bluuebunny/crossref_metadata_2025 huggingface dataset:
Processing of Hemojuvelin Requires Retrograde Trafficking to the Golgi in HepG2 Cells.
Authors: Zhang An-Sheng , Maxson Julia Julia , Enns Caroline A
Published: April 2020

Hemojuvelin (HJV) was recently identified as a critical regulator of iron homeostasis. It is either associated with the cells through a GPI-anchor or released as a soluble form. The cellular form acts as a co-receptor for bone morphogenic proteins (BMPs) and activates the transcription of hepcidin, a hormone that regulates iron efflux from cells. Soluble HJV antagonizes BMP signaling and suppresses hepcidin expression. Secretion of HJV requires binding to the transmembrane receptor neogenin. In this study we examined the trafficking and processing of HJV. Cellular HJV reached the plasma membrane without obtaining complex oligosaccharides, indicating that HJV avoided Golgi processing. Secreted HJV, in contrast, had complex oligosaccharides and could be derived from the pool of HJV at the plasma membrane. Neogenin did not play a role in HJV trafficking to the cell surface but was necessary for secretion of HJV, suggesting that it could be involved in either retrograde trafficking of HJV or in cleavage leading to secretion.
Predictors for the Efficacy of Peripheral Blood Progenitor Cell (PBPC) Collection–Results from 4050 Harvests Performed in a Single Centre
Authors: Hoelig Kristina , Kroschinsky Frank , Kramer Michael , Blechschmidt Matthias , Zimmer Kristin , Oelschlaegel Uta , Rutt Claudia , Bornhaeuser Martin , Ehninger Gerhard
Published: April 2020

Background : Peripheral blood progenitor cells (PBPCs) are routinely applied worldwide for allogeneic related and unrelated stem cell transplantation. Factors influencing the mobilisation of PBPC’s in healthy donors are poorly understood. The database of the Apheresis Centre at the University of Dresden was evaluated to identify predictors of PBPC mobilisation.

Methods : 4050 PBPC collections (3928 first donations, 122 second donations) of healthy unrelated donors between 1/1996 and 1/2008 were carried out according to a standardized protocol and prospectively documented in a database. Peripheral blood CD 34 counts were performed before each leukapheresis. CD 34 yield of every PBPC product was calculated in absolute numbers and per kg body weight of the recipient. All parameters are presented as median and range. Mann-Whitney test was performed for discrete variables. Correlation analysis by Pearson was applied for continuous variables. Multivariate regression analysis was carried out to detect factors independently predicting mobilization efficacy.

Results : The range of peripheral CD 34+ haematopoietic stem cells obtained on day 5 was 8.7–285/μl (median 67.5/μl) in male and 6–282/μl, (median 51/μl) in female donors. The median CD 34 yield from the first leukapheresis was 5.88 ×108. Inadequate CD34-yields (< 2 × 106/kg) were obtained only in 18 donors (0.45%). Peripheral CD 34 count at day 5 is significantly correlated with (positive linear regression): BMI, baseline platelet count, male sex, G-CSF application (split dose), baseline leukocyte count. Peripheral CD 34 count at day 5 correlates negatively with: female sex, single dose G-CSF application, regular alcohol consumption and regular smoking status. Linear regression analyses revealed no significant influence of donor age. Multivariate correlation analysis included sex, BMI, baseline platelets, G-CSF-application, baseline leukocytes, age and smoking. This model explained 21.2 % of the variabilityA strong correlation exists between peripheral CD 34-counts at day 5 and apheresis yield (70% of the variability explained).

Conclusions : A dose of 7.5 μg/kg/d lenograstim proved to be safe and effective in a large cohort of unrelated donors for mobilizing sufficient haematopoietic progenitor cells for allogeneic transplantation. Our analysis of 4050 donations revealed significant, but very weak correlations of the peripheral CD 34-counts with donor characteristics and life style parameters. No single parameter derived from donor baseline investigation reliably predicts CD 34 yield. Further evaluation of healthy donors including comprehensive genomic linkage analysis is necessary to elucidate the variable efficiency of PBPC mobilization
Early Events in the Clonal Evolution in Patients with Chronic Lymphocytic Leukemia Carrying TP53 Mutations and 17p Deletion
Authors: Ghia Emanuela M. , Rassenti Laura , Kipps Thomas J
Published: April 2020

The leukemia cells of patients with chronic lymphocytic leukemia (CLL) may experience clonal evolution of TP53 mutations and/or deletions in the short arm of chromosome 17 (del(17p)). Such changes are associated with poor prognosis and resistance to standard chemotherapy. We evaluated for TP53 mutations and p53 protein expression before and after irradiation in leukemia cells with del(17p) of 14 patients at various times prior to therapy. The proportion of leukemia cells harboring del(17p) ranged from 3% to 99.5%, as assessed by fluorescence in situ hybridization (FISH). Four cases had less than 5% CLL cells with del(17p) at the initial sample date. Ten out of 14 patients had leukemia cells that used unmutated IGHV (71%), and 9 (64%) or 6 (43%) had CLL cells with high-level expression of ZAP-70 or CD38, respectively. The median time from diagnosis to sample collection was 2.6 years and the median follow up was 3.8 years. We found 10 of 14 (71%) had proportions of CLL cells with del(17p) ranging from 3% to 55%, but no detectable TP53 mutations in exons 5 through 8, where cancer-related mutations in TP53 are most common. These cases also had functional TP53, as assessed by leukemia-cell induction of p53 and p53-target genes in response to ionizing radiation. Four cases of 14 (28.6%) had TP53 mutation within one of the exons studied and constitutive p53 protein expression both before and after irradiation. In particular, 2 patients with del(17p) detected in 83.5% and 93% of the CLL cells, had leukemia-cell somatic mutations in exon 7 of TP53 at codon 244 and codon 242, respectively. A third patient with 99.5% of the CLL cells with del(17p) had a mutation in exon 5 codon 174. We identified heterozygosity in exon 5 at codon 179 associated with constitutive expression of dysfunctional p53 protein in a fourth patient in which only 14% of the CLL cells had del(17p). The mutation observed at codon 179 of exon 5 resulted in an amino acid change from histidine to leucine. Collectively, even though located in the DNA-binding domain of p53, none of the mutations identified were common to hot spot p53 mutations identified in other types of cancer. However similar mutations (histidine to arginine or histidine to tyrosine) at codon 179 were observed in 3 CLL cases recently published (Zenz T. et al. prepublished Blood 2008) on samples collected at 0, 5, 9 months from diagnosis, respectively. This study reveals early events in the clonal evolution of CLL leading to loss of p53 function, which can be found even in cases that have less than 20% leukemia cells harboring del(17p), as assessed by FISH. It also suggests that mutations at codon 179 might be a common hot spot in CLL, which potentially could provide for another means with which to evaluate for cases at risk for developing drug-resistant disease either spontaneously or following standard chemotherapy.
The CD4+CD25highregulatory T Cell Reconstitution after Allogenetic Stem Cell Transplantation and Its Correlations with aGVHD
Authors: Li Caixia , Depei Wu , Sun Dao ping , Liu Yuejun , Chang Weirong , Zhu Ming qing , Cen Jiannong , Qiu Qiaocheng
Published: April 2020

Background CD4+CD25+ regulatory T cell (Treg), the most significant subset of regulatory T cells, has been found to play an important role in suppressing allogenetic immune response and controlling GVHD recently. The present single-center study monitored the ratio of CD4+CD25high T cells and the expression of FOXP3 gene in peripheral blood post-transplant to assess the reconstitution of CD4+CD25high Treg early after allogenetic stem cell transplantation and its correlations with aGVHD.

Methods: 22 patients undergoing allo-HSCT were enrolled. To detect the CD4+CD25+T cells and the CD4+CD25high T cells subpopulations ratio in CD4+ T lymphocytes(CD4+CD25+/CD4+ ACD4+CD25high/CD4+) Athe CD4+CD25high T cells subpopulations ratio in CD4+CD25+ T cells(CD4+CD25high/CD4+ CD25+) Aand the expression of FOXP3 mRNA in the donor graft and periperal blood frequently after transplantation with the flow cytometry and real-time PCR assays.

Results: \|[Dagger]\|@CD4 +CD25high/CD4+ and CD4+CD25high/CD4+CD25+ ratios in the donor graft were significantly lower in the aGVHD group(P<0.05)‡AEarly after transplantation, Both groups’CD4+CD25+/CD4+ ratio in peripheral blood increased significantly than that in the donor graft(P<0.05).CD4+CD25high/CD4+ and CD4+CD25high/CD4+CD25+ ratios were also significantly higher than that in donor graft in the aGVHD group, but not in the no-aGVHD group.‡BThe expression of FOXP3 in two groups after transplantation increased gradually, until six months after transplantation, its expression was still below the control level(P<0.05). The expression of FOXP3 was lower in the aGVHD group.

Conclusion: \|[Dagger]\|@The early activation and proliferation of the CD4+CD25high regulatory T cells is critical for the maintainance of immune hemostasis and the control of aGVHD.‡AThe occurrence of aGVHD is related with lower ratios of CD4+CD25high regulatory T cells in CD4+T cells and activated CD4+ effector T cells.‡BaGVHD may affect the quantities of CD4+CD25high regulatory T cells and the expression of FOXP3.
Highlighting TH-1 Rather Than TH-17 Cytokine Network in Acute Cutaneous Gvhd
Authors: Keerthivasan Shilpa , Xin Hong , Qin Jian-Zhong , Miele Lucio , Stiff Patrick , Nickoloff Brian J
Published: April 2020

Graft versus Host disease (GVHD) is a major lethal complication of allogeneic hematopoietic stem cell transplantation (HSCT). As skin is one of the major organs involved and the most accessible, it serves as an ideal organ site to further understand the pathogenesis and progression of acute GVHD and could serve as a window into pathophysiology of other organ sites. Cytokine network aberrations are a central feature of the pathogenesis of acute cutaneous GVHD, terminating in the hallmark of premature apoptosis of keratinocytes. The purpose of this study is to delineate the cytokine network aberrations at the molecular and cellular level in affected skin from patients with acute GVHD with the goal of developing novel therapeutic approaches to defined targets with the goal of preventing or reducing acute GVHD. We performed immuno-phenotypic profiling of acute cutaneous GVHD biopsies determining relative numbers of various types of immunocytes including T cells, dendritic cells, and macrophages. Cytokine expression (IFNγ and TNFα) was determined by RT-PCR on RNA extracted from eight different lesional skin and two non-lesional biopsies. We analyzed for TH-1 (IFN γ and TNFα), TH-2 (IL-4) and TH-17 (IL-17A, IL-22) cytokine mRNAs. We observed that TH-1 cytokine mRNAs were detected in 7 out of 8 different lesional skin biopsies but none of two nonlesional skin biopsies. Interestingly TH-2 and TH-17 cytokine mRNAs were not detectable in acute cutaneous GVHD lesions or nonlesional skin biopsies. RNA extracted from a 2-way mixed lymphocyte reaction (MLR) served as a positive control for these RT-PCR results. Given recent data that suggested that Notch-1 regulated TH-1 polarization by transcriptionally controlling the Tbx gene (MinterLM et al 2005), we hypothesized that inhibiting Notch signaling might be of potential therapeutic importance given these results of TH-1 polarization in acute skin GVHD. To block Notch signaling we utilized several pharmacological gamma secretase inhibitors (which may have therapeutic applications), and supplemented this pan-Notch receptor inhibition approach by use of a Notch-1 specific siRNA. Preliminary studies suggest that both approaches reduce IFN γ levels in allogeneic T cell responses in-vitro. Further experiments are in progress to confirm these findings and to determine the molecular mechanism. This study begins to define the cytokine network operative within acute cutaneous GVHD lesions highlighting the finding that TH-1 rather than TH-17 cytokines may be the more important target.
Serum Levels of Soluble Annexin II in Patients with APL
Authors: Kawasugi Kazuo
Published: April 2020

Acute promyelocytic leukaemia (APL, M3) is associated with both a characteristic t(15;17) and severe bleeding diathesis caused by disseminated intravascular coagulation (DIC) and/or hyperfibrinolysis. It has been suggested that annexin II, a coreceptor for tissue plasminogen activator (t-PA) and plasminogen (PLG), is overexpressed on the surface of promyelocytes, leading to an increased fibrinolytic potential. However, little is known about the serum levels of annexin II in patients with APL. Therefore, we measured concentrations of annexin II in sera from patients with APL (n=10). The serum annexin II levels were determined by following methods (sandwich ELISA).

The EIA plates coated for 48h at 4· with 2ug human annexin II antibodies (monoclonal) in carbonate buffer pH 9.4 respectively. The plates were then washed once with PBS and were done for non-specific blocking with 200ul/well PBS containing 1% BSA (PBSA) for 2h at room temperature (RT). After blocking, the plates were washed once with PBS. Subsequently, 100ul of samples diluted by ten times were added to the wells. After 2h incubation at RT, the plates were washed twice with PBS containing 0.05% Tween20 (PBST) and human annexin II rabbit antibodies (polyclonal) diluted 1:250 with PBSA were added to the wells. After 2h incubation at RT, the plates were washed twice with PBST and then incubated for one hour at RT with 100ul/well HRP-conjugated goat anti-rabbit Ig diluted 1:250 in PBSA. The plates were washed twice with PBST and reaction was developed by adding 100ul/well of substrate solution (0.04% O-phenylenediamine in phosphate/citrate buffer pH 5, with 0.04% H2O2). The reaction was stopped after 10 min by adding 50ul/well 2.5M H2SO4, the absorbance was read at 492nm using a spectrophotometer In the patients with APL, we found significant elevations in serum annexin II levels as compared with normal controls.

In other words, serum levels of annexin II were 0.109 (mean OD values, n=10) from patients with APL.

On the other hand, concentrations of annexin II in sera from normal controls were 0.092 (mean OD values, n=10). There was correlation between the FDP and annexin II levels in the APL patients with DIC The present work demonstrates significant elevations in serum annexin II levels in patients with APL. Therefore, we suggest that elevation of annexin II may be associated with the development of an increased fibrinolytic potential.
Autoimmune Thrombocytopenic Purpura in a Patient with Renal Clear Cell Carcinoma
Authors: Xu Li , Ying Shaoxu , Li Xiao
Published: April 2020

A case of renal clear cell carcinoma associated with autoimmune thrombocytopenic purpura is reported. A 56-year-old man presented as thrombocytopenia (10,000/microl) was diagnosed as idiopathic thrombocytopenic purpura after certain tests. He was started on large dose of steroid, intravenous gamma globulin, TPO and multiple platelet transfusion. Platelet count was not stable. Then computerized tomography revealed a left renal tumor 46×25×31mm3. Based on the diagnosis of left renal neoplasm, left nephrectomy with preoperative high-dose intravenous gamma globulin and platelet transfusion was performed. Pathological examination revealed clear cell carcinoma. After the operation, the platelet count increased gradually. But the tumor metastasized 4 months later. Therefore, we consider that extensive tests shall be run to exclude cancer before confirming diagnosis of ITP, the elder patients, or the patients with poor therapeutic efficacy or cortisone dependent. We do not recommend using Cs as the treatment for Cancer-related ITP-like Syndrome. The tumor should be controlled first as soon as possible. If the cancer is localized, IVIG and platelet transfusion can be an appropriate initial treatment for upgrading platelet counts quickly to win time for surgery.
Characterization and Prevalence of Anemia in Subjects with Spinal Cord Injury
Authors: Santos Filipe Nadir Caparica , Pithon Karla ROCHA , Cliquet Marcelo G , Cliquet Alberto
Published: April 2020

INTRODUCTION: Anemia has been considered to be very prevalent in chronic diseases, commonly due to blood loss or inflammatory reasons; as so, it has been considered common sense to imply that anemia is also frequent among subjects with spinal cord injury. Likewise, malnutrition, reenforced by the common association between depression and spinal cord injury, justify the study of the prevalence of anemia in this population.

OBJECTIVE: To evaluate the prevalence of anemia among subjects with spinal cord injury, and its relation with the injury level and also if there is any difference when comparing patients from the Spinal Cord Injury Rehabilitation Ambulatory of UNICAMP (SCIRAU) and outpatients that weren’t previously followed by us, considered as controls; if anemia is found to be present, we intend to evaluate its etiology.

METHODOLOGY: Patients with spinal cord injury from SCIRAU and controls were randomly selected to be enrolled in this study, being 77,3% male (n=17) and 22,7% female (n=5). These patients were evaluated as of their level of injury (separated into paraplegic vs. tetraplegic) and age, being invited to offer a blood sample for analysis of hematological parameters; hemoglobin (Hb) and hematocrit were evaluated. To be considered as being anemic, the cutoff point was 12,0 g% for females and 13,0 g% for males.

RESULTS: The male group had a median age of 31.9 years, significantly lower then the female group, with a median of 46.4 years [p=0.02]. The median value of hemoglobin in the male group (M=14.72, SD=1.23) was statistically higher than the female group (M=12.78, SD=0.17) [p=0.00001]. Comparing the results of Hb between the paraplegic (M=14.48, SD=1.67, N=10) and tetraplegic (M=14.125, SD=1.10, N=12) patients, there were no statistically significant difference [p=0.56]. Such difference also was not confirmed when comparing SCIRAU patients (M=14.44, SD=1.43, N=18) and outpatients from the control group (M=13.58, SD=0.77, N=4) [p=0.16].

CONCLUSION: Surprisingly, none of the patients involved in this study had anemia. The difference between the hematological parameters when comparing male and female subjects was already expected and should not be attributed to the age difference. No significant relation between the level of injury and hemoglobin level were observed. As such, no difference could be seen among patients followed in our SCIRAU and control outpatients. Hence, even though spinal cord injury is considered to be a chronic disease and scientific literature suggest that these patients are at risk for malnutrition and depression (both associated with a higher prevalence of anemia), the hypothesis that these subjects present with a higher prevalence of anemia could not be seen in our study.
Donor Versus No Donor Analysis of Newly Diagnosed Myeloma Patients Included in the HOVON 50/54 Study
Authors: Lokhorst Henk , Sonneveld Pieter , van der Holt Bronno , van Oers Marinus , Raymakers Reinier , Zweegman Sonja , Minnema Monique C. , Zijlmans Mark
Published: April 2020

The value of allo-SCT in myeloma is disputed. Prospective randomized trials are the gold standard for the evaluation of treatment efficacy; however such a design is difficult to perform in an allo-setting. As the availability of a matched sibling donor is a random process, the presence or absence of a donor can be used as a surrogate for randomization. Patients with an HLA–identical sibling donor, included in the phase-III HOVON50 study, that was designed to assess the effect of thalidomide in induction treatment and as maintenance after high-dose therapy (HDM 200 mg/m2) and auto-SCT could proceed to the Hovon 54 study in which an Allo-SCT after conditioning with low dose TBI only was performed between 2–6 months after HDM. In the first survey performed in a subset of patients that had received at least 1 course of HDM and for whom tissue typing data were already available, 124 could be classified as having a sibling donor and 124 as having no sibling donor. Tissue typing data of another 81 patients are still waited for. Both groups were comparable with regard to age, myeloma stage, and prognostic factors including cytogenetics and ISS stage. 94% of the patients in the no donor group achieved at least a PR (35% CR, 32% VGPR and 26% PR), versus 96% of the patients in the donor group (40% CR, 34% VGPR and 22% PR). After a median follow-up of 38 months after HDM, PFS and OS were also comparable between the two groups. Median PFS was 32 versus 28 months for the donor group and no donor group respectively (P=0.44), while median OS was 61 and 54 months (P=0.14). Among the 98 patients in the donor group who received an allo-SCT non relapse mortality was 10% at 1 year and 13% at 3 years after allo-SCT. This first preliminary analysis showed no improvement of tandem Auto Allo-RIC as part of first line therapy in myeloma as compared Auto-SCT followed by maintenance therapy. A longer follow-up may be needed for definite conclusions. An update of the results including those patients with yet unknown tissue typing status and longer follow up will be presented
Lentivirus-Mediated RNA Interference Targeting Vascular Endothelial Growth Factor Gene Enhances the Sensitivity of K562 Cells to Imatinib
Authors: Li Li , Zhang Ri , Cen Jiannong , Zhu Ziling
Published: April 2020

Background: Vascular endothelial growth factor (VEGF), one of the most potent mediators of angiogenesis, plays an important role in tumor cell proliferation and migration. Elevated VEGF levels are found in both the plasma and the bone marrow of patients with chronic myelogeneous leukemia(CML). Moreover, the CML-associated oncogene Bcr-Abl induces endogenous secretion of VEGF. The aim of this study was to construct a lentiviral vector-mediated RNA interference (RNAi) of vascular endothelial growth factor (VEGF) gene and to investigate the effects of RNAi inhibiting VEGF gene alone or in combination with imatinib on the proliferation and apoptosis of K562 leukemic cell line.

Methods: A lentiviral expression vector containing short hairpin RNA (shRNA ) targeting VEGF was constructed and cotransfected with the pPACKH1 packaging plasmids mixture into 293T cells by Lipofectamine 2000. K562 cells were infected with the packaged lentivirus. The levels of VEGF mRNA and protein were detected by real-time quantitative RT- PCR, Western blot and ELISA. Cellular proliferation was determined by trypan blue exclusion and MTT assay. imatinib -induced apoptosis was analyzed by flow cytometry.

Results: The lentiviral shRNA vector targeting VEGF has been constructed and transfected into K562 cells successfully. The expression of VEGF mRNA and protein in K562-shVEGF cells transfected with pRNAT-shRNA were significantly inhibited when compared with those of K562 and K562–con cells (mock transfection). The proliferation rate of K562-shVEGF cells slowed down. After imatinib treatment, the percentages of apoptotic cells in K562-shVEGF cells increased more significantly than those of K562 and K562–con cells.

Conclusion: Suppression of VEGF by lentivirus-mediated RNAi could effectively inhibit proliferation and increase the sensitivity of K562 cells to imatinib. These results indicate that Bcr/Abl-targeting compound in combination with anti-angiogenic therapies may provide a potent therapeutic strategy to CML.
Small-Molecule Screen Identifies ROS as Key Regulators of Actin Dynamics in Neutrophils
Authors: Hattori Hidenori , Subramanian Kulandayan K , Luo Hongbo R.
Published: April 2020

Precise spatial and temporal control of actin polymerization and depolymerization is essential for mediating various cellular processes such as migration, phagocytosis, vesicle trafficking and adhesion. In this study, we used a small-molecule functional screening approach to identify novel regulators of actin dynamics during neutrophil migration. Here we show that NADPH-oxidase dependent Reactive Oxygen Species act as negative regulators of actin polymerization. Neutrophils with pharmacologically inhibited oxidase or isolated from Chronic Granulomatous Disease (CGD) patient and mice displayed enhanced F-actin polymerization, multiple pseudopods formation and impaired chemotaxis. ROS localized to pseudopodia and inhibited actin polymerization by driving actin glutathionylation at the leading edge of migrating cells. Consistent with these in vitro results, adoptively transferred CGD murine neutrophils also showed impaired in vivo recruitment to sites of inflammation. Together, these results present a novel physiological role for ROS in regulation of action polymerization and shed new light on the pathogenesis of CGD.
Clinical Characteristics in Neonates with Alloimmune Neutropenia: Significance of the Detection of Antineutrophil Antibodies
Authors: Mizoguchi Yoko , Nakamura Kazuhiro , Karakawa Shuhei , Sato Takashi , Onodera Rie , Kurita Emi , Hiraoka Asako , Taniguchi Kikuyo , Kobayashi Masao
Published: April 2020

Neonate alloimmune neutropenia (NAIN) is caused by the transplacental transfer of maternal alloantibodies directed against antigens on the infant’s neutrophils. To date, there are scant studies about its clinical characteristics and characterization of antineutrophil antibodies though some case reports are found. In this study we analyzed 11 cases with NAIN from January 2005 to December 2007. The diagnosis of NAIN was confirmed by the transient neutropenia less than 500/μl of absolute neutrophil count (ANC), the detection of maternal antineutrophil antibody, the incompatibility of neutrophil antigens between parents, and their mothers without autoimmune diseases. Antineutrophil antibodies were detected by granulocyte indirect immunofluorescence test using flow cytometry. To quantify the strength of the antibodies, the ratio of the mean fluorescence channel of each sample to that of control serum was expressed as relative fluorescence intensity according to the method reported previously (Blood99: 3468, 2002). The median age at diagnosis in NAIN patients was 8 days after birth ranged from 0 to 30 days. The average of ANC at the presentation was 170/μl ranged from 0 to 500/μl. All antineutrophil antibodies detected in sera of both neonates and their mothers were against HNA-1 antigens. The alloantibody against HNA-1a was found in 2 cases, that against HNA-1b was in 6 cases, and that against FcγR IIIb was found in 3 cases. The fact that the frequencies of homozygote of HNA-1a and HNA-1b in Japanese population were approximately 50% and 12%, respectively may reflect the frequency of alloantibody specificity in NAIN. During the neutropenic period, 7 cases with NAIN showed mild to moderate infections associated with neutropenia, such as pyrexia and pyodermia. In contrast, 4 of 11 cases with NAIN did not have any infectious episodes in their clinical course. In all patients, the spontaneous recovery of neutropenia with the disappearance of alloantibody was observed within 6 months (median 85 days ranged from 3 weeks to 6 months). The duration until spontaneous resolution of neutropenia was dependent on the strength of alloantibody found in sera of the mothers and neonates. Two patients with significantly high strength of alloantibodies had the relatively long duration to restore the neutropenia(4 months and 6 months). In conclusion, the specificity of antineutrophil antibodies in patients with alloimmune neutropenia is dependent on the frequencies of neutrophil antigens in Japanese population. The quantification of alloantibodies in neonates and their mothers may be useful in considering the clinical course of neutropenia in neonates.
Folate Receptor Saturation, Recycling, and Release Kinetics in Activated Macrophages
Authors: Varghese Bindu , Vlashi Erina , Low Philip S.
Published: April 2020

Activated macrophages over-express a receptor for the vitamin, folic acid. Conjugation of folic acid to low molecular weight drugs, genes, liposomes, nanoparticles, and imaging agents does not significantly compromise the vitamin’s affinity for its receptor, thus facilitating both therapeutic and imaging agents to be targeted as folate conjugates to activated macrophages. In these studies the in vivo kinetics and binding characteristics of the folate receptor on macrophages has been characterized with the objective of optimizing targeted drug delivery. Our results show that saturation of the receptor in inflamed tissues can be reached at a dose of 100 nmol/kg. Furthermore, the rapid recycling characteristics of the receptor (every 10–20 minutes) suggests that frequent dosing will allow for maximal uptake of folate conjugates by activated macrophages accumulated in inflamed tissues. Finally, the short circulation half-life (30 minutes) of the water-soluble folate conjugate used in this study minimizes the possibility of nonspecific uptake by receptor-negative tissues. The kinetic observations made in this study can be utilized to optimize the therapeutic efficacy and imaging agent delivery of folate conjugates to activated macrophages in vivo.
Single Use of Rasburicase (rasb) Leads to Insufficient Tumor Lysis Syndrome (TLS) Control in Adults: Outcome and Resource Utilization in a One Year Real-Life Observational Study
Authors: Krych Matthaeus , Ostermann Julia , Hiddemann Wolfgang , Ostermann Helmut
Published: April 2020

TLS due to the rapid lysis of tumor cells following initiation of chemotherapy can lead to the release of intracellular components into the extracellular space. Among these hyperuricemia might exceed the excretory capacity of the kidneys and lead to renal failure. Rasb is a recombinant urate oxidase given during the first 3–5 days of chemotherapy to effectively lower uric acid serum levels. Limited data suggest that a single prechemotherapy dose of rasb might be sufficiently efficacious and this single dose regimen is widely used to reduce treatment costs.

We analyzed the clinical and economical data of all adults who received rasb for prophylaxis or treatment of TLS at a university hospital during a one year period. The goal was to describe treatment patterns and outcome outside of clinical trials and to verify whether patients with TLS can be treated cost-effectively. 38 patients (pts) received rasb, however 1/3 of the pts were treated off-label for other reasons then TLS. 24 pts (median age 66 yrs, 21 male) received rasb for malignancy-associated hyperuricemia and were eligible for further clinical and economical evaluation. 2/3 of these pts were treated for hematological malignancies (acute leukemia 6pts, lymphoma 8 pts) and 8 pts for solid tumors. Apart from hydration (mean volume 2 l per day; all pts), furosemide (mean dose 80 mg/day; all pts) and urinary alkalinization with sodium bicarbonate (pH > 7; 7 pts), 20 patients received a single 7,5 mg dose of rasb and 4 pts received multi dose. In 14 pts a tumor lysis was observed. 6 pts died, three fatalities were directly associated with TLS. Mortality was higher among pts with single dose rasb vs. multi dose rasb (30% vs. 0 %) and higher among patients with urinary alkalinization (71% vs. 5%). No anaphylaxis, hemolysis or methemoglobinemia was observed in this cohort.

The mean duration of hospitalisation was 23 days, the mean treatment costs were 16.200 €. The costs exceeded the revenues based on diagnosis related groups for Germany (G-DRG) in 18 pts and resulted in a 7% funding gap. 107.000 € were spent for drugs for these patients, 10% of these for rasb.

We conclude from this observational study that

TLS is a menacing disease, resulted in a 12,5 % mortality and should therefore be cautiously monitored and treated, single dose rasb was associated with a higher mortality rate then multi dose, urinary alkalinization cannot be recommended when rasb is used, TLS leads to a 7 % underfunding in the respective G-DRGs, for TLS pts. the usage of rasb elevates the total drug expenses by 10%.
Differential Regulations and Roles of Mcl-1 and Bcl-Xl during Megakaryopoiesis
Authors: Oh Doyeun , Yoon Seongmin , Park Seonyang , Bae Jeehyeon
Published: April 2020

Backgrounds: Platelet plays an essential role in thrombosis and hemostasis and is produced from hematopoietic stem cells through a serious of differentiation and maturation processes called megakaryopoiesis. The major factor known to control platelet formation is thrombopoietin (TPO), but recently more proteins including apoptosis regulators have been reported to involve in megakaryopoiesis. Evolutionally conserved Bcl-2 family proteins are central regulators of apoptosis. Antiapoptotic Bcl-2 subfamily comprised of Bcl-xL, Mcl-1, Bcl-2, Bcl-w, and Bfl-1 plays a pivotal role in controlling cell death and survival under various conditions. According a recent study, Bcl-xL is a key molecular clock that determines the life span of platelets, but the role and regulation of Bcl-2 members in megakaryopoiesis are largely unknown.

Methods and Results: We have established an in vitro system of megakaryopoiesis and performed the profiling of Bcl-2 family genes during megakaryocytosis. We found that Bcl-xL and Mcl-1 were predominant molecules among other members of the pro-survival proteins as determined by quantitative RT-PCR. TPO differentially regulates Bcl-xL and Mcl-1 in Meg-01 cells, in which Bcl-xL protein was significantly up-regulated while the level of Mcl-1 was attenuated. Furthermore, the roles of Bcl-xL and Mcl-1 during megakaryopoiesis were determined by modulating the expression levels of two proteins. Overexpression of Mcl-1 prominently enhanced the viability of the cells, whereas the knockdown of Mcl-1 promoted apoptosis of the cells. In contrast, forced expression of Bcl-xL did not affect the cell survival but rather significantly stimulated differentiation of megakarycytes.

Conclusion: Bcl-xL and Mcl-1 are likely essential molecules during megakaryopoiesis. Moreover, the present study implies that although both Bcl-xL and Mcl-1 are members of antiapoptotic Bcl-2 family that promote the survival of different cells, these two proteins have distinctive and non-redundant functions in megakaryocytes.
Mobilization of Hematopoietic Progenitors Following Myocardial Ischemic Injury Underpins the Physiological and Therapeutic Benefits of G-CSF on Myocardial Repair
Authors: Curtis Davi D J. , Kanellakis Peter , Bobik Alex
Published: April 2020

Despite promising pre-clinical results, administration of granulocyte-colony stimulating factor (G-CSF) for improving heart function in humans after acute myocardial infarction has been disappointing. Therefore, a better understanding of the mechanism(s) of heart repair by G-CSF is urgently required. To address the potential mechanism of G-CSF, we have generated chimeric mice using G-CSF receptor knockout mice to distinguish the hematopoietic benefits of G-CSF from the direct effects of G-CSF on cardiomyocytes and blood vessels. Lethally irradiated wild-type mice were reconstituted with G-CSFR −/− bone marrow (BM) cells to generate G-CSFR BM −/− chimeras. Alternatively, lethally irradiated G-CSFR −/− mice were reconstituted with wild-type BM cells to generate G-CSFR cardiac −/− chimeras. Myocardial ischemic injury was induced by transient (60 minutes) occlusion of the left coronary artery. Mice were treated daily with saline or G-CSF for 5 days beginning 4 hours after ischemic injury. Mice were sacrificed 28 days post-injury for assessment of cardiac function and cellular repair. As previously reported in wild-type mice, G-CSFR −/− cardiac chimeras had 30–50% improved cardiac function compared with saline-treated G-CSFR −/− cardiac chimeras (ANOVA; P<0.05). This suggests that the benefits of G-CSF are NOT due to direct effects on cardiomyocytes or blood vessels, as previously proposed by Harada et al. (Nature Medicine2005; 11: 305–311). In contrast, G-CSF BM −/− chimeras had dramatically impaired cardiac repair following ischaemic injury. The most striking finding was that saline-treated G-CSFR BM −/− mice had 20% worse cardiac function and cellular repair than saline-treated control G-CSFR BM +/+ mice (ANOVA; P<0.05). Furthermore, G-CSF treatment in G-CSFR BM −/− mice produced no significant benefit on cardiac function or cellular repair. The lack of benefit of G-CSF in these mice was due to an inability of G-CSF to mobilise hematopoietic progenitors because treatment of G-CSFR BM −/− mice with the CXCR4 antagonist, AMD3100 improved cardiac function and cellular repair as well as G-CSF in control G-CSFR BM +/+ mice. Together, these results suggest that mobilization of hematopoietic progenitors is a critical component of not only the therapeutic but also physiologic effects of G-CSF for repair after acute myocardial ischemic injury.
Multiple Site Gfi1b Self-Regulation and GATA1/SCL Pentameric Complex: Shifting the Equilibrium towards Repression
Authors: Hernández Aurora , Villegas Ana , Iborra Francisco , Wood William G. , Anguita Eduardo
Published: April 2020

Transcription factor Gfi1b is required for erythroid and megakaryocytic differentiation. Gfi1b is over-expressed in patients and cell lines of erythroid and megakaryocytic leukemias. This indicates that this gene must be tightly regulated to achieve normal erythropoiesis and megakaryopoiesis. The mechanism of it is largely unknown. We have searched for conserved noncoding sequences from multiple evolutionary diverse species (multispecies conserved sequences, MCSs) between Gfi1b neighboring genes and localized several elements containing multiple highly conserved erythroid specific transcription factor binding sites. These MCSs localize with erythroid specific DNasa1 hypersensitive sites. Chromatin immunoprecipitation (ChIP) assays demonstrated GATA1/SCL/E2A/Ldb1/LMO2 pentameric complex and NF-E2, together with Gfi1b, binding in human and mouse cell lines and primary cells representing different stages of erythropoiesis and megakaryopoiesis. Analysis of histone modifications and cofactors binding in relation with Gfi1b expression and protein level gives clues about how activating and repressive complexes attach to Gfi1b promoter and potential long distance regulatory elements maintaining an equilibrium that changes with gene downregulation.
Absence of Association Between TNF α Polymorphysm and Cerebral Large Vessel Abnormalities in Adults with Sickle Cell Anemia
Authors: Vicari Perla , Silva Gisele Sampaio , Noguti Maria A E , Neto Faustino Moreira , Santos Normelia Jesus , Massaro Ayrton Roberto , Figueiredo Maria Stella
Published: April 2020

Purpose: Stroke is a common and serious complication of sickle cell disease (SCD) affecting children as well as adults. Recent reports suggested that promoter region polymorphism in the tumour necrosis factor alpha (TNF-α) gene at position −308 is an important risk factor for large vessel stroke in children with SCD. The role of TNF-α polymorphism in the frequency of magnetic resonance angiography (MRA) abnormalities in adults with SCD is still uncertain. Our objective was to evaluate TNF-α polymorphism in adults with SCD from a tertiary University-based hospital in Sao Paulo, Brazil and correlate them to brain magnetic resonance imaging (MRI) and MRA findings.

Casuistic and Methods: The determination of the G-308A polymorphism of the TNF-α gene was performed in forty-nine adult patients with SCD followed in our outpatient clinic. All subjects were evaluated with brain MRI and MRA to determine the presence of previous stroke, arterial tortuosity and intracranial stenosis

Results: Thirty-three patients (67.3%) had abnormal brain MRA scans (intracranial stenosis or arterial tortuosity). Eight patients (16.3%) had intracranial stenosis on MRA and 29 (59.2%) showed arterial tortuosity. Forty one patients (83.7%) had the GG TNF-α (−308) genotype and eight had the GA genotype. There were no cases of AA genotype. There was no correlation between homozygosis for the TNF-α (−308) G allele and MRA abnormalities.

Conclusion: Although TNF-α (−308) polymorphism has been considered a potential predictor of genetic risk for stroke in children with SCA, we found no association between the polymorphism and large vessel abnormalities in adults with sickle cell disease. (FAPESP: 04/04498-4)
HIF-2alpha Associated Familial Erythrocytosis Supports the PHD2-HIF- 2alpha-VHL Axis as the Major Regulator of Erythropoietin Production
Authors: Percy Melanie , Furlow Paul W , Beer Philip A , Campbell Gavin , Dekker Adriaan W. , Green Anthony R. , Li Xiping , Sutherland Scott , Oscier David , Rainey M. Glenn , Rivera Enid , van Wijk Richard , Wood Marion , Lappin Terence R.J. , McMullin Mary Frances , Lee Frank S
Published: April 2020

Transcriptionally erythropoietin (Epo) synthesis is tightly regulated by the hypoxia inducible factor (HIF), which is composed of one alpha and one beta subunit that are constitutively expressed. The beta subunit is non-variable, but three different alpha subunits give rise to three isoforms of HIF. The alpha subunit is proteasomally regulated in the presence of oxygen by hydroxylation of the proline in the LXXLAP motif of the oxygen dependent degradation (ODD) domain of HIFalpha, catalysed by members of the prolyl hydroxylase domain (PHD) family of enzymes. This allows the von Hippel Lindau (VHL) protein to associate with the alpha subunit, which is subsequently tagged with ubiquitin and degraded by the proteasome. Any defect in the oxygen sensing pathway that allows the alpha subunit to escape proteasomal regulation leads to elevated expression of HIF target genes.

Recently mutations in both VHL and PHD2 have been identified in a cohort of patients with erythrocytosis, but no mutations were found in the ODD domain of HIF1alpha. Instead, investigation of the homologous region in HIF-2alpha revealed four different mutations, Pro534Leu, Met535Val, Gly537Arg and Gly537Trp in seven individuals/families. Affected individuals presented at a young age with elevated serum Epo. Several individuals have a clinical history of thrombosis, but no evidence of a von Hippel Lindau-like syndrome.

To define how the four mutations relate to the erythrocytosis phenotype functional assays were performed in vitro. Binding of PHD2 to the four HIF-2alpha mutants was impaired to varying degrees, with both the Gly537 mutants showing the greatest reduction. The association of VHL with the hydroxylated Met535Val mutant peptide was similar to wild type HIF- 2alpha, but was decreased in the other three HIF-2alpha mutants. Expression of three HIF- 2alpha target genes, adrenomedullin, NDRG1 and VEGF, was significantly up-regulated in cells stably transfected with the mutants under normoxia compared to wild type HIF-2alpha. Mutations in the ODD domain of HIF-2alpha disrupt proteasomal regulation by reducing the association with PHD2 and hence hydroxylation. Furthermore the binding of VHL is also impaired, even when HIF-2alpha is hydroxylated. Examination of the three-dimensional structure of hydroxylated HIF-1alpha bound to VHL confirms that amino acids close to site of hydroxylation (Pro-531 in isoform 2) are important for this association. These observations, together with recent studies utilising murine models of erythrocytosis, support the PHD2-HIF-2alpha-VHL axis as the major regulator of erythropoietin.
Smoking and Hodgkin Lymphoma: A Meta-Analysis
Authors: Pascual Sheila , Milani Cannon , Mitri Joanna , Castillo Jorge
Published: April 2020

INTRODUCTION: The etiology of Hodgkin lymphoma (HL) is largely unknown. However, certain associations have been noted, such as familial factors and infection with viruses. Smoking has been associated with the development of multiple malignancies and some studies have reported an association between HL and smoking but the relationship of tobacco use with lymphomas in largely unclear.

OBJECTIVE: To investigate the potential relationship between tobacco use and the development of HL using a meta-analysis methodology of retrospective, case-control studies.

METHODS: An extensive search was conducted using Pubmed/MEDLINE through July 2008. Case-control studies that reported odds ratio (OR) and 95% confidence intervals (CI) or allow for those values to be calculated were included in our analysis. Case reports, editorials, letters to the editor, review articles and prospective studies were excluded. The smoking status was then subdivided in three groups: never smokers, former smokers and current smokers. Meta-analyses were performed comparing the risks of former and current smokers against the risk of never smokers of developing HL. Fixed and random effects models were used to assess for heterogeneity.

RESULTS: Seven case-controls studies, accounting for 3201 cases and 15268 controls were included in the analysis. Most of the articles reported OR adjusted for age, sex and educational level. In former smokers, the OR was 0.73 (95% CI, 0.65 – 0.82) when compared to never smokers; no heterogeneity was detected. The current smoker group had an OR of 1.70 (95% CI, 1.36 – 2.13) when compared to the never smoker group; some heterogeneity was detected in this group (p < 0.003, I2 = 69.6%).

CONCLUSIONS: Despite the heterogeneity observed in the analysis, the current smoker group seems to have a 70% increased risk of developing HL. Although a cause-effect linkage between tobacco use and HL is difficult to prove, further basic and translational research is necessary to clarify the potential etiological role of smoking in HL.
FLT3 Internal Tandem Duplication Is Associated with High Relapse Rate and Central Nervous System Involvement in Acute Promyelocytic Leukemia
Authors: Tashiro Haruko , Oka Yoko , Sugao Toshihiko , Shirasaki Ryosuke , Noguchi-Mizutani Mitsuho , Akiyama Nobu , Kawasugi Kazuo , Shirafuji Naoki
Published: April 2020

The internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3/ITD) is now accounted as one of important risk factors for the prognosis in acute myelogenous leukemia (AML) patients with normal karyotype. In all-trans retinoic acid era, acute promyelocytic leukemia (APL) has become the most curative subtype of adult AML, and usually hematopoietic stem cell transplantation is not selected for APL patients in 1st complete remission (CR) or 2nd CR without minimal residual disease (MRD) at the polymerase chain-reaction level; however, relapses are still reported to occur in 15–25% of cases after achieving 1st CR without MRD, and some risk factors are proposed including the difference of breakpoint region of the translocation, and leukocyte count at the onset. The incidence of FLT3/ITD in APL has been reported 17–35%; however, it is still a matter of debate that the presence of FLT3/ITD is associated with poor prognosis in APL patients. Thus, we focused on the incidence, clinical features, and prognostic implications of FLT3/ITD in adult newly diagnosed 17 APL patients (median age of 45 years, range 22–67 years, 11 males and 6 females, and median total leukocyte count (TLC) of 2.0 × 109/L, range 0.4– 156.1 × 109/L). All patients achieved CR. The incidence of the presence of FLT3/ITD was 29.4%. The median TLC was significantly higher in FLT3/ITD positive group (median TLC of 9.4 × 109/L) as compared in the negative group (median TLC of 1.2 × 109/L). Higher LDH level in blood was also observed in FLT3/ITD positive group. The rate of central nerves system (CNS) involvement was significantly higher in FLT3/ITD positive group than that in the negative group (60 % vs. 0%). Relapse rate was also significantly higher in FLT3/ITD group (100 % vs. 0%). These data indicate that the presence of FLT3/ITD is associated with the high relapse rate and CNS involvement in APL patients, and implies one of poor prognostic factors.