bluuebunny/crossref_metadata_2025 huggingface dataset:
Cbl-Deficient Mice Display Erythropoietin Hypersensitivity and Enhanced Phosphorylation of Protein Kinase B/Akt.
Authors: Richmond Terri D. , Bailey Monica L. , Langdon Wallace Y. , Barber Dwayne L.
Published: April 2020

Erythropoietin (EPO) is the primary cytokine regulator of erythropoiesis, stimulating growth, preventing apoptosis, and promoting differentiation of red blood cell progenitors. The critical importance of EPO, EPO receptor (EPO-R) and JAK2 to erythropoiesis is demonstrated by the fatal embryonic anemia that develops in EPO, EPO-R or JAK2 knockout mice. Intracellular signal transduction pathways regulating growth, differentiation and cell survival downstream of the EPO-R and JAK2 are well documented. However, activation of the EPO-R is transient and down regulated by several negative regulators including tyrosine phosphatases, inositol phosphatases and ubiquitin ligases.

The negative regulator, Cbl, has been implicated as a tumour suppressor in murine sarcoma, B cell leukemia, and erythroleukemia. More recently, Cbl was found in a de novo form of acute myeloid leukemia and has been implicated in the formation of gastric tumours. Cbl is known to bind, ubiquitinate, and downregulate signaling from numerous activated hematopoietic and non-hematopoietic receptors. The discovery that Cbl is a target of EPO-dependent tyrosine phosphorylation, together with the finding that the EPO-R is ubiquitinated in vivo, led us to hypothesize that Cbl deficiency leads to altered murine erythropoiesis.

Resting C57Bl/6 Cbl-/- mice display normal hematologic parameters with the exception of an increased platelet count. However, Cbl deficient mice respond to phenylhydrazine-mediated anemia with increased reticulocyte production and hematocrit recovery. The hypersensitivity of Cbl deficient mice to anemia may be explained by a three-fold enhancement of splenic colony forming unit-erythroid (CFU-E) and an overall increase in burst forming unit-erythroid (BFU-E) and CFU-E. Furthermore, the elevated sensitivity of Cbl deficient mice to anemia is echoed by increased EPO-R and protein kinase B (PKB)/Akt phosphorylation in splenic erythroblasts at high levels of EPO stimulation.

Erythrocyte differentiation was examined by monitoring the expression of the erythroid markers CD71 (Transferrin Receptor) and Ter119. Cbl deficient mice do not have significantly more proerythroblasts than wild-type mice. Interestingly, Cbl deficient mice show impaired erythroid maturation with a 1.7 fold decrease in orthochromatophilic erythroblast levels, elevated erythroid apoptosis in the bone marrow, and no compensation by splenic erythroblast production.

These data (as well as earlier studies from our laboratory with STAT1-/- and SHIP-1-/- mice) illustrate the remarkable ability of the spleen to compensate for alterations in bone marrow erythropoiesis. It also suggests that Cbl regulates pathway(s) associated with regulation of erythroid cell survival through regulation of the PI 3 kinase, PKB/Akt signaling cascade.
GATA Motif on the Erythropoietin Gene Promoter Is Essential for Repression of Ectopic Constitutive Erythropoietin Production.
Authors: Obara Naoshi , Suzuki Norio , Ki-Bom Kim , Imagawa Shigehiko , Nagasawa Toshiro , Yamamoto Masayuki
Published: April 2020

In response to anemia erythropoietin ( Epo) gene transcription is markedly induced in the kidney and liver. This regulation has been ascribed for the promoter GATA motif and 3′ enhancer. To elucidate how the Epo gene expression is regulated in vivo, we established transgenic mouse lines expressing green fluorescent protein (GFP) under the control of 180-kb mouse Epo gene locus. GFP expression was induced by anemia or hypoxia specifically in peritubular interstitial cells of renal cortex and hepatocytes surrounding the central vein. Surprisingly, the renal Epo-producing cells were identified with neuron-like morphology and expression of neuronal markers. We also examined the regulatory mechanism of Epo gene using the transgenes in which mutations had been introduced in the GATA promoter motif, since we have reported the motif as a negative regulatory element for the inducible Epo gene expression. A single nucleotide mutation in the motif resulted in constitutive ectopic expression of GFP in the renal distal tubules, collecting ducts and certain epithelial cells of other tissues. Since both GATA-2 and GATA-3 bind to the GATA box in the distal tubular cells, these factors are likely to constitutively repress ectopic Epo gene expression in these cells. Thus, GATA-based repression is essential for the inducible and cell-type specific expression of the Epo gene.
The Sickle Cell-Pulmonary Hypertension Screening Study: ECHO Findings at Two-Years of Follow Up.
Authors: Castro Oswaldo , Kato Gregory , Sachdev Vandana , Machado Roberto , Ernst Inez , Coles Wynona A. , Nichols James S. , Hunter Lori A. , Gladwin Mark T.
Published: April 2020

In a prospective study started in 2001 echocardiography (ECHO) was used to screen 195 unselected adult sickle cell patients for pulmonary hypertension (PHTN). The prevalence of PHTN [tricuspid regurgitant jet velocity (TRV) of 2.5 m/sec or higher, corresponding to a pulmonary artery systolic pressure (PAs) of at least 35 mm Hg] was 32 %. Markers of hemolysis (low Hb, and high reticulocytes and LDH) were associated with PHTN risk. Although the pulmonary pressures in sickle-related PHTN were not as high as in other forms of PHTN, they were predictive of short survival (NEJM2004;350:886).

We now report the TRV results in 113 patients who had repeat ECHO exams at 2.1 years (range 1.2–4.1 y) after study enrollment. Their mean age was 36.2 ± 10.3 y and 59 % were female. Their hemoglobin types were: SS, 77.7 %; SC, 14.3 %; Sickle-thal.+, 6.3 %; and Sickle-thal.0, 1.8 %. At baseline, 40 patients had PHTN (mean TRV 2.75 m/sec) and their mean TRV at the 2 y follow up was 2.71 m/sec (p=0.45). In 6 subjects the TRV decreased to normal values from baselines of 2.5 – 3 m/sec. Nevertheless, the mortality of the patients diagnosed with PHTN at baseline currently stands at 40% at 40 months of follow-up. Seventy-three patients did not have PHTN at enrollment (mean TRV 2.0 m/sec) and as a group their TRV at the 2 y follow up had not changed significantly (mean 2.1, p=0.1). However, 11 of the 73 patients (15.1%) with initially normal TRV developed PHTN at 2 years (mean TRV 2.70, range 2.5–3.5 m/sec). The table shows that age, sex, Hb type, Hb F %, and hydroxyurea treatment of the patients who developed PHTN at the 2 y follow up were not significantly different from those who did not.

However, patients who developed PHTN were significantly more anemic, had higher reticulocyte counts, and higher serum ferritin values. A trend toward an increased bilirubin and LDH was observed. Hence, hemolysis probably played a pathogenetic role in these patients’ increased pulmonary pressures, as is also postulated for sickle patients who had PHTN at baseline.

These preliminary results suggest that the incidence of PHTN in adult sickle patients could be as high as 7 % per year and that ECHO screening of these patients every two years is probably indicated.

No PHTN PHTN P value N 62 11 Mean age, y (±SD) 34.1 ± 8.9 34.6 ± 13.4 0.85 Sex (% female) 56 64 0.75 Non-SS (%) 27 18 0.5 On hydroxyurea (%) 41 25 0.5 Hb (g/dl, mean±SD) 9.98 ± 1.74 8.68 ± 1.91 0.04 Hb F (%,mean±SD) 7.9 ± 6.8 7.5 ± 7.4 0.869 Abs. Retics (x1000/mm3, mean±SD) 205 ± 107 288 ± 151 0.045 LDH (U/L, mean±SD) 286 ± 107 359 ± 179 0.09 Bilirubin (mg/dl, mean±SD) 2.5 ± 1.88 3.7 ± 2.58 0.10 Ferritin (mg/L, mean±SD) 632 ± 925 1439 ± 1849 0.046 Creatinine (mg/dl, mean±SD) 0.76 ± 0.23 0.69 ± 0.25 0.36
Insulin-Like Growth Factor II Is an Autocrine Growth Factor To Prevent Apoptosis as Well as To Enhance Proliferation and Maturation of Erythroid Progenitor Cells.
Authors: Nagatomo Taro , Muta Koichiro , Ohga Shouichi , Ochiai Masayuki , Hikino Shunji , Hara Toshiro
Published: April 2020

To reveal the novel function of erythroid progenitor cells in fetal erythropoiesis, the gene expression pattern in umbilical cord blood (CB)-derived CD36+ erythroid progenitor cells (EPCs) was analyzed using cDNA microarray containing 240 cytokine and growth factor related genes. Among the genes analyzed, insulin-like growth factor II (IGF-II) gene showed a 124-fold higher level of expression in CB-EPCs, compared with that seen in phytohemagglutinin (PHA)-stimulated adult peripheral blood mononuclear cells (PBMCs) (table1). Real-time PCR revealed that the IGF-II mRNA levels in CB-EPCs were higher than those seen in lymphocytes or monocytes separated from CB or PBMCs. When CB-EPCs were cultured with erythropoietin (EPO) in serum-free medium, the number of erythroid colonies was decreased in the presence of IGF-II-neutralizing antibody. To further assess the role of IGF-II in erythropoiesis, we purified erythroid colony-forming cells (ECFCs) from human umbilical cord blood by magnetic selection and liquid culture with IL-3, stem cell factor and EPO. The expression levels of IGF-II, type 1 or 2 IGF receptor in the mature ECFCs were higher than those in the immature ECFCs. Addition of IGF-II-neutralizing antibody decreased the number of ECFCs in liquid culture with EPO. The anti-proliferative effect of IGF-II-neutralizing antibody was evident in both dimethylthiazole tetrazolium bromide (MTT) and bromodeoxyuridine (BrdU) incorporation assays. When apoptosis of cells was examined using Annexin V, the addition of IGF-II-neutralizing antibody increased apoptosis, thereby indicating the anti-apoptotic effects of IGF-II secreted from erythroid cells. Furthermore, ECFCs failed to undergo normal erythroid maturation in the presence of IGF-II-neutralizing antibody, as assessed by flow cytometric and morphologic analyses. These findings indicate that IGF-II, which is produced by erythroid progenitor cells themselves, has crucial roles in controlling erythropoiesis by modulating apoptosis, proliferation and maturation via an autocrine mechanism.

Cytokine and growth factor genes highly expressed in CB-EPCs selected by cDNA microarray analyses ratio gene 124.0 insulin-like growth factor II 7.9 vascular endothelial growth factor 3.9 interleukin 8 3.4 GRO2 oncogene 3.0 GRO1 oncogene 1.6 transforming growth factor, beta 1 1.4 vascular endothelial growth factor B
Delta-1 Promotes Soluble IL-6R-Mediated Action of IL-6 on Human Erythropoiesis While Counteracting the Effect of IL-6 on the Generation of Myeloid-Lineage Cells.
Authors: Yamamura Kentaro , Ohishi Kohshi , Katayama Naoyuki , Shiku Hiroshi , Nishikawa Mitsuo , Nakahata Tatsutoshi
Published: April 2020

Notch ligand Delta-1 is shown to influence proliferation and differentiation of hematopoietic progenitors by interacting with various cytokines. IL-6 exerts various effects on human hematopoiesis by activating gp130 via soluble IL-6 receptor (R) as well as membrane-bound IL-6R. However, little is known about the interaction between Delta-1 and gp130 activation in human hematopoiesis. We previously reported that Delta-1 in combination with IL-6R/IL-6 fusion protein FP6, which directly and agonistically activates gp130, enhances the production of mixed and pure erythroid progenitors from cord blood CD34+CD38− cells, while not significantly affecting the generation of myeloid progenitors (ASH 2004). Here, we examined the interaction between Delta-1 and gp130 activation in the generation of erythroid cells and myeloid-lineage cells from cord blood CD34+CD38− cells in serum-free suspension cultures. In the presence of SCF, Flt3L, TPO, and IL-3 (4GF), Delta-1 acted synergistically with FP6 to promote the production of CD36+glycophorin A (GPA)high erythroid cells from CD34+CD38− cells, whereas few CD36+GPAhigh erythroid cells were generated by Delta-1, IL-6, and Delta-1 plus IL-6. When we assessed the stage of erythroid cells affected by Delta-1 and FP6, Delta-1, in conjunction with FP6, augmented the generation of CD36+GPAhigh erythroid cells from CD34+CD38− cell-derived CD36+GPA− erythroid progenitors equivalent to BFU-E stage but not from CD34+CD38−cell-derived CD36+GPAlow erythroid progenitors equivalent to CFU-E stage. On the other hand, IL-6 and FP6 increased the production of CD14+ monocytic cells and CD15+ granulocytic cells from CD34+CD38− cells. Delta-1 had opposite effect and antagonized the stimulatory effect of IL-6 and FP6 on the generation of monocytic and granulocytic cells. Although the development of CD1a+CD14− dendritic cells from CD34+CD38− cells was suppressed by IL-6 and FP6 but instead potentiated by Delta-1, Delta-1 competed with the inhibitory effect of IL-6 and FP6 on the generation of dendritic cells. These data indicate that Delta-1 modulates the effect of IL-6 on human hematopoiesis by enhancing its soluble IL-6R-mediated action on early erythropoiesis while antagonizing its effect on the generation of myeloid-lineage cells.
Gfi-1 Represses C/EBPe and Is Required for the Survival and Proliferation of Myeloid Cells Stimulated by G-CSF.
Authors: Dong Fan , Zhuang Dazhong , Qiu Yaling
Published: April 2020

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic cytokine that plays a major role in granulopoiesis. G-CSF supports the proliferation, differentiation and survival of myeloid progenitor cells. Treatment of cells with G-CSF activates multiple intracellular signal transduction pathways leading to alterations in the activities of transcription factors involved in myeloid development. Gfi-1 is a zinc-finger transcriptional repressor that is required for granulopoiesis. Mutations in Gfi-1 have been associated with severe congenital neutropenia (SCN). How Gfi-1 acts in myeloid cells is still poorly understood. Expression of Gfi-1 was upregulated during granulocytic differentiation induced by G-CSF in myeloid 32D cells. Truncation of the carboxy terminus of the G-CSF receptor, as seen in patients with acute myeloid leukemia (AML) evolving from SCN, disrupted the ability of the G-CSF receptor to upregulate Gfi-1. Ectopic expression of a naturally occurring dominant negative Gfi-1 mutant, N382S, identified in patients with SCN, resulted in premature apoptosis and reduced proliferation of cells induced to differentiate with G-CSF. The expression of neutrophil elastase (NE) and C/EBPe was significantly increased in 32D cells expressing N382S. In contrast, overexpression of wild type Gfi-1 abolished G-CSF-induced upregulation of C/EBPe, but had no apparent effect on NE upregulation by G-CSF. Notably, G-CSF-dependent proliferation and survival were inhibited upon overexpression of C/EBPe, but not NE. These data suggest that a key role of Gfi-1 in granulopoiesis is to repress C/EBPe and that overt C/EBPe overexpression may be deleterious to the proliferation and survival of myeloid cells. Our data may explain why loss of Gfi-1 function, either as a result of gene knockout in mice or gene mutations in human, results in a block of granulocytic differentiation and severe neutropenia.
The IL-7 Receptor alpha Promoter Is Activated at Distinct Stages during B Cell Development by the Ets Transcription Factors PU.1 and GABP.
Authors: DeKoter Rodney P. , Schweitzer Brock L. , Jones Darrel , Hildeman David A. , Huang Kelly J.
Published: April 2020

The cytokine interleukin-7 (IL-7) is required for B and T lymphocyte development, and for the survival and maintenance of both naive and memory T lymphocytes. The receptor for IL-7 (IL-7R) is heterodimeric, consisting of a common gamma chain (γc) and an alpha subunit (IL-7Rα). The γc is expressed in most hematopoietic cell types, but the IL-7Rα gene is regulated in a cell type and developmental stage-specific manner. We have previously shown that the Ets-family transcription factor PU.1 is required to activate transcription of the IL-7Rα gene during fetal lymphocyte development. However, several questions remain unanswered. First, the IL-7Rα promoter is poorly characterized. Second, the IL-7Rα is expressed at high levels in the T cell lineage where PU.1 is not expressed. Third, the transcription factor early B cell factor (EBF) can activate IL-7Rα transcription in developing B cells in the absence of PU.1. To address these questions, we have characterized the IL-7Rα promoter region in detail. First, we determined that the major transcription start sites in the IL-7Rα gene are downstream of an Ets/PU.1 binding site. We found that the intact Ets site is required for IL-7Rα promoter activity, as well as to mediate enhancer action from a distance. IL-7Rα promoter activity depends on the proper orientation of the Ets site relative to functional initiator sequences. We found, using gel shift analysis, that both PU.1 and the Ets transcription factor GA binding protein (GABP) are expressed in developing B cells, and can interact with the Ets binding site in the IL-7Rα promoter. However, the function of PU.1 is distinct from GABP during B cell development. Retroviral transduction of PU.1 mutant progenitor cells with a PU.1 retrovirus robustly rescues IL-7Rα transcription and IL-7-dependent B cell development. In contrast, transduction with GABPα and GABPβ1 subunits fails to activate IL-7Rα transcription in PU.1 mutant progenitor cells. We conclude that activation of the IL-7Rα gene requires PU.1 during the earliest stages of lymphocyte development, but is alternatively utilized by PU.1 and GABP after commitment to the B cell lineage.
Asthma Is Associated with Mortality in Children with Sickle Cell Anemia.
Authors: Boyd Jessica H. , Macklin Eric , Strunk Robert C. , DeBaun Michael R.
Published: April 2020

Background: Acute chest syndrome (ACS) is one of the leading causes of premature death among people with sickle cell anemia (SCA). Children with SCA and asthma have an increased risk of ACS (Boyd et al, Pediatric Pulmonology, 2004). The Cooperative Study of Sickle Cell Disease (CSSCD) has previously reported the median age at death to be approximately 45 years, 42 years for males and 48 years for females (Platt et al, New England Journal of Medicine, 1994). The objective of this study is to determine if asthma in patients with SCA is associated with mortality.

Methods: The cohort was comprised of African American participants with SCA anemia enrolled in the CSSCD from birth to 66 years of age who were followed beyond five years and were evaluated for asthma. Patients who died before age 5 years were not included in the cohort. Asthma was classified by physician diagnosis, documentation of an acute asthma event, and/or record of use of prescribed asthma medication. Mortality was determined for patients with and without asthma. Cox regression analysis included the following established risk factors for death (Platt et al, 1994): covariates age, white blood cell count level, renal failure, hemoglobin F level, seizure, and acute chest syndrome.

Results: A total of 1963 African American individuals with SCA were classifiable for asthma, enrolled in the study and followed for a total of 18,496 patient-years. The average age at study entry was 13.9 years, and the mean length of follow-up was 9.4 years. A total of 138 individuals (7.0%) had asthma; 70% were classified by a physician diagnosis, 10% by documentation of an acute asthma event, and 20% by record of using a prescribed asthma medication. Individuals with asthma had a median lifespan of 52.5 years in comparison to 64.3 years and over a two-fold higher risk of mortality after controlling for age, white blood cell count, renal failure, hemoglobin F level, seizure, and acute chest syndrome rate (hazard ratio 2.36 by Cox regression, 95% CI 1.21 to 4.62, p=0.01).

Conclusion: Asthma was associated with a significant increase in the risk of all-cause mortality among patients with SCA who lived to 5 years of age. In addition to previously identified risk factors, asthma is an independent predictor of mortality.
Fetal Hemoglobin Protects Against Activation of Hemostasis Endothelial and Circulating Blood Cells in Sickle Cell Anemia.
Authors: Setty Yamaja , Vaidyula Vijender R. , Dampier Carlton D. , Rao A. Koneti , Stuart Marie J.
Published: April 2020

To determine whether fetal hemoglobin (HbF) protects against activation of hemostasis, endotheliium, and circulating blood elements, infants, children and young adults with homozygous SS disease (n=35) were studied. F-cell numbers, biomarkers of endothelial activation (including sVCAM-1, sE- and sP-selectin), white cell and platelet activation (sL-selectin and sP-selectin respectively) and markers of hemostatic activation, including D-Dimer and prothrombin fragment F1.2 were analyzed. Subjects with HbSS demonstrated significant increases (P<0.001) in all cell activation and hemostatic biomarkers. Analyses revealed a dominant, statistically significant correlation between F-cell and biomarkers of endothelial, white cell and hemostatic activation. F-cells correlated inversely with sVCAM-1 (R = −0.64, P<0.00005), sE-selectin (R = −0.51, P = 0.03) and sP-selectin (R = −0.41, P<0.02). Since HbF protects against HbS polymerization, our data suggests a role for the sickle erythrocyte in endothelial activation and is consistent with reported decreases in sVCAM-1 following hydroxyurea therapy or a chronic transfusion regimen. sP- and sE-selectin levels correlated with neutrophil counts (R = 0.63 and R = 0.42, P<0.00006 and P<0.02 respectively) while no relationship between platelets and sP-selectin was noted. An additional correlation between sP- and sE-selectin (R = 0.50, P<0.003) was also observed, reflecting enhanced neutrophil-endothelial interaction as part of an inflammatory and prothrombotic HbSS phenotype. Surprisingly, a positive correlation between sL-selectin and F-cells occurred (R = 0.4, P<0.02) with the young infant with HbSS demonstrating higher levels of this leucocyte activation marker than the older subject. A dysregulation of L-selectin shedding following neutrophil activation has been proposed to occur in sickle cell anemia. Our results suggest that HbF protects against this dysregulation. D-Dimer levels correlated negatively with F-cells (R = −0.76, P<0.000001), with a striking relationship between D-Dimer and markers of endothelial activation, including sVCAM-1 (R = 0.53, P<0.001), sE-selectin (R = 0.39, P<0.03) and sP-selectin (R = 0.41, P<0.02). Such findings are in keeping with D-Dimer’s well-documented role as a biomarker in other inflammatory and ischemia-related disorders including coronary, cerebrovascular and peripheral arterial ischemic states. This report expands our knowledge regarding the protective effects of HbF against endothelial and hemostatic activation, and suggests a role for D-Dimer as a prothrombotic and proinflammatory marker in sickle cell anemia.
Do Pediatric Sickle Cell Disease Patients Develop Diastolic Dysfunction?.
Authors: Zilberman Mark V. , Du Wei , Whitten-Surney Wanda J. , Sarnaik Sharada A.
Published: April 2020

Sickle cell disease (SCD) is a known cause of chronic volume overload. Although systolic dysfunction is rare, as many as 80% of adult patients with SCD have an echocardiographic mitral valve inflow pattern suggestive of abnormal left ventricular (LV) diastolic function (DF). DF has not been studied in pediatric SCD patients. Therefore, the objective of this study was to evaluate DF in pediatric SCD patients using echocardiographic velocities of mitral inflow and tissue Doppler (TDI) indices. Echocardiograms were performed in 79 SCD patients (ages 2 – 18 years), and 84 controls matched for age and race. LV and left atrial (LA) volumes, LV mass, early (E) and late (A) mitral inflow velocities, and TDI velocities at the septal and lateral aspects of the mitral valve were obtained. LV dilatation was defined as LV end-diastolic dimension more then 2 standard deviations above the median (z-value >2). LV hypertrophy was defined as LV mass/volume index>1.15 g/ml. For SCD patients, average hemoglobin (Hb) levels for the year prior to the study were recorded. Data were analyzed using t-test and Spearman correlation analysis.

Of 79 SCD subjects, 32 (43%) had LV dilatation, 11(14.9%) had LV hypertrophy and 3 had both. LV z-scores were inversely related to Hb (r −0.62, p<0.0001) but unrelated to age. The early mitral inflow velocities E were negatively correlated with Hb levels(r − 0.34, p<0.04) and were positively correlated with LV z-scores (r 0.41, p<0.04) reflecting pre-load dependency of mitral inflow indices. DTI diastolic velocities were unrelated to Hg or LV size reflecting pre-load independent nature of these indices. DTI systolic velocities S’ correlated positively with LV mass/volume index (r 0.28, p<0.02).

SCD patients had significantly larger LV and LA volumes than controls (p<0.01). The early E and late A mitral inflow velocities were higher in the SCD group than the control, although the differences were not statistically significant. DTI indices did not differ between SCD patients and the control.

Conclusion: Despite high incidence of left ventricular dilatation, pediatric SCD patients don’t demonstrate diastolic dysfunction when evaluated using pre-load independent echocardiographic tissue Doppler indices.
The Determination of the Inhibitory Activity of Some Factor VIII Type II Inhibitor Antibodies Is Biased by the Type of Factor VIII Assay.
Authors: Jacquemin Marc G. , Lamprecht Seb , Lavend’homme Renaud , Roodt Jan , Chatelain Bernard , Deneys Veronique , Dewaele Jeroen , Stassen Jean-Marie , Meiring Muriel , Saint-Remy Jean Marie , Pieters Henry , Badenhorst Philip
Published: April 2020

Although type II Factor VIII (FVIII) inhibitor antibodies inhibit only partially FVIII activity, even when such antibodies are in very large excess over FVIII, they frequently cause severe bleedings in patients with acquired or congenital hemophilia A. During the preclinical evaluation of anti-FVIII antibodies as potential antithrombotic agents in baboons, we observed severe bleedings in animals treated with an antibody (mAb-LE2E9) inhibiting in vitro about 70% baboon FVIII. The administration of another antibody (mAb-LE2E9Q), inhibiting less than 20% baboon FVIII in vitro did not induce any significant hemoglobin drop by comparison to control animals. Surprisingly, mAb-LE2E9Q significantly reduced thrombus development in extracorporeal circulation models of venous and arterial thrombosis. The discrepancy between the levels of FVIII inhibition observed with these two antibodies and their in vivo activity prompted us to use modified types of FVIII assays to further evaluate in human plasma the inhibitory activity of these antibodies. When a FVIII chromogenic assay was performed with a low phospholipid concentration rather than with the standard concentration, the inhibitory activity of mAb-LE2E9 increased from 83 to 93% and that of mAb-LE2E9Q from 36 to 49%. Similarly, when activated platelets were used as a source of phospholipid, the maximal inhibitory activity of both antibodies increased to the same levels. Likewise, the use of different one stage and two stages commercial FVIII assays allowed to record maximal inhibitory activities ranging from 20 to 59% in a reference human plasma spiked with mAb-LE2E9Q. Thus, type II anti-FVIII antibodies induce qualitative FVIII alterations that are poorly detected by some FVIII assays. Variability inherent to the FVIII assays may therefore contribute to the large interlaboratory variability encountered even with a standardized method such as the Nijmegen modification of the Bethesda assay. This phenomenon may also account for the discrepancies frequently observed between severity of bleedings and residual FVIII activity in patients with type II inhibitor antibodies.
Evidence for an Inhibitor-Blocking Antibody in an "Acquired Hemophiliac".
Authors: Litwiller Robert , Pruthi Rajiv , Fass David
Published: April 2020

Factor VIII (FVIII) inhibitors have been classified as Type I, linear inactivation kinetics and complete inhibition of FVIII at saturation, or Type II, non-linear kinetics and residual FVIII activity. To investigate the hypothesis that "inhibitor-blocking antibodies" contribute to the characteristics of Type II inhibitors, mixing studies were performed between fractionated Type II antiserum and various Type I inhibitor preparations. Type II IgG fractions were tested for inhibitory capacity and ability to prevent inhibition mediated by inhibitors from other hemophiliacs. The IgG from Type II plasma (II) (29 BU, 8% FVIII) and several Type I plasmas ((I) plasmas) were fractionated with DEAE resin. Late-eluting Type II fractions (II):6 and (II):7, showed slight protection of FVIII from the inhibiting activity of several Type I plasmas and additive inhibition with others. When tested against Type I IgG fractions instead of type I plasmas, (II):6 and (II):7 provided significant protection of FVIII from the inhibitory action of some immunoglobulins and additive inhibition with others. When (II):6, which contained slightly less than 1 BU of inhibitory activity, was preincubated with normal plasma, it blocked about 3 BU of inhibitory activity in an equal volume of fraction (I):1; residual FVIII increased from 11.4% to 27.2%. IgG fraction (II):6 provided protection from DEAE fraction 2, (I):2, from another type I patient (residual FVIII went from 15.1% to 31.7%) but was additive with (I):4 from the same patient (residual FVIII went from 8.0% to 3.2%). Preincubation with (II):7 increased residual FVIII activity in normal plasma incubated with (I):1 from 10.1% to 20.7%. Fraction (II):7 was processed further by hydrophobic interaction chromatography on phenyl sepharose and two fractions, Phen(II):5 and Phen(II):6 provided significant protection of FVIII from inhibition by (I):1; residual FVIII activity increased from 11.5% to 34.1% and from 7.9% to 28.2%, respectively. DEAE fractions (II):6 and (II):7 were shown to contain anti-FVIII IgG. Depletion of mixtures of plasma or rFVIII and (II):6 with immobilized Protein A, reduced FVIII activity by 42.8% and 68.8%, respectively. In addition, adsorption of (II):6 with immobilized Protein A, eliminated its ability to block FVIII inhibition by (I):1. The co-existence of these antibodies in "acquired hemophiliac" plasma would contribute to the complex kinetics seen in these patients. If a sufficiently diverse panel of such blocking IgGs occurred naturally, or could be produced in the laboratory, and their interaction with the target antigen did not adversely affect the half-life, then these blocking antibodies might prove useful to alleviate the effects of inhibitory antibodies in hemophilia and other diseases.
Hemophilia and the Dynamics of Hemostasis.
Authors: Brummel-Ziedins Kathleen E. , Branda Richard F. , Butenas Saulius , Mann Kenneth G.
Published: April 2020

The conversion of fibrinogen to fibrin and cross-linking to form a stable clot are key processes in effective hemostasis. We investigated fibrinopeptide (FP) A and FPB release, factor XIII (FXIII) activation and fibrin mass in tissue factor initiated coagulation in whole blood from individuals with hemophilia (n=8) and healthy subjects (n=35). For hemophilia whole blood, the rates and patterns of fibrin formation are altered when compared to blood from healthy individuals. The rate of FPA release is decreased from 2.3μ M/min in healthy individuals to 1.5μ M/min in hemophilia and more significantly the rate of FPB release is decreased from 1.3μ M/min to 0.19 μ M/min in hemophilia whole blood. The activated form of FXIII in hemophilia whole blood is formed at a rate of 4nM/min versus healthy individuals where the rate is accelerated to 7nM/min. Prior to clot time in hemophilia whole blood, FPB release is reduced. Whereas more FPA is released in hemophilia whole blood prior to clot time than in whole blood from healthy individuals, 67% versus 47%, respectively. FXIII activation is delayed relative to FPA release. Around clot time in hemophilia whole blood, FPB levels are only ~1μ M and FPA levels are 8 fold higher. In normal whole blood at clot time, FPA release is only 2.5 fold higher than FPB release, potentially suggesting that there is an essential correlation of time dependence between FPA, FPB and FXIII activation to form a stable clot. Whereas, this pattern of fibrin formation is altered in the hemophilia state. Clots from hemophilia whole blood are more soluble as evidenced by a 43% decrease in fibrin mass compared to clots from healthy individuals. Overall, data indicate that hemophilia clots are weaker due to decreased lateral association (FPB release) and are mostly composed of fibrin units that are only partially cross-linked. Thus, suggesting that FXIII activation and FPB release play crucial roles in the pathology of hemophilia and the fragile nature of blood clots in hemophiliacs.
National Responses to HIV Versus HCV-Infection from Virally Contaminated Blood Products among Persons with Hemophilia (PWH): More Different Than Alike.
Authors: Bennett Charles L. , Frohlich Lauren E. , McCaffrey Kathryn R. , McKoy June M. , Ramsey Glenn E. , Lindenberg Julia S.
Published: April 2020

Background: 95% of HIV- and HCV-infections among PWHs occurred with use of contaminated blood products prior to 1985. Overall, 20% to 90% of PWHs in developed countries have HIV- and/or HCV-infection. We compared country-specific public health approaches, judicial activities, and compensation for these viral infections.

Methods: Reports from hemophilia organizations, national ministries of health, published articles, and the World Federation of Hemophilia were reviewed.

Results: Except for the United States, the number of PWHs who developed HCV-infection from contaminated blood products was 1.5 to 3-fold as great as for HIV-infection- as a result of delayed use of heat-treated blood products, importation in late 1984 of HCV-infected non-heat treated blood products from the United States, and failure to use surrogate laboratory marker hepatitis screening tests. Compensation funds for HIV-infected PWHs were established in Japan ($521,000 at Dx); France ($305,000 at Dx; $102,000 for AIDS); the United States ($115,000 at Dx); Ireland ($106,000 at Dx); the United Kingdom ($55,000 at Dx); Australia ($48,000 at Dx); Canada ($13,000 at Dx/$18,000/yr); Germany ($12,000/yr for HIV; $24,000/yr for AIDS); and Italy ($6,000/yr; $82,000 at death). Compensation has also been provided to HCV-infected PWHs in Ireland ($266,000 at Dx); Canada ($251,000 at Dx); the United Kingdom ($33,000 at Dx; $42,000 if w/liver damage); and Italy ($10,000/yr; $37,000 at death).

Conclusions: In most developed countries, despite a greater number of HCV-versus HIV-infected PWHs, markedly less attention has been paid to HCV-infected PWHs. All countries should review HCV-related blood safety decisions made in the 1980s and consider providing compensation to HCV-infected PWHs.

A comparison of national responses to HIV and HCV infections from blood products Country -PWH (thousands) % PWH with HIV:HCV Man-dated HIV ELISA (date) Man-dated heat Rx factor (date) Anti-HBc marker screening (date) Nat’l Funds for HIV/HCV among PWHs (year) Nat’l Panels for HIV/HCV decisions (year) USA-20 50%:30% Mar 85 Oct 84 Oct 84 96/none 95/none Italy- 8.7 23%:55% Mar 85 Jul 85 None 92/98 92/05 GDR- 6 47%:90% Oct 85 Oct 85 None 95/none 94/none UK-6 28%:80% Oct 85 Jun 85 None 88/03 87/05 France-4 50%:90% Aug 85 Oct 85 None 89/none 91/none Japan-3.4 60%:90% Nov 86 Jun 86 None 88/none 96/none Canada-2 40%:88% Nov 85 Jul 85 None 89/98 97/none Australia-1.5 31%:90% May 85 Jan 85 None 89/none 88/none Ireland-0.3 36%:76% Oct 85 Feb 85 None 91/97 91/97
Anti-HCV Agent, Ribavirin, Decreases Events of Bleeding in Hemophilia Patients, Possibly by Elevating the Clotting Activity of Factor VII.
Authors: Yamamoto Koji , Honda Takashi , Suzuki Satoshi , Toyoda Hidenori , Matsushita Tadashi , Kojima Tetsuhito , Goto Hidemi , Takamatsu Junki
Published: April 2020

Ribavirin has been used in combination with interferon-alpha (IFN-α) to treat chronic hepatitis C. This combination therapy has been reported to be more effective than IFN-α monotherapy for eradicating hepatitis C virus (HCV), including patients with concomitant hemophilia. Eight consecutive hemophilia patients were treated for HCV infection with IFN-α and ribavirin between June 2002 and May 2005 at Nagoya University Hospital as outpatients.

Characteristics of patients and responses to anti-HCV treatment Patient Age (yrs) Hemophilia type Severity of hemophilia HIV infection Ribavirin load (mg/day) HCV genotype Eradication of HCV The eradication of HCV was considered positive when the absence of serum HCV RNA was maintained for 24 weeks after treatment was completed. 1 28 A moderate N 800 3a Yes 2 61 A severe N 800 3a Yes 3 50 A severe N 600 --> 400 1b No 4 42 B mild N 800 2a +1b Yes 5 44 A severe N 800 3a Yes 6 52 A mild N 600 2b Yes 7 37 A mild P 800 --> 600 1a No 8 44 B moderate N 800 1a Yes

All patients were men with a mean age (SD) of 44.8 (10.0) years. Seven patients had hemophilia A, and 2 had hemophilia B. Hemophilia was severe in 4 patients, moderate in 2 and mild in 3. Four patients had been previously treated with IFN-α-2b alone (Intron A®, Schering Plough, K.K., Osaka, Japan) but HCV had not been eradicated. During this study, all patients were treated with the same 24-week regimen of IFN-α. Oral ribavirin (Rebetol, Schering-Plough, Kenilworth, N.J.) was administered at a dose of 600 mg/day for patients who weighed 60 kg or less and 800 mg/day for those who weighed more than 60 kg during 24 weeks. We observed the reduction of the frequency and dose of infusion with clotting factors as a hemostatic therapy in HCV-positive hemophilia patients who were administered with ribavirin.

Figure Figure

(Use of clotting factor concentrates 6 months before, during and 6 months after combination therapy with ribavirin and IFN-α. Use of clotting factors is presented as the average use per month.) In order to investigate the mechanism of this prophylactic effect of ribavirin to bleeding in hemophiliacs, we analyzed ribavirin-induced changes in the activity of factor VII in patients’ plasma. The clotting activity of factor VII in plasma has been elevated at 15% on an average in 9 HCV-positive hemophilia patients during treatment with ribavirin (without ribavirin: 86.3±7.6%; with ribavirin: 102.0±10.3%). Furthermore, a significant induction of factor VII mRNA was demonstrated in cultured normal human hepatocytes or HepG2 cells when treated with ribavirin at the therapeutic concentration. These observations indicate that ribavirin can elevate factor VII procoagulant activity in plasma, possibly due to the induction of factor VII in hepatocytes, thus, contributing to decreased events of bleeding in HCV-positive hemophiliacs.
A Multi-Institution Retrospective Case Report Analysis of Emergence of Breakthrough Zygomycoses in Patients on Prior Voriconazole Therapy.
Authors: Trifilio Steven M. , Balasubramanian Lakshmi , Mehta Jayesh , Tallman Martin S. , Kennedy LeAnne , Bennett Charles L. , Kontoyiannis Dimitrios P.
Published: April 2020

Background: The incidence of Zygomycete infection has been increasing, almost exclusively in patients with hematologic malignancy with or without history of stem cell transplantation. In a single institution case control study, voriconazole was identified as an independent risk factor for the development of Zygomycete infections. A small number of single institution case reports have also been noted.

Methods: Investigators for The Research on Adverse Drug Events and Reports (RADAR) project requested collaboration from cancer centers regarding the identification and characterization of Zygomycete infections in patients with prior exposure to voriconazole. 12 centers participated in this study project. Medical records were reviewed and data collected on a standard case report form including diagnosis, transplant type, concomitant risk factors, exposure times, concurrent infection and outcomes. A minimum of 5 days exposure was noted between clinical diagnosis and confirmation with either tissue or histology.

Results: 54 cases were identified from 12 centers and with notably almost 75% mortality rate in individuals who were diagnosed with Zygomycoses with prior exposure to voriconazole.Of interest 31/54 cases received voriconazole for prophylaxis. Presenting characteristics were similar to those reported previously with lung as major site of infection. Concomitant risk factors included steroid use, immunosupression, GVH, neutropenia and diabetes. Simultaneous co-infections from diverse bacterial, viral and fungal infections were noted in 60% of patients signifying a compromised immune status. Drug therapy with an amphotericin product used to treat the identified Zygomycosis was highly ineffective with failure in 26/32 patients with resultant mortality.

Conclusion: Rates of breakthrough zygomycoses with high mortality rates with prior voriconazole therapy are on the increase. Appropriateness of prophylactic use as well as early diagnosis and institution of antifungal therapy in these patients with Zygomycoses is essential to combat this devastating infection.
Induction and Capture of CD4+ Cytotoxic Adenoviral Specific T-Cells in Response to pan-DR Binding Adenoviral Epitopes; towards Immunotherapy.
Authors: Haveman Lianne M. , Prakken Berent , Klein Mark R. , Albani Salvo , Bierings Marc
Published: April 2020

In the immunocompromised host adenovirus may cause fatal infections, especially after stem cell transplantation. No effective antiviral medication exists for severe adenoviral infection. Adoptively transferring adenoviral (Adv) specific T-cells from the donor into the patient may be a promising treatment, but requires the identification of these cells. The aim of the study was to induce Adv-specific cells in response to recently detected pan-DR binding CD4+ T-cell epitopes of adenovirus serotype 5. Epitopes were selected by using a computer algorithm designed to predict HLA pan-DR binding T-cell epitopes. Peripheral blood mononuclear cells (PBMCs) of 26 healthy adults were incubated with 19 different 15-mer peptides. Proliferation expressed as Stimulation Index (SI) was determined. Five peptides with highest SI derived from fiber protein, E1B protein, hexon protein (2 peptides) and DNA-polymerase were selected. To induce Adv-specific T-cells PBMCs from healthy subjects were either cultured with complete inactivated adenovirus for 11 days and restimulated with the different peptides for 3 days (n=10) or directly cultured with the peptides for 7 days (n=10). The cytokine profile induced by these epitopes was determined with multiplex immunoassay (MIA). By using the T-cell capture (TCC) method1 and FACS-sorting it was possible to capture the Adv-specific T-cells for further characterization by PCR and FACS analysis. In comparison with medium and irrelevant Adv-peptides PBMCs cultured with the 5 selected Adv-peptides induced significant larger amounts of Adv-specific CD4+ T-cells and showed a predominant pro-inflammatory cytokine profile. This suggests that peptides are naturally processed. By using TCC Adv-peptide specific CD4+ T-cells were identified and sorted. The Adv-specific T-cells displayed a high expression of TGF-1b, IFNg and Tbet (Th1 response), but also in lesser extent GATA3 and IL10 (Th2 response). A majority of the CD4+ Adv-specific cells expressed perforin and granzyme B, indicating that these CD4+ T-cells play an essential role in adenoviral infections. The induction of a specific immune response to adenovirus and subsequent capture of the Adv-specific T-cells is an important step towards adoptive immunotherapy in case of Adv-infections in the immunocompromised host.

Figure 1. Induction of adenoviral specific T-cells (CD4, CTB double positive cells) in response to Adv-peptides. 1A) Induction of CD4+ Adv-specific in response to medium stimulated PBMCs and 1B) in response to a peptide derived from hexon protein. Figure 1. Induction of adenoviral specific T-cells (CD4, CTB double positive cells) in response to Adv-peptides. 1A) Induction of CD4+ Adv-specific in response to medium stimulated PBMCs and 1B) in response to a peptide derived from hexon protein.
Fatal Infectious Complications Developing Late after Allogeneic Stem Cell Transplantation.
Authors: Bjorklund Andreas , Aschan Johan , Ringden Olle , Winiarski Jacek H. , Ljungman Per T.
Published: April 2020

Background and aim: The procedure of allogeneic stem cell transplantation (SCT) has evolved during the past decades. Infectious complications are still a major problem contributing to the transplantation related mortality (TRM). The epidemiology and outcome of early infections after SCT are well described. However, less is known has about late infections after SCT. Thus, the aim of this study was to determine risk factors for fatal infections occurring later than 6 months after allogeneic SCT.

Material and methods: Our study is based on 938 consecutive SCT patients transplanted 1976–2003 of whom 688 (73%) had survived for at least 6 months after SCT. A retrospective chart review was performed identifying 44 (6.4%) patients surviving for at least 6 months, having died from infection. Patients who had relapsed in their malignant disease were excluded. A control group of 176 patients (4 per case) was identified using relapse-free survival for at least 6 months and year of SCT as the matching criteria. Five controls were excluded leaving 171 patients in the control population. Risk factors for death from late infections were identified by logistic regression.

Results: 29 patients (66%) developed their fatal infection within 18 months and 37 (84%) within 5 years after SCT. 37 patients (84%) had ongoing chronic graft versus host disease (GVHD) and 36/44 (82%) had ongoing immunosupression at the time of death. 57 controls had died after 6 months from SCT; 32 of 57 from relapse. Comparing patients and controls in univariate analyses, the mean age was 30.6 years in the cases and 26.5 years in the controls (p=.13). 22/44 (50%) cases had been transplanted from an unrelated or mismatched donor, compared to 57/171 (33%) controls, p=.053; and 35/44 (80%) cases had received a conditioning regimen including myeloablative dose of TBI compared to 113/171 (66%) in the control group, p<.05). Regarding post-transplant complications 40/44 (91%) cases had experienced cGVHD compared with 101/171 (59%) controls, p<.001. 21/44 (48%) cases had developed obstructive respiratory dysfunction compared with 46/171 (27%) controls, p=.01; and more cases (33/44; 75%) than controls (85/171; 50%;) had experienced CMV infection. In multivariate analysis chronic GVHD (OR 9.2; p<.001), use of a mismatched or unrelated donor (OR 4.8; p<.001), and having had a CMV reactivation (OR 8.3; p=.004) increased the risk. Age, acute GVHD, TBI or obstructive respiratory dysfunction had no significant impact on the risk for late fatal infection.

Conclusion: Infections later than 6 months after SCT are important contributors to late TRM. Risk factors for late fatal infections include chronic GVHD, use of alternative donors and CMV infection.