bluuebunny/crossref_metadata_2025 huggingface dataset:
Proteomic Identification of TAL1/SCL-Interacting Proteins: ETO-2 and MTGR1 Interact with TAL1 in Erythroid Progenitors.
Authors: Cai Ying , Xu Zhixiong , Xie Jingping , Koury Mark J. , Hiebert Scott W. , Brandt Stephen J.
Published: October 2019

The TAL1/SCL gene, originally identified from its involvement by a recurrent chromosomal translocation in T-cel acute lymphoblastic leukemia, encodes a basic helix-loop-helix (bHLH) transcription factor essential for hematopoietic and vascular development. Although TAL1 is believed to regulate transcription of specific sets of target genes, the mechanisms underlying TAL1-directed gene expression are poorly understood. Previous studies have shown, in fact, that it can act as either an activator or repressor depending on the coregulator(s) with which it interacts. To comprehensively identify TAL1’s interaction partners in erythroid cells, we stably expressed a tandem epitope-tagged mouse TAL1 protein in murine erythroleukemia (MEL) cells and determined the composition of affinity-purified TAL1-containing complexes by multidimensional mass spectrometry. From this analysis, we identified all known members of a TAL1-containing DNA binding complex previously characterized in erythroid cells, including TAL1, its E protein DNA-binding partners, the zinc finger transcription factor GATA-1, the LIM-only protein LMO2, and the LIM domain-binding protein Ldb1, as well as proteins described to interact with GATA-1 (FOG-1), LMO2 (ELF2A2), and Ldb1 (SSDP2 and SSDP3). In addition, we identified a number of other DNA binding proteins, chromatin modifying proteins, and transcriptional regulators, including the ETO family members ETO-2 and MTGR1. TAL1 interaction with ETO-2 and MTGR1 was verified by coimmunoprecipitation analysis in MEL cells expressing these proteins at endogenous levels, in MEL cells stably expressing an epitope-tagged TAL1 protein, and in COS cells transiently transfected with TAL1 and ETO-2 or MTGR1 expression vectors. Mapping analysis with GAL4 fusion proteins identified the bHLH domain as the region in TAL1 responsible for interaction with these ETO family proteins. Significantly, expression of MTGR1 enhanced ETO-2 interaction with TAL1-GAL4 protein. Finally, transient transfection analysis with a luciferase reporter construct linked to multiple GAL4 DNA binding sites showed greater than additive augmentation of TAL1-directed gene repression with coexpression of the two ETO-related proteins compared to that observed with ETO-2 or MTGR1 transfected individually. These results identify ETO-2 and MTGR1 as authentic TAL1 interacting proteins and suggest that a hetero-oligomeric complex of the two contributes to TAL1-directed repression in erythroid progenitors.
Increased Generation of Hepatocytes Expressing Bone Marrow-Derived Markers after Hematopoietic Progenitors’ Mobilization in a Murine Model of Hepatic Damage.
Authors: Fellow Oscar Quintana-Bustamante , Alvarez-Barrientos Alberto , Fabregat Isabel , Bueren Juan A. , Segovia Jose C.
Published: October 2019

The contribution bone marrow (BM) cells to the repair of a damaged liver has been described in animal models and in humans. This process has been explained both as a result of hematopoietic stem cell (HSC) trans-differentiation, as well as a result of cell fusion between HSC-derived cells with cells from non-hematopoietic tissues. In the present work we have investigated whether the mobilization of HSCs can increase the generation of BM- derived hepatocytes (BM-DHs) in damaged livers. With this purpose, Ly-5.1 mice were myeloablated and transplanted with 107 BM cells from Ly-5.2 congenic mice, which also express the green fluorescent protein (EGFP) under the control of the b-actin promoter. Three months after transplantation, groups of 4–6 animals were injected with saline or with carbon tetrachloride (CCL4) for three more months to induce a cirrhosis-like damage. A marked dysbalance in serologic parameters, together with an altered hepatic structure was observed in the CCL4-treated animals. Part of the animals corresponding to each group were mobilized with G-CSF for one, two or three weeks. No difference in the mobilization of hematopoietic cells was observed in CCL4-treated versus non-treated animals. One month after G-CSF treatment, animals were sacrificed and the presence of BM-DHs, identified as EGFP+CD45− hepatocyte-like cells, with ability to accumulate glycogen, was evaluated by fluorescent immuno-histochemistry using specific antibodies for EGFP and the murine Ly-5.2 panleukocyte antigen and successive PAS staining. BM-DHs were clearly observed after hepatic damage (4 BM-DH/106 cells) at rates similar to results already published by others. Moreover, the G-CSF mobilization significantly augmented the percentage of BM-DHs in the liver of CCl4-treated and non CCl4-treated mice (69/106 and 23/106 cells, respectively). Whether these BM-DHs result from the trans-differentiation of HSCs or from the in vivo fusion of HSC-derived cells, and whether the mobilization treatment improve the recovery of the damaged liver are now under study. Taken together, this data demonstrate that the mobilization of HSCs from the BM to the periphery markedly increases the presence of BM-DHs in the liver of transplanted mice, opening the possibility of using hematopoietic growth factors in the treatment of non-hematopoietic degenerative diseases.
Safety and Efficacy of Alternate Desferrioxamine and Deferiprone Compared to Desferrioxamine Alone in the Treatment of Iron Overload in Transfusion-Dependent Thalassemia Patients.
Authors: Galanello Renzo , Kattamis Antonios , Piga Antonio , Tricta Fernando
Published: October 2019

The safety and efficacy of alternating desferrioxamine and deferiprone for the treatment of iron overload in patients with transfusion-dependent anemias was studied in 60 thalassemia patients regularly treated with desferrioxamine. Patients were randomized to continue desferrioxamine alone (20–60 mg/kg/day, 5–7 days/week) or to alternate desferrioxamine (20–60 mg/kg/day, 2 days/week) with oral deferiprone (25 mg/kg tid, 5 days/week). Both treatment groups were similar for age (19.8 ± 6.1 years for desferrioxamine alone and 18.7 ± 4.8 years for alternate therapy) as was gender distribution and mean standard dose of desferrioxamine at the time of study initiation. Over the following 12 months, all patients were monitored weekly for adverse events and for their white blood cell count. Efficacy of the chelation was evaluated by measurement of the serum ferritin, liver iron concentration (magnetic susceptometry by SQUID), and by Non-Transferrin Bound Iron (NTBI). Compliance was comparable for both arms (96.1 ± 5.0% for alternate therapy vs 95.7 ± 5.7 % for desferrioxamine alone; p=0.7883). There was no significant difference in the proportion of patients with adverse events in the two therapy groups but the chelation regimens were associated with distinct adverse events. The alternate therapy was associated with transient gastrointestinal symptoms, such as vomiting in 5 patients (17%), abdominal pain in 3 patients (10%), or diarrhea in one patient (3%), or transient increase of serum ALT levels in one patient (3%), occurring mainly in the first weeks of therapy and were mild/moderate in severity. Daily infusions of desferrioxamine were associated with abscess at the site of infusion in one patient (3%), and allergic reactions in another patient (3%). Mean serum ALT levels were not significantly different between the two therapies. There were no episodes of agranulocytosis and only one patient, treated with desferrioxamine alone, experienced milder neutropenia.

Both therapies resulted in similar decreases of serum ferritin (−349 ± 573 mg/L for the desferrioxamine arm; −248 ± 791 for the alternate arm; p=0.5802), and of liver iron concentrations (−239 ± 474 μg/g wet weight for the desferrioxamine arm; −65 ± 615 μg/g wet weight for the alternate therapy arm; p=0.2263) by the end of the treatment period. No significant changes in NTBI were observed between the two treatment arms (1.10 ± 7.19 μmol/L for the desferrioxamine arm; −0.03 ± 8.13 μmol/L for the alternate arm; p=0.5775). In conclusion, this 12 month study in transfusion-dependent thalassemia demonstrated that the alternating therapy with deferiprone and desferrioxamine is not associated with a significant increase in the incidence of adverse events and that it has comparable efficacy to desferrioxamine alone in controlling iron overload.
Analytical Performance Evaluation of a Linear Array-Based β-Hemoglobinopathy Mutation Assay.
Authors: Jarvis Michael , Komaromy-Hiller Gabor , Amos Jean
Published: October 2019

Routine clinical evaluation of β-globin phenotypes usually includes HPLC screening and alkaline/acid electrophoresis and/or isoelectric focusing. Although common structural variants are easily identified in the routine environment, specific β-thalassemia mutations are not and these can be the basis for prognosis and also for subsequent prenatal diagnosis for at-risk family members. Herein, we report evaluation of the performance of a linear array-based β- hemoglobinopathy mutation assay provided by Roche Molecular Diagnostics (Pleasanton, CA) that detects 6 β-globin variants (S, C, E, Knossos, D-Los Angeles and O-Arab) and 42 β-thalassemia mutations (90% predicted detection in any at-risk population group). The assay consists of one multiplex PCR with biotinylated primers, amplicon hybridization to a reverse line blot array, and colorimetric detection of a streptavidin-HRP conjugate. We tested 50 archived, abnormal and 92 sequential clinical samples, all previously tested in our routine assays. The results of the DNA and HPLC/electrophoresis were 100% consistent with the linear array, although Hb-S, C, D-Los Angeles and E were the only structural variants present in our collection. We verified the presence of Hb-S and excluded Hb-O-Arab in an ambiguous compound heterozygote for which HPLC and electrophoresis could not distinguish possible Hb-C-Harlem from Hb-O-Arab. Of 15 putative β-thalassemia minor samples, 10 had 6 different β-thalassemia mutations. Based on our experience, the daily throughput for a single technologist performing this mutation analysis is approximately 200 samples; it is, thus, an appropriate second-tier assay for newborn screening programs. The assay is robust, reliable and is also an excellent screen prior to sequencing for rare mutations.
Lack of Correlation between Serum Ferritin and Liver Iron Concentration in Beta-Zero Thalassemia Intermedia.
Authors: Galanello Renzo , Giagu Nicolina , Barella Susanna , Maccioni Liliana , Origa Raffaella
Published: October 2019

Serum ferritin and liver iron concentration (LIC) are the most commonly used methods for assessment of iron overload in thalassemia. While in patients with thalassemia major a significant correlation has been found between these two parameters, data are lacking in patients with thalassemia intermedia. In this study we measured the serum ferritin and LIC in 22 adult patients with beta-zero thalassemia intermedia never transfused (14 patients) or sporadically transfused, i.e. less than 10 units in total (8 patients), who maintained a mean hemoglobin of 8.8 ± 1.1 g/dl. Serum ferritin levels were measured by an automated chemiluminescence immunoassay analyzer, whereas LIC was determined by atomic absorption in liver biopsies. We compared the results obtained in those patients with those obtained in 22 regularly transfused (mean annual Hb = 11.3 ± 0.3 g/dl) and iron chelated thalassemia major patients, matched by sex, age and liver iron concentration. We also determined serum erythropoietin (s-epo) and serum transferrin receptor (s-TfR) in a cohort of the two patient groups (12 thalassemia intermedia; 15 thalassemia major). Mean LIC was 11.3 ± 6 mg/g dry weight tissue in thalassemia intermedia, and 11.8 ± 7 mg/g d.w. in thalassemia major group. Mean serum ferritin (at least 2 determinations from each patient within ± 2 months of liver biopsy) was 627 ± 309 ng/ml in thalassemia intermedia and 2748 ± 2510 ng/ml in thalassemia major. The difference was statistically significant (p = 0.0001). LIC was weakly correlated with serum ferritin in thalassemia major patients (r2=0.46; p=0.001) and uncorrelated in patients with thalassemia intermedia (r2=0.04; p=0.37) (Figure). S-epo and s-TfR were significantly higher in thalassemia intermedia than in thalassemia major [s-epo 467 ± 454 mU/ml versus 71 ± 44 mU/ml (p<0.001); s-TfR 43 ± 13 mU/ ml versus 13 ± 6 mU/ml (p<0.0001)]. The discrepancy between LIC and serum ferritin in thalassemia intermedia patients may be due to the higher levels of s-epo (secondary to anemia) in those patients, which through the iron regulatory protein 1 determine an up-regulation of s-TfR and a repression of ferritin translation (Weiss et al 1997). The mechanism of iron overload may also be mediated by hepcidin, whose synthesis could be suppressed as a consequence of anemia. The observation reported has important implications for iron chelation in patients with thalassemia intermedia. In such patients serum ferritin levels have little value for the monitoring of iron overload.

Figure Figure
Relevance of Routine Bacterial Screening of Pooled Random Platelet Concentrates with the Bactalert® System.
Authors: te Boekhorst Peter A.W. , Beckers Erik A.M. , Vos Greet C. , Vermeij Hans , Leebeek Frank W.G. , van Rhenen Dick J.
Published: October 2019

Background: Transfusion of bacterial contaminated platelet (PLT) units can cause serious transfusion reactions. To prevent this complication, bacterial screening by the automated BacTalert® system of platelet concentrates was implemented in the southwest part of the Netherlands since october 2001. Despite this system, two severe transfusion reactions caused by bacterial contaminated PLT units occurred. Therefore, evaluation of the efficacy of this approach was done.

Material and Methods: PLT products in the Netherlands are produced by the Sanquin Bloodbanks. In the Southwest part of the Netherlands PLT products mainly consist of pooled products derived from five random donors. Of all produced platelet products, samples were taken and cultured (aerobic and anaerobic) for seven days using the automated BacTalert® system. The PLT products were issued to the hospitals on a "negative-to-date" basis. In case of positive cultures, conformation and identification cultures were taken. At the same time, involved and related products thet were derived from the same donor were also withdrawn. In case of delivery of possibly contaminated products to the hospitals, these were notified and the products were recalled. If the products were already transfused, treating physicians were notified and asked if adverse reactions had occurred in these patients.

Results: From October 2001 to 2003, a total of 28,104 produced pooled PLT units were tested for bacterial contamination using the BacTalert® system. Positive signals were found in 203 (0,72%) samples. At the time of a positive signal, 125 PLT products were already issued to the hospitals and of these 125 products, 113 units were already administered to patients. Analysis of all positive cultures revealed mainly skin-derived bacteria, but in 15 sample Bacillus cereus (7%). In 9.4% (=19) of the samples with a postive BacTalert® signal, bacterial growth could not be confirmed. None of the PLT units with a postive bacterial culture that were already administered to patients caused clinically significant transfusion reactions. Nevertheless, two neutropenic patients due to leukemia treatment, suffered from life-threatening B. cereus sepsis after PLT transfusion. In both cases, B.cereus was cultured from the PLT bags, whereas the BacTalert® tested samples remained negative.

Conclusions: Issuing pooled PLT units on a negative-to-date basis for bacterial screening resulted in a significant amount of recall procedures. The BacTalert® system failed to detect B. cereus contamination in at least two cases that resulted in sever transfusion reactions. Other strategies, including pathogen reduction, might be applied to guarantee a better degree of bacterial safety.
Complications of Pediatric Apheresis.
Authors: Michon Bruno , Duval Michel , Winikoff Rochelle , Champagne Martin A.
Published: October 2019

Objectives

Even if the practice and applications of apheresis have grown over the last decade, the data in pediatrics are limited. The frequency of the complications is low in adults but there is a lack of clinical trials assessing the nature and the frequency of the complications in children.

Methods

We have revised the medical files of all patients (0–18 years old) who have undergone an apheresis at Ste-Justine Hospital (pediatric hospital in Montreal, Canada) between November 1994 and July 2002. The reported complications were: symptoms of hypocalcemia, hypotensions (drop of systolic BP ≥10 mmg Hg), complications of the vascular access (thrombosis and infection), reactions to blood products and severe anemia.

Results

A total of 1632 aphereis were performed on 186 different patients (32 distinct diagnosis). The procedures were plasma exchange (67%), peripheral blood stem cells collect (18%), red cell exchange (6,9%), cytoreduction leucapheresis (0,7%) and immunoadsorption (6,7%). Adverse reactions, although mostly minor, were reported in most of the patients. The more frequent complications were hypotensions (14% of apheresis and 48,4% of patients), hypotensions which needed bolus (4,8% of apheresis and 26,9% of patients) , symptoms of hypocalcemia (9,7% and 28,5%), allergic reactions (4,4% and 5,9%) and troubles related to the vascular access: infection (2,1% and 16,1%) and thrombosis (1,7% and 12,4%). A severe anemia (Hb < 70g/L) complicated 2,5% of apheresis (17,2% of patients). There were two deaths related to procedures. With a multivariate analysis, we have identified independent risk factors for occurence of complications: lower weight, lower hemoglobin pre-pheresis, pheresis in ICU and number of pheresis.

Conclusion

The incidence of complications of apheresis in our pediatric cohort is much higher than what is reported in adults. The adverse reactions can be explained by the citrate toxicity, the extravascular volume shift and the vascular access installed. We consider that apheresis in pediatrics present unique features and should be done in specialized centers.
Increased Clearance Explains the Ultra-Large Multimers in Von Willebrand Disease Type 2M Vicenza; Lessons from Recombinant VWF Vicenza and Modeling of Multimer Catabolism.
Authors: Gézsi András , Balázsi Gábor , Sallai Krisztina , Mohl Adrienn , Nagy Eszter , Szabó Teréz , Sadler J. Evan , Bodó Imre
Published: October 2019

The pathogenesis of VWD Vicenza has remained elusive. VWD Vicenza is characterized by low plasma but normal platelet VWF concentration, the presence of ultra-large plasma multimers, and a heterozygous R1205H mutation. VWF Vicenza is reported to have a decreased half-life in the circulation. When we expressed rVWF Vicenza R1205H in 293T cells, it was secreted with wild type efficiency and multimer distribution, suggesting that the primary defect is accelerated clearance. To evaluate this hypothesis, we developed a pharmacokinetic model of VWF multimer catabolism. The initial assumptions are: 1. Secretion occurs at a fixed rate with the initial “ultra-large” multimer distribution seen in platelet alpha granules. 2. Cleavage of multimers occurs with a probability p that increases with increasing multimer size. 3. Clearance occurs with a time constant determined by the plasma half life and is independent of multimer size. Modeled multimer distributions were compared to those determined experimentally for patient plasma samples. The effects of DDAVP infusion also were modeled and compared to published data (Casonato et al, Blood2002; 99:180). For p = 7.5 x 10−4 min−1 and a half life of 12 h, the modeled multimer patterns were comparable to the observed steady-state distribution of normal VWF. Decreasing the half life to 2 hours produces a low plasma concentration of “ultra-large” multimers typical of VWD Vicenza without a change in any other parameter. Conversely, increasing the probability of cleavage by only thirty percent produces typical VWD 2A multimer distributions. The model also reproduces the triplet patterns of normal and type 2A VWF. Finally, the DDAVP simulations reproduced the time course of VWF plasma concentrations and multimer distributions of DDAVP-treated patients. We conclude that increased clearance alone can explain the ultra-large multimer distribution of VWD Vicenza. Similar modeling should allow the estimation of VWF cleavage and clearance rates in other variants of VWD and in other clinical situations including thrombotic thrombocytopenic purpura.

Figure Figure Figure Figure
Effects of Hepcidin, Ferritin, and Iron Deprivation on Erythroid Colony Formation in Vitro.
Authors: Means Robert T. , Dallalio Gail A. , Fleury Thomas W.
Published: October 2019

The anemia of chronic disease (ACD) results from a combination of three pathologic processes. In ACD, a modest shortening of red cell survival creates an increased demand for red cell production, which is not met because of an impaired erythropoietic response and defects in reticuloendothelial iron mobilization and utilization. The impaired erythropoietic response, in turn, has two components: a blunted erythropoietin response, and an impaired response of erythroid progenitors to erythropoietin. Recombinant human erythropoietin (rhEPO) can reverse this impaired progenitor response in vitro, and can also correct ACD in patients. These processes have generally been considered effects of the cytokines which mediate the immune and inflammatory response, such as tumor necrosis factor, interleukin-1, and the interferons. It has recently been proposed that hepcidin, a mediator of innate immunity with the iron regulatory properties, is the factor responsible for ACD. If this is the case, then hepcidin should be able to induce the pathophysiologic mechanisms implicated in ACD. We therefore evaluated the effects of hepcidin and associated phenomena on human CFU-E colony formation in vitro. All CFU-E cultures were performed in plasma clots in serum-containing medium with rhEPO 1 U/mL. Hepcidin at concentrations 10 ng/mL -10 μg/mL had no effect on CFU-E colony formation. A number of studies have demonstrated that increased hepcidin message expression and protein production are strongly associated with increases in serum ferritin concentrations, and so the effect of added ferritin on erythroid colony formation was studied. Neither ferritin nor apo-ferritin 10 – 1000 ng/mL had inhibitory effects on CFU-E colony formation. The effect of iron deprivation on erythroid colony formation was evaluated with using desferrioxamine. Desferrioxamine 0.01mM decreased CFU-E colony formation to 60% of control values, while higher concentrations completely ablated colony growth. In summary, hepcidin does not appear to inhibit CFU-E colony formation directly or indirectly through ferritin. It may exert such an effect by decreasing availability of iron for erythropoiesis; however, such a finding would be difficult to reconcile with the observed clinical response of ACD to rhEPO, given that iron availability is typically a limiting factor in the erythropoietic response to rhEPO. The role of hepcidin in the overall pathogenesis of ACD remains to be fully determined.
Molecular Basis of Congenital Dyserythropoietic Anemia Type I in French Patients.
Authors: Tamary Hannah , Dgany Orly , Proust Alexis , Krasnov Tanya , Avidan Nili , Tchernia Gil , Geneviève David , Comier-Darre Valérie , Le Merrer Martine , Bader-Meunier Brigitte , Ferrero-Vacher Corinne , Yaniv Isaac , Delaunay Jean
Published: October 2019

Congenital dyserythropoietic anemia type I (CDA I) is an inherited disorder characterized by macrocytic anemia and occasionally also by distal bone malformations. The disease has recently been shown to be caused by mutations in the CDAN1 gene, encoding codanin-1. The aim of the study was to characterize the CDAN1 mutations in 12 French patients with CDA I. The clinical data of the 12 French patients (10 kindreds) with CDA I were reviewed. Each of the 28 CDAN1 exons was amplified and sequenced. Half of the patients had a severe disease with prominent neonatal manifestations, complex bone disease, or both. Nine disease-causing mutations were identified: 6 described previously (P1130L, P671L, F869I, R681X, R713W, S1034F) and 3 novel mutations (R687W, F52L and IVS 8 G to A). Seven were missense mutations located in exons 14 to 28. Twenty seven per cent were identified in exon 14. No patient was homozygous for null type mutations. Only in two monozygotic twin patients did we fail to uncover a mutation. Mutations P671L, F52L and R713W were found on four, two and two alleles, respectively. One of the P671L alleles carried a second mutation (R687W). In conclusion the CDAN1 gene is apparently involved in most cases of CDA I in the French patients explored. Our study supports the previous findings that homozygosity for the null type mutations may be lethal, and that CDAN1 mutations are localized mainly in exons 12–28. In the present small group of patients, no apparent phenotype-genotype correlations were observed, in particular concerning abnormalities of the bones. Analysis of a larger number of patients from different ethnic groups is required to further characterize phenotype-genotype correlation in CDA I.
Quantity and Functional Changes of Subsets of Th Cells in the Bone Marrow of Severe Aplastic Anemia Patients.
Authors: He Guangsheng , Shao Zhonghong , Wu De Pei , Ma Xiao , Sun Aining
Published: October 2019

Objective To evaluate the quantity and functional changes of subsets of Th cells in the bone marrow of severe aplastic anemia (SAA) patients and the relationship between these changes and the patient hematopoietic function.

Methods By FACS, the quantity and ratio of Th1 cells and Th2 cells, the percentage of CD3+CD8+ cells in the bone marrow were detected in 24 patients with SAA at active phase, 15 patients with SAA at recovery phase, and 16 normal controls respectively. By radioimmunoassay, the levels of cytokines of Th1 type (TNF-α), or Th2 type (IL-4) in the serum of 20 patients with SAA at active phase, 12 patients with SAA at recovery phase, 16 normal controls were measured respectively. The relationships between CD3+CD8+ cells, TNF-α and Ret, ANC were evaluated; the relationships between Th1 cells and CD3+CD8+ cells, TNF-α or Ret, ANC were evaluated; the relationships between IL-4, balance of Th1/Th2 and Ret, ANC were also evaluated.

Results The percent of Th1 cells, Th2 cells, and ratio of Th1/Th2 in bone marrow of patients with SAA at active phase was: 4.87±2.64%, 0.41±0.26%, 21.22±5.07, were higher than those of normal controls: 0.42±0.30% (p<0.01), 0.24±0.17% (p<0.05), 1.57±0.93(p<0.01) respectively, and all of these decreased to normal levels at recovery phase (p>0.05); the percent of CD3+CD8+ cells decreased from 32.32±18.69% at active phase to 13.76±2.96% at recovery phase significantly (p<0.01); the serum levels of TNF-α, IL-4 at active phase was 4.29±3.15ng/ml and 1.24±0.73 ng/ml respectively, higher than those of normal controls(1.21±1.16 ng/ml, 1.18±0.97 ng/ml), but only the difference of TNF-α was significant (p<0.01); in recovery SAA patients, the serum levels of TNF-α significantly decreased to 1.46±1.41 ng/ml (p<0.01), and the levels of IL-4 increased continually markedly to 3.05±1.94 ng/ml; the CD3+CD8+ cells and TNF-α of patients correlated with Ret (p<0.05;p<0.05) and ANC (p<0.05;p<0.05) negatively, Th1 cells correlated with CD3+CD8+ cells and TNF-α positively (p<0.01;p<0.05), the Ret and ANC negatively(p<0.01;p<0.01), IL-4 and the balance of Th1/Th2 correlated with Ret and ANC positively (p<0.05;p<0.01) (p<0.01;p<0.01).

Conclusion The formation of bone marrow failure in SAA might be caused not only by the increase of Th1 cells, Th1 type effective cells and cytokines, but also by unsufficient compensation of Th2 cells and Th2 type cytokines, which shifted the balance of Th1/Th2 to Th1.
Transcranial Doppler in Sickle Cell Anemia Adult Patients: Brazilian Experience.
Authors: Silva Gisele S. , Figueiredo Maria S. , Vicari Perla , Massaro Airton R. , Filho Adauto Castelo , Gabbai Allan A. , Bordin Jose Orlando
Published: October 2019

Sickle cell anemia (SCA) may cause a variety of neurological complications, including stroke and headaches. Stroke occurs in up to 9% of children with SCA, and transcranial Doppler (TCD) studies have demonstrated that increased velocities are related to higher stroke risk. Throbbing headache occurs in SCA but its cause, frequency, and relationship to TCD velocities have received little attention. On the other hand, there are few TCD studies in adult patients. Our aims were: 1) to describe the main features of TCD in adult SCA patients, and 2) to investigate if there were correlation between TCD features and presence of headache. TCD was performed in 56 adult SCA patients (≥ 16 years old) and in 56 healthy individuals (HI), matched by age and race. There were 6 patients with a remote history of stroke but none were on chronic transfusion. The SCA group was submitted to a neurological evaluation and specifically asked about the occurrence of headache and its characteristics. The highest flow velocity (maxFV) recorded for each artery was considered the most representative. We analyzed the frequency of FV asymmetry (side-to-side difference > 20%) and focal FV changes. The mean maxFV was significantly higher in patients (117.7 ± 21.6 cm/s) than in HI (72.45 ± 11.48 cm/s) (p<0.005). Only one patient had maxFV higher than 170 cm/s. The frequencies of asymmetry and of focal FV changes were significantly higher in SCA. Forty-one patients (73.2%) reported having headaches. Twenty-eight patients (50%) had severe (= 5 for pain intensity at a 1–10 scale) and frequent headaches (at least once a month). This group of patients presented TCD velocities significantly higher than patients without or with milder headaches (p=0.035). In conclusion, TCD maxFV was significantly higher in adult patients with SCA than HI, however, only one patient was considered at risk of stroke according to TCD criteria described in children. FV asymmetry and focal FV changes may be markers for arterial disease in adult SCA patients, and need to be further confirmed by neuroimaging and clinical follow up studies. The patients with severe headaches presented TCD velocities significantly higher than patients without or with milder headaches, but this finding needs to be confirmed by more and larger studies.
Global Gene Expression Profile of Human Bone Marrow before and after Hydroxyurea Administration in Sickle Cell Anemia.
Authors: Costa Flavia C. , Cunha Anderson F. , Fattori Andre , Peres Tarcisio S. , Costa Gustavo G.L. , Saad Sara T.O. , Costa Fernando F.
Published: October 2019

The levels of HbF in sickle cell disease (SCD) can be increased by pharmacological agents such as hydroxyurea (HU), which has been shown to reduce the frequency of pain crises, hospitalizations and acute chest events requiring blood transfusions in adults with SCD. However, there is some evidence to suggest that some SCD patients show benefits from HU treatment without increase in their HbF levels. Thus, the molecular mechanism by which HU increases HbF levels and improves the clinical evolution in SCD remains unclear. This study aims to provide the global gene profile of human bone marrow of a homozygous patient three months before beginning treatment and after the initial administration of HU and to investigate groups of differentially expressed genes that could be involved in the pathways by which HU improves the clinical evolution in SCD. Using the Serial Analysis of Gene Expression (SAGE) technique, two libraries, before HU administration (HbS profile) and after HU administration (HbSHU) were performed. A total of 47.192 and 46.697 tags were analyzed for the HbS and HbSHU profiles and represented 15.735 and 15.901 distinct tags, respectively. Among these, 4.151 and 3.817 tags were no match tags that could represent new genes that remain to be identified. When both profiles were compared, 518 transcripts were determined to have statistically significant differential levels of expression (P value < 0.05). The functional annotation of transcripts, according to the Gene Ontology Consortium, showed that the categories of binding and structural molecule activity were up-regulated following HU treatment. For example, genes associated with nucleic acid binding such as Signal Transducer and Activator of Transcription 5 A, v-fos FBJ murine osteosarcoma viral oncogene homolog, Early Growth Response 1 and several ribosomal and zinc finger proteins were induced by HU treatment. Conversely, the transporter activity category was down regulated by HU treatment. Genes associated with oxygen transporter activity and other genes associated with ion binding, like S100 calcium binding protein A8 (calgranulin A) and transferrin were found to be down regulated by HU treatment. Taken together, these results strongly suggest that HU produces a significant change in the expression of bone marrow cells. Future studies of these described genes that are differentially expressed during HU treatment may contribute to further the understanding of the mechanism by which HbF acts in SCD and improves the clinical evolution of the disease. The description of new genes involved in these pathways may also represent a potential tool to identify new targets for the therapy of SCD.
Study on Hepatitis C Treatment Using Ribavirin and Pegylated Interferon in Thalassemia Patients (SCRIPT): One Year Report.
Authors: Inati Adlette , Sharara Ala , Ghorra Sandy , Aoun Elie , Taha Marwan , Koussa Suzanne , Taher Ali
Published: October 2019

BACKGROUND: Treatment of thalassemia patients with HCV using combination interferon and ribavirin is limited to few case series.

DESIGN: Multicenter randomized single-blinded trial comparing PEG-IFN a-2a +/− ribavirin in thalassemia major patients with chronic HCV.

METHODS: 20 patients with HCV genotypes 1 and 4 were randomized into Group A (PEG-IFN 180 mg SC once weekly) and B [PEG-IFN and ribavirin (≥10.6 mg/kg/d)] for 48 weeks. Baseline studies included liver biopsy (histology and iron concentration), quantitative PCR, and HCV genotyping. The primary end point was a SVR 24 weeks post-treatment. Secondary end points were transfusion requirements, EVR, ETR, and biochemical response.

RESULTS: Baseline characteristics of both groups were comparable. The mean age was 21.6 ± 11 years in group A and 16.5 ± 3.9 in group B. Fibrosis grade >1 was present in 42% in group A and 50 % in group B. Mean viral load was 137,570 IU/ml and 178,087 IU/ml respectively. Genotype 1 was found in 66% in group A and 87% in group B. EVR occurred in 8/12 in group A and 6/8 in group B (67 and 75% respectively). ETR was observed in 7/12 in group A and 6/8 in group B (59.3% and 75% respectively). Transfusion requirements increased by 55% in group B versus 21% in group A (p=0.08). Adverse events were not significantly different between treatment groups.

CONCLUSIONS: This interim report suggests that combination PEG-IFN and ribavirin is effective at achieving ETR in thalassemics, is associated with an increase in transfusion requirements but no other significant side effects. Additional follow up and patient recruitment is needed to evaluate long term safety and sustained viral response.
HB Florida: A New Elongated C-Terminal β-Globin Variant Causing Dominant β-Thalassemia.
Authors: Sonati Maria F. , Weinstein Beatriz I. , Erramouspe Beatriz , Albuquerque Dulcineia M. , Kimura Elza M. , Oliveira Denise M. , Costa Fernando F.
Published: October 2019

An elongated C-terminal β-globin variant, due to the deletion of one nucleotide (-C) in between codons 140/141 (GCC/CTG→GCC/TG), which modified the C-terminal sequence and added 10 more residues to the β-chain [(141)Trp-Pro-Thr-Ser-Ile-Thr-Lys-Leu-Ala-Phe-Leu-Leu-Ser-Asn-Phe-(156)Tyr-COOH], was found in an 8-year-old Argentine girl of Spanish descent with clinical picture of β-thalassemia intermedia. The patient presented chronic moderate hemolytic anemia (RBC=3.8x10¹²/L, Hb=8.6 g/dL, Hct=28%), with pallor, jaundice and liver and spleen enlargement, having required blood transfusion for 5 times, during viral and bacterial infections; peripheral blood analysis revealed a remarkable degree of anisocytosis with microcytosis (RDW=28%, MCV=73.0 fl), poikilocytosis (with ovalocytes and schistocytes), hypochromia (MCH=22.6pg), 13% of reticulocytes and 2% of erythroblasts, punctate basophilia, elevated Hb A2 and Hb F levels (5% and 13%, respectively), without any detectable abnormal Hb (Hb and globin chain electrophoreses/HPLC). Tests for unstable hemoglobins were weakly positive, but the staining of the bone marrow cells with methyl violet allowed the visualization of many inclusion bodies, suggesting that this is probably a hyperunstable variant whose proteolysis in the bone marrow precursors results in dominant β-thalassemia phenotype, since the other β-locus showed no alteration. Mother’s carrier was completely normal and the father, although not available for studying, was also probably normal.
Pegfilgastrim in Autoimmune Neutrophenia.
Authors: Hopkins Tamara V. , Floyd Justin D. , Mirza Irfan A. , Lobins Raymond L. , Bashir Qaiser , Shahab Nasir
Published: October 2019

A 50-year-old Caucasian male with chronic renal failure with two failed cadavaric kidney transplants was evaluated after multiple admissions for life-threatening febrile neutropenia. The absolute neutrophil counts (ANC) had ranged from 165–988/mm3 in the previous year. No cause was identified. A bone marrow aspirate revealed normal trilineage hematopoiesis without dysplasia and cytogenetic analysis demonstrated 46 XY. Core biopsy revealed adequate cellularity. A blood antineutrophil antibody test was positive confirming a diagnosis of autoimmune neutropenia (AIN). Treatment with prednisone was initiated. Although the patient responded to prednisone (100mg/day), evidenced by the ANC increasing to 5544/mm3, he developed massive shifts in weight secondary to fluid retention which complicated dialysis. Additionally he had mild to moderate steroid induced psychosis and sleep disturbances. Attempts at decreasing the dose of prednisone resulted in a decrease in the ANC as well. Therefore, treatment with filgastrim at 300 mcg SQ daily was initiated. The ANC rapidly increased from 571 to 16,578/mm3. Treatment was then switched to pegfilgastrim 6 mg SQ every 3 weeks for ease of administration. An initial ANC increase to 16,000/mm3 was noted; however, by week 3 the ANC had decreased to 448mm3. As a result, the treatment schedule was changed to pegfilgastrim 6 mg SQ every 2 weeks. Since this adjustment, the ANC has consistently ranged from 1500–20,000/mm3. Additionally, there have been no other infections. Pegfilgastrim has already been shown to be as effective as filgastrim in cancer chemotherapy patients, providing a more convenient and tolerable alternative to filgastrim. To date there have been no documented uses of pegfilgastrim in AIN patients. It is logical to assume that pegfilgastrim would have similar efficacy in AIN as well. It was this assumption that first lead to its use in our patient. However, dosing every two weeks rather than the standard every 3 weeks was necessary to achieve the desired effect.
Extracellular Nucleotide UTP Regulate Lipopolisaccharide-Induced Cytokine Production in Human Neutrophils.
Authors: Garcia Analia E. , Tuluc Florin , Meshki John , Bredetean Ovidiu , Kunapuli Satya P.
Published: October 2019

Extracellular nucleotides can function as paracrine and autocrine mediators that signal through P2Y nucleotide receptors. Human polymorphonuclear (PMN) cells express P2Y receptors on the cell surface but the role of these G protein-coupled receptors in human neutrophils is not well established. Interleukin-8 (IL-8), which is a CXC-chemokine that induces adherence of neutrophils to vascular endothelium and extravasation into tissue, is produced by macrophages and endothelium. Additionally, neutrophils can synthesize and release interleukin-8 upon stimulation with lipopolisacharides (LPS) but the intracellular signaling pathways involved in this process are not clearly understood. LPS is a potent activator of macrophages and is responsible for the initiation the septic shock, increasing the secretion of various cytokines. Here we present evidence that LPS (1 ng/ml to 100 ng/ml) causes IL-8 production in a dose- and time- dependent manner and demonstrate that LPS itself is responsible for mitogen-activated protein kinase (MAPK) ERK1/2 phosphorylation. We used an enzyme immunosorbent-linked assay (ELISA) to determinate the amount of IL-8 released by isolated human neutrophils and present evidence that extracellular nucleotide uridine triphosphate (UTP) (1 μM/ml to 100 μM/ml) can inhibit IL-8 production in human neutrophils in a dose- and time- dependent manner. The addition of ATP had a similar inhibitory effect suggesting that nucleotides act on neutrophils via the P2Y2 receptor, which is equally activated by UTP and ATP. A cytokine array was used to detect the release of 42 different cytokines in the extracellular medium. IL-8 and IL-6 ere the major cytokines detected in the medium, though other cytokines were detected in lower concentrations. In the current study, we demonstrated that extracellular nucleotides may have an important role in regulating IL-8 production in neutrophils and potentially modulating the inflammatory response.
The Role of Serum Procalcitonin and Interleukin-6 in the Differentiation and Surveillance of Sepsis and Non-Infectious Systemic Inflammatory Response Syndrome.
Authors: Liu Xiaoli , Du Bin , Pan Jiaqi , Hua Baolai
Published: October 2019

To evaluate the discrimination of serum procalcitonin (PCT) and interleukin-6 (IL-6) between patients with sepsis and non-infectious inflammatory response syndrome (SIRS) and the pridicton power of clinical outcome, a perspective study was performed in 27 patients with sepsis and 30 patients with non-infectious SIRS. The serum concentrations of PCT, IL-6, and C-reactive protein (CRP), white blood cell count, percentage of neutrophil, and maximal body temperature were obtained less than 24 hours after clinical onset of SIRS. The serum levels of PCT and IL-6, and percentage of neutrophil were significantly higher in patients of sepsis than in those of SIRS (PCT 5.54 [1.20, 32.74] μg/L vs 0.77 [0.22, 3.90] μg/L, P=0.001; IL-6 163.66 [33.60, 505.26] ng/L vs 37.72 [22.52,110.78] ng/L, P=0.004; CRP 15.28±8.41 g/L vs 9.51±7.65 g/L, P=0.010; and percentage of neutrophil 91%±4% vs 88%±4%, P=0.010). Receiver operating characteristic curves showed that the power of PCT and IL-6 were the best of all above. There was significant correlation between serum concentrations of PCT or IL-6 and the APACHE II or SOFA score, so was between serum PCT concentration and the ICU length of stay. Serum concentrations of PCT and IL-6 are more reliable indicators to differentiate sepsis and non-infectious SIRS than the conventional inflammatory markers, and correlate with the disease severity. And PCT levels were significantly correlated with ICU length of stay. (Supported by research grants from PUMCH, China)
High Molecular Weight Kininogen Fragments Stimulate the Secretion of Interleukin 1β through the Urokinase-Type Plasminogen Activator Receptor (uPAR) and CD11b/CD18 (Mac-1) in Human Blood Mononuclear Cells.
Authors: Khan Mohammad M. , Bradford Harlan N. , Isordia-Salas Irma , Espinola Ricardo , Colman Robert W.
Published: October 2019

High molecular weight kininogen (HK) is known to bind specifically and saturably to Mac-1 with a Kd = 9–18 nM for neutrophils and to uPAR with a Kd =30 nM for endothelial cells. However, the functional results of HK interaction with Mac-1 or uPAR on leukocytes is not fully understood. Kallikrein cleavage of single chain HK to a two chain form (HKa) with release of bradykinin (BK) occurs in sepsis, arthritis, and inflammatory bowel disease. We hypothesized that HKa stimulates secretion of inflammatory cytokines. Mononuclear cells were isolated from normal subjects by a Histopaque density gradient. We have expressed kininogen domain 3 (D3) and a fragment of domain 3, coded for by exon 7, E7P (aaG235-Q292), in E. Coli as glutathione S-transferase (GST) fusion proteins. HK and HKa were purified proteins. GST was recombinant. All proteins contained <0.01 EU/ml endotoxin. For all experiments, 2 X 106/ml mononuclear cells/ml were preincubated with monoclonal antibodies, murine IgG (both at 1.8 mM) or HANKS buffer containing 0.15 M NaCl, pH 7.4 for 30 minutes at 37°C. HK, HKa, GST-D3, GST-E7P, GST-D5 or GST all at 600 nM were added. Centrifugation allowed separation of the mononuclear cell suspension into cells and supernatant. The latter was used for assay of interleukin-1β (IL-1β) by ELISA. HK and all fragments tested stimulated secretion of IL-1β of 84.8 to 306.3 pg/ml when incubated with mononuclear cells for 30 minutes at 37°C. Anti-Mac-1 antibody inhibited IL-1β secretion by HK 100%, by HKa 89%, by GST-D3 78%, by GST-E7P 94% and by GST-D5 98%. Anti-uPAR antibody inhibited IL-1β release by HK 88%, by HKa 77%, by GST-D3 95%, by GST-E7P 85%, and by GST-D5 76%. Inhibition by both receptor antibodies is consistent with their known complex formation. A monoclonal antibody (mAb) to HK D5 (C11C1) and a mAb to HK D3 (2B5) both inhibited IL-1β release by HK, HKa, GST-D5 and GST-D3 indicate that both D3 and D5 are important in cytokine release. Murine IgG gave 0% inhibition in all studies. These results indicate that kininogen may contribute to the pathogenesis of inflammatory diseases by releasing IL-1β from human blood mononuclear cells.
Src Homology 2 Domain-Containing Inositol-5-Phosphatase 1 (SHIP1) Negatively Regulates TLR4 Mediated LPS Signaling through Phosphatase Activity and PI3K Independent Mechanism.
Authors: An Huazhang , Xu Hongmei , Zhou Jun , Zhang Minghui , Cao Xuetao
Published: October 2019

Lipopolysaccharide (LPS) is a cell wall component of Gram negative bacteria. LPS activates immune cells and triggers production of proinflammatory cytokines and other mediators through Toll-like receptor (TLR) 4. Src homology (SH) 2 domain-containing inositol-5-phosphatase 1 (SHIP1) plays important roles in negatively regulating the activation of immune cells primarily via phosphoinositide 3-kinase (PI3K) pathway by catalyzing PI3K product PtdIns-3,4,5P3 into PtdIns-3,4P2. However, little is known about the role of SHIP1 in TLR4-mediated LPS responses. We investigated the role of SHIP1 in LPS-induced MAPKs activation and IkappaB-alpha degradation by overexpression and RNA interfering experiments. SHIP1 becomes tyrosine phosphorylated and membrane translocated upon LPS stimulation in RAW264.7 macrophages. Stable SHIP1 transfection inhibits LPS-induced TNF-alpha and IL-6 production by 60% and 70% respectively. Transient SHIP1 transfection inhibits LPS-induced phospharylation of extracellular signal-regulated kinase (ERK1/2), p38, c-Jun NH2-terminal kinase (JNK) and degradation of IkappaB-alpha by about 20%, 50%, 40% and 40% respectively at the time point of 20 min after LPS stimulation. Disruption of SHIP1 phosphatase activity enhances LPS-induced activation of Akt, a downstream molecule of PI3K pathway, but fails to reverse SHIP1 mediated inhibition of LPS-induced activation of MAPKs and degradation of IkappaB-alpha. Consistently, LY 294002 and Wortmannin sufficiently inhibit LPS-induced Akt activation but can not diminish SHIP1 mediated inhibition of LPS-induced activation of MAPKs and degradation of IkappaB-alpha. On the contrary, SHIP1-specific RNA interfering inhibits SHIP1 expression and enhances LPS-induced phospharylation of ERK1/2, p38, JNK and degradation of IkappaB-alpha by about 2.6, 2.6, 3.0 and 8.0-fold respectively at the time point of 20 min after LPS stimulation. Even in the presence of LY 294002, SHIP1 RNA interfering could increase LPS-induced IL-6 production. Furthermore, SHIP1 overexpression nearly completely abolished TLR4 reconstitution conferred LPS responsiveness in COS7 cells. These results demonstrated that SHIP1 negatively regulates LPS responses in macrophage via phosphatase activity and PI3K-independent mechanism.