bluuebunny/crossref_metadata_2025_split huggingface dataset:
Educating the Innate Immune System
Authors: Worthen Scott , Mei Junjie , Liu Yuhong , Deshmukh Hitesh 
Published: October 2019

Abstract
The microbiome regulates the development of innate immunity G. Scott Worthen, Junjie Mei, Yuhong Liu, Ning Dai, Hitesh Deshmukh Circulating granulocytes are maintained within a fairly narrow window for each individual during homeostasis. This regulatory system is nonetheless capable of dramatic shifts during stress. The mechanisms that govern control over granulocyte number remain incompletely understood. Recent information, however, provides clues to the feedback control systems that regulate homeostatic and emergency granulopoiesis. The first clues have come from adult mice with abnormalities in granulocyte function. Defects in Leukocyte Integrins or chemokine receptors result in marked increases in circulating (?2 integrin) or bone marrow (Cxcr2) neutrophils, associated with increased circulating IL-17 and G-CSF. This result appears to be due to a functional inability to arrive at sentinel site(s) whence they are attracted by (among others) Cxcl5. Strikingly, a single infusion of normal WT neutrophils resets this feedback loop. One signal for expression of IL-17 and G-CSF appears to be the gut microbiome. Antibiotic treatment also resets the system, reducing neutrophil numbers and cytokine expression. Thus, at sentinel site(s), the supply of functional neutrophils is balanced against perceived threat from the microbiome. In neonates, exposure to commensal organisms is an immediate result of birth from a sterile environment into a dirty one. We have described postnatal granulopoiesis in murine neonates, a dramatic increase in circulating and bone marrow neutrophils, that lasts for 5-7 days. Human infants similarly have been shown to demonstrate a postnatal increase in circulating neutrophils for 72 hrs after birth. Antibiotic-exposed mouse pups fail to develop postnatal granulopoiesis, as do mice deficient in IL17ra, MyD88, TLR4, or G-CSF even if not exposed to antibiotics, indicating a pathway that requires exposure to LPS that induces IL17 and G-CSF, as in adults. In contrast to adults, however, commensal bacteria are required for host defense. Antibiotic-exposed pups are exquisitely sensitive to E. coli sepsis, which can be partially reversed by transfer of cecal contents or exogenous G-CSF. Furthermore, small quantities of LPS, fed by gavage, can also partially protect. Thus, neonates use exposure to commensal bacteria and their products to trigger the rapid expansion and functional maturation of granulocyte development, and in so doing, prepare the neonate for potential exposure to pathogens. Adults maintain this system, where it is at least partially responsible for maintenance of granulocyte homeostasis.

Disclosures
No relevant conflicts of interest to declare.
Reacting to Inflammatory Signals
Authors: Carlesso Nadia 
Published: October 2019

Specialized cellular niches in the bone marrow (BM) modulate critical functions of the hematopoietic stem and progenitor cells, such as self-renewal, cell-fate decisions and the balance between proliferation and differentiation. Constituents of the BM niche, such as osteoblasts, endothelial cells and macrophages, respond to inflammation with secretion of pro-inflammatory cytokines, and serve as effectors of the inflammatory circuitry; thus their excessive activation can potentially impact on the regulation of hematopoietic cells, and on the initiation and progression of myeloid malignancies.
Our current understanding of the mechanisms for Bcr/Ab-negative myeloproliferative neoplasia (MPN) involves recognition of driver mutations (targeting i.e. Tet2, Jak2, Mpl, or Asxl) and of an inflammatory microenvironment. However, despite accumulating evidences on the role of inflammation and of the microenvironment in the maintenance and progression of myeloid malignancies, it is still unclear how the inflamed BM niche contributes to disease establishment and progression. Similarly, the causes leading to an inflammatory microenvironment in MPN are not fully understood.
We use genetically-controlled animal model of BM inflammation to study the contribution of the inflammatory microenvironment to MPN. We found that loss of Notch signaling in the BM microenvironment results in an inflammatory state of the BM niche promoting myeloproliferation and marrow fibrosis. Using this model, we discovered that loss of Notch/RBPJ signaling leads to transcriptional upregulation of the pro-inflammatory microRNA miR-155, especially in BM endothelial and mesenchymal cells, resulting in miR-155-dependent inhibition of the NF-κB inhibitor κB-Ras1. Decreased levels of kB-Ras1 resulted in the heightened and persistent activation of NF-κB and in the increased production of NF-kB dependent pro-inflammatory cytokines, leading to uncontrolled myeloproliferation. Deletion of miR-155 in the stroma of RBPJ-/- mice prevented the development of the myeloproliferative disease, and patients affected by MPN exhibit elevated expression of miR155 in their BM. Therefore, we hypothesize that the Notch/miR155/NF-kB axis regulates the level of the inflammatory tonus in the BM niche, and that persistent deregulation of this pathway may contribute to the establishment and progression of MPN. In this presentation, we will discuss our findings and ongoing studies in the context of other recent insights on the crosstalk between tumor cells and the BM niche, and their implications for disease initiation, progression and therapeutic approaches.

Disclosures
No relevant conflicts of interest to declare.
Disease Response Guiding Therapy in Acute Lymphocytic Leukemia: Pivotal Role of Minimal Residual Disease
Authors: Pulsipher Michael A. 
Published: October 2019

In spite of an explosion of data regarding mutations associated with childhood ALL, to date these key genetic changes rarely have been the driver of therapy. Clinical parameters at presentation (WBC, age, T- vs. B-lineage, etc.) have dictated initial risk stratification and induction approaches, followed by risk-adapted therapy based upon leukemic response measured by minimal residual disease (MRD, either PCR- or flow cytometry-based). With minor variations, rapid disappearance of peripheral MRD, followed by significant clearance from the marrow after induction, and most importantly, the level of MRD after consolidation have allowed clear distinctions in outcomes that have driven intensification or de-intensification of therapy resulting in improved outcomes. Although specific gene mutations have been associated with risk, MRD has further identified better risk patients within genetic subgroups. For patients noted to be very high risk who are candidates for hematopoietic cell transplantation (HCT), the presence of MRD both pre- and post-transplant has been associated with increased risk of relapse; the risk being modified by level of MRD, whether or not GVHD occurs after HCT, and timing after HCT when MRD is measured. In lower risk patients being treated with chemotherapy and higher risk patients eligible for HCT, more sensitive approaches to flow cytometry and PCR, as well as next-generation sequencing (NGS) MRD approaches (sensitive to 1/10^7 cells) are currently being tested. It is not clear yet whether NGS-MRD offers substantial improvements in patients treated with chemotherapy, as broad-based testing is underway; the latest comparative outcomes will be presented. There is evidence of a striking improvement in our ability to define patients who will do very will after transplant (not relapse), and preliminary evidence that post-HCT NGS MRD testing is more sensitive that other methodologies in defining risk of relapse after transplant. As the latest information about the ability of different approaches to MRD is shown in this session, we will also present how response to therapy based upon MRD interacts with various genetic subtypes (Ph+ ALL, extreme hypodiploidy, etc.). Even in subclasses that are considered very high risk based solely upon genetics, measurement of MRD can define higher and lower risk groups. Going forward, as more and different types of patients are subcategorized and treated with targeted agents based upon specific mutations, it is likely MRD response will continue to be important in mapping intensity of approach and defining children at highest risk of relapse who might benefit from HCT or other cellular therapeutic approaches.

Disclosures
Pulsipher: Novartis: Membership on an entity's Board of Directors or advisory committees.
The Role of Hepcidin in Health And Disease
Authors: Muckenthaler Martina U. 
Published: October 2019

Imbalances of iron homeostasis account for some of the most common human diseases. Pathologies result from both, iron deficiency or overload and frequently affect the hepcidin/ferroportin regulatory system that balances systemic iron metabolism. The small hepatic peptide hormone hepcidin orchestrates systemic iron fluxes and controls plasma iron levels by binding to the iron exporter ferroportin on the surface of iron releasing cells, triggering its degradation and hence reducing iron transfer to transferrin. Hepcidin thus maintains transferrin saturation at physiological levels assuring adequate iron supplies to all cell types. My presentation will focus on mechanisms that control hepcidin and ferroportin expression as well as on pathologies that arise when this key regulatory circuitry underlying systemic iron homeostasis is disrupted.

Disclosures
No relevant conflicts of interest to declare.
Vascular and Endothelial Biology of Aging
Authors: Faraci Frank M. 
Published: October 2019

Abstract
Although aging is one of the greatest risk factors for vascular disease, very little is known regarding mechanisms that control the progression of vascular aging at the level of the endothelial cell. Endothelial dysfunction - a critical element of carotid artery and cerebrovascular disease - progresses with age, contributing to hypoperfusion, increased risk for ischemic stroke, and cognitive decline. Studies from several laboratories support the concept that age-induced endothelial dysfunction may occur earlier and be larger in magnitude in the cerebral circulation than in blood vessels outside of the brain. Thus, the circulationof the brain may be particularly sensitive to age-induced endothelial dysfunction. In relation to underlying mechanisms, angiotensin II type 1 receptors, NADPH oxidase, and oxidative stress appear to play a key role in age-related vascular dysfunction. The nuclear receptor peroxisome proliferator-activated receptor-g (PPARg) exerts protective effects in the vasculature when pharmacologically activated. We recently examined the hypothesis that endothelial PPARg protects against vascular aging. We studied carotid arteries from adult and old transgenic mice with endothelial specific expression of a human dominant negative mutation in PPARg driven by the vascular cadherin promoter (designated E-V290M), along with non-transgenic littermates. Endothelial function was similar in arteries from adult non-transgenic and E-V290M mice as well as old non-transgenic mice. In contrast, there was a marked reduction in endothelial function in old E-V290M mice. This augmented endothelial dysfunction was not altered by inhibition of cyclooxygenase, but was restored to normal by a superoxide scavenger, an inhibitor of NADPH oxidase, or inhibition of Rho kinase. Oxidant and inflammatory related mechanisms often interact. Vascular expression of interleukin-6, another mediator of vascular disease, was increased 1.6-fold in old non-transgenic mice, but almost 9-fold in old E-V290M mice. Expression of CDKN2A, a molecular marker of senescence, was ~two-fold greater in old E-V290M mice compared to controls. These findings provide the first evidence that senescence and age-related vascular dysfunction is accelerated following cell-specific interference with endothelial PPARg through mechanisms that involve oxidative stress, inflammation, and Rho kinase. This critical role for endothelial PPARg has implications for vascular pathophysiology as well as therapeutic approaches for age-induced large and small vessel disease.

Disclosures
No relevant conflicts of interest to declare.
Fresh Frozen Plasma Transfusion Ameliorates INR and aPTT but Does Not Increase Thrombomodulin-Corrected Thrombin Generation in Patients with Cirrhosis
Authors: Rassi Amanda Bruder , Rocha Tania RFR , Farias Alberto Q , Mariano Livia Caroline Barbosa , D'Amico Elbio Antonio 
Published: October 2019

Abstract

Introduction: In vitro studies have shown that transfusion of fresh frozen plasma (FFP) has limited efficacy in correcting coagulopathy in cirrhosis. This study aims to assess the effect of FFP transfusion on endogenous thrombin potential (ETP) in patients with cirrhosis with abnormal coagulation test results. Methods: Using a stringent protocol for control of pre-analytic variables, blood samples were collected before and up to 8 hours after FFP transfusion. All samples were tested for INR, aPTT and ETP. The ETP assay was performed in platelet-poor plasma, using an automated fluorometer (CAT, Netherlands), using the technique developed by Hemker and modified by Tripodi. ETP was defined as the area under ROC curve and results were expressed as the ratio of ETP with thrombomodulin addition to ETP without (used to mimic in vivo conditions when patients are transfused). Results: 42 patients were included (male n=28; Child-B n= 11; Child-C n=31). Patients underwent FFP transfusion prior to high-risk invasive procedures (n=25), for treatment of bleeding (n=10) or for both reasons (n=6). The mean dose of FFP was 11.3 ml/kg (95% CI 9.16 - 20.46). After transfusion, INR decreased from 2.6±1,4 to 2.09±0.5 (p<0.0001) and aPTT from 1.47±0.5 to 1.27±0.26 (p<0.0001). At baseline, normal or above normal values of ETP ratio (³0.66) were found in 37 (88%) patients, and mean values remained largely unchanged after FFP transfusion (0.75±0.22; median 0.82 before to 0.76±0.16; median 0.81 after transfusion; p=0.56). The results for INR, aPTT and ETP are shown in figure 1. During the one-week follow-up period, 4 patients (9,5%) had acute reactions to transfusion, including fluid overload and allergic reactions. Of the 31 patients that underwent invasive procedures, four had severe bleeding. Discussion: FFP transfusion at standard doses ameliorated INR and aPTT, but did not increase thrombin generation corrected by thrombomodulin. That might be explained by the fact the former tests detect only 5% of the amount of thrombin generated. Besides, they are performed without the addition of thrombomodulin, an endothelial protein with anticoagulant effects, to which patients with cirrhosis have shown to be resistant. Patients were exposed to the risks of transfusions, without a clear benefit in the improvement in thrombin generation and decreasing the risk of bleeding. Our results call into question the common use of INR and aPTT for guiding the transfusion policy of FFP when treating coagulopathy in cirrhosis.
Figure 1: Results showing comparison before and after FFP transfusion
for (A) INR, (B) aPTT and (C) ETP ratio with and without thrombomodulin.

Disclosures
No relevant conflicts of interest to declare.
IFNγ-Induced Necroptosis Contributes to Hematopoietic Stem and Progenitor Cell Death and Bone Marrow Failure
Authors: O'Donnell Joanne , Roderick Justine E. , Pasparakis Manolis , Kelliher Michelle 
Published: October 2019

Abstract
RIPK1 has important kinase-dependent and kinase-independent scaffolding functions that prevent or activate necroptosis or apoptosis. Complete RIPK1 deficiency results in cell death and widespread inflammation yet tissue specific RIPK1 deletion can result in apoptosis, necroptosis and/or systemic inflammation, depending on the cell type. We have previously demonstrated that a hematopoietic RIPK1 deficiency results in constitutive activation of RIPK3 and MLKL and induction of necroptosis (Roderick et al, PNAS, 2014). These mice exhibit elevated serum TNFα and IFNγ levels, hematopoietic stem and progenitor cell (HSPC) loss, and ultimately succumb to bone marrow failure (BMF). When mice with a hematopoietic RIPK1 deficiency were placed on a RIPK3 deficient background, plasma pro-inflammatory cytokine and chemokine levels were reduced, HSPC numbers increased and BMF was significantly delayed. These mouse genetic data demonstrate that necroptotic death contributes to BMF in the mouse. To identify the receptor/ligands that trigger necroptosis, we generated mice with a hematopoietic RIPK1-deficiency on the Tnfr1-/-and Tnfr1-/-Tnfr2-/-genetic backgrounds. An absence of TNF signaling failed to prevent necroptosis and consequently, vav-iCre Ripk1f/f Tnfr1-/- Tnfr2-/-mice succumbed to BMF. Because type II interferons can also induce necroptosis, we generated vav-iCre Ripk1f/f Ifngr1-/- mice. These mice appear phenotypically normal demonstrating that an absence of IFNγ signaling prevents HSPC necroptosis and BMF.Collectively, these data may implicate IFNγ-mediated, RIPK3-dependent necroptosis in human BMF syndromes and raise the intriguing possibility that the progressive HSPC elimination observed in these patients reflects in part, IFNγ-induced necroptotic death.

Disclosures
No relevant conflicts of interest to declare.
Telomere Length and Somatic Mutation in Aplastic Anemia- Correlation with Response to Treatement
Authors: Park Hee Sue , Park Si Nae , Im Kyoungok , Kim Jung Ah , Hwang Sang Mee , Lee Dong Soon 
Published: October 2019

Abstract
Background: Telomere length (TL) of cell reflects the cellular age and short TL suggest the potential susceptibility to DNA damage. We investigated whether 1) the treatment response is associated with TL of hematopoietic cells and 2) whether somatic mutations are associated with TL in patients with aplastic anemia (AA).
Methods: We measured the mean TL and heterogeneity of bone marrow nucleated cells at the single cell level by quantitative fluorescence in situ hybridization. A total of 146 AA patients were enrolled; AA (n=146) and normal peripheral blood control (n=147). TL was expressed as telomere/centromere ratio. We analyzed the average and the distribution of TL belonging to the lowest 10th percentile of cell population. Along with measurement of TL, target sequencing for 88 hematopoiesis-related genes was performed to detect somatic mutations.
Results: We investigated the frequencies of cytogenetic aberrations and somatic mutations in 437 patients with aplastic anemia (AA) with prognostic relevance. Clonality was determined by G-banding/ fluorescent in situ hybridization (FISH) (n=280 patients), and targeted capture sequencing for 88 hematopoiesis-related genes (n=77). Of 437 patients, 20 (4.6%) showed disease progression and monosomy 7(50%) was the most predominant cytogenetic evolution at disease transformation. One third of patients (25/77, 32.4%) presented at least 1 mutation and the number of mutated genes was 18 and frequently mutated genes was NOTCH1 (3/25, 12%), NF1 (3/25, 12%), SCRIB (2/25, 8%), BCOR (2/25, 8%), DIS3 (2/25, 8%), DNMT3A (2/25, 8%), MED12 (2/25, 8%). In contrast to that 4.6% of AA patients showed disease progression, 4.0% of patients with SM showed disease progression. The telomere length of bone marrow nucleated cells was measured at single cell level by FISH (n=146) patients. Mean TL of AA (T/C ratio 6.99) was significantly shorter than normal control (T/C ratio 11.24). Patients with cytogenetic aberrations (CA) and/or somatic mutation (SM) showed significantly shorter TL compared to AA with normal karyotypes. However, the severity of AA did not correlate with TL. Patients with TL<6.88 showed significantly lower CR to IST (p=0.008) (Fig.1)
Conclusion: Patients with TL attrition showed poor response to IST. Short TL can be used not only as a biomarker for AA, but also as a predictive marker for treatment response to IST. In addition, CA and/or SM was strongly associated with adverse prognosis or treatment response, so we strongly suggest to adopt more sensitive test for the detection of minor clonal cell population in AA.
Figure 1 Figure 1.

Disclosures
No relevant conflicts of interest to declare.
Oncogenic Splicing of the Apoptosis-Stimulating Protein of p53-2 (ASPP2) Is an Initiating Step in Leukemogenesis Facilitating Acquisition of Structural Genomic Mutations
Authors: Kampa-Schittenhelm Kerstin Maria , Mau-Holzmann Ulrike , Lopez Charles , Schittenhelm Marcus M. 
Published: October 2019

Abstract
ASPP2 is an independent haploinsufficient tumor suppressor which initiates induction of apoptosis after cellular damage in a TP53-dependent manner. We recently reported the identification of two C-terminally truncated splice variants of ASPP2 with high frequency in acute leukemias. Tantalizingly, these variants lack the functionally relevant TP53 binding sites, suggesting a dominant-negative, oncogenic function. The most prevalent isoform lacking exon 17 (ASPP2k) is detected in up to 60% of acute leukemias. A cell sort for the CD34+ stem cell fraction confirmed ASPP2k-expression in the leukemia-initiating clone. Immunoblotting revealed translation of ASPP2k into a genuine protein isoform.
Generation of isoform-specific antibodies allows to discriminate ASPP2k from the wildtype isoforms. Interestingly, ASPP2k is the sole isoform expressed in some native leukemia samples. Other samples reveal a heterozygous expression pattern suggesting a dominant-negative function of the C-terminally truncated ASPP2k isoform.
Forced expression of ASPP2k using a HisMax plasmid vector in murine IL3-dependent pro-B Ba/F3 cells induced a more aggressive phenotype with mitotic failure and perturbed cellular proliferation compared to the empty vector controls. Of note, IL3 was successfully weaned in the ASPP2k cells - indicating that autoactivating genomic alterations must have occurred upholding cellular integrity and viability. Fractionated gamma irradiation accelerated this process. In contrast, the empty vector strains did not survive IL3 weaning. Chromosomal analyses of IL3-independent Ba/F3 ASPP2kcells revealed structural alterations including monosomies and additional marker chromosomes.
Furthermore, we demonstrate that ASPP2k is stress-inducible. Changing culture conditions (e.g. temperature) of cell lines or ex vivo native AML samples resulted in a dramatic increase of the ASPP2k isoform. Consecutive treatment of cells with daunorubicin lead to relative resistance of pre-stressed cells compared to their counterparts cultured under standard conditions. Vice versa, specific knock-down of ASPP2k resulted in a significant increase in induction of apoptosis upon chemotherapy compared to empty vector controls.
Our data demonstrate that ASPP2k plays a distinctive role as an anti-apoptotic regulator of the TP53 checkpoint rendering cells to a more aggressive phenotype as evidenced by proliferation and apoptosis rates - and facilitates acquisition of genomic mutations, a first initiating step in leukemogenesis.

Disclosures
No relevant conflicts of interest to declare.