bluuebunny/crossref_metadata_2025_split huggingface dataset:
Elevated End-Tidal Carbon Monoxide Concentration in Children with Sickle Cell Anemia
Authors: Lal Ashutosh , Yen Kristen , Patterson Lasandra , Goldrich Alisa , Marsh Anne M , Bhatnagar Anish 
Published: October 2019

Abstract
Background: Carbon monoxide (CO) produced during oxygen-dependent cleavage of porphyrin ring of heme is excreted in exhaled breath. The catabolism of heme is increased when red blood cells are destroyed at an accelerated rate. Thus, quantifying CO in exhaled breath could serve as an indicator of hemolysis. However, the requirement for forced breath sample has limited the measurement of exhaled CO in young children.
Objective: To assess end-tidal CO concentration (ETCOc) in children with sickle cell anemia (SCA).
Design/Methods: ETCOc was measured using the CoSense ETCO Monitor (Capnia Inc. Palo Alto, CA). Children between 5-14 years with SCA (Hb SS) who were not on chronic transfusions were eligible. Healthy children served as age-matched controls. Children with exposure to second-hand smoke, acute respiratory infection or symptomatic asthma were excluded. End-tidal breath samples were collected by placing the tip of a nasal cannula 5 mm into the nares. Up to 3 measurements were taken for each subject and the highest ETCOc value was used for analysis. (ClinicalTrials.gov: NCT01848691)
Results: The mean (range) age of 16 children with SCA and 16 controls was 9.7 years (5-14 years) and 9.9 years (5-14 years), respectively. The mean (± s.d.) ETCOc for SCA was 4.85 ± 2.24 ppm versus 0.96 ± 0.54 ppm for control group (p<0.001). The ETCOc in the control group ranged from 0.2 to 2.3 ppm, but was ≤1.2 ppm in 14/16, which is suggested as the upper limit of normal for healthy children. In the SCA group, the ETCOc range was 1.8 to 9.7 ppm, with values ≥2.4 ppm in 15/16 subjects. A threshold ETCOc value of >2.1 ppm provided both sensitivity and specificity equal to 93.8% (69.8-99.8%) for distinguishing SCA from healthy children. Children with SCA who had higher absolute reticulocyte count also demonstrated higher ETCOc (r=0.62, p=0.011). Patients with severe anemia (hemoglobin <8 g/dL) had a higher mean ETCOc (5.43 ppm) than the rest (4.40 ppm) but the difference was not significant. ETCOc level tended to increase with age in SCA (r=0.45, p=0.08).
Conclusions: Carbon monoxide in exhaled breath can be measured in young children in the clinic using a portable monitor. ETCOc may be a valuable tool for non-invasive monitoring of the severity of hemolysis in SCA. The mean ETCOc was 5-fold higher in SCA compared with controls, with little overlap seen between the groups. This suggests a potential use for ETCOc as a point-of-care screening test for SCA in children.
Figure 1 Figure 1.
Figure 2 Figure 2.

Disclosures
Lal: Capnia, Inc: Research Funding. Yen:Capnia, Inc. : Employment. Bhatnagar:Capnia, Inc: Employment.
Dasatinib Is Effective in the Treatment of Mice Models with Immune Thrombocytopenia
Authors: Shimoyama Tadashi , Okano Yoshiaki , Fujishima Yukiteru , Oyake Tatsuo , Kowata Shugo , Murai Kazunori , Ito Shigeki , Ishida Yoji 
Published: October 2019

Abstract
Background: Immune thrombocytopenia (ITP) is an autoimmune disease in which anti-platelet antibody (APA) is produced. APA-coated platelets are captured and phagocytized by macrophages in the spleen. Recent study revealed that spleen tyrosine kinase (Syk) inhibitor is effective in the treatment of ITP, because Syk phosphorylation is the key step of the phagocytosis by macrophages. Activated Fc receptor signal transduction is initiated by phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) tyrosine residues by SRC family kinases. Recruitment of Syk to dually phosphorylated ITAMs triggers the activation of Syk. To prove the hypothesis that the inhibition of SRC family kinase induces the decreased phosphorylation of Syk, resulting in decreased phagocytosis by macrophages, a SRC family kinase inhibitor, dasatinib, was used in the experiments.
Methods, Results and Discussion:
In vitro study; Murine macrophage cell line, RAW, was incubated with APA coated murine platelets for 30 minutes. Phagocytosis by RAW was significantly decreased with dasatinib (100nM, p<0.01), indicating SRC family kinase activity is required for efficient phagocytosis. Phosphorylated Syk was decreased in RAW, incubated with anti-Fc receptor antibody (rat IgG) and anti-rat IgG antibody with dasatinib (100nM), shown in the Western blot analysis (Figure 1). These results suggest that Syk phosphorylation is the key step in phagocytosis.
In vivo study; (1) Three hours before APA intra-peritoneal injection, dasatinib (2.5mg/kg) was oral-administrated. Six hours after APA injection, platelet counts were measured. The platelet counts were 366 ± 164 x109/L with dasatinib (n=4, mean ± SD) and 114 ± 51 x109/L without dasatinib (n=4)(P=0.026)(Figure 2). (2) Osmotic pump, filled with APA, were inserted in murine intra-peritoneal cavity and dasatinib (2.5mg/kg) was oral-administered once daily for 7 days. The platelet counts were 499 ± 98 x109/L with dasatinib (n=4, mean ± SD) and 82 ± 131 x109/L without dasatinib (n=4) at day 7 (p<0.0022) (Figure 3). These results strongly suggest that dasatinib inhibit the phagocytosis in vivo.
Conclusion: Dasatinib inhibits phosphorylation of Syk, inducing decreased phagocytosis of APA-coated platelets via decreased SRC family kinase activity. These findings reveal that SRC family kinase controls the efficiency of phagocytosis in part through the regulation of Syk function. Dasatinib might be effective in the treatment of ITP.

Disclosures
No relevant conflicts of interest to declare.
Immune Response to FIX Is Dependent on Mouse Background Strain and Modulated By TLR4
Authors: Markusic David M 
Published: October 2019

Abstract
Introduction
Many investigators rely on mouse models to study immune responses against factor IX protein (FIX) products. While it is recognized that there are distinct differences in immune responses towards FIX in different mouse strains, there is an absence of studies conducted on mice with a F9 gene deletion. Our laboratory has developed protocols to prevent or reverse ongoing anti-hFIX IgG inhibitors in hemophilia B mice with a F9 gene deletion (F9-/Y) on both a BALB/c and C3H/HeJ background. Interestingly C3H/HeJ F9-/Y mice develop high titer anti-FIX IgG1 inhibitors and anaphylaxis, while BALB/c F9-/Y mice have mild anti-hFIX IgG1 inhibitors without anaphylaxis. The C3H/HeJ mouse strain is deficient for the innate immune sensor toll like receptor 4 (TLR4) while a congenic C3H/OuJ strain has functional TLR4. Several recent studies have indicated an important link between coagulation and innate immunity and TLR4 has been linked to sensitivity in food allergy models. Together these studies suggest that TLR4 may have a role in modulating FIX immunity. Here we report results from a study exploring B and T cell responses against FIX protein therapy on two different mouse strains BALB/c and C3H/HeJ with the same F9 gene deletion and the role of TLR4 in modulating FIX immunity.
Methods
BALB/c and C3H/HeJ F9-/Y mice were challenged weekly with recombinant FIX protein. Humoral immune responses were determined by IgG1 and IgG2a anti-hFIX ELISA, Bethesda assay for inhibitors and B cell ELISpot on bone marrow and spleen cells to measure FIX specific antibody secreting B cells. T cell studies measured the TH1 (IFN-γ) and TH2 (IL-4) cytokine responses in splenocytes at the mRNA and protein level in response to FIX protein. Antibody responses were also measured in C3H/HeJ/OuJ F9-/Y mice with restored TLR4 function following FIX challenge.
Results
BALB/c F9-/Y mice have a TH2 skewed response and a reduction in anti-hFIX secreting plasma cells in the bone marrow. Independent antigen challenge revealed both strains generated equivalent IgG1 antibody titers to an intravenously delivered antigen suggesting the reduced anti-FIX response in BALB/c mice is antigen specific. C3H/HeJ F9-/Y mice have a mixed TH1 and TH2 response (mainly TH2). Importantly TLR4 signaling has an important modulatory role in the C3H background on the levels of anti-hFIX IgG1 and incidence of anaphylaxis.
Conclusions
The background strain has an important impact on FIX immune responses, which in permissive strains can be modulated by mutations in innate immune sensors.

Disclosures
No relevant conflicts of interest to declare.
Validation of the rAHF-PFM PK Guided Dose Calculator Medical Device Using an Unrelated Clinical Dataset
Authors: Steinitz Katharina Nora , Spotts Gerald , Loew-Baselli Alexandra , Oh Myungshin , Penny Mark , Gringeri Alessandro , Reininger Armin J 
Published: October 2019

Abstract

Introduction: Prevention of bleeding represents the standard of care for patients with hemophilia without inhibitors. However, standard prophylactic dosing regimens with FVIII are currently based on body weight alone and do not take into account considerable inter-individual variability in FVIII PK profiles. In order to facilitate personalization of prophylaxis to individual needs and individual PK response we have developed a device that allows PK guided dosing in routine clinical care. In fact, the major barrier of PK-guided dosing in clinical practice is the necessity of multiple blood samples (6 to 11 over 48 hours) associated with a single infusion and the lack of easy-to-use statistical software to generate classical PK profiles. Leveraging the vast rAHF-PFM clinical database with more than 150 classical PK assessments, a Population PK model for children and adults was combined with a Bayesian algorithm to create a user friendly medical device. This methodology allows the use of only a few samples after different infusions to estimate individual PK parameters and help to calculate dosing regimens.
Methods: In order to carry on an external validation of the Bayesian population PK model of the device, individual 10 point PK assessment data from an unrelated source, i.e. not included in the base population PK model, (rAHF-PFM prophylaxis study, Valentino et al 2012) was utilized to validate the accuracy of the calculation. First, we compared predicted post infusion FVIII levels with observed FVIII levels using all the available post infusion samples. In a second step we compared individually estimated PK parameters as obtained by using 10 post infusion samples with a progressively reduced number of post infusion samples.
Results: The external validation study indicated a good agreement between the observed and predicted FVIII levels (Figure) and PK parameters. Furthermore it showed that the rAHF-PFM medical device had a good predictive performance even when using only 2 sampling time-points.
Figure 1 Figure 1.
Conclusion: An easy-to-use medical device that can estimate PK parameters and thus assists in designing prophylactic dosing regimens based on individual- PK characteristics represents a groundbreaking innovation. This literally shifts the treatment paradigm from one-size-fits-all prophylaxis towards a more personalized therapy in hemophilia A and allows further optimization of dosing in patients in order to improve outcome in a sustainable fashion.

Disclosures
Steinitz: Baxter: Employment. Spotts:Baxter Healthcare Corporation: Employment. Loew-Baselli:Baxter Innovations GmbH: Employment, Equity Ownership. Oh:Baxter Healthcare Corporation: Employment. Penny:Baxter Healthcare Limited: Employment, Equity Ownership. Gringeri:Baxter Innovations GmbH: Employment. Reininger:Baxter Innovations GmBH: Employment, Equity Ownership.
Absolute Lymphocyte Count As an Independent Prognostic Factor of IPI and NCCN-IPI in DLBCL Patients
Authors: Okada Kazuya , Ochi Shinichi , Nagayama Takashi , Nabe Shogo , Sakai Kazuya , Muranushi Hiroyuki , Okamoto Yusuke , Sugiura Hiroyuki , Sato Aki , Sato Takayuki , Maeda Takeshi , Onishi Tatsuhito , Ueda Yasunori 
Published: October 2019

Abstract
Background: Previous studies have shown that the absolute lymphocyte count (ALC) in peripheral blood at diagnosis may be an independent prognostic factor of IPI for patients with diffuse large B-cell lymphoma (DLBCL). In the rituximab era, the U.S. National Comprehensive Cancer Network International Prognostic Index (NCCN-IPI) was developed to improve the risk stratification of DLBCL in comparison to the existing IPI. Therefore, the aim of this study was to clarify the impact of ALC at diagnosis on event free survival (EFS) and overall survival (OS) on analysis performed with factors included in NCCN-IPI.
Patients and methods: We retrospectively reviewed the ALC of 413 patients with newly diagnosed DLBCL treated with R-CHOP at our hospital between January 2005 and March 2013. Primary central nervous system lymphoma patients were excluded from this study. ALC was determined in all patients from complete blood count with differential white blood count at the time of diagnosis, and prior to therapy administration. EFS and OS were estimated according to the Kaplan-Meier method. Multivariate analysis was performed with the proportional hazard Cox model.
Results: The median ALC was 1.2x10E9/L (range, 0.06-9.0). We set an ALC cut-point at 1.0x10E9/L based on previous studies. The median follow-up duration was 40 months. Baseline characteristics according to ALC (<1.0x10E9/L[n=145] and >1.0x10E9/L[n=268]) are summarized in Table1. Patients with ALC<1.0x10E9/L had a significantly poorer EFS and OS than patients with ALC>1.0x10E9/L (5-year EFS, 37.0% versus 68.9%, p<0.001; 5-year OS, 46.3% versus 80.0%, p<0.001). On multivariate analysis performed with factors included in IPI and NCCN-IPI, ALC remained an independent predictor of EFS (IPI: hazard ratio [HR] 1.95; 95% confidence interval [CI] 1.43-2.68; p<0.001, NCCN-IPI: HR 1.94; 95%CI 1.42-2.65; p<0.001) and OS (IPI: HR 2.35; 95%CI 1.61-3.42; p<0.001, NCCN-IPI: HR 2.29; 95%CI 1.57-3.33; p<0.001) (Table2). Importantly, within the poor R-IPI group, ALC distinguished patients with different 5-year EFS (24.4% versus 50.4%, p<0.001) and OS (35.7% versus 65.7%, p<0.001). For the high NCCN-IPI group also, ALC distinguished patients with different 5-year EFS (14.8% versus 39.8%, p<0.01) and OS (17.5% versus 54.5%, p<0.001) (Figure1).
Conclusions: According to our results, ALC<1.0x10E9/L is an adverse prognostic factor and independent of IPI and NCCN-IPI. ALC might be more successful in identifying high-risk patients in which IPI and NCCN-IPI analysis was unrevealing. Our results suggest that other therapeutic strategies may be more effective in high-risk patients with DLBCL.

Disclosures
No relevant conflicts of interest to declare.
Azacitidine Treatment in High Risk Myelodysplastic Patients in Complete Haematological Remission Reverts Mesenchymal Stem Cells to a Normal Phenotype
Authors: Poloni Antonella , Maurizi Giulia , Mattiucci Domenico , Costantini Benedetta , Mariani Marianna , Mancini Stefania , Fanelli Mirco , Olivieri Attilio , Leoni Pietro 
Published: October 2019

Abstract
Introduction. Myelodysplastic syndromes (MDSs) are a heterogeneous group of clonal hematopoietic stem cell (HSC) malignancies that are characterized by ineffective bone marrow hematopoiesis, peripheral blood cytopenias, and a substantial risk for progression to acute myeloid leukemia. Mesenchymal stem cells (MSCs) isolated from bone marrow of patients affected by myelodysplastic syndromes (MDS) play a critical role in myelodysplastic microenvironment showing altered structural epigenetic and functional features.
Methods. In this work we evaluated the effect of azacitidine treatment on MSC-MDS. In particular, we analyzed MSC-MDS from 24 high-risk patients at diagnosis and after azacitidine treatment, studying their morphology, proliferative potential, cell cycle activity and their capacity to support haematopoiesis.
Results. MDS-MSCs at diagnosis appeared larger and flattened, achieved confluence at a significantly lower rate than donors and displayed reduced proliferative capacity. In particular 40% of samples were unable to expand.
This reduced proliferative capacity of MSC-MDS at diagnosis suggested changes in the cell cycle activity. Therefore we studied the gene expression profiles of 37 regulatory genes, observing CDKN2B up-regulation in MDS-MSCs (8 times higher than donors).
Notably, after azacitidine treatment MDS-MSCs of patients who reached complete haematological remission (MDS-MSCs-CR) reverted to the typical BM-MSC morphology and recovered a proliferative potential similar to normal BM-MSC achieving confluence at a significantly higher rate.
Molecular analysis on MDS-MSC-CR revealed a significant reduction in the expression level of CDKN2B showing correlation between cell cycle progression and expression level of this gene.
Moreover, to study the long-term hematopoietic maintaining ability, MDS-MSCs at diagnosis were cultured with CD133+ cells, and they showed a decreased ability to support the growth of myeloid and erythroid progenitors. Conversely, MSC-MDS-CR showed an increased capacity to support haematopoiesis similar to healthy donors.
Conclusion. We showed that MDS-MSCs at diagnosis were structurally and functionally altered while MSC-MDS-CR after azacitidine revert to a normal phenotype. It has been supposed that healthy MSCs adopt MDS-MSCs like molecular features when exposed to haematopoietic MDS cells.
Our results may confirm these data suggesting that myelodysplastic cells can alter bone marrow microenvironment interacting with MSC and affecting their normal role and functionality.

Disclosures
No relevant conflicts of interest to declare.
Circulating Multiple Myeloma Cells (CMMCs): A Novel Method for Detection and Molecular Characterization of Peripheral Blood Plasma Cells in Multiple Myeloma Precursor States
Authors: Weiss Brendan , Sasser Kate , Rao Chandra , Foulk Brad , Gross Steven , Mallon Gayle , Erb Colleen , Berkowitz Anne , Mangan Patricia A. , Shelly Brenda K. , Cohen Adam D. , Vogl Dan T. , Garfall Alfred L. , Stadtmauer Edward A. 
Published: October 2019

Abstract
Background Circulating plasma cells (PCs) have been identified as a prognostic factor in patients with myeloma precursor states (MGUS and SMM) and active multiple myeloma (MM). Enumeration of circulating PCs by available methods is not suitable for widespread use and does not provide molecular characterization. We developed and evaluated a novel method for enumeration and molecular characterization of circulating PCs (circulating multiple myeloma cells, “CMMC”), based on the CELLSEARCH® System (Janssen Diagnostics LLC, Raritan, NJ), an automated technology for the capture, enumeration and characterization of rare cells in the peripheral blood.
Methods We are performing a prospective study of patients with MGUS and SMM to evaluate CMMCs as biomarker for progression to active MM. Utilizing the CELLSEARCH® System CMMCs were captured by CD138 ferrofluid magnetic particles and identification was defined as CD38+ and CD19-, CD45-. Nonviable cells were excluded by DAPI. Isolated CMMCs were stored and FISH for t(4:14), t(14;16) and del17 was performed.
Results We have enrolled 16 patients, MGUS = 3, SMM = 11, and newly diagnosed MM = 2. The Mayo Risk stratification for MGUS patients was: low risk = 2, low-intermediate = 1. All SMM patients were low risk by Mayo Model incorporating serum free light chains. The median number of bone marrow plasma cells for MGUS patients was 7 (range 7-9) and for SMM patients was 15 (range 10-40). The median CMMCs for MGUS = 6 (range 2-55), median CMMCs for SMM = 31 (5-1918). The two patients with NDMM had 5870 and 5 CMMCs, respectively. A single patient with SMM progressed with a symptomatic solitary lumbar plasmacytoma and had CMMCs of 5 and 3 at baseline and progression, respectively. Abnormalities by FISH were detected in both bone marrow and CMMCs. Accrual is ongoing and additional data will be presented at the meeting.
Conclusions The CELLSEARCH® CMMC assay can detect, quantify and provide molecular characterization of circulating PCs in MGUS/SMM/MM; longer prospective follow-up is needed to test the prognostic value of CMMCs.

Disclosures
Weiss: Janssen: Consultancy, Research Funding. Sasser:Janssen: Employment. Rao:Janssen: Employment, Equity Ownership. Foulk:Janssen: Employment. Gross:Johnson & Johnson: Employment, Equity Ownership. Cohen:Janssen: Membership on an entity's Board of Directors or advisory committees. Vogl:Celgene Corporation: Consultancy; Amgen: Consultancy; Millennium/Takeda: Research Funding; GSK: Research Funding; Acetylon: Research Funding. Stadtmauer:Janssen: Consultancy.
PRL-3 Mediates Survival of Primary Myeloma Cells
Authors: Rø Torstein Baade , Slordahl Tobias Schmidt , Abdollahi Pegah , Vansemb Esten , Hagen Lars , Baranowska Katarzyna Anna , Misund Kristine , Slupphaug Geir , Borset Magne 
Published: October 2019

Abstract
Introduction:Phosphatase of regenerating liver-3 (PRL-3) is a marker of aggressive and metastatic disease in a number of solid tumors. PRL-3 is expressed in primary multiple myeloma (MM) cells and MM cell lines, but not in normal plasma cells. We have previously shown that PRL-3 is induced in MM cells by interleukin (IL)-6 and other growth factors, and that PRL-3 promotes MM-cell migration (Fagerli et al, Blood, 2008). A study by Broyl et al (Blood, 2010) identified PRL-3 as a marker gene for a subgroup of patients with MM. In this study we examined the effect of the small molecular inhibitor PRL-3 Inhibitor I on primary MM cells, and we examined possible cellular targets of PRL-3 using a proteomics approach.
Methods: Co-cultures of primary myeloma cells and pooled bone marrow stromal cells from several MM patients were grown for 3 days in the presence or absence of PRL-3 Inhibitor I. Apoptosis was determined by staining cells with YO-PRO-1. To examine cellular targets of PRL-3 we made PRL-3 overexpressing cells from the human MM cell line INA-6 by retroviral transduction (INA-6-PRL-3) and control cells (INA-6-MOCK). Stable isotope labeling by amino acids in cell culture (SILAC) was used for in vivo incorporation of 12C-Lysine (“light”) and 13C-Lysine (“heavy”) in these transduced cell lines, preparing them for Orbitrap mass spectrometry based quantitative proteomics. The experiments were performed three times. The results were analyzed using Ingenuity Pathway Analysis software (Quiagen).
Results: PRL-3 Inhibitor I significantly reduced viability in all 9 primary MM cell samples tested, ranging from 5-70 % absolute reductions in viability at the top concentration of 40 µM. Proteomics analysis was performed on INA-6-PRL-3 and INA-6-MOCK cell lines at baseline and following incubation in IL-6-free media for 12 hours to deplete INA-6-MOCK cells of PRL-3. At 12 hours, a total of 6747 proteins were identified. 481 proteins were upregulated and 22 proteins downregulated by more than 2-fold in the INA-6-PRL-3 cells compared to INA-6-MOCK cells in 2 out of 3 experiments. Several proteins known to be important for cell survival and migration were upregulated in INA-6-PRL-3 cells, like CD44, Myc assosciated factor X (MAX) and Lyn. Pro-apoptotic proteins Caspase-1 and Caspase-2 were downregulated. Among canonical signaling pathways, there were most proteins belonging to the protein ubiquitination pathway (9,8 % of proteins; p<0,001).
Conclusion: PRL-3 Inhibitor I induced apoptosis in primary MM cells. Quantitative proteomics analyses comparing PRL-3 overexpressing INA-6 cells to normal INA-6 cells identifies several proteins important for MM cell migration and survival. This study indicates that PRL-3 could be important in the pathogenesis of MM and a potential target in the treatment of MM.

Disclosures
No relevant conflicts of interest to declare.