Total Bone Marrow Cells Research Articles

The characterization analysis of pathogenic T cells in immune-mediated aplastic anemia mouse model

目的通过DsRed小鼠淋巴细胞输注加全身辐照（TBI）建立免疫介导的再生障碍性贫血（AA）C.B10-H2b/LilMcd (C.B10)小鼠模型，分析C.B10 AA小鼠模型中病理性T细胞在不同器官的动态归巢过程及特征。方法通过异体淋巴细胞输注加TBI建立AA小鼠模型，以TBI组为对照，分析外周血细胞和骨髓单个核细胞数，验证造模效果。在造模的第3、6、9、12天，处死小鼠并收集骨髓、脾脏、淋巴结和胸腺单个核细胞，流式细胞术分析DsRed+ T细胞的动态变化及效应记忆T细胞（TEM）、中央记忆T细胞（TCM）、初始T细胞（naïve T）和效应T细胞（TEFF）的比例。PCR Array分析AA小鼠不同组织内DsRed+病理性T细胞活化相关基因的表达谱。结果与TBI组相比，AA组小鼠在造模第9、12天出现严重骨髓衰竭，第12天时骨髓有核细胞数、外周血细胞数均显著减少（P值均<0.05）。AA组小鼠骨髓、脾脏、淋巴结内DsRed+ T细胞比例随时间增加而增加。骨髓中的DsRed+ T细胞比例在第3、6天低于脾脏与淋巴结，但在第12天时高于脾脏与淋巴结（P值均<0.05）。整个骨髓衰竭形成过程中，胸腺中DsRed+ T细胞比例均最低。在第12天时，AA小鼠骨髓、淋巴结和脾脏单个核细胞中，DsRed+CD3+CD4+ T细胞比例分别为（91.38±2.10）％、（39.78±6.98）％、（67.87±12.77）％，三者之间两两比较均有统计学意义（P值均<0.05）；DsRed+CD3+CD8+ T细胞比例分别为（98.21±1.49）％、（94.06±4.20）％、（96.29±1.23）％，差异均无统计学意义（P值均>0.05）。第9、12天，AA组小鼠骨髓中几乎所有的DsRed+CD4+ T或DsRed+CD8+ T细胞均为TEM，而淋巴结中包括TEM、TCM以及naïve T。PCR Array结果显示：骨髓中DsRed+CD4+或DsRed+CD8+ T细胞中CD38、IFN-γ、LAG3、CSF1、SPP1及TNFSF13B表达增高，但 DsRed+CD4+ T细胞中FOXP3、CTLA4表达降低。结论DsRed小鼠淋巴细胞输注可导致C.B10小鼠发生骨髓衰竭，并可以在该AA模型小鼠中追踪DsRed+病理性T细胞。在C.B10 AA模型小鼠体内，DsRed+CD8+和DsRed+CD4+ T细胞先归巢到脾脏与淋巴结，在其中扩增分化，最终转运至骨髓并转化为TEM。胸腺对异体T细胞的归巢过程更具抵抗性，提示胸腺在免疫介导AA模型的骨髓衰竭过程中可能是一个免疫豁免部位。在C.B10 AA模型小鼠的骨髓衰竭形成过程中，归巢到骨髓的DsRed+病理性T细胞较迁移到脾脏与淋巴结的细胞具有更强的免疫活性，而免疫抑制活性则减弱。

Assessment of the Toxicity of Aluminum Oxide and Its Nanoparticles in the Bone Marrow and Liver of Male Mice: Ameliorative Efficacy of Curcumin Nanoparticles.

The potential influence of nanoparticles (NPs) on the liver and bone marrow has received attention. The aim of this work was to evaluate the effect of nanocurcumin on the oxidative stress, apoptosis, and toxicity induced by Al2O3 and its NPs. The experimental animals (n = 72 mice) were divided into the following groups: group I, as a control; groups II and III, as aluminum oxide and its NPs (6 mg/kg); group IV, as aluminum oxide + nanocurcumin (Al2O3 + N-Cur, 20 mg/kg); and group V, as aluminum oxide NPs + nanocurcumin (Al2O3-NP + N.Cur., 20 mg/kg). Al2O3 and its NP groups significantly increased p53, Nrf2 levels, and the white blood cell count. They also decreased the Hsp70 level, antitrypsin, immunoglobulin G, and the red blood cell count. In addition, they significantly decreased the total and differential bone marrow cell counts and the maturation index ratio (MIR). Nanocurcumin (N.Cur.) reverted the previous proteins, blood parameters, total bone marrow cell count, and the MIR as M/E, I/Mg, MMI, I/Me, and EMI to normal. Furthermore, N.Cur. prevented apoptosis and reduced the histopathological score and collagen fiber percentage caused by Al2O3 and its NPs in the liver. Nanotechnology was used to increase the therapeutic efficiency of curcumin against the harmful effects of oxidative stress associated with Al2O3 NPs.

Clinical Outcome and 8-Year Follow-Up of Alveolar Bone Tissue Engineering for Severely Atrophic Alveolar Bone Using Autologous Bone Marrow Stromal Cells with Platelet-Rich Plasma and β-Tricalcium Phosphate Granules.

Background: Although bone tissue engineering for dentistry has been studied for many years, the clinical outcome for severe cases has not been established. Furthermore, there are limited numbers of studies that include long-term follow-up. In this study, the safety and efficacy of bone tissue engineering for patients with a severely atrophic alveolar bone were examined using autogenous bone marrow stromal cells (BMSCs), and the long-term stability was also evaluated. Methods: BMSCs from iliac bone marrow aspirate were cultured and expanded. Then, induced osteogenic cells were transplanted with autogenous platelet-rich plasma (PRP) and β-tricalcium phosphate granules (β-TCP) for maxillary sinus floor and alveolar ridge augmentation. Eight patients (two males and six females) with an average age of 54.2 years underwent cell transplantation. Safety was assessed by monitoring adverse events. Radiographic evaluation and bone biopsies were performed to evaluate the regenerated bone. Results: The major population of transplanted BMSCs belonged to the fraction of CD34−, CD45dim, and CD73+ cells, which was only 0.065% of the total bone marrow cells. Significant deviations were observed in cell growth and alkaline phosphatase activities among individuals. However, bone regeneration was observed in all patients and the average bone area in the biopsy samples was 41.9% 6 months following transplantation, although there were also significant deviations among each case. No adverse events related to the transplants were observed. In the regenerated bone, 27 out of 29 dental implants were integrated. Dental implants and regenerated bone were stable for an average follow-up period of 7 years and 10 months. Conclusions: Although individual variations were observed, the results showed that bone tissue engineering using BMSCs with PRP and β-TCP was feasible for patients with severe atrophic maxilla throughout a long-term follow-up period and was considered safe. However, further studies with a larger number of cases and controls to confirm the efficacy of BMSCs and the development of a protocol to establish a reproducible quality of stem cell-based graft material will be required.

Residual effects of busulfan and irradiation on murine hematopoietic stem and progenitor cells

Exposure of young C57BL/6 (B6) mice to two courses of busulfan (BSF) injections or two rounds of sublethal total-body irradiation (TBI) induced significant damage to the function of hematopoietic stem and progenitor cells (HSPCs). Fifteen weeks after treatment, BSF- and TBI-treated mice had reduced white blood cells without significant change in red blood cells or platelets, indicating that BSF and TBI hematotoxicity was chronic, with leukocytes being the major targets. Hematopoietic damage induced by BSF or TBI persisted long term. Residual adverse effects were reflected by significantly decreased CD45R B cells and reduced recovery of total bone marrow cells, especially HSPCs carrying markers for KSL (Kit+Sca-1+Lin-) cells, multipotent progenitor (MPP) cells (KSLCD34+CD135+), myeloid progenitor (MP) cells (Kit+Sca-1-Lin-), and common lymphoid progenitor (CLP) cells 62 wk posttreatment. Transplantation of bone marrow (BM) cells from BSF and TBI donors at 49 weeks after treatment into lethally irradiated hosts resulted in decreased engraftment of CD45R B cells in blood and reduced reconstitution of BM HSPCs including KSL cells, short-term hematopoietic stem cells (KSLCD34+CD135-), MPP cells, and MP cell subsets. TBI donor had better reconstitution of CLP cells in recipients posttransplantation than did BSF donor, suggesting an impact of TBI and BSF on B cells at different development stages. In summary, BSF and TBI exposure produced long-lasting adverse effects on hematopoiesis with pronounced effects on mature B cells, immature ST-HSCs, and hematopoietic progenitor cells. Our results may have implications for therapy of human diseases.

Granulocytic Myeloid-Derived Suppressor Cells in Murine Models of Immune-Mediated Bone Marrow Failure

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of immature myeloid cells with immunoregulatory function. Limited published studies have reported conflicting data concerning the effects of MDSCs on autoimmune diseases and graft-versus-host disease. MDSCs can be divided into two major subsets, more abundant granulocytic (G-MDSCs) and monocytic (M-MDSCs). We examined G-MDSCs in murine models of human bone marrow failure (BMF).We first characterized bone marrow (BM) MDSCs from C.B10 mice. CD11b +Ly6G +Ly6C low G-MDSCs suppressed in vitro proliferation of both CD4 and CD8 T cells from C57BL/6 (B6) mice, while Ly6G +Ly6C - cells had no effect and Ly6G -Ly6C + cells increased T cell proliferation (Fig. 1A). We then tested G-MDSCs in vivo utilizing antibody-mediated cell depletion. Lymph node (LN) cells from B6 donor mice were injected into sub-lethally irradiated major histocompatibility-mismatched CByB6F1 mice to induce BMF. Anti-Ly6G antibody injection worsened cytopenias and BM hypoplasia, and they increased BM CD4 and CD8 T cell infiltration. In contrast, anti-Ly6G antibody injection in the minor histocompatibility-mismatched C.B10 BMF model improved platelet counts and reduced BM CD8 T cells. The pathogenic and protective effects in the two models correlated with differential anti-Ly6G antibody modulation of G-MDSCs: in the CByB6F1 model, anti-Ly6G antibody eradicated G-MDSCs in blood and BM while in the C.B10 model the same antibody generated a novel G-MDSC cell population, of identical Ly6C lowCD11b + phenotype but intermediate Ly6G expression, which was not present in the CByB6F1 animals after antibody injection. When we examined the efficacy of G-MDSCs in C.B10 BMF: Ly6G + cells were enriched from BM of normal C.B10 donors (94%-97% Ly6C lowLy6G +CD11b +), and injected at the time of marrow failure initiation. Mice infused with Ly6G + cells had significantly higher levels of WBC, RBC, platelets, and total BM cells, decreased BM CD4 and CD8 T cell infiltration, and improved BM cellularity. These results indicated a protective role of G-MDSCs. When G-MDSCs were injected at day 3 after LN cell infusion, treated mice again had higher levels of WBC, RBC, platelets, and total BM cells at day 14, alleviating BMF. As both prophylaxis and therapy, G-MDSCs decreased Fas expression and Annexin V binding of residual BM cells, suppressed intracellular levels of gamma interferon and tumor necrosis factor alpha, as well as cell proliferation protein Ki67 levels in BM CD4 and CD8 T cells, relative to BMF control mice. TotalSeq simultaneously detecting surface proteins and mRNA expression in whole BM mononuclear cells in the therapy model showed an increased proportion of myeloid cells and reduced proportion of T cells in marrow from G-MDSC-treated mice based on cell surface markers and marker gene expression (Fig. 1B). Gene pathway analysis revealed down-regulation of Fas expression and reduced program cell death in total BM cells and decreased expression of genes related to cell cycle in infiltrating T cells from Ly6G + cell-treated mice-both results consistent with suppression by G-MDSCs of T cell proliferation and protection of target BM cells from apoptosis. In vitro culture of T cells from B6 mice with G-MDSCs which had been isolated from C.B10 BM cells showed dose-dependent suppression of T cell proliferation.In conclusion, our results demonstrate an active role of G-MDSCs in protecting BM from immune-mediated destruction, by suppression of T cell proliferation in the BM. G-MDSCs might have clinical application as treatment in human aplastic anemia and other immune-mediated and autoimmune diseases. [Display omitted] DisclosuresYoung: Novartis: Research Funding.

Histone Hypoacetylation-Driven CD9 Repression Arrests Differentiation and Evades Immunosurveillance in Pediatric Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a genetically heterogeneous hematologic malignancy characterized by uncontrolled proliferation of myeloid progenitor cells accompanied by impaired terminal differentiation. Despite intensive treatment regimes, the clinical outcomes remain poor, underscoring the need to decipher the underlying pathology and implement therapeutic interventions. Emerging evidence suggest myeloblasts could evolve machineries to evade T cell patrol and hinder immunotherapies. Here, we present a new mechanism driving immune escape in the context of pediatric AML, based on our discoveries of CD9 in hematopoietic stem cells (HSC; Leung et al, Blood, 2011) and acute lymphoblastic leukemia (ALL; Leung et al, Leukemia, 2020).We first examined CD9 expression and its prognostic impact in patient cohorts of childhood leukemia. The expression of cell surface CD9 on blasts of pediatric AML patients (13.2%, n=81) was significantly lower than that of pediatric ALL patients (90.4%, n=181, P<0.001) or that on CD34+ HSC of normal bone marrow donors (48.4%, n=22, P=0.014). Among pediatric AML cases, the blasts of 32 patients (39.5%) were CD9+. The 5-year RFS rate of CD9- patients was significantly lower than CD9+ patients (34.1% vs. 61.2%, P=0.018).Enforced CD9 expression in MV4-11 cells significantly suppressed proliferation (P<0.01), Ki-67 expression (P=0.041) and colony formation (P=0.002). NOD/SCID mice transplanted with CD9+ cells exhibited a drastic reduction of leukemic load in the bone marrow, spleen, blood and liver by 70.7-91.8% (P<0.05), a significantly prolonged survival duration (P<0.001), and a marked regression of extramedullary myeloid sarcoma when compared with animals transplanted with CD9- MV4-11 cells.A marked decrease of H3K9/27Ac occupancy in the CD9 locus was observed in AML than in ALL cells (4.8-14.2-fold, P<0.05), and strongly correlated with CD9 repression (r=0.585-0.719, P<0.01). Exposure of CD9- AML cell lines (n=8) and samples (n=9) with a histone deacetylase inhibitor panobinostat significantly elevated CD9 mRNA and protein expression (3.1-32.2-fold, P<0.05), restored activating histone acetylation marks (4.1-41.6-fold, P<0.05) and suppressed myeloblast proliferation ex vivo (median IC50: 21.4 nM).Mechanistically, global transcriptome profiling of pediatric AML (n=31) revealed decreased stemness (NES: -1.7, P=0.01) and increased monocyte (NES: 1.8, P=0.034) gene signatures in CD9+ patient samples. Moreover, single-cell transcriptomic analyses of total bone marrow cells from MV4-11-tranplanted mice detected a significant enrichment of differentially regulated genes functioning in the antigen processing and presentation pathway. Concordantly, we observed a profound up-regulation of CD9 (9.4-51.1-fold, P<0.001) in PMA-mediated monocyte/macrophage-like AML differentiation cultures preceding the appearance of lineage markers CD14, CD36 and iCD68 as well as the antigen presentation molecule MHC-I. In the overexpression system, CD9 not only elevated the expression of monocytic markers, but also promoted basal and IFNγ-induced MHC-I/II expression (P<0.01) through the JAK/STAT axis. Inter-patient comparisons of bone marrow samples (n=27) revealed a higher MHC-I expression in CD9+ than CD9- AML (MFI: 61820 vs. 18601, P<0.001). Interestingly, tetraspanin CD9 physically bound to MHC-I/II and formed an immune complex as revealed by co-immunoprecipitation. In NSG mice, co-transplantation of human PBMCs mounted an effective immunity against CD9+ but not CD9- AML (MV4-11 and MOLM-13), concomitant with a robust bone marrow infiltration of cytotoxic T cells. Syngeneic transplantation in immunocompetent mice, AML/T cell co-cultures, antigen-specific assays and panobinostat priming/T cell adoptive transfer are currently underway to fully dissect the role of CD9 in leukemia immunity.Taken together, our data provided molecular, cellular and clinical evidence showing the plausible function of CD9 as a key driver intertwining differentiation and immunosurveillance in pediatric AML, and inspired a new combinatorial epigenetic/immunotherapy for this rare but aggressive malignancy. DisclosuresNo relevant conflicts of interest to declare.

Abstract 1887: Anti-CD38/CD28xCD3 trispecific T cell engager induces proliferation of primary T cells and mediates potent killing of primary malignant plasma cells isolated from Multiple Myeloma bone marrow aspirates

Abstract Despite recent breakthrough in the treatment of multiple myeloma (MM) relapses are frequent, highlighting the need for novel approaches. Here we describe a novel anti-CD38/CD28xCD3 trispecific T cell engager (TCE) that targets CD38 expressed by malignant plasma cells and activates T cells by engaging CD3 and CD28 (1). Using bone marrow (BM) aspirates from MM patients, we confirmed that binding to malignant plasmocytes is primarily driven by the expression of CD38 and showed ex vivo that CD38/CD28xCD3 TCE induces a dose-dependent proliferation of CD4+ and CD8+ effector T cells. We also showed that MM patients' primary T cells were stimulated by CD38/CD28xCD3 TCE resulting in RPMI 8226 MM cell line lysis in co-culture assays. Killing of tumor cells was accompanied by MM effector CD8+ T cell degranulation and immunostimulatory cytokine production (IFN-γ and TNF-α). Finally, using MM patients' total BM cells, we showed that CD38/CD28xCD3 TCE induced the activation and cytokine production by cytotoxic CD8+ T cells and the CD38-dependent depletion of autologous primary malignant plasmocytes. Importantly, treatment of total BM cells with CD38/CD28xCD3 TCE also led to a CD38-dependent depletion of immunosuppressive myeloid derived suppressor cells (MDSC) that express CD38. Overall, these data show that CD38/CD28xCD3 trispecific TCE is active against primary myeloma cells and support the current evaluation of this next generation anti-CD38 multi-specific antibody in phase I clinical trial in patients with relapsed/refractory MM. (1) Wu, L., Seung, E., Xu, L. et al. Trispecific antibodies enhance the therapeutic efficacy of tumor-directed T cells through T cell receptor co-stimulation. Nat Cancer 1, 86-98 (2020). Citation Format: Nizar El-Murr, Sahar Kassem, Nadège Carrié, Christophe Henry, Angela Virone-Oddos, Zhi-yong Yang, Dmitri Wiederschain, Helgi Van de Velde, Marielle Chiron, Ludovic Martinet. Anti-CD38/CD28xCD3 trispecific T cell engager induces proliferation of primary T cells and mediates potent killing of primary malignant plasma cells isolated from Multiple Myeloma bone marrow aspirates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1887.

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.

Role of GATA2 in the Maintenance of the Bone Marrow Microenvironment

(Background) The bone marrow (BM) microenvironment comprises multiple stem cell niches derived from BM mesenchymal stem cells (BM-MSCs). Previous in vitro analyses have suggested that transcription factor GATA2 plays an important role in adipocyte differentiation and hematopoietic supporting activity of BM-MSCs (Kamata et al. Haematologica 2014). However, the role of GATA2 in BM-MSCs in vivo remains uncharacterized.(Method) Mice harboring a Gata2 exon 5 flanked by loxP sites (Gata2 -floxed mice, designated Gata2flox) were a kind gift from Dr. Camper (Charles et al. Molecular Endocrinology 2006). Gata2 was deletedin under the MSC-related promoters (Nestin , Prx1 , and Lepr ) (Matsuzaki et al. Cell Stem Cell 2014) based on the Cre-loxP system (Gata2f/f/ Nes -ER-Cre, Gata2f/f/ Prx1 -Cre, and Gata2f/f/ Lepr -Cre mice) (The Jackson Laboratory). For transplantation analysis, 2x105 BM cells from SJL (CD45.1+; The Jackson Laboratory) mice were intravenously injected into lethally irradiated CD45.2+Gata2flox/flox/ER-Cre or CD45.2+Gata2flox/flox mice (a total of 10 Gy). The recipient mice were injected intraperitoneally with 1 mg of tamoxifen (Sigma, USA) on days 29-31 and 36-38 after transplantation and subsequently analyzed on days 50-51. For the analysis, we focused on the CD45.1 donor cell fraction. To isolate MSCs, the compact bones were crushed with a pestle and treated with collagenase (Wako, Japan), after which Ter119-CD45- MSC fraction was separated using EasySep Mouse Mesenchymal Stem/Progenitor Cell Enrichment Kit (StemCell Technologies, Canada). BM hematopoietic progenitors were separated and evaluated with both MACS (Miltenyi Biotech) and BD FACSAria II (BD Biosciences). For transcription profiling, a GeneChip Mouse Gene 2.0 ST Array (Affymetrix, USA) was used and the data was subsequently analyzed with the DAVID Bioinformatics Program (http://david.abcc.ncifcrf.gov/).(Results) After confirming Gata2 deletion under each BM-MSC-related promoter (Nestin , Prx1 , and Lepr ), we evaluated the hematological profile, total BM cell counts, BM progenitor frequency (LSK, CMP, GMP, MEP, and CLP), colony assay with sorted LSK fraction, BM histology, and colony-forming cell assay of BM-MSCs. Whereas we found a significant decrease in the colony number of Gata2flox/flox/ Prx1 -Cre mice, the phenotypic alterations in BM-MSC-specific conditional Gata2 knockout mice are somewhat weak, implying that Gata2 deletion with a single marker might be insufficient. Thus, we next transplanted normal BM cells into irradiated Gata2flox/flox/ER-Cre mice, after which Gata2 was knocked out by tamoxifen administration. In this case, Gata2 would be deleted in all cell types constituting the BM microenvironment but not the hematopoietic cells. Based on the analyses of the CD45.1+ donor cell fraction, we found a significant decrease in the percentage of the CMP fraction, whereas that of CLP was marginally increased (p = 0.081) by Gata2 knockout, suggesting that GATA2 expression in BM-MSCs might function in the differentiation of LSKs to CMPs.To elucidate the underlying molecular mechanisms, we performed expression profiling with the MSCs obtained from Gata2flox/flox/ER-Cre mice. Prior to the profiling analysis, RT-PCR analysis confirmed that the separated MSCs abundantly expressed MSC-related genes, including Nestin , Prx1 , Lepr , Cd29 , and Sca-1 . Based on the average of 2 independent datasets, we demonstrated that GATA2 knockout activated 110 and repressed 141 genes (> 2-fold). The repressed gene ensemble included adhesion molecules (Itga11 , Itgb3 ), extracellular matrix (Prg3 ), and genes involved in hematopoietic progenitor mobilization (Mmp9 , Elane , Ctsg ). Activated genes included Fabp5 , involved in fatty acid metabolism, suggesting that GATA2 knockout might accelerate adipocyte differentiation in vivo . Gene Ontology analysis revealed significant enrichment of genes related to cell adhesion (P = 4.8E−05) and chemotaxis (P = 2.7E−08), suggesting that the expressions for extracellular signals in BM-MSCs might be regulated by GATA2.(Conclusion) Besides its critical role in hematopoietic stem cells, GATA2 could also affect the function of BM-MSCs in vivo . Our data might offer a better understanding of the pathophysiology of bone marrow failure syndrome. 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.

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.

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.

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.

Insulin-like growth factor-1 short-period therapy stimulates bone marrow cells in obese swiss mice.

Bone marrow cells (BMCs) from obese Swiss mice fed with Western diet show mitochondrial dysfunction. Obesity interferes with BMCs disrupting energetic metabolism, stimulating apoptosis, and reducing cell proliferation since adipose tissue releases inflammatory adipokines into the medullar microenvironment. These changes lead to reduction of BMC differentiation capacity and hematopoiesis impairment, a process responsible for blood cell continuous production through hematopoietic stem cells (HSCs). This work aimed to analyze the effects of IGF-1 therapy on BMC viability in Western diet-induced obesity, in vivo. We observed that after only 1week of treatment, obese Swiss mice presented reduced body weight and visceral fat and increased mitochondrial oxidative capacity and coupling, indicating mitochondrial function improvement. In addition, IGF-1 was able to reduce apoptosis of total BMCs, stem cell subpopulations (hematopoietic and mesenchymal), and leukocytes, restoring all progenitor hematopoietic lineages. The treatment also contributed to increase proliferative capacity of hematopoietic stem cells and leukocytes, keeping the hematopoietic and immune systems balanced. Therefore, we conclude that IGF-1 short period therapy improved BMC survival, proliferation, and differentiation capacity in obese Swiss mice.

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.

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.

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.

Megakaryocyte-Derived TGF-β1 Negatively Regulates Megakaryopoiesis in Mice

Megakaryocytes (MK) and platelets contain a high concentration of transforming growth factor β1 (TGFβ1). Mice with conditional deletion of Tgfb1 in megakaryocytes (PF4Cre/Tgfb1flox/flox) resulted in &amp;gt;90% reduction of TGFβ1 in platelets and 50% reduction of TGFβ1 in plasma. TGFβ1 has been shown to play an inhibitory role in megakaryopoiesis in vitro, and inhibiting TGFβ1 increased megakaryopoiesis in vivo. However, the source of TGFβ1 in megakaryopoiesis is unknown. In this study, we tested whether megakaryocyte-derived TGFβ1 contributes to megakaryopoiesis in bone marrow (BM) by comparing three groups of mice: PF4Cre/Tgfb1flox/flox, littermate control Tgfb1flox/flox, and WTC57Bl/6 mice. Bones (femurs) from these mice (n=12) (age 15-30 weeks, males 60% and females 40%) were harvested, fixed, decalcified, sectioned, and H&amp;E stained. Whole stained BM areas of the sectioned femurs were scanned with an Aperio slide scanner to quantify the number of megakaryocytes and the demarcation membrane system (DMS) and ploidy (nucleus size) of the megakaryocytes were quantified by manually counting megakaryocytes and tracing their DMS and nucleus. The percentage of MK among total BM cells was calculated by dividing total numbers of BM cells in the total area of a BM section with the number of MK in the section. Freshly isolated BM cells were cultured in vitro in culture medium (DMEM+10%FBS) in the presence of thrombopoietin (TPO, 100 ng/ml) with and without TGFβ1 (20 ng/ml) or with a neutralized antibody against the active form of TGFβ1 (AF-101; 2 ug/ml). TGFβ1 and TPO levels in plasma, BM exudates, and cells were measured by ELISA. PF4Cre/Tgfb1flox/floxmice had &amp;gt;50% reduction in TGFβ1 levels in BM cells and exudates (TGFβ1 levels in BM exudates were 1.4 ± 0.033 ng in WT and 0.68 ± 0.065 ng in PF4CreTgfb1flox/floxmice, p&amp;lt;0.01; and in BM cells 50 ± 9 ng/ml in WT and 22 ± 4.2 ng/ml in PF4CreTgfb1flox/flox; p&amp;lt;0.001). MK numbers were ~25% higher in PF4Cre/Tgfb1flox/floxmice (n=6) compared to combined littermate controls (n=3) and WT (n=3) (MK was 0.30 ± 0.02% in PF4Cre/Tgfb1flox/flox and 0.23 ± 0.16% in combined controls; p&amp;lt;0.001 (n=6), whereas blood platelet counts were only marginally higher in PF4Cre/Tgfb1flox/flox (1114 ± 300) vs. controls (806 ± 116; p&amp;lt;0.05). There was a ~2-fold higher plasma TPO levels in PF4CreTgfb1flox/floxmice vs. WT (p=0.04, n=4). Increased DMS and nucleus areas in MK have been shown to correlate with proplatelets formation and platelet production. However, DMS and nuclear areas remained unchanged between genotypes [(DMS area was 197 ± 46 in PF4CreTgfb1flox/flox and 228 ± 50 um2 in combined WT and littermate controls (p=0.3), and nucleus size was 154 ± 23 in PF4CreTgfb1flox/flox and 160 ± 33 um2 in controls (p=0.7)], indicating that the role of TGFβ1 is limited to megakaryopoiesis. To test whether the in vivo phenotype was recapitulated, we cultured whole BM isolated from WT and PF4Cre/Tgfb1flox/flox mice, which showed a ~2.5-fold increase in MK numbers vs. WT when cultured for 5 days in TPO-supplemented medium. The addition of recombinant TGFβ1 in culture medium inhibited MK numbers, and a neutralizing antibody against TGFβ1 resulted in increased MK numbers. We conclude that MK-derived TGFβ1 negatively regulates megakaryopoiesis in mice. Further investigation is needed to determine the mechanism by which TGFβ1 regulates TPO-induced megakaryopoiesis. Our study may be important in megakaryocyte generation in vitro and may have important implications in vivo under normal and stress-inducing conditions where variable megakaryopoiesis is observed, such as essential thrombocythemia and primary myelofibrosis. Disclosures No relevant conflicts of interest to declare.