bluuebunny/crossref_metadata_2025 huggingface dataset:
Intravenous Hemin as Prophylaxis of Allograft Function Following Second Liver Transplant for Erythropoietic Protoporphyria.
Authors: Schiller Gary J. , Goldstein Leonard , Finn Richard , Busuttil Ronald
Published: October 2019

Erythropoietic protoporphyria (EPP) is a rare disorder of heme biosynthesis caused by an inherited deficiency of mitochondrial ferrochelatase. This deficiency results in accumulation of protoporphyrin, a heme precursor, in bone marrow, skin, nervous system, and liver. In some patients, toxic levels of protoporphyrins in the liver cause chronic liver disease, cirrhosis, and hepatic failure. Liver transplantation is the only treatment available; however, the risk of allograft dysfunction is high as a result of continued, excessive protoporphyrin production due to the underlying systemic enzyme deficiency. We present the case of a 40 year-old man with EPP, diagnosed at age 6, who underwent orthotopic liver transplantation (OLT) at age 25. By age 31, biopsy-proven recurrent EPP with liver dysfunction was identified in the allograft with subsequent hepatic failure seven years later at age 38. A second liver transplant was complicated by a biliary anastomotic leak requiring further surgery and prolonged hospitalization. Six months prior to a second liver transplant, the patient was started on 4 mg/kg hemin infusions every week; following second liver transplant, the frequency was increased to twice weekly to maintain graft function. Infusions have been well tolerated and administered at home through a central venous catheter and there has been no evidence of iron overload. Three months following second transplant, blood alkaline phosphatase, ALT, AST, and bilirubin were 1009, 53, 64, and 11.5, respectively. Fourteen months after the second allograft, these levels are 234, 65, 65, and 1.2, respectively. Prior to hemin therapy, erythrocyte protoporphyrin level was 6600 mg/dl and was 5573 mg/dl immediately before second allograft. With twice weekly hemin, erythrocyte protoporphyrin level fourteen months after transplantation is 1240 mg/dl - greatly reduced but still elevated above the normal range. A liver biopsy three months after the second allograft showed cholestasis and biliary obstruction consistent with recurrent porphyria, but no subsequent biopsy has been performed yet despite improvement in alkaline phosphatase, bilirubin, and transaminase levels. This case study confirms an earlier report of clinical graft stabilization with hemin infusions after orthotopic liver transplantation for erythropoietic protoporphyria (Dellon et al., Transplantation2002; 73:911–5) and to our knowledge is the first case of long-term hemin use to maintain the integrity of a second liver allograft. Further follow-up and biopsy will be needed to determine the impact of this strategy on preserving graft function.
Parental Ferric Hydroxide Saccharate Therapy for Iron Deficiency Anemia: Safety and Efficacy.
Authors: Abdel-Razeq Hikmat , Radwi Ghazala , Khalil Manar , Bajouda Ali , Macalintal Marifi
Published: October 2019

Background: Iron deficiency remains the most common cause of anemia both locally and worldwide. In adults, the most common etiology of iron deficiency is blood loss; in men and postmenopausal women, iron deficiency almost inevitably signifies gastrointestinal (GI) blood loss. In women of childbearing age, genitourinary blood loss is the usual culprit. The goal of iron therapy for iron deficiency anemia (IDA) is to supply sufficient iron to repair the hemoglobin deficit and replenish storage iron. Iron can be given in either oral or parenteral forms. Because of the local and systemic side effects linked to parenteral iron therapy, it has been the less favored form of treatment of IDA. In this retrospective review, we report our experience using parenteral iron therapy for certain indications in patients with severe IDA.

Methods: Total number of patients was 57, 54 were females. Mean age was 31 years (12–60 yrs). Iron deficiency anemia was found to be due to: menorrhagia (46%), pregnancy (23%), malabsorption (22%), GI loss (7%) and poor dietary habits (4%). Patients presented with the following symptoms: dizziness (84%), exertional dyspnea (40%), palpitations (28%) and fatigue (23%). The indications for parenteral iron therapy were: severe symptomatic anemia (58%), pregnancy (25%), poor response to oral iron (14%), noncompliance to oral therapy (10%) and bleeding (7%).

Treatment plan: The form of iron used was Ferric Hydroxide Saccharate Complex. Iron requirement was calculated based on the following formula: Total iron deficiency in mg = [body weight (kg) x (normal Hb -actual Hb in g/L)x 0.24] + 500.The mean iron requirement was 1200mg (700mg – 2400mg). This was given in an outpatient setting as daily intravenous infusions starting with a dose of 100mg on the first day. The rest of the requirement was divided into doses of 200mg given over consecutive days.

Results: The mean Hb pre-treatment was 7.96g/dL (4.9–10.7g/dL) and the mean Hb post treatment was 10.6g/dL. (8.2–12.3g/dL). The rise in Hb was at a rate of 0.76/dL per week (0.17– 1.9). All patients tolerated the treatment well with only one complaining of burning sensation at the site of the IV line. There were no other side effects including anaphylactic reactions. In conclusion, the above data demonstrates that parenteral iron therapy, in the form and schedule given, is a fast, convenient, effective and safe means of treating severe, symptomatic IDA.
Ferroportin1-Deficient Zebrafish Treated with Iron Dextran Develop Anemia Despite Increased Macrophage Iron Stores.
Authors: Fraenkel Paula G. , Traver David , Donovan Adriana , Zon Leonard I.
Published: October 2019

Patients with type 4 hemochromatosis exhibit iron accumulation in the reticuloendothelial system and a tendency to develop anemia following venesection. These patients are heterozygous for mutations in the intestinal and macrophage iron exporter, ferroportin1 (fpn1), however it is not known whether these mutations cause a gain or loss of function. Of current animal models, only the Tp85c allele of the zebrafish anemia mutant weissherbst (weh) encodes a missense mutation (L167F) in the conserved region of fpn1, between transmembrane helix 3 and 4, where several missense mutations have been identified in individuals with type 4 hemochromatosis. Tp85c homozygote zebrafish die within 10–14 days post-fertilization, however administering iron dextran injections allows them to survive. To explore the effects of fpn1 deficiency on blood development and iron homeostasis in adult zebrafish, we raised Tp85c homozygotes along with control cohorts of injected and uninjected heterozygotes and wild types. Iron dextran injections were discontinued at 4 months of age in all cohorts. Although iron accumulation was observed in all iron-injected fish, only homozygotes exhibited iron accumulation in the intestinal epithelium on Perl’s staining, consistent with a block in enterocyte export. At 6 months of age, none of the cohorts were anemic, however at 12 months of age, only the homozygotes developed hypochromic anemia, characterized by reduced levels of hemoglobin per erythrocyte and impaired late erythroid maturation. Histologic evaluation of the anemic homozygotes revealed increased numbers of iron-loaded macrophages in the liver and in the kidney, the zebrafish bone marrow equivalent. Quantitative realtime rt-PCR, performed on liver and intestinal tissues at 1 year of age, revealed that homozygotes had increased intestinal expression of fpn1 compared to the other cohorts. Hepatic transcript levels of the secreted iron-regulator hepcidin were profoundly decreased in homozygotes, consistent with response to anemia. In contrast, hepatic transcript levels for transferrin, fpn1, and DMT1 were not significantly different among the cohorts. In summary, we provide evidence that Tp85c is a loss of function mutation affecting enterocytes and macrophages. After iron dextran injections were discontinued, Tp85c homozygotes developed anemia, despite increased macrophage iron stores. These studies support the hypothesis that fpn1-deficiency impairs iron cycling and suggest this as a possible mechanism in type 4 hemochromatosis.
Management of Severe Intractable Sickle Cell Pain with Intrathecal Opioid Analgesia.
Authors: Ballas Samir K. , Kucler Marcia , Tabbaa Kutaiba
Published: October 2019

Management of severe Sickle Cell Pain (SCP) often entails the utilization of opioids via the intravenous route. In this abstract, we describe the utilization of Intrathecal Morphine Analgesia (IMA) infusion in the management of severe SCP. Two African American women were the subjects of this study: a 50-year-old patient with sickle cell anemia and a 51-year-old patient with sickle-b+-thalassemia. Complications of their disease included frequent acute painful episodes, S/P cholecystectomy, iron overload, and severe avascular necrosis (AVN) of the left hip in the first patient and frequent acute painful episodes, S/P cholecystectomy, retinopathy, and severe AVN of the right hip in the second patient. Both patients were admitted frequently to the hospital due to severe acute painful episodes involving mostly the hips and lower extremities. In both patients, a Medtronic Indura Catheter was inserted intrathecally under fluoroscopy guidance. The entry level was at the L3–L4 lumbar space and the tip of the catheter reached the T8 level. The catheter was attached to an Isomed pump that was filled with a mixture of preservative free morphine and clonidine and placed in a subcutaneous pouch in the abdominal wall. The first patient required 0.5mg of morphine and 25mg of clonidine daily, and the second patient required 1.5mg of morphine and 150mg of clonidine daily. The pumps were refilled every 2–3 months. No side effects were noted and the vital signs of both patients remained stable. Both patients continue to use IMA for 2–3 years to date with no complications. The utilization of IMA resulted in significant decrease in the frequency of acute painful episodes treated in the emergency room or hospital, as well as the number of hospital days and the amount of oral opioid consumption expressed in morphine sulfate (MOSO4) equivalent as shown in the table. Moreover, the quality of life of both patients improved: the first patient resumed social and family activities and the second patient could be gainfully employed. The data show that utilization of IMA in selected patients with severe intractable pain below the diaphragm has a significant salutary effect on the frequency and duration of hospital admissions and the total amount of opioids consumed.

Effect of IMA on Annual Utilization of Medical Facilities and Opioid Consumption

Hospital Amissions Hospital Days ER Visits PO MOSO4 Equivalent Patient #1 Before IMA 15 222 14 17585 mg After IMA 7 88 7 6885 mg Patient #2 Before IMA 9 19 21 8415 mg After IMA 5 18 9 3310 mg
Neutropenia in Patients with Thalassemia Major.
Authors: Galanello Renzo , Origa Raffaella
Published: October 2019

Monitoring of the neutrophil count has become an important issue in the management of thalassemia patients after the introduction of the new oral chelator deferiprone, as this chelator has been associated with agranulocytosis and milder neutropenias. Although a clear relation between deferiprone and agranulocytosis has been demonstrated, it is unclear whether milder neutropenias are due to deferiprone use or to other factors such as hypersplenism, infections, autoantibodies, or other drugs. In this study we evaluated 7,708 consecutively blood counts and leukocyte differential obtained between August 2003 to June 2004 from 344 patients with thalassemia major, aged 2 to 40 years, treated with deferiprone (N=39), desferrioxamine (N=179), ICL670 (N=45), or a combination of deferiprone and desferrioxamine (N=81). Complete blood count and differential were performed with Coulter LH 750 automated haematology analyzer (Beckman Coulter, Miami, FL), which can detect nucleated red blood cells (NRBC) and correct the white blood cell (WBC) count in the presence of NRBC, platelets clumps, unlysed or fragmented RBC. The capability to correct the WBC count is particularly relevant in the monitoring of the neutrophil counts of patients with excessive NRBC, which could lead to potential errors in the differrential count. Neutropenia was defined as a neutrophils count below 1500 cells/μL, whereas agranulocytosis was defined as a neutrophil count below 500 cells/μL. No episodes of agranulocytosis were observed. Episodes of neutropenia (range 850 – 1490 neutrophils/μL) were observed in 29 (8,4%) patients. Repeated episodes of neutropenia were observed in 7 of the 29 patients. At the time of neutropenia, 23 (79%) of the patients were being chelated with desferrioxamine, 2 (7%) with deferiprone, 3 (10%) with a combination of both chelators, and 1 (3%) with ICL670. Considering the type of chelation, neutropenia was observed in 12,8% of patients being treated with desferrioxamine, 5% in patients treated with deferiprone, 3,7% in patients treated with combination therapy, and 2.2% in patients treated with ICL670. The severity of neutropenia, assessed as absolute neutrophil count, was not different on the basis of the type of chelation (p=0,26). Neutropenia was more frequent in children than in adults (31% vs 7.8%, respectively, p=0.0001). No statistically significant difference in the incidence of neutropenia was found between males and females (10.2% vs 6.5% respectively, p=0.2), anti-HCV positive and negative (7.7% vs 10.3% respectively p=0.43), or splenectomized and non-splenectomized patients (11.1% vs 8.1% respectively, p=0.5). Neutropenia appears to be a common event in patients with thalassemia major, irrespectively of the chelation therapy used. The higher incidence in children may be secondary to a greater frequency of viral infections at this age, which may produce neutropenia with several mechanisms including redistribution, sequestration and destruction of neutrophils.
Molecular Analysis of Alpha Hemoglobin Stabilizing Protein (AHSP) in Caucasian Patients with different Beta-Thalassemia Phenotypes.
Authors: Cappellini M. Domenica , Refaldi Chiara , Bignamini Daniela , Zanaboni Laura , Fiorelli Gemino
Published: October 2019

Beta-thalassemia is a inherited hemoglobin disorder characterized by absent or reduced synthesis of the b globin chains. The pathophysiology and the severity of b-thalassemias reflect the degree of globin chain imbalance and the excess of free a globin chains that precipitate and cause oxidative damage in red cell precursors inducing their premature destruction in the bone marrow (ineffective erythropoiesis). Although the phenotype of b thalassemias can be modified by inherited factors such as different number of a globin genes or increased fetal hemoglobin production, other mechanisms appear to be involved. Recently, a protein, named alpha hemoglobin stabilizing protein (AHSP), that acts as a molecular chaperone specifically for free a globin chains, preventing their precipitation in red cell precursors, has been identified.

To establish whether AHSP might have a role in modifying the clinical outcome of b thalassemias, we have analyzed the AHSP gene in 70 Caucasian b thalassaemic subjects: 26 patients with b°/b° genotype (Thalassaemia Major),24 patients with Thalassemia Intermedia (b°/b+ or b+/b+) and 20 patients with a Thalassaemia Intermedia phenotype but with only one mutation in the b globin gene, a normal a globin genotype and no other causes of anemia. In all the subjects, we have performed Denaturing High-Performance Liquid Chromatography (DHPLC) of the three exons and the direct genomic sequencing of coding and noncoding regions (~ 1.5 kb) of AHSP gene. No mutations able to modify the structure or function of AHSP have been found, however we identified eight single nucleotide polymorphisms (SNPs) spanned along the whole gene that segregate in four different aplotypes. To evaluate a possible relationship between a particular aplotype and b thalassemia severity, the allele frequency of each single aplotype in the tree groups has been established and compared to that of 33 Caucasian normal controls: no statistically significant association has been proved. Even though the loss of AHSP aggravates the b thalassaemia phenotype in mice, in Thalassemic Caucasian population the AHSP apparently doesn’t make changes in the clinical severity of b thalassemia confirming the results recently found in Thai population.
Massive and Selective Ex Vivo Generation of Matured and Functional Human Red Blood Cells (RBC) from Hematopoietic Stem Cells of Diverse Origins: Towards the New Concept of “Cultured RBC”.
Authors: Douay Luc , Kobari Ladan , Lapillonne Helene , Chalmers David , Kiger Laurent , Cynober Therese , Marden Michael C. , Wajcman Henri , Giarratana Marie-Catherine
Published: October 2019

We report a technological approach permitting, for the first time, the massive (up to 2x106-fold cell expansion) and selective (100%) ex vivo production of mature RBCs (cRBCs) starting from CD 34+ cells from peripheral blood (PB), bone marrow (BM) or cord blood (CB) into mature red cells in three steps: firstly, cell proliferation and erythroid differentiation were induced in serum free media supplemented with SCF, IL-3 and Epo for 8 days. Secondly, cells were co-cultured with additional Epo alone on either the murine MS-5 stromal cell line or human mesenchymal cells for 3 days. In the third step, all exogenous factors were withdrawn and cells were incubated on a simple stroma for 4 to 10 days.

These cultured erythroid cells (reticulocytes and mature RBCs) displayed characteristics identical to those of native cells, in terms of MCV, MCH, MCHC, enzyme content (G6PD and PK) and deformability. The nature of the Hb produced depended on both the origin of the CD34+ cells and the culture conditions. cRBCs derived from PB or adult BM contained adult Hb (95±1%) whereas cRBCs derived from CB contained essentially HbF (64±13%). As for native RBCs, Hb was able to fix and release oxygen. CFSE-labelled-reticulocytes ex vivo generated from leukapheresis were injected into NOD-SCID mice. The transfused reticulocytes were found in the circulation to the same extent as native RBCs and fully matured into RBCs. This methodology is applicable for fundamental analysis of the mechanisms of terminal erythropoiesis and hemoglobin synthesis. Moreover, large scale cRBCs production could be possible with such a protocol. It can therefore be extrapolated to a wide range of clinical applications in the field of gene therapy, infectious diseases and particularly transfusion medicine with a pointed interest for the generation of a cell population homogeneous in age, thus achieving the new concept of cultured RBCs transfusion.
Identifying Mechanisms of Enhanced NK Cell Cytotoxicity Using Permanent NK Cell Lines.
Authors: Suck Garnet , Branch Donald R. , Vergidis Joanna , Fahim Soad , Keating Armand
Published: October 2019

Although NK cells are promising candidates for adoptive immunotherapy and at least one permanent cell line is in clinical trials, further studies evaluating efficacy and mechanisms of action are warranted. As a first step towards identifying the most potent effector cells, we investigated the molecular mechanisms of cytotoxicity of the three natural killer lines, KHYG-1, NK-92 and YT, and the NK-T cell line, SNT-8, under standardized culture conditions with human serum as the only serum source. We confirmed the previously established differential killing potential of the 4 cell lines against target K562 cells using a new method based on detecting Annexin V (+) target cells by flow cytometry. By labeling the NK cells with specific antibodies, the assay is designed to screen any target cell for NK cytotoxicity. In contrast to previous reports, we found KHYG-1 the most cytotoxic, followed by NK-92, SNT-8 and YT. Genotypic and transcriptional phenotypic analysis of the cell lines for killer cell Ig-like receptors (KIRs) by SSP-PCR showed that inhibitory KIRs outnumbered activating KIRs in all cases but did not explain the differential cytotoxicity. A correlation with cytotoxicity was found with expression of the activating type II C lectin-like receptor, NKG2D: KHYG-1, 99%+; NK-92, 91%+; SNT-8, 6%+ and YT, 2%+. Moreover, the ITAM-bearing adaptor molecule DAP12, involved in the alternative activation signaling pathway via NKG2C-CD94 and activating KIRs, was detected only for KHYG-1 by immunoblotting, These data suggest that the superior cytotoxicity of KHYG-1 may be due, in part, to the additional activation of this alternative pathway that is not triggered in the other lines. The downstream signaling molecules involved in NK cytotoxicity, including the tyrosine phosphatases SHP-1, SHP-2 and SHIP-1 (inhibitory), as well as SHIP-2, the tyrosine kinases ZAP-70, Syk, PI3K and the MAP kinase phospho-ERK-2 (activating) were compared among the lines by immunoblotting followed by densitometry normalized to b-actin or ERK-2 for phospho-ERK-2. We found that the activating kinase Syk was expressed only in NK-92 and KHYG-1 at even higher levels. Also, phospho-ERK-2, was hyperphosphorylated only in KHYG-1. Perforin, granzyme A and granzyme B, present in cytotoxic granules, were compared by RT-PCR and intracellular flow cytometry and/or immunoblotting. Perforin was found to be almost exclusively fully processed to the active 60 kD form only in KHYG-1, in contrast to the other lines, which displayed approximately half the levels of the active form. These data provide a further explanation for the superior cytotoxicity of KHYG-1 and demonstrate the value of comparing cell lines with diverse cytotoxic potential as a means of elucidating cell killing mechanisms. It is conceivable that targeted modifications to the signaling pathways for cytotoxicity in this model will lead to the generation of activated NK cells with even greater efficacy.
Continuous Versus Subsampling Analysis of Thrombin Generation.
Authors: Lau Alice , Berry Leslie , Chan Anthony K.C.
Published: October 2019

Thrombin generation measurement is becoming a useful technique to assess the hemostatic system. Subsampling methods though labour intensive, measure activities directly. The continuous method has become an alternative option. Studies by Hemker et.al. (1) showed that both methods gave comparable results but had some differences. We decided to further assess the variation between the methods. For the continuous method, 125μl of plasma, 15μl of Pefabloc (50mg/ml) and 12.5μl of Pefachrome TG5134 (0.5mM) were added to a 96 well ELISA plate and prewarmed to 37°C. The reaction was started by adding 97.5μl of 1/12.5 Thromborel S in 25.64mM CaCl2/Tris buffer. The optical density at 405nm was recorded at an interval of 15sec for 15 min. For the subsampling method, 125μl of 1/16 Thromborel S in 20mM CaCl2/Tris buffer was added to 125μl of defibrinated (Arvin) plasma. Subsamples were taken into 5mM EDTA solution at 15sec intervals. Chromogenix S2238 was used for measuring thrombin in the subsampling method. The continuous method gave a peak height of ~410nM and area under the curve of 860–900 nM.min whereas the subsampling method gave a peak height of ~360nM and an area under the curve of 550–600nM.min. This difference disappeared when PefachromeTG5134 was added into the reaction mixture prior to running subsampling assays. Thus, substrate likely slowed inhibition of generated thrombin in the continuous assay, which confirms previous findings by Hemker et.al. (1). Further examination revealed that the concentration of α2M-IIa complex was very different between the two methods. α2M-IIa complex calculated from the continuous method (80nM) was much greater than α2M-IIa complex obtained experimentally by the subsampling method (38nM) even in the presence of TG5134. However, addition of a large excess of antithrombin and heparin at the final equilibrium time point of the continuous assay reduced the α2M-IIa complex to the same level (38nM) as that of the subsampling method. We have shown that the slow-acting substrate Pefachrome TG5134 gave an increased estimate of both free and α2M-IIa complex. Thus, a significant amount of free thrombin remains at the end of thrombin generation that is not accounted for in the continuous method. This estimate of final α2M-IIa complex concentration may be important in determining the relative protection against long-term plasma thrombin activity in pediatric patients with elevated α2M levels.
SB 497115-GR, a Low Molecular Weight TPOR Agonist, Does Not Induce Platelet Activation or Enhance Agonist-Induced Platelet Aggregation in Vitro.
Authors: Erhardt Joseph , Erickson-Miller Connie L. , Tapley Peter
Published: October 2019

SB-497115-GR is a low molecular weight, orally active molecule that requires Tpo receptor (TpoR) expession for activity and activates JAK/STAT signalling in platelets. The ability of recombinant Tpo to enhance agonist-induced platelet aggregation is well-documented, thus this study was undertaken to compare the ability of rhTpo (150 ng/ml) and SB-497115 (0.01–10 uM) to affect platelet activation. Platelets were obtained from healthy human volunteers, following written informed consent, by standard venipuncture technique. Citrated whole blood was used directly in platelet activation and P-selectin assays within 15 minutes. Platelet aggregation was assessed by light transmittance aggregometry. Aggregation was induced by a submaximal concentration of adenosine diphosphate (ADP) (1.0 or 1.5 uM) in experiments examining synergy with rhTpo (150ng/mL) or SB-497115 (0.01–10 uM). For examination of potential inhibitory actions of SB-497115 on platelet function, aggregation was induced by ADP (3 uM), collagen (2 ug/mL), or the thrombin receptor activating peptide (TRAP; 20 uM). Pre-treatment of platelet samples with rhTpo, but not SB-497115, potentiated the effects of 1.0 or 1.5 uM ADP. No inhibitory effect on normal ADP, Collagen or TRAP-induced aggregation was seen with either SB-497115-GR or rhTpo. In experiments investigating the expression of platelet P-selectin, an early marker of platelet activation, platelets labeled with anti-CD62P antibody were analyzed by flow cytometry. Data was expressed as percent of platelets positive for CD62P within the defined platelet gate. Treatment with SB-497115 (0.1 – 10 uM) demonstrated no induction of platelet CD62P expression above background levels. In contrast, rhTpo produced a modest but significant and reproducible increase in the percent of platelets positive for CD62P. This study demonstrates that, in contrast to rhTpo, the orally active small molecular weight agonist of the Tpo receptor, SB-497115-GR, does not mimic the ability of rhTpo to enhance ADP-induced aggregation of platelets. SB-497115-GR also did not directly induce P-selection expression on human platelets. Finally, SB-497115 has no antagonistic effect on ADP-, TRAP-, or collagen-induced platelet aggregation. Thus, SB-497115 is differentiated from rhTpo with respect to platelet activation.
ATRA Resolves the Differentiation Block in t(15;17) Myeloid Leukemia by Restoring PU.1 Expression.
Authors: Mueller Beatrice U. , Pabst Thomas , Osato Motomi , Tenen Daniel G.
Published: October 2019

The transcription factor PU.1 is crucial for hematopoiesis. pu.1 knockout mice lack mature neutrophils, macrophages, B- and T-cells. PU.1 regulates important taget genes, like the M-CSF-, the G-CSF-, and the GM-CSF receptor promoter. Heterozygous mutations of the PU.1 gene have been found in some patients with AML, but not in AML with the t(15;17) encoding the PML-RARA fusion protein. These facts let us to hypothesize that decreased expression of PU.1 might contribute to the block in myeloid differentiation in t(15;17) AML. We therefore screened 141 AML patients for PU.1 mRNA expression using real-time PCR technology. We detected that t(15;17) AML patients (n=18) had significantly less PU.1 mRNA than AML-M2 patients (n=35), and 16fold less PU.1 mRNA than mature neutrophils. Moreover, the amount of PML-RARA transcripts in APL patients was inversely correlated to PU.1 mRNA levels. Conditional expression of PML-RARA in U937 cells suppressed PU.1 mRNA and PU.1 protein. HT93A cells derived from a t(15;17) AML-M3 patient nearly lack PU.1 mRNA and protein expression, and binding activity to the PU.1 site in the M-CSF-Receptor promoter is suppressed. Treatment of HT93A cells with all-trans retinoic acid (ATRA) powerfully restores PU.1 mRNA and protein and induces neutrophilic differentiation. We identified a region of extending −122 bp upstream of the translation start site in the PU.1 promoter mediates ATRA-induced activation of the PU.1 promoter in HT93A cells. Finally, conditional expression of PU.1 in HT93A cells is sufficient to trigger neutrophilic differentiation. This is the first report demonstrating that PU.1 is a critical target of the PML-RARA protein and an early ATRA-responsive gene.
The Role of Rho/Rac Pathways in Proplatelet Formation of Megakaryocytes.
Authors: Murai Kazunori , Kowata Shugo , Ito Shigeki , Sato Mamiko , Ishida Yoji
Published: October 2019

We reported that Rho and/or Rac signal pathways were involved in proplatelet formation (PPF) of megakaryocytes in the last ASH Meeting. Recently Nagata et al. reported that 3β-hydroxysteroid dehydrogenase (3β-HSD) was a target of p45 NF-E2 and that estradiole, synthesized by 3β-HSD in megakaryocytes, stimulated PPF (GENES & DEVELOPMENT 2003). In this study we focused on the signal pathways, activated by estradiole, and the predominant pathway in the process of PPF using murine megakaryocytes. Murine megakaryocytes were isolated from bone marrow cells by Percoll® density centrifugation and BSA gradient method. The purity of the isolated megakaryocytes was more than 40% in all experiments.

Isolated megakaryocytes (300/well) were cultured in serum free condition with 17β-estradiol (E2) or/and Y27632 (Rho kinase inhibitor) or PP1(Src family kinase inhibitor) or LY294002 (PI3K inhibitor). Y27632 inhibits Rho pathway and the 2 latters inhibits Rac activation. Y27632 alone stimulated PPF of murne megakaryocytes (0 μM:100%, 1μM:116.2±0.2%,10μM:129.2±4.25% (p<0.01),100μM: 159.8±13.3% (p<0.01))(n=4). E2 alone stimulated PPF of murne megakaryocytes in the dose-dependent manner (0μM:100%,1μM:108.1±4.2%,10μM:118.7±9.5%(p<0.05),100μM: 123.7±3.7% (p<0.01))(n=4). While, PP1 or LY294002 alone inhibited PPF of murine megakaryocytes (PP1; 0μM: 100%, 2μM: 82.1±7.3%(p<0.05), 20μM: 36.2±4.5%(p<0.01), LY294002; 0μM: 100%, 1μM: 94.8±2.3%, 10μM:66.5±4.7%(p<0.01),100μM:43.9±4.2%(p<0.01))(n=4). Based on these results, Rho pathway inhibition or activated Rac stimulated PPF. The addition of E2 to Y27632 resulted in no inhibition/stimulation of PPF (E2/Y27632; 0 nM/10 μM: 100%, 2.3 nM/10 μM: 113±3.5%, 23 nM/10 μM 116.9±9.0%, 230 nM/10 μM: 115.8±4.6 %, 0 nM/100 μM:100%, 2.3 nM/100 μM: 97.2±6.9%, 23 nM/100 μM: 93.1±5.1%, 230 nM/100 μM: 99.4±5.7%)(n=4).The addition of E2 to PP1 or LY294002 resulted in the dose-dependent manner reduction of PPF (E2/PP1; 230 nM/0 μM: 100%, 230 nM/2 μM: 61.9±8.5%, 230 nM/20 μM: 51.0±4.1%, E2/LY294002; 23 nM/0μM: 100%, 23 nM/10μM: 66.8±2.0%, 23 nM/100μM: 68.4±3.3%)(n=4). The degree in reduction with E2/PP1 or E2/LY294002 was similar to that with PP1 or LY294002 alone.These results strongly suggested that that PPF of megakaryocytes, stimulated by E2, might not be involved in Rho but Rac pathway.

Next, we investigated the predominance between Rho and Rac pathways in the process of PPF.The addition of PP1 or LY294002 to Y27632 resulted in no inhibition of PPF (PP1/Y27632; 0μM/100 μM:100%, 2μM /100 μM:105.6±6.0%, 20 μM/100 μM: 107.5±4.7, LY294002/Y27632; 0μM/100 μM:100%, 1μM/100 mμM:92.5±13.8%, 10μM /100 μM:92.6±6.6%, 100μM/100 μM: 97.8±8.0%, ( n=4)).

These results indicated that Rho pathway inhibition stimulated PPF of megakaryocytes, even if activated Rac decreased. Therfore, Rho pathway is more predominant than Rac pathway in PPF of megakaryocytes.
Angiogenic Cytokines: Vascular Endothelial Growth Factor and Basic Fibroblast Growth Factor Levels in Serum of Children with Hematopoietic Malignancies and Solid Tumors.
Authors: Palgan Izabela , Wysocki Mariusz , Palgan Krzysztof , Debski Robert , Styczynski Jan
Published: October 2019

Angiogenesis is a process of blood vessel formation from the preexisting capillaries. It plays an essential role of growth and development of cancer. The most important angiogenic stimulators are Vascular Endothelial Growth Factor (VEGF) and basic Fibroblast Growth Factor (bFGF). The aim of the study was to evaluate the serum levels of VEGF and bFGF in children with solid tumors, hematological malignancies and in healthy controls. Patients: 110 children (33 with solid tumors and 77 with hematological malignancies, including 46 with acute lymphoblastic leukemia and 17 with acute myeloblastic leukemia) (56 male and 54 female) aged 2–18 years (median 8.5 years) and 70 healthy controls (31 male and 39 female) aged 3–18 years (median 13 years). For the quantitative determination the Quantikine VEGF and bFGF immunoassay, a solid-phase enzyme-linked immunosorbent assay method was used. Elevated VEGF and bFGF were defined as being higher than the 95 percentile value in control group. Results: At the time of diagnosis the range of VEGF serum levels in all cancer patiens was 0–2691.3 pg/ml, and in remission 6.08–967.67 pg/ml; while the range of bFGF at diagnosis was 0–64.48 pg/ml, and in remission 0–32.52 pg/ml. In all children with cancer, serum levels of VEGF and bFGF were significantly more often elevated than in healthy controls (p<0.004; p<0.0005). At diagnosis, almost 12% patients had simultaneously elevated serum levels of VEGF and bFGF. Among patients with solid tumors at the time of diagnosis, elevated levels of VEGF and bFGF was observed in 42% and 60%, respectively. There were no differences between serum levels of these factors in patients in remission and in control group. In patients with progression, serum levels of VEGF and bFGF were higher, when compared to levels of these factors at diagnosis. The range of serum levels of VEGF in children with solid tumors at the time of diagnosis was 85.96–2691.3 pg/ml (median 365.48 pg/ml), and in remission 6.08–787.16 pg/ml (median 204.1 pg/ml). The median level of VEGF at diagnosis in children with hematopoietic malignancies (range 0–1869.6 pg/ml, median 111.6 pg/ml) was lower than in children with solid tumors (p<0.0005). In children with acute leukemias at diagnosis, serum levels of VEGF were significantly lower than in children with lymphomas (p<0.0003) and in controls (p<0.05). These results suggest that evaluation of serum levels of VEGF and bFGF can be helpful for monitoring activity of cancer, particularly in solid tumors and in the future can be a target of anti-angiogenic therapy.
Microgranular Variant (M3V) of Acute Promyelocytic Leukemia (APL) Does Not Have a Worse Prognosis than Classical APL in the Atra Era: A Report of 153 Patients Treated on Intergroup 0129 and Pethema LPA96 and LPA99.
Authors: Tallman Martin S. , Kim Haesook T. , Schiffer Charles A. , Appelbaum Frederick R. , Feusner James H. , Ogden Angela K. , Shepherd Lois , Willman Cheryl L. , Bloomfield Clara D. , Rowe Jacob M. , Larson Richard A. , Wiernik Peter H. , Martin Guillermo , Rayon Chelo , de la Serna Javier , Rivas Concha , Gonzalez-San Miguel Jose D. , Deben Guillermo , Sanz Miguel A.
Published: October 2019

The microgranular variant (M3V) accounts for 15–20% of patients (pts) with acute promyelocytic leukemia (APL). Historically, it has been associated with a higher white blood cell count (WBC) at diagnosis and an inferior outcome compared to classical morphology. Pts with APL treated with ATRA plus chemotherapy strategies have a 3–5 year DFS of 70–80%. The prognosis of pts with M3V treated with ATRA-containing chemotherapy strategies has not been adequately studied. This analysis included 153 patients with M3V entered on the North American Intergroup protocol 0129 (INT0129), a randomized phase III study of either ATRA (INT, ATRA)or daunorubicin/cytarabine for induction (INT, DA) and ATRA maintenance or observation after intensive consolidation or PETHEMA protocols LPA96 and LPA99 in which all pts received ATRA plus idarubicin for induction, intensive consolidation and ATRA plus low-dose chemotherapy for maintenance (PETHEMA, ATRA). The median age was 39 years (range 3–79), the median WBC was 14.5 K/μL (range 0.4–550), and the median platelet count was 25 K/μL (range 1.8 – 207.0). The median follow-up time for pts on INT0129 is 9.7 years, and for pts on PETHEMA LPA96 and LPA99 is 3.2 years. The 3-year disease-free (DFS) and overall survival (OS) analyses are shown in the table below.

N Median (in mo) (95% CI) DFS p-value N Median (in mo) (95% CI) OS p-value *comparison between ATRA (INT+PETHEMA) and DA; **comparison among WBC groups. NE: not estimable or not reached in this analysis     INT+PETHEMA, ATRA 106 NE 77% <0.0001* 128 NE 75% <0.0001*     INT, ATRA 19 NE 63% 24 NE 71%     PETHEMA, ATRA 87 NE 81% 104 NE 76% INT, DA 20 8.3(6.7, 12.6) 15% 25 19.4(10.4, 41.8) 32% INT+PETHEMA, ATRA     0<WBC<10K 47 NE 89% 0.01** 52 NE 90% 0.07**     10K≤WBC<50K 37 NE 78% 48 NE 69%     WBC≥50K 22 NE 54% 28 NE 58% INT, DA     0<WBC<10K 8 8.4(3.0, 24.6) 13% 0.94** 10 19.5(8.6, 41.8) 30% 0.32**     10K≤WBC<50K 6 9.2(5.2, 12.6) 17% 8 56.6(10.3, NE) 50%     WBC≥50K 6 7.9(7.4, 15.9) 17% 7 13.6(6.8, 22.2) 14%

In a Cox regression model, when age, gender, WBC, platelet, Hgb, ATRA treatment (ATRA vs DA), and source of pts (INT vs. PETHEMA) are included, only ATRA induction treatment (HR=0.16, p-value=0.0005) appears to be a significant prognostic factor for DFS, and age, WBC, and ATRA induction treatment (HR=0.38, p-value=0.03) for OS. These data suggest that the outcome for pts with APL M3V is excellent, influenced by the WBC at diagnosis and is not different than that of pts with classical morphology when treated with ATRA-containing strategies.
Study on the Relations between HLA-Class II Antigens and Idiophatic Thrombocitopenic Purpura in Adults.
Authors: de Paz Raquel , Cano Pedro , de la Concha Emilio Gomez , Montes Jose Antonio Rodriguez , Hernandez-Navarro Fernando
Published: October 2019

Background: The mechanism of autoimmunity of chronic idiopathic autoimmune thrombocytopenia (cAITP) is not fully understood but may involve binding of antigenic peptides to HLA antigens. We studied MHC class II phenotypes in adult patients with cAITP.

Methods: We have typed 64 adult Caucasian (Spanish) patients with cAITP (17 male and 59 female; median age 47 years range 17–77 years), and compared with the data from to control groups with geographic and ethnic differences. 200 Spanish control samples and 617 American control samples were analyzed. Patients with cAITP were evaluated according to their response to treatment with steroids, splenectomy, dapsona and Anti-CD20. According to the clinical response to the therapy we were able to form two subgroups; patients with good response, normal platelet count during therapy/after splenectomy and poor, less than 50x109/L or no response. Splenectomy was performed in 19 patients and resulted in a bad response in 11 patients of them. 25 of these patients showed a good and 39 a poor response. Class II typing was performed using polymerase chain reaction (PCR) with sequence specific primers.

Results: The comparison of antigen frequencies of the whole group of patients with cAITP and the two groups of healthy controls (Spanish and American samples) revealed significant increase of the HLA-DR12 and HLA-DR10 alleles of the MHC class II especifities (p<0.05). Moreover, the HLA-DR2 and HLA-DR4 phenotype was significantly decreased in the subgroup of patients with good response to treatment (p<0.05), with a high incidence in the subgroup of patients with bad response to all treatment, including progression to splenectomy and refractory to new treatments like Anti-CD20. No other significance was found.

Conclusions: Our data showed that DRB12 and DRB10 were more prevalent in ITP patients than in the two different control groups. Moreover, the difference in the genetic distribution of HLA-DR2 and HLA-DR4 in patients with good and poor response to treatment may indicate that ITP is a more heterogeneous condition than previously suspected, and this genetic marker may improve the early management of patients by better predicting prognosis.
Activation of the Extrinsic Coagulation Pathway in Inflammatory Exudates from Acute Lung Injury: Influence of Hydration on Factor Xa Generation and Inhibition.
Authors: McGee Maria P. , Hite Robert D. , Morris Peter E.
Published: October 2019

The exudative phase of ALI (acute lung injury) is characterized by loss of the endothelial/alveolar barrier, disruption of local water homeostasis, and intra-alveolar activation of blood coagulation. In previous studies, ALI exudates sampled by BAL (bronchoalveolar lavage) demonstrated increased tissue factor and antiproteinase activities compared to BAL from healthy lungs. This study applied osmotic stress techniques to explore the hypothesis that local water activity influences the procoagulant and antiproteinase activities recovered in human BAL fluids. Samples were prepared from 8 injured and 7 normal lungs by sequentially centrifuging BAL fluid at 200 and 40,000g to remove cells and surfactant particles, respectively. Factor Xa generation and inhibition were measured by chromogenic assay under standard (buffer pH 7.4, 0.13 NaCl, 1.3 mM CaCl2, 0.5mg/ml ovalbumin) and osmotic stress (~0.6 atm in standard buffer with polyethylene glycol 8000, ~26 Å radius) conditions by adding either purified factor X (200 nM) or factor Xa (50 nM) to the samples. Factor Xa generation curves had a sigmoidal shape, and the yields in 25 min-long reactions were variable, as expected, and ranged from 4 to 47 nM. Average activity values were 16-fold higher in ALI than in normal lungs, while tissue factor antigen levels measured by ELISA were 6-fold higher than in normal and ranged from 3.5 to 108 ng/ml. Under osmotic stress, the yields and rates increased in samples from healthy lungs, while the responses in ALI samples were mixed. Four showed decreases; 2 increases and 2 showed no change. Antiproteinase capacity, measured as the decrease in factor Xa activity, was variable but higher in ALI samples, and in both ALI and normal lungs, it increased in response to osmotic stress. These results imply that local hydration and dehydration directly influence procoagulant/anticoagulant activities in bronchoalveolar exudates, regardless of other factors, and that impaired alveolar fluid clearance may contribute directly to coagulation imbalances in ALI.
Gene Expression Profiles Distinguish Children Who Develop Therapy-Related Myeloid Leukemia.
Authors: Bogni Alessia , Cheng Cheng , Liu Wei , Yang Wenjian , French Deborah , Mukatira Suraj , Downing James R. , Pui Ching-Hon , Relling Mary
Published: October 2019

In children with acute lymphoblastic leukemia (ALL), failure due to therapy-related myeloid leukemia (t-ML) is a devastating complication. Using a target gene approach, only a few host genetic risk factors for t-ML have been defined. Microarray analysis of gene expression allows for a more genome-wide approach to identify possible genetic risk factors for t-ML. We assessed gene expression profiles (12625 gene probe sets) using oligonucleotide-based arrays in diagnostic ALL blasts from 228 children treated on St. Jude ALL protocols (Total XIII) that included etoposide; 13 of these children developed t-ML. A group of 83 probe sets were significantly related to the time-dependent risk of t-ML, with principal component analysis plot (right panel) separating patients who developed t-ML from the others. Hierarchical clustering of the 83 probe sets grouped patients into 3 clusters (n=163, n=52, n=13), with the cumulative incidence of t-ML being significantly higher in the last cluster (p < 0.0001, left panel) compared to those of the other gene-expression-defined clusters.

Figure Figure

A permutation test indicated that probe sets selected by chance are unlikely to obtain the observed distinct clusters (p=0.045). Distinguishing genes included transcription-related oncogenes (v-Myb, Pax-5), cyclins (CCNG1, CCNG2 and CCND1) and Histone H4. Common transcription factor recognition elements among similarly up- or down-regulated genes included several involved in hematopoietic differentiation or leukemogenesis (Maz, PU.1, FOXO4). This approach has identified several genes whose expression differentiates patients at risk of t-ML, and provides targets for assessing the germline predisposition to leukemogenesis.
Haemophilia A Genotype and Inhibitor Risk: Preliminary Results of a French Haemophilia Centre.
Authors: Borel-Derlon Annie , Slaoui Mounia , Costa Jean Marc , Lavergne Jean Maurice
Published: October 2019

Caen Haemophilia centre monitored 343 haemophilia A patients, 62 of them with a severe defect. We report here the preliminary results of genotypes and factor VIII (FVIII) inhibitors response for the first 59 and particularly for 15 patients with FVIII inhibitors. For severe haemophilia A, the first step is a long PCR for intron 22 inversion, then PCR for intron 1 inversion. The second step is a direct sequencing of the FVIII gene in severe form without inversion and in moderate or mild disease. 41% (16 / 39) of the patients with severe haemophilia A have an intron 22 inversion, the remainder patients have single point mutations (17), principally on exon 14, large deletion (1), small deletion (3) or splice mutations (3). In the severe form, 10 of these patients have developed inhibitors: 6 with intron 22 inversion, 3 non sense mutations on exons 14 or 18, 1 large deletion on exon 14 and 1 splice mutation on intron 22. In the moderate or mild form, 20 patients have missense mutations, 5 of them have developed inhibitors. The genotype of three patients has been previously reported to be associated with a high incidence of inhibitors in moderate and mild form: Arg 593 Cys by loss of tolerance to exogenous and endogenous FVIII (1), Arg 2150 His by reducing the binding of FVIII to von willebrand factor (2). For the other two patients, we identified two missense mutations: Val 2017 Ala, Tyr 1786 Ser. We are processing genotypes for each haemophilia A patients to evaluate the genotype - phenotype relationship and the FVIII inhibitor risk related to genetic and environmental factors.