bluuebunny/crossref_metadata_2025_split huggingface dataset:
Disruption of SOCS3 Promotes the Anti-Cancer Efficacy of Primary NK Cells
Authors: Naeimi Kararoudi Meisam , Elmas Ezgi , Lamb Margaret , Chakravarti Nitin , Trikha Prashant , Lee Dean Anthony 
Published: October 2019

Abstract

Genetic modification of NK cells to enhance cancer immunotherapy has potential application to treat a wide range of cancers. Recently, we developed a new strategy in which CRISPR/Cas9 elements are introduced into NK cells as ribonucleoproteins (RNPs) via electroporation, followed by expansion on feeder cells expressing 4-1BBL and membrane-bound IL-21 to generate large numbers of genetically modified NK cells. This method was used to genetically modify several genes in primary and expanded NK cells including suppressor of cytokine signaling 3 (SOCS3). SOCS3 negatively regulates cytokine signaling through the JAK/STAT pathway. We hypothesized that disruption of SOCS3 in primary NK cells using Cas9/RNPs could potentially maintain STAT3 signaling levels and subsequently increase their proliferation and cytotoxic function.
We designed gRNAs to target exon 2 of the SOCS3 gene and electroporated them along with Cas9 protein as Cas9/RNPs into primary NK cells using the Lonza 4D electroporator. We tested 6 different conditions of gRNAs alone or in combination. The NK cells in the control group were electroporated with no Cas9/RNPs. After electroporation, the cells were rested in culture media supplemented with 100 IU of human IL-2 for 48 hours and then expanded using irradiated feeder cells. At day 7, an equal number of cells were restimulated with irradiated feeder cells to test the effect of SOCS3 KO on proliferation. Western blot was used to assay the knock out efficacy at the protein level. Calcein assay and IncuCyte Zoom (Essen) were performed to measure cytotoxicity against two cancer cell lines, K562 and Daoy.
Our results showed a significant reduction in SOCS3 protein levels in 3 conditions (gRNA1, gRNA3 and gRNA1+ gRNA3) in comparison to the control group. The calcein assay and IncuCyte zoom showed the modified SOCS3 KO NK cells could kill the tumors more efficiently in comparison to the control. Proliferation data showed the SOCS3 KO cells can grow faster than the control group.
In conclusion, our data demonstrate the role of SOCS3 and JAK/STAT pathway in NK cell function and suggests that SOCS3 may be a good target for genetic modification to improve cancer immunotherapy using NK cells.

Disclosures
Lee: CytoSen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck, Sharp, and Dohme: Consultancy; Courier Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
A Highly Efficient and GMP Compliant Protocol to Manufacture CCR5 Edited Cells to Treat HIV Infection
Authors: Romito Marianna , Meyer Emily , Poddar Sushmita , Heinz Helena , Rositzka Julia , Hildenbeutel Markus , Gautron Anne-Sophie , Busser Brian W , Kok Yik Lim , Turchiano Giandomenico , Haas Simone , Schäfer Richard , Smith Julianne , Metzner Karin J , Mussolino Claudio , Duchateau Philippe , Cathomen Toni , Cornu Tatjana I 
Published: October 2019

Abstract
Targeted genome editing in blood and immune cells enable new therapeutic applications, especially for infectious diseases. We present a GMP-compliant protocol to manufacture CCR5-edited CD34+ hematopoietic stem and precursor cells (HSPCs) with the goal to cure patients suffering from chronic infection with human immunodeficiency virus type 1 (HIV1). We hypothesize that genetic disruption of the CCR5 gene, which encodes the major HIV1 co-receptor, in HSPCs will give rise to an HIV-resistant immune system after transplantation. We have developed engineered nucleases based on transcription activator-like effector nucleases (TALENs) targeting CCR5. Electroporation of CD4+ T-cells and CD34+ HSPCs with mRNAs encoding TALENs revealed disruption of up to 80% of CCR5 alleles in CD4+ T-cells and over 90% of alleles in HSPCs. The high gene editing frequencies in T-cells and HSPCs were confirmed by deep sequencing, and no cleavage activity above background levels were detected at the top 20 predicted off-target sites. CCR5-edited CD4+ cells preserved their proliferation capacity and their biological function. Importantly, these cells showed significantly reduced CCR5 expression and became resistant to infection with the R5-tropic HIV-1JR-FL virus. The CCR5-edited HSPCs maintained their proliferation potential and their capacity to differentiate into the various blood lineages in vitro and in vivo, and clonal analysis revealed bi-allelic CCR5 disruption in more than 75% of cells. In summary, our developed protocol enables highly efficient and GMP-compliant knockout of the CCR5 locus in clinically relevant cells, so forming the foundation for a planned phase I/II clinical study.

Disclosures
Gautron: Cellectis SA: Employment. Busser:Cellectis: Employment, Patents & Royalties: Cellectis. Smith:Cellectis. Inc: Employment, Patents & Royalties. Duchateau:Cellectis: Employment, Patents & Royalties: Cellectis. Cathomen:TRACR Hematology: Consultancy; Cellectis: Research Funding; Miltenyi Biotec: Research Funding. Cornu:Cellectis: Research Funding; Miltenyi Biotec: Research Funding.
In Elderly Patients with Newly Diagnosed Aggressive B-Cell Lymphoma, the Rate of Change in Body Weight after the First Course R-CHOP (-like) Chemo Therapy Has Effect on Their Survival
Authors: Ayako Muramatsu , Hiroaki Nagata , Kodai Kuriyama , Yoshiko Hirakawa , Muneo Oshiro , Toshiki Iwai , Hitoji Uchiyama 
Published: October 2019

Abstract
Background
The incidence of B-cell lymphoma increases with age, and over 40% occurs in patients at age of 70 years old or more. Aggressive B-cell lymphoma was often treated with R-CHOP (-like) regimen. However, in elderly B-cell lymphoma patients, treatment intensification often must be lowered due to the risks of comorbidities and organ function deterioration, and treatment outcomes are worse compared to younger patients. The optimal dose of R-CHOP (-like) therapy is necessary to improve the outcome of the elderly patients with B-cell lymphoma. We conducted a retrospective cohort study examining the influence of the rate of change in body weight after the first chemo therapy on their outcomes and survival.
Methods
Clinical records of 111 patients who had received R-CHOP (-like) regimen were retrospectively analyzed.
They were all over 73 years old, and newly diagnosed with aggressive B-cell lymphoma by WHO 2008 criteria.
They were treated at Japanese Red Cross Kyoto Daiichi Hospital between January 1st 2008 and December 31st 2017.
Data on clinical characteristics and treatment modalities were obtained through the review of medical charts.
Progression-free survival (PFS) and overall survival (OS) were estimated using the Kaplan-Meier method.
The impact of variables on PFS and OS was evaluated by univariate log-rank tests and by multivariate analysis using the Cox proportional hazards model.
Results
Median age at diagnosis was 78 years old (73-94).
The 2-year OS rate was 77.5% (95%Cl: 68.3-84.3%), PFS rate was 77.4% (95%Cl: 68.2-84.3%) in all patients.
The 5-year OS rate was 62.3% (95%Cl: 50.2-72.2%), PFS rate was 55.5% (95%Cl: 43.4-66%) in all patients.
The average rate of change in body weight after the first therapy was 4.59%.
Large changes in body weight ( >9.3%) after the first therapy had worse clinical outcomes with shorter median OS (1.43 years vs. NA, P <0.001 HR 4.39, 95% CI 2.14 to 8.99, see figure 1) and median PFS (1.43 years vs. 6.9 years, P<0.001, HR 3.65, 95%CI 1.82to 7.29, see figure 2).
Conclusion
Large changes in body weight ( >9.3%) after the first therapy were associated with poor outcomes in elderly people with newly diagnosed aggressive B-cell lymphoma.
This suggests that adjusting drug dosage on and after the second therapy in those patients has possible to be improve their survival.

Disclosures
No relevant conflicts of interest to declare.
Efficacy and Safety of Ruxolitinib in Treatment of Steroid Refractory/Refractory Acute Graft-Versus-Host Disease: A Prospective Multicenter Research
Authors: Fan Zhiping , Deng Lan , Li Xudong , Zhang Qing , Xuan Li , Huang Fen , Lin Ren , Jin Hua , Xu Na , Shi Pengcheng , Wang Zhixiang , Ye Jieyu , Jiang Ling , Sun Jing , Liu Qifa 
Published: October 2019

Abstract
Objectives To explore the efficacy and safety of ruxolitinib in the treatment of steroid refractory/refractory acute graft versus host disease (aGVHD).
Methods A prospective analysis was conducted in the 31 steroid refractory/refractory aGVHD cases who treated with ruxolitinib from October 2017 to June 2018. The first dose of ruxolitinib was 5-10mg bid, and the dose was adjusted to 5mg qd for maintenance treatment when complete remission (CR) status lasted for 2 weeks after steroid refractory/refractory aGVHD turned into CR.
Results Of all the 29 steroid refractory/refractory aGVHD patients, 15 patients were treated with ruxolitinib in case of disease progression after receiving at least one second-line treatment, and 14 cases directly with ruxolitinib after the occurrence of corticosteroid refractoriness. The time of starting ruxolitinib treatment after the occurrence of aGVHD was 10 (4-81) d, the median time of onset was 8 (3-18) d. The overall response rate was 89.6% (26/29) including 22 complete responses (75.9%), and 4 partial responses (13.8%). 12 patients with thrombocytopenia need to reduce the dose of ruxolitinib, and 1 patient withdrew treatment for obvious bleeding tendency. With the median follow-up 3 (2-8) months after treatment with ruxolitinib, 28 patients survived and 1 died of severe pneumonia related to aGVHD, and the overall survival was 96.6% (28/29).
Conclusion Ruxolitinib is safe and effective in the treatment of steroid refractory/refractory aGVHD, and can be used as a second-line treatment for refractory aGVHD.

Disclosures
No relevant conflicts of interest to declare.
Abnormal Microenvironment of Bone Marrow B Lymphocytes in Patients with Primary Immune Thrombocytopenia
Authors: Yu Tianshu , Hou Ming , Liu Xinguang , Han Panpan 
Published: October 2019

Abstract
BACKGROUNDS:
Primary immune thrombocytopenia (ITP) is an autoimmune disorder characterized by peripheral blood platelet count<100x10^9/L and increased risk of bleeding, but its exact pathogenesis remains unclear. Previous researches mostly emphasized on periphral blood, in order to elucidate the pathogenesis and provide new strategies for treatment, a series of studies about bone marrow B cells were carried out in our investigation.
METHODS:
5ml bone marrow blood samples from 11 newly diagnosed ITP patients and 7 allo-HSCT healthy donors were collected into heparin anticoagulant tubes , bone marrow mononuclear cells (BMMCs) were separated with Ficoll density gradient-centrifugation in two hours. Different subsets of B lymphocytes were determined by multicolor flow cytometry including naive B cells, total memory B cells , plasma cells and regulatory B cells (Bregs) ,as well as some chemokine receptors of B cells (CXCR5, BAFFR, BCMA, TACI) . RNA were extracted from BMMCs using Trizol reagent, transcription factors related to development and differentiation of B cells (PRDM1, Pax5, IRF-4, XBP1) were detected by real-time PCR.
RESULTS:
The percentage of B cells (CD19+) in bone marrow lymphocytes in ITP patients was significantly lower than that in healthy donors (6.16±0.74% vs. 17.28±2.43%, P < 0.0001). The proportion of naive B (CD19+CD27-) in B cells was also lower compared with normal controls (59.11±7.60% vs. 81.58±4.00%, P=0.041), and the proportion of memory B (CD19+CD27+) was higher(40.17±7.67% vs. 18.01±3.89%, P=0.045),but there was no significant difference in plasma cells (CD19-CD138+). Besides,there was a decrease of Breg (CD19+CD24highCD38high) in ITP patients compared with healthy donors (20.33±5.05% vs. 57.98±9.76% , P = 0.008), and its percentage of total lymphocytes was also significantly lower unsurprisingly (1.38±0.38% vs. 12.23±2.88%, P < 0.001). The level of BAFFR in mature B cells was elevated in ITP patients (80.72±4.53% vs. 45.81±8.49%, P = 0.002). However, no significant difference was observed in other three chemokine receptors. All four Transcription factors related to B cell development and differentiation was not found to be significantly different between ITP patients and healthy controls.
CONCLUSIONS:
Our results showed that memory B cells which represent the active form were increased, and Bregs which mediate immune tolerance were much more decreased in ITP patients. As BAFFR is the only specific receptor of B cell activating factor(BAFF), its elevated expression suggested the BAFF-BAFFR system enhanced chemotactic function of B cells in ITP patients. All those results indicated that the bone marrow B cells in patients with ITP were in a state of immune overstimulation, this may potentially constitute a novel therapeutic target.
Figure. Figure.

Disclosures
No relevant conflicts of interest to declare.
Profile of Capillary-Leak Syndrome in Patients Received Chimeric Antigen Receptor T Cell Therapy
Authors: Hu Yongxian , Feng Jingjing , Shao Mi , Huang He 
Published: October 2019

Abstract
Background: Autologous T cells modified to express a chimeric antigen receptor (CAR-T) has demonstrated exciting efficacy in treating leukemia and there has been some reports about the toxicities recently. However, the spectrum of capillary-leak syndrome (CLS) associated with CAR-T cell therapy has not been systematically evaluated, which can be a life threatening complication as results of the cytokine release syndrome (CRS). Therefore, as the use of CAR-T therapy continues to expand to broader applications, it is prudent to characterize the profile of CLS to help providers guide safe management.
Method: We reviewed all acute lymphoblastic leukemia (ALL) patients who had participated in the clinical trial from our center to receive CAR-T therapy between 2016-2018. Patients analyzed in the study received either CD19 CAR-T cells or CD19 plus CD22 CAR-T cells. The diagnosis of CLS includes edema, acute hypotension and hemoconcentration with hypoproteinemia or hypoalbuminemia. CRS grading was evaluated with Lee's criteria for CRS.
Result: 42 ALL patients were included in this study with the mean age of 27(8-52) years old. 11(11/42, 26.2%) patients were diagnosed as CLS and 31 were not. It was observed that CLS was more common in patients who developed severe CRS. Patients with CLS was found to have high rate of hypotension and use of gamma globulin.(Table 1) Top level concentration of serum IL-6 in CLS patients was much higher than that in non-CLS patients (16438.7 vs 3292.7 pg/mL, p=0.0016), which is consistent with the well recognized concept of IL-6 as an indicator of CRS.(Figure 1) It is important to notice that CLS patients had lower levels of serum total protein (TP, 43.7 vs 52.8 g/L, p=0.0005) and serum albumin (ALB, 27.4 vs 33.8 g/L, p=0.0011), while the hemoglobin (HGB) concentration showed no difference, suggesting that TP and ALB might be better indicators for CLS than HGB, although hemoconcentration, hypoproteinemia and hypoalbuminemia are both important in diagnosis.(Figure 2) Moreover, there was no significant difference in age, gender, Ph type of ALL, type of CAR-T cells infused and death ratio.(Table 1) Although CRS has been reported to be related with disease burden before the therapy, our data showed no difference of it between the patients with and without CLS.
Conclusion: In conclusion, we have evaluated a basic profile of CLS among CAR-T patients in our center and the study indicates that CLS warrants extra attention for patients who receive CAR-T therapy. Further investigations are required to elucidate best practices for prevention and management of CLS in CAR-T therapy.

Disclosures
No relevant conflicts of interest to declare.
Impact of Body Mass Index (BMI) on Survival Endpoints in Multiple Myeloma
Authors: Kocoglu Mehmet Hakan , Patel Ameet , Rapoport Aaron P. , Ma Ning , Ding Jennifer , Elder Simran , Hossain Aneesha , Badros Ashraf Z. 
Published: October 2019

Abstract
Background: The role of metabolism in outcomes of cancer patients is under investigation. A higher body mass index (BMI) substantially increases the risk of cancer and impacts the treatment. High BMI is also associated with increased risk of MM (Euro J Cancer 2011). However no data is available in the effect of BMI on myeloma survival after autologous stem cell transplantation (ASCT)
Methods: We retrospectively reviewed patient charts who had ASCT between Jan 2010 - November 2011. Patients were selected if they have adequate data, two lines of induction treatment prior to transplantation. Most of the patients received maintenance treatment. IRB granted exemption for anonymous data collection. We collected demographic data, cytogenetic risk, response with induction and after transplant, progression free and overall survival. BMI was estimated at the time of ASCT.
Results: A total of 223 post ASCT myeloma patients met inclusion criteria. Median age of the cohort was 58 years, 48.8% were African-American, 51% Caucasian, 1% Asian, and 0.5% Hispanic. Median BMI was 29.7 and 30 in female and males respectively. Median BMI was 29.4 in Caucasians and 29.6 in African-American cohorts. 24% of patients had high risk cytogenetics. 72% of patients experienced very good partial response (VGPR) or better post-transplant.
On multivariate analysis, PFS and OS showed no association at any of the four BMI levels (p=0.573), age, gender, race, or depth of response. High risk cytogenetics was associated with PFS (p= 0.022). There was no association with high risk cytogenetic features at any BMI level, age, gender, race (p=0.245). For patients with BMI > 40, PFS differed significantly when compared to patients with normal BMI: 37 months versus 50, p=0.52. Unadjusted hazard ratio (HR) 2.10 (95% CI: 0.62-7.18) with respect to patients with normal BMI. Subgroup multivariate analysis with patients with morbid obesity did not show any association with depth of response (partial response or VGPR or better) or high-risk cytogenetic features.
Conclusion: BMI as a surrogate marker does not significantly impact long term survival outcomes in patients with multiple myeloma in the post-transplant setting and thus should not factor in transplant decision-making. Patients with morbid obesity appear to have a trend towards shortened progression free survival and may warrant further investigation.
Figure. Figure.

Disclosures
Badros: Karyopharm: Research Funding; Celgene: Consultancy, Research Funding; GSK: Research Funding.
Coagulation Factor XII Plays an Important Role in Procoagulant Activity of Apoptotic Cells
Authors: Yang Aizhen , Wu Yi 
Published: October 2019

Abstract
Apoptosis can be induced in a variety of pathological disorders, including inflammation, autoimmune diseases, and chemotherapy. When cells undergo apoptosis, they express phosphatidylserine (PS) on cell membrane surface and thus become procoagulant. Although it has been known that the procoagulant activity of apoptotic cells are tightly associated with thrombotic disorders, such as atherothrombosis and Trousseau syndrome, the mechanisms by which apoptotic cells activate the coagulation system and enhance blood clotting are largely unknown. In this study we investigated which coagulation factor(s) is involved in this process. Using western blotting and chromogenic substrate assay, we found that incubation with apoptotic cells induced by Dexamethasone (DXMS), but not with viable cells, resulted in rapid cleavage and activation of FXII. Moreover, apoptotic cells-mediated FXII activation was significantly increased in the presence of prekallikrein (PK) and high molecular weight kininogen (HK), other two components of plasma contact system. However, incubation of apoptotic cells did not cause dramatic changes of other coagulation factors, suggesting a selective association of FXII activation with apoptotic cells. Activation of FXII by apoptotic cells was markedly inhibited by a specific anti-kallikrein antibody, indicating the activation of the contact system by apoprotic cells. Flow cytometric measurement showed that FXII bound to apoptotic cells in a concentration-dependent manner, which was inhibited by annexin V and PS liposome. A surface plasmon resonance assay showed a direct binding of FXII to PS (KD=3.9E-9 M). When challenged by apoptotic cells, clotting time of plasma from FXII-knockout mice was significantly prolonged, which was reversed by replenishment with human FXII. Moreover, an inhibitory anti-FXII antibody completely prevented apoptotic cells-induced intrinsic tenase complex formation. Consistently, apoptotic cells significantly increased thrombin production in normal plasma, which were attenuated by PS blocker annexin V, an inhibitory anti-FXII antibody, and the deficiency of FXII, respectively. Addition of human FXII to XII-deficient plasma recovered thrombin generation. As evaluated by ELISA, the levels of thrombin-antithrombin complex in circulation were significantly increased when apoptotic cells were intravenously injected into wild-type mice, but not in FXII-knockout mice. In conclusion, FXII plays an important role in apoptotic cells-mediated procoagulant activity.

Disclosures
No relevant conflicts of interest to declare.
Combined Single-Cell and Spatial Transcriptomics to Deconvolute the Hematopoietic Stem Cell Niche
Authors: Haas Simon , Baccin Chiara , Al-Sabah Jude , Velten Lars , Lars Steinmetz , Trumpp Andreas 
Published: October 2019

Abstract
Coordinated interaction of many cell types is required to facilitate hematopoietic and mesenchymal stem cell maintenance and differentiation in the bone marrow. However, the molecular factors and cell types involved in this complex interplay remain poorly understood. Here we developed a combined single cell and spatial transcriptomics approach to address this problem.
Large-scale single-cell transcriptional profiling in conjunction with a multi-layered sorting approach allowed us to generate a complete and evenly sampled transcriptional map of all major bone and bone marrow populations. Our dataset covers all cell types or differentiation trajectories involved in mesenchymal and hematopoietic stem cell differentiation, osteogenesis, adipogenesis, myelopoiesis, erythropoiesis, lymphopoiesis, memory T cell formation as well as bone marrow neural innervation and vascularization at the single cell level. Using this data, we derive fundamental properties of the described cell types, clarify the cellular source of signals affecting stem cell differentiation processes and provide a systems view on putative intercellular interactions. Systematic spatial transcriptomics, using laser-capture microdissection of selected bone marrow niches followed by transcriptional profiling and bioinformatic cellular deconvolution, allowed us to confirm predicted interactions and map the cellular composition of distinct bone marrow niches.
Our analyses highlight the importance of pre-adipogenic CXCL12 abundant reticular cells as key niche cells for stem cell maintenance, provides a holistic systems view of the hematopoietic stem cell niche and offers a novel approach to systematically deconvolute the molecular, cellular and spatial composition of complex tissues.

Disclosures
No relevant conflicts of interest to declare.