bluuebunny/crossref_metadata_2025_split huggingface dataset:
Decreased Numbers of Tregs in Aplastic Anemia Is Detected by Immunohistochemistry and Flow Cytometry.
Authors: Serio Bianca , Peerwani Ziad , Tiu Ramon , Powers Jennifer , Hsi Erik , Maciejewski Jaroslaw M.D. 
Published: April 2020

Abstract
Idiopathic aplastic anemia (AA) is characterized by immune-mediated destruction of hematopoietic stem cells, leading to peripheral pancytopenia. Immune pathogenesis in AA is supported by experimental data, as well as clinical observations and may be related to the breach of peripheral or central tolerance. Regulatory T cells (Treg) constitute one of the most important mechanisms of central tolerance engaged in the down-modulation of autoreactive T cells. Tregs have been found to be reduced in several autoimmune diseases and decreased frequencies of Tregs were also reported in AA and MDS. Overexpression of the high affinity IL-2 receptor alpha chain (CD25) and the forkhead family transcription factor P3 (FoxP3), required for the development and function of Tregs, serve as phenotypic markers for Tregs. We investigated Treg levels in a cohort of AA patients (N=21) and healthy individuals (N=15); flow cytometric quantification of Treg was carried out after surface/intracellular staining of whole blood for Treg markers (CD3, CD4, CD25, FoxP3). After proper gating (light scatter properties, CD3, CD4, CD25), CD4+ T cells were subdivided into CD25−, CD25int and CD25hi populations, and the co-expression of CD25hi and Foxp3 was analyzed. In comparison to controls, AA patients (N=12) show not only lower frequencies of CD4+CD25hi+ T cells within the total lymphocyte population (median 0.07% vs. 0.21%; p=.03), but also absolute lower absolute numbers (1.31/uL vs. 5.78/uL, p=.0002). Similarly, CD4+CD25hi+FoxP3+ T cells were found to be depressed in untreated AA patients in comparison to controls (median 0.07% vs. 0.21% and 1.06/uL vs. 4.76/uL; p=.03 and p=.003). While Tregs were lower in patients with active disease unresponsive to immunosuppressive treatment (responder 0.1% vs non responder 0.07%, CD4+CD25hi Tcells, p=.02), serial testing performed in 6 patients treated with ATG/CsA did not reveal correlation between hematologic improvement and recovery of Treg numbers. When double immunohistochemical staining for CD3 and Foxp3 was performed in pre-treatment bone marrow core biopsies of AA patients (N=3) and controls (N=2) a mean of 3 CD3+Foxp3+ cells/10 high power fields (hpf) were counted (vs. mean 28/10 hpf, p&lt;.05 in controls), suggesting that lower numbers of Tregs were also present in the bone marrow of AA patients. In conclusion, our results suggest that Tregs are decreased in blood and marrow of patients with idiopathic AA, consistent with the breach of peripheral tolerance in AA. In addition to flow cytometry, immunohistochemical staining of histologic specimens can be used for the quantitative analysis of Tregs in bone marrow failure syndromes and other immune-mediated conditions such as GvHD.
A Role for Survivin in Enucleation of Erythroid Progenitors.
Authors: Keerthivasan Ganesan , Kang Jeong Ah. , Seferos Dwight S. , Mirkin Chad A. , Wickrema Amittha , Crispino John D. 
Published: April 2020

Abstract
Enucleation is the final step in the development of reticulocytes and this process is unique to mammalian red blood cells among vertebrates. Even though enucleation of erythroblasts was structurally studied by electron microscopy half a century ago, little is known about the underlying mechanism. Initially it was thought that enucleation involved degradation of DNA and other apoptotic processes. More recently it has been shown that enucleation of erythroid progenitors does not require major apoptotic machinery including caspase-3 activation (Krauss et al, 2005; Yoshida et al, 2005). Alternatively, it has been suggested that enucleation in erythroblasts is a result of asymmetric cytokinesis. In support of this later model, survivin, a regulator of chromosomal segregation and cytokinesis, which also has an anti apoptotic role, is highly expressed in orthochromatic erythroblasts, cells at the stage of differentiation just prior to enucleation (Gurbuxani et al, 2005). Moreover conditional heterozygous deletion of survivin in hematopoietic cells caused a decrease in enucleated cells in the spleen (Leung et al, 2007). This has led us to hypothesize that survivin has a role in enucleation of erythroblasts. To test this prediction, we first over expressed survivin in MEL (murine erythroleukemia) cells and then subjected them to DMSO induced differentiation. Normally MEL cells decrease their survivin expression level upon differentiation and at the same time undergo limited enucleation (&lt;4%) even after 5 days of differentiation. In contrast, survivin overexpressing clones consistently enucleated 3–5 fold more than control cells, suggesting that levels of survivin are likely to be important for enucleation. We are currently harnessing the power of survivin antisense nanoparticle agents to assess the specific requirement of survivin in enucleation of primary human orthochromatic erythroblasts. Separately, we evaluated the localization of survivin and its known partners in enucleating primary human erythroid cells by confocal microscopy. Survivin is predominantly localized to the cytoplasmic compartment and to our surprise did not colocalize with Aurora B kinase and INCENP, two of its partners in the chromosomal passenger complex, which is essential for mitosis. On the contrary, we noticed that survivin partially colocalized with actin at the junction between the extruding nucleus and cytoplasm, as well as in the periphery of cytoplasm in these enucleating cells. Moreover enucleation assays performed in the presence of various inhibitors of cell cycle and cytokinesis did not prevent enucleation in erythroblasts. Taken together, these findings demonstrate that enucleation is not a form of cytokinesis and further suggest that survivin participates in enucleation by a novel mechanism.
A Novel Mutation of the α Spectrin Gene in a Family of Northern European Descent Is Associated with Three Different Phenotypes.
Authors: Agarwal# Neeraj , Swierczek# Sabina , Naidoo Kubendran , Prchal Josef T. , Coetzer Theresa L. 
Published: April 2020

Abstract
Mutations of α spectrin (Sp) involving the Sp heterodimer self-association site (the α I domain of Sp) represent the most common group of membrane skeletal defects in hereditary elliptocytosis (HE) and a closely related disorder, hereditary pyropoikilocytosis (HPP). HPP is characterized by extreme anisocytic microcytosis, a severe spectrin dimer self-association defect and spectrin deficiency. The conventional explanation for the different phenotypes is that HPP subjects are compound heterozygotes for an α sp defect that interferes with sp tetramer assembly and a second defect which results in decreased synthesis of functional α sp. In contrast, HE subjects have normal spectrin content and a less severe sp self-association defect. The clinical expression of HE/HPP is influenced by the inheritance of modifying factors such as the α Sp hypomorphic mutation, α LELY. α LELY is a low expression allele of the α Sp gene characterized by a C&gt;G mutation in codon 1857 of exon 40 and a C&gt;T-12 mutation in intron 45 that is responsible for partial skipping of exon 46, which is essential for the functional assembly of α/b sp dimers. Here we describe a family of northern European descent in which members had different erythrocyte morphology ranging from atypical HPP to HE to normal and a novel αSp mutation. Quantitation of RBC membrane proteins of the propositus with atypical non-microcytic HPP revealed 48% spectrin dimers (control 10%) due to a marked increase in the 74kD αI Sp peptide. There was only a slight decrease in the spectrin/band 3 ratio, which correlated with the normocytic morphology. There was also an abnormal α Sp peptide at 41kD suggesting presence of α LELY. Sequencing of his α Sp gene revealed heterozygosity for a novel mutation in exon 2, codon 34: CGG-&gt;CCG (Arg&gt;Pro) and heterozygosity for α LELY. These mutations were also present in his brother and daughter who have HE, while another son, who had Arg34Pro, but not α LELY has repeatedly confirmed normal morphology (data in pedigree, figure 1). Ongoing clinical, α Sp peptide, DNA and quantitative Real Time PCR mRNA studies of extended family reveal a complex interaction of codon 34: G&gt;C mutation with α LELY and different levels of transcripts of α Sp alleles. #equal contribution.
Figure Figure
Dematin and Adducin Provide a Critical Link between the Spectrin Cytoskeleton and the Erythrocyte Membrane Via Glucose Transporter-1.
Authors: Khan Anwar A. , Hanada Toshihiko , Gaetani Massimiliano , Li Donghai , Reed Brent C. , Speicher David W. , Chishti Athar H. 
Published: April 2020

Abstract
There is considerable interest in the elucidation of the mechanism that governs the linkage of elongated spectrin molecules to the erythrocyte plasma membrane. The mechanism by which the “head” region of the spectrin dimer, which participates in tetramer formation, binds to the membrane via ankyrin and band 3 has been reasonably well characterized. However, the mechanism by which the tail end of the spectrin dimer is anchored to the plasma membrane is not completely understood. Dematin and adducin are actin binding proteins located at the spectrin-actin junctions or “junctional complex” in the erythrocyte membrane. Individual suppression of their function in mice by the gene deletion exerts a modest effect on erythrocyte shape and membrane stability. In contrast, the combined deletion of dematin and adducin genes results in severe defects of erythrocyte shape, membrane instability, and hemolysis. Based on these findings, we proposed a model whereby dematin and adducin could function as a molecular bridge linking the junctional complex to the plasma membrane. Using a combination of cell surface labeling, immunoprecipitation, and vesicle proteomics, we have identified glucose transporter-1 as the receptor for dematin and adducin in the human erythrocyte membrane. This finding is the first description of a transmembrane protein that binds to dematin and adducin, thus providing a rationale for the attachment of the cytoskeletal junctional complex to the lipid bilayer via glucose transporter-1. Since homologues of dematin, adducin, and glucose transporter-1 exist in many non-erythroid cells, we propose that a conserved mechanism may exist that couples sugar and other related transporters to the actin cytoskeleton.
Comparable Outcome after Allogeneic Hematopoietic Cell Transplantation from Matched Unrelated and Sibling Donors in Elderly Patients with Acute Myeloid Leukemia - Results of a Retrospective Analysis in 368 Patients.
Authors: Schetelig Johannes , Bornhäuser Martin , Schmid Christoph , Hertenstein Bernd , Schwerdtfeger Rainer , Martin Hans , Stelljes Matthias , Hegenbart Ute , Schäfer-Eckart Kerstin , Füssel Monika , Wiedemann Beate , Thiede Christian , Baurmann Herrad , Ganser Arnold , Kolb Hans-Jochem , Ehninger Gerhard 
Published: April 2020

Abstract
Purpose: In patients with acute myeloid leukemia (AML) differential indications for matched sibling and unrelated hematopoietic stem cell transplantation (HCT) are considered and arbitrary age limits for HCT exist. We sought to determine whether donor type is a prognostic factor in elderly patients in the era of high-resolution DNA-based HLA-typing.
Patients and Methods: We performed a retrospective cohort analysis in patients with AML older than 50 years who had received an allogeneic HCT between 1995 and 2005. If available, DNA from donors and recipients of unrelated HCT was used for molecular retyping in order to get information on HLA-A, -B, -C, -DRB1 and DQB1 at the allele-level. Donor-recipient pairs with fully matched donors or one mismatch out of ten alleles were considered well-matched.
Results: We identified 368 patients with cytogenetic intermediate or high risk AML who fulfilled the entry criteria. The median age of this cohort of patients was 57 years (range 50 to 73 years). 46% of patients had matched sibling donors, 3% related non-sibling donors, 41% well-matched unrelated donors and 10% poorly matched unrelated donors. In the respective period the percentage of patients with unrelated donors increased from 0% in 1995 to 64% in 2004. High risk features were a history of prior myelodyplasia in 34% of patients, poor risk cytogenetic abnormalities in 33% of patients and a disease status beyond CR1 in 62% of patients. 72% of patients received reduced-intensity conditioning regimens. Peripheral blood stem cells were used as graft in 84% of patients. In multivariate analysis disease status at HCT (p&lt;0.001) and cytogenetic risk (p&lt;0.001) proved to be highly significant predictors, both, for EFS and OS. Whereas, the relative risk of a patient with a well-matched unrelated donor compared to a sibling donor was 0.9 (95% CI, 0.6 to 1.2) for EFS and 1.0 (95% CI, 0.7 to 1.4) for OS. In subgroup analyses EFS was better in AML patients with cytogenetic high risk disease beyond first remission (CR1) (p=0.0147) who had well-matched unrelated donors compared to those with sibling donors and not inferior in any of the other subgroups.
Conclusions: Allogeneic HCT from matched unrelated donors (&gt;=9/10) should be considered equivalent to sibling HCT in terms of survival for patients above the age of 50 years with intermediate or high risk AML. In advanced stages of AML with high risk cytogenetics patients with matched unrelated donors may even have better EFS compared to those with sibling donors.
Nutritional Deficiencies in Chronically Transfused Hemoglobinopathy Patients.
Authors: Claster Susan , Wood John , Carson Susan , Hofstra Thomas , Nord Anne , Khanna Rachna , Coates Thomas 
Published: April 2020

Abstract
Vitamin and mineral deficiencies have been well described in non-transfused patients with sickle cell disease (SCD). Levels of biomarkers of oxidant damage are increased in both SCD and thalassemia major (TM), and levels of antioxidants have been shown to be lower in both diseases. Hypermetabolism, increased hemolysis and denaturation of unpaired globin chains contribute to oxidative stress in SCD and TM. Chronic transfusions, initially thought to be protective against oxidation by marrow suppression, may have the opposite effect due to the well-known propensity of iron to promote free radical generation. Methods: To test this hypothesis, we measured levels of vitamins A, B1, B6, C, D, E as well as selenium, zinc, copper and carnitine and compared them to liver iron concentrations in chronically transfused SCD and TM patients. All patients were required to fast overnight and to hold their iron chelator for 24 hours prior to blood draws. Samples were processed by routine clinical processing (Quest Nichols Institute, San Juan Capistrano, CA). Results were expressed as the percentage of patients outside the reference range for the referral laboratory. Twenty four patients with TM and 43 patients with SCD were studied.
Abnormal Values In Transfused Patients SC (%abn) TM (%abn) A 73.7 52.4 B1 38.5 37.5 B6 34.2 34.8 Folate 28.6 37.5 C 56.7 66.7 D25 74.4 50.0 D1, 25 48.6 39.1 Se 67.5 75.0 Zn 24.3 8.3 Carnitine (Free) 22.2 41.7
Results: The results in the table demonstrate that over half the patients were deficient in A, C, D and selenium. A third of the patients had low levels of B vitamins and folate (despite replacement doses in the latter), as well as carnitine. B12 levels(not shown) were normal. E levels (not shown) were low in only a few patients. Deficiency profiles were remarkably similar between the two diseases: only copper was significantly higher in the SCD patients (34.2% elevated vs 0% in TM). B6 and folate were weakly correlated with hepatic iron concentration (r2 = −0.12, −0.18). B6, folate, D25-OH, and D1,25 OH levels declined with age. These results demonstrate that chronically transfused patients have significant deficiencies in nutrients which are involved in buffering oxidant stress, regardless of their diagnosis. Severity of iron overload was not a strong predictor of these abnormalities; although the precise explanation is not known, chronic anemia may be the unifying factor. The magnitude of B1, B6, C and D deficiencies in some patients was well into the range known to cause serious complications. These combined abnormalities may contribute to the morbidity of chronically transfused patients with SCD and TM.
Rituximab Therapy for Autoimmune Hemolytic Anemia and Immune Thrombocytopenia: A Belgian Registry.
Authors: Dierickx Daan , Mineur Philippe , Triffet Agnes , Kentos Alain , Keppens Carine , Pierre Pascal , Ferster Alina , Boulet Dominique , Vermylen Christiane , Deprijk Bernard , Beghin Yves , Dresse Marie-France , Kargar Khalil , Bries Greet , Verhoef Gregor , Delannoy Andre 
Published: April 2020

Abstract
Background. Immune thrombocytopenic purpura (ITP) and autoimmune hemolytic anemia (AIHA) may respond to the chimeric anti-CD20 monoclonal antibody rituximab, even when refractory to conventional therapy.
Aims. To collect data on Belgian patients given rituximab in the setting of ITP or AIHA in order to assess the response rate and the factors predictive for response in a multicenter study.
Method. Belgian hematology centers were invited to fill a questionnaire specifying the major characteristics and quality of response of ITP and AIHA patients given rituximab. For ITP, complete response (CR) was defined as a platelet count &gt;150,000/μL, and a partial response (PR) as a platelet count 50–100,000/μL. For AIHA, Response (R) was defined as a 2g increase of the hemoglobin concentration and achievement of transfusion independence.
Results. All the patients were given rituximab after relapse or after failing at least one previous line of treatment. Except in 10 cases, rituximab was given at the dose of 375mg/m2 weekly for 4 weeks. In 31 assessable episodes of ITP a CR was achieved in 18 patients (58%) and a PR in 2 patients (6%). In 53 episodes of AIHA, R was achieved 42 times (79%). In both ITP and AIHA patients we could find no significant correlation between response and sex, age, prior splenectomy, platelet count or hemoglobin concentration when rituximab was started, number of previous treatments, response to previous treatments, duration of disease before rituximab was given, presence of an underlying malignant or autoimmune condition. In patients with AIHA, response rates were similar in cold agglutinin disease (8/10) and in warm antibody mediated hemolysis (29/38). Progression-free survival in responding patients is presented graphically. At one year, 83% of ITP patients and 61% of AIHA patients were alive without progression.
Conclusion. In this still open registry, we could confirm that rituximab induces responses in a majority of previously treated patients with ITP or AIHA. Responses could not be predicted from pre-treatment patient characteristics. In most patients response duration exceeded one year.
Progression-free survival of responding patients Progression-free survival of responding patients
Coexistence of Congenital Red Cell Pyruvate Kinase Deficiency and Hereditary Stomatocytosis.
Authors: Marcello Anna P. , Vercellati Cristina , Fermo Elisa , Bianchi Paola , Barcellini Wilma , Zanella Alberto 
Published: April 2020

Abstract
We describe a case of chronic hemolytic anemia due to the co-presence of pyruvate kinase (PK) deficiency and Hereditary Stomatocytosis (HSto). The propositus was a 30 years old adopted male with no known family history; he had severe neonatal jaundice requiring exchange transfusion, followed by a life-long history of moderate to severe chronic hemolytic anemia (Hb 7–10 g/dL), with jaundice and splenomegaly. At the age of 6 months hemoglobin screening was made and a beta trait was found. At the age of 20 splenectomy and cholecystectomy were performed. Surgery resulted in an increase of 1.5 g/dL in haemoglobin, and in a conspicuous rise of reticulocytes (from 125×109/L to 562×109/L). Two thrombotic events occurred thereafter, the former 6 days after surgery, and the latter two years later, during a toxoplasmosis infection. At the time of the study Hb was 10.8 g/dL, MCV 82.2 fL, reticulocytes 562×109/L, unconjugated bilirubin 2.19 mg/dL, LDH 335 U/L, haptoglobin &lt;20 mg/dL, serum ferritin 342 ng/mL and transferrin saturation 71%. The peripheral blood smear examination showed the presence of echinocytes (13%), stomatocytes (11%), acantocytes (10%), schistocytes (7%), elliptocytes (6%), spherocytes (4%), target cells (4%) and a few erythroblasts. Erythrocyte osmotic fragility was decreased; screening test for unstable hemoglobins and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of red cell membrane gave normal results. The study of the most important red cell enzymes revealed reduced PK activity (6.0 UI/gHb, normal range 11.1–15.59 UI/gHb) and thermal stability (43%, normal range 57–100%). Direct sequencing of PK-LR gene showed a compound heterozygosity for mutation 1456T (Arg486Trp) and the new variant −73g&gt;c. Mutation −73g&gt;c occurs in the most proximal of the four GATA motifs in the R-type promoter region and possibly result in a decrease of mRNA synthesis, as already reported for the variant −72a&gt;g (Manco et al, 2000). Molecular analysis of HFE gene showed heterozygosity for H63D mutation. The history of post splenectomy thrombosis and the presence of stomatocytes in peripheral blood smear prompted us to investigate for the coexistence of hereditary stomatocytosis. The determination of plasma potassium and sodium concentration revealed an increase in intracellular sodium (16.3 mmol/LRBC, reference range 5.0–12.0) and a decrease in intracellular potassium (74.73 mmol/LRBC, reference range 90–103), suggestive for a diagnosis of dehydrated HSto, or hereditary xerocytosis. This defect likely accounts for the thrombophilic state in this case, since HSto is known to be associated with hypercoagulability, particulary after splenectomy.