bluuebunny/crossref_metadata_2025_split huggingface dataset:
Stop-Codon Mutation In The Extracellular Part Of G-CSFR Leading To G-CSF Unresponsiveness Due To Defects In Erk1/2 Phopsphorylation In a Patient With Severe Congenital Neutropenia
Authors: Klimenkova Olga , Zeidler Cornelia , Skokowa Julia , Welte Karl 
Published: October 2019

Abstract
We identified a 10 year old female severe congenital neutropnia patient who did not respond to the treatment with G-CSF (Filgrastim) up to 100 µg/kg/day, s.c. By search of possible reasons of G-CSF unresponsiveness, we detected by deep sequencing of the CSF3R (SOLID 5500xl, 3125 reads) a novel mutation within the extracellular part of CSF3R. The mutation was also confirmed by convential Sanger sequencing. It is a heterozygous stop-codon mutation C1641T (NM_000760), p.W547* (NP_000751) which is localized proximal to the cell membrane in the fifth fibronectin type III domain of the CSF3R. This is a first report describing a stop-codon mutation in the extracellular part of CSF3R in severe congenital neutropenia. FACS analysis revealed severely diminished surface expression levels of G-CSFR on CD33+ bone marrow myeloid progenitor cells of this patient as compared to G-CSFR levels on CD33+ cells of healthy individuals. In line with reduced expression of G-CSFR, we found disturbed G-CSFR-triggered intracellular signaling pathways activated by G-CSFR. MAPK/Erk1/2 activation by phosphorylation is known to be crucial for G-CSF- induced myeloid differentiation and STAT5 signaling is important for proliferation and survival of myeloid cells upon stimulation with G-CSF. We demonstrated abrogated phosphorylation of ERK1/2 and of STAT5 in bone marrow CD33+ myeloid cells of CN patient treated with G-CSF in vitro for 2, 5, 10 and 15 minutes, comparing with healthy donor. Since this patient has no known inherited CN-associated mutations (e.g. ELANE, HAX1, G6PC3), it may represent a new subgroup of CN patients characterized by non-responsiveness to G-CSF due to lack of binding of G-CSF to its receptor. Since the majority of investigations on CSF3R mutations are targeting the cytoplasmic part of the receptor only, we suggest that CN patients who do not respond to G-CSF treatment should be tested also for mutations within the extracellular part of the CSF3R.

Disclosures:
No relevant conflicts of interest to declare.
Ratios Of FcγRIIa and FcγRIII Binding Affinities Govern The Efficacy Of PMN-Mediated Cytotoxicity – Implications For Fc-Engineering Approaches Of Therapeutic Antibodies
Authors: Derer Stefanie , Glorius Pia , Schlaeth Martin , Lohse Stefan , Muchhal Umesh , Desjarlais John , Humpe Andreas , Valerius Thomas , Peipp Matthias 
Published: October 2019

Abstract

Introduction
Antibody-dependent cell-mediated cytotoxicity (ADCC) has been suggested to be an essential effector mechanism for the in vivo activity of tumor-targeting therapeutic monoclonal antibodies (mAbs). Thus, enhancing the affinity of human IgG1 mAbs to NK cell-expressed FcγRIIIa by glyco- or protein-engineering of their Fc moiety has been demonstrated to improve NK cell-mediated ADCC and represents a promising strategy to improve antibody therapy. However, human polymorphonuclear (PMN) cells express the homologous FcγRIIIb isoform, which does not trigger ADCC. The aim of the present study was to analyze a panel of distinct IgG1 mAbs, displaying different affinities for FcγRIIa and FcγRIII, with respect to PMN recruitment for tumor cell destruction.

Methods
Affinities of analyzed mAbs to distinct FcγR were determined by surface plasmon resonance technology. Induction of ADCC was assessed by 51Cr release experiments in the absence or presence of FcγRIII- or FcγRII-blocking agents to unravel the contribution of both receptors in these assays.

Results
Non-fucosylated or protein-engineered IgG1 variants with optimized FcγRIII binding capacities demonstrated the expected benefit in triggering NK cell- mediated ADCC but did not mediate ADCC by PMN, which could be restored by FcγRIIIb blockade. Additionally, eosinophils as well as PMN from paroxysmal nocturnal hemoglobinuria (PNH) patients – expressing no or low levels of FcgRIIIb – mediated effective ADCC with FcgRIII-optimized mAbs. Additional experiments with Fc variants displaying enhanced FcγRIIa binding or with double FcγRIIa/FcγRIII-optimized constructs demonstrated enhanced PMN-mediated ADCC compared to control mAbs. Statistical analyses revealed that FcγRIIa/FcγRIIIb affinity ratios correlated with the extent of human PMN-mediated ADCC.

Conclusions
To conclude, the present study represents novel findings concerning recruitment of PMN for tumor cell destruction by Fc-engineered antibodies. While PMN-mediated ADCC was completely abolished by FcγRIII-optimized antibodies through predominant FcγRIIIb binding, it was potently enhanced by optimization of FcγRIIa binding affinity. Importantly, functional analyses unraveled the ratio between FcγRIIa and FcγRIII binding affinities to control PMN-mediated ADCC activity and therefore opened new alleys in engineering of therapeutic antibodies.

Disclosures:
Muchhal: Xencor Inc.: Employment. Desjarlais:Xencor Inc.: Employment.
Identification Of Factors That Can Predict a Response To Intravenous Immunoglobulin Treatment In Children With Immune Thrombocytopenic Purpura
Authors: Yoshida Nao , Morimoto Yoshihito , Kawashima Nozomu , Matsumoto Kimikazu , Kato Koji 
Published: October 2019

Abstract
Immune thrombocytopenic purpura (ITP) is a common benign bleeding disorder characterized by isolated thrombocytopenia caused by the immune-mediated destruction of platelets. The 2004 Japanese Guidelines for Childhood ITP recommended intravenous immunoglobulin (IVIG) as first-line therapy for children with ITP, because this strategy results in more rapid increases in platelet counts when compared with oral steroid therapy. However, predictive markers of the response to IVIG have not been well defined. The present study therefore evaluated whether clinical and laboratory findings before treatment could predict the response to IVIG and progression to chronic ITP.
We reviewed the clinical data of 49 newly diagnosed patients (< 18 years) with ITP who were given IVIG as initial treatment at Japanese Red Cross Nagoya First Hospital from 1997 to 2011, and evaluated the short-term response to IVIG as outcomes at 2 weeks after IVIG and the long-term response to IVIG as thrombocytopenia-free survival (TFS). TFS was defined as the probability of survival without treatment failure (death, relapse, disease progression requiring other therapeutic intervention, or progression to chronic ITP) after initial IVIG and was estimated by the Kaplan-Meier method.
Short-term response to IVIG was observed in 40 patients (82%), and long-term treatment response, defined as 1-year TFS, was 65%. At 6 months after initial IVIG treatment, seven patients (14%) progressed to chronic ITP. In multivariate analysis, lower white blood cell (WBC) count at diagnosis was identified as the only independent risk factor for the short- or long-term response to IVIG (P=0.007 or P=0.043, respectively) and the progression to chronic ITP (P=0.034). Patients with WBC count<7.0×109/L had a lower probability of TFS (41% vs. 77%, P=0.003) and a higher rate of progression to chronic ITP (29% vs. 6%, P=0.040) than those with WBC count ≥7.0×109/L. None of the other variables, including gender, age at diagnosis, season of onset, initial platelet count, initial hemoglobin level, preceding infection, fever at diagnosis, H. pylori infection, total and daily dose of IVIG, product of IVIG, and laboratory findings including CRP, TP, T-bil, LDH, AST, ALT or BUN, were significantly associated with the response to IVIG and the progression to chronic ITP in multivariate analysis.
In conclusion, this study showed that WBC count at onset of ITP is a strong predictor of short- and long-term treatment response to IVIG and the development of chronic ITP in pediatric patients with ITP. For patients with low WBC count, the addition of other agents, such as steroids, to IVIG at the time of initial diagnosis might improve the treatment outcomes.

Disclosures:
No relevant conflicts of interest to declare.
ADAMTS13 Expressed In Platelets Offers Systemic Protection Against Arterial Thrombosis and Therapeutic Benefits In Murine Models Of Thrombotic Thrombocytopenic Purpura
Authors: Pickens Brandy , Mao Yingying , Zheng X. Long 
Published: October 2019

ADAMTS13, a plasma metalloprotease, cleaves von Willebrand factor (VWF) and inhibits arterial thrombosis and inflammatory response. Deficiency of plasma ADAMTS13, due hereditary mutations in ADAMTS13 gene or autoantibodies against ADAMTS13 protease, results in a potentially fatal syndrome, thrombotic thrombocytopenic purpura (TTP). Plasma infusion or exchange is the only effective therapy available to date. To develop novel therapeutics against TTP, we tested a hypothesis that recombinant ADAMTS13, expressed specifically in platelets, may offer systemic protection against arterial thrombosis and therefore provide therapeutic benefit for TTP. To test this hypothesis, we first generated transgenic mice (JAX B6SJL) carrying a human full-length ADAMTS13 gene under a murine platelet glycoprotein 1b (CD41) promoter. The transgenic mice were then bred with Adamts13-/- (CAST/Ei) mice for >4 generations. By Western blotting and activity assay, we show that a full-length human ADAMTS13 protein (∼195 kDa) and its proteolytic activity toward a FRETS-hVWF73 peptide are detected in platelet lysate obtained from transgenic (rA13-PltTG) mice but not in Adamts13-/- mice or wild-type mice. Little to no ADAMTS13 activity was detected in plasma in transgenic mice, suggesting the expressed human ADAMTS13 is stored inside platelets. ADAMTS13 was releasable upon stimulation with thrombin (1 U/ml), collagen (10 µg/ml), and AYPGKF (0.5 mM). More significantly, rA13-PltTG mice had higher baseline platelet count than Adamts13-/- mice, but exhibited a dramatically reduced rate of thrombus formation in mesenteric arterioles after oxidative injury with 10% ferric chloride as compared with Adamts13-/-mice and wild-type mice. Finally, rA13-PltTG mice were protected from Shigatoxin-2 (Stx-2) or murine recombinant VWF (mVWF)-induced “TTP-like” syndrome, as demonstrated by fewer rA13-PltTG mice having thrombocytopenia (defined by a 40% drop in platelet count from the baseline after challenge with Stx-2 or mVWF), faster and more complete recovery of thrombocytopenia, and significantly higher survival rate than Adamts13-/- mice. In summary, we have generated transgenic mice expressing human ADAMTS13 in platelets. Platelet ADAMTS13 is releasable upon stimulation by agonists and biologically functional in proteolytic cleavage of VWF in vitro. The platelet-derived ADAMTS13 offers systemic protection against arterial thrombosis after oxidative injury and provide a therapeutic benefit to murine models of TTP, resulting from hereditary ADAMTS13 deficiency. We are now in the process testing the efficacy of this strategy as a novel therapeutic for acquired TTP with inhibitors.

Disclosures:
No relevant conflicts of interest to declare.
Increased Functional T Cell Defects in Patients with low CD4 Counts after Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Holtick Udo , Frenzel Lukas P. , Alexander Shimabukuro-Vornhagen , Theurich Sebastian , Claasen Julia , Scheid Christof , von-Bergwelt-Baildon Michael , Hallek Michael , Wendtner Clemens , Chemnitz Jens M. 
Published: October 2019

Background
The recovery of the host immune system after allogeneic hematopoietic stem cell transplantation is pivotal to prevent infections, relapse and secondary malignancies. In particular, numerical CD4 T-cell reconstitution is delayed and CD4-helper cell function considered impaired as consequence of the transplant procedure and concommitant immunosuppressive medication. From HIV/AIDS patients it is known that numerical and functional CD4 defects increase the risk of opportunistic infections. Therefore, even in the absence of immunosuppressants and graft-vs-host disease, anti-infective prophylaxis is usually given for at least six months. We hypothesized that the numerical CD4 defect in patients may be reflected by immunosuppressive RNA fingerprints previously established for certain immuno-inhibitory molecules and tested whether the functional CD4 capacity was different according to the CD4 cell number.

Methods
RNA was separated from CD4 T-cells of 10 patients with CD4 counts >500/µl, 10 patients with CD4 counts <200/µl and four healthy controls. All patients had to be off immunosuppression and without any clinical signs of graft-vs-host disease. Transcriptional activity was assessed with regard to previously defined fingerprints motives for CTLA-4, IL-10, PD-1, TGF-β and PGE-2. CD4 T-cells from all groups were further tested for their proliferative capacity and cytokine production.

Results
Hierarchical clustering segregated the three groups. Applying the immunosuppressive fingerprints, patients with CD4 T-cells >500/µl were demonstrated to be under the influence of PGE2, whereas patients with CD4 T-cells <200/µl were demonstrated to be under the influence of PGE2 and CTLA-4. In normal controls, no association was found. The proliferative capacity of patient CD4 T-cells upon CD3-CD28-bead stimulation was not significantly different from healthy controls. The production of IL-2 by stimulated CD4 T-cells was significantly downregulated in patients with CD4 T-cells <200/µl, while there was no difference in IFN-ƴ and TNF-α secretion.

Conclusion
The severity of the CD4 numerical defect reflects the state of immunosuppression as demonstrated by RNA immuno-inhibitory fingerprint motives. This partially translates into functional differences as measured by decreased IL-2 secretion. In addition to time after transplant, CD4 T-cell numbers should be considered for the decision to stop or maintain anti-microbial prophylaxis in patients after allogeneic stem cell transplantation.
(UH, LPF and CW, JMC contributed equally to this work.)

Disclosures:
No relevant conflicts of interest to declare.
Monitoring Early CMV-Specific CD8+ T-Cell Responses After Allogeneic Stem Cell Transplantation By Pentamer and Streptamer Staining
Authors: Beloki Lorea , Ciaurriz Miriam , Ramirez Natalia , Zabalza Amaya , Mansilla Cristina , Bandres Eva , Calvillo Mercedes Rodriguez , Olavarría Eduardo 
Published: October 2019

We present the results of a pilot study using pentamer (PM) and streptamer (ST) multimer complexes for monitoring CMV-specific CD8+ T-cells (CTLs). We analysed 15 patients that underwent allogeneic Stem Cell Transplantation (HSCT). Patient characteristics are summarized in Table 1. All patients and donors were positive for the HLA-A*02:01 allele. PM and ST were directed against the epitope NLVPMVATV (495-503) of the CMV phosphoprotein 65 (pp65). Samples were obtained at 15-day intervals until day +90 and monthly thereafter.PatientCMV status (D/R)GenderAgeDiagnosisDonorConditioningEngraftment (day)Follow up (months)GVHD (day)1+/+F43NHLSIBRIC2415-2-/+F33MDSURDRIC1912-3+/+M55AMLSIBMAC1714474-/+F41AMLURDMAC2121-5+/+F32AMLSIBMAC2013256+/+F64MDSSIBRIC2714897-/+M58CLLURDRIC2313928+/+M57ALLURDMAC155-9+/+F39AMLURDMAC2012-10-/+M42ALLURDMAC2125-11-/+M44AMLURDMAC12815312+/+F65AMLSIBRIC258-13-/+M27AMLSIBMAC3031814+/+M65AMLSIBRIC1966215+/-F30SAASIBRIC133-
Three patterns were observed. In 3 patients (20%) no CMV-specific-CTLs could be detected despite several CMV reactivations, requiring prolonged cumulative antiviral therapy (median 68 days; range 67-136).
In 7 patients (47%) CMV reactivation occurred at a mean of 41 days (10-94) and triggered a rapid increase of CMV-specific-CTLs with a median of 22.7 x 105/L (range 1.3-279.7). The CMV-PCR became immediately negative and antiviral therapy was stopped promptly after a median of 15.5 days (6-23).
Finally, 5 patients (33%) showed an early immune reconstitution with CMV-specific-CTLs detected with a median of 0.7 x 105/L (range 0.2-2.8) in the absence of CMV-PCR reactivation at a median of 21 days (10-34) post-SCT. No CMV-PCR reactivation was observed in this group with a median follow-up of 12 months (5-14).

Discussion
Monitoring CMV-specific-T-cells might be able to distinguish patients at higher risk of recurrent virus reactivation and in need of prolonged antiviral therapy. Patients with increasing CMV-specific-CTLs detectable at the time of CMV-PCR reactivation may only need a short course of antiviral therapy, while those with early CMV-specific-CTLs may be protected from CMV reactivation.

Conclusion
Using Multimer-based (Pentamer and Streptamer) monitoring of CMV-specific T-cell immune reconstitution after allogeneic HSCT may contribute to the clinical decision regarding when and for how long to commence anti-CMV therapy.

Disclosures:
No relevant conflicts of interest to declare.
C-Reactive Protein As a Predictor Of Bacterial Disease In Children With Sickle Cell Disease and Fever
Authors: Olivares Maria , Richardson Matthew W , Visintainer Paul 
Published: October 2019

Introduction
Children with sickle cell disease (SCD) are at increased risk of serious bacterial disease compared to children without SCD. Children with SCD and fever are recommended to undergo prompt evaluation, including a physical exam, complete blood count, blood culture, and sometimes chest x-ray. These children often receive empiric parenteral antibiotics until blood cultures show no bacteremia for forty-eight hours. Studies in this population have identified clinical and laboratory features that are associated with low rates of bacteremia or that predict bacteremia. C-reactive protein is an acute phase reactant that has been studied as a predictor for bacteremia in a variety of clinical situations. Crp, however, has not been evaluated as a predictor of serious bacterial disease in children with SCD.

Method
Retrospective chart review of children age 0 – 18.9 year old admitted to Baystate Children’s Hospital with a diagnosis of SCD and fever who had crp and blood culture drawn between January 1, 2009 and December 31, 2011. SCD included homozygous SS disease, SC disease, or Sβ-thalassemia. Bacterial disease was defined as bacteremia, osteomyelitis, pneumonia/ acute chest syndrome, pyelonephritis. Parental report of fever was accepted. Age, maximum temperature, white blood count (WBC), absolute neutrophil count (ANC), percent of neutrophils (ANC/WBC), and crp were evaluated.

Results
80 cases met inclusion criteria. 18 cases had bacterial disease. Compared to children without bacterial disease, those with bacterial disease were older (mean age 11 vs. 4.8 yr, p < 0.001), had higher mean crp (5.7 vs. 1.6 mg/dL, p <0.001), higher mean WBC (20.9 vs. 15.6 x 103/mm3, p = 0.004), and higher mean ANC (13.5 vs. 8.7 x 103/mm3, p = 0.002). There were no significant differences between maximum temperature and percent neutrophils between the two groups. After regression analysis for all variables, age and crp remained significant between the two groups (p = 0.001 for both). A receiver operator characteristic (ROC) curve using crp and age produced an area under the curve of 0.87.

Conclusions
Children with SCD hospitalized for fever and diagnosed with bacterial illnesses are older and have higher crp compared to those without bacterial illness. The ROC curve based age and crp has characteristics of a good screening test. Because children with SCD and fever will usually receive empiric antibiotics regardless and the repercussions of a missed bacterial illness are great, using crp to determine antibiotic administration is likely limited. Rather, a low crp, especially in a younger child, may be helpful to determine patients who are candidates for early discharge or those who can be managed as an outpatient. Prospective use of crp for fever in this population, including outpatients, is warranted in order to identify the best sensitivity and specificity.

Disclosures:
No relevant conflicts of interest to declare.
Hemoglobinopathies Of Difficult Diagnosis. Case Reports From a Main Referral Pediatric Hospital In Venezuela
Authors: Bello Adriana C. 
Published: October 2019

Hemoglobinopathies, sickle cell disease and thalassemias, are the most common genetic disorders worldwide, affecting millions of individuals. Diagnosis can take place very early in life, ideally through newborn screening, sometimes later in life. Disease modifying, acute and long term management strategies are provided for specific genotypes, and can be challenging for the treating hematologists.
It is essential the adequate diagnosis for the optimal management of these patients. Most diagnosis are made through hemoglobin electrophoresis, high performance liquid chromatography (HPLC) in the majority of cases of thalassemias. However, in some cases the correct diagnosis cannot be made by these methods, and more complex studies are needed, at the molecular level to genetically characterize the disorder.
We report a series of 12 patients from the pediatric hematology department from Hospital de Ninhos JM de los Rios, affected by severe congenital hemolytic anemias, who where assessed and managed at this tertiary Referral Center, who did not have definitive diagnoses by conventional methods, in September 2010. They all had presented symptoms from severe anemia since very early age, and where of difficult management through strategies like chronic transfusions, splenectomy or hydroxycarbamide, because lack of definitive diagnosis.
Samples were shipped after appropriate consentment to a highly specialized referral laboratory, (Hemoglobin Diagnostic Reverence Laboratory, Boston University School of Medicine), free from any charge, for its molecular characterization.
We obtained very accurate and complex results, also very interesting cases, like one unstable hemoglobinopathy from Heinz bodies (Hb Volga) and never before described thalassemic mutations, both homozygous and heterozygous. This was assuring, also reflected in a more appropriate and safer management of our patients.
We conclude it is always necessary to establish adequate diagnosis in our patients affected by hemoglobinopathies, for their adequate characterization and management. This will be reflected in a better quality of life.

Disclosures:
No relevant conflicts of interest to declare.