bluuebunny/crossref_metadata_2025 huggingface dataset:
Host Tissue PD-L1 Separates GVL Effect from Gvhd after Temporary Depletion of Donor CD4+ T Cells Early after HCT
Authors: Ni Xiong , Song Qingxiao , Deng Ruishu , Jin Hua , Cassady Kaniel M. , Zhang Mingfeng , Forman Stephen J. , Martin Paul J. , Wang Jianmin , Zeng Defu
Published: October 2019

Recipient tissue expression of programmed death-ligand 1 (PD-L1, B7H1) down-regulates graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT), but this information has not been translated clinically. Here we demonstrate a critical role for recipient PD-L1 expression in preventing both acute and chronic GVHD while preserving graft-versus-leukemia (GVL) effects when donor CD4+ T cells are temporarily depleted in vivo early after HCT. Depletion of donor CD4+ T cells increases serum IFN-γ concentrations and enhances recipient tissue expression of PD-L1 and donor CD8+ T cell expression of the PD-L1 receptors CD80 and PD-1. In GVHD target tissues, depletion of CD4+ T cells increases anergy and apoptosis of infiltrating CD8+ T cells in a manner that depends on recipient PD-L1 expression, thereby preventing damage to intestinal Paneth cells and stem cells, hepatocytes, and thymic medullary epithelial cells, and preventing both acute and chronic GVHD. In lymphoid tissues, depletion of CD4+ T cells augments CD8+ T cell proliferation without increasing CD8+ T cell anergy or apoptosis, resulting in expansion of donor CD8+ T cells and strong GVL effects. Therefore, temporary depletion of donor CD4+ T cells early after HCT represents a novel approach for preventing GVHD while preserving GVL effects. This work was supported by NIH R01 AI066008 and Nesvig Lymphoma Fundation (to D.Z.) and by NSFC 81090413, 81270638 (to J.W.).

Disclosures

Forman: Amgen: Consultancy; Mustang: Research Funding. Martin:Neovii: Research Funding; RegImmune: Research Funding; Enlivex: Consultancy; Janssen: Consultancy; Pfizer: Consultancy; Pharmacyclics: Consultancy.
Effect of Transfusion of the Third Party Umbilical Cord Blood on Haplo-Identical Hematopoietic Stem Cell Transplantation
Authors: Wang Jing Bo , Fei Xin Hong , Cheng Hao Yu , Yin Yu Ming , Zhang Wei Jie , Zhang Shu Qin , Li Qian , Xue Song , Gu Jiang Ying , He Jun Bao , Yang Fan , Liu Meng Qi , Zhao Jie , Liu Dai-Hong
Published: October 2019

Objective To retrospectively evaluate the effect of the third party umbilical cord blood on haplo-identical hematopoietic stem cell transplantation.

Methods From June 2012 to May 2015, 125 leukemia patients were enrolled, including 41 cases of ALL, 62 cases of AML, 12 cases of MDS, 7 cases of CML-BP, 2 cases of acute mixed leukemia and 1 case of Mother cell dendritic cell tumor. Inclusion criteria: 1) AL patients; 2) halpo-identical HSCT; 3) 3/6 matched cord blood was available. Patients were divided into two groups, ie. group A (HSCT, n=65) and group B (HSCT plus umbilical cord blood transfusion group, n=60). Myeloablative conditioning regimens consisted of BuCy, TBI/FLAG, TBI/Cy, and FLAG that followed by reduced-intensified BUCY. The median dose of mononuclear cells in group A and B were 8.58×108/kg and 9.01×108/kg, respectively. The median dose of CD34+ cells for transfusion in each group were 3.67×106/kg and 2.94×106/kg, respectively. The dose of grafted UCB MNCs and CD34+ cells for group B were 3.5×107/kg and 2×105/kg, respectively. All patients received cyclosporineA, MMF and methotrexate for GVHD prophylaxis. The endpoints of this study were hematological engraftment, incidence of acute and chronic GVHD, incidence of relapse, transplant-related mortality(TRM), non-relapse mortality(NRM), Overall survival(OS) and Disease-free survival(DFS) in each group.

Results The median follow-up time was 17(3-29) months in group A and 18(3-35) months in group B. Patients in group A reached a sustained ANC of more than 0.5*109/L at a median of 11 days, whereas 14 days in group B. Platelet more than 20*109/L occurred at a median of 19 days in group A, whereas 17 days in group B (P= .4). The rate of aGVHD was not significantly different in the two groups, 56.9% in group A and 48.3% in group B (P= .21). The accumulative incidence of II-IV grade aGVHD was 35.4% in group A and 30% in group B (P= .42). The incidence of chronic GVHD was 79.2% in group A and 71% in group B (P= .47). The incidence of extensive type was lower in group B, 69.2% Vs 35%, P=0.09. The incidence of CMV was lower in group B, 80% Vs 60% (P= .01). The accumulative incidence of EBV was lower in group B, 35.4% Vs 3.3% (P<0.01). At two years, the accumulative incidence of relapse was 24.4% in group A and 17.2% in group B, P=0.13. The two-year accumulative incidence of OS was 72.9% in group A and 84.8% in group B, P=0.07. The one-year accumulative incidence of DFS in each group were 65.7% and 79.4%, respectively, P=0.03. Conclusion Our clinical results have shown that HSCT with transfusion of the third party umbilical cord blood is a promising modality for induction of immunity reconstitution. Better survival results may benefit from lower incidence of GVHD and virus infection.

Disclosures

No relevant conflicts of interest to declare.
Trends in Financial Toxicity for Patients of a Community Cancer Clinic
Authors: Santhosh-Kumar Cherupolil R. , Struve Stephanie , Freier Sue
Published: October 2019

Background: Rising health care costs - especially those of new medications for cancer treatment - have caused a severe financial strain on patients, leading to significant alteration of lifestyles. Published information regarding financial distress is scant with data only from large tertiary care institutions that may not be relevant to smaller communities. We sought to analyze the extent of financial toxicity over time in a community cancer clinic staffed by two medical oncologists.

Methods: Since 2010, a team of two oncology social workers prospectively collected data on financial assistance for patients treated at our community cancer clinic. Data regarding medication assistance is available for 2012-2014.

Results: Approximately 500 new patients and 1600 unique follow-up patients are evaluated and treated at the clinic annually. From January 2010 through December 2014, 1,012 requests for assistance were processed. The number of requests increased from 134 in 2010 to 320 in 2014. Most common requests were for medications and financial aid. Trends are shown in the table. Though the number of patients applying for medication assistance increased in 2014, the amount requested was less than previous years because of the influx of specialty pharmacies in the market with their own assistance programs.

Conclusions: Financial toxicity for cancer patients is a real issue in the community clinic setting.Requests for financial assistance have increased over time. The number of FDA approvals for new cancer medications seem to correlate with the increase. Studies are in progress to further clarify the financial impact of cancer on patients.Table.Trends in requests for assistance and aid and FDA approval for new medications.Requests for20102011201220132014Financial aid6411686101143Service311112733Equipment1311111823Transport312357Medications51534449114Patient cost of medications for which aid requested$731,891$1,554,684$1,253,827FDA approvals for new Oncology medications61181513

Disclosures

No relevant conflicts of interest to declare.
Changes in Gene Expression during G-CSF-Induced Emergency Granulopoiesis in Humans
Authors: Pedersen Corinna Cavan , Borup Rehannah , Fischer-Nielsen Anne , Mora-Jensen Helena , Fossum Anna , Cowland Jack B. , Borregaard Niels
Published: October 2019

Emergency granulopoiesis refers to the increased production of neutrophils in bone marrow and their release into circulation induced by severe infection. Several studies point to a critical role for granulocyte colony-stimulating factor (G-CSF) as the main mediator of emergency granulopoiesis. However, the consequences of G-CSF stimulation on the transcriptome of neutrophils and their precursors have not yet been elucidated in humans. Here, we investigate the changes in mRNA and miRNA expression in successive stages of neutrophil development following in vivo administration of G-CSF in humans, mimicking emergency granulopoiesis. Blood samples were collected from healthy individuals after five days of G-CSF administration. Neutrophil precursors were sorted into discrete stages of maturation by flow cytometry and extracted RNA was subjected to microarray analysis. mRNA levels were compared to previously published expression levels in corresponding populations of neutrophil precursors isolated from bone marrow of untreated, healthy individuals. miRNA expression was investigated in the most mature cell population to determine G-CSF-induced changes in circulating neutrophils. G-CSF substantially affected mRNA and miRNA expression patterns, demonstrating significant impact on neutrophil development and function. 1110 mRNAs were differentially expressed more than 2-fold with G-CSF while the treatment induced changes in the levels of 73 miRNAs in the mature population. In addition, G-CSF treatment reduced the levels of four out of five measured granule proteins in mature neutrophils including hCAP-18, which was completely deficient in neutrophils from G-CSF-treated donors. Cell cycle analysis pointed towards an induced proliferative capacity of myelocytes. These results indicate that multiple biological processes are altered in order to satisfy the increased demand for neutrophils during G-CSF-induced emergency granulopoiesis.

Disclosures

No relevant conflicts of interest to declare.
Transcutaneous Immunization with a Solid Nanoscopic Imiquimod Suspension Enhances Tumor Rejection
Authors: Stein Pamela , Gogoll Karsten , Peters Tanja , Denny Mark , Tenzer Stefan , Stevanovic Stefan , Schild Hansjoerg , Theobald Matthias , Langguth Peter , Radsak Markus
Published: October 2019

Introduction:

Transcutaneous immunization (TCI) is a novel vaccination strategy to induce strong therapeutic cytotoxic T-lymphocyte (CTL) responses by directly targeting skin-resident professional antigen-presenting cells (APC). This vaccination approach is very promising to overcome current limitations of standard vaccination approaches that are mostly effective in prophylaxis, but not in the treatment of diseases. In this context, we have developed a TCI method based on a synthetic TLR7 agonist imiquimod that partial tumor protection in experimental rodent models. In our present work, we describe a novel optimized formulation of imiquimod in a solid suspension of crystalline imiquimod (IMI-Sol) that induces superior CTL responses and enhanced tumor protection.

Methods:

We determined imiquimod-penetration into the skin as well as systemic distribution by HPLC in C57BL/6 mice (at 6-12 weeks). For TCI, C57BL/6, Tlr7 (TLR7-/-) or Myd88 (MyD88-/-) gene deficient mice IMI-sol or imiquimod (in the commercially available formulation Aldara®) together with the CTL epitope SIINFEKL (derived from chicken ovalbumin) was applied on two consecutive days on the dorsum of the animals after hair removal by electric clippers. Induction of CTL responses was characterized after 7 days by the analysis of peptide-specific CTLs (ELISpot, tetrameric MHC I complexes) and in vivo cytolytic activity by flow cytometry. Tumor protection was assessed using the B16OVA tumor model.

Results:

Despite equal imiquimod penetration into the skin, we found superior vaccination capacity of IMI-Sol compared to Aldara® leading to enhanced tumor-protection. Using TLR7-/- or MyD88-/- mice, we confirmed that the induction of CTL responses with IMI-Sol was completely dependent on the TLR/MyD88 pathway. The enhanced immune response are due to more efficiently activated skin-derived DCs upon IMI-Sol treatment found in skin-draining lymph nodes.

Conclusions:

We developed of a superior imiquimod formulation for transcutaneous vaccination that boosts vaccination efficacy by the enhanced activation of skin-resident APCs leading to the induction of therapeutic CTL responses underscoring the importance of pharmaceutical aspects affecting vaccination potency beyond understanding of the underlying basic mechanisms. Combined efforts are necessary to establish TCI as a promising novel next generation vaccination platform that can be used for the treatment of persistent infections and cancer.

Disclosures

No relevant conflicts of interest to declare.
Identification of the Protein S Binding Site in the Factor IXa Serine Protease Domain Presents Therapeutic Possibilities
Authors: Plautz William E. , Sekhar Satish , Majumder Rinku , Sheehan John P.
Published: October 2019

Protein S (PS) has been shown to inhibit Factor IXa (FIXa); however, its binding sites on FIXa have not been fully elucidated. By structural analysis of FIXa in conjunction with previous studies of FIXa's ability to bind heparin, an FIXa inhibitor, we identified that multiple residues that make up the heparin binding exosite, including Lys126, Asn129, Lys132, Arg150, Arg165, and Arg170 (chymotrypsin numbering), might contribute to binding PS; these residues were substituted with either Ala or Leu in separate FIXa mutants. Factor Xa (FXa) generation assays along with fluorescence analyses provided both the catalytic efficiencies and dissociation constants for the FIXa mutants under conditions of varied PS. Furthermore, anisotropic measurements of the fluorescent DEGR-FIXa's binding interaction with PS, when titrated against each of the FIXa mutants, supported the data found in the previous assays. These studies suggested that multiple residues, which are critical for the binding of heparin, such as Lys126, Lys132, Arg165, and Arg170, are also required in binding PS. To additionally verify this data, further ex vivo studies, including co-immunoprecipitation and flow cytometry of PS and FIXa are underway. Although previous studies have shown that the first EGF-like domain of FIXa is required to bind PS, these novel results have not only localized the main inhibitory site of PS on FIXa, but also provided enough information to propose a binding mechanism, which may rely on the reciprocal association between the head groups of PS and FIXa with the other's EGF-like domains, forming a bridging structure upon the cell membrane. Further studies are required to verify this hypothesis, but when clarified, it could provide immense benefits in the production of therapeutics for thrombosis.

Disclosures

No relevant conflicts of interest to declare.
Super Cross-Presentation of Tumor Antigens to Elicit Anti-Lymphoma Immunity By Synthetic Design of an Anti-Phosphatidylserine Bridge Protein
Authors: Corey Daniel , Weissman Irving L
Published: October 2019

Cell loss by apoptosis is a common feature in tumors. Dying tumor cells induce immune tolerance within the tumor microenvironment largely through highly conserved homeostatic clearance programs that restore tissue immune homeostasis and contribute to the formation of an immunosuppressive niche. The translocation of phosphatidylserine (PS) on cellular membranes, during the initial phases of apoptosis, functions as a recognition and removal signal that limits the immunogenicity of cell death. We examined whether altering clearance of dying cancer cells to elicit inflammatory turnover potentiates immune responses against lymphoma cells. To remove inhibitory signals in the homeostatic clearance pathway we utilized a molecular bridge scaffold to engineer a modified phosphatidylserine bridge protein (FA58C2-hIgG1 or C2-hIgG1) that works as a bridge between apoptotic cells expressing aminophospholipids and phagocytes bearing Fc receptors. In vitro administration of C2-hIgG1 to murine bone marrow derived macrophages promotes engulfment of apoptotic murine lymphoma cells (38C13 cell line) and ablates the secretion of the anti-inflammatory cytokine interleukin-10 (IL-10) and suppression of pro-inflammatory cytokines tumor necrosis factor (TNF-α) and IL-12p40 to the presence of apoptotic cells. Similarly, uptake of C2-hIgG1 treated lymphoma cells triggers upregulation of the costimulatory markers CD80, CD86, and MHC class II on macrophages and promotes secretion of Th1-recruiting lymphocyte chemokines CXCL9, CXCL10, and CCL5. Accordingly, in vivo administration of C2-hIgG1 partially restores immune responses to dead lymphoma cells in antigen cross presentation assays and promotes recruitment and retention of tumor antigen specific CD8+ T cells, dendritic cells, and natural killer cells into tumors. These effects combine to elicit anti-lymphoma immunity, improve responses to immune checkpoint inhibitors, and enhance the effectiveness of adoptive T cell transfers using engineered T Cell Receptors (TCRs) but not CD19-directed chimeric antigen receptor engineered (CAR-T) T cells.

Disclosures

No relevant conflicts of interest to declare.
NEDD4-1 Modulates the Resistance of Multiple Myeloma to Bortezomib
Authors: Huang Xi , Zhang Enfan , Guo Xing , Chen Jing , Lin Xuanru , Chen Qingxiao , Yan Haimeng , Yang Li , Li Yi , He Jingsong , Huang He , Cai Zhen
Published: October 2019

Background: Multiple myeloma (MM) is among the most common hematologic malignancies. Proteasome inhibitor bortezomib (Bor) is one of the most effective drugs for treatment of MM. However, during long-term Bor treatment, MM cells may eventually develop acquired-resistance to Bor which results in recurrence and a poor prognosis of MM. Several researches show that E3 ubiquitin ligases (E3s) primarily determine the substrate specificity of ubiquitin proteasome system and play an essential role in Bor resistance of MM. NEDD4-1 E3s, a founding member of the Neural precursor cell-Expressed Developmentally Downregulated gene 4 (NEDD4) family, was proved to involve in the proliferation, migration, invasion of cancer cells and the sensitivity of anticancer therapies. Our current study aims to explore the role and underlying mechanism of NEDD4-1 in acquired resistance of Bor in MM.

Methods: The mRNA and protein levels of NEDD4-1 and its substrates in MM cell lines (H929, LP-1, RPMI8226, OPM-2 and ARP-1) and MM patients were detected by Quantitative Realtime PCR and Western Blotting. Lentiviral plasmids containing shRNA against NEDD4-1 were transfected into MM cells. Cell viability, proliferation and apoptosis of MM cells were measured by Cell Counting kit8 (CCK8) and flow cytometry. Gene array was used to compare the gene expression profiles of a panel of Bor treated MM cells vs vehicle-treated MM cells.

Results: Gene array showed NEDD4-1 was significantly increased in MM cells treated with Bor. MM cells (CD138+ plasma cells of the bone marrow) from refractory/recurrence patients expressed lower NEDD4-1 than primary patient myeloma cells. Also, MM cell lines H929, ARP-1, LP-1 highly expressed NEDD4-1 at mRNA and protein levels. RPMI8226 and OPM-2 were relatively low expressed. Cell growth assay displayed no significant difference in proliferation between the NEDD4-1 knockdown (KD) and the control group (P>0.05). After suppression of NEDD4-1 using shRNAs, the killing effect of Bor in MM was significantly weaker than the control group (P<0.05). We also found that PTEN was decreased in the NEDD4-1 KD H929 cell line. Otherwise, phospho-STAT3 (ser727) and oncoprotein c-Myc and Bcl-2 were upregulated.

Conclusion: Collectively, our study reveals that inhibition of NEDD4-1 can reduce MM sensitivity to Bor via regulating PTEN, c-Myc and Bcl-2, may be related to JAK/STAT signaling pathway, which suggests that NEDD4-1 probably acts as a novel drug target and therapeutic paradigm in the battle against multiple myeloma.

Disclosures

No relevant conflicts of interest to declare.
Impact of Cardiac Stage and Hematologic Response on AL Amyloidosis Patients with Renal Involvement
Authors: Wong Sandy W. , Toskic Denis , Warner Melissa , Moreno-koehler Alejandro , Fein Daniel , Fogaren Terry , Lee Lisa , Oliver Colin , Guthrie Spencer D. , Comenzo Raymond L.
Published: October 2019

Cardiac stage and depth of hematologic remission are major predictors of survival for AL amyloidosis patients (Wechalekar et al., Blood, 2013; Dispenzieri et al., JCO, 2004; Palladini et al., JCO, 2012). Renal staging in AL amyloidosis (AL) has been studied in the context of renal survival (Palladini et al., Blood 2014). Influences on survival for renal patients have yet to be fully defined. We performed a retrospective study of all AL patients with renal involvement diagnosed at our center between 7/1/08 and 6/30/15. In this cohort of consecutive patients (n=80) median age was 63 (IQR 55-70) and 56% were men. Eighty-eight percent had lambda plasma cell disease and median involved FLC was 140mg/L (69-485). Thirty-nine percent were renal stage 1, 44% stage 2, and 16% stage 3. Median 24-hour proteinuria and serum creatinine were 6.23 g (3.47-10.70) and 1.03 mg/dL (0.80-1.80) respectively, and median eGFR was 72 mL/min (41-90). Fifty-eight percent had cardiac involvement, of whom 11% were cardiac stage 1, 54% stage 2, and 34% stage 3, while 18% had GI and 9% peripheral nerve involvement.

As first-line therapy, 70% received bortezomib-based regimens and 25% melphalan-based autologous stem cell transplant. By intention-to-treat, at 6 months after beginning therapy, 54% of patients had a hematologic response of PR or better, and renal and cardiac responses occurred in 13% and 14% of patients respectively, while renal progression occurred in 6%. Median overall survival (OS) for this cohort (n=80) was 67 months. Those with cardiac involvement (n=45) had a median OS of 41 months and, while median OS was not reached for cardiac stage ≤ 2, it was 31 months for those who were stage 3 (P<0.05) (Figure). Median OS was also not reached for patients achieving hematologic response ≥ VGPR with a median follow-up of 19 months.

In conclusion, for AL patients with renal involvement, both cardiac stage and depth of hematologic response are important contributors to overall survival. Furthermore, as this real world intention-to-treat analysis demonstrates, there is a continuing need for better therapies for both the hematologic disease and the organ damage associated with AL.

Figure. Figure.

Disclosures

Oliver: Prothena Biosciences, Inc.: Employment, Equity Ownership. Guthrie:Prothena: Employment, Equity Ownership, Other: Leadership. Comenzo:Takeda: Consultancy, Research Funding; Prothena: Consultancy, Research Funding; Karyopharm: Research Funding; Janssen: Consultancy, Research Funding.
Beta-Globin Gene Targeting in Human Hematopoietic Stem Cells Using Cas9 Ribonucleoprotein and rAAV6
Authors: Dever Daniel Patrick , Porteus Matthew
Published: October 2019

The b -hemoglobinopathies including sickle cell disease (SCD) and b -thalassemia ( b -thal) affect millions of people worldwide . SCD and b -thal are caused by mutations in the b -globin gene (HBB) resulting in either abnormal sickling or severely reduced protein production, respectively. A curative strategy for the b -hemoglobinopathies would be ex vivo HBB gene correction in patient-derived hematopoietic stem and progenitor cells (HSPCs) followed by autologous hematopoietic stem cell transplantation (auto-HSCT). We report the first CRISPR/Cas9 gene-editing platform for achieving homologous recombination (HR) at the HBB gene in long-term repopulating HSCs derived from mobilized peripheral blood. We combine electroporation of Cas9 protein complexed with chemically modified sgRNAs and delivery of a HR donor by recombinant adeno-associated viral vectors, serotype 6 (rAAV6). Notably, by including a reporter gene in the HR donor, we are able to identify and purify a population of HSPCs with >90% of cells having targeted integration at the HBB gene. These cells can be identified because HR-mediated integration causes the reporter gene to be expressed at log-fold higher levels than the non-integrated reporter. When transplanted into immunodeficient mice, the purified population gives rise to engraftment of HBB-edited human cells in primary and secondary recipients, confirming the presence of long-term repopulating hematopoietic stem cells (LT-HSCs). Importantly, we show efficient correction of the SCD-causing E6V mutation in SCD patient-derived CD34+ HSPCs by either editing the nucleotide mutation or knocking in an anti-sickling b -globin cDNA. Edited SCD CD34+ cells were shown to express adult b -globin (HbA) mRNA after HSPCs were differentiated into erythrocytes in vitro, confirming intact transcriptional regulation of the edited HBB allele. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting HSCs for HR at the HBB locus to advance the development of next generation therapies for b -hemoglobinopathies.

Disclosures

Porteus: CRISPR Therapeutics: Consultancy, Equity Ownership.
Specific Deletion of Keap1 in Endothelial Cells or/and Macrophages of SCD Mice Induces Nrf2 to Protect Tissues Against Oxidative Insults
Authors: Keleku-Lukwete Nadine , Harit Panda , Naganuma Eriko , Suzuki Mikiko , Yamamoto Masayuki
Published: October 2019

Sickle cell disease (SCD) model mice frequently suffer from hemolysis. Endothelial cells face hemolysis, and they activate inflammatory cells following their own activation. Heme and iron discharge into blood stream enhanced by the following ischemia reperfusion injury as consequence of intermittent vaso-occlusion, jeopardize functions of SCD mouse organs. Transcription factor Nrf2 regulates expression of the genes involved in antioxidant cell defense. The roles that Nrf2 plays in pathological conditions in association with oxidative insults make the Nrf2 pathway attractive for developing new drugs for SCD treatment. Recently, we demonstrated that systemic Nrf2 induction significantly relieves inflammation and organ damage of SCD mice, while hemolysis is still unaffected (PNAS 2015). To examine the mechanisms underlying the contribution of Nrf2, we analyzed SCD mice by means of cell type-specific activation of Nrf2 through conditional loss of its repressor Keap1. We generated compound SCD mice with Keap1 floxed and cell-specific Cre expression alleles (SCD::Keap1F/F::Tie1-Cre and SCD::Keap1F/F::LysM-Cre mice targeting endothelial cells and macrophages, respectively). In lungs of SCD::Keap1F/F::Tie1-Cre mice, expression of Nrf2 and Nrf2-target genes were upregulated, while expression of inflammatory cytokines were significantly repressed. In both genotypes, necrosis and inflammation of liver and lungs were significant improvement, compared to littermates control mice. Plasma leucocytes counts, plasma heme level, total bilirubin and indirect bilirubin levels, were significantly reduced in both genotypes compared to controls, and these levels were slightly higher in the SCD::Keap1F/F::LysM-Cre mice compared to SCD::Keap1F/F::Tie1-Cre mice. 8-OHdG staining of lungs showed smaller number of positive cells in SCD::Keap1F/F::Tie1-Cre mice. These results indicate that Nrf2 activation in both endothelial cells and macrophages contributes to relieve chronic complications of SCD.

Disclosures

No relevant conflicts of interest to declare.
Hydrogen Exchange Mass Spectrometry Reveals Allosteric Interactions Between the Distal and the Proximal Domains of Human ADAMTS13
Authors: Zander Catherine B. , Mayne Leland , Englander S. Walter , Zheng X. Long
Published: October 2019

Background: Deletion of the distal C-terminal domains, or a decrease in pH, or the binding of a monoclonal antibody to the C-terminal domains of ADAMTS13 increases its proteolytic activity towards VWF peptide substrate, VWF73, by approximately 2-fold; this suggests an allosteric interaction between the distal and proximal domains of ADAMTS13. However, neither the underlying mechanism of the allosteric interaction nor the exact sites of interaction between the proximal domains and the distal C-terminal domain are fully understood.

Objective: To determine the binding sites and kinetic interactions between the proximal and distal domains of ADAMTS13 and to gain insight into the mechanism of allosteric regulation of ADAMTS13 function.

Methods: Hydrogen deuterium exchange plus LC-mass spectrometry (HX-MS) and surface plasmon resonance (SPR) were employed to determine conformational changes and direct binding interactions between the proximal and distal domains of ADAMTS13.

Results: Recombinant full-length (rFL-A13) and an ADAMTS13 variant truncated after the spacer domain (rMDTCS) were purified to homogeneity. After incubation with deuterium for various times (0-6 hours), both rFL-A13 and rMDTCS were digested with pepsin and fungal protease-13 and then partially resolved by liquid chromatography before being injected into a LTQ ObitrapXL mass spectrometer for peptide identification. Overall, 188 of the total 338 peptides in rFL-A13 were identified, spanning the metalloprotease, cysteine-rich, and spacer domain. Only 16 peptides were found to be less accessible (or protected from deuterium-hydrogen exchange) in rFL-A13 than in rMDTCS. Two of these regions reside in the metalloprotease domain (residue 78-127 and 282-304). The next region is at a loop on the cysteine-rich domain (residue 446-473 and 495-501). The next peptides are in the adjacent loops of the spacer domain with one peptide 596-603, and five overlapping peptides spanning from 632-642, and then finally one peptide from the C-terminal of the MDTCS variant (residue 676 to 688). These results indicate that the distal C-terminal domains interact with multiple discrete sites in the proximal domains of ADAMTS13, resulting in protection from hydrogen-deuterium exchange. Such an interaction was further demonstrated by direct binding experiments with SPR. The distal domains including CUB, T2-CUB, and T5-CUB bound directly to M, MDT, and MDTCS under flow with high affinity.

Conclusion: Our findings demonstrate the allosteric interactions between the distal domains and the proximal domains at multiple discrete sites. Such an interaction may regulate catalytic efficiency and substrate specificity of the metalloprotease domain of ADAMTS13 under various conditions.

Disclosures

Zheng: Ablynx: Consultancy; Alexion: Research Funding.
Platelet Alpha-Storage Pool Defect Combined with Non-Classic Delta-Storage Pool Deficiency - with a Severe Bleeding Tendency
Authors: Bergmann Frauke , Schwierczek Kathrin , Sollfrank Stefanie , Czwalinna Andreas , Erkel Joseph , Lackner Karl , Walter Ulrich , Rossmann Heidi , Jurk Kerstin
Published: October 2019

Aims :We report about a 35 year old male patient with severe and frequent epistaxis and hematoma since infancy. He presented with mild thrombocytopenia and increased mean platelet volume. Von Willebrand's disease and subhemophilia had been excluded. Previously, he was diagnosed with immune thrombocytopenic purpura. He never underwent elective surgery. His parents were asymptomatic. However, his young daughter also suffers from severe bleeding symptoms and was referred to our coagulation outpatient clinic to investigate the bleeding disorder.

Methods:Platelet function was assessed by light transmission-, lumi-aggregometry and flow cytometry. Lysates of gel-filtered platelets were analyzed for total granule P-selectin, CD63 and von Willebrand factor (VWF) content by Western blotting and for serotonin levels by ELISA, respectively. Molecular genetic analysis was performed using whole exome sequencing and pyrosequencing.

Results: Platelet aggregation in response to ADP was not inducible. Platelet aggregation in response to epinephrine resulted in monophasic curves. Platelet aggregation in response to TRAP-6 or collagen was inducible but impaired and accompanied by disaggregation. In contrast, arachidonic acid- and U46619-induced platelet aggregation as well as ristocetin-induced platelet agglutination/aggregation was within the normal range. Also platelet surface expression of GPIIb/IIIa, GPIb/V/IX, GPVI and CD29 was normal. Activation of GPIIb/IIIa in response to ADP, convulxin or TRAP-6 was inducible but diminished and in response to epinephrine or arachidonic acid normal. P-selectin (alpha-granule marker) and CD63 (dense body/lysosome marker) surface expression was not inducible by thrombin or convulxin and markedly reduced in response to TRAP-6 and arachidonic acid. Immunoblot analysis of isolated platelets demonstrated pronouncedly decreased content of total P-selectin, VWF and CD63. Fluorescent staining of adenine nucleotides by mepacrine in patient's platelet dense bodies and secretion of ATP induced by arachidonic acid (normal aggregation) were absent. However, patient's platelet serotonin levels were within the normal range. The molecular genetic diagnostic showed an undescribed mutation in the GATA1 gene (c.886A>C hemizygot, p.T296P). The bioinformatic check confirmed a pathogenetic importance of this mutation located in the domain I of the C-terminal zinc finger.

Conclusion: We describe the first case of a combined platelet alpha- and partial delta-storage pool disorder where the dense granules lack adenine nucleotides but contain normal levels of serotonin - causing a severe bleeding disorder.

Disclosures

No relevant conflicts of interest to declare.
Natural IgM Against Oxidation-Specific Epitopes Inhibit Microvesicle-Driven Coagulation
Authors: Obermayer Georg , Afonyushkin Taras , Puhm Florian , Schwameis Michael , Jilma Bernd , Binder Christoph J
Published: October 2019

Increasing evidence demonstrates an important role for immune functions in coagulation and thrombosis, and both microbial and sterile triggers have been implicated in these responses. Oxidation-specific epitopes (OSE) are products of lipid peroxidation that are present on oxidized low-density lipoproteins and dying cells, and represent important sterile triggers of inflammation in cardiovascular diseases. We have previously shown that a large part of naturally occurring IgM antibodies have specificity for OSEs and that 50% of circulating procoagulant microvesicles (MV) carry OSEs, such as malondialdehyde-adducts (MDA), on their surface. Because OSE-specific natural IgM bind MVs and increased levels of circulating MVs have been implicated in an increased thrombotic risk, we studied the capacity of these antibodies in directly modulating the procoagulant potential of MVs in vitro and in vivo. We first showed that increasing concentrations of three different monoclonal IgM antibodies with specificity for MDA (LR04, NA17, E014) significantly delayed MV-induced fibrin formation in plasma, while an MDA-specific IgG antibody or F(ab')2 fragments of LR04 had no effect. Moreover, peak thrombin generation of normal, factor VII- or factor XII- deficient microvesicle poor plasma (MVPP) substituted with tissue factor positive MVs was significantly reduced in the presence of LR04 but not a control IgM antibody, indicating that it exerts its effect on the common pathway of the coagulation cascade. A similar anticoagulatory effect of LR04 was observed in freshly drawn whole blood analyzed by rotational thrombelastometry. Addition of LR04, compared to a control IgM antibody, increased clotting time, clot formation time, and the time to reach maximum clot firmness, and decreased maximum clot firmness. Finally, in a murine pulmonary embolism model, we could show that 30-minute survival of mice that received intravenous injections of epinephrine together with pro-coagulant MVs derived from the pancreatic cancer cell line HPAFII was significantly increased in mice co-injected with LR04 compared to controls. Our study identifies an inhibitory role of MDA-specific IgM antibodies in MV-induced coagulation. These effects are exerted on the common pathway of the coagulation cascade and require the intact structure of IgM antibodies. The findings may offer novel diagnostic and therapeutic approaches in the prevention and treatment of thrombosis.

Disclosures

No relevant conflicts of interest to declare.
Increased Vascular Permeability in the Bone Marrow Microenvironment Contributes to Acute Myeloid Leukemia Progression and Drug Response
Authors: Passaro Diana , Di Tullio Alessandro , Abarrategi Ander , Rouault-Pierre Kevin , Foster Katie , McNaughton Linda Ariza , Lassailly Francois , Bonnet Dominique
Published: October 2019

The biological and clinical behavior of hematological malignancies are not only determined by the properties of the leukemic cells themselves, but are also highly affected by interaction with the microenvironment, pointing to the existence of an active crosstalk between the two compartments. Previous studies showed that acute myeloid leukemia (AML) cells actively modify endothelial cells ex vivovia several pathways, mainly mediated by VEGF. However, as anti-VEGF therapies haven't produced successful results in clinical trials, an extensive study of the crosstalk between AML and the vascular niche in the bone marrow (BM) is required to provide new therapeutic strategies. In the present study we combined the use of mouse models of AML, human AML patient-derived xenografts (PDX) and direct analysis on patient-derived BM biopsies to provide a global, reliable picture of the bone marrow vasculature in AML disease. We found several abnormalities in the vascular architecture and function in PDX, such as increased number of endothelial cells, increased microvascular density (MVD), decreased vascular mean diameter and increased hypoxia. Furthermore, using two-photon confocal intravital imaging we witnessed increased vascular permeability upon AML engraftment, observed homogeneously among different PDX. Interestingly, induction chemotherapy failed to normalize the vascular permeability in the BM, despite significant reduction in AML engraftment. We identified increased nitric oxide (NO) as a major mediator of the AML-induced vascular leakiness in the BM. Increased levels of NO and activated NOS3 were found in PDX and in an independent cohort of patient-derived BM biopsies. Strikingly, inhibition of NO production using genetic and pharmacological approaches reduced the vascular permeability, potentiated the normal HSC function and significantly improved treatment response in PDX. These results strongly support the notion of a primary function of the vascular permeability in AML progression, drug response and in affecting normal stem cell function, and they call for clinical trials incorporating NOS inhibitors during the remission phase to target the abnormal vascular niche and improve the treatment response.

Disclosures

No relevant conflicts of interest to declare.
Identification of New Multiple Myeloma-Specific Cell Surface Antigens As Immunotherapeutic Targets
Authors: Hosen Naoki , Hasegawa Kana , Aoyama Yasutaka , Ichihara Hiroyoshi , Mugitani Atsuko , Nakao Takafumi , Manabe Masahiro , Oji Yusuke , Sugiyama Haruo
Published: October 2019

Cancer-specific cell surface antigens are ideal targets for therapies using monoclonal antibodies (mAbs) and their derivatives, such as chimeric antigen receptor (CAR)-T cells. However, such antigens are not likely to remain unidentified following extensive searching by transcriptome or proteome analyses. However, we hypothesized that cancer-specific antigens formed by post-translational events, such as glycosylation, complex formation, or conformational changes, might have been missed in previous screens. Such antigens could be discovered by thoroughly searching for cancer-specific mAbs and characterizing the antigens recognized by these mAbs. To test our hypothesis, we applied this strategy to identify novel therapeutic targets specific for multiple myeloma (MM), a major hematological cancer. We first identified two MM-specific mAbs designated as MMG49 or R8H283 after screening more than 10,000 anti-MM mAb clones. Then, we identified the antigens recognized by these mAbs by an expression cloning method. Interestingly, genes identified as antigens for both mAbs were not specific to MM cells, suggesting that both mAbs recognize MM-specific epitopes formed by post-translational events. Finally, we showed that these mAbs or CAR-T cells derived from them could reduce tumor burden in MM-xenograft models, but did not damage normal hematopoietic cells. These results not only demonstrate that these mAb or CAR-T cell therapy is promising for MM, but also suggest that MM-specific immunotherapetic target antigens formed by post translational events may be still missed.

Disclosures

Aoyama: Alexion: Honoraria.
Gene Disruption of Zebrafish Sbds Phenocopies Human Shwachman-Diamond Syndrome but Suggests More Global and Lineage Defects
Authors: Oyarbide Usua , Kell Margaret J , Farinas Javier , Topczewski Jacek , Corey Seth
Published: October 2019

Introduction: Shwachman-Diamond Syndrome (SDS) is a complex disorder resulting in short stature, neutropenia, bony dysplasia, and pancreatic malabsorption. Individuals with SDS are also at increased risk for developing myelodysplastic syndrome and/or acute myeloid leukemia (AML). Its estimated incidence of 1 in 77,000 births makes SDS difficult to study and develop newer therapies. SDS results from mutations in the SBDS gene. Genetic ablation of Sbds results in early embryonic lethality (ED 7.5) in mice. Zebrafish provide an attractive, alternative model organism to study human diseases. Genome analysis of zebrafish (Danio rerio) revealed the presence of a single sbds gene, encoding a protein 90% identical to the human orthologue.

Methods: We used CRISPR/Cas9 genome editing technique to generate indel mutations in sbds andestablished multiple zebrafish lines. We used zebrafish sbds mutants to analyze the function of this gene during vertebrate development and in the pathophysiology of SDS.

Results: Western blotting showed progressive decrease of Sbds expression with no detectable protein at 8 days post fertilization (dpf). These results suggest that the presence of Sbds during early stages of the development is due to maternal deposition, which was confirmed by qPCR and immunoblot demonstration of sbds transcript and proteins in in all stages of the development including the 1 cell-stage. Unlike Sbds-/- mice, homozygous mutant sbdsnu132live up to 6 weeks. They display marked growth retardation. We also observed a significantly low number of neutrophils at 5 dpf and 15 dpf. No differences were found in macrophages and hemoglobinization. Interestingly, multiple organs, especially the pancreas, liver, digestive tract and eye showed histologic evidence of atrophy at 21 dpf. Differences were also observed in fat deposition and bone formation. These defects were not seen at 15 dpf, suggesting a critical stage in development dependent on functional sbds.

Conclusions: To our knowledge, we established the first zebrafish model of SDS, making it a highly relevant model to investigate its pathophysiology and develop new small molecule therapies. Comparison of null states between mice and zebrafish suggest that loss of Sbds results in two phenotypes: an early embryonic stage required for viability and development and a post-embryonic (larval) stage critical for granulocyte production and normal size. Data will be presented on bone formation, neutrophil chemotaxis, metabolomics, and RNAseq in the zebrafish model.

This work was supported by grants from the NIH R21CA15920, Shwachman-Diamond Syndrome Foundation and the Alex Turnquist Memorial Research fund of Shwachman-Diamond America.

Disclosures

No relevant conflicts of interest to declare.
Recipient and Donor KIRs Ligands Match Prevents CMV Reactivation Post-Haploidentical T Cell-Replete Transplantation
Authors: Zhao Xiang-Yu , Luo Xue-Yi , Yu Xing-Xing , Zhao Xiaosu , Lv Meng , Han Ting-Ting , Chang Ying-Jun , Huo Ming-Rui , Xu Lan-Ping , Zhang Xiao-Hui , Liu Kai-Yan , Li Dan , Huang Xiao-Jun
Published: October 2019

Licensed NK cells have been demonstrated to have anti-cytomegalovirus (CMV) activity. We prospectively analysed the HLA typing of donor-recipient pairs and the KIR typing of the donors in 180 leukaemia patients to assess the predictive roles of licensed NK cells and donor-activating KIR genes on CMV reactivation post-T cell-replete haplo-SCT. Multivariate analysis showed that donor and recipient KIR ligand-ligand graft-versus-host direction or host-versus-graft direction mismatch were associated with the incidence of CMV reactivation [HR=1.663, 95%CI, 1.161-2.383, P=0.006] and refractory CMV infection [HR=2.34, 95%CI, 1.28-4.27, P=0.006] post-haploidentical T cell-replete transplantation. Donor and recipient KIR ligand-ligand match decreased CMV reactivation (51.65% [46.67, 56.62%] vs. 75.28% [70.87, 79.69%], P=0.012), refractory CMV infection (17.58% [13.77, 21.40%] vs. 35.96% [31.09, 40.82%], P=0.004) and CMV disease (3.30% [1.51, 5.08%] vs. 11.24% [8.04, 14.43%], P=0.024), respectively. Meanwhile, there was a significantly increased risk of CMV reactivation in patients who accepted a KIR2DS2-positive donor compared those who accepted a KIR2DS2-negative donor (80% [71.93, 88.07%] vs. 63.87% [60.18, 67.56%], P=0.039), especially in recipient HLA-C1/C2 and donor HLA-C1C2 with KIR2DL1/2DL2/2DL3/2DS2 genotype (100%).

Conclusions: These findings suggested that donor and recipient KIR ligand-ligand match might promote the NK cell licensing process, thereby increasing NK cell-mediated protection against CMV reactivation. However, roles of donor positivity for the KIR2DS2 against CMV reactivation need to be further explored in T cell-replete haplo-SCT.

Disclosures

No relevant conflicts of interest to declare.
PRE-Transplant Positron Emission Tomography (PET) Predicts Outcomes of Patients with Hodgkin disease Undergoing Reduced Intensity Conditioning Haploidentical Transplant
Authors: Marani Carlo , Morbelli Silvia , Raiola Anna Maria , Dominietto Alida , Ferrarazzo Giulia , Nati Sandro , Congiu Marina , Bregante Stefania , Varaldo Riccardo , Gualandi Francesca , Angelucci Emanuele
Published: October 2019

Background. In recent years, the number of Hodgkin Lymphoma (HL) patients submitted to reduced intensity haploidentical allogeneic transplant (RIC haplo SCT) consistently increased. [Sureda et al. Curr Treat Options Oncol 2014] However, with a post alloSCT relapse rate so high as 40-60%, disease control after autoSCT relapse remains a clear unmet medical need. In respect of improving transplant outcome identification of factors influencing outcomes are warranted

Methods.We reviewed outcomes obtained in 44 consecutive HL patients who received RIC haplo SCT between September 2009 and June 2015 at our institution. The donor was haploidentical relative in all cases. All patients had received at least 3 conventional chemotherapy lines before RIC haplo SCT; most of them (72%) were refractory to front-line chemotherapy and all but one had received an autologous SCT. All patients received the same reduced intensity conditiong regime, and GvHD prophilaxis with post-transplant cyclophosphamide (Raiola et al. BBMT 2008). The median age was 31years (range 18-60). Deauville five-points scale were used to interpretate the results of pre-transplant positron-emission tomography (PET) studies, with a threshold of 4 or above considered positive. Survival curve and GvHD incidence were calculated with the method of Kaplan and Meier. Cox regression model was performed to identify pre-transplant factors influencing outcomes.

Results. All patients engrafted but 1 who showed autologous recovery. The median day for 500 neutrophils was 15 days (13-23). Three patients died in remission at 8 (cGvHD), 16 (myocarditis) and 51(pneumonia) months from SCT, respectively. Three years non relapse mortality = 8%. Risk of grade II-IV acute GvHD and 3-year chronic GvHD were 22 and 21%, respectively. With a median follow-up for surviving patients of 38 months (range 13-83), 21 relapsed (47%). All relapsed patients were treated with further therapy, mainly immuno-chemotherapy and donor lymphocyte infusion (DLI). The 3-year overall (OS) and progression free survival (PFS) were 77% and 39%, respectively. Pre-transplant PET status was the only factor influencing outcomes of the procedure (OR 5.4; 95% CI 1.7 - 16.6). In the 41 patients with available pre-transplant PET status, 19 were positive and 22 negative. Outcomes accordingly to pre-transplant PET status are shown in the following table.

Conclusions.Pre-transplant PET status is a factor influencing outcomes of relapsed/refractory HL submitted to RIC haplo SCT. Grade II-IV aGvHD was markedly reduced in patients with metabolic remission at time of transplantation.

Table Table.

Disclosures

Angelucci: Novartis oncology, celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.