bluuebunny/crossref_metadata_2025_split huggingface dataset:
Clonality in Granulocytes Detected by an Improved HUMARA Assay: A Good Prognostic Marker in Bone Marrow Failure Patients Exhibiting Chromosomal Abnormalities of Indefinite Significance.
Authors: Ishiyama Ken , Sugimori Chiharu , Yamazaki Hirohito , Takami Akiyoshi , Nakao Shinji 
Published: October 2019

Abstract
Some patients with aplastic anemia (AA) and approximately 40% of patients with refractory anemia (RA) of myelodysplastic syndrome exhibit karyotypic abnormalities in bone marrow dividing cells. Although some of the patients undergo evolution to acute myeloid leukemia (AML), others follow a clinical course similar to AA patients without chromosomal abnormalities. Except for several abnormalities such as −7 and 5q-, the clinical significance of such chromosomal abnormalities in bone marrow failure patients remains unclear. We recently developed a reliable HUMARA assay capable of detecting a clonal population in granulocytes which constitutes 30% or more of total granulocytes (Blood. 2003;102:1211–1216). Studying correlation between chromosomal abnormalities and the presence of clonality may help in understanding the pathogenetic role of chromosomal abnormalities in AA and RA. We thus analyzed 50 acquired AA and 28 RA female patients who were heterozygous for the HUMARA gene. Chromosomal abnormalities such as add(5)(q13), 9q–9q+ and del(7)(q14q22) were found in 8% of AA and 21% of RA patients. Clonality was detected in 38% of AA patients and 39% of RA patients. Incidence of chromosomal abnormalities in patients with clonality (27%) was higher than that in patients without clonality (4%, p<0.01). In two AA patients who respectively exhibited add(5)(q13) in 10% and +8 in 38% dividing cells, clonality was not detected and these abnormal clones became undetectable at the time of subsequent bone marrow examination. Clonality was detected in the other 2 AA patients respectively exhibiting 9q–9q+ in 40% and del(7)(q14q22) in 25% dividing cells, and in all 5 RA patients respectively exhibiting +8 in 10%, del(5)(q13q31), dup(1)(q32q12) in 90%, del(5)(q13), add(11)(q23), inv(9) in 65% and X,-X in 100% of dividing cells. None of the 50 AA patients including 2 patients with clonality and chromosomal abnormalities underwent evolution to AML during 2-year follow up while one of 28 RA patients who exhibited del(5)(q13q31) progressed to AML. The proportion of clonal granulocytes in total granulocytes estimated by the HUMARA assay remained unchanged in most patients with clonality except for the transformed one. These data indicate that the chromosomal abnormality in bone marrow dividing cells is not necessarily associated with presence of clonal granulocyte population in peripheral blood and that detection of clonality in granulcytes in bone marrow failure patients with chromosomal abnormalities of indefinite significance is useful in predicting prognosis of these patients.
Global ENU Mutagenesis Screen for Genetic Modifiers in Sickle Cell Disease Mice.
Authors: Ryan Thomas M. , Huo Yongliang , McConnell Sean 
Published: October 2019

Abstract
Genetic modifiers of sickle cell disease (SCD) will be identified in an animal model of SCD after mutagenesis with the chemical mutagen N-ethyl-N-nitrosourea (ENU). This phenotype driven approach utilizes a third generation knock-in (KI) mouse model of SCD that reproduces most if not all of the pathology of the disorder. This model was produced by targeted gene replacement of the murine α-globin genes with human α-globin and the murine β globin genes with a human γ- and βs-globin gene cassette that mimics the fetal to adult hemoglobin switch that occurs in man. Sickle embryonic stem (ES) cells were derived from developing blastocysts isolated from female sickle mice that were mated with sickle males. Sickle ES cells were treated with ENU and a mutant library of independent subclones was established and archived. The optimal ENU dosage was empirically determined through a series of pilot experiments that measured the HPRT mutation frequency and the efficiency of producing sickle mice from the mutagenized cells by tetraploid embryo complementation. Animals harboring mutations that affect hematological indices, kidney function, or liver function are identified in the mutagenized sickle mice by comparison to cloned control sickle mice. Microsatellite linkage analyses of mutant offspring outcrossed to congenic SCD mice and direct sequence comparison to the murine genome will allow the positional cloning of modifier genes. Putative modifying factors will be positively confirmed by introducing the exact germline modification discovered during the ENU screen into the unmutagenized ES cells, followed by the direct examination of the phenotype in mice generated from the modified cells by cloning. These studies will define gene(s) responsible for the phenotypic variation in disease severity that is observed in the SCD population. By experimental design, the therapeutic benefit or detriment associated with each modifying gene(s) on the in vivo pathophysiology of sickle cell anemia will be tested directly in our animal model of this disorder.
Renal Involvement in Saudi Patients with Sickle Cell Disease. A Single Institution Experience.
Authors: Abdel-Razeq Hikmat N. , Bajouda Ali , Khalil Manar , Al-Shareef Muneera , Siddiqi Nauman 
Published: October 2019

Abstract
Background: Renal involvement in patients with sickle cell disease has been reported to be as high as 20%. This can take the form of proteinuria, decreased GFR, isothenuria or hematuria. Our own clinical observation of patients attending our sickle cell clinic or admitted to the medical ward at our institution suggests lower rates of renal involvement than previously reported. In this report, we evaluate Saudi patients with sickle cell disease (SS) at our institution for renal involvement.
Patients and Methods: Consecutive afebrile patients attending sickle cell clinic or admitted to inpatient wards were screened. Serum creatinine, urine analysis and 24 hr urine collection for creatinine clearance (GFR) and proteinuria were performed. Data on demographics and co-morbidities (DM, HTN, hepatitis B and C) and medications were obtained. Analysis was performed using SPSS 10.0®.
Results: 62 patients were available for study; results are summarized in tables.
Seven of 48 patients (15%) had proteinuria >300mg/d while two (4.2%) had proteinuria of more than 1.0 gram/24 hrs. Nearly 20% had GFR < 60ml/min. None of the patients had renal tubular acidosis. Serum creatinine was normal in all patients including those with low GFR and proteinuria.
Conclusions: Contrary to our initial believes, significant percentage of Saudi sickle cell patients, like others, have renal involvement with low GFR and proteinuria. Serum Creatinine alone doesn’t indicate this decline in renal function. We recommend annual evaluation for proteinuria and GFR determination in all sickle cell patients, and those at risk should be aggressively managed with nephroprotective agents like angiotensin converting enzyme (ACE) inhibitors.
Age (yrs) M:F BMI Hepatitis C Hydroxyurea NSAID 22.1+/−8.1 23:39 19.3+/−5.1 9 (17.3%) 29 (58%) 33 (66%) Urine Volume (ml/24 hrs) Specific Gravity Serum Creatinine (u mol/L) GFR Proteinuria (mg/24hrs) 1927 +/− 780 1.011 +/− 0.005 40.6 +/− 12.9 113.7 +/− 47.7 256 +/− 371 (20–2390)
A Tool for Predicting Haemoglobin Variants - an Aid to DNA Diagnostics.
Authors: Clark Barnaby E. , Cunningham Juliette C. , Wild Barbara J. , Thein Swee Lay 
Published: October 2019

Abstract
The hemoglobin (Hb) variants represent a well-documented array (>800 in number) of amino acid substitutions, hybrid Hbs and Hbs with extended/shortened amino acid sequences. The majority of these structural alterations have little or no impact on the well being of the individual. However, some can result in severe disease when inherited in a homozygous or compound heterozygous state (e.g. sickle cell disease), while others can give a thalassaemic picture if the globin variant is highly unstable. Kings’s College Hospital (KCH), which is a referral centre for Hb variants, uses automated HPLC (Biorad Variant II) as the first line screening test, followed by further electrophoretic separations (isoelectric focusing (IEF), cellulose acetate membrane (CAM) and acid gel electrophoresis (AGE)) if a variant is present. Definitive diagnosis is then achieved by either mass spectrometry or DNA analysis of the suspected globin gene. The former method is quick and uses small quantities of blood but the equipment is expensive and requires a high degree of expertise, making it unavailable to many institutions. PCR based DNA diagnostics is routine in many service pathology laboratories but it is hampered by the cost of consumables and the time it takes. To minimise these KCH has developed a database that statistically evaluates the phenotypic separations and predicts which Hb variant is present based on past data. A single entry from any one of the tests (HPLC, IEF, CAM, AGE or mass difference) is enough to generate a prediction. Using this prediction only a small region of the putative globin gene will need sequencing or analysis by restriction digest, thereby reducing costs and turnaround times. The database contains the phenotypic separation data of >700 samples collected over six years. Variants were definitively diagnosed using electrospray ionisation mass spectrometry, complemented in some cases by DNA analysis. KCH will continue to enter variant data to increase the data set and improve the tool’s accuracy and aims to make the prediction tool available online so that other laboratories may obtain predictions and add their data. Laboratories will then be able to retrieve predictions based on their own past data or the entire database.
Predictors of Renal Insufficiency in Sickle Cell Disease.
Authors: Keller Margaret A. , Borowski Meggin K. , Devoto Marcella , Li Hui-Hua , Brown Morgan , Surrey Saul , Ballas Samir K. 
Published: October 2019

Abstract
Renal insufficiency is the source of significant morbidity and mortality in adults with sickle cell disease (SCD). A population of adult patients with SCD and end-stage renal disease (ESRD) requiring hemodialysis was examined for factors that predict ESRD. ESRD patients (mean age 36.8 +/− 7.74) were compared with patients without ESRD over 50 years old (mean age 59.3 +/− 8.31). We found that the ESRD population was characterized by more males (14 males, 8 females ESRD vs. 4 males, 15 females non-ESRD), a higher occurrence of the CAR beta-like globin cluster haplotype (44 % ESRD vs. 11 % non-ESRD) and relatively lower fetal hemoglobin levels (3.6 +/− 2.16 ESRD vs. 8.6 +/− 6.35 non-ESRD). In order to further examine potential predictors of renal disease, including genetic modifiers unlinked to the beta-globin cluster, we examined a broader population of patients with elevated creatinine, defined as levels greater than 1.0 mg/dl, who did or did not require hemodialysis. We hypothesize that gender, beta-like globin cluster haplotype, fetal hemoglobin levels and polymorphisms in critical regulators of vasomotor tone are predictors of renal insufficiency in SCD. Genotyping of a single nucleotide polymorphism (SNP) in endothelial nitric oxide synthase (eNOS or NOS3) and an insertion/deletion polymorphism (I/D) in the angiotensin-converting enzyme (ACE) gene will allow us to determine if these polymorphisms are over-represented in this cohort. The eNOS T-786C SNP alters the level of transcription of the eNOS gene and the amount of eNOS activity. We recently showed that this polymorphism was associated with a history of acute chest syndrome in females with SCD (Br. J. Haematol., 2004, 124:240). The ACE I/D polymorphism has been associated with altered plasma ACE levels. Twenty-one adult patients with sickle cell disease with renal insufficiency and 25 age- and gender-matched control patients seen at Thomas Jefferson University are being examined. Genotype results and statistical analysis will be presented. It is hoped that identification of predictors of renal insufficiency in patients with SCD might lead to early identification of at-risk patients, allowing for therapeutic intervention.
Two Novel Mutations in the α-Globin Genes Leading to α-Thalassemia.
Authors: Rund Deborah , Koren Ariel , Zalman Lucia , Ben Barak Ayelet , Oppenheim Ariella , Filon Dvora 
Published: October 2019

Abstract
Although deletional α-thalassemia is the most common form of the disease worldwide, in Israel, point mutations leading to α-thalassemia are relatively common. We routinely sequence the α-globin genes when we fail to find deletions which can account for the hematological phenotype. Our PCR is specific for sequencing either the α1 or α2 globin gene. We here report two novel mutations, one in α1 and the other in α2, which lead to a thalassemia phenotype. Case 1. A 5 yr old Druze girl from northern Israel was referred due to microcytic anemia. Her hematological values were: RBC 5x109/L, Hb 8.8 gr%, MCV 55 fl, MCH 17.6 pg, MCHC 31.7gr/dl, RDW 16.2. HbA 2: 2.2%, HbF: 1%. Iron deficiency was excluded (serum iron: 58 mg/dl, transferrin: 247mg%, and ferritin: 46.2 ng/ml) The patient’s physical examination and mental development were normal, without splenomegaly or jaundice. No therapy was required, and transfusion was unnecessary. The parents had very mild microcytic anemia (mother Hb 11 gr% MCV 75.5, father Hb 13.8, MCV 78.6) with normal Hb electrophoresis. DNA of the parents and propositus was sequenced. The child was found to be homozygous (and the parents, heterozygous) for a deletion of a single cytosine (within a stretch of four) at codons 118/119 of the α2 globin gene, leading to a frameshift at codon 119. Case 2. This family is of mixed ancestry, the mother is from Bukhara. The hematological data is presented in the table below. Sequencing of the α-globin genes of the mother revealed an A to G change at the acceptor site of IVS1 at nt 116 in the α1 gene in compound heterozygosity with the well known 5 nt deletion in IVS1 of α2 (HphI). The same IVS1 nt 116 mutation at this exact nucleotide was previously reported by Harteveld (1996) to be found in the α2 globin gene, causing Hb H inclusion bodies when found in combination with 3.7 kb deletional α-thalassemia. We conclude that rare point mutations can still be identified in the α-globin genes which can be the cause of otherwise unexplained α-thalassemia phenotypes.
Family 2 hematological data and alpha globin genotype
Family member RBC Hb MCV MCH RDW chromosome 1 chromosome 2 del=deletion Mother 5.68 10.7 63.2 18.8 16.4 α2 HphI α1 IVS1-116 Father 6.15 13.4 70.7 21.8 13.9 del 3.7kb del 3.7 kb Son (9 yrs) 5.59 11.1 64.4 19.9 13.7 α2 HphI del 3.7 kb Daughter (7 yrs) 5.44 10.9 64.3 20.0 13.5 del 3.7 kb α1 IVS1-116 Son (2 yrs) 5.55 10.5 61.6 18.9 14.3 del 3.7 kb α1 IVS1-116
Reappraisal of Symptoms and Signs of Uncomplicated Beta Thalassemia Trait.
Authors: Premawardena A. P. , Malewana T. , Arambepola M. , Olivieri N. F. , Weatherall D. J. 
Published: October 2019

Abstract
Although considered a mild disorder, beta thalassemia trait (uncomplicated by any other hematologic abnormality) has been reportedly associated with symptoms of anemia, while in other series hepatosplenomegaly has been reported as associated with this diagnosis. No controlled study has examined the symptoms and signs of individuals with beta thalassemia trait and compared these with age- and sex-matched normal individuals. We administered a questionnaire to 397 parents (67% females) of children with beta thalassemia major attending the National Thalassemia Centre in Kurunagala, Sri Lanka, and to 87 normal volunteers (90% females). All individuals underwent physical examination by the same investigator, as well as testing by automated cell counter (Coulter), and HPLC (Bio Rad). The mean age of the parents (33 years) and controls (29.5 years) was not significantly different. In parents with thalassemia trait, 20% of males and 42% of females complained of reduced exercise tolerance compared to 12.5% of males and 38% of females in the controls (P= NS). The questionnaire revealed that 41% of parents, and 36% of controls, complained of recurrent headache; 18% of parents, and 15% of controls, complained of lethargy (P=NS). A history of malaria infection was reported in 30% of parents with thalassemia trait, and in 24% of controls. Mean quality-of-life score, recorded formally by a linear analog scale, from 0 (poorest) to 10 (greatest) was 7.1 in parents and 7.2 in controls. Splenomegaly was detected in 6% of parents with beta thalassemia trait (mean spleen size 1.5 cm below the costal margin), and 5% of normal controls (mean spleen size 1 cm below the costal margin). These findings suggest that, by contrast to many reports in the older literature, in uncomplicated beta thalassemia trait there appears to be no symptoms or signs greater than those observed in a normal control population and that fatigue, lethargy, exercise intolerance, or splenomegaly in a patient with beta thalassemia trait should not be attributed to the underlying hemoglobinopathy.
Preferential Uptake of Gamma Tocopherol by Mast Cells. Does This Uptake Affect Mast Cell Cytokine Production?.
Authors: Aljitawi Omar S. , Fitzgerald Mathew , Qui Min , Krishnan Koyamangalath , Stone William L. , Krishnaswamy Guha 
Published: October 2019

Abstract
Vitamin E, the main lipid soluble antioxidant, exists in eight different forms, of which α-tocopherol and γ-tocopherol are the two major forms. Previous experiments showed vitamin E uptake by macrophages that contribute to inflammation and immunity. On the other hand, vitamin E has structural similarity to the thiazolidinedione, troglitazone, a peroxisome proliferator-activated receptor (PPAR) agonist. In previous experiments we found that troglitazone (TGZ), a PPAR gamma agonist, had a negative effect on mast cell cytokine production. We therefore wondered whether vitamin E enters human mast cells, and if so, does this modulate mast cell cytokine production? We choose (interleukin) IL-6 for its pro-inflammatory properties and because it’s known to be produced by mast cells in response to stimulants used in the experiment. In this study we try to answer these two questions. Cultured human mast cell line (HMC-1) was first incubated for 24 hrs with pharmacological concentrations of both alpha and gamma forms of vitamin E (10 μM). The mast cells were then activated with phorbol-12-myristate-13-acetate [PMA (50ng/ml)] and ionomycin (5μm) for 24 hours and cell-free supernatants collected. In additional experiments, IL-1ß (10ng/mL) was added to activate mast cells. IL-6 levels in the supernatants were determined in each well utilizing ELISA. Mast cell concentrations of alpha or gamma tocopherol were measured by high pressure liquid chromatography [HPLC] and electrochemical analysis. Mast cells pre-incubated in alpha and gamma forms of vitamin E at 10 μM did not affect mast cell IL-6 production. Mast cells, however, showed uptake of both forms of tocopherols but more pronounced uptake of the gamma form (13181.05 pmole/well of gamma compared to 8742.99 pmole/well of alpha tocopherol). We conclude that mast cells appear to store both alpha and gamma tocopherols but preferentially more gamma tocopherol. Though Vitamin E and PPAR agonists have similar structures, they did not show similar effect on mast cell cytokine production, suggesting they might have different mechanisms of action.
IL-6 Produced by Type I IFN DC Controls IFN-γ Production during the MLR by Blocking the Suppressive Effect of Regulatory T Cells.
Authors: Detournay Olivier , Mazouz Naima , Goldman Michel , Toungouz Michel 
Published: October 2019

Abstract
The dendritic cell family is composed of different subsets able to differentially govern the immune response. Their potent antigen presenting properties make them an attractive candidate for immunization against pathogens or cancer. In that setting, the recently characterized type I IFN DCs present interesting features including a higher expression of molecules involved in antigen presentation and the ability to trigger both the cellular and humoral arms of the immune responses. In view of the pivotal role of regulatory T cells in limiting the effectiveness of effector cells, we analyzed the interactions between these cells and type I IFN DC. DC generated from monocytes in the presence of IFN-β and IL-3 (DCI3) were activated by the maturation agent poly I:C and compared with the classical myeloid DC generated in the presence of GM-CSF and IL-4 (DCG4). Despite the release of lower amounts of IL-12 after maturation, DCI3 were able to induce a higher IFN-γ production by T lymphocytes during the MLR. Analysis at the mRNA level disclosed that DCI3 over transcribed the IL-6 gene leading to the release of high amounts of the protein both after the maturation process and during the MLR itself. Neutralization of IL-6 revealed that this cytokine specifically contributed to the IFN-γ release induced by DCI3. Finally, depletion of CD25+ T cells prior to the MLR identified these cells as a target for IL-6. We conclude from these results that DCI3 are endowed with the unique property of blocking the suppressive effect of regulatory T cells through high IL-6 production during the MLR. This novel mechanism of T cell control is relevant for the use of this DC type in vaccination strategies.
Clinical-Grade Type I IFN-DC Differentiated in the Presence of Either IL-3 or GM-CSF Show Similar Phenotypic and Functional Proprieties.
Authors: Mazouz Naima , Detournay Olivier , Rennesson Joelle , Lambermont Micheline , Marchant Arnaud , Goldman Michel , Toungouz Michel 
Published: October 2019

Abstract
Despite limited clinical efficacy in large trials, dendritic cells (DC)-based immunization has yielded impressive responses in some patients. Key questions remain to be solved in order to optimize this therapeutic vaccine. Among them, the nature of the DC used and its state of maturation are pivotal. Besides myeloid DCs which are the cells essentially used in clinical trials, a new type of DC has been described resulting from the differentiation of monocytes in the presence of type I IFNs. In the present study, we analyzed the features of clinical grade type I IFN DC generated in the presence of either IL-3 (IL-3- DCs) or GM-CSF (GM-CSF-DCs) and compared their capacity to respond to poly I:C, a double-stranded RNA analog that mimics viral infection. The two DC types disclosed a similar immunophenotype characterized by high levels of costimulatory molecules, CCR7, HLA-class-I and class-II molecules. After poly I:C maturation, both DC types displayed a marked upregulation of CD80, CD83 and CD86. In addition, poly I:C stimulated them to secrete IFN-α IL-12 p70, IL-12 p40 and IL-6. Both DC types elicited potent allogeneic reactions. Priming of autologous T cells by IL-3-DCs or GM-CSF-DCs pulsed with an HLA-A2 restricted melan-A derived peptide, lead to expansion of melan-A specific CTL secreting high levels of IFN-γ and displaying a phenotype of memory cells. We conclude that mature clinical grade IL-3-DCs and GM-CSF-DCs share similar phenotype and functional properties including the capacity to prime Ag-specific CTL.