bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression Pattern of TCR-zeta Chain in Patients with Aplastic Anemia and Polycythemia Vera.
Authors: Yang Lijian , Li Yangqiu , Li Chuang , Chen Shaohua , Chen Si , Lin Chen
Published: April 2020

TCR zeta chain of TCR-CD3 receptor complex is though to be critical for T cells development and T cell effector function. The absence of TCR zeta chain not only influences the TCR expression on the cell membrane, but also impairs the proliferative response and the level of activation of mature T cells. Recently, the abnormal expression of TCR zeta chain was found different disease, for example in hematological malignancy. In the present study, we analyzed the expression level of TCR zeta chain gene in T cells from patients with aplastic anemia (AA) and polycythemia vera (PV), thereby to estimate the feature of T cells activation status. Real-time PCR with SYBR Green I technique was used for detecting the expression level of TCR zeta chain gene in peripheral blood mononuclear cells of patients with AA (25 cases),PV (12 cases), and 30 normal individuals. β2-microglobulin gene was used as an endogeneous reference. Evaluation of TCR zeta chain gene expression level were used the 2−ΔΔCt method. Compared with the normal control, TCR zeta chain gene expression level could be divided into two groups: 10 patients with AA (40%) and 5 patients with PV (41.7%) were over expression, while 15 patients with AA (60%) and 7 patients with PV (58.3%) were down expression. The expression level of TCR zeta gene is nonsignificat age-associated in both AA and PV patients. Moreover, the expression level of TCR zeta gene is nonsignificant age-associated or gender-link in T cells from patients with AA. The signaling pathway of T cells activation might display some disorder in patients with AA or PV. The effect of the change of TCR zeta chain gene expression pattern in AA and PV patients should be further confirmed.
A Retrospective Chart Review of the Hematologic Consequences of Roux-en-y Gastric Bypass Surgery.
Authors: Costantino Christina L. , Kruse Susan , Henry David H.
Published: April 2020

Roux-en-y gastric (RYGB) bypass surgery has become increasingly common as a procedure to control overweight/obesity. Due to the resulting bypass of significant amounts of gastric and duodenal mucosa, patients may develop anemia. We reviewed charts of 28 patients who had undergone RYGB surgery in the past 5 years who were referred to us for evaluation. Eighteen of these patients had no hematologic consequences as a result of the surgery. Five patients were anemic but were excluded because they had other causes for anemia than the bypass surgery: myeloma (1), pregnancy (1), breast cancer (1), and heavy menstruation (2). The remaining 5 patients however revealed hematologic abnormalities an average of approximately 23 months following RYGB surgery. In our retrospective chart review, we found anemia most commonly caused by iron deficiency although 1 patient had B12 deficiency, and we suggest periodic follow up of patients months to years after such surgery to screen for anemia from potential malabsorption of iron or B12 from this surgical alteration of gastrointestinal anatomy.

Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Months since RYGB & first heme evaluation 24 months 28 months 39 months 11 months 11 months Pre/Post Menopausal pre pre pre pre pre Hemoglobin 8 12.9 9.8 6.7 9.9 B12 165 501 233 340 660 Ferritin 14 9 7 <1 5 Folate 20.8 >24.0 16.3 16.4 >24.0 Creatinine 0.6 0.9 0.9 1.0 0.8 WBC 7.7 8.5 9 6.9 5.6 Platelets 364 300 473 378 462 Reticulocyte Count 1.00% 1.30% 1.20% 1.30% 1.30%
Succesful Treatment with Sirolimus in a Refractory Case of Pure Red Cell Aplasia (PRCA).
Authors: Ovilla Roberto
Published: April 2020

A patient with severe anemia since her birth was established a diagnosis of PRCA. Bone marrow aspirate showed an absence of red cell lineage, with lymphocyte hyperplasia. She was initiated with high dose of steroids without response. Two cycles of antithymocyte globulin (ATG) was administered, the first ATG 30mg/kg/5d with short response mainly evidenced by reticulocyte count, the second was established as ATG 15mg/kg/5d plus cyclosphosphamide 120mg/kg with a very transitory response. In November 2002 a Cord Blood Transplant was done, with a conditioning regimen based in Busulphan plus Cyclophosphamide and a prophylaxis for GVHD based on Cyclosporine plus Methylprednisolone. After a very rapid hematopoyesis recovery was discharged, but with no evidence of engraftment and autologous hematopoietic recovery, she received red cell transfusions monthly for about 36 months until December 2004 with desferroxamine as chelating drug. In December 2004 she started with Sirolimus 1mg a day orally with a very impressive response, red cells, hemoglobin levels, hematocrit, reticulocyte count improvement until normal levels. Red Cell transfusions were discontinued and two years later Sirolimus was also discontinued. She is in a very good Health Condition without any treatment in the last 8 months. We report this case in order to inform this successful treatment and in the order to use the same therapy in other cases.
A GATA-1-Regulated microRNA Cluster That Is Essential for Erythropoiesis.
Authors: Dore Louis C. , dos Santos Camila O. , Amigo Julio , Dorman Christine , Wu Weisheng , Hardison Ross C. , Paw Barry H. , Weiss Mitchell J.
Published: April 2020

The nuclear factor GATA-1 controls erythropoiesis by regulating transcription of numerous protein-encoding genes. Here we show that GATA-1 also activates the expression of a critical erythroid microRNA (miR) locus. MicroRNAs regulate gene expression and tissue differentiation by binding the 3′ untranslated regions of mRNA transcripts to inhibit their translation or stability. We used an oligonucleotide-based microarray to analyze miR expression in G1E cells, a GATA-1-null erythroid line that undergoes terminal maturation when GATA-1 activity is restored. We identified 8 miRs that are significantly upregulated during GATA-1-induced erythroid maturation. Two of the most highly induced miRs, 144 and 451, are encoded in tandem on mouse chromosome 11 and are transcribed on the same primary microRNA transcript. Northern blot studies showed that the miR144/451 locus is strongly induced by GATA-1 and specifically expressed at high levels in human and murine erythroid precursors. Bioinformatic analysis of the miR144/451 locus identified a conserved region containing three closely-spaced GATA binding motifs located about 2.7 kb upstream of the transcriptional start. In erythroid cells, this region exhibits enhancer activity and binds GATA-1 and its cofactor FOG-1, as determined by chromatin immunoprecipitation studies. MicroRNAs 144 and 451 are conserved between mammals and zebrafish, facilitating further in vivo studies. Whole mount in situ hybridization of zebrafish embryos showed both miRs to be exclusively expressed in the developing blood islands (intermediate cell mass) at 24h post-fertilization (hpf), colocalizing with gata-1 and β-globinE1 expression. Both miRs are deficient in the gata-1 null mutant, vlad tepes, consistent with their epistatic relationship to gata-1. Transient inactivation of miR-451 function using anti-sense morpholino oligomers selectively ablated hemoglobin staining cells in embryos at 48 hpf. These morphant embryos also exhibited dramatically reduced β-globinE1 mRNA expression. In contrast, there were no quantitative effects on thrombocytes (platelet-equivalent cells) in morphant embryos. Together, our data define a new regulatory axis through which GATA-1 promotes red cell development by directly activating the transcription of miR144/451, a critical erythroid-specific microRNA locus.
Usual Clinical Parameters Underestimate the Incidence and Significance of Erythropoietin Deficiency as a Risk Factor for Death in Sickle Cell Disease.
Authors: Saraf Santosh , Sidhwani Seema , Farooqui Mohammed , Vara Stephen , DeSimone Joseph , Molokie Robert , Saunthararajah Yogenthiran
Published: April 2020

A robust reticulocytosis is necessary to maintain hemoglobin levels compatible with life in patients with sickle cell disease. Relative reticulocytopenia, defined as a absolute reticulocyte count less than 250 x 109/L despite hemoglobin <9g/dl, has been identified as a risk factor for early death. RR could result from chronic bone marrow vaso-occlusive insult or a renal endocrine defect leading to erythropoietin (EPO) deficiency. In non-SCD patients, EPO levels and creatinine clearance are clinical parameters that are used to define patients with anemia from EPO deficiency. The normal ranges for these parameters may not apply to SCD, and therefore the incidence and significance of EPO deficiency in SCD may have been underestimated, compromising rational EPO replacement therapy. In a retrospective analysis of 417 patients with HbSS or S-β-thalassemia, confirming previous findings, RR was a risk factor for early death. There was a significant association between RR and proteinuria. Usually, there is an inverse correlation between hemoglobin and EPO levels (negative feed-back), this was seen in patients without RR. However, in patients with proteinuria, the correlation between hemoglobin and EPO levels was a positive one, suggesting hemoglobin dependence on EPO levels. Proteinuria or increased creatinine levels were a risk factor for early death but not if RR was included in the model, suggesting the increased risk of death associated with proteinuria and creatinine was mediated by RR. RR was also associated with decreased platelet and WBC counts, suggesting a contribution by cumulative vaso-occlusive bone marrow damage to RR. However, platelet and WBC counts were not risk factors for early death within the cohort. Damage to renal endocrine function/EPO deficiency should be added to the list of clinically significant chronic organ damage in SCD. EPO deficiency may precede and be causative of other end-organ damage, such as cardiac diastolic dysfunction and renal exocrine failure. Proteinuria should be recognized as a clinical parameter that should prompt consideration of EPO deficiency. Since EPO replacement is clinically feasible, renal endocrine dysfunctiton is a cause of symptoms, clinical deterioration and death that could be rationally addressed.
Clinical Utility and Safety of Methacholine Challenge in Children with Sickle Cell Disease.
Authors: Brown Michael S. , Boyd Jessica H. , Bates Pamela , DeBaun Michael R. , Strunk Robert C.
Published: April 2020

Introduction: Lung disease is major cause of morbidity in children with sickle cell disease (SCD), yet understanding of lung pathophysiology is lacking. The diagnosis of asthma in children with asthma has become an important consideration in clinical management because of its association with a twice greater the rate of painful and acute chest syndrome (ACS) episodes when compared to children without asthma. Provocation challenges with methacholine have been used in both clinical and research settings to evaluate the presence of degree of airway hyperresponsiveness (AHR) in studies of asthma; however, no such evaluation has been described in children with SCD.

Methods: As part of routine clinical care, 21 children with SCD multiple or severe acute chest syndrome (ACS) episodes were challenged with methacholine to determine if AHR was a component of their recurrent respiratory tract symptoms.

Results: A total of fourteen of the children had a positive challenge, none of whom had evidence of obstruction or bronchodilator reactivity on spirometry. Ten of fourteen patients were previously on inhaled corticosteroids and had their asthma diagnosis confirmed and four were given a new diagnosis of asthma. In each positive challenge, forced expiratory volume in one second reversed to at least 90% of baseline after bronchodilator treatment. No patient developed a painful or ACS episode temporally related to the challenge.

Conclusion: Evaluation of AHR with methacholine challenge in patients with SCD appears to be well tolerated and may elucidate the basis of lung morbidity in a subgroup of children with repetitive or serious ACS episodes.
ADAMTS13 Antigen, Activity, Autoantibody and ADAMTS13/fXI Complex Levels May Be Useful Diagnostic Biomarkers for Patients with Sickle Cell Disease.
Authors: Fryer Hugh J.L. , Grafos Nicholas , Guinto Enriqueta R. , Wu Jogin , Bai Bing , Greenfield Robert S.
Published: April 2020

Severe reduction of ADAMTS13 activity or high titers of ADAMTS13 autoantibody levels have been implicated as causative factors in Thrombotic Thrombocytopenia Purpura (TTP). Ultralarge von Willebrand factor (UL-VWF) has been shown to mediate the binding of platelets and sickle red blood cells to the endothelium. Thus, ADAMTS13, which cleaves UL-VWF, may play a role in thrombotic events in patients with sickle cell disease (SCD). In this preliminary study, patients with SCD were compared with normal donors for plasma levels of ADAMTS13 antigen, activity, autoantibody, and ADAMTS13/fXI complex. ADAMTS13 antigen was measured using IMUBIND® ADAMTS13 ELISA, ADAMTS13 activity using the ACTIFLUOR™ ADAMTS13 FRET, ADAMTS13 autoantibody titers using IMUBIND® ADAMTS13 Autoantibody ELISA, and complex using IMUBIND ADAMTS13/fXI. Patients with SCD included in this study (n=10) exhibit a number of thrombotic symptoms including deep vein thrombosis, pulmonary embolism, and pulmonary hypertension. Some were found to have autoantibodies to Protein S or alloantibodies to red blood cell proteins. Patients were treated with hydroxyurea, coumadin, as well as various pain medications. Patients in a crisis stage of SCD were included in this study. The four ADAMTS13-related markers were found to be abnormal in patients with SCD compared with normal donors. Cutoff levels for each of the markers, determined based upon normal donor plasmas, were found to be as follows: for ADAMTS13 activity, values lower than 510 ng/ml were considered below normal; for ADAMTS13/fXI complex, values exceeding 360% of the normal were considered to be elevated; for ADAMTS13 autoantibody, values above 14.8 AU/ml were considered above normal. Using these cutoff levels, none of the normal donors (n=47) had abnormal activity or complex levels. One normal donor (2.1%) had elevated autoantibody levels. In comparison, for patients with SCD, 7/10 (70%) had reduced activity levels, 8/10 (80%) had elevated levels of ADAMTS13/fXI complex, and autoantibody was detected in 6/10 (60%) of the patients. These data suggest that ADAMTS13 may play a role in thrombotic complications associated with SCD. ADAMTS13-related biomarkers measured herein may aid in the diagnosis of patients with SCD and may prove useful in monitoring the treatment of SCD.
Specific Micro-RNA Expression Profile in Hodgkin Lymphoma.
Authors: Ping Tan Lu , Harms Geert , Blokzijl Tjasso , Schakel Rikst Nynke , Gibcus Johan , Möller Peter , Küppers Ralf , Reijmers Rogier M. , Pals Steven T. , Poppema Sibrand , Kroesen Bart-Jan , van den Berg Anke
Published: April 2020

Introduction Classical Hodgkin lymphoma (cHL) and nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) differs not only in the form of histology and reactive background but also in the phenotypes of the tumor cells. Although tumor cells from both HL subtypes are originated from the germinal center (GC) B cell, gene expression studies show that lymphocytic and histiocytic (L&H) cells from NLPHL resembles normal B cells while Hodgkin/Reed-Sternberg cells (H/RS) from cHL demonstrate a loss of B cell phenotype and have significant overlap with primary mediastinal B cell lymphoma (PMBL). Recently, a new class of small RNAs, namely the micro-RNAs (miRNAs), has been identified. It is now known that at the post-transcriptional level, miRNAs negatively regulate gene expression in a sequence specific manner. Unique miRNAs expression patterns have been reported in various tissue types and also during a wide range of physiological states, such as cell proliferation, development, differentiation, apoptosis and hypoxia. As miRNAs play important roles in many cellular processes, it is proposed that there is a link between aberrant miRNA expression and loss of B cell phenotype in cHL.

Methods In this study, miRNA profiles from cell lines of various B cell lymphoma subtypes were examined by qRT-PCR. Also, several B cell subsets were sorted from tonsil by FACS and the miRNA profiles studied by qRT-PCR. Some of the miRNAs are analyzed by in situ hybridization (ISH) in both HL tissue and tonsil samples.

Results The miRNA profiling data indicated that cHL cell lines cluster together with PMBL while DEV, an NLPHL cell line, clusters together with CB. Upon validation of differentially expressed miRNAs on a cell line panel of 33 cell lines by monoplex qRT-PCR, 5/8 miRNAs identified as differentially expressed between cHL and GC B cells, were confirmed. Four out of six miRNAs differentially expressed between cHL and PMBL, were also confirmed as being differentially expressed in a larger cell panel. A high degree of overlap was observed between the most abundantly expressed miRNAs in the four HL cell lines. Expression of these miRNAs in HRS cells was verified by ISH in HL tissue samples. miRNA profiles of naive, GC and memory B cells display unique patterns. The overall miRNA expression levels were much lower than observed in the cell lines. Results of miRNA ISH in tonsil tissue demonstrated a specific staining pattern for each miRNA. These data indicate that miRNAs are particularly important for subsets of lymphocytes.

Conclusion Several miRNAs that are expressed specifically in Hodgkin lymphoma have been identified. However, the effect of the aberrant expressions of these miRNAs in HL is yet to be elucidated, as the targets of these miRNAs remain unknown.
Neutrophil Gelatinase-Associated Lipocalin Levels in Patients with Thalassemia Intermedia: Lack of Association with Erythropoiesis, Iron Metabolism, Renal Dysfunction and Inflammation.
Authors: Papassotiriou Ioannis , Margeli Alexandra , Drakatos Antonios , Delikou Sophia , Kassiou Korina , Kattamis Christos , Ladis Vassilios , Kattamis Antonios
Published: April 2020

Neutrophil Gelatinase-Associated Lipocalin (NGAL, 24p3, SIP24, lipocalin-2 or siderocalin) is a novel iron-carrier protein exerting pleiotropic properties. The physiological role of NGAL is not fully elucidated though a few pivotal functions, such as regulation of apoptosis of leukocytes have been described recently. It has also been demonstrated that NGAL is abundantly expressed in erythroid progenitor cells inducing apoptosis and inhibiting their differentiation. NGAL is reduced in the erythroid cells of mice with acute anemia and injection of recombinant lipocalin-2 delayed the recovery of red blood cells suggesting a negative effect of NGAL in accelerating erythropoiesis. The recently reported functions of NGAL on erythroid progenitor cells and its relation to iron transport prompted us to study the pattern of NGAL levels in Thalassemia Intermedia (TI), which is characterized by erythroid progenitor cells hyperactivity and irregularities in iron metabolism (especially iron transport). To this end we measured NGAL levels in 30 patients with TI and in 20 apparently healthy controls matched for age. We also evaluated other parameters that could be possibly related to NGAL’s functions as: soluble transferrin receptor (sTfR) for eythroid marrow activity; NTBI concentration for non-transferrin-bound mediate iron delivery pathway; creatinine, cystatin C and β2-microglobulin for renal dysfunction and CRP and IL-6 for low grade inflammation. The main results of the showed that: a) NGAL levels were significantly higher in patients with TI compared to controls (139.1±86.1 vs 51.2±11.8 μg/L, p<0.0001), while 4 patients presented with NGAL levels within normal range, b) in patients with TI no correlation was found between NGAL and the erythropoiesis’ parameters Hb, Hb F and sTfR, (p>0.66, p>0.67 and p>0.81 respectively), the parameters of iron metabolism, ferritin and NTBI (p>0.90 and p>0.95 respectively) the markers of renal dysfunction and/or those of low-grade inflammation (p>0.50). The increased NGAL levels observed in TI patients in this study are in agreement with the elevated expression of NGAL observed in experimental models of thalassemia. We postulate that the induction of NGAL in these patients may represent either a response facilitating the survival of the less damaged thalassemic erythroid precursors, or a consequence due to the abnormal iron regulation in patients with TI.
Cucurbitacin D Upregulates Fetal Hemoglobin Expression in K562 Cells and Human Erythroid Progenitors.
Authors: Xing Hongtao , Zhang Siwei , Koeffler H. Phillip , Fung Ming Chiu
Published: April 2020

The search for novel therapeutic candidates causing reactivation of fetal hemoglobin (a2g2; HbF) to reduce the imbalance of globin gene expression is important in order to develop effective approach for the clinical management of sickle cell anemia and b-thalassemia. For the first time, we have identified cucurbitacin D (CuD), a naturally occurring oxygenated tetracyclic triterpenoid, as a molecular entity inducing g-globin gene expression and HbF synthesis in K562 cells and human erythroid progenitors from either peripheral blood or bone marrow. The upregulation of HbF induced by CuD was dose- and time- dependent. CuD was compared to hydroxyurea (HU), 5-azacytidine, amifostine, recombinant human erythropoietin (rhEPO), and sodium phenylbutyrate. At their optimal dosage, CuD (12.5 ng/mL) and HU (25.0 μg/mL) induced nearly 70% K562 cells to express total hemoglobin after 6 days culture, which was higher than the induction by Amifostine (30%), 5-azacytidine (36%), rhEPO (16%), sodium phenylbutyrate (23%) at their optimal concentrations and negative control (11%). Fetal hemoglobin ELISA showed that CuD (12.5 ng/mL) and 5-azacytidine (400 ng/mL) induced higher levels of fetal hemoglobin in K562 cells (15.4 ng/μL and 29.3 ng/μL, respectively), compared to HU (10.3 ng/μL), amifostine (7.8 ng/μL), rhEPO (10.9 ng/μL), sodium phenylbutyrate (9.9 ng/μL) at their optimal concentrations and negative control (5.3 ng/μL). CuD induced a significantly higher fetal cell percentage than HU in K562 cells (65% vs 37% maximum) and primary erythroid progenitors (36% vs 21% maximum) based on the immunofluorescence imaging and flow cytometry analysis. Real-time PCR results showed that the amount of γ-globin mRNA increased from 2.5-fold in CuD-optimal-treated cells (12.5 ng/mL, 48 hours) compared with 1.5-fold in HU-optimal-treated cells (25.0 μg/mL, 48 hours). Growth inhibition assay (MTT) demonstrated that CuD at its optimal γ-globin inducing dosage (12.5 ng/mL) inhibited proliferation of K562 by less than 10% of untreated control cells; while hydroxyurea at its optimal dosage (25.0 μg/mL) inhibited 80% of cell division. The in vitro therapeutic index (calculated by dividing the dose inhibiting 50% cell growth (IC50) by dose inducing 50% maximal HbF production (ED50)) of CuD was 40-fold greater than HU. Taken together, the results suggest that CuD has the potential to be a therapeutic agent for treatment of sickle cell anemia and b-thalassemia.
Idiopathic Hypereosinophilic Syndrome in a Child.
Authors: Ferrara Mara , Capozzi Laura , Save Giuseppina , Esposito Luigi
Published: April 2020

The idiopathic hypereosinophilic syndrome (HSE) is a new heterogeneus disorder characterized by persistent unxplained hypereosinophilia (eosinophil count > 1.5 x 10 9 /L) persisting at least 6 months with possibile organ involvment. The diagnosis of HSE is based on exclusion criteria of other causes of eosinophilia. We reporte a case of an atopic 3 year-old male child with a history of eczema since first years of life and hypereosinophilia. At admission he showed severe generalized eczema with pruritus pain in the jonts, fever, several athsma, mild hepatosplenomegaly and lymphoadenomegaly. Biological findings included mild hypochromic microcytic anemia (Hb 10.5 g/dl, MCV 65 μ3, MCH 23 pg) due to heterozygous α thalassemia -delection (α) −37-, hyperleukocitosis (WBC 45.5 x 10 3 / μL) and hypereosynophilia (20.9 x 10 3 / μL). Bone marrow aspiration revealed absence of blast cells, mild reduction of myeloid (40%) and erythroid cells (10%) with increased M/E ratio 6/1. The eosynophilic cells represented 35%. Some of these cells presented cytoplasmatic atypia. Blood lymphocyte immunophenotypical analysis showed increased values of T lymphocytes CD3+, B lymphocytes CD19+ and T helper lymphocytes CD3+CD4+ Serum IgE levels were increased. Cytokines serum levels were undectable. Karyotype was 46XY. Non cytogenetic abnormalities were found in myeloid cells. Specific viral antibodies as well as autoantibodies and parasitologic assessements were negative. Corticosteroids were used with mild clinical improvement, followed by worsenings when the drug was stopped. The absence of blast cells or cytogenetic abnormality favor the diagnosis of HSE. However some patients in whom a diagnosis of idiopathic HSE appears appropiate may subsequently develope malignancies. A systematic survey of a large group of patients with otherwise unexplained eosinophilia is needed as well as a prolonged follow up of patients to early evaluate in each case neoplastic risk.
Induction of Apoptosis in Neutrophils from Septic Patients by Proinflammatory Molecules.
Authors: Gomez Luz S. , Arango Julian C. , Matute Juan D. , Gomez Ivan D. , Lopez Juan A. , Toro Fabiola , Jaimes Fabian , Patino Pablo J.
Published: April 2020

Neutrophils are in part responsible of the tissue damage accompanying the systemic inflammatory response produced during sepsis. A mechanism to limit their activity is apoptosis, however neutrophil apoptosis appear to be delayed in sepsis, which could increase the abnormal inflammatory response. On the other hand, it has been described that the proinflammatory molecules involved in delaying neutrophil apoptosis can induce apoptosis in other cells. Moreover there is not studies evaluating early apoptotic response in neutrophils from septic patients. In order to study the early apoptic response (30, 60 and 180 min) of neutrophils isolated from septic patients and healthy volunteers, these cells were isolated from peripheral blood, cultured with not stimulus or with LPS or fMLP and then analyzed by flow cytometry using DiOC6 and Annexin V/propidium iodide. Our data show that spontaneous apoptosis in neutrophils isolated from septic patients is increased during the first 3 h of in vitro culture in comparison to apoptosis from healthy volunteers neutrophils; however, this response is delayed when neutrophils isolated from septic patients were cultured during 24 h. Furthermore neutrophils isolated from healthy controls and septic patients stimulated with LPS or fMLP evidenced an apoptotic response at early incubation times in comparison with untreated cells. Previous studies have demonstrated a delay of apoptotic response of neutrophils obtained from patients with sepsis, which has been explained by the induction of anti-apoptotic proteins or inhibition of expression of apoptotic genes by LPS and other proinflammatory mediators. On the contrary our results suggest that there is an early increase in apoptosis when neutrophils isolated from septic patients are cultured without stimulus or in presence of microbial molecules such as fMLP and LPS. This contradictory evidence might be explained if we take in account the heterogeneity of circulatory neutrophils. At early time after LPS incubation a susceptible population of neutrophils activates different apoptotic responses, however the cells that are resistant to this effect activate a cascade of intracellular events that finally induces the transcription of several genes with anti-apoptotic and inhibits the expression of those pro-apoptotic. As consequence of these differences new pathogenic processes and therapeutic targets should be considered in sepsis.
Conservative Whole Blood Extended Differential Method.
Authors: Paul Ronald , Lopez Lidice L. , Careaga Diana B. , Rubio Oilda , Mylvaganam Ravindra
Published: April 2020

The neutrophilic precursors (excluding bands), blast cells and erythroblasts normally reside in the bone marrow. The presence of any of these precursors as well as atypical/variant lymphocytes in peripheral blood is indicative of a diseased or disordered state. Therefore, it is of clinical importance to identify and enumerate these atypical cell populations. In general, commercial hematology systems do not have the ability to enumerate all of the nucleated atypical cells that are traditionally identified by manual differential. However, the manual differential is labor intensive and can be highly variable between qualified readers resulting in significant cost and quality assurance issues for the laboratory. We have developed a unique fluorescent based Extended Differential method that uses a minimum number of monoclonal antibodies in combination with a cell permeant metachromatic nucleic acid dye to detect circulating atypical cells. Data will be presented comparing the results of this approach with the manual differential. The current study was comprised of 579 clinical samples containing a variety of atypical cell types as determined by morphological examination. Analysis of difference plots comparing the Extended Differential method with the manual differential revealed good agreement between the percent of immature granulocytes (IGs) and the number of nucleated red blood cells (NRBCs) per 100 white blood cells (WBCs). The same analysis applied to the determination of blast percent exposed a greater frequency of disparate results. These disparate results were further investigated using a comprehensive monoclonal antibody based reference method incorporating a variety of specificities including anti-CD45 and anti-CD34. The data demonstrated that cells classified as atypical/variant lymphocytes or smudge cells by morphological examination were classified as blasts utilizing CD45 and/or CD34 expression with the Extended Differential and flow reference methods. In addition to the difference analyses, the results obtained with manual differential and Extended Differential methods were subjected to Receiver Operating Characteristics (ROC) analysis in order to determine the overall analytical sensitivity and specificity using a threshold of 1% or greater as the criteria for the presence of atypical cells. The ROC analysis demonstrated areas under the curve (AUC) of 0.9743, 0.9831 and 0.9643 for IGs, blasts and NRBCs, respectively showing good agreement for the presence or absence of the respective atypical cell populations (p < 0.0001).
Analyses of Immune Defense Mechanisms of Human Polymorphonuclear Neutrophils Co-Cultivated with Aspergillus fumigatus Germ Tubes.
Authors: Loeffler Juergen , Mezger Markus , Einsele Hermann
Published: April 2020

Invasive fungal infections with the opportunistic pathogen Aspergillus fumigatus show an increasing incidence due to a higher number of patients with hematological malignancies. Polymorphonuclear neutrophils (PMNs), as part of the innate immunity, recognize fungal pathogens at an early stage after infiltration. Besides phagocytotic mechanisms, PMNs kill pathogens by the release of reactive oxygen species (ROS). Human PMNs were isolated from blood of healthy donors and co-cultivated with A. fumigatus germ tubes for defined time points. Oxidative burst was determined in a kinetic measurement by the use of dichlorfluorescein. In parallel, PMNs were co-cultivated with A. fumigatus germ tubes, followed by whole genome expression analyses (Affymetrix U133 Plus 2.0 Array). We could demonstrate that A. fumigatus germlings of the clinical relevant strain ATCC 9197 represented a strong stimulus for the release of ROS. PMNs actively tracked germlings and directly attached to fungi as demonstrated by real-time microscopy. In addition, co-cultivation of PMNs with A. fumigatus germ tubes resulted in a strong upregulation of genes involved in self-protection against radicals (hämoxygenase, heat shock 70kDa protein HSPA8, thioredoxin, HSPA1B, HSP90AB1, Ferritin). After 6h of co-cultivation, 195 genes showed an at least 4fold altered gene expression. Therein, 4 genes encoding for cytokines and chemokines (IL-8, CCL3, CXCL2, IL1RN) were significantly upregulated. Luminex ELISA analyses confirmed array data and revealing IL-8 to be strongly released (5fold) by PMNs after fungal co-culturing. In conclusion, A. fumigatus had a substantial effect on the activity of human PMNs. In consequence, various defence strategies were activated, including phagocytosis, ROS release and mobilization of other immune effector cells by secretion of chemoattractant cytokines. A better understanding of innate immune defense mechanisms may provide new directions for antifungal therapies.
The Value of CT-Guided Percutaneous Lung Biopsy in the Diagnosis of Pulmonary Fungal Infections in Patients with Hematologic Diseases.
Authors: Cai Zhen , Shi Jimin , Huang He , Lin Yu , Ye Xiujin , He Jingsong , Xie Wanzhuo , Zhang Jie , Li Li , Zheng Weiyan , Wei Guoqing , Luo Yi , Zhang Buwen , Lin Maofang
Published: April 2020

Introduction: Patients with hematologic diseases are at high risk for invasive fungal infections (IFI).The diagnosis of IFI is difficult with specificity as low as 20%–30%. The currently used diagnostic methods for IFI such as CT scan, fungal cultures, and antigen detection are insufficient in reaching a definitive diagnosis. Histopathology with tissue diagnosis is the method of choice. CT-guided percutaneous lung biopsy (CT-PCLB) can provide pathological diagnosis and hence significant guidance for clinical management.

Objectives: To evaluate the sensitivity and safety of CT-PCLB for the diagnosis of pulmonary fungal infections in patients with hematologic diseases.

Patients and Method: We retrospectively studied 16 patients hospitalized in our department from April 2005 to December 2006 who were clinically regarded as having pulmonary fungal infections by clinical symptoms and imaging and who subsequently underwent CT-PCLB. Among the 16 patients, 10 had AML, 2 had ALL, 2 had ITP, 1 had MDS and 1 had multiple myeloma. At the time of percutaneous lung biopsies, median WBC was 3.2 k/ul (range 0.7–7.2 k/ul), median platelets were 82 k/ul (range 32–453 k/ul). Peripheral blood cultures were performed in all patients. Eight patients who had symptoms of cough and expectoration also received sputum culture for at least three times. Of the 16 patients, 10 were diagnosed with fungal infections (8 aspergillus’s and 2 mold fungi), 4 with chronic organizing pneumonitis, 1 with tuberculosis, and 1 with pneumocystis carinii. All tissue diagnoses were from histological examination with samples obtained from CT-PCLB. However, the results of blood cultures and sputum cultures were negative in these 8 patients. CT-PCLB diagnosed 10 out of 16 patients for a fungal infection rate of 63%. The biopsy-induced complications encountered were pneumothorax in 3/16 (19%) and hemoptysis in 6% (1/16). No surgical interventions were needed for the 3 patients with pneumothoraxes that resolved spontaneous under close observation. Antifungal agents (such as voriconazole) had been given to 8 patients diagnosed as aspergillus infection and all patients had a complete or partial response by CT imaging. However, 2 patients who received broad-spectrum antibacterial antibiotics showed clinical improvement, which indicated possible false negative diagnoses for fungal infections by CT-PCLB.

Conclusion: CT-guided percutaneous lung biopsy (CT-PCLB) was a safe, sensitive and specific method for the diagnosis of pulmonary fungal infections in patients with hematologic diseases. Its definitive value should be tested in a prospective clinical trial.
Iron Chelator ICL670 Inhibits HIV-1 Transcription.
Authors: Debebe Zufan , Ammosova Tatyana , Nick Hanspeter , Niu Xiaomei , Jerebtsova Marina , Ray Patricio , Gordeuk Victor R. , Nekhai Sergei
Published: April 2020

HIV-1 replication is induced by the excess of iron and iron chelation by desferrioxamine (DFO) inhibits viral replication in HIV-1 infected CEM T cells [1]. Treatment of cells with DFO or 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone inhibits expression of proteins that regulate cell-cycle progression, including cycle-dependent kinase 2 (CDK2) [2]. HIV-1 transcription is activated by Tat protein, which recruits transcriptional co-activators to the HIV-1 promoter. Elongation of HIV-1 transcription is mediated by the interaction of HIV Tat with host cell cycle-dependent kinase 9 (CDK9)/cyclin T1, which phosphorylates the C-terminal domain of RNA polymerase II. Our recent studies showed that CDK2 participates in HIV-1 transcription by phosphorylating Tat [3]. Thus inhibition of CDK2 by iron chelators might present a new approach to inhibit HIV-1 transcription. We evaluated the effect of a clinically approved orally effective iron chelator, 4-[3,5-bis-(hydroxyphenyl) -1,2,4-triazol-1-yl]-benzoic acid (ICL670 or deferasirox) on HIV-1 transcription. ICL670 inhibited Tat-induced HIV-1 transcription in CEM, 293T and HeLa cells at concentrations that did not induce cytotoxicity. The chelator decreased cellular activity of CDK2 but not its protein level and reduced HIV-1 Tat phosphorylation by CDK2. ICL670 did not decrease CDK9 protein level but significantly reduced association of CDK9 with cyclin T1 and reduced phosphorylation of Ser-2 residues of RNA polymerase II C-terminal domain. In conclusion, our findings add to the evidence that iron chelators may inhibit HIV-1 transcription by deregulating CDK2 and Cdk9. Further consideration should be given to the evaluation of ICL670 for future anti-retroviral therapeutics and to the development of iron chelators specifically as anti-retroviral agents.
A Multi Center Trial of hyperCVAD+Rituxan in Patients with Newly Diagnosed Mantle Cell Lymphoma.
Authors: Epner Elliot M. , Unger Joseph , Miller Thomas , Rimzsa Lisa , Spier Catherine , Leblanc Michael , Fisher Richard
Published: April 2020

BACKGROUND: A Phase II Study multinstitutional study by the lymphoma group within the Southwest Oncology Group (SWOG 0213) was performed to determine the 1 year progression free survival of patients with newly diagnosed mantle cell lymphoma (MCL). Previously, this regimen was utilized in a similar group of newly diagnosed MCL patients in a single institution study by Romaguera et al (JCO200523:7013) at the MD Anderson Cancer Center. They reported a 97% response rate, a 87% CR rate, and a 3 year failure free survival rate of 73%.

PATIENTS AND METHODS: This study was a prospective phase II trial of rituximab plus fractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone (hyperCVAD; considered one cycle) alternating every 21 days with rituximab plus high-dose methotrexate-cytarabine (considered one cycle) for a total of eight cycles per the MD Anderson protocol. Newly diagnosed MCL patients under age 70 with adequate organ function and measurable disease were eligible. All cases were histologically confirmed.

RESULTS: 49 eligible patients were enrolled. There was one probable treatment-related death due to colitis. Grade 4 hematologic toxicity was reported for 87% of patients. 40 patients have been evaluated for response. 3 patients had inadequate assessment and are assumed to be non-responders.16 patients (40%) had complete responses, 7 patients (18%) had unconfirmed complete responses, and 12 patients (30%) had partial responses for an overall response rate was of 88% (35/40; 95% CI, 73%–96%). Median follow-up among patients still alive is 1.6 years (range: 0.3–4.1 years). The progression-free survival estimate at 1-year is 89% (95% CI: 80%–98%) and at 2 years is 64% (95% CI: 46%–82%). The overall survival estimate at 1 year is 91% (95% CI: 82%–99%) and at 2 years is 76% (95% CI: 60%–91%). 13 of the 49 eligible patients have progressed or died. One year overall survival was 91% and one year progression free survival is 89%. Two year PFS is 63% (95% CI: 46%–82%). Two year OS is 76% (95% CI: 60%–91%). The 1-year progression-free survival estimate is 89% (95% CI: 80%–98%). The 1-year overall survival estimate is 91% (95% CI: 82%–99%). The overall response rate was 88% (35/40; 95% CI, 73%–96%).

CONCLUSIONS: The hyperCVAD + rituximab regimen is active as reported in MCL with a one year progression free survival of 89%. However, similar to the reported MD Anderson experience, this regimen is toxic and a continuous pattern of relapse over time is observed.
The Impact of P2Y-Mediated Activation on Release of Angiogenic Proteins by Platelets from Healthy Individuals.
Authors: Bambace Nadia M. , Pace Thomas R. , Schneider David J. , Holmes Chris E.
Published: April 2020

The storage and release by platelets of the pro-angiogenic protein, vascular endothelial growth factor (VEGF) and the anti-angiogenic protein, endostatin may influence not only wound healing but also tumor angiogenesis and metastasis. The intra-platelet mechanisms and platelet activators that control angiogenic protein release are incompletely elucidated. Previous work demonstrated that the proteinase-activated receptors (PARs), PAR-1 and PAR-4 differentially impact platelet release of these angiogenic proteins (Ma et al., PNAS 102:216). We determined the influence of the adenosine diphosphate (ADP) receptors, P2Y1 and P2Y12 on platelet release of VEGF and endostatin. Minimally altered whole blood from healthy volunteers was incubated with ADP alone [12.5 μM] or in combination with a reversible P2Y1 antagonist [MRS2179 (1.25 mM)] or a reversible P2Y12 antagonist [cangrelor (1mM)]. VEGF and endostatin release was measured with the use of an ELISA assay. ADP-induced activation of platelets increased the concentration of VEGF released by an average of 19.7 +/− 14 ng/mL (p= 0.019) but did not change the concentration of endostatin. Inhibition of the P2Y12 receptor prevented the ADP mediated release of VEGF and decreased VEGF release by 27.9 +/− 14 ng/ml (p=0.005) whereas inhibition of the P2Y1 receptor did not significantly reduce the release of VEGF (p=0.3, mean decrease 14.3 +/− 30 ng/ml) relative to ADP stimulated control. In summary, ADP-induced activation of platelets leads to the release of VEGF but not endostatin. Release of VEGF is attenuated by a P2Y12 but not a P2Y1 receptor antagonist. Accordingly, activation of platelets by ADP appears to be pro-angiogenic and inhibition by a P2Y12 antagonist should attenuate this effect.
G-Quartet Oligonucleotide T70923 Blocks GP Ib-VWF Mediated Platelet Aggregation under Static and Flow Conditions.
Authors: Zhou Zhou , Bernado Aubrey , Guan Yongli , Ball Chalmette , Jing Naijie , Dong Jing-fei
Published: April 2020

Hemostasis at site of the vessel injury is initiated by platelets being tethered to exposed subendothelium. The tethering events are mediated by interaction between subendothelial von Willebrand factor (VWF) and its platelet receptor GP Ib-IX-V complex. If the ligand-receptor interaction, when it occurs in intact vascular lumen or at the site of ruptured atherosclerotic plaques, results in life-threatening thrombosis found in patients with thrombotic thrombocytopenic purpura and myocardial infarction. The development of these thrombotic events suggests that the VWF-GP Ib interaction may be a potential target for therapeutic intervention. To explore such potentials, we have developed and examined the effects of a G-quartet oligonucleotide (T30923, GGGTGGGTGGGTGGGT) on the VWF-GP Ib interaction. G-rich DNA sequences, from which the G-quartets derive, were originally identified in the telomeres DNA and have the ability to form rigid G-quartet structures that interact with proteins. The T30923 structure was designed and determined by nuclear magnetic resonance to form an intramolecular G-quartet that is composed of two identical G-quartets in the center and two GTGT loops on the top and bottom of stacked G-quartets, respectively. Platelet aggregation was induced by four agonists: ristocetin, botrocetin, collagen, and ADP in the presence of either 20 mM of T30923 or the control oligonucleotide. We found that T30923 specifically and dose-dependently blocked platelet aggregation induced by ristocetin, but not by other agonists, with a maximal inhibition achieved at 20 mM. Computer-simulation based on the crystal structure of the VWF A1 domain and the N-terminal VWF-binding domain of GP Iba suggested that the G-quartet binds to multiple sites on VWF-A1. Since the action of ristocetin mimics that of fluid shear stress, we tested the ability of T30923 to block VWF-GP Ib interaction under flow condition. Shear-induced platelet aggregation at 100 dyn/cm2 was blocked 59.3%±11.8% by 20 mM of T30923, but not a control oligonucleotide. Furthermore, the G-quartet T30923 blocked over 80% of platelet adhesion to immobilized VWF, but not collagen under a constant wall shear stress of 30 dyn/cm2. These results demonstrate the potential of T30923 to serve as a VWF antagonist. Furthermore, alterations of DNA sequences and tertiary structure provide a large number of G-quartet variants to be generated and then screened for specific phenotypes. The approach may lead to a new class of potential G-quartet based antithrombotic agents.
Quantitative Analysis of Angiogenesis Inhibition by Cleaved High Molecular Weight Kininogen Using VESGEN.
Authors: Ribita Daniela , Zlotoff Benjamin , McKay Terri L. , Gedeon Daniel J. , Hylton Alan G. , Rasin Arkady , McCrae Keith R. , Parsons-Wingerter Patricia
Published: April 2020

Angiogenesis requires specific molecular regulation of complex vascular branching morphology. The cleaved form of high molecular weight kininogen (HKa) has been shown to cause apoptosis of proliferating endothelial cells, and inhibit angiogenesis in vivo. However, critical morphological effects of HKa on the complex branching morphology of angiogenic vascular trees have not been defined. We used the quail chorioallantoic membrane (CAM) angiogenesis assay, in which angiogenesis agonists or antagonists cause uniform perturbation of angiogenesis throughout the entire vascular tree, to assess the effects of HKa on branching morphology. HKa (1.56 to 50 micrograms) was applied uniformly to the CAM, and its effect on angiogenesis determined after 24 hours of incubation. VESGEN vascular analysis software, currently under development at NASA Glenn Research Center, was used to quantify major vessel parameters in low-magnification images of vascular trees automatically extracted by VESGEN. Results derived from qualitative observation and initial quantification of two experiments demonstrates that HKa significantly inhibits vascular growth. Vessel length density (Lv) and branch point density (Br) in CAM specimens treated with 25 micrograms of HKa decreased by 23% and 31% respectively (24 ± 1 cm/cm2 and 441 ± 43 /cm2, compared to 31 ± 0 cm/cm2 and 677 ± 51 /cm2 in vehicle-treated controls; mean +/− S.E. for n = 3). The fractal dimension (Df) of skeletonized vascular images is a highly sensitive indicator of vessel density that ranges from 1.35 for strong angiogenesis inhibition, to 1.40 and 1.48 for controls and strong angiogenesis stimulation, respectively. Df decreased to 1.35 ± 0.01 in the HKa-treated specimens, relative to 1.39 ± 0.00 in controls. However, thickening of large vessels in response to HKa resulted in a virtually equivalent vessel area density (Av) of 0.167 ± 0.012 cm2/cm2, relative to 0.174 ± 0.015 cm2/cm2 in controls. Further studies of the site-specific effects of HKa on branching morphology within the vascular tree are in progress. These results demonstrate that the antiangiogenic effects of HKa result in a unique antiangiogenic ’fingerprint’ vascular morphology in comparison to other angiogenesis inhibitors that include transforming growth factor-β1 and angiostatin.