bluuebunny/crossref_metadata_2025 huggingface dataset:
Mesenchymal Stem Cell As a Salvage Treatment For Refractory Acute Graft-Versus-Host Disease: a Matched Pair Analysis
Authors: Zhao Ke , Lou Rui , Huang Fen , Peng Yanwen , Jiang Zujun , Huang Ke , Zhang Yu , Fan Zhiping , Zhou Hongsheng , Liu Can , Xiao Yang , Sun Jing , Xiang Peng , Liu Qifa
Published: October 2019

Background

Refractory acute graft versus host disease (aGVHD) is a major cause of death after allogenetic haematopoietic stem cell transplantation (allo-HSCT). This study evaluated the efficacy and safety of mesenchymal stem cells (MSCs) from bone marrow of a third-party donor to refractory aGVHD.

Methods

Twenty-five patients with refractory aGVHD were enrolled in this prospective study. MSCs were given at a median dose of 1×106 cells/kg once weekly until aGVHD got complete response (CR) or MSCs were infused for a total of 8 doses. In order to evaluate infections, tumor relapse and cGVHD, 25 refractory aGVHD patients with MSCs treatment was matched with grade II-IV aGVHD patient without MSCs in the corresponding period

Results

After a total of 112 doses of MSCs were administered, with a median of 4 (range:2-12) doses per patient, the overall response (OR) rate for aGVHD was 72%,including CR in 56% and partial response (PR) in 16%. The efficacy of MSCs to aGVHD was significantly related with the severity and the number of involved organs of aGVHD, but unrelated with the onset timing of MSC. The incidence of CMV and EBV infections, including viremia and associated disease, was not different between MSCs and non-MSCs groups during aGVHD treatment and follow-up. At a median follow-up time of 126 (range: 49–1067) days post-transplantation, only two patients relapsed in MSCs group, compared with four relapsed in the control group. Tumor relapse was not different between the two groups (P=0.771). No toxic side effects and other secondary tumors were observed after MSCs treatment except for one EBV-associated post-transplant lymphoproliferative disorders (PTLD). The incidence of cGVHD in MSCs group, especially extensive cGVHD, was lower than that in non-MSCs group (25.0% Vs 68%, P=0.001; 20.0% Vs 64%, P=0.043, respectively). The ratio of CD3+CD4+/CD3+CD8+ T cells and the proportion of CD4+CD25+ regulatory T cells (Tregs) at 8 weeks after MSCs were higher than that before treatment (P=0.037 and P=0.020, respectively), and there were not different from the healthy people at 36 weeks after treatment.

Conclusions

MSCs derived from bone marrow of third-party donors are effective to refractory aGVHD. It might not increase the risks of infections, tumor relapse, but reduce the incidence and severity of cGVHD.

Disclosures:

Liu: Science and Technology Program of Guangzhou of China (11A72121174): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647): Research Funding; National Public Health Grand Research Foundation (Grant No. 201202017): Research Funding; 863 Program (No. 2011AA020105): Research Funding.
Pre-Transplant Weight Loss and Total Serum Protein Predict Relapse Of Acute Myeloid Leukaemia After Allogeneic Stem Cell Transplantation
Authors: Luft Thomas , Dietrich Sascha , Radujkovic Aleksandar , Stölzel Friedrich , Falk Christine , Benner Axel , Schaich Markus , Bornhäuser Martin , Ehninger Gerhard , Kraemer Alwin , Hegenbart Ute , Ho Anthony D , Dreger Peter
Published: October 2019

Background

Although allogeneic stem cell transplantation (alloSCT) represents a curative chance for higher risk acute myeloid leukaemia (AML), relapse occurs in a significant proportion of patients. The impact of nutritional status on the outcome of allogeneic stem cell transplantation (alloSCT) is controversial. This study investigates the influence of pre-transplant body mass index (BMI), weight loss, and serological indicators of nutritional homeostasis on relapse and death of acute myeloid leukemia (AML) after alloSCT.

Methods

Pre-transplant weight loss and BMI along with serum levels of total serum protein (TSP), albumin, C-reactive protein, and leptin were collected retrospectively in a training cohort of 149 patients allografted for AML and correlated with clinical outcome. A metabolic risk score was established and tested in an independent validation cohort (n=167).

Results

Pre-transplant weight loss exceeding 2%, TSP lower than 70 g/L, and decreased leptin levels were associated with a significantly increased risk of relapse and death. In contrast, we did not observe a consistent pattern for the impact of nutritional indicators on non-relapse mortality (NRM). Multivariate analyses adjusting for age, cytogenetic risk, treatment line, blast count at alloSCT, donor, and conditioning confirmed weight loss and low TSP as independent risk factors of relapse and overall survival, but not of NRM. Weight loss >2% and low TSP were used to build a metabolic score for prediction of relapse risk and mortality. In multivariate analyses with clinical confounders, patients with both metabolic risk factors had a strongly increased risk of relapse (p=0.0003, HR 15.62, 95%CI 3.51-69.71) and death (p=0.002, HR 3.64, 95%CI 1.62-8.18) compared to patients without metabolic risk factor. The risk prediction of the metabolic score could be confirmed in the validation cohort (Figure 1).

Conclusions

Altered nutritional homeostasis prior to alloSCT predicts the risk of recurrence of AML after transplantation. Studies addressing pretransplant nutritional interventions in order to reduce AML relapse rates are warranted.

Disclosures:

No relevant conflicts of interest to declare.
Autologous Transplantation Is Safe and Effective For Patients With Multiple Myeloma Related Renal Insufficiency
Authors: Greene Claire C , Hwang Jimmy , Lee Wolf Jeffrey , Damon Lloyd E , Mahindra Anuj , Martin Thomas G
Published: October 2019

Introduction

Patients (pts) with MM often present with varying degrees of renal insufficiency (RI) which can cause significant morbidity. In younger pts with MM, high-dose chemotherapy with autologous stem cell transplantation (ASCT) has been shown to enhance remission and possibly improve survival. In pts with RI there has been concern about the safety and efficacy of proceeding to ASCT. At our center, ASCT has been routinely offered to pts with RI. Herein we report the results of MM pts with RI who received ASCT since 2000.

Patients and Methods

Outcomes of 50 MM pts with RI were compared to 100 matched ASCT recipients. The matched transplant pts studied were similar with respect to subject-, disease-, and transplant-related characteristics. All pts met standard eligibility criteria for ASCT and pts with RI had a serum creatinine (Cr) level of >2 mg/dL (average Cr = 6.07 with a range of 2 to 11.79 mg/dL), at the time of ASCT. 14 of the 50 pts were on dialysis at the time of ASCT. All patients had active MM requiring therapy and all pts had evidence of stable disease or treatment response prior to transplant. For pts with RI, the average age at transplant was 56 years (range 33 to 69 yrs) and the median time between diagnosis and ASCT was 302 days. The preparative therapy consisted of single agent Melphalan, given on Day (-3) or (-2) with a median administered dose of 140 mg/m2 (range 100-200 mg/m2) while the dose for the matched patients was 200mg/m2. All pts were hospitalized for ASCT and received standard supportive care; >2 x 10e6/Kg CD34(+) cells, G-CSF, transfusions as necessary and appropriate anti-infectives.

Results

The 30-day and 100-day treatment related mortality (TRM) rates for both the patients with RI and matches were 0%. Amongst the patients with RI, 4 patients required ICU care and the average duration of hospitalization was 23.36 days (range: 14 - 48 days) and duration of hospitalization for matches 19.4 days (range: 11-58 days). Time to ANC > 0.5 x10e9/L was 12 days (range 10-21 days) and 12 days (range 10-22 days) in the RI and matched cohort respectively. Time to platelets > 20 x 10e9/L was 11 days (range: 6-22 days) and 14 days (range 8-51days) in the RI and matched cohort respectively. 32% of pts with RI were in very good partial response (VGPR)+ complete remission (CR) of which 12% were in CR at ASCT. After ASCT, 50% of pts achieved a VGPR+CR of which 34% pts achieved a CR. The mean overall survival (OS) for patients with RI was 4.2 yrs and for controls was 6.5 yrs [Figure 1. HR 2.036, 95% CI (1.204-3.443) p=0.0084].

Conclusion

ASCT is a safe and effective treatment option for MM pts with RI including those on dialysis with engraftment characteristics similar to pts without RI.

Disclosures:

Wolf: Onyx: Honoraria; Millenium: Honoraria; Celgene: Honoraria, Research Funding.
Interleukin-6 Reduces Erythroid Development and Mitochondrial Membrane Potential In Human Erythroleukemic Cells
Authors: McCranor Bryan J. , Kim MinJung , Cruz Nicole M , Xue Qian-Li , Berger Alan E. , Walston Jeremy D. , Civin Curt I. , Roy Cindy N.
Published: October 2019

Introduction

Anemia of inflammation or chronic disease is common and is associated with diseases such as cancer, chronic kidney disease, autoimmune disease, and acute and chronic infections. Concentration of the inflammatory cytokine interleukin-6 (IL-6) is known to negatively correlate with hemoglobin concentration in many of these disease states. Whether IL-6 promotes anemia of inflammation outside of the IL-6-hepcidin antimicrobial peptide axis has not been extensively studied. We previously reported that chronic inflammation affected maturation of erythroid progenitors in a mouse model of chronic inflammation but over expression of hepcidin, alone, did not. We hypothesized that IL-6 may directly impair erythroid maturation, independent of iron restriction.

Methods

We utilized the human erythroleukemia TF-1 cell line, treated with recombinant human erythropoietin, to model erythroid maturation and exposed TF-1 cells to increasing doses (0, 1, 10, 100ng/ml) of IL-6 over six days. TF-1 erythroid maturation was determined by flow cytometry (based on CD44, CD71, and CD235a expression), and benzidine staining. In addition, expression levels of genes representing early [glycophorin A (GPA)], mid [aminolevulinic acid synthase 2 (ALAS2) and hemoglobin adult major beta chain (HBB)], and late [erythrocyte membrane protein Band 3 (SLC4A1)] stages of erythropoiesis were analyzed by qRT-PCR. Finally, mitochondrial mass, membrane potential, and oxidative stress were measured using fluorescent indicators.

Results

IL-6 significantly repressed erythropoietin-dependent TF-1 erythroid maturation at concentrations at or above 10 ng/ml (p=0.001, Cuzick test). We observed impaired hemoglobin synthesis as demonstrated by decreased benzidine staining (p=0.022, Cuzick test), but this did not correspond to detectable decreases in ALAS2 or HBB expression. However, IL-6 down regulated expression of SLCA4A1 which is expressed late in erythropoiesis (p=0.005, Welch t-tes). The mitochondrial membrane potential was decreased, at all IL-6 treatment doses (p=0.05, Student’s t-test), and mitochondrial mass was significantly decreased at the highest dose (p=0.05, Student’s t-test).

Conclusions

These data demonstrate that IL-6 can impair mitochondrial membrane potential, hemoglobin production, and erythroid maturation in an in vitro setting. Our findings suggest that IL-6 affects cells relatively late in erythropoiesis, after they are primed for hemoglobin synthesis. We hypothesize that the effect of IL-6 on the mitochondria of maturing erythroid cells could be a mechanism behind the impaired maturation phenotype that we observed. These results may indicate a novel pathway of action for IL-6 in the anemia of inflammation, and demonstrate potential for the opportunity to develop new therapeutic targets that affect late erythroid development.

Disclosures:

Roy: Celgene Corp.: Research Funding.
Ligand-Regulated and Developmental Stage-Specific Expression Of The EPO Receptor During Human Erythroid Progenitor Cell Formation
Authors: Rainville Nicole , Kuhrt David , Su Su , Jachimowicz Edward , Ruth Asch , Li Lei , Green Jennifer M , Wojchowski Don M.
Published: October 2019

Upon engagement of its cell surface receptor, EPO sharply regulates erythroid progenitor cell (EPC) development. Little is known concerning the dynamics of EPOR expression, however, due to its rarity in primary cells. Using EPO dosing and response studies together with a highly specific and sensitive hEPOR antibody, we provide new insight into stage and ligand dose-dependent regulation of cell surface EPOR expression among human bone marrow derived primary EPCs. Developmentally, EPOR levels peaked within a KITposCD36posGPAneg proerythroblast cohort. In contrast to prior studies using transfected murine myeloid cell lines, the limiting of EPO substantially increased EPOR levels, demonstrating ligand dependent modulation of endogenous EPOR in hEPCs. EPO dosing and withdrawal studies further showed that in early EPCs EPO efficiently supported survival but not proliferation. In GPApos erythroblasts EPOR levels were diminished, but GPApos cells nonetheless exhibited strong EPO dependency for both survival and growth. Investigations provide novel insight into regulated EPOR surface expression among developing primary EPCs, and may also aid in understanding the action modes and effects of emerging EPOR agonists and erythropoiesis stimulating agents.

Disclosures:

No relevant conflicts of interest to declare.
Cathelicidin Mediates Anti Lymphoma Cytotoxicity By Human Macrophages
Authors: Bruns Heiko , Fabri Mario , Büttner Maike , Mougiakakos Dimitrios , Pasemann Shirin , Amann Kerstin , Mackensen Andreas , Gerbitz Armin
Published: October 2019

In high-grade lymphatic malignancies, such as Burkitt’s lymphoma (BL), macrophage infiltration represents a characteristic morphological hallmark. Although macrophages can be directly cytotoxic against tumor cells, tumor associated macrophages (TAMs) regularly fail to kill them. The mechanism responsible for this loss of function remains unknown. Here, we firstly demonstrate that M1 (classically activated) macrophages selectively kill proliferating lymphoma cells by releasing the antimicrobial peptide cathelicidin in a vitamin D dependent fashion. Moreover, we show that cathelicidin leads to cell death by targeting the mitochondria. In contrast, M2 (alternatively activated) macrophages, and M2-like TAMs in BL exhibit an altered vitamin D metabolism that leads to reduced production of cathelicidin and consequently failure to lyse lymphoma cells. However, treating M2 macrophages with 1,25D or a vitamin D receptor agonist efficiently triggers cathelicidin-mediated tumoricidal activity against BL cells. Taken together, these observations indicate that a therapeutic activation of the vitamin D pathway could restore antitumor activity of TAMs, which are an important player in the tumor stroma.

Disclosures:

Mackensen: Eli Lilly: Consultancy.
Tyrosine Phosphatase SHP-1 Acts As a Key Regulator Of Alloresponses In Primary Murine T Cells
Authors: Kardinal Christian , Herbst Jessica , Diekmann Ulf , Sauer Martin G.
Published: October 2019

Background

T cell-mediated alloresponses may result in severe transplantation-related morbidity and mortality such as graft rejection or graft versus host disease. After the adhesive interaction of a T cell and an allogeneic antigen-presenting cell an integrin-mediated, detrimental response against tissues expressing the respective alloantigen is being initiated. So far, the molecular processes underlying signaling events between T cell receptor activation and cellular adhesion are not fully understood. The vast majority of published studies was done with various tumor-derived cell lines, thus producing controversial results depending on the cell line used.

Methods

To overcome these drawbacks, we used in vitro generated polyclonal primary T cells based on the priming of naïve T cells from B10.A mice (responder) with denditic cells from C57BL/6 mice (allogeneic stimulator) or from B10.A mice (syngeneic stimulator) to study the lymphocyte function-associated antigen 1 (LFA-1)-mediated adhesion.

Results

We identified the protein tyrosine phosphatase SHP-1 as a key regulator of LFA-1-mediated adhesion. Upon alloactivation of primary T cells LFA-1-mediated adhesion was significantly higher when compared to syngeneically stimulated cells. Furthermore, elevated adhesion of alloactivated cells was accompanied by a strong decline of SHP–1 activity. To reinforce these findings, we examined the influence of a siRNA-based knockdown of SHP-1 from syngeneically and allogeneically stimulated T cells. Most notably, transfection of syngeneically stimulated cells with SHP-1-specific siRNA resulted in a two-fold increase of adhesion, when compared to cells transfected with nonsense siRNA under identical conditions. In contrast, transient overexpression of wild–type SHP-1 in allostimulated cells strongly reduced LFA–1-mediated adhesion.

Since our experiments have ascertained SHP–1 as a negative regulator of the adhesion-associated signaling cascade SLP-76 → ADAP → LFA–1, we investigated the tyrosine phosphorylation of ADAP. Western blots revealed strong tyrosine phosphorylation of ADAP in allogeneically stimulated cells, but not in syngeneically stimulated cells. Furthermore, SHP-1 binding with ADAP was significantly stronger in syngeneically stimulated cells, when compared to allogeneically stimulated cells. Besides these experiments, substrate specificity of SHP–1 to ADAP confirmed by in vitro dephosphorylation asssays with GST-SHP–1 and GST-SHP–2 fusion proteins indicate that SHP–1 modulates the binding of SLP–76 with ADAP by tyrosine dephosphorylation of ADAP.

Conclusion

These findings might be of clinical relevance, since the decisive role of SHP–1 in the regulation of LFA–1-mediated adhesion in alloresponses allows the development of novel target-specific immunosuppressive agents.

Disclosures:

No relevant conflicts of interest to declare.
Development Of Mature T Lymphocytes Requires Alpha-Synuclein
Authors: Shameli Afshin , Xiao Wenbin , Harding Clifford , Meyerson Howard , Sumodi John , Maitta Robert
Published: October 2019

Synucleins (including α-, β- and γ-synucleins) are a group of proteins that are expressed at high levels in the central nervous system. The physiologic function of these proteins is unknown. Alpha-synuclein has been implicated in the pathogenesis of neurodegenerative disorders such as Parkinson's disease and Lewy body dementia, as it is highly expressed in the Lewy bodies from both disorders. The expression of α-synuclein in hematopoietic system has been shown in erythroid precursors and megakaryocytes in bone marrow, as well as erythrocytes and platelets in peripheral blood. Moreover, some studies demonstrated the expression of α-synuclein on peripheral blood mononuclear cells (PBMC), including B and T lymphocytes, NK cells and monocytes; and its expression is shown to be higher in PBMCs of individuals with Parkinson's disease compared to healthy controls.

In order to study the role of α-synuclein in development of different hematopoietic elements, we compared bone marrow, peripheral blood and lymphoid organs of age and sex-matched α-synuclein knock-out (KO) mice and wild type (WT) animals of the same genetic background (n=10). Flow cytometric analysis of bone marrow elements did not show differences in the percentages and absolute numbers of erythroid, megakryocytic and myeloid lineages (data not shown). However, differential complete blood cell count (CBC) showed statistically significant decrease in red blood cell (RBC) count, hemoglobin (Hb) and hematocrit (Hct) in KO mice compared to WT mice. No difference was noted in other RBC indices (Table 1). However, platelets were smaller in KO mice as measured by the mean platelet volume (MPV). There was no difference in the number of platelets and white blood cell (WBC) counts. There was a significant reduction in the percentage of circulating lymphocytes, and associated increase in the percentage of neutrophils and monocytes in KO mice compared to WT mice, although the difference in the number of lymphocytes did not reach statistical significance (Table 1).

Flow cytometric analysis of T lymphocytes in thymus and peripheral lymphoid organs demonstrated marked defect in development of mature T cells. There was a significant increase in the number of double negative thymocytes in KO mice associated with significant decrease in the number of single positive T cells. Furthermore, splenic CD4+ and CD8+ T cells were markedly decreased in KO mice, indicating that α-synuclein is required for T cell development (Table 2).

In summary, our findings indicate an absolute requirement for α-synuclein in development of mature T lymphocytes. The underlying mechanism for this function is subject of future studies. Moreover, while α-synuclein-deficiency does not affect the development of myeloid lineage and platelets, lack of this protein is associated with lower number of erythrocytes, suggesting its role in development and/or survival red blood cells.

Disclosures:

No relevant conflicts of interest to declare.
Engineering The Human Thymic Microenvironment To Support Thymopoiesis
Authors: Montel-Hagen Amelie , Chung Brile , Ge Shundi , Blumberg Garrett , Klein Sam , Yuhua Zhu , Parekh Chintan , Arumugam Balamurugan , Yang Otto , Crooks Gay M.
Published: October 2019

A system that allows manipulation of the human thymic microenvironment is needed both to elucidate the extrinsic mechanisms that control human thymopoiesis, and to develop potential cell therapies for thymic insufficiency. Implantation of intact fetal thymic fragments into immune deficient mice can be used to model human thymopoiesis in vivo, but this approach has limited capacity for cell or molecular manipulation for either experimental or translational application. We developed an implantable thymic microenvironment composed of two human thymic stroma populations critical for thymopoiesis, thymic epithelial cells (TECs) and thymic mesenchyme (TM). TECs and TM from postnatal human thymi were cultured in specific conditions, allowing cell expansion and manipulation of gene expression, prior to re-aggregation into a functional thymic unit. Human CD34+ hematopoietic stem and progenitor cells (HSPC) differentiated into T cells in the aggregates in vitro and in vivo following inguinal implantation of aggregates in immune deficient mice. Cord blood HSPC previously engrafted into murine bone marrow, were able to migrate to the implants and differentiate into functional human T cells with a broad T cell receptor repertoire. Furthermore, lentiviral mediated expression of vascular endothelial growth factor in TM enhanced TEC survival and function, resulting in significantly greater thymocyte production relative to control implants. These results demonstrate an efficient in vivo system for the generation of T cells from human HSPC, and represent the first model to allow manipulation of gene expression and cell composition in the microenvironment of the human thymus.

Disclosures:

No relevant conflicts of interest to declare.
Malleable Extracellular Domains In Platelet GPIb-IX Complex
Authors: Zhou Liang , Yang Wenjun , Li Renhao
Published: October 2019

Background

Glycoprotein (GP)Ib-IX complex is critical to hemostasis and thrombosis. Its proper assembly is closely correlated with its surface expression level and requires cooperative interactions among extracellular and transmembrane domains of GPIbα, GPIbβ, and GPIX subunits. Two interfaces have been identified between 2 GPIbβ and GPIX extracellular domains.

Objective

To understand how extracellular domains interact in GPIb-IX.

Methods

The GPIbβ extracellular domain (GPIbβE) or the GPIX counterpart (GPIXE) in GPIb-IX was replaced simultaneously or individually with a GPIbβE/GPIXE chimera called GPIbβEabc (Fig1), and the effect of domain replacement on assembly and expression of the receptor complex in transiently transfected Chinese hamster ovary cells was analyzed.

Results

Five chimeric complexes, along with wild-type complex shown in Fig1, are characterized. All five chimeric complexes formed wild-type-like GPIbα-GPIbβ disulfide bonds, indicating that the interactions between extracellular domains do not affect formation of the GPIbα-GPIbβ disulfide bonds. Replacing GPIXE with GPIbβEabc in GPIb-IX retained interface 1, but not interface 2. While this domain replacement retained the ability of GPIbβ to aid expression of GPIX and the overall complex organization, the reciprocal enhancing effect of GPIX on expression of GPIbβ and GPIbα was lost. However, when the extracellular domain in GPIbβ was replaced with GPIbβEabc, surface expression level of each subunit in chimeric complex 3 and chimeric complex 4 expressing cells, was slightly lower than those in wild-type complex expressing cells, but significantly higher than those in other chimeric complexes expressing cells, indicating that chimeric complexes 3 and 4 share the same stability, but adopt different conformations. After replacing the ectodomains in both GPIbβ and GPIX with GPIbβEabc, the expression levels of GPIbα and associated subunits in chimeric complex 5 expressing cells were as low as those in chimeric complex 1 expressing cells, despite the different ability of GPIbβEabc and GPIbβE to self-associate.

Conclusions

The results demonstrate the importance of the association between GPIbβ and GPIX extracellular domains for assembly and efficient expression of GPIb-IX. In particular, interface 2 is critical for the stability of GPIbβ in the complex. However, not all the effects of extracellular domain replacement on GPIb-IX expression can be explained by the two separate interfaces, suggesting that GPIbβ and GPIX extracellular domains are malleable to accommodate and propagate potential conformational changes therein. This finding has significant implication on the molecular mechanism underlying the GPIb-IX-mediated signaling in platelets.

Disclosures:

No relevant conflicts of interest to declare.
The Parallel Signaling Pathways Of Phosphatidylserine (PS) Exposure Downstream Of Platelet FcγRIIa
Authors: Andre Pierrette , McKenzie Steven E. , Bergmeier Wolfgang
Published: October 2019

Platelet FcγRIIa is an important component of heparin-induced thrombocytopenia and other immune-mediated thrombocytopenia and thrombosis syndromes. Platelet FcγRIIa, an ITAM receptor, signals not only to cause aggregation and secretion, but also to PS exposure in the platelet procoagulant response when platelets are co-stimulated via the GCPR PARs. The molecular mechanisms downstream of FcγRIIa that lead to PS exposure are incompletely understood. Recently, we (WB; Ahmad et al., JTH 2011; 9:2077) demonstrated that downstream of PAR and GPVI/FcRγ, another ITAM receptor, there are CalDAG-GEF I (CDGI)-dependent and CDGI-independent, ADP/P2Y12-dependent parallel signaling pathways to PS exposure. In this study, we investigated the molecular signaling requirements for PS exposure downstream of FcγRIIa + PAR dual stimulation.

We studied the exposure of PS in FcγRIIa transgenic (tg) mouse platelets following dual stimulation through FcγRIIa via anti-mouse CD9 and through PAR4 via PAR4 activating peptide (PAR4AP; AYPGKF). Washed platelets from FcγRIIa-tg mice and FcγRIIa-tg/CDG1-/- mice were stimulated with varying concentrations of anti-mouse CD9 and PAR4-AP (200uM) under static conditions and immediately measured for PS exposure by labeled Annexin-V in flow cytometry. We observed that at 0.5ug/ml of anti-mouse CD9 that the PS exposure of the FcγRIIa-tg/CDG1-/- platelets is approximately 70% of the FcγRIIa-tg platelets. However, when the platelets are pre-incubated with P2Y12 inhibitor MesAMP at 100uM, the PS exposure of the FcγRIIa-tg platelets is decreased by approximately 50% (n=3). For the FcγRIIa-tg/CDG1-/- platelets in the presence of MesAMP, PS exposure is completely abolished (n=3). This indicates that CDG1 contributes part of the signal that leads to PS exposure, while ADP/P2Y12 contributes the other CDGI-independent part of PS exposure downstream of FcγRIIa and GPCR dual stimulation. At a lower concentration of anti-mouse CD9 (0.25ug/ml; near threshold), the level of PS exposure of the FcγRIIa-tg/CDG1-/- platelets is approximately 80% of the FcgRIIa-tg platelets. In addition, at 0.25 ug/ml of anti-CD9, FcγRIIa-tg/CDG1-/- platelets pre-incubated with the P2Y12 inhibitor revealed no stimulated PS exposure. This observation indicates that ADP/P2Y12 plays a more significant role in PS exposure as the concentration of FcγRIIa stimulant nears threshold.

Eradication of procoagulant PS exposure may require targeting of both the CDGI-dependent and CDGI-independent pathways for optimum therapeutic benefit in HIT and other immune-mediated thrombocytopenia and thrombosis disorders. CDGI inhibitors useful in human platelets will allow translation of these findings.

Disclosures:

No relevant conflicts of interest to declare.
Utility Of The Immature Platelet Fraction In Predicting Recovery With Or Without Treatment In Pediatric Immune Thrombocytopenia
Authors: Ghemlas Ibrahim A. , Klaassen Robert J. , Barrowman Nick , Leung Elaine W.
Published: October 2019

Background

The Immature Platelet Fraction (IPF) is a novel parameter available on the Sysmex XE-Series Hematology analyzers. It can be expressed as both IPF percent (IPF %) and absolute IPF count (AIPF#). It has been shown that IPF% is higher in Immune Thrombocytopenia (ITP) than in the normal population, and correlates with bone marrow platelet production or thrombopoiesis.

Objectives

To evaluates the utility of the IPF parameters to predict treatment response or recovery in pediatric ITP patients.

Method

This is a retrospective, single institution study performed at a 165 bed tertiary care pediatric hospital (Children's Hospital of Eastern Ontario, Ottawa, Canada). We reviewed the medical charts and electronic databases of all patients with ITP who had measured IPF parameters between June 2011 and June 2013. The standard definitions and terminology of the International Working Group were used.

Patient age, phase of ITP at the time of the study (acute, persistent, and chronic), initial platelet count, IPF%, AIPF#, and type of therapy given (including observation alone) were recorded. For patients who responded to treatment or had spontaneous recovery without treatment, we analyzed the platelet count, IPF% and AIPF# on subsequent CBCs, then looked at the course of IPF% during platelet count recovery.

Comparisons between groups were performed using Kruskal-Wallis tests or Mann-Whitney tests, as appropriate. Association between measured blood parameters was measured using the Spearman correlation coefficient. Within-patient Spearman correlations were also used to study the association between IPF% and platelet counts during recovery. The combined significance of these associations was determined using Stouffer’s method.

Results

Conclusion

IPF measurements are easily available parameters that are useful in the management of ITP in pediatric patients. This study suggests the use of AIPF# as a predictive factor for recovery in pediatric ITP patients with and without treatment, and IPF% as a predictive factor for early recovery.

Disclosures:

No relevant conflicts of interest to declare.
Angelica Polysaccharide and TPO Have a Protective Effect On Hcmv-Induced Apoptosis In Megakaryocytes
Authors: Yang Mo , Chen Jian Liang , Ye Jie yu , Li Su yi , Liang En yu , Chong Beng
Published: October 2019

Human cytomegalovirus (hCMV) infection is often associated with thrombocytopenia. Megakaryocytes may be one of the major sites of hCMV infection, then inducing this cell apoptosis. Angelica Sinensis (Danggui) is an important ingredient of many commonly used herbal Medicine for promoting blood production. Our previous study has showed that the hematopoietic effect of Angelica Sinensis is related to its constituent, angelica polysaccharide (APS) (Yang M et al, J Ethnopharma, 2009). This present study investigated the anti-apoptotic effect of APS and TPO on hCMV-induced apoptosis in megakaryocytes. Human bone marrow mononuclear cells (MNC) or megakaryocytic cell line CHRF-288-11 and hCMV AD169 strain were co-cultured in this study. hCMV significantly inhibited the formation of CFU-MK as shown in three different concentrations of viral infection groups (103, 104 and 105 pfu/ml), compared with blank control and mock control (n=10, P<0.05). hCMV also significantly inhibited the growth of CHRF cells in these three different concentrations after incubation for 3 days, which compared with control group (n=10, P<0.01). hCMV DNA and mRNA were also positively detected in CHRF cells and the cells of CFU-MK with IS-PCR and RT-PCR respectively, while it was negative in blank and mock control groups. We further studied the effect of APS and TPO on CFU-MK formation. Results showed that APS (50 ug/ml) like TPO (50 ng/ml) enhanced hCMV-reduced CFU-MK (P=0.05, n=6). CHRF cells were also analyzed by Annexin V/PI with flow cytometry at day 3 after infection with hCMV AD169. The percentage of apoptotic cells in group of 103 pfu/ml was 19.0 ± 2.0%; The group of 104 pfu/ml was 23.0 ± 1.5%; The group of 105 pfu/ml was 28.0 ± 3.0%. The control group was 2.0 ± 0.5%. The apoptotic cells were confirmed by morphologic observation. In addition, apoptotic signals from megakaryocytic surface, cytoplasma and mitochondria were detected in hCMV infected cells by flow cytometry with Caspase-3 and JC-1 assay. Compared to mock infection control at day 5, Annexin-V positive cells population increased by 58%; active caspase-3 signal increased by 120% in viable cell population; and cell population with damaged mitochondial membrane showed a 5-times increase. Moreover, the anti-apoptotic effect of APS and TPO on CHRF cells was also demonstrated by using Annexin-V assay. Our studies showed that hCMV induces the apoptosis in megakaryocytes via mitochondrial and caspase-3 signaling, and angelica polysaccharide (APS) like TPO has a protective effect on hCMV-induced apoptosis in these cells.

Disclosures:

No relevant conflicts of interest to declare.
Blocking Protein Secretion and Degradation Is a Novel Treatment Strategy For Malignant Cells With High Protein Load
Authors: Lowe Eric , Maglathlin Rebecca , Lerner Alana , Taunton Jack , Kirk Christopher J. , Arastu-Kapur Shirin
Published: October 2019

Multiple myeloma (MM) is the result of deregulated proliferation in malignant plasma cells that secrete an overabundance of a monoclonal immunoglobulin (Ig). The high level of Ig production in MM cells places a unique burden on the ubiquitin proteasome system (UPS) to maintain protein homeostasis and may explain why MM cells are among the most sensitive cell type to proteasome inhibition. We hypothesized that blocking protein secretion would further increase the protein load in MM cells and induce an anti-tumor response. In H929 and U266 cells, exposure to the proteasome inhibitors carfilzomib (CFZ) or bortezomib for 1 hour resulted in ubiquitination of several components of the Sec61 translocon, a hetero-trimeric channel present in the ER membrane central to protein secretion. Recently, cotransins were identified as a class of selective Sec61 inhibitors that block the translocation of specific nascent peptides into the ER. We determined the sensitivity to dual inhibition of protein secretion and degradation in 3 MM lines (NCI-H929, RPMI-8226, U266) and two plasma cell leukemia lines (JJN3, ARH-77) using the combination of CFZ and the cotransin CT08. We observed that 8226, H929, and JJN3 cells were sensitive to single agent CT08, with cell viability IC50 values ranging from 120 to 170 nM, while U266 and ARH-77 showed little sensitivity. CFZ IC50 values ranged from 1.5 to 6 nM across all 5 cell lines. CT08-sensitive cells treated with both agents, across a matrix of concentrations, showed a significant decrease in cell viability. For most concentrations, combination index (CI) values fell below 0.9, indicating synergistic activation of cell death, and reached levels of 0.19 (H929), 0.277 (JJN3), and 0.536 (8226). In resistant cells, CT08 potentiated (CI not evaluable) an anti-myeloma response when co-treated with CFZ, suggesting a significant interaction effect. CT08 also potentiated the cytotoxic effect of CFZ in the small cell lung cancer line DMS114 despite these cells being 10-fold less sensitive to CFZ than MM cell lines. In addition, CT08/CFZ combination treatment of U266 cells in co-culture with the bone marrow stromal cell line HS-5 overcame the protective effect of the stromal cells, as measured by Annexin V and propridium iodide staining. Taken together, these data suggest that dual Sec61 and proteasome inhibition represents a novel treatment strategy for malignant cells with high protein secretion like multiple myeloma.

Disclosures:

Lowe: Onyx Pharmaceuticals, Inc.: Employment, Equity Ownership. Lerner:Onyx Pharmaceuticals, Inc.: Employment, Equity Ownership. Kirk:Onyx Pharmaceuticals: Employment, Equity Ownership. Arastu-Kapur:Onyx Pharmaceuticals, Inc.: Employment, Equity Ownership.
Host and Donor B Cell-Derived IL-10 Ameliorates Murine Graft-Versus-Host Disease
Authors: Weber Michael , Stein Pamela , Fillatreau Simon , Roers Axel , Schild Hansjörg , Radsak Markus P
Published: October 2019

Allogeneic hematopoetic stem cell transplantation (HSCT) is the treatment of choice for a variety of hematologic malignancies. Graft-versus-Host disease (GvHD) is a key contributor to treatment related morbidity and mortality and consequently limits the efficacy of allogeneic HSCT. Interleukin 10 (IL-10) is a well-known cytokine with immunoregulatory and anti-inflammatory properties, also important in context of GvHD. B cells have been described as potent IL-10 producers in various situations. Here we show how host as well as donor derived B cells contribute to GvHD amelioration through IL-10 production.

We address the role of IL-10 in GvHD in an acute murine MHC mismatch model: Mice on a C57BL/6 background received bone marrow and CD90+ T cells from mice on a BALB/c background or vice versa. Transplantation experiments with IL-10 deficient donor or host cells clearly show the importance of donor derived IL-10 in general. To further dissect the cells contributing to IL-10 production in this situation we employed an IL-10 knock-in reporter mouse in which expression of eGFP is under control of the Il-10 locus. Lethal irradiation as used in the conditioning regiment before transplantation revealed B cells as major contributors of host derived IL-10. In addition, transfer of cells from reporter mice into preconditioned recipients showed also donor B cells as contributors to IL-10 production. A phenotypical characterization of the eGFP+ B cells exhibited a CD1d+TIM-1+CD5int phenotype, in line with immunoregulatory B cells. To finally confirm the relevance of B cells derived IL-10 in GvHD, we employed B6.B-IL-10-/- mice that have a B cell specific IL-10 knock-out as donors or recipients. Here we found a reduced survival associated with the incapability of the B cells to produce IL-10 in both cases.

Taken together, our results provide new insights in the mechanisms and the variety of cells contributing to the course of GvHD. An improved understanding of these aspects might help to pave the way for new treatment options to overcome current limitations of allogenic HSCT.

Disclosures:

No relevant conflicts of interest to declare.
Potential Role Of RUNX1 In The Pathogenesis Of Juvenile Myelomonocytic Leukemia (JMML)
Authors: Huang Hui , Bauer Daniel E. , Loh Mignon L. , Bhagat Govind , Cantor Alan B. , Kung Andrew L.
Published: October 2019

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of young children. The only current curative treatment is bone marrow transplantation. Yet even with this aggressive therapy, ∼50% of children still die from their disease. Somatic mutations leading to constitutive activation of the tyrosine phosphatase Shp2 (also called PTPN11) or of RAS signaling occur in ∼90% cases of JMML. However, the transcription factors that act downstream of these aberrant signaling events have not been identified. We recently showed that RUNX1 is a direct interacting partner of Shp2 in megakaryocytic cells (Huang et al. 2012. Genes Dev 26: 1587-1601). Moreover, we showed that RUNX1 is normally negatively regulated by src-family kinase (SFK) mediated tyrosine phosphorylation in megakaryocytes and T-lymphocytes, and that Shp2 contributes to RUNX1 tyrosine dephosphorylation. We now show that overexpression of a mutant RUNX1 (RUNX1Y260F, Y375F, Y378F, Y379F, Y386F, “RUNX1-5F”), which is expected to mimic constitutive dephosphorylation by Shp2 in murine Lin- Sca-1+ c-kit+ (LSK) bone marrow cells is resistant to SFK-mediated tyrosine phosphorylation and leads to a dramatic expansion of CFU-M/CFU-GM and Gr1+Mac1+ cells in vitro and in vivo. In contrast, these effects are not seen when wild type RUNX1 or RUNX1Y260D, Y375D, Y378D, Y379D, Y386D (“RUNX1-5D”; mimicking constitutive RUNX1 tyrosine phosphorylation) are overexpressed. The RUNX1-5F expressing cells also have increased replating activity in serial colony forming assays, increased proliferation (BrdU incorporation), decreased apoptosis, and reduced cytokine dependence. This partially phenocopies conditional knock-in mice that express JMML associated activating Shp2 mutations. Flow sorted Gr1+Mac1+ cells from the RUNX1-5F transduced cultures expressed higher levels of the direct RUNX1 target gene PU.1, which plays a role in myelomonocytic growth, and Cyclin D1. To test whether RUNX1 is required for the myelomonocytic hyperproliferation in JMML, CD34+ peripheral blood cells from a patient with JMML and known activating Shp2 mutation (Shp2E76G) were lentivirally transduced with doxycycline-inducible RUNX1-5D or RUNX1-5F expression constructs and cultured under myeloid growth conditions. Upon doxycycline induction, the RUNX1-5D overexpressing cells (resistant to Shp2) exhibited at 32% reduction in BrdU incorporation. In contrast, the control RUNX1-5F expressing cells had no significant reduction in proliferation. These results are consistent with RUNX1 acting as an essential downstream target of activated Shp2 in JMML. As ERK mediated phosphorylation (downstream of RAS/MEK) is also known to increase RUNX1 activity, we propose that RUNX1 may be a common downstream transcriptional target of both activated Shp2 and RAS signaling in the pathogenesis of JMML.

Disclosures:

No relevant conflicts of interest to declare.
Donor-Recipient Matching For Killer Immunoglobulin-Like Receptor Genotypes Confers Protection Against Graft Versus Host Disease Without Affecting Disease Relapse After Allogeneic Hematopoietic Cell Transplantation
Authors: Faridi Rehan M , Kemp Taylor J , Lewis Victor A. , Berka Noureddine , Storek Jan , Khan Faisal
Published: October 2019

Background

Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative therapy for malignant and non-malignant hematological disorders. Unfortunately, complications of HCT, primarily graft versus host disease (GVHD) and relapse of the underlying disease are substantial. In Alberta, all HCTs are performed with antithymocyte globulin conditioning and >95% of these use HLA matched donors. However, in spite of that approximately 35% Albertan HCT recipients die or suffer long-term from GVHD while another 20% die due to disease relapse. Unfortunately, strategies/treatments aiming to control GVHD in most cases lead to increase in the rate of disease relapse and infections. In the recent years, the natural killer (NK) cell genetic system has garnered substantial research interest as an immunogenetic system that significantly influences HCT outcomes. The complexity of NK cell function is modulated by a series of activating and inhibitory cell surface receptors known as the Killer Immunoglobulin-like Receptors (KIR). Here we set out to determine the influence of KIR matching between HCT donor and recipient pairs on allogenic HCT complications.

Study design

Hypothesizing that donor-recipient KIR gene/profile mismatch would affect the allo-HCT outcomes, we genotyped 92 (discovery cohort) and 111 (validation cohort) allo-HCT pairs by Luminex-based rSSO method. The KIR genotypes were classified into AA and Bx genotypes on the basis of KIR gene constitution of the individual. Donor (D) and recipients (R) were ‘matched' for KIR genotypes when both carried either AA (two copies of group A haplotypes) or B/x (at least one copy of group B haplotype) genotypes. Effect of KIR matching on significant GVHD (described as grade 2-4 acute GVHD or chronic GVHD needing systemic therapy) as well as disease relapse was analyzed using binomial regression and Kaplan-Meier statistics.

Results

As observed in both the discovery and validation cohorts, a significant protection against GVHD was conferred upon when both the donor and recipient were matched for the KIR genotypes (HR=2.224; p=0.01) without any effect on disease relapse (HR=1.098; p=0.934) (figure1).

Conclusions

In spite of D-R matching for major immunogenetic determinants like HLA, complications of allogeneic HCT are significant. Since GVHD represents a flip side to graft-vs-leukemia (GVL) reaction, strategies to decrease GVHD in most cases have resulted in increased relapse. Matching donor and recipients for KIR genotypes can offer a significant protection against GVHD without increasing the risk of disease relapse.

Disclosures:

No relevant conflicts of interest to declare.
Nitrite Levels Are Associated To Fetal Hemoglobin Concentration In Patients With Sickle Cell Anemia: Implications In Inflammatory Process
Authors: Pitanga Thassila Nogueira , Cerqueira Bruno A. V. , Boas Wendell Vilas , Santana Sanzio S. , Reis João O. , Goncalves Marilda Souza
Published: October 2019

Introduction

Sickle cell anemia (SCA) is a genetical hemolytic disorder defined as chronic inflammatory disease. Nitrite (NO-2) in SCA (HbSS) patients may be associated with the hemolytic process while fetal hemoglobin (Hb F) presents protective effect in these patients. Serum NO-2 interferes with the role of Hb F in reducing the hemolytic process contributing to vaso-occlusive crises.

Aims

The aim of this study was to evaluate the correlation of the serum levels of nitrite with fetal hemoglobin, low density lipoprotein cholesterol (LDL-C) and triglycerides levels in HbSS patients in steady-state of the Centro de Referência em Doença Falciforme de Itabuna (CERDOFI), Bahia, Brazil.

Methods

Forty-two patients diagnosed with SCA were included at baseline. Diagnosis was confirmed by high-performance liquid chromatography (HPLC). Serum levels of nitrite were performed using Griess reaction, and fetal hemoglobin, LDL cholesterol and triglycerides levels were measured by biochemical methods. The experimental protocol was approved by the Researcher Board Committee on Human Research, Gonçalo Moniz Research Center and informed consents were signed by patients.

Results

Our results showed a significant increase in NO-2 (p<0.0001) in adult patients with SCA compared to the control group (healthy volunteers). Data demonstrated a negative correlation between nitrite and fetal hemoglobin (p=0.004, r=-0.627). No correlation was found between fetal hemoglobin or nitrite with LDL-Cl and triglycerides.

Conclusion

The results reinforce that the serum nitrite can predict the clinical disease course.

Disclosures:

No relevant conflicts of interest to declare.