bluuebunny/crossref_metadata_2025_split huggingface dataset:
Longitudinal Changes in Iron Overload Parameters and Iron Chelation Therapy in Young Patients with Thalassemia Major,
Authors: Kattamis Antonis , Stokidis Konstantinos , Delaporta Polyxeni , Dimitra Kyriakopoulou , Petropoulou Theoni , Boiu Sorina , Ladis Vassilis 
Published: October 2019

Abstract
Abstract 3202

Background:
New modalities for the assessment of iron overload and the availability of new oral chelators have led to important changes in the iron load status and its treatment for patients with thalassemia major (TM). The goals of this retrospective analysis were to evaluate the changes that occurred in regards to the degree of iron overload, as well as to the therapeutic regimen of iron chelation over the last decade in young patients with TM.

Methods:
All patients with TM followed in our unit, who were <18 years at certain time points, were included in this study. Group A included all patients who were younger than 18 years old on 1/1/2001, while group B, C, D, E and F on 1/1/2003, 1/1/2005, 1/1/2007, 1/1/2009, and 1/1/2011, respectively. Liver iron concentration (LIC) and cardiac siderosis (T2*) were evaluated by MRI. Cardiac iron concentration (CIC) was calculated based on the recently prescribed formula CIC= 45 × (T2*Heart)−1.22.The closest MRI, which was <12 months from the time point, was recorded for each patient at each group. The therapeutic regimen for iron chelation, being deferoxamine (DFO), deferiprone (DFP), combination therapy of DFO and DFP (DFO+DFP) and deferasirox (DFX), used at the different time points were also recorded.

Results:
The results of the analysis are shown in the following table:
Ferritin levels did not change significantly over the last decade (p>0.05). There was a trend for decreasing values of LIC (Independent Samples Kruskal-Wallis test, p=0.075) with the mean LIC of group E and F being significantly lower than group C (Mann-Whitney test, p<0.05). Similarly, there was a trend for improvement in the indexes of cardiac iron load. Of note is, that cardiac overload was not documented in this group of patients. None of the patients has significant (T2*<10 msec), and only 3 patients had moderate cardiac siderosis (T2*>10 <20 msec).

Conclusions:
A steady decrease in the number of young patient with thalassemia has been observed, reflecting the efficacy of the thalassemia prevention program. As expected, the utilization of MRI to evaluate iron overload has increased significantly especially in the second part of the last decade, but it remains limited mainly to older children and teenagers. Oral chelation has become the preferable mode of treatment of hemosiderosis in young patients with TM. While DFX is, currently, the most used iron chelator, the use of DFO is becoming limited as an additive therapy to DFP. Despite presumed better compliance with oral chelation therapy, the iron overload indexes have not improved dramatically. This may reflect the short period of using the oral chelators or/and the need for further treatment intensification.

Disclosures:
Kattamis: Novartis Oncology: Honoraria, Research Funding, Speakers Bureau; Apopharma: Honoraria.
Regulation of Plasmacytoid Dendritic Cells by Commonly Used Anti-Viral Vaccines,
Authors: Jahrsdörfer Bernd , Nussbaum Benedikt , Mandel Birgit , Busch Daniel , Panitz Verena , Vollmer Angelika , Beyer Thamara , Debatin Klaus-Michael , Fabricius Dorit 
Published: October 2019

Abstract
Abstract 3221
Human plasmacytoid dendritic cells (pDC) play a central role in regulating adaptive T cell responses in the course of neoplastic, viral and autoimmune disorders. Recently, we demonstrated that apart from their pro-inflammatory effects, which are mainly mediated by secretion of interferon-alpha (IFN-a), pDC can also exhibit potent anti-inflammatory functions borne by active secretion of granzyme B (GrB), a serine protease classically known from CTL, NK cells and regulatory T cells. Here, we hypothesized, that commonly used anti-viral vaccines may affect pDC on several levels including their immunophenotype as well as their capacity to secrete either IFN-a or GrB. Using various methods including FACS, ELISpot, ELISA, spinning-disk confocal microscopy and RT-PCR, we could demonstrate that various anti-viral vaccines including vaccines against TBEV, yellow fever, polio, measles, rotavirus, varicella and hepatitis B were able to affect pDC by modulating expression of a series of surface molecules involved in cell adhesion, antigen-presentation and co-stimulation. In addition, major differences between the vaccines were found in terms of their effects on secretion of IFN-a and GrB. Interestingly, while only one vaccine, FSME Immun (TBEV) induced substantial IFN-a responses in pDC, all others tested did not. In contrast, virtually all vaccines tested induced more or less strong suppression of GrB secretion by maturing pDC. Of note, pDC that secreted high amounts of GrB induced by far lower allogeneic T cell proliferation as compared to pDC that secreted little or no GrB. Moreover, suppression of pDC-derived GrB by a substrate-specific GrB inhibitor resulted in significant enhancement of T cell proliferation in co-cultures of GrB-secreting pDC with allogeneic T cells. Our data demonstrate 1) that anti-viral vaccines may have distinct effects on both the immunophenotype and the secretory potential of pDC, and 2) that GrB is an important novel variable affecting the capacity of pDC to either trigger or dampen adaptive T cell responses. Our results may have implications for further study of the role pDC play in the regulation of adaptive immune responses, and for the potential application of this knowledge in the development of novel adjuvants admixed to anti-viral vaccines.

Disclosures:
No relevant conflicts of interest to declare.
HIV-Infected T Cells Induce Granzyme B-Secreting Regulatory B Cells in An Interleukin 21-Dependent Fashion,
Authors: Kaltenmeier Christof , Dahlke Karen , Gawanbacht Ali , Beyer Thamara , Hofmann Stefanie , Härter Georg , Grüner Beate , Kern Peter , Kirchhoff Frank , Schrezenmeier Hubert , Jahrsdörfer Bernd 
Published: October 2019

Abstract
Abstract 3222
We and others have recently provided evidence that a series of lymphocyte subsets including, plasmacytoid dendritic cells, B cells and regulatory T cells are able to secrete the cytotoxic serine protease granzyme B (GrB) into the extracellular compartment, where it contributes to the suppression of T cell proliferation by a so far undefined GrB-dependent mechanism. For B cells, we found that viral antigens in the context of the acute phase cytokine interleukin (IL-) 21 can potently induce GrB. Here, we demonstrate that infection of CD4+ T cells with HIV-1 (NL4-3), but not mock infection, induces strong expression of IL-21 on both mRNA and protein levels in CD4+ T cells. Moreover, we found that HIV-infected CD4+ T cells are able to induce high levels of GrB in co-cultured B cells and that inhibition of IL-21 with specific antibodies abrogates T cell-mediated GrB induction in B cells. In support of these data, serum levels of both IL-21 and GrB are significantly higher in patients acutely infected with HIV as compared to healthy control subjects. Surprisingly, co-culture of CD4+ T cells with B cells during HIV-1 infection strongly suppressed both, proliferation of T cells as well as virus replication as indicated by significantly reduced p24 levels in culture supernatants. Notably, this effect was enhanced by external stimulation of B cells with IL-21, and was reduced by inhibition of GrB using specific GrB inhibitors. To further explore the underlying mechanisms of our findings, we performed confocal microscopy of T cell-B cell co-cultures and demonstrated that GrB-secreting B cells directly interact with CD4+ T cells, thereby transferring active GrB to them. Moreover, we found that GrB+ B cells decreased CD4+ T cell expression of the T cell receptor-zeta chain, a known GrB target, which is required for T cell proliferation, and known to be suppressed in HIV patients. In summary, we provide evidence that HIV induces the acute phase cytokine IL-21 in infected CD4+ T cells, thereby indirectly triggering the expression of GrB by B cells. GrB-secreting B cells may play a so far unappreciated role in decelerating the expansion of HIV, particularly in the early phase of acutely infected patients. Our study reveals a novel pathogenetic mechanism in HIV infection with potential relevance for the development of novel immunotherapeutic approaches.

Disclosures:
No relevant conflicts of interest to declare.
The Therapeutic Effect of Umbilical Mesenchymal Stem Cells Injected Directly Into Medullary Cavity of Ilium to Treat Refractory Aplastic Anemia and Bone Marrow Fibrosis,
Authors: Zheng Zhuanzhen , Qiao Zhenhua , Chen Wenliang , Gong Rong , Wang Yalin , Xu Lianrong , Ma Yanping , Hou Lihong , Zhang Li , Lu Yujin , Jiang Bo , Li Guoxia , Dong Chunxia 
Published: October 2019

Abstract
Abstract 3226

Objective
To evaluate mesenchymal stem cells(MSCs) combination with routine therapy to treat refractory aplastic anemia and bone marrow fibrosis, observe the course of the hematopoitic reconstitution and evaluate the security and curative effect. Methods 12 primary myelofibrosis and 19 chronic aplastic anemia patients who had all accepted routine therapy more than 6 months,because of pathogenetic condition, they needed infusing constituent blood such as at least condensed red blood cells 6 units and platelet 4 units per months, and despite this, whole blood cells counts were very lower. Those patients all had intermittent dizzy, fatigue, bad appetite and purpura. Because of age, complication, and psychological reasons, they refused Allogenic stem cell transplantation. So besides routine therapy, they received umbilical MSCs 1×107, every two weeks one time, those cells were injected directly into medullary cavity of ilium. Results 28 recipients showed hematopoietic reconstitution when PLT was up to 20×109/L and granulocytes was up to 2.0×109/L on day 28 post MSCs infusion. Bone marrow biopsy showed hyperplasia from hypoplasia to evident hyperplasia. On day 28, Th cells ascended from 23.0 to 36.2, Ts cells descended from 24.2 to 14.6, Th.. MTs from 0.95 to 2.47:1, IgG from 6.6 to 13.3, NK cells from 2.40 to 13.5, indicating earlier immune recorrect. At the last follow-up of 12 months, 17 patients showed normal hemoglobin and platelet counts, immune globulin was in the normal level, indicating the hematopoietisis and immune persistent reconstitution. 5 patients still needed infusing constituent blood cells, although, the interval of platelet and red blood cells infusion obviously prolonged, and the times to see doctor decreased and life quality elevated greatly. Conclusion This is a novel clinical research of successful treatment of refractory CAA and MF, it tell us infusion HLA-mismatched allogenic MSCs directly into medullary cavity of ilium is efficiency and security.

Disclosures:
No relevant conflicts of interest to declare.
Immunosuppressive Drugs Upregulate the Immunoinhibitory Receptor Osteoactivin in Monocyte-Derived Dendritic Cells,
Authors: Schwarzbich Mark-Alexander , Gutknecht Michael , Guettler Lisa , Kanz Lothar , Salih Helmut R. , Gruenebach Frank , Rittig Susanne Malaika 
Published: October 2019

Abstract
Abstract 3232
Osteoactivin, also known as transmembrane glycoprotein NMB (GPNMB) and dendritic cell-associated transmembrane protein (DC-HIL), is a type I transmembrane glycoprotein that is detected abundantly in dendritic cells (DC), but not or only at low levels in monocytes. Its expression on antigen-presenting cells (APC) can inhibit T cell activation by binding the type I transmembrane proteoglycan syndecan-4 (SD-4) on T cells. We here studied the influence of different therapeutically used immunosuppressive agents such as the corticosteroid prednisolone and the calcineurin inhibitors tacrolimus or cyclosporine A on the expression of osteoactivin in human monocyte-derived DC. Therefore, DC were generated from blood monocytes isolated by plastic adherence and exposed to GM-CSF and IL-4. Prednisolone, tacrolimus or cyclosporine A were added to the culture medium at concentrations obtained upon clinical application in patients (cyclosporine A: 1μg/ml, prednisolone: 3.5μg/ml, tacrolimus: 10ng/ml) every second day starting from the first day of culture. Cells were harvested for immunophenotyping and osteoactivin expression analysis by immunostaining, western-blotting and real-time RT-PCR or for functional analyses in mixed lymphocyte reactions (MLR) on day 7 of culture. DC generated in the presence of therapeutic concentrations of the immunosuppressant drugs showed a reduced expression of CD1a. Furthermore, we observed a clear upregulation of osteoactivin on DC that had been treated with the immunosuppressive agents. In line with osteoactivin up-regulation, exposure to the immunosuppressive drugs resulted in reduced stimulatory capacity of the DC in MLR with allogenic T cells, and this could be restored by addition of blocking osteoactivin antibody. In summary, our results demonstrate that upregulation of osteoactivin is critically involved in the inhibition of DC function upon exposure to different classes of immunosuppressive agents. These findings suggest that upregulation of osteoactivin in DC constitutes a ubiquitous mechanism that may underlie inhibition of T cell activation by these drugs.

Disclosures:
No relevant conflicts of interest to declare.
Platelet Generation Under Shear Force Is Modulated by Site-Specific Phosphorylation of Myosin-II Heavy Chain,
Authors: Spinler Kyle R , Shin Jae-Won , Discher Dennis E 
Published: October 2019

Abstract
Abstract 3262
Platelets are generated from megakaryocytes (MKs) under blood shear. When activated, platelet aggregration and contraction result in part from non-muscle myosin-IIA (NMM-IIA), which is the most abundant isoform of MKs. Defects in this motor protein are a common basis of May-Hegglin disease in which thrombocytopenia is a common symptom. Recently, we showed that reversible inhibition of NMM-IIA – rather than irreversible perturbations – together with soft 2D matrix increases polyploid MKs and also platelet generation (Shin et al., PNAS, 2011; 108:11458–63). While we identified specific phosphorylation sites in NMM-II heavy chain that can modulate MK polyploidization physiologically, it remained to be investigated whether the same sites can also influence platelet generation and activation. Since we showed previously that cytoskeletal proteins undergo forced disruption of specific domains and specific interactions within shear-stressed cells (Krieger et al., PNAS, 2011; 108:8269–74), it is possible that NMM-II heavy chain phosphorylation sites are also exposed under physical forces. Here, intravenous blood shear forces simulated by a cone and plate rheometer are shown to induce the de-activating and isoform-specific phosphorylation at Ser1943 (pS1943) of NMM-IIA in MKs. MEG01 cells show that the fraction of pS1943 NMM-IIA increases up to a low shear stress (0.5Pa), while further increases in shear stress lead to cell fragmentation and a decay in pS1943. Mutational analysis of this site in MEG01 cells together with knockdown and overexpression addresses whether pS1943 is both sufficient and necessary for cellular fragmentation under shear. The role of pS1943 in platelet activation will also be discussed in relation to May-Hegglin disease.

Disclosures:
No relevant conflicts of interest to declare.
CCR2 and CCR5 Co-Expressing CD11b+ Monocytes Affect Neovascularization in An Ischemic Hind Limb Mouse Model,
Authors: Kim Jeong-A , Noh Kyoungmi , Kim Hyunkyung , Broxmeyer Hal E. 
Published: October 2019

Abstract
Abstract 3270
Monocyte/myeloid -lineage cells are known to be involved in neovascularization. However, the specific monocyte /myeloid population that participates in neovascularization has not been clearly identified. Several subsets of monocyte/myeloid cells have been proposed to be important for this process. For example, CD11b+CXCR1hiCCR5+ resident monocytes regulate tissue healing by increasing angiogenesis, and CD11b+CXCR1loCCR2+ inflammatory monocytes are mobilized to the inflammation site. Interestingly, we found that CD11b+ myeloid cells isolated from ischemic muscle of post-femoral artery dissected C57BL/6 mice are positive for both CCR2 and CCR5. To study the relationship between CD11b+CCR2+CCR5+ monocytes and neovascularization in an ischemic hind-limb model of C57BL/6 mice, we addressed the following questions:(1) Are the number of tissue-resident CD11b+CCR2+CCR5+ monocytes increased in ischemic muscle, post-ischemic injury?; (2) Are CD11b+CCR2+CCR5+ monocytes recruited to the ischemic muscle and do they incorporate into the blood vessels in the ischemic site?; (3) Do CD11b+CCR2+CCR5+ monocytes modulate neovascularization in ischemic hind-limbs of C57BL/6 mice upon direct injection of these cells into ischemic muscle. Results demonstrate that numbers of CD11b+CCR2+CCR5+ monocytes markedly increase in ischemic muscle by day 4 after the surgery (n=5 each; 0.8±0.4×105 cells/g tissue in non-surgically treated mice vs 9.5±1.7×105 cells/g tissue in the ischemic muscle, p<0.05) then return to basal levels at day 21. Following immunostaining of ischemic muscles, CD11b+CCR2+CCR5+ monocytes localize in perivascular areas of non-surgically treated C57BL/6 mice. At day 4 after the surgery, these cells infiltrate into ischemic muscles. Infiltrated CD11b+CCR2+CCR5+ cells make numerous vessel-like structures 21 days after the surgery. Following direct injection of purified CD11b+CCR2+CCR5+ monocytes from ischemic muscle at day 4, an increase in blood flow was manifest by 28 days in the CD11b+CCR2+CCR5+ monocyte-injected group (n=7 each, 67.6±5.7% in the CD11b+CCR2+CCR5+ monocyte-injected group vs 39.9±6.7% in the control group, p<0.05). Thus, we found that CD11b+CCR2+CCR5+ monocytes infiltrated and formed numerous vessel-like structures in the ischemic muscle, and additional injection of CD11b+CCR2+CCR5+ monocytes increased the blood flow. These findings strongly suggest that CD11b+CCR2+CCR5+ monocytes play an important role in neovascularization.

Disclosures:
No relevant conflicts of interest to declare.
Splenectomy and the Incidence of Venous Thromboembolism and Sepsis In Patients with Immune Thrombocytopenia,
Authors: Wun Ted , Boyle Soames F. , Brunson Ann , White Richard H. 
Published: October 2019

Abstract
Abstract 3284

Introduction:
Splenectomy is efficacious for many patients with chronic immune thrombocytopenia (ITP) refractory to initial corticosteroid therapy. However, there are concerns about short and long term complications, especially venous thromboembolism (VTE) and sepsis. Information regarding the incidence and risk factors for these complications might inform therapeutic decisions.

Methods:
Using the California Office of Statewide Health Planning and Development Patient Discharge Dataset, we identified all the records of Californians discharged with a diagnosis of ITP from 1991–2009. Similarly, all records with a procedure code for splenectomy, or principal or secondary diagnosis of VTE or sepsis were identified. The resultant datasets were then merged with the ITP dataset so that incident splenectomy, VTE, and sepsis could be determined for the ITP cohort.

Results:
A total of 37,780 unique patients were initially identified with an ITP code, of which 13,798 were in the principal position. Records where the first ITP code was on the same date as a splenectomy were then excluded as we wished to study a cohort of ITP cases that warranted admission but may or may not have undergone splenectomy later. This resulted in 10,752 cases that constitute the ITP analysis cohort. This cohort was 60% female, 57% White, 23% Hispanic, 9% Asian, and 8% African-American with a median age of 54 years. One thousand nine hundred fifty-five ITP patients underwent splenectomy subsequent to their first ITP hospitalization; 8,797 did not. The cumulative incidences of VTE were 4.4% and 1.8%, and for sepsis were 7.9% and 6.7%, for patients who did and did not undergo splenectomy, respectively. Cox proportional hazard models for VTE and sepsis are given in the tables.

Discussion:
In this large cohort of cases with ITP, splenectomy was associated with hazard ratios (HR) of 2.6 to 4.8 for VTE and a HR of 8.2 in older patients for sepsis, confirming the results of smaller studies. Age and diagnosis of lupus also increased the risk of VTE. Asians had a lower risk of VTE, consistent with previous findings. The absolute risk of VTE was < 5% and would not seem to warrant long-term primary prophylaxis. Age, lupus, and co-morbidities also increased the risk of sepsis in patients with ITP. The increased risk of sepsis in Asians is unexplained. Of note, the cumulative incidence of sepsis in patients that did not undergo splenectomy was still considerable and may have reflected the use of immunosuppressive therapy.

Disclosures:
Wun: Glycomimetics, Inc.: Research Funding; Eli Lilly, Inc.: Research Funding.
A Novel Missense Mutation Asp69Gly in the Platelet Glycoprotein Ib(beta) Gene in Two Families with Bernard-Soulier Syndrome,
Authors: Hassenpflug Wolf A , Weber Belinda , Obser Tobias , Budde Ulrich , Schneppenheim Reinhard 
Published: October 2019

Abstract
Abstract 3300
Bernard-Soulier Syndrome (BSS) is characterized by macrothrombocytopenia and impaired platelet function due to defects of the GpIb-IX-V complex. Patients with Bernard-Soulier Syndrome are rare and only few disease causing mutations have been identified in the platelet gylcoprotein Ib(alpha), Ib(beta) or glycoprotein IX genes, respectively.
We characterized 2 independent families with Bernard-Soulier Syndrome. Affected members of both families presented with a bleeding history including gross hematoma after minimal trauma, recurrent epistaxis, menorrhagia and prolonged bleeding after surgery. Routine laboratory tests showed reduced platelet numbers and increased platelet volume. Consistent with the diagnosis of Bernard-Soulier syndrome FACS analysis demonstrated reduced GpIb-IX expression on the platelet surface. Platelet agglutination in the presence of ristocetin (RIPA) was impaired.
Using standard techniques we sequenced the GpIb(alpha), GpIb(beta) and GpIX genes. We identified a novel mutation Asp69Gly in the GpIb(beta) gene in both families. The mutation was homozygous in two children of the first family and one child of the second family. As expected, the parents of the affected index patients were heterozygous mutation carriers. To further investigate the impact of the Asp69Gly mutation on GpIb expression we used structure prediction software and homology-based protein modeling. Asp69Gly lies within an extracellular region flanking a leucin-rich element and cysteine-cluster. It affects the highly conserved center aspartic acid of a CDC motif that is thought to form one of two cystein-loops close to the transmembrane domain. We propose that this mutation disrupts the regular conformation of the cystein loop, potentially leading to abnormal protein folding and protein decay in the endoplasmic reticulum.
In addition to a mere defect of biosynthesis our data hint at additional functional consequences of the Asp69Gly mutation. The father and the mother of the index patient in the first family, who are heterzygous for the Asp69Gly mutation, express only slightly reduced levels of platelet GpIb-IX based on FACS analysis. Nevertheless both of them suffer from epistaxis and menorrhagia, respectively. Expression studies are underway to clarify the influence of the Asp69Gly mutation on GpIb expression and function. Preliminary data show reduced cell surface expression of the GPIb-IX complex and reduced binding to von Willebrand-factor (VWF).
While the functional and structural effects of mutations in the GpIb(alpha) and GpIX genes have been thoroughly studied, the impact of mutations in the GpIb(beta) gene is less clear. Our results could help to elucidate how the GpIb(beta) protein contributes to the assembly and surface expression of the GpIb-IX-V complex.

Disclosures:
No relevant conflicts of interest to declare.