bluuebunny/crossref_metadata_2025 huggingface dataset:
Allografts Selectively Photodepleted of GvHD Causing T Cells and Followed by Low-Level Immunosuppression: A Novel Method to Improve Disease Control After HLA-Matched Sibling Transplantations.
Authors: Mielke Stephan , Shenoy Aarthi , Rezvani Katayoun , Yong Agnes S. M. , McIver Zachariah A. , Fellowes Vicki S. , Savani Bipin N. , Kurlander Roger , Khuu Hanh , Boss Carol , Barrett A.John
Published: October 2019

Abstract 515

We established a highly efficient GMP-grade, ex-vivo selective allodepletion process where host-activated donor T cells are eliminated based on their preferential retention of the photosensitizer 4,5-dibromorhodamine 123 (TH9402) and exposure to visible light (Kiadis Pharma, The Netherlands). As relapse of disease largely impairs the overall success of allogeneic stem cell transplantation we aimed to improve this outcome by using selectively T cell depleted allografts in order to reduce post transplant immunosuppression and thereby enhance graft versus malignancy effects. To determine the appropriate level of post transplant immunosuppression we designed a three sequential de-escalation stage trial with grade III-IV acute GvHD as the primary endpoint involving 17 patients per study cohort. Here we report on the first completed study cohort of NIH trial 07-H-0136 where seventeen patients (median age 44 (28-68) years) with hematological malignancies received a CD34-selected (Miltenyi, Germany) stem cell allograft together with 5 × 106/kg selectively depleted donor T cells following an age-adapted, radiation-based preparative regimen (FluCyTBI). Eleven patients had high risk disease (including ALL (Ph+, CR>1), refractory NHL, AML/MDS, AML with chloroma and blast crisis CML). Low-dose cyclosporine was used as sole immunosuppression for 90 days post transplant in the absence of GvHD. At a median follow-up of 385 (119-714) days actuarial probabilities (±SEM) of acute GvHD were 35±12% for grade II-IV and 0% for grade III-IV. Non-relapse mortality (NRM) was low with 17±11%. Overall survival (OS) was 73±12% and relapse-free survival (RFS) was 65±13% with a relapse probability of 21±11% (Figure). A low relapse incidence in a high-risk population suggests functionality of selectively allodepleted T cells. The absence of severe GvHD reflects the efficacy of the allodepletion process. Based on these findings we have initiated recruitment for the next study cohort where post transplant immunosuppression will be limited to 45 days only. Ultimately the aim is to achieve further reduction in immunosuppression in the absence of severe GvHD in order to enhance graft versus malignancy effects and thereby improve the outcome after allogeneic stem cell transplantation especially for patients at high risk for relapse.

Disclosures:

Mielke: Kiadis Pharma, The Netherlands: Research Funding, The current trial is supported under a clinical trial agreement between NHLBI and Kiadis.. Savani:Kiadis Pharma Inc., The Netherlands: Consultancy. Barrett:Kiadis Pharma, The Netherlands: The current trial is supported under a clinical trial agreement between NHLBI and Kiadis..
Comparable Disease-Free and Overall Survival After “Well-Matched” Unrelated Donor and Matched Sibling Donor Transplantation in Acute Myeloid Leukemia with Adverse Risk Karyotype in First Complete Remission: A Report From the Acute Leukemia Working Committee of the Centre for International Blood and Marrow Transplant Research.
Authors: Gupta Vikas , Tallman Martin S. , He Wensheng , Logan Brent , DiPersio John F. , Bunjes Donald W. , Weisdorf Daniel J.
Published: October 2019

Abstract 526

In patients with acute myeloid leukemia (AML) in first complete remission (CR1), the indications for matched sibling donor (MSD) transplants and unrelated donor (URD) haemopoietic stem cell transplantation (HSCT) are different. We sought to determine the prognostic impact of donor type on the outcomes of AML with adverse risk karyotype in CR1, a high-risk AML population considered as a standard indication for MSD and URD HSCT. We evaluated the outcomes of 584 patients undergoing allogeneic HSCT for AML with adverse risk karyotype in CR1 between 1995 and 2006, reported to the CIBMTR. Adverse risk karyotype was defined according to SWOG/ECOG classification. Cytogenetics abnormalities were further classified as: complex karyotype (3 or more abnormalities), 32%; and Non-complex divided as abnormal chromosome 7, 25%; chromosome 5, 9%; MLL gene rearrangements, 18%; t (6;9), 5%; and others, 10%. 226 patients underwent MSD and 358 URD. URD were classified based on high resolution typing as:” well matched” [n=254 (71%)] with no known disparity at HLA A, B, C, DRB1; and, “partially matched” [n=104 (29%)] with one locus known or likely mismatched. Previous MDS was present in 19% and 14% had therapy-induced (t-AML). Conditioning regimens were myeloablative and reduced intensity in 74% and 26%, respectively. At 3 years treatment-related mortality (TRM) incidence was 28% (95% CI 24-31); relapse 36%(32-40); disease-free survival (DFS) 36%(32-41) and overall survival (OS) 39%(35-44). Multivariate analyses are summarized in the table. “Well matched” URD and MSD yielded similar DFS and OS, while outcomes were significantly inferior for “partially matched” URD. Cytogenetically defined subsets had similar outcomes. Evaluated as a time-dependent covariate, chronic GVHD had a significantly lower risk of relapse (RR 0.68, p=0.046), while acute GVHD had no effect (RR 0.99, p=0.96). “Well matched” URD and MSD lead to similar DFS and OS in AML CR1patients with adverse risk karyotype. The pool of patients who may benefit from graft-vs-leukemia effect generated with allogeneic HSCT may be considerably expanded with “well-matched” URD HSCT. If a suitable MSD is not availabel, “well-matched” URD should be strongly considered where a MSD HSCT would otherwise be undertaken.

Disclosures:

No relevant conflicts of interest to declare.
Allogeneic Hematopoietic Cell Transplantation Can Cure Some Patients with Acute Leukemia in Relapse or Primary Induction Failure: A CIBMTR Study.
Authors: Duval Michel , He Wensheng , Klein John P. , Tallman Martin S. , DiPersio John F. , Bunjes Donald W. , Weisdorf Daniel J.
Published: October 2019

Abstract 528

Acute leukemia refractory to chemotherapy is uniformly fatal without hematopoietic cell transplantation (HCT). However, the benefit of transplantation for patients not in complete remission (CR) is controversial.

PATIENTS AND METHODS:

2,255 patients transplanted (at 252 centers in 38 countries) with an allogeneic donor after myeloablative conditioning regimen between 1995 and 2004 for acute leukemia in relapse or with primary induction failure were reported to the Center for International Blood and Marrow Transplant Research (CIBMTR). 1,673 had acute myeloid leukemia (AML) and 582 acute lymphoblastic leukemia (ALL). Median age was 38 and 29 years for AML and ALL patients, respectively. Presence of circulating blasts, >25% marrow blasts, Karnofsky score < 90 %, or transplant in first relapse was observed in nearly half of the patients (see Table).

RESULTS:

100 day mortality was 39% in AML and 41% in ALL. The median follow-up of survivors was 5 years. Overall survival (OS) was 19% (95% confidence interval (CI) 17-21) for AML and 16% (13-20) for ALL patients. For AML patients, five adverse pre-transplant variables significantly impacted OS : first CR duration < 6 months, blasts in the blood, donor other than HLA-identical sibling or partially matched unrelated, Karnofsky score < 90%, poor-risk cytogenetics. 106 AML patients had none of these high risk criteria, with a 3 years OS of 44% (35-54). For ALL patients, survival was worse with either: 1st refractory or ' 2nd relapse, ≥ 25% marrow blasts, CMV seropositive recipient, age ≥10 years. 8 ALL patients had no high risk criteria, with a 3 years OS of 85% (53-100). Grade 3-4 acute graft-vs-host disease (GVHD) occurred in 23% of AML and 27% of ALL patients. Chronic GVHD developed in 27%. Leukemia was the cause in 42 % of AML and 37% of ALL patients' deaths.

CONCLUSIONS:

1. HCT can cure some patients with acute leukemia in relapse or with PIF, particularly those lacking defined high risk features. 2. These pre-transplant variables should be considered when deciding whether or not to offer HCT to such patients.

Disclosures:

No relevant conflicts of interest to declare.
Total Therapy 3 Successor (TT3-S) Protocol (2006-66)–Confirming Results Reported for Initial TT3 (TT3-I) Protocol (2003-33) for Newly Diagnosed Multiple Myeloma (MM).
Authors: Nair Bijay , Alsayed Yazan , Waheed Sarah , Anaissie Elias , van Rhee Frits , Szymonifka Jackie , Hoering Antje , Crowley John , Barlogie Bart
Published: October 2019

Abstract 531

Introduction:

TT3-I imparted high CR rates and superior overall survival (OS) and event-free survival (EFS). Bortezomib pharmaco-genomic studies, performed concurrently, revealed that gene expression profiling (GEP) changes 48hr after a bortezomib test-dose had major prognostic significance, which we wished to validate in TT3-S. The purpose of this work is to compare clinical outcomes among the 303 TT3-I and 177 TT3-S patients.

Methods:

Our MM data base was scrutinized for patient characteristics and outcomes of the 2 cohorts in terms of CR rates and duration, as well as OS and EFS, in the context of major prognostic variables (cytogenetic abnormalities (CA), B2M, LDH, GEP risk).

Results:

Patients in the 2 studies were comparable except for a higher incidence of several adverse features in TT3-S: albumin <3.5g/dL in 45% v 25% (p<0.001), B2M >=3.5mg/L in 57% v 45% (p=0.012) and GEP-defined high-risk in 22% v 15% (p=0.035). Outcome comparisons for TT3-S v TT3-I were almost identical: 3-year estimates of OS 80% v 82%, EFS 81% v 77%, sustained CR 61% v 62%; in GEP low-risk/no CA subgroup: OS 94% v 91%, EFS 90% v 87%, sustained CR 98% v 94% (Figure 1a); GEP low-risk/CA subgroup: OS 81% v 79%, EFS 81% v 74%, sustained CR 86% v 90%; GEP high-risk/no CA subgroup: OS 70% v 82%, EFS 75% v 46%, sustained CR 75% v 30%; GEP high-risk/CA subgroup: OS 48% v 41%, EFS 52% v 38%, sustained CR 60% v 55% (Figure 1b). None of the Kaplan-Meier plots for these GEP-risk/CA constellations were different between TT3-S and TT3-I.

Conclusions:

We have validated the remarkable success of the TT3 principle in low-risk MM, whereas high-risk disease deserves novel strategies as being pursued in TT5 with dose-dense and less dose-intense therapies.

Disclosures:

van Rhee: Genzyme Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
Long-Term Survival Analysis of the North American Intergroup Study C9011 Comparing Fludarabine (F) and Chlorambucil (C) in Previously Untreated Patients with Chronic Lymphocytic Leukemia (CLL).
Authors: Rai Kanti R. , Peterson Bercedis L. , Appelbaum Frederick R. , Tallman Martin S. , Belch Andrew , Morrison Vicki A. , Larson Richard A.
Published: October 2019

Abstract 536

Long-term outcomes following novel therapies for CLL have rarely been reported. Between 10/90 and 12/94, 509 eligible, untreated patients (pts) with symptomatic CLL were enrolled by 4 cooperative groups onto study C9011; 179 were randomized to F, 193 to C, and 137 to F+C. After slightly more than 5 years median follow up, with the time of last follow-up in June 1999, we reported in 2000 (NEJM 343:1750) that F provided significantly higher response rates and longer remission duration and progression-free survival (PFS) than C (p<0.001 for all 3 endpoints). The combination arm with F+C was stopped early because of high morbidity and mortality. There was no difference in overall survival (OS) among the 3 groups. Nearly 10 years have now elapsed since this report. Therefore, with the time of last follow-up in January 2009, we analyzed the long term outcomes of pts enrolled on the study. PFS was defined as the time between randomization and the occurrence of progressive disease or death due to any cause.

Results:

Of the 509 pts, 85% have now died; among pts on the F and C arms, 92% have progressed. We found that F treatment resulted in significantly longer PFS than did C (p < 0.001), with notable differences in PFS at 2, 3, and 4 years (Table). While the F and C arms had the same OS during the initial 5 years following randomization, our current analysis with longer follow-up shows that pts treated on the F arm had better survival than did those on the C arm during the ensuing years (Figure). The p-values for this difference are 0.04 (unadjusted for covariates) and 0.07 (covariate-adjusted). The emergence of improved survival following initial F treatment, appearing only after 5-6 years, is an unexpected and noteworthy finding. Reporting second malignancies was required on this study. There were 27 epithelial cancers reported (9 on F, 11 on C, 7 on F+C), involving colon, lung, breast, prostate, pancreas, liver, bladder and skin (6 squamous, 2 melanoma). Seven therapy-related myeloid neoplasms (t-MN) were reported; 6 were on F+C; 1 on F. Richter's transformation to non-Hodgkin lymphoma was reported in 34 pts; prolymphocytic leukemia occurred in 10; Hodgkin lymphoma in 6; myeloma in 2; hairy cell leukemia in 1. These cases were distributed with 18 on F, 18 on C, and 17 on F+C. Thus, the overall incidence of second malignancies reported was 17%.

Conclusion:

Initial treatment with F provides better long-term outcomes than initial treatment with C. Second malignancies are common, but the overall incidence is not increased on the F-containing treatment arms except for t-MN.

Disclosures:

No relevant conflicts of interest to declare.
Comparative Analysis of Aberrant Autophagy in HAX1-Mutated and ELANE/ELA2-Mutated Neutrophil Granulocytes.
Authors: Appaswamy Giridharan , Boztug Kaan , Rittner Rebecca Elisabeth , Brandes Gudrun , Welte Karl , Zeidler Cornelia , Klein Christoph
Published: October 2019

Abstract 550

Severe congenital neutropenia is a heterogeneous disorder with the phenotypic hallmark of ‘myeloid maturation arrest‘. Recent work in our group identified that HS-1 associated protein X1 (HAX1), a mitochondrial protein is essential for survival of neutrophils through maintenance of mitochondrial membrane potential (MMP). A large group of patients have mutations in ELANE/ELA2 or HAX1, respectively, causing increased susceptibility to apoptosis. Although phenotypically indistinguishable, it is controversial whether HAX1 and ELA2 mutant cells share a joint pathway. To further address the biological abnormalities, we measured reactive oxygen species (ROS) production and autophagy in primary human neutrophil granulocytes from SCN patients. HAX1 deficient neutrophils, but not SCN neutrophils expressing functional HAX1 showed evidence of enhanced production of ROS, a continuous byproduct of the oxidative phosphorylation. Antioxidant defense system in a cell constitutes proteins that restrain ROS mediated cellular damage. The degradation of catalase, a primary antioxidant defense protein was accelerated in HAX1 deficient neutrophils as compared to healthy controls. This effect was not observed in ELA2 deficient neutrophils or in SCN neutrophils expressing functional HAX1. Oxidative phosphorylation coupled ATP synthesis; a prime function of the mitochondria is hampered with mitochondrial dysfunction, resulting from loss of MMP. We observed that the HAX1-deficient neutrophils but not ELA2-deficient neutrophils have an elevated AMP/ATP ratio as measured by the increased activation of AMP activated protein kinase α (AMPKα). In view of increased ROS production hampering the cellular organelles, and decreased levels of cellular ATP, we hypothesized that autophagy may play a role in HAX1-deficient neutrophils. Autophagy describes a process of organelle digestion within a cell aiming at sparing energy and disposal of damaged organelles. In case these rescue mechanisms are futile, the cell undergoes apoptosis. Expression of beclin-1, an autophagy essential protein was increased in HAX1-deficient neutrophils, accompanied by an increase in the ATG12-5 complex formation as compared to healthy control cells. Furthermore, transmission electron microscopy studies revealed evidence of increased autophagy in HAX1-deficient neutrophils. These observed effects are specific to HAX1 deficiency, as these biochemical aberrations were not observed in ELA2-deficient patients or SCN patients expressing functional HAX1. Moreover the phenotype was observed only in neutrophils and not in HAX1-deficient lymphoid or monocytic cells. Our results unravel a novel role of the antiapoptotic protein HAX1 in maintenance of cellular homeostasis in neutrophil granulocytes by regulating the autophagic machinery through control over ROS production.

Disclosures:

No relevant conflicts of interest to declare.
Ectopic Human Mesenchymal Stem Cell-Coated Scaffolds Create An In Vivo Leukemia Niche in NOD/SCID Mice.
Authors: Vaiselbuh Sarah Rivkah , Edelman Morris , Lipton Jeffrey Michael , Liu Johnson M.
Published: October 2019

Abstract 559

Introduction:

Different cellular components of the normal hematopoietic niche have been identified. However, the niche for malignant hematopoiesis remains to be elucidated. Recent work of other groups has suggested that hematopoietic stem cells (HSC) within the bone marrow anchor themselves in place by attaching to osteoblasts and/or vascular sinusoid endothelial cells. We have recently identified mesenchymal stem cells (MSC) as niche-maker cells and found a crucial role of the SDF-1/CXCR4 axis in this process. Stromal Derived Factor-1 (SDF-1/CXCL12) regulates stem cell trafficking and the cell cycle via its receptor CXCR4.

Methods:

Polyurethane scaffolds, coated in vitro with human bone marrow MSC, were implanted subcutaneously in non-irradiated NOD/SCID mice. CD34+ HSC or primary AML cells (from a leukapheresis product) were injected either in situ or retro-orbitally in the mice and analyzed for engraftment. The mice were treated twice per week with in situ injections of SDF-1, AMD3100 (a CXCR4 antagonist) or PBS (control). After 2 to 4 weeks, the scaffolds were processed and evaluated for cell survival in the mesenchymal niche by immunohistochemistry.

Results:

We created in vitro MSC-coated scaffolds that retained inoculated AML cells in the presence of SDF-1, while AML cells seeded on empty scaffolds were not retained. In vivo in NOD/SCID mice, the MSC-coated scaffolds, in the presence of SDF-1 enabled homing of both in situ injected normal CD34+ HSC and retroorbital- or in situ injected primary human AML cells. The scaffolds were vascularized and showed osteoclasts and adipocytes present, suggestive of an ectopic human bone marrow microenvironment in the murine host. Finally, the SDF-1-treated scaffolds showed proliferation of the MSC stromal layer with multiple adherent AML cells, while in the AMD3100-treated scaffolds the stromal lining was thin and disrupted at several points, leaving AML cells free floating in proximity. The PBS-treated control-scaffold showed a thin single cell MSC stromal layer without disruption, with few AML cells attached.

Conclusion:

The preliminary data of this functional ectopic human microenvironment in NOD/SCID mice suggest that AMD3100 (a CXCR4 antagonist) can disrupt the stem cell niche by modulation of the mesenchymal stromal. Further studies are needed to define the role of mesenchymal stem cells in maintaining the hematopoietic/leukemic stem cell niche in vivo.

In Vivo Leukemia Stem Cell Niche:

(A) Empty polyurethane scaffold. (B)Vascularization in SQ implanted MSC-coated scaffold (s) niche in NOD/SCID mice. (C) DAB Peroxidase (brown) human CD45 positive nests of AML cells (arrows) 1 week after direct in situ AML injection. (D) Human CD45 positive myeloid cells adhere to MSC in vivo (arrows).

Disclosures:

No relevant conflicts of interest to declare.
Expression of Mir-196b Is Not Exclusively MLL-Driven but Especially Linked to Activation of HOXA Genes in Children with Acute Lymphoblastic Leukemia.
Authors: Schotte Diana , Lange-Turenhout Ellen , Stumpel Dominique JPM , Stam Ronald W. , Buijs-Gladdines Jessica , Meijerink Jules P.P. , Pieters Rob , Den Boer Monique L.
Published: October 2019

Abstract 580

MicroRNAs (miRNAs) play important roles in diverse biological processes including hematopoiesis. As a consequence, aberrant expression of miRNAs may contribute to hematopoietic malignancies. It has been reported that miR-196b is transcriptionally activated by MLL and MLL-fusion genes and is therefore highly expressed in MLL-rearranged leukemia. In order to investigate whether high expression levels of miR-196b are restricted to MLL-rearranged leukemia cases, we measured the expression in samples of 72 selected pediatric acute lymphoblastic leukemia (ALL) cases i.e. MLL-rearranged and non-MLL-rearranged precursor B-ALL and T-ALL patients. MiR-196b was highly expressed in 9/12 MLL-rearranged precursor B-ALL patients, but also in 14/22 T-ALL patients. In particular, 100% of T-ALL cases carrying CALM-AF10 (n=5), MLL-AF6 (n=2), SET-NUP214 (n=3) and an inversion of chromosome 7 (n=1) showed high expression levels of miR-196b comparable to the high levels found in MLL-rearranged ALL. Like MLL-rearrangements, these genetic abnormalities have been functionally linked with upregulation of HOXA cluster genes. MiR-196b expression levels in these patients were strongly correlated with the expression of HOXA family but not with HOXB and HOXC cluster genes (Rs ≥ 0.7, P≤0.003). Since miR-196b is located between HOXA9 and HOXA10 on chromosome 7, our data suggest co-activation of miR-196b and HOXA family genes in pediatric ALL. In parallel to the high expression level of miR-196b we found decreased methylation at CpG islands located 5' of miR-196b in MLL-rearranged cases compared to normal bone marrow cells, which suggests an epigenetic origin for the high expression level of miR-196b in these patients. Despite the fact that MLL-rearranged ALL patients often respond poorly to prednisolone and L-asparaginase, upregulation of miR-196b was not indicative for the resistance to these drugs in pediatric ALL. In conclusion, high-level expression of miR-196b is not only MLL-driven, but can also be found in other types of leukemia that display aberrant activation of HOXA genes.

Disclosures:

No relevant conflicts of interest to declare.
Manipulating Higher Order Chromatin Structure of the β-Globin Locus by Targeted Tethering of a “looping” Factor
Authors: Deng Wulan , Gregory Philip D , Reik Andreas , Blobel Gerd
Published: October 2019

Abstract 647

The mammalian β-globin locus is under the coordinated control of multiple transcription factors to ensure the correct expression of the globin genes during development. The distal β-globin locus control region (LCR) physically interacts with β-like globin promoters to form developmentally dynamic chromatin loops. The hematopoietic transcription factor GATA-1 and its associated cofactor Ldb1 bind to the LCR and the β-globin promoter and are essential for loop formation and β-globin expression. However, the molecular basis of chromatin looping and its cause-effect relationship with transcriptional activation are unclear. Here, we examined whether Ldb1 is an effector of GATA-1 during loop formation. Specifically, we tested whether artificial tethering of Ldb1 to the endogenous β-globin promoter and LCR can substitute for GATA-1 function. Ldb1 was fused to artificial zinc finger proteins (ZFP) designed to bind to the LCR and β-globin promoter. Ldb1-ZFPs were introduced pairwise into murine GATA-1 null erythroid cells in which the β-globin locus is relaxed and transcriptionally silent. In vivo binding of the Ldb1-ZFPs to their targets was verified by ChIP assay. Strikingly, expression of Ldb1-ZFPs but not Ldb1 or ZFPs alone led to substantial activation of β-globin transcription in the absence of GATA-1. Moreover, chromosome conformation capture experiments showed that Ldb1-ZFPs triggered physical association between the LCR and the β-globin promoter. Recruitment of RNA polymerase II (Pol II) and its phosphorylation at serine 5 are critical LCR-dependent regulatory steps in β-globin transcription. We found that Ldb1-ZFP expression facilitated Pol II recruitment at the β-globin promoter and serine 5 phosphorylation to the same level as GATA-1-expressing erythroid cells. This is consistent with an Ldb1-ZFP-induced LCR-β-globin promoter chromatin loop. In concert, these results indicate that Ldb1 is a critical effector for GATA-1 by mediating enhancer-promoter loops. In broader terms, our results suggest that chromatin loop formation can be sufficient for gene activation in the absence of an essential transcription factor. We are currently in the process of examining whether targeting of the LCR to embryonic and fetal globin genes can be used to activate them in adult cells. Targeted chromatin loop formation may provide a method to activate fetal or adult hemoglobin expression in individuals with β-thalassemia or sickle cell anemia.

Disclosures:

No relevant conflicts of interest to declare.
Transfusion of Fresh but Not Stored Blood Reduces Infarct Size and Improves Cardiac Function Following Acute Myocardial Infarction In Anemic Rats
Authors: Xenocostas Anargyros , Hu Houxiang , Chin-Yee Nicolas J , Chin-Yee Ian , Feng Qingping
Published: October 2019

Abstract 661

Background:

Anemia is a predictor of poor outcomes in acute myocardial infarction (MI) and may increase the degree of ischemia, resulting in greater tissue injury. The only existing therapeutic intervention used to acutely correct anemia is red blood cell (RBC) transfusion, although its effectiveness at improving outcomes in anemic subjects following MI remains controversial. We have previously shown that transfusion of fresh blood in anemic rats following MI decreased infarct size, and improved cardiac function and survival (Hu et al. Transfusion 2010; 50:243-251). However, it was not known if the transfusion of stored blood, which develops a series of chemical and corpuscular changes collectively known as the “storage lesion”, would be equally effective in this model.

Experimental Design:

Acute MI was induced by coronary artery ligation in 49 male Sprague-Dawley rats, 38 of which were made anemic (Hb = 80–90 g/L) using a combination of dietary iron deprivation and phlebotomy, and 11 with normal Hb levels. Rat blood was stored for 7 days in CPDA-1 to induce a storage lesion similar to the one seen in human blood stored under identical conditions for 28 days (d'Almeida et al. Transfusion Medicine 2000; 10:291-303). Anemic animals were randomly assigned to one of three groups: i) fresh blood transfusion (stored for <4 hours) to increase the Hb to 100 g/L, ii) transfused with stored blood to increase the Hb to 100 g/L, or iii) no transfusion. At 24 hours post-MI, infarct size, cardiac function and survival rates were determined.

Results:

A similar degree of left ventricular (LV) myocardial ischemia (area at risk) was induced in all groups (P=NS). In agreement with previous results, the infarct size to area at risk ratios were significantly increased in the anemic (Hb 80–90 g/L) animals compared to non-anemic MI controls, and transfusion of fresh blood to a target Hb level of 100 g/L after MI decreased infarct size (P<0.05) and improved both systolic (+dP/dtmax) and diastolic (−dP/dtmin) LV function compared to anemic animals (P<0.05). However, the transfusion of stored blood into anemic animals post-MI did not have these effects when compared to the group receiving fresh blood (P<0.05). Furthermore, survival was increased in anemic animals receiving fresh blood (9/11; P<0.05) but not stored blood (7/14; P=NS) compared to the non-transfused group (5/13).

Conclusions:

The prolonged storage of blood negates the beneficial effects of fresh blood transfusion, which include reductions in infarct size, and improvements in cardiac function and short-term survival following acute MI in this animal model.

(This study was supported by the Canadian Institutes of Health Research, Canadian Blood Services and the Bayer Partnership Fund).

Disclosures:

No relevant conflicts of interest to declare.
Dendritic Cell Activation by PF4/H Multimolecular Complexes
Authors: Afonina Galyna , Joglekar Manali , Qi Rui , Arepally Gowthami M.
Published: October 2019

Abstract 722

Heparin-Induced Thrombocytopenia (HIT) is caused by antibodies to multimolecular complexes of Platelet Factor 4 (PF4) and heparin (H). Little is known about the cellular mechanisms underlying the PF4/H immune response. Our previous studies have shown that mice injected with murine (m) PF4/H complexes develop a de novo immune response to mPF4/H, but do not respond to injections of mPF4 alone or H alone. In other studies using this model, we have shown that the HIT immune response is T-cell dependent, requires the presence of PF4/H multimolecular complexes and does not engage TLRs via MyD88. To examine the cellular basis of the HIT immune response, we performed studies addressing requirements for antigen presentation. We first isolated splenocytes from non-immunized C57Bl/6 mice and incubated 4×105 splenocytes with mPF4 alone (10mg/ml, final concentration.), heparin alone (0.4U/ml, final), mPF4/H complexes (10mg/ml and 0.4U/ml, final), buffer or LPS (1 mg/ml, final, positive control). We noted significant levels of IL-12 in wells incubated 24hrs with mPF4/H (109 ± 7 pg/mL) or LPS (256 ± 22 pg/mL) but not wells containing mPF4 (33 ± 7 pg/mL), H (5 ± 7 pg/mL), or buffer (9 ± 13 pg/mL). In other studies, we noted that splenic dendritic cells (DCs) were primarily activated by mPF4/H complexes and that cellular activation, as gauged by IL-12 (Figure 1A) or IFN-g (data not shown) occurred in a heparin-dependent manner. DCs activation by mPF4/H complexes was not dependent on CXCR3 or pattern recognition receptors, such as receptor for dectin, mannose or complement receptor 3 (CR3 or CD11b antibody). To determine the effect of DC activation on T-cell responses, we performed mixed lymphocyte reaction assays using DCs isolated from C57Bl/6 mice (3×105 cell/well) and naïve T-cells from Balb/c mice (1:5 cellular ratio). Pre-incubation of Bl/6 DCs with mPF4/H complexes, but not mPF4, H or buffer alone resulted in Balb/c T cell activation and release of cytokines indicative of T helper (Th) 1 immune response (Figure 1B). In summary, we show that DC s are directly activated by PF4/H multimolecular complexes, and that cellular activation by complexes results in a predominant Th1 polarization. Additional studies are underway to identify the relevant receptor for DC activation, and additional pathways of antigen processing and presentation that are necessary for the initiation of a PF4/H specific adaptive immune response.

Disclosures:

No relevant conflicts of interest to declare.
Deficiency In Stress Response Gadd45a and Gadd45b Alters MAPK Kinase Signaling Leading to Functional Defects In Innate Immune Responses of Neutrophils and Macrophages In Vitro and In Vivo
Authors: Salerno Dominic M. , Tront Jennifer , Hoffman Barbara , Liebermann Dan
Published: October 2019

Abstract 927

Stress sensor GADD45 proteins modulate p38-NF-Kb and JNK signaling; which play major roles in leukocyte activation and innate immunity. We have previously documented that GADD45a-deficient and GADD45b-deficient mice exhibit impaired responses to hematological stress, including acute stimulation with inflammatory cytokines (Gupta et. al, Oncogene. 25:5537, 2006). Whether GADD45 genes modulate myeloid compartment function, notably the response of macrophages & granulocytes to inflammatory stress, remains largely unexplored. Data obtained thus far indicate that bone marrow (BM) derived neutrophils and macrophages lacking GADD45 family members exhibit defects in chemotaxis towards inflammatory stimuli. BM derived neutrophils and macrophages lacking GADD45 family members were also observed to exhibit defects in the production of reactive oxygen species (ROS) upon treatment with LPS (500ng/mL) in vitro. Furthermore, BM derived macrophages from mice lacking GADD45a and GADD45b were found to be defective in phagocytosis and cellular adhesion upon stimulation with LPS (500ng/mL) in vitro. Additionally, it was observed that BM derived neutrophils and macrophages lacking GADD45 family members exhibit defects in inflammatory cytokine secretion in response to activation with LPS. Notably, GADD45a, GADD45b & GADD45g null mice injected intraperitoneally with sublethal doses of LPS were significantly more susceptible to septic shock compared to wt mice, showing significantly increased morbidity. Moreover at 18 hrs. post-injection, the spleens of KO mice harbored apoptotic foci in the white pulp, identified as tingible body macrophages ingesting dying cells. To dissect the signaling pathways involved, MAPK specific chemical inhibitors were used to correlate MAPK signaling alteration to defects in innate immune responses of neutrophils and macrophages lacking GADD45a or GADD45b. LPS stimulated wt neutrophils treated with MAPK inhibitors exhibited defects in signaling and innate immune responses mimicking neutrophils and macrophages lacking GADD45a or GADD45b. Collectively, these novel data provide evidence to show that deficiency in GADD45a and GADD45b leads to functional defects in the myeloid innate immune responses of neutrophils and macrophages in vitro and in vivo. These findings increase our understanding of innate immunity and have the potential to contribute towards development of novel approaches to clinical treatment of sepsis.

Disclosures:

No relevant conflicts of interest to declare.
Calorie Restriction and Aging
Authors: de Cabo Rafael
Published: October 2019

Abstract SCI-2

A prominent manifestation of aging is a reduced ability to respond to environmental stressors, including heat and oxidative stress. Reduced stress tolerance and decreased ability to maintain homeostasis are at least partially responsible for the increased morbidity and mortality that occurs with advancing age. The age-related attenuation of stress pathways and increased expression of stress-response genes with aging are examples of the growing body of evidence linking reduced stress responsiveness to aging. In 1935, McCay and colleagues first reported that reducing the caloric intake of rodents could significantly lengthen their mean and maximal life span, slowing down basic aging processes. The effect of calorie restriction (CR) on delaying aging has been replicated in many animal species including nonhuman primates, although in these, potential life span alterations cannot be ascertained for several more years due to their longevity CR causes a reduction in body weight, tissue growth, blood glucose, insulin levels and body temperature. In addition, CR prevents the age-related decline in tolerance to different stressors such as oxidative and heat, and the age-related reduction in expression of protective heat shock and oxidative stress proteins. While CR is the only intervention that has consistently been shown to increase maximum life span and prevent or delay the onset of age-associated pathophysiological changes in laboratory rodents, the underlying mechanisms remain elusive. Using calorie restriction (CR) as their benchmark research tool, gerontologists are making progress in identifying dietary and pharmacologic interventions that may be applicable to retarding aging processes in humans.

Disclosures:

No relevant conflicts of interest to declare.
Non-Myeloablative Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) for Patients with Severe Sickle Cell Disease (SCD)
Authors: Hsieh Matthew M , Fitzhugh Courtney D , Weitzel R. Patrick , Coles Wynona , Link M Beth , Tisdale John F.
Published: October 2019

Abstract 10

Allo-HSCT remains the only curative approach for patients with SCD, yet a high risk of procedural toxicities and graft-versus-host disease (GvHD) limit this approach in adult patients with end organ damage from SCD. We chose a low-dose radiation approach utilizing sirolimus based upon its unique ability to promote T cell tolerance. Entry criteria include irreversible SCD-related complications (stroke, nephropathy, or tricuspid regurgitant jet velocity >2.5 m/s), or reversible complications not ameliorated by a 6-month course of hydroxyurea (frequent vaso-occlusive crises or acute chest syndrome). Conditioning was achieved with 1mg/kg of alemtuzumab divided over 5 days, a single total body irradiation dose of 300cGy, and oral sirolimus targeting trough levels between 10–15 ng/ml. Donor chimerism was measured by microsatellite PCR among CD3 and CD14/15 positive white cells. Twenty-three patients have been transplanted to date, and their ages ranged from 17 to 64 years (median 28). All are alive at 2 months to 7 years post allo-HSCT. Conditioning was well-tolerated. All received unmanipulated G-CSF mobilized peripheral blood progenitors obtained from 8/8 HLA-matched siblings. CD34+ cell doses ranged from 5.5 to 31 × 10e6/kg (median 14.4), and CD3+ cell doses ranged from 1.6 to 5.4 × 10e8/kg (median 3.4). Three patients engrafted temporarily but lost their grafts between the 2nd and 3rd months post transplant and had recurrent SCD. Twenty patients engrafted with mean myeloid chimerism of 97.5% (median 89%) and CD3 chimerism of 42% (median 49%). Improvements in laboratory parameters of engrafted patients are as follows: pre vs post HSCT hemoglobin, 8.7 vs 12.1 g/dL; reticulocyte count, 181 vs 74 k/uL; LDH, 328 vs 202 units/L; total bilirubin, 2.87 vs 1.0 mg/dL. Hemoglobin electrophoresis revealed replacement by donor type hemoglobin in 19 patients by 1 year, with amelioration of the SCD phenotype allowing for therapeutic phlebotomy. In 17 patients at 1 year or more post allo-HSCT, 5 had CD3 chimerism >50% which allowed complete withdrawal of immunosuppression; they have maintained stable mixed chimerism. Two patients received preemptive treatment with ganciclovir or foscarnet for presumed CMV reactivation (blood PCR >300 genome/mL) before day 30. Treatment in both patients was discontinued 1 week later with negative blood PCR and prophylaxis with acyclovir was resumed. There were 2 patients with zoster at 2 and 3 years post allo-HSCT. Finally, no engrafted patient to date has developed any evidence of acute or chronic GVHD. Our results demonstrate that this relatively simple conditioning regimen is well-tolerated across a broad age range of adults with severe SCD, is effective in achieving stable mixed chimerism without the development of GVHD, and is sufficient to induce functional tolerance.

Disclosures:

No relevant conflicts of interest to declare.
B Cell Receptor-ERK1/2 Signal Cancels PAX5-Dependent Repression of BLIMP1 Probably Through PAX5 Phosphorylation: A Mechanism of Antigen-Triggering Plasma Cell Differentiation
Authors: Yasuda Takahiko , Hayakawa Fumihiko , Kurahashi Shingo , Naoe Tomoki
Published: October 2019

Abstract 1109

Plasma cell differentiation is initiated by antigen stimulation of B cell receptor (BCR). Until BCR stimulation, BLIMP1, a master regulator of plasma cell differentiation, is suppressed by PAX5, a key transcriptional repressor to maintain B cell identity. After BCR stimulation, upregulation of BLIMP1 and subsequent suppression of PAX5 by BLIMP1 are observed and thought to be the trigger of plasma cell differentiation; however, the trigger that derepresses BLIMP1 expression is yet to be revealed. Here, we demonstrated PAX5 phosphorylation by ERK1/2, the main component of BCR signal, in vitro and in vivo. The sites of PAX5 phosphorylation were identified by PCR mutagenesis assay. In luciferase reporter assays, transcriptional repression on BLIMP1 promoter by PAX5 was canceled by PAX5 phosphorylation. Furthermore, transcriptional repression by phosphorylation-defective mutant of PAX5 was attenuated by CA-MEK1 co-expression to a significantly lesser extent than that by wild-type PAX5, indicating its resistance to ERK1/2 signal-dependent cancelation of the transcriptional repression (Figure A). Finally, BCR stimulation induced strong ERK1/2 activation, phosphorylation of endogenous PAX5 (Figure B), and upregulation of BLIMP1 mRNA expression in B cells. These phenomena were inhibited by U0126, MEK1 inhibitor. These data imply that PAX5 phosphorylation by BCR signal is the initial event in plasma cell differentiation (Figure C).

Disclosures:

Naoe: Kyowa-Hakko Kirin.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Chugai Pharma.: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Zenyaku-Kogyo: Research Funding; Otsuka Pharma.: Research Funding.
KSHV Latency Locus Augments B Cell Response In Vivo
Authors: Sin Sang-Hoon , Dittmer Dirk P
Published: October 2019

Abstract 1115

A small subset of genes is transcribed during latency in Kaposi sarcoma-associated herpesvirus (KSHV) lymphomas. To better understand viral lymphomagenesis we generated a transgenic mouse expressing all viral latent genes including LANA, vFLIP, vCyc, kaposin and all viral miRNAs. All viral miRNAs were expressed in B cells. The complete latency locus induced activation of mature B cells more robustly than a single LANA protein transgenic mouse. It led to the specific expansion of marginal zone (MZ) B cells. The transgenic mice showed an augmented primary response to T-dependent antigen leading to acute MZ activation and germinal center (GC) activation. Furthermore, they exhibited an augmented response to TLR4 stimulation. Our data indicates that the KSHV latency locus fuels transformation processes as a chronic antigenic stimulator. These data supports a model of lymphomagenesis, in which virally infected B cells become hyperresponsive to B cell stimulation, thus providing an expanded pool for secondary genetic alterations.

Disclosures:

No relevant conflicts of interest to declare.
FAK Regulates Progenitor B Cell Growth, Localization and Retention in Bone Marrow Microenvironments
Authors: Park Shin-Young , Wolfram Peter , Manis John , Beggs Hilary , Nombela-Arrieta Cesar , Pivarnik Gregory , Silberstein Leslie E
Published: October 2019

Abstract 1119

Progenitor B cells are thought to reside in complex bone marrow microenvironments, e.g. niches, where they receive signals for growth and maturation. Here we have conditionally targeted in B cells the non-receptor tyrosine kinase, Focal Adhesion Kinase (FAK), because of its function as an integrator of cell extrinsic signals including CXCL12 and VCAM-1. The number of progenitor (pro-, pre- and immature) B cells is reduced by 30–40% in CD19-Cre Fak fl/fl mice compared to wild type mice, and ex vivo cultured, Fak deleted pro-B cells exhibit impaired IL-7 ± CXCL12 mediated proliferation and survival. A novel quantitative laser scanning cytometry approach was used to demonstrate that Fak deletion also disrupts the non-random gradient distribution in femoral bone marrow of progenitor B cells, which normally are preferentially localized in endosteal regions of the metaphyses and to a lesser degree of the diaphysis. Increased numbers of pro-B cells are detected in the periphery of Fak deleted mice, and mobilization of pro-B cells induced by the inflammatory antigen NP-CGG-alum is increased 3-fold in Fak-deleted versus wild type mice. In addition, intravenously transferred Fak deleted pro-B cells have defective homing specifically to the extra-vascular compartment of the bone marrow. Collectively, these studies illustrate the importance of FAK in regulating progenitor B cell homeostasis and maintenance of their spatial distribution in bone marrow niches.

Disclosures:

No relevant conflicts of interest to declare.
Abnormalities of the Hematopoietic Stem Cell Compartment in Children After in Utero Exposure to AZT
Authors: Andre-Schmutz Isabelle , Cortivo Liliane Dal , Six Emmanuelle M. , Kaltenbach Sophie , Radford-Weiss Isabelle , Cagnard Nicolas , Cocchiarella Fabienne , Benachi Alexandra , Mandelbrot Laurent , Mavilio Fulvio , Cavazzana-Calvo Marina , Blanche Stéphane
Published: October 2019

Abstract 1123

Mother to child HIV-1 transmission prophylaxy with antiretroviral (ARV) during pregnancy is remarkably effective. Although now based on various ARV-combinations, azydothymidine (AZT) remains the cornerstone drug in all guidelines. AZT hematologic toxicity after in utero exposure consisted in a reversible anemia but also in a persistant slight decrease on platelet, neutrophils and lymphocytes count up to 2 years of age. This long lasting biological effect evokes an alteration of the hematopoietic stem cell compartment. The very long term impact is unknown.

Twenty six neonates from HIV-1 infected mothers treated with an AZT based combination during pregnancy in Paris area were included in the study and compared to 20 neonates from healthy mothers. Umbilical cord blood was sampled after obtaining written informed consent from the mother. The experimental scheme included phenotypical and functional analysis of the hematopoietic compartment as well as transcriptome analysis of hematopoietic progenitors. Phenotypic analysis revealed a tendancy to higher numbers of CD34+ cells in the AZT group as compared to the control group. A similar finding was observed for hematopoietic stem cells defined on the basis of a Lin-CD34+ CD38loCD90+CD45RA- phenotype. Multipotent progenitors Lin-CD34+ CD38loCD90-CD45RA- were also increased at a statistically significant level. Affymetrix analysis of gene expression profiles was performed on CD34+ cells sorted from 8 AZT and 6 controls. Ingenuity analysis revealed significant differences in genes involved in hematopoiesis and cell cycle. Among hematopoietic genes for which differences of expression were noted between the two groups, 14 have a known role in the maintenance and differentiation of hematopoietic stem cells. Quantitative RT-PCR are actually ongoing to further characterize the gene expression profile of AZT CD34+ cells.

These results support the hypothesis of an alteration of the hematopoietic stem cell compartment in children after in utero exposure to AZT. Potential hematotoxicity of other ARV drugs must be evaluated to delineate the safest as possible mother to child HIV-1 transmission prophylaxy.

Disclosures:

No relevant conflicts of interest to declare.
The Role of Akt3 in Mediating Outside-in Signaling of the Platelet Integrin αIIbβ3
Authors: O'Brien Kelly A , Hay Nissim , Du Xiaoping
Published: October 2019

Abstract 1134

Ligand binding to integrins mediates cell adhesion and transmits “outside in” signals that lead to cell spreading, migration, and proliferation. In platelets, the prototype integrin aIIbb3-mediated outside-in signaling is required for platelet spreading and retraction, and greatly amplifies platelet activation. Previous studies suggest that phosphoinositide 3-Kinases (PI3K) are activated upon binding of integrin αIIbβ3 to its ligand fibrinogen, and is important in outside-in signaling leading to platelet spreading. However, the mechanism by which PI3K transmits outside-in signals has been unclear. A major known downstream effector of PI3K is the Akt (protein kinase B) family of serine/threonine kinases, including Akt1, Akt2, and Akt3. We have recently shown that platelets not only express Akt1 and Akt2 as previously reported, but also express a substantial amount of Akt3. To investigate whether Akt3 is a downstream effector mediating PI3K-dependent integrin outside-in signaling, platelets from Akt3 knockout mice were compared with wild type platelets for their spreading on fibrinogen. Platelets from Akt3−/− mice showed partially, but significantly reduced spreading on fibrinogen, indicating that Akt3 is important in integrin-mediated outside-in signaling leading to platelet spreading. Consistent with the results of Akt3 knockout, treatment of platelets with a pan Akt inhibitor also significantly inhibited spreading of human and mouse platelets on fibrinogen. Akt becomes phosphorylated upon platelet spreading on fibrinogen, which is significantly reduced in Akt3 knockout platelets, and is abolished by PI3 Kinase inhibitor, wortmannin, or Src Family Kinase (SFK) Inhibitor, PP2, suggesting that Akt activation is downstream from PI3K, and SFK during integrin outside-in signaling. Thus, our data reveals that Akt3 is an important downstream effector of PI3K-dependent integrin outside-in signaling promoting platelet spreading.

Disclosures:

No relevant conflicts of interest to declare.