Vs-Host Disease of the Bone Marrow Post Allogeneic Hematopoietic Cell Transplantation in Mice

Graft-vs-Host Disease (GVHD) is a common complication post allogeneic hematopoietic cell transplantation (allo-HCT). In contrast to classical target organs alloreactivity directed against hematopoietic and non-hematopoietic bone marrow (BM) structures has been poorly characterized, even though hematopoietic dysfunction following allo-HCT is frequently observed.Here, we studied in MHC-matched, minor antigen mismatched HCT models (C3H-SW > C57BL/6 (B6)) the effects of lethal total body irradiation and HCT of HSC (cKIT+Sca1+Lin-) alone or in combination with Tc / Tc subsets (CD4+, CD8+ or total (CD4+CD8) T cells (Tc)) on the hematopoietic compartment (HC) and non-hematopoietic compartment (NHC) of the BM. Up to 8 weeks (w) post-HCT bones and marrow were analyzed by flow cytometry and 3D-confocal microscopy imaging.Post allo-HCT there was a transient weight loss and decrease of total BM cell counts in all experimental groups, but no signs of overt GVHD. At 2w mice given HSC had significantly higher absolute BM counts compared with HSC+Tc/Tc subset recipients. Most pronouncedly affected were B-cells that recovered promptly in HSC recipients but were severely delayed beyond w8 in the HSC+Tc groups (particularly in CD4 recipients). Likewise, BM granulocyte recovery during the first 4w was significantly better in HSC vs. HSC+Tc recipients. Moreover, alloreactive (but not congenic) Tc severely disrupted the NHC: at 2w post allo-HCT HSC recipients showed prompt recovery of BM endothelial cells (EC; CD45-Ter119-CD31+), and CXCL12-abundant reticular cells (CAR; CD45-Ter119-CD31-CD140B+). In contrast, HSC+Tc recipients had significantly lower EC and CAR cells counts and their recovery was significantly delayed. 3D-confocal microscopy confirmed these observations and revealed rapid recovery of extracellular matrix and sinusoidal vascular structures, with simultaneous disappearance of adipocytes at 2w post-HCT in the HSC group, whereas HSC+Tc recipients displayed severe disruption of the structural integrity with impaired recovery of the BM microvessels but rather occupation of space by adipocytes. To visualize CAR cells by 3D imaging we used B6.CXCL12-GFP recipients and found, again, impaired CAR cell regeneration in HSC+Tc, but not pure HSC recipients. We next addressed the question whether alloreactive Tc exert direct GVH-related damage to NHC structures, or alternatively, only the recipient's HC is attacked whereas suppression of EC and CAR cells results from the inflammatory milieu. By transplanting C3H.SW BM into lethally irradiated B6 recipients chimeras with a C3H.SW HC and B6 NHC were generated. In a 2nd HCT HSC +/- Tc were transplanted from (i) C3H.SW (allo to NHC; syngeneic to HC) or (ii) B6 (congenic to NHC, allo to HC) donors into these chimeras. (i) At 2w post-2nd HCT BM cellularity, EC and CAR cell numbers were decreased when donor Tc were allogeneic to the NHC, but syngeneic to the HC. NHC populations were normal in number when pure allogeneic HSC (without Tc) were given. (ii) When grafts for 2nd HCT were from B6 donors (allo to HC, congenic to NHC), donor Tc had no effect on EC numbers. CAR cells were reduced in numbers, but less severely compared with after transfer of allogeneic HSC+Tc. Together, these findings suggest, that EC can be directly targeted by alloreactive Tc; CAR cells are affected by both, alloreactive Tc, and inflammatory conditions generated by attack of the HC. Finally, to test the influence of GVH-related cytokines on the NHC we used B6.TNFaR-/- and B6.IFNyR-/- mice as recipients of C3H.SW HSC +/- Tc. Recovery of HC and NHC was identical in WT and TNFaR-/- recipients of C3H.SW grafts. In contrast, IFNyR-/- recipients of allogeneic HSC+CD4 grafts had significantly higher CAR cell counts compared with WT.B6 recipients (but lower than recipients of pure HSC). These findings support the hypothesis that CAR cells are also susceptible to inflammatory damage.In clinical HCT delayed hematopoietic reconstitution presents a major problem contributing to increased morbidity and mortality. GVH reactions of the BM have been poorly characterized. Our data show that not only host hematopoiesis, but also non-hematopoietic stromal and vascular compartments of the BM are affected by both alloreactivity of donor Tc and their inflammatory microenvironment, even in the absence of clinical GVHD. DisclosuresNo relevant conflicts of interest to declare.

Next Generation Flow (NGF) Cytometry for Minimal Residual Disease (MRD) Evaluation in Multiple Myeloma (MM) Patients with Sustained Complete Response (CR) after Frontline Therapy: Results of a Prospective Single-Center Analysis

MRD evaluation is included in the novel IMWG response criteria with a minimum sensitivity level for MRD- that of 10-5. However, several data suggest a sensitivity level of at least 10- for MRD- may be more appropriate. Such high sensitivity would be particularly relevant to identify, among MM patients who remain at sustained response for several years, those who may still be at risk of relapse due to persistent MRD and for whom closer monitoring could be beneficial. Thus, the primary endpoint of this prospective study was to evaluate the incidence of MRD- in patients who remained in CR for ≥2-years after first line therapy, using NGF. Secondary endpoints included the detection level of MRD, the reproducibility of the method, and the correlation of MRD status with the hematopoietic bone marrow (BM) profile defined by NGF as well as with prognostic factors at diagnosis. Fifty-two patients who were treated at the Department of Clinical Therapeutics (Athens, Greece) and were in sustained CR for ≥2-years (median period of CR: 58 months, range: 24-197 months) were evaluated for the presence of MRD using NGF, according to the EuroFlow guidelines. BM samples were labeled using two independent 8-color panels, both containing CD19-PEC7, CD27-BV510, CD38-FITC, CD45-PERCP, CD56-PE, CD138-BV421 and additionally CD81-APCC750 and CD117-APC for the surface tube or CyIgκ-APC and CyIgλ-APCC750 for the intracytoplasmic tube. A minimum of ten million events were recorded per patient-sample with a BD FACSCantoII and data analysis was conducted with Infinicyt software, allowing merging of the two panels and detection of MRD with a median sensitivity level of 2.2x10-6 (range, 2.1x10-6 - 2.8x10-6). Two independent experts evaluated the results for reproducibility assessment. Using the same markers, we identified the major BM cell subsets (erythroid and myeloid progenitors, erythroblasts, T, NK and NKT cells, naive B cells, memory B cells and B-cell precursors, neutrophils, monocytes, basophils, eosinophils and mast cells) and analyzed the hematopoietic profile of each patient. Our analysis revealed 30 (58%) MRD- and 22 (42%) MRD+ patients, mostly at the level of 10-5 (n=7/22, 32%) and 10-6 (n=5/22, 23%). The reproducibility of the method was 98%, as there was only one disagreement between the two independent researchers (one patient who was MRD+ at the level of 10-6) and MRD levels were significantly correlated (r=0.892, p The MRD status was not associated with any of the prognostic clinical parameters at diagnosis (age, ISS, cytogenetic profile or LDH), although the majority of MRD+ had ISS-1 disease (58%). Three (10%) MRD- patients and 3 (14%) MRD+ had high-risk cytogenetics. Nineteen (61%) MRD- patients and 13 (62%) MRD+ had received ASCT. There were no differences regarding frontline therapy among MRD- and MRD+ patients (PI- vs IMiDs- vs PI+IMiD-based therapies). We demonstrated that the NGF method by EuroFlow can be standardized, and provide a detection MRD level of 2.1x10-6 with high reproducibility rate. We also showed that 40% of MM patients in prolonged CR have persistent MRD+. Interestingly, such patients displayed more erythroblasts and monocytes together with increased T, NK and NK-T cell numbers. By contrast, no significant clinical differences were observed between MRD+ and MRD- patients with ≥2-years in CR. Thus, NGF-based MRD assessment is warranted to discriminate independently of treatment or disease risk, patients reaching sustained CR that could have higher risk of relapse due to persistent MRD and may benefit from closer monitoring. Disclosures Terpos: Janssen: Honoraria, Research Funding; Amgen: Honoraria, Other: SC member, Research Funding; Abbvie: Honoraria; BMS: Honoraria; Genesis/Celgene: Honoraria, Other: DMC member, Research Funding; GSK: Honoraria; Takeda: Honoraria, Other: SC member. Kastritis: Genesis pharma: Honoraria; Janssen: Honoraria; Prothena: Honoraria; Takeda: Honoraria; Amgen: Honoraria, Research Funding. Paiva: EngMab: Research Funding; Amgen: Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding; Novartis: Honoraria; Sanofi: Consultancy, Honoraria, Research Funding; Janssen: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Merck: Honoraria. Dimopoulos: Amgen Inc, Celgene Corporation, Janssen Biotech Inc, Onyx Pharmaceuticals, an Amgen subsidiary, Takeda Oncology: Consultancy, Honoraria, Other: Advisory Committee: Amgen Inc, Celgene Corporation, Janssen Biotech Inc, Onyx Pharmaceuticals, an Amgen subsidiary, Takeda Oncology; Novartis: Consultancy, Honoraria; Genesis Pharma: Research Funding.

Acute Deletion of Tet2 or Tet3 Transiently Dysregulate Murine Erythropoiesis

The majority of palindromic CpG dinucleotides in mammalian DNA are symmetrically methylated on cytosine (5mC). Erythroid differentiation is associated with replication-dependent genome-wide loss of ~30-50% of all 5mC (1). One potential mechanism of demethylation involves Tet dioxygneases, which oxidize 5 methylcytosine (5mC) to 5-hydroxy-methylcytosine (5hmC), thereby interfering with maintenance methylation by DNMT1 (2). Tet2 and Tet3 are the two major Tet proteins expressed in hematopoietic cells. Tet2 is one of the most commonly mutated genes in hematological malignancies. Germline deletion of Tet2 results in increased hematopoietic stem cell self-renewal, erythroid and myeloid hyperplasia and myeloid and lymphoid malignancies (3, 4). Germline deletion of Tet3 is embryonic lethal (5). Here we investigated the potential role of Tet2 and Tet3 in the global loss of DNA methylation during erythroid differentiation, using mouse genetic models.We bred Tet2fl/fl and Tet3fl/fl mice onto the Rosa26-CreERT2 background, enabling acute deletion in vivo of either Tet2, Tet3 or both genes in adult and fetal mice. Within 4 to 10 days of deletion, we noted a rapid decline in total bone marrow cellularity in all genotypes; bone-marrow erythroblasts decreased to 15% of control value in Tet2-deleted (Tet2del) mice (p<0.0001), and to 43% (p=0.05) and 20% (p<0.0001) of controls in the Tet3 del and Tet2/Tet3 double deleted mice (DKO), respectively. These findings were associated with decreased reticulocyte counts in peripheral blood, and a decline in hematocrit, in Tet2del (36% vs. 46% in controls, p<0.004) and the DKO (31%, p=0.03). Bone-marrow CFU-e progenitors, but not BFU-e progenitors, also decreased in number. These losses partially improved over the next 2 to 4 weeks in mice deleted for either Tet2 or Tet3, but not in the DKO mice, in which anemia persisted. Megakaryocyte CFU-Mk progenitors increase in the DKO but are unaffected in the single deletions. Lymphoid lineage cells (CD4+, CD8+ and CD19+) showed similar transient losses, but the numbers of granylocytic and monocytic progenitors in the bone marrow was not affected. Similarly, fetal liver cell number decreased in all genotypes within 4 days of deletion, compared with controls, in all genotypes, and most severely in the DKO.We used three approaches to determine the potential effect of Tet proteins on erythroid DNA demethylation: Mass spectrometry, to measure global levels of 5mC and 5hmC in fetal liver erythroblasts; 5hMe-DIP-seq (5hyrdoxymethyl-DNA immune precipitation- sequencing), to determine the genomic distribution of hydroxymethylation in erythroblasts; and pyrosequencing for DNA methylation/hydroxymethylation at specific genomic loci including repetitive LINE_1 elements. We found that 5hmC is highly enriched in previously identified erythroid enhancers. Tet2 accelerates, but is not essential, for DNA demethylation of specific erythroid enhancers. Neither Tet2 nor Tet3 are required for global DNA demethylation.Taken together, we conclude that Tet2 and Tet3 are required during erythropoiesis, where they play partially redundant roles, possibly contributing to the changing chromatin environment during activation of erythroid terminal differentiation. Their acute deletion leads to transient loss of erythroblast viability and to anemia, but this is followed by a process of adaptation and chronically compensated erythropoiesis. These results raise the possibility that the adaptation required for the rapid partial recovery in the erythroid and lymphoid lineages following acute Tet2 loss may contribute, over the long term, to the development of myeloid and lymphoid malignancies.1. J. R. Shearstone et al., Global DNA demethylation during mouse erythropoiesis in vivo. Science334, 799-802 (2011).2. M. Ko et al., Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2. Nature468, 839-843 (2010).3. M. Ko et al., TET proteins and 5-methylcytosine oxidation in hematological cancers. Immunol Rev263, 6-21 (2015).4. J. An et al., Acute loss of TET function results in aggressive myeloid cancer in mice. Nature communications6, 10071 (2015).5. T. P. Gu et al., The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes. Nature477, 606-610 (2011). DisclosuresNo relevant conflicts of interest to declare.

The Role of Cyclophosphamide in the Conditioning Regimen of Stem Cell Transplantation in Iron Overload Mice

Background: Hematopoietic stem cell transplantation(HSCT) is the only effective and reliable method for the treatment of severe thalassemia. Most of the transplant centers adopted Busulfan(Bu)/cyclophosphamide(Cy) pretreatment program, the pediatric Transplantation Center of Southern Medical University used “NF-08-TM” pretreatment program based on Cy/Bu achieved remarkable results.Whether the significant achievements related to cyclophosphamide mobilizing hematopoietic stem cells resulting in an increase efficiency of hematopoietic stem cell clearance, has not yet been reported.METHODS: Establish three-levels risk grading standards of iron overload with C57 mice by intraperitoneal injection of dextran iron, based on the domestic recognition three-levels risk grading standards of thalassemia major. Eight groups(d1,d2,d3,d4,d5,d6,d7,d14) were injected intraperitoneally with Cy 50mg / kg for two consecutive days to establish the pretreatment model of transplantation. Peripheral blood cells, BMNNCs, frequencies of different HPCs, HSCs, LT-HSCs in BMMNCs were monitored. The distribution in the cell cycle of HSCs, the level of reactive oxygen species and the microenvironment of HSCs were analyzed by flow cytometry. After N-acetylcysteine anti-oxidation, the above indicators were measured.RESULTS: Compared with the control group, the Serum ferritin in the low, medium and high dose iron group was increased by 7.6 times, 12.8 times and 15.2 times. And the Hepatomegaly Index was increased by 46.9%, 96.5% and 160.2%. Pathological sections showed a large number of iron deposits in the liver, spleen and bone marrow cells, and positively correlated with the dose of iron. Similar to the risk criteria of thalassemia classification, three-level risk model of iron overload was successfully established.Compared with normal mice, the peripheral blood of iron overload model mice did not change significantly, while the number of WBCs and YM% 1d-4d in cyclophosphamide group were significantly decreased (P <0.05),and 7d back to normal; the number of PLT was higher than that of model group and the difference was statistically significant (P <0.05), but there was no significant difference in RBC and HBG.The number of BMNNCs and HPCs in iron overload model mice did not change significantly compared with normal mice,but the number of HSC and LT-HSCs decreased significant (P <0.05); The change rules of BMNNCs and WBC in cyclophosphamide group were the same, and the number of HSCs increased from the first day to the highest in the third day (P <0.05).Compared with normal mice,the cell cycle distribution of HSCs iniron overload model mice did not change significantly, while the proportion of G0 / G1 phase cells in cyclophosphamide group was significantly decreased at the 1st day to the third day(P <0.05). But S and G2 / M phase change the opposite,the difference was statistically significant (P <0.05).After the treatment with NAC, the decrease of G0 / G1 in 1d-3d could be partially reversed(P <0.05). Compared with normal mice,the ROS level of the total bone marrow cells ,HSCs were significantly higher in moderate-risk model mice and cyclophosphamide group, respectively (P <0.05), and the level of ROS in cyclophosphamide groups increased from the first day ,highest in the second day,and then back to normol. After NAC treatment, the level of ROS in cells decreased compare with before respectively (P <0.05).CONCLUSION:The three-level risk model of iron overload was successfully established by treated with different doses of dextran iron intraperitoneally. Iron deposit could lead to long-term hematopoietic stem cell damage, but did not cause significantly changes in peripheral blood.The effect of Cyclophosphamide on mobilization of HSCs in thalassemia transplantation pretreatment regimen may be related to oxidative stress.This study proved initially the advantages of “NF-08-TM” pretreatment in the transplant of thalassemia inour center compared with other transplant centers ,and provided a theoretical basis for further development of individualized pretreatment programs. DisclosuresNo relevant conflicts of interest to declare.

IGF Signaling Predicts Outcomes and Is a Promising Target Therapy for Acute Myeloid Leukemia

Background : Despite the growing knowledge about acute myeloid leukemia (AML) pathophysiology, it remains a disease with high mortality rates, and the early recognition of patients at higher risk allows adjusted risk- or target-specific therapies. In AML, autocrine insulin like growth factor type 1 (IGF1) and its receptor (IGF1R) activation has been previously described. Free IGF mediate cellular functions through binding to the two IGF receptors, IGF1R and IGF2R. IGF2R binds to and induces degradation of IGF2, preventing IGF2-IGF1R signaling activation, IGF1R activation induces cell proliferation and prevents cell death. Therefore, IGF2R may act as a tumor suppressor and IGF1R has opposite effects. IGF1R pharmacological inhibitors have recently been investigated in the clinical setting for solid tumors, but still underexplored in hematological neoplasms. Aims : To investigate IGF1R and IGF2R mRNA expression in primary samples from AML patients and their impact in clinical outcomes, and to analyze the effects of the IGF1R pharmacological inhibitor linsitinib in AML cell lines. Material and methods : IGF1R and IGF2R mRNA expression levels in AML patients (n=172) were obtained from TCGA AML study (2013) available online on CBioPortal for Cancer Genomics (Cerami et al. 2012; Gao et al. 2013). Gene expression was investigated, by qPCR, in a subset AML cohort (n=101; APL=48, CBF-leukemia=53 [t(8;21)=30, Inv16=23]), and in total bone marrow samples from healthy donors (HD) (n=23). The study was approved by the ethics committee of the Institution. NB4, NB4-R2 and Kasumi-1 cell lines were submitted to linsitinib (0.25, 0.5, 1.0, 2.5, 5.0, 10, 20 or 40 µM) for 72 hours and were evaluated for cell viability (MTT assay), apoptosis (Annexin V/PI), autophagy (acridine orange staining), and protein expression/activation (western blot). Statistical analyses were performed using Student's t-test, Mann-Whitney test, Kruskal-wallis test and Spearman correlation test, as appropriate. For survival analysis, Kaplan-Meyer curves were compared with log-rank test and cox regression analyzes were applied. Results :Patients were dichotomized according to the median IGF1R and IGF2R expression values, and the IGF1R / IGF2R ratio was calculated by division of absolute number of reads coming from RNA-seq data. Patients with high IGF1R / IGF2R showed poorer overall survival (OS) (29% vs. 56%, P <0.01), and disease free survival (DFS) (31% vs. 45%, P =0.015). Individually, high IGF1R (33% vs. 51%; P =0.042) and low IGF2R (32% vs. 52%; P =0.012) were associated with a shorter OS. By Cox regression analysis, IGF1R / IGF2R (P= 0.039), cytogenetic risk stratification (P <0.001), age (P <0.001) and white blood cell counts (P =0.003) independently predicted poorer OS. AML patients harboring t(15;17) or t(8;21) presented higher levels of IGF1R / IGF2R when compared with other AML patients (P≤ 0.01). Using a subset of APL and CBF-leukemia, a higher IGF1R / IGF2R ratio was observed in AML patients with t(15;17) or t(8;21) compared with healthy donors (HD) (P <0.01). IGF1R and IGF2R expression positively correlated in HD (r =0.9296; P <0.001), and negatively correlated in AML TCGA study (r =-0.2366; P =0.0018). In NB4, NB4-R2 and Kasumi-1 cells, linsitinib ≥1 µM reduced cell viability (P <0.05) and significantly increased apoptosis, as demonstrated by increased cleaved caspase 3 and annexin V+ cells. The mean percentage of annexin V+ cells for control, linsitinib 10, 20 and 40 µM were 3,9%, 14%, 32% and 93% for NB4, 6%, 24%, 53% and 96% for NB4-R2 and 7%, 19%, 49% and 96% for Kasumi-1 (all P <0.05), respectively. Linsitinib 10 µM increased autophagy in at least 3 fold-change in all cell lines tested (all P <0.05). Linsitinib 10 µM plus the autophagy inhibitor chloroquine 20 µM increased apoptosis when compared with monotherapy in NB4 cells (45% vs 15%), NB4-R2 cells (68% vs 15%) and Kasumi-1 (35% vs 15%) (all P <0.05). The IGF1R pharmacological inhibitor reduced phosphorylation/activation of IGF1R (Tyr1135), IRS1/2 (Tyr612), AKT1/2/3 (Ser473), P70S6K (Thr421/Ser424), 4EBP1 (Thr70) and ERK1/2 (Thr185/Tyr187). Conclusions :In AML,increased IGF1R / IGF2R predicts poorer survival outcomes and the pharmacological IGF1R inhibitor linsitinib exerts an anticancer activity and downregulates PI3K/AKT and MAPK signaling. In the concept of multi target treatment, IGF1R arise as a promising therapeutic target for patients with AML. DisclosuresNo relevant conflicts of interest to declare.

An Analysis for Therapeutic Doses of Patients with Neuroendocrine Tumor Treated with Lutetium 177 (177Lu)-DOTATATE.

Background: The aim of this study is to clarify the critical organs that limit treatment scheme and also evaluate the validity of currently used critical organ threshold values in neuroendocrine tumor (NET) patients, receiving peptide receptor radionuclide therapy (PRRT) with Lutetium 177 (177Lu)-DOTATATE. Materials and Methods: Thirty-six NET patients (ages 16-73 years) who received 177Lu-DOTATATE treatment were evaluated retrospectively in this study. Dosimetric calculations were made using medical internal radionuclide dose method. For calculation of organ doses, Internal Dose Assessment at Organ Level/Exponential Modelling 1.1 software program was used. Follow-up data were used to determine the organ failure. Results: A total of 141 cycles and mean of 3.91 (±1.33) cycles were applied to the patients. A mean of 691 mCi (±257 mCi) 177Lu-DOTATATE infusion in total and a dose between 70 and 200 mCi per treatment was applied to patients. Seven of 36 patients reached 23 Gy renal dose limit. In these patients, although kidney doses were between 23 and 29 Gy, there was no diminution in renal functions during follow-up. Two of 36 patients reached total bone marrow dose of 2 Gy limit. Bone marrow suppression did not develop in these patients. Conclusion: The critical organs that seem to affect the treatment scheme in PRRT with 177Lu-DOTATATE are kidney and bone marrow. Although there are established threshold levels, derived from radiotherapy experience, more studies are needed to clarify these dose limits in systemic radionuclide therapies such as PRRT.

Antioxidant Fusion Protein SOD1-Tat Increases the Engraftment Efficiency of Total Bone Marrow Cells in Irradiated Mice.

Total body irradiation is a standard procedure of bone marrow transplantation (BMT) which causes a rapid increase in reactive oxygen species (ROS) in the bone marrow microenvironment during BMT. The increase in ROS reduces the engraftment ability of donor cells, thereby affecting the bone marrow recovery of recipients after BMT. In the early weeks following transplantation, recipients are at high risk of severe infection due to weakened hematopoiesis. Thus, it is imperative to improve engraftment capacity and accelerate bone marrow recovery in BMT recipients. In this study, we constructed recombinant copper/zinc superoxide dismutase 1 (SOD1) fused with the cell-penetrating peptide (CPP), the trans-activator of transcription (Tat), and showed that this fusion protein has penetrating ability and antioxidant activity in both RAW264.7 cells and bone marrow cells in vitro. Furthermore, irradiated mice transplanted with SOD1-Tat-treated total bone marrow donor cells showed an increase in total bone marrow engraftment capacity two weeks after transplantation. This study explored an innovative method for enhancing engraftment efficiency and highlights the potential of CPP-SOD1 in ROS manipulation during BMT.

Measured Distribution of Total Red Bone Marrow in Young Children

Measured Distribution of Total Red Bone Marrow in Young Children

Megakaryocyte progenitor cell function is enhanced upon aging despite the functional decline of aged hematopoietic stem cells.

SummaryAge-related morbidity is associated with a decline in hematopoietic stem cell (HSC) function, but the mechanisms of HSC aging remain unclear. We performed heterochronic HSC transplants followed by quantitative analysis of cell reconstitution. Although young HSCs outperformed old HSCs in young recipients, young HSCs unexpectedly failed to outcompete the old HSCs of aged recipients. Interestingly, despite substantial enrichment of megakaryocyte progenitors (MkPs) in old mice in situ and reported platelet (Plt) priming with age, transplanted old HSCs were deficient in reconstitution of all lineages, including MkPs and Plts. We therefore performed functional analysis of young and old MkPs. Surprisingly, old MkPs displayed unmistakably greater regenerative capacity compared with young MkPs. Transcriptome analysis revealed putative molecular regulators of old MkP expansion. Collectively, these data demonstrated that aging affects HSCs and megakaryopoiesis in fundamentally different ways: whereas old HSCs functionally decline, MkPs gain expansion capacity upon aging.

Enhancing Cystic Fibrosis Immune Regulation.

In cystic fibrosis (CF), sustained infection and exuberant inflammation results in debilitating and often fatal lung disease. Advancement in CF therapeutics has provided successful treatment regimens for a variety of clinical consequences in CF; however effective means to treat the pulmonary infection and inflammation continues to be problematic. Even with the successful development of small molecule cystic fibrosis transmembrane conductance regulator (CFTR) correctors and potentiators, there is only a modest effect on established infection and inflammation in CF patients. In the pursuit of therapeutics to treat inflammation, the conundrum to address is how to overcome the inflammatory response without jeopardizing the required immunity to manage pathogens and prevent infection. The key therapeutic would have the capacity to dull the inflammatory response, while sustaining the ability to manage infections. Advances in cell-based therapy have opened up the avenue for dynamic and versatile immune interventions that may support this requirement. Cell based therapy has the capacity to augment the patient’s own ability to manage their inflammatory status while at the same time sustaining anti-pathogen immunity. The studies highlighted in this manuscript outline the potential use of cell-based therapy for CF. The data demonstrate that 1) total bone marrow aspirates containing Cftr sufficient hematopoietic and mesenchymal stem cells (hMSCs) provide Cftr deficient mice >50% improvement in survival and improved management of infection and inflammation; 2) myeloid cells can provide sufficient Cftr to provide pre-clinical anti-inflammatory and antimicrobial benefit; 3) hMSCs provide significant improvement in survival and management of infection and inflammation in CF; 4) the combined interaction between macrophages and hMSCs can potentially enhance anti-inflammatory and antimicrobial support through manipulating PPARγ. These data support the development of optimized cell-based therapeutics to enhance CF patient’s own immune repertoire and capacity to maintain the balance between inflammation and pathogen management.

130 Burn Injury Reduces Bone Marrow Mesenchymal Stem Cells and Sensitizes Their Adrenergic Receptor Subgroups in a Murine Model

Abstract Introduction Previous burn and traumatic injury studies have established that adrenergic signaling is increased after burn injury and may lead to an impairment of hematopoietic cell development in the bone marrow (BM). Nonetheless, mesenchymal stem cells (MSCs), which have gained momentum in regenerative medicine also play a predominant role in the BM niche. Understanding the propensity of the adrenergic receptor (AR) response by MSCs can be utilized for devising targeted therapies. However, the traditional plastic adherence procedure using ex vivo culture of BM cells for several weeks may skew the actual characteristics of MSCs. Our current study focused on isolating MSCs from freshly obtained BM in a murine scald burn model with a goal to characterize the expression pattern of native AR subgroups present on BM MSCs as compared to sham mice. Methods Eight, two-month-old adult female mice were subjected to a 15% total body 3rd degree burn or sham burn. The mice were sacrificed 7 days later. Femurs were removed and total bone marrow cells were flushed out. Multi parametric flow cytometry was used to gate for cells negative for hematopoietic cell markers (CD45, CD11B) and positive for MSC markers (CD105, CD106, SSEA, Ly6A) and AR subgroups (α1, α2, β1, β2, β3). We measured the number of BM MSCs, quantified the subtypes of ARs present on MSCs, and compared the ratio of AR antibody binding per total MSC population. Results Overall the frequency of MSCs per million total BM cells decreased by 48% post-burn injury with165,300 ± 194 in sham versus 110,000 ± 30 in burn displayed as bar graph in Panel A. Over 90% of MSCs consistently express β2 AR and only 10% express α2 AR subgroup in both scald and sham burn. Presence of other subgroups ranged from 50% to 80% of MSCs as seen in histograms to the right of dotted line in Panel B. Our AR propensity score based on AR mean fluorescence intensity adjusted to total number of MSCs present was increased by 2.8-fold for α1, 2.5-fold for β1, 1.6-fold for β3, and 1.3-fold for β2 AR subgroups (Panel C). These findings indicate burn injury not only decreases the frequency of BM MSCs but also increases the affinity of certain AR subgroups present on MSCs. Since BM MSCs are the major source of cytokines, chemokines and growth factors; detailed studies on AR mediated signaling in BM MSCs is warranted. Conclusions Polarization of AR signaling in BM MSCs by burn-induced catecholamines may have broader implications for comorbidities such as bone resorption and muscle wasting observed in human patients post burn trauma.

In vivo HSPC gene therapy with base editors allows for efficient reactivation of fetal γ-globin in β-YAC mice

AbstractBase editors are capable of installing precise genomic alterations without creating double-strand DNA breaks. In this study, we targeted critical motifs regulating γ-globin reactivation with base editors delivered via HDAd5/35++ vectors. Through optimized design, we successfully produced a panel of cytidine and adenine base editor (ABE) vectors targeting the erythroid BCL11A enhancer or recreating naturally occurring hereditary persistence of fetal hemoglobin (HPFH) mutations in the HBG1/2 promoter. All 5 tested vectors efficiently installed target base conversion and led to γ-globin reactivation in human erythroid progenitor cells. We observed ~23% γ-globin protein production over β-globin, when using an ABE vector (HDAd-ABE-sgHBG-2) specific to the –113A>G HPFH mutation. In a β-YAC mouse model, in vivo hematopoietic progenitor/stem cell (HSPC) transduction with HDAd-ABE-sgHBG-2 followed by in vivo selection resulted in >40% γ-globin+ erythrocytes in the peripheral blood. This result corresponded to 21% γ-globin production over human β-globin. The average –113A>G conversion in total bone marrow cells was 20%. No alterations in hematological parameters, erythropoiesis, and bone marrow cellular composition were observed after treatment. No detectable editing was found at top-scoring, off-target genomic sites. Bone marrow lineage–negative cells from primary mice were capable of reconstituting secondary transplant-recipient mice with stable γ-globin expression. Importantly, the advantage of base editing over CRISPR/Cas9 was reflected by the markedly lower rates of intergenic HBG1/2 deletion and the absence of detectable toxicity in human CD34+ cells. Our observations suggest that HDAd-vectorized base editors represent a promising strategy for precise in vivo genome engineering for the treatment of β-hemoglobinopathies.

Trilaciclib dose selection: an integrated pharmacokinetic and pharmacodynamic analysis of preclinical data and Phase Ib/IIa studies in patients with extensive-stage small cell lung cancer

PurposeTrilaciclib is a first-in-class CDK4/6 inhibitor that transiently arrests hematopoietic stem and progenitor cells (HSPCs) in the G1 phase of the cell cycle to preserve them from chemotherapy-induced damage (myelopreservation). We report integrated analyses of preclinical and clinical data that informed selection of the recommended Phase II dose (RP2D) used in trilaciclib trials in extensive-stage small cell lung cancer (ES-SCLC).MethodsA semi-mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model developed from preclinical data guided selection of an optimal dose for G1 bone marrow arrest in a first-in-human Phase I study (G1T28-1-01). PK, PD, safety, and efficacy data from G1T28-1-01 and two Phase Ib/IIa studies (G1T28-02/-03) in ES-SCLC were analyzed to support RP2D selection.ResultsModel simulation of bone marrow arrest based on preclinical data predicted that a ≥ 192 mg/m2 dose would induce a 40–50% decrease in total bone marrow proliferation in humans and almost 100% cell cycle arrest of cycling HSPCs. Consistent with this model, analysis of bone marrow aspirates in healthy volunteers after trilaciclib 192 mg/m2 administration demonstrated almost 100% G1 arrest in HSPCs and 40% decrease in total bone marrow proliferation, with minimal toxicity. G1T28-02/-03 reported similar PK parameters with trilaciclib 200 mg/m2 but slightly lower exposures than expected compared with healthy volunteers; consequently, 240 and 280 mg/m2 doses were also tested to match healthy volunteer exposures. Based on PK and relevant safety data, 240 mg/m2 was selected as the RP2D, which was also favored by myelopreservation endpoints in G1T28-02/-03.ConclusionIntegrated PK/PD, safety, and efficacy data support 240 mg/m2 as the RP2D for trilaciclib.ClinicalTrials.gov IdentifiersNCT02243150; NCT02499770; NCT02514447.

Flow Cytometry-Based Analysis of the Mouse Bone Marrow Stromal and Perivascular Compartment.

Bone marrow stromal cells (BMSCs) account for an extremely small percentage of total bone marrow cells; therefore, it is technically challenging to harvest a good quantity of BMSCs with good viability using fluorescence-activated cell sorting (FACS). Here, we describe the methods to effectively isolate BMSCs for flow cytometry analyses and subsequent FACS. Use of transgenic reporter lines facilitates FACS-based isolation of BMSCs, aiding to uncover fundamental characteristics of these diverse cell populations.

The state of the red bone marrow in rats depending on age in case of poisoning with lead acetate and potassium dichromate

Introduction: The current ecological situation is described by a rapid increase in air pollution through various chemical emissions. Aim: The study aims to define how the level of lead and chromium compounds affects the parameters of red bone marrow and blood in animals and how these processes depend on their age. Material and methods: To study the toxic intraday effect of heavy metals, a metal probe was used for combined priming of rats with lead acetate and potassium dichromate. The indicators were analyzed with the use of a cytogram, and the indices of the ratio between young and mature forms were calculated. The obtained factual material was subjected to computer processing with the calculation of the Student’s criterion and confidence intervals. Results and discussion: It was established that in young rats there is an accumulation of polychromatophilic cells with a large amount of hemoglobin, which leads to a violation of their maturation – with that, the subsequent stage of division passes very quickly, when, upon transforming into orthochromatic normoblasts, they pass into the blood after 15–20 h, forming a variant of terminal division. In old animals, there was a decrease in total bone marrow cellularity and severe anemia. Conclusions: After the experiments and profound analysis, the author of the study defined the difference of toxic effects on young and old animals.

Which Bone Marrow Sparing Strategy and Radiotherapy Technology Is Most Beneficial in Bone Marrow-Sparing Intensity Modulated Radiation Therapy for Patients With Cervical Cancer?

BackgroundTo evaluate the dosimetric parameters of different bone marrow sparing strategies and radiotherapy technologies and determine the optimal strategy to reduce hematologic toxicity associated with concurrent chemoradiation (cCRT) for cervical cancer.MethodsA total of 15 patients with Federation International of Gynecology and Obsterics (FIGO) Stage IIB cervical cancer treated with cCRT were re-planned for bone marrow (BM)-sparing plans. First, we determined the optimal BM sparing strategy for intensity modulated radiotherapy (IMRT), including a BMS-IMRT plan that used total BM sparing (IMRT-BM) as the dose-volume constraint, and another plan used os coxae (OC) and lumbosacral spine (LS) sparing (IMRT-LS+OC) to compare the plan without BM-sparing (IMRT-N). Then, we determined the optimal technology for the BMS-IMRT, including fixed-field IMRT (FF-IMRT), volumetric-modulated arc therapy (VMAT), and helical tomotherapy (HT). The conformity and homogeneity of PTV, exposure volume of OARs, and efficiency of radiation delivery were analyzed.ResultsCompared with the IMRT-N group, the average volume of BM that received ≥10, ≥20, ≥30, and ≥40 Gy decreased significantly in both two BM-sparing groups, especially in the IMRT-LS+OC group, meanwhile, two BMS-IMRT plans exhibited the similar effect on PTV coverage and other organs at risk (OARs) sparing. Among three common IMRT techniques in clinic, HT was significantly less effective than VMAT and FF-IMRT in the aspect of BM-Sparing. Additionally, VMAT exhibited more efficient radiation delivery.ConclusionWe recommend the use of VMAT with OC and LS as separate dose-volume constraints in cervical cancer patients aiming at reducing hematologic toxicity associated with cCRT, especially in developing countries.

Adult blood stem cell localization reflects the abundance of reported bone marrow niche cell types and their combinations

AbstractThe exact localization of hematopoietic stem cells (HSCs) in their native bone marrow (BM) microenvironment remains controversial, because multiple cell types have been reported to physically associate with HSCs. In this study, we comprehensively quantified HSC localization with up to 4 simultaneous (9 total) BM components in 152 full-bone sections from different bone types and 3 HSC reporter lines. We found adult femoral α-catulin-GFP+ or Mds1GFP/+Flt3Cre HSCs proximal to sinusoids, Cxcl12 stroma, megakaryocytes, and different combinations of those populations, but not proximal to bone, adipocyte, periarteriolar, or Schwann cells. Despite microanatomical differences in femurs and sterna, their adult α-catulin-GFP+ HSCs had similar distributions. Importantly, their microenvironmental localizations were not different from those of random dots, reflecting the relative abundance of imaged BM populations rather than active enrichment. Despite their functional heterogeneity, dormant label-retaining (LR) and non-LR hematopoietic stem and progenitor cells both had indistinguishable localization from α-catulin-GFP+ HSCs. In contrast, cycling juvenile BM HSCs preferentially located close to Cxcl12 stroma and farther from sinusoids/megakaryocytes. We expect our study to help resolve existing confusion regarding the exact localization of different HSC types, their physical association with described BM populations, and their tissue-wide combinations.

Immunomodulatory Activity of the Marine Sponge, Haliclona (Soestella) sp. (Haplosclerida: Chalinidae), from Sri Lanka in Wistar Albino Rats: Immunosuppression and Th1-Skewed Cytokine Response.

Natural secondary metabolites of sponges of the genus Haliclona are associated with an array of biological activity with therapeutic usage. We investigated the immunopharmacological properties of a presumably novel marine sponge species from Sri Lanka, Haliclona (Soestella) sp. Sponge material was collected from southern Sri Lanka by scuba diving. Sponge identification was based on spicule and skeleton morphology using light microscopy. Selected in vivo and ex vivo tests investigated nonfunctional and functional immunomodulatory activity of the Haliclona (Soestella) sp. crude extract (HSCE) in the Wistar rat model. Compared to the controls, rats orally gavaged daily for 14 consecutive days with 15 mg/kg dose of the HSCE manifested a significant reduction of immune cell counts of total WBCs (by 17%; p < 0.01), lymphocytes (38%), platelets (52%), splenocytes (20%), and bone marrow cells (BMC; 60%) (p < 0.001), with a concurrent increase in the neutrophil : lymphocyte ratio (p < 0.05); RBC counts abated by 53% (p < 0.001). A significant reduction of the splenosomatic index was evident with the 10 and 15 mg/kg doses (p < 0.001). Rat plasma TNF-α cytokine level was augmented by tenfold (p < 0.001), IL-6 level by twofold (p < 0.01) with the 15 mg/kg HSCE treatment, while IL-10 was detectable in rat plasma only with this treatment; the corresponding Th1 : Th2 cytokine ratio (TNF-α : IL-10) was indicative of an unequivocal Th1-skewed cytokine response (p < 0.01). Ex vivo bone marrow cell and splenocyte proliferation were significantly and dose dependently impaired by HSCE (IC50 0.719 and 0.931 μg/mL, respectively; p < 0.05). Subacute toxicity testing established that HSCE was devoid of general toxic, hepatotoxic, and nephrotoxic effects. In conclusion, HSCE was orally active, nontoxic, and effectively suppressed nonfunctional and functional immunological parameters of Wistar rats, suggestive of the potential use of the HSCE as an immunosuppressant drug lead.