bluuebunny/crossref_metadata_2025 huggingface dataset:
Thrombophilic Genetic Mutations Have No Impact in the Pathogenesis of Thromboembolism in Gastro-Intestinal Cancer Patients.
Authors: Olimpieri Odoardo M. , Schiavon Gaia , Giannetti Barbara , Tirindelli Maria C. , Santini Daniele , Valeri Sergio , Vincenzi Bruno , Coppola Roberto , Tonini Giuseppe , Avvisati Giuseppe
Published: October 2019

Deep venous thrombosis and pulmonary embolism are well recognized and feared complications in cancer patients, with a considerable impact on the overall survival and the quality of life. During the last years many prothrombotic genetic mutations have been described, of these the most common are: Factor V Leiden, prothrombin G20210A gene mutation and the C677T mutation in the 5,10 methylenetetrahydrofolate reductase (MTHFR) gene. Aim of this study was to asses the impact of these thrombophilic gene mutations on the development of VTE (Venous ThromboEmbolism) in patients with gastro-intestinal neoplasms. Moreover, we have tried to determine whether or not the C677T polymorphism of the MTHFR gene was associated with a higher toxicity during chemotherapy with antimetabolites such as 5-FU. From October 2002 to June 2004 48 patients (aged more than 18 years) not assuming anticoagulant therapy, with a new histopathologic diagnosis of gastro-intestinal cancer and no known hematologic or immunologic diseases were investigated by standard polymerase chain reaction techniques for the presence of the above thrombophilic gene mutations. Of the 48 tested patients, a homozygous or heterozygous Factor V Leiden was present respectively in 1 and 3, while the G20210A Factor II mutation was observed in 6 patients (all heterozygous). The C677T mutation in the MTHFR gene allelic frequency in the studied population was 46%. A VTE was observed in 7/48 patients (14.5%) and none of these patients had Factor V Leiden or G20210A Factor II mutations while 4 had a C677T MTHFR mutation. Therefore, none of the thrombophilic genetic mutations studied was associated to the development of VTE in the cohort of gastro-intestinal cancer patients. On the contrary chemotherapy (P=0.007) and advanced inoperable disease (P=0.009) were the only risk factors statistically significant for the development of VTE. As for toxicity during chemotherapy with antimetabolites, it was present in 25% of patients with the C677T polymorphism of the MTHFR while none of the patients without mutation had a toxicity (P=0.06). In conclusion, thrombophilic genetic mutations have no effect on the development of VTE in gastro-intestinal cancer patients. Moreover, we confirm that chemotherapy and an advanced disease play an important role in the pathogenesis of VTE in cancer patients. Furthermore, patients carrier of the C677T mutation in the MTHFR gene have a higher risk (OR: 1.4; C.I. 95%: 0.88–2.24) of toxicity during an antimetabolites-based chemotherapy.
A Dynamic Regulation of Platelet Secretion by Septin Proteins.
Authors: Martinez Constantino , Dent Judith A. , Russell Susan , Ware Jerry
Published: October 2019

Septins form a cytosolic protein family gaining recognition as important effectors in molecular mechanisms involving membrane rearrangements, such as cytokinesis and exocytosis. So far, twelve different members of the family (named SEPT1 to SEPT12) have been characterized in mammals, and their roles are not limited to those mentioned above, but embrace a large number of physiological and pathophysiological events. In particular, reports have associated septins with cancer, apoptosis and neurodegenerative diseases. In this study, we further characterize the platelet septin, SEPT5 (formerly CDCrel-1) as a regulator of platelet physiology. SEPT5 is highly expressed in megakaryocytes, heart and brain and is directly linked to secretion mechanisms in both platelets and neurons. In neurons, SEPT5 negatively modulates neurotransmitter release and has recently been implicated in mechanisms leading to development of schizophrenia in rat models. In platelets, we and others have shown that SEPT5 is preferentially located around α-granules and associates with syntaxin 4 and other members of the septin protein family (SEPT4 and SEPT8) to form a cytoplasmic macromolecular complex. Work from other laboratories has linked SEPT5 to different members of the SNARE family such as syntaxin 1A, the sec6/8 complex, and SNAP25. We have found the overexpression of mouse SEPT5 is associated with fewer α-granules but those present are larger in size suggesting levels of Sept5 maintain normal α-granule size. In a murine model of SEPT5 deficiency, platelets display an increased sensitivity to platelet agonists such as collagen and the thromboxane analogue U46619. To date, SEPT5 is the only mammalian septin whose relevance has been tested in a mouse knockout model. Our current studies use a lumi-aggregometer to document the active secretion of ATP in SEPT5 deficient platelets. Platelets lacking SEPT5 are more responsive to submaximal concentrations of acid insoluble fibrillar type I collagen (< 2.5 mg/mL). We observe a 2- to 3-fold increase in ATP secretion in deficient platelets as compared to their wild-type littermates. The increased dense granule release in the absence of SEPT5 also coincides with increased platelet aggregation. A maximum release of ATP was determined in the presence of the calcium ionophore (A23187, 40μM). Higher concentrations of collagen (5 μg/ml) produced a maximum level of ATP release in SEPT5 deficient mice unlike their wild-type littermates. Our current hypothesis places SEPT5 as a scaffolding protein that serves to anchor other proteins into higher-ordered molecular complexes. SEPT5 biology in platelets needs to be further studied to fully understand its role in granule secretion and formation. While important for platelet biology, these studies may also have implications for mechanisms associated with neurotransmitter release. As such, the platelet becomes an excellent model for analyzing SEPT5 function with implications for hemostasis and beyond.
Proteomic Pathway Discovery Reveals That C/EBPalpha Disperses PML Nuclear Bodies To Recruit Its Own Coactivators in a MAPK-Dependent Manner.
Authors: Meisel A. , Kachirin R. , Christopeit M. , Hiddemann W. , Behre G.
Published: October 2019

The downregulation of the tumor suppressor protein C/EBPalpha is a critical event during cancerogenesis. In contrast restoration of its expression leads to differentiation and ultimately apoptosis. To investigate the mechanisms mediating the antiproliferative and differentiation-inducing activity of C/EBPalpha, we performed a proteome-wide screen for C/EBPalpha target proteins using 2-D-gel electrophoresis and MALDI-TOF masspectrometry. Besides 80 other target proteins we identified the tumor suppressor PML as a protein being regulated by C/EBPalpha. In fluorescence microscopy studies we could show that the induction of C/EBPalpha leads to a dramatic decrease in the number of PML nuclear bodies. We found out that these changes occur due to a reduction in the slower migrating sumoylated isoforms of PML. In reporter assays overexpression of PML was able to increase the trans-activation capacity of C/EBPalpha. CBP was further augmenting this coactivating effect. Cadmium, which is like C/EBPalpha desumoylating the PML protein and dispersing the PML nuclear bodies was having the same activating effect. We could show that p38 was mediating the desumoylation of PML, while the p38 inhibitor SB203580 could partially abrogate the C/EBPalpha-induced effects on PML nuclear bodies. Taken together our investigations could identify a mechanism how C/EBPalpha is targeting PML and CBP to sites of active transcription.
The Role of CCAAT Displacement Protein in Neutrophil-Specific Gene Expression.
Authors: Lee Aimee , Khanna-Gupta Arati , Zibello Terry , St.Clair Caryn , Berliner Nancy
Published: October 2019

CCAAT displacement protein (CDP) is a highly conserved, ubiquitously expressed homeodomain protein with extensive homology to the Drosophila cut protein. CDP contains three highly conserved DNA-binding repeats called cut repeats, as well as a conserved homeodomain sequence. CDP is a transcriptional repressor of several developmentally regulated genes, including the phagocyte-specific cytochrome heavy chain gene, gp91-phox, and CCAAT enhancer binding protein epsilon (C/EBPε)and its downstream targets the neutrophil secondary granule proteins (SGPs), including lactoferrin (LF). We have previously shown that CDP binds to and represses both the C/EBPε and lactoferrin (LF) gene promoters thereby preventing expression of secondary granule proteins (SGPs) both directly and indirectly. CDP represses expression of SGPs in 32Dcl3 cells, an IL-3 dependent murine myeloid cell line that undergoes differentiation in response to IL-3 withdrawal and G-CSF stimulation. Moreover, LF is not expressed in NB4 cells, a human acute promyelocytic cell line which contains the t(15;17) PML-RARα translocation. CDP has been found to bind persistently to the LF promoter in NB4 cells after morphological differentiation following ATRA induction. Several attempts at generating a CDP knockout mouse have been performed, but all have produced incomplete knockouts. We have generated short hairpin RNA (shRNA) constructs to knock down mouse CDP in myeloid cells by RNA interference. Single cell clones stably expressing the mouse shRNA have been generated in 32Dwt18 cells, and have been shown to decrease CDP expression. Knockdown of CDP does not change the phenotype of the cells, which remain IL3 dependent and undergo phenotypic maturation upon G-CSF induction. However, increased LF gene expression can be seen in uninduced cells expressing CDP shRNA, and the level of LF expression in single cell clones expressing the shRNA for CDP appears to correlate with the level of CDP repression. Control clones do not express LF until induced with G-CSF for several days. The knockdown of CDP does not appear to affect the expression of C/EBPε, suggesting that LF expression reflects direct modulation of CDP binding to its promoter and is not an indirect effect of increased C/EBPε expression. This suggests that CDP can function as the sole negative regulatory element for LF gene expression, and that relief of CDP repression can increase LF expression independent of increased binding of positive regulatory factors.
An Effective Program to Resolve Ambiguous Results from State Newborn Hemoglobinopathy Screening.
Authors: Hoppe Carolyn , Azimi Mahin , Aslanian Sharon , Lubin Bertram , Vichinsky Elliott , Therrell Brad
Published: October 2019

Newborn screening (NBS) for hemoglobinopathies is an important mechanism for identification of affected individuals so that prophylactic treatment and comprehensive care are delivered before life-threatening complications occur. Whilst more common Hb variants, such as Hb S and Hb C, are readily identified, many children with other potentially significant Hb variants never have a conclusive diagnosis made by state newborn screening programs. In conjunction with the National Newborn Screening and Genetics Resource Center (NNSGRC), we investigated whether unconfirmed or ambiguous samples could be rapidly diagnosed and reported, using only a portion of the original dried blood spot (DBS) specimen.

State NBS programs were approached about sending DBS samples from newborns with ambiguous Hb results for further testing. Using a single 6 mm hole punch from the original DBS, the primary screening results were first confirmed with HPLC, IEF and citrate agar electrophoresis. With the same sample, genotyping for Hb S, C, E, Knossos, D-LA, and O-Arab, and > 95% of the most common β-thalassemia mutations, was performed using a novel linear array platform. Samples revealing Bart’s Hb were tested for common deletional and non-deletional α-thalassemia mutations using gap-PCR or DNA sequencing. The test results, along with a clinical interpretation, were reported back to the NBS programs within 2 weeks.

During a one-year period (08/03–08/04), a total of 309 newborn samples from 30 participating states were received for definitive diagnostic testing. Of these, 115 (37%) revealed a clinically significant genotype. Specifically, 102 newborns had sickle cell disease (55 HbSS, 27 HbSC, 16 HbS/β-thal, 3 HbSE, 1 HbS/O-Arab); 6 newborns had β-thalassemia (2 β0-thal, 4 E/β-thal); and 7 had α-thalassemia (5 HbH, 1 HbH-CS, 2 α-triplication). Compound heterozygosity for 2 Hb variants was found in 15 cases and interpreted as clinically insignificant. Another 14 samples revealed a clinically benign HbEE genotype. A Hb variant trait was identified in 124 samples and 41 samples had a normal Hb genotype.

This NNSGRC-sponsored pilot study demonstrates that a centralized referral laboratory providing definitive diagnostic testing for hemoglobinopathies is not only feasible, but a much needed resource for many state newborn screening programs. Using rapid and efficient molecular methods, our laboratory identified a clinically significant sickling of thalassemic disorder in 37% of previously unconfirmed newborn samples. Based on these results, the implementation of a referral laboratory to provide prompt definitive diagnosis of clinically relevant hemoglobin variants would be an invaluable extension of existing newborn screening programs.
Increased GM-CSF Levels in Sickle Cell Disease Are Associated with Increased Leukocyte Counts and Are Reversed by Hydroxyurea.
Authors: Conran Nicola , Ikuta Tohru , Saad Sara T.O. , Costa Fernando F.
Published: October 2019

The hematopoietic cytokine, GM-CSF, stimulates the proliferation and differentiation of progenitor cells to macrophages, eosinophils and megakaryocytes. Increased levels of GM-CSF have previously been reported in sickle cell disease (SCD) patients with low fetal hemoglobin (HbF) levels (Croizat, Br J Haematol. 87:592; 1994); however, a clearcut association between GM-CSF and HbF levels in SCD has yet to be established, since there is some evidence that levels may depend on other factors (Haider et al., Acta Haematol. 102:140; 1999). To further investigate the possible role of GM-CSF in SCD, plasma samples were collected from normal individuals (AA), steady-state sickle cell anemia patients (SS) and sickle cell patients on hydroxyurea therapy (SSHU; 20-30 mg/kg/day). Plasma GM-CSF levels were measured using a specific immunoenzymatic assay. HbF levels were quantified by ion-exchange HPLC and hematological measurements obtained using an Advia Hematology System. Plasma GM-CSF levels seem to be higher in SS patients than in normal controls (0.828 ± 0.215 pg/ml, n=39; 0.230 ± 0.081 pg/ml, n=9; respectively). In contrast, GM-CSF was significantly lower in SSHU patients than in SS without HU (0.288 ± 0.104 pg/ml; n=14; P = 0.045 compared to SS). A negative correlation between plasma GM-CSF and HbF levels was seen in patients (r = −0.332; P = 0.026; n=45; SS and SSHU groups) and, notably, a positive correlation between GM-CSF and white blood cell counts was observed (r = 0.302; P = 0.037; n= 48; SS and SSHU groups). Furthermore, GM-CSF (2.5 ng/ml) abolished the production of γ globin during erythropoietin-stimulated differentiation of TF-1 hematopoietic cells. These results provide further support to the hypothesis that plasma GM-CSF correlates negatively with HbF in sickle cell disease. Interestingly, in vitro data suggest that increased GM-CSF may, in fact, diminish HbF levels rather than reflect increased haematopoietic stress as a consequence of low HbF. Importantly, GM-CSF appears to correlate with leukocyte numbers in SCD, and levels were significantly decreased in patients taking HU, indicating that this cytokine may contribute to the leukocytosis seen in some SCD patients and may play a role in leukocyte count reduction by HU.
Generation of Transgenic Mice Expressing Human Hemoglobin E.
Authors: Chen Qiuying , Bouhassira Eric E. , Besse Arnaud , Suzuka Sandra M. , Fabry Mary E. , Nagel Ronald L. , Elison-Hirsch Rhoda
Published: October 2019

HbE (β26 Glu → Lys) is the most common pathogenic Hb variant in the world and is found with the greatest frequency in Southeast Asia. The HbE mutation at codon 26 of the βE-globin leads to an alternative splicing site, making this globin both a structural as well as a thalassemic mutation; furthermore, βE globin chains are unstable. HbE trait is asymptomatic and the HbEE genotype has only mild clinical manifestations. On the other hand, HbE/thalassemia presents a panoply of phenotypes from very severe to a mild beta/+ thalassemia. To date, there is no animal model of HbE disease. An animal model of this disorder could lead to better understanding of the effects of modifier genes and some of the pathogenic features of this disease. In addition, an animal model can facilitate the development of gene therapy. We report here the creation of four lines of transgenic mice that are PCR positive for HbE: two with founders that express high levels of HbE (32% human α, 26% human βE), one with low expression of HbE (9% human α, 8% βE), and one without detectable expression, but PCR positive. Knockouts (KOs) for mouse α and β globins have been bred into the founders. To date, the most severe mouse generated has a full KO of mouse α and a partial KO of mouse β. RBCs from all of the partial KOs generated to date have normal MCHC and minimally elevated reticulocyte counts (less than 1% greater than the C57Bl background). We previously demonstrated that HbEE RBCs are microcytic, but have normal MCHC. We found that HbE mice with full α-KO and partial β-KO, are microcytic (MCH 10.9 vs 14.2 for C57Bl and MCV 35.4 vs 47.5 for C57Bl), but have normal MCHC (MCHC 30.0 vs 30.5 for C57Bl). Smears from these mice exhibit numerous target cells that are not necessarily associated with low MCHC. As reported for human red cells containing HbE, elevated levels of hemoglobin oxidation products were detected in founder mice expressing HbE, but not in negative litter mates, and were found, at an even higher level, in partial KO mice expressing HbE. The next step will be to generate a mouse similar to the HbE/thalassemia phenotype by generating mice expressing exclusively HbE from either the lines expressing high levels of HbE or the line expressing low levels of HbE. These models will help elucidate several of the challenging features of the phenotypes described in humans and allow for the development of the potential cure or amelioration of HbE/thalassemia.
Histone Acetyltransferase Activity of p300 Is Required for Transcriptional Repression by the Promyelocytic Leukemia Zinc Finger Protein.
Authors: Guidez Fabien , Howell Louise , Isalan Mark , Cebrat Marek , Alani Rhoda M. , Ivins Sarah , Pierce Sarah , Cole Philip A. , Licht Jonathan D. , Zelent Arthur
Published: October 2019

The Promyelocytic Leukemia Zinc Finger (PLZF) gene was identified in a rare case of acute promyelocytic leukemia (APL) with translocation t(11;17)(q23;q21) and resistance to therapy with all-trans-retinoic acid. Recent studies have indicated a critical role of PLZF in maintenance of spermatogonial stem cells. Prominent expression of PLZF in hematopoietic stem cells, suggest that it may also play a similar role in this compartment. The wild type PLZF protein is a DNA sequence-specific transcription repressor containing nine Krüppel-like C2-H2 zinc fingers and an N-terminal BTB/POZ repression domain. Transcriptional repression by PLZF is mediated through recruitment of the nuclear receptor co-repressor (N-CoR/SMRT)/histone deacetylase (HDAC) complexes to its target genes, such as c-MYC and HOX genes. We now show that transcriptional repression by PLZF is surprisingly also dependent on the histone acetyl transferase (HAT) activity of the p300 protein. PLZF associates with p300 in vivo and its ability to repress transcription is specifically dependent on acetylation of PLZF on lysines in its C-terminal C2-H2 zinc-finger motifs. Acetylation of PLZF enhances its ability to bind its cognate DNA binding site in vitro as determined by EMSA and in vivo as measured by chromatin immunoprecipitation. An acetylation site mutant of PLZF fails to repress transcription despite retaining its abilities to interact with co-repressor/HDAC complexes, due to inefficient DNA binding. Inhibitors of p300 abolish transcriptional repression by PLZF and mutants of PLZF that mimic acetylation were insensitive to these inhibitory effects. Acetylation of PLZF by p300 was specific since over-expression of another HAT, p/CAF or the selective inhibition of p/CAF had no effect on PLZF activity despite the ability of the proteins to associate with each other. Taken together, our results indicate that a histone deacetylase dependent transcriptional repressor can be positively regulated through acetylation and point to an unexpected role of a co-activator protein in transcriptional repression.
Generation of Robust Numbers of Functional Human Hepatocytes by Human Hematopoietic Stem Cells in the Non-Injury Fetal Sheep Model.
Authors: Torabi Alireza , Almeida-Porada Graca , Zanjani Esmail D.
Published: October 2019

We have previously shown that human hematopoietic stem cell preparations (HSCs) are able to engraft and transdifferentiate into human hepatocyte in the human/sheep xenograft model (Blood. 2004 Jul 1 Epub). In the present study 1x106 human CD34+ cells were transduced with GFP expressing RD114 pseudotyped spleen fociforming retroviral vector in vitro and injected into pre-immune fetal sheep. Two months post-transplantation, immunohistochemistry confirmed production of human hepatocytes that were double positive for GFP and Hepar1. These data indicate that transduced human CD34+ cells are able to transdifferentiate into human hepatocyte and still be able to express GFP. In order to retrieve human hepatocytes from the liver of chimeric sheep, we used c-met antibody to sort out human hepatocytes. Flowcytometric analysis demonstrated 8% c-met+ cells in the liver of chimeric sheep. Preliminary data using PCR analysis from c-met+ cells for human beta-2 microglobulin demonstrated presence of human cells in c-met+ population. Livers from chimeric sheep were treated with collagenase to obtain single cell suspension and 3x105 cells/ml were placed in T75 flask covered with 0.2% gelatin and cultured in optimized media designed to support the proliferation and maintenance of human and sheep hepatocytes. In this system, using a weekly passage protocol, we were able to maintain/expand human (and sheep) hepatocytes for up to 3 months. The cells are able to produce human albumin in culture and are CK19 positive, a-fetoprotein negative, and cytochrome P450 negative. These results demonstrate that in this non-injury large animal model human HSCs generate robust numbers of functional human hepatocytes apparently via a “transdifferentiation” mechanism.
Immune-Mediated Hepatitis Drives Bone marrow to Hepatocyte Plasticity.
Authors: Dahlke Marc H. , Popp Felix C. , Piso Pompiliu , Schlitt Hans J. , Bertolino Patrick
Published: October 2019

Liver dysfunction is a major health burden world-wide. Future cell-based therapies for liver regeneration may benefit from the fact that bone marrow cells can fuse with or transdifferentiate into hepatocytes. All models demonstrating bone marrow to hepatocyte plasticity presented so far, however, have used highly artifical conditions of liver regeneration - applying toxins, genetic pressure models or liver resection. We have set up a model of transgenic T cell induced bystander hepatitis in bone marrow chimeras to assess the effect of hepatitis, a common liver pathology in humans, as an enhancer of bone marrow to hepatocyte plasticity events.

MHC haplotype (Kb) transgenic bone marrow from 178.3 mice or control bone marrow from B10.BR (Kk) mice was transplanted into sublethally irradiated B10.BR (Kk) mice. Hepatitis was induced by repeated injections of Des Kk T cell receptor transgenic T cells against the Kb antigen. In additonal groups Retrorsine was used as an agent inhibiting endogenous hepatocyte proliferation and GCSF for mobilisation of bone marrow stem cells.

Repeated injections of transgenic T cells induced subsequent waves of hepatitis in recipients of MHC haplotype transgenic bone marrow but not in control animals confirmed by serum ALT levels. Hepatocyte single cell suspensions from animals suffering from hepatitis revealed an increased expression of donor bone marrow derived antigen. This could be further enhanced by either increasing the number of circulating stem cells or by inhibiting the endogenous response of resident hepatocytes. FISH analysis showed fusion nuclei on a single cellular level.

T cell receptor transgenic T cells induce bystander hepatitis in an antigen specific manner. This inflammatory response drives the plasticity of bone marrow cells to hepatocytes and their potential contribution to liver regeneration. Fusion between donor cells and resident hepatocytes is the underlying mechanism of liver regeneration in this model mimicking a common liver pathology.
Low Bone Mass in Thalassemia: The Thalassemia Clinical Research Network (TCRN) Experience.
Authors: Vogiatzi Maria G. , Lane Joseph , Fleisher Martin , Macklin Eric A. , Fung Ellen B. , Kirby Melanie , Kwiatkowski Janet , Holm Ingrid , Olivieri Nancy , Vichinsky Elliott , Giardina Patricia J.
Published: October 2019

Low bone mass is emerging as a frequent and debilitating morbidity in Thalassemia (thal). The TCRN conducted a cross-sectional observational study to determine the prevalence and factors contributing to bone disease among North American thal patients (pts). Spinal (L1-L4) Bone Mineral Density (BMD) Z- and T-score measurements by DXA (Hologic 4500 and Delphi models) were read centrally. Each subject’s weight, height, hematologic, endocrine and genetic parameters, iron chelation and transfusion regimens, dietary calcium intake, history of fractures and bone pain, self-reported physical activity and bone turnover markers were assessed. BMD was measured in 302 pts: 207 Thal Major (TM), 37 Thal Intermedia (TI), 35 Beta E, 7 Hemoglobin H disease (HbH), 2 homozygous alpha (α) and 14 HbH/Constant Spring (HbH/CS). Among all diagnostic groups, the prevalence of low bone mass (LBM; Z/T <-2), reduced bone mass (RBM; Z/T -2 to -1) and normal bone mass (NBM; Z/T >-1) was 52%, 27% and 21%, respectively. LBM prevalence was 55% in TM, 53% in TI, 51% in Beta E, 0% in HbH, 50% in α and 43% in HbH/CS. RBM prevalence was 26% in TM, 22% in TI, 31% in Beta E, 71% in HbH, 50% in α and 29% in HbH/CS. Pt groups aged: 6–11 yrs, 11–20 yrs, 20+ yrs had Z/T-scores mean±SD[n] were: −1.32±0.82[51], −1.73±1.08[77] and −2.43±1.14[174], respectively. Z/T-scores were significantly lower among older pts (p<.001) and significantly higher among heavier pts after controlling for Tanner stage. Mean age-adjusted Z/T-scores of thal diagnostic groups and their slopes vs. age did not differ significantly although the samples of some groups were small. Among TM and TI pts, those with genotype β°/β° tended to have lower age- and weight-adjusted Z/T-scores (mean [95% CI]: −2.25 [−2.65 to −1.86], n=39). Less than 1% had hypoparathyroidism, 4% vit D deficiency, 8.5% diabetes mellitus, 8.5% hypothyroidism and 11.5% growth hormone deficiency (GHD). Only GHD was significantly correlated with decreased Z/T-scores after controlling for age and diagnosis. Urinary N-telopeptide (NTx) is elevated across all three age groups (median[IQR] mM BCE/mM creatinine: 664[456–930], 302[90–624], 69[34–124]), respectively. Preliminary analysis of bone turnover markers in a subset of subjects (n=114) suggests that NTx, urinary or serum was a significant independent predictor of spine Z/T-scores controlled for age and age-adjusted weight. There was no relationship between Z/T-score and serum osteocalcin. This large and comprehensive study of thal bone disease has demonstrated that decreased bone mass occurs with high frequency, worsens with age, is affected by weight and GHD and is associated with elevated NTx, i.e. increased bone resorption. Future studies are needed to identify efficacious long-term therapies to improve thal bone disease.
Stabilization of Human Erythrocytes for Lyophilization: Recovery of Biochemical Activities and Membrane Filterability.
Authors: Xu Shang-Zhi , Bode Arthur P.
Published: October 2019

It has much more advantages to preserve human red blood cells (RBC) in freezing and drying form than conventional blood storage, even though it is still under developing. In the present study, we successfully used 0.045% glutaraldehyde(GA) to fix RBCs under appropriate conditions prior to lyophilization and regained the bulk of RBCs membrane filterability. The phlebotomized whole blood was anti-coagulated with CPDA-1 and the plasma was removed by centrifugation. White blood cells were removed using Leucocytes filter (Pall Corporation). 1 x 10 9 cells/ml were separately incubated with 0.03%, 0.045% and 0.05% GA at 37 °C for 10 min; RBCs at concentrations 0.5 x 10 9, 1 x 10 9 and 2 x 10 9 cell/ml were incubated with 0.045% GA at 37 °C for 10 min; Bovine serum albumin (BSA), trehalose, hydroxyethyl starch and dextran were used as protective additive in freezing and drying RBCs, respectively; ethanolamine, glycine, lysine, BSA, glutamic acid and homocysteic acid were used to recover membrane deformability due to GA fixation. Recovery RBCs yield after reconstituion was determined by a cell counter. Fee hemoglobin , glutathione (GSH), 2,3-diphosphoglycerate (2,3-DPG), ATP, and Glucose-6-phosphate dehydrogenase (G-6-PDH) in RBCs were determined before and after lyophilization. Membrane flexibility was assayed by osmotic fragility test. RBCs filterability was determined using positive filter apparatus through 5 micrometer pore size nitrocellulose membrane. Our results suggested that RBCs at 1 x 10 9 cells/ml fixed by 0.045% GA for10 min at pH 7.8 were enough to resist freezing and drying damages and the bulk of RBCs’s membrane filterability were remained. The effective fixation of GA is dependent on GA’s concentration, RBCs concentration, incubation period and pH etc. BSA is the most potential additive in preserving RBCs. More interestingly, the following reagents can recover the reduced RBCs membrane filterability by GA fixation. The potency of recovery is in the order of : 10 mM ethanolamine > 5 mM lysine > 0.5%BSA > 10 mM glutamatic acid > 5 mM homcysteic acid > 2% glycine. After reconstitution, 85 ± 2.3% RBCs yield is achieved, 71 ± 4.1% of these RBCs can freely pass through 5 micrometer pore size filter membrane. Biochemical function indexes as GSH, 2,3-DPG, ATP, and G-6-PDH are saved by 20 ± 1.2%.

Lyophilized human red blood cells yields from reconstitution by different treatment.

1x 109 cells/ml fixed at 0.03% glutaraldehyde 44.7 ± 4.1 0.045%glutaraldehyde 84.7 ± 5.8 0.05%glutaraldehyde 88.7 ± 7.6 0.045% glutaraldehyde fix: 0.5x 109 cells/ml 91 ± 4.3 1x 109 cells/ml 84.7 ± 5.8 2 x 109 cells/ml 35.2 ± 2.3

Effects of various treatment on lyophilized RBCs after reconstitution.

Group Filterability(%) 1x9 cells/ml fixed by: N = 5. 0.03%glutaraldehyde 88.3 ± 1.2 0.045%glutaraldehyde 73.3 ± 1.8 0.05%glutaraldehyde 44.7 ± 2.8 0.045%glutaraldehyde fixation at: 0.5x109 cells/ml 0 1x109 cells/ml 73.3 ± 1.8 2x109 cells/ml 83.9 ± 4.6
The High Frequency of HLA-A*0206 Allele in Japanese Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) and Its Significance.
Authors: Ikeda Kazuhiko , Shichishima Tsutomu , Akutsu Kazuko , Maruyama Yukio
Published: October 2019

PNH is an acquired hematologic disorder which is characterized by complement-mediated hemolysis, thrombosis, and bone marrow failure. Also, PNH is one disorder of bone marrow failure syndromes, including aplastic anemia (AA) and myelodysplastic syndrome (MDS). It is well known that immunologic mechanisms by cytotoxic T lymphocytes (CTLs) contribute to pathophysiology of these disorders. In fact, some reports (Maciejewski et al, Blood, 2001; Shichishima et al, Blood, 2002) showed that HLA-DR*1501 is related with clinical pathophysiology of PNH. In this study, to clarify significance of CD8+ CTLs in pathophysiology of PNH, we investigated HLA class I (A and B) alleles in Japanese patients with PNH (female: male=7:17), AA (female: male=14:15), and MDS (female: male=6:16) by high-resolution method using polymerase-chain reaction after obtaining informed consent and approval from the institutional Human Research Committee. Mean age ± standard deviation of PNH, AA, and MDS patients was 52 ± 16, 54 ± 20, and 59 ± 18, respectively. HLA genotyping showed that the frequency of HLA-A*0206 allele in PNH patients (22.9%) was significantly different from those in 309 unrelated Japanese individuals (Saito et al, Tissue Antigens, 2000) (7.7%; p<0.02) and AA patients (5.2%; p<0.01). In contrast, we found no significant differences in the frequencies of the other alleles between PNH, AA, or MDS patients and the controls or between these disorders, except for high frequency of HLA-B40 alleles in AA patients (Nakamura et al, Blood, 2003). Then, various clinical parameters, including peripheral blood, bone marrow blood, and laboratory findings, proportions of glycosylphosphatidylinositol protein-negative population in erythrocytes, granulocytes, and monocytes, findings of chromosomal analyses and HLA-DR alleles, transfusion requirements, past history of AA, and history of thrombosis, were statistically compared between HLA-A*0206-positive group (n=10) and -negative group (n=14) in PNH patients. Statistical analyses showed that the reticulocyte counts , the values of lactate dehydrogenase, and the frequency of PNH patients with over 30% of CD59− erythrocytes in HLA-A*0206-positive group were significantly higher than in HLA-A*0206-negative group (121 ± 49 x 109/L vs 76.9 ± 43.9 x 109/L, p<0.03; 2866 ± 2606 IU/L vs 938 ± 775, p<0.02; and 80.0 % vs 28.6 %, p<0.02, respectively). Moreover, we found no AA (n=3) and MDS (n=5) patients with both the HLA-A*0206 allele and more than 1% of CD59− granulocytes. In conclusion, our findings suggest that the HLA-A*0206 allele in PNH may be correlated with the grade of the disease by complement-mediated hemolysis during negative selection of PNH clones, probably due to immunologic mechanisms by CD8+ CTLs.
The Role of α-Thalassemia and Iron Deficiency in the Difference between Hemoglobin Levels of African-Americans (AA) and Whites.
Authors: Beutler Ernest , West Carol
Published: October 2019

The fact that the average hemoglobin concentration (Hb) of AA is lower than that of whites has been documented extensively. Several investigations have shown that this difference of approximately 0.8 g/dL is due neither to iron deficiency nor to socioeconomic status. Its cause remains unknown. We compared the Hb of 1,493 AA and 31,029 white anonymized patients attending a Health Appraisal Clinic and confirmed the known difference in Hb, both for females and males (0.79 and 0.47 g/dL) respectively. The difference persisted when a subset of the subjects were paired by age and narrowed slightly in females when those with serum ferritin levels of <10 ng/ml or transferrin saturations of <16% were excluded (difference in females 0.59 g/dL; males 0.47). We determined the α-thalassemia −3.7 genotype of 298 AA. The gene frequency was found to be 0.17, and the distribution of genotypes fit the Hardy-Weinberg equilibrium. However, in a sample of 155 white subjects only one α-thalassemia allele was found (gene frequency=0.003). Among the AA subjects, the Hb and MCV values were lower in homozygotes (−a/−a) and heterozygotes (aa/−a) for α-thalassemia than in the aa/aa subjects. The table presents data for AA and white subjects after excluding all who did not have a documented serum ferritin level of >9 ng/ml and a transferrin saturation of >16%. Excluding subjects with sickle trait had no effect.

Ethnic Group Genotype n Mean Hb SE Hb Mean MCV SE MCV −a/−a 3 11.87 0.418 72.23 2.32 F AA aa/−a 20 12.69 0.202 85.22 0.86 aa/aa 65 13.17 0.127 90.43 0.61 White 2917 13.60 0.016 90.85 0.07 −a/−a 2 13.85 0.550 83.05 1.65 M AA aa/−a 36 14.37 0.161 85.81 0.78 aa/aa 86 14.75 0.123 89.78 0.53 White 5335 15.09 0.013 90.35 0.06

As shown in the table, the average Hb of non-iron deficient AA females and males who had 4 normal α loci (aa/aa) was 0.43 and 0.34 g/dL lower respectively than those of whites, the difference being significant with p<0.01. We conclude that one cause of the lower Hb of AA compared to white subjects is the high prevalence of α-thalassemia in the AA population, but that it accounts for only about one-quarter of the difference after iron deficiency has been excluded. There are other, as yet undefined, causes that play a role. These may include the lower ATP (Biochem. Genet.1:25, 1967) and higher 2,3 BPG (Transfusion18:108, 1978) levels that have been documented in the red cells of AA subjects.
Correlation between Leg Ulceration and Perforator Venous Insufficiency (PVI) in Patients (Pts) with Sickle Cell Disease (SSD).
Authors: Braverman Albert , Nehra Anupama , Gillette Peter , Weedon Jeremy , McGagh Deidre , Olivier Tracy , Gwertzman Gary , Deitch Jonathan
Published: October 2019

Chronic or recurrent leg ulceration occurs in 25% of adult SSD pts, but as many as 75% never develop leg ulcers. PVI correlates with leg ulceration in non-SSD pts. We studied 46 SSD pts: 40 with hemoglobin (Hb) SS, 3 with sickle-b thalassemia, 2 with Hb SC and 1 with Hb SD. Chronic and/or recurrent leg ulceration was present on at least 1 leg of 25/46 (ulcer+) and absent in 21/46 (ulcer-) SSD pts. Their ages ranged from 19–67 years, with a median of 31. Using color-flow venous duplex (7–5 mega-Herz probe; Advanced Technology Laboratory 5000), the status of perforator veins in both legs of each pt was determined (only one of a single pt’s two ulcerated legs was studied). Perforator veins were considered incompetent (PVI) if their diameter was >3 mm, and their reflux times were >0.35 seconds. For the 36/91 ulcer+ and 55/91 ulcer- legs, there was a significant association between PVI and ulceration in the same leg (generalized mixed linear model, p=0.002). Estimated probability of ulceration in a non-PVI leg was 0.20 (95% confidence interval [0.10, 0.36]); estimated probability of ulceration in a PVI leg was 0.61 (95% CI [0.41, 0.79]). Odds ratio was 6.2 (95% CI [2.0, 19]). The prevalence of ulceration in either leg, among 22 pts with PVI in at least 1 leg, was compared with that among 23 pts with PVI in neither leg (omitting the pt with one unstudied leg). There was significant association between ulceration in either leg and PVI in either leg (Fisher’s exact test p<0.001). Estimated probability of ulceration in either leg when neither leg had PVI was 0.22 (95% confidence interval [0.07, 0.44]); estimated probability of ulceration in either leg when either leg had PVI was 0.86 (95% CI [0.65, 0.97]). Odds ratio was 23 (95% CI [4.7, 110]). PVI may be involved in the pathogenesis of leg ulceration in pts with SSD.
Blood Mononuclear Cells and Platelets Have Abnormal Fatty Acid Composition in Homozygous Sickle Cell Disease.
Authors: Okpala Iheanyi , Ren Hongmei , Ghebremeskel Kebreab , Ugochukwu Cynthia , Ibegbulam Obike , Crawford Michael
Published: October 2019

Leukocyte adhesion to vascular endothelium contributes to vaso-occlusion and widespread organ damage in sickle cell disease (SCD). Previously, we found high expression of the adhesion molecules αMβ2 integrin and L-selectin in HbSS individuals with severe disease. The n-6 and n-3 polyunsaturated fatty acids (FA) are vital structural and functional components of cell and sub-cellular membranes. They modulate cell adhesion, inflammation, aggregation and vascular tone. We investigated the FA composition of mononuclear cells (MNC) and platelets of HbSS patients in steady-state (n = 28); and racially matched, healthy HbAA controls (n = 13). MNC phospholipids of the patients had low levels of docosahexanoic acid (DHA, p<0.01), n-3 metabolites (p<0.05) and total n-3 polyunsaturated FA (p<0.05); table 1. In contrast, arachidonic (AA, p<0.005), AA:DHA ratio (p<0.005, fig 1) and total n-6 metabolites (p<0.05) were increased in the patients. Similarly, platelets from HbSS patients had low levels of eicosapentanoic acid (EPA, p<0.05), and raised AA (p<0.05) in choline phosphoglycerides (CPG); with reduced linoleic acid (LA, p<0.005) and DHA (p<0.05) in ethanolamine phosphoglycerides. Platelet CPG had lower DHA levels in HbSS individuals with complications of SCD compared to those who had no complications (p<0.05, fig.2). Reduced EPA and DHA relative to AA favours the production of aggregatory and pro-inflmmatory eicosanoids that activate leukocytes and platelets. This may lead to enhanced inflammation, leukocyte adhesion, platelet aggregation and vaso-occlusion in SCD.

Table 1: Fatty Acid Composition of MNC Total Phospholipids in HbSS Patients and HbAA Controls

Fatty Acids HbSS Patients HbAA Controls Values are Means [SD}. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 24:0 0.71 [0.30]*** 1.3 [0.4] saturates ∑ 38.2 [3.6] 39.1 [1.7] 16:1 0.69 [0.45] 0.56 [0.11] 18:1 14.4 [1.8]* 12.9 [1.9] 24:1 1.2 [0.3] 1.1 [0.4] ∑monoenes 16.2 [2.1]** 14.3 [1.4] 18:2n-6 6.1 [0.9] 7.0 [1.4] 18:3n-6 0.11 [0.04]* 0.23 [0.17] 20:2n-6 0.56 [0.18]** 0.83 [0.32] 20:3n-6 (DHGLA) 1.2 [0.2]* 1.4 [0.2] 20:4n-6 20.2 [1.7]*** 18.1 [1.8] 22:4n-6 1.7 [0.4] 1.6 [0.4] 22:5n-6 0.3 [0.21] 0.24 [0.14] n-6 metabolites ∑ 24.1 [1.9]* 22.6 [1.7] n-6 ∑ 30.2 [2.0] 29.7 [2.2] 20:5n-3 (EPA) 0.43 [0.16] 0.61 [0.35] 22:6n-3 (DHA) 1.9 [0.4]** 2.5 [0.6] ∑n-3metabolites 4.3 [0.9]* 4.8 [0.4] n-3 ∑ 4.5 [0.9]* 5.0 [0.4] DHGLA:AA ratio 0.06 [0.01]**** 0.08 [0.01] AA:EPA ratio 52.4 [20.9] 38.6 [18.4]
Detection of Acute Chest Syndrome by MRI in a Sickle Transgenic Mouse Model.
Authors: Kennan Richard P. , Alleyne Dawn , Aldrich Thomas K. , Suzuka Sandra M. , Renick Robert , Nagel Ronald L. , Fabry Mary E.
Published: October 2019

Acute chest syndrome (ACS) is a potentially deadly complication in sickle cell patients, particularly in young adults. In patients younger than 17 years, ACS is frequently associated with infection; however, in adults, association with vasocclusive crisis and fat and bone marrow embolism is more common. We have developed a sickle transgenic mouse model for ACS that consists of injecting a mixture of bone marrow and mouse fat into the tail vein of a sickle transgenic mouse. The mouse used was a NY1 partial KO that expresses 100% human α and 55% βS. Mice were injected with bone marrow (BM) from two femurs (about 15 mg) and, as murine BM lacks fat, purified peritoneal fat (about 15 mg). Creatine kinase (CK), predominantly the MM form, and sVCAM increased above baseline levels at 6, 24, and 48 hours in NY1 partial KO mice, but not in control C57Bl mice. Magnetic resonance imaging (MRI) was performed at 48 hours after the initial injection. It is normally difficult to perform MR imaging of lung due to its inherently short transverse relaxation time and relatively low proton density relative to other tissue. However, these difficulties can be overcome through the use of ultra short echo time spin echo sequences at high field strength. Spin echo images were obtained using a respiratory-gated spin echo sequences at TE=3 msec and at TE=10 msec. From this data one can calculate a quantitative relaxation rate (1/T2) map and an average value of R2. Decreased values for mean R2 are an indication of fluid accumulation. In NY1 partial KO mice examined 48 hours after injection, the 1/T2 map revealed focal patches with high water content and a mean R2 of 131 vs R2 = 220 for an uninjected control mouse; when the same animals were examined three months later R2s were found to be 181 and 124. C57Bl control mice were also examined at 48 hours post injection, and R2 values of 211 and 178 were found. Pulmonary edema is a defining finding in ACS and its presence in sickle mice, but not in control mice is strong support for this model. The mice survived both the fat/BM injection and the MRI, indicating that it will be possible to obtain repeated images from the same animal to study the natural course of lung injury and interventions that may ameliorate injury.
Barriers to Deferoxamine Adherence for Adults with Sickle Cell Disease.
Authors: Treadwell Marsha , Sung Jennifer , Murray Eileen , Hagar Robert , Major Kimberly , Hackney-Stephens Ekua , Glendenning Alastair , Hoehner Christy , Vichinsky Elliott P.
Published: October 2019

Background: The barriers to adherence with chelation therapy for chronically transfused and iron overloaded patients with sickle cell disease (SCD) have been described only anecdotally.

Objectives: To describe barriers to home deferoxamine (DFO) administration adherence among adults with SCD. It was hypothesized that barriers would include limited patient education on the importance of chelation and perceived aversiveness of the regimen.

Methods: Medical records were reviewed for 189 adult patients seen at a comprehensive sickle cell center. Patients with transfusion induced hemosiderosis, defined as a serum ferritin ≥ 1500 ng/ml, were administered a four item interview asking if iron overload had ever been discussed with them; if they had been informed they were iron over loaded; if chelation therapy had been offered; and if not currently home chelating, why not. Patients not interviewed were deceased (3); unavailable (10); or declined (3). A study coordinator who did not provide clinical care conducted the interviews.

Results: 54 of the 189 patients (29%) had a history of intermittent or chronic transfusion, or pheresis. 45 of these patients were iron overloaded. 29 of these patients agreed to complete the interview; 22 (76%) were female. Average age was 41.5 years (range 22.4 – 58.4 years) and average serum ferritin was 4240.8 (range 1547 – 9420). 23 of the 29 patients (79%) reported that their physician or nurse had discussed iron overload and chelation with them. 16 of these (55%) reported that they were currently receiving home DFO therapy. Reasons given for not administering home DFO included:

Reason Number (%) “Don’t want to stick self” 3 (23) No longer being transfused or being exchanged 3 (23) Awaiting clinical trial for oral chelator 2 (15) Home situation too complex 2 (15) Don’t want to (no further explanation) 2 (15) Too many adverse effects 1 (8)

Discussion: Life threatening levels of iron overload were observed in intermittently transfused adult sickle cell patients. Contrary to expectations, iron overload and its treatment had been discussed with most patients. However, just over half were currently chelating at home. Toxicity of DFO and misunderstanding that iron overload is no longer a problem if chronic transfusion therapy stops are the most common reasons for non-compliance. Repeated patient counseling are essential in order to prevent progressive iron toxicity in sickle cell disease.

Reason Number (%) “Don’t want to stick self” 3 (23) No longer being transfused or being exchanged 3 (23) Awaiting clinical trial for oral chelator 2 (15) Home situation too complex 2 (15) Don’t want to (no further explanation) 2 (15) Too many adverse effects 1 (8)