bluuebunny/crossref_metadata_2025 huggingface dataset:
Polychemotherapy in patients with primary CNS lymphoma.
Authors: Glasmacher Axel , Pels Hendrik , Helmstaedter Christoph , Deckert Martina , Kroschinsky Frank , Schackert Gabriele , Reichmann Heinz , Egerer Gerlinde , Ho Anthony , Schulz Holger , Engert Andreas , Diehl Volker , Bode Udo , Klockgether Thomas , Schlegel Uwe , Schmidt-Wolf Ingo G.H.
Published: October 2019

In primary CNS lymphoma (PCNSL) irradiation alone is no longer the primary treatment and combination with chemotherapy has significantly improved response and survival. However, patients treated with combined modality therapy suffer from significant neurotoxicity. Therefore, studies with chemotherapy alone have been carried out. We recently reported on a phase II multicenter trial to evaluate event-free survival, overall survival, response rate, response duration and toxicity of combination chemotherapy in patients with previously untreated PCNSL. 65 consecutive patients with histologically proven PCNSL received a total of 6 courses of combination chemotherapy with 3 different courses of chemotherapy. In patients with response these 3 cylces were repeated. The combination chemotherapy included high-dose methotrexate (MTX) (5g/m2, course 1,2,4,5) and cytarabine (ara-C) (3g/m2/d, d1-2, courses 3,6) combined with dexamethasone (courses 3–6), vinca alkaloids and ifosfamide alternating with cyclophosphamide. In addition, MTX, prednisolone and ara-C were applied via an Ommaya or Rickham reservoir in each course. The study population included 65 consecutive patients with a median age of 62 years with a range between 27 and 75. 62 patients are evaluable for response. There were 9% therapy-related deaths mainly due to neutropenic infections. Ommaya reservoir infections occurred in 12/64 (19%) and acute transient MTX related encephalopathy in 2 (subacute in 1) patients. The 5-year-survival of patients below 61 years was 75%, of patients of 61 years or older 17% (Pels H et al., J Clin Oncol21(24), 2003). The results for patients younger than 60 years could be superior to previously reported therapy regimes. The survival rates for patients above 60 years are comparable to those reported for combined radiochemotherapy. However, in contrast to radiochemotherapy permanent treatment-associated neurotoxicity is infrequent. Based on these data we constructed a follow-up trial. Patients below the age of 61 are treated as in the antecedent protocol without intraventricular therapy. Patients above 60 are treated in a protocol modified by the addition of procarbazine in 2 cycles and a maintenance therapy with procarbazine. First results of this follow-up trial will be presented.
Inv (16) Suppresses CEBPA in Acute Myeloid Leukemia by Activation of Calreticulin.
Authors: Helbling Daniel , Mueller Beatrice U. , Timchenko Nikolai A. , Betts David R. , Jotterand Martine , Meyer-Monard Sandrine , Fey Martin F. , Pabst Thomas
Published: October 2019

Inversion of chromosome 16 (inv(16)) results in the reciprocal chromosomal rearrangement of the CBFB and MYH11 genes and it is the hallmark of malignant cells observed in patients with acute myeloid leukemia (AML) subtype M4Eo. Alterations of structure or expression of CEBPA - a key myeloid transcription factor - have been implicated in particular subtypes of AML. To investigate the effects of inv(16) on CEBPA we conditionally expressed inv(16) in U937 cells and found that CEBPA mRNA levels remained unchanged. However, CEBPA protein and binding activity were suppressed by 100% and 71%, respectively. In parallel, analysis of patients with inv(16) (n=12) as compared to normal karyotype AML M4 patients (n=6) showed that CEBPA protein and binding activity were significantly reduced (100% and 71.6%, respectively) whereas CEBPA mRNA levels remained similar. Calreticulin, an inhibitor of CEBPA translation, was induced on mRNA and on protein level in inv(16) AML patients as well as after conditional expression of inv(16) in a U937 cell system. Furthermore, inhibition of Calreticulin by siRNA powerfully restored CEBPA levels. Our results identify CEBPA as a key target gene of the leukemic fusion protein inv(16) and suggest that suppression of CEBPA by Calreticulin contributes to the differentiation block in AML with inv(16).
Identifying Hypercoagulable States with the Overall Haemostatic Potential (OHP), a Simple Global Haemostasis Assay.
Authors: Curnow Jennifer L. , Morel-Kopp Marie-Christine , Aboud Margaret , Ward Christopher
Published: October 2019

Hypercoagulable states typically result in arterial and/or venous thromboembolism. Routine coagulation tests do not identify affected patients and thrombophilia testing detects, at best, 50% of patients at risk. We hypothesise that the OHP can differentiate patients with various hypercoagulable states from a control group. The OHP is a global haemostasis assay of fibrin generation and lysis in citrated plasma. It produces a number of parameters for comparison. We established reference ranges with plasmas from 100 normal donors, then assayed plasmas from 90 clinically hypercoagulable patients being tested for a lupus anticoagulant. A further 81 patients with demonstrable antiphospholipid antibodies (APLAs) were then assayed. Statistical analysis involved calculation of means, medians, standard deviations and Mann-Whitney testing. All parameters tested were significantly different in the clinically hypercoagulable group (p<0.001), demonstrating accelerated fibrin generation and markedly reduced fibrinolysis. On subgroup analysis patients with arterial (8) or venous thromboembolism (30), antiphospholipid antibodies (9), normal or complicated pregnancy (22) and autoimmune diseases (12) all showed significantly increased fibrin generation and lysis compared with controls. The subsequently tested 81 patients with APLAs also showed a significantly hypercoagulable OHP compared with controls (p<0.01). APLA positive patients with thromboembolism showed a non-significant trend to accelerated fibrin generation and delayed fibrinolysis when compared to those without thromboembolism. APLA positive patients taking warfarin showed significant hypercoagulability when compared with APLA negative patients on long term warfarin therapy (p < 0.01). The OHP clearly identifies patients with clinically hypercoagulable states, even in the presence of warfarin. Fibrin generation is increased and fibrinolysis delayed. We postulate that the OHP may identify patients at high risk of thromboembolism.
The Effect of Patient Age and Calendar Year on the Incidence of Idiopathic vs. Secondary Venous Thromboembolism (VTE): A Population-Based Cohort Study.
Authors: Kobbervig Catie E. , Heit John A. , Petterson Tanya M. , Christianson Teresa J. , Bailey Kent R. , Melton L. Joseph
Published: October 2019

If the observed dramatic increase in VTE incidence with advancing age is due to increased VTE risk factor exposure (i.e., secondary VTE), the incidence of idiopathic VTE should not vary with age or calendar year.

Objective: To estimate the incidence of idiopathic and secondary VTE by age and by calendar year.

Methods: Using the resources of the Rochester Epidemiology Project, we identified the inception cohort of Olmsted County, MN, residents with a first lifetime VTE during the 30-year period, 1966–1995 (n=2761). For each case, we reviewed the complete medical records in the community for 48 baseline clinical characteristics that are commonly-accepted risk factors for VTE. We categorized VTE cases as idiopathic (n=305) if no such characteristics were present; the remaining cases were categorized as secondary. Age- and sex-specific incidence rates were calculated using idiopathic or secondary VTE cases as the numerator, and age-, sex- and calendar year-specific estimates of the population of Olmsted County as the denominator.

Results: The incidence of both idiopathic and secondary VTE increased exponentially with age for both men and women (p<0.001). Over the 30-year study period, the age-adjusted incidence of idiopathic VTE was essentially constant among men (from 19.0 to 17.1 per 100,000 men-years for 1966–70 and 1990–95, respectively), but decreased markedly among women (from 18.5 to 3.6 per 100,000 woman-years for 1966–70 and 1990–95, respectively; p=0.005 for the interaction).

Conclusions: The dramatic increase in VTE incidence with age likely reflects the biology of aging, although as yet unidentified VTE risk factors cannot be excluded. The reason for the decreased incidence of idiopathic VTE over the last 30 years among women is unclear.
Use of Intravenous Immunoglobulin for Treatment of Neurological Conditions: A Systematic Review.
Authors: Hutton Brian , Fergusson Dean , Sharma Michael , Tinmouth Alan , Wilson Kumanan , Cameron Bill , Hebert Paul C.
Published: October 2019

Purpose: Given the increasing use of intravenous immunoglobulin for various neurological conditions, its significant cost and the uncertainty pertaining to its benefits and harms, we conducted a systematic review of randomized controlled trials that compared intravenous immunoglobulin to other current therapies.

Methods: A systematic search strategy of Medline (1966-June 2003) and the Cochrane Registry of Controlled Trials (June 2003 edition) was developed to identify randomised controlled trials. Two authors (BH, DF) independently reviewed all citations retrieved from the electronic search to identify all potentially relevant trials for this review. In cases where a unanimous decision could not be reached, a third party was consulted. To be eligible, studies had to be randomised controlled trials comparing intravenous immunoglobulin to either placebo or an active control and evaluating clinical outcomes. Measures of effect were calculated for each trial independently, and studies were pooled based on clinical and methodologic judgment as to its appropriateness. Fixed effects and random effects models were selected for each meta-analysis based on appraisal of the clinical homogeneity of patients across studies, as well as from testing for heterogeneity between studies. In circumstances where pooling of trials was inappropriate or not feasible, a qualitative discussion of the findings was developed.

Results: Thirty-seven trials representing 14 conditions were identified. Results from meta-analysis suggest that intravenous immunoglobulin therapy is effective for the treatment of multiple sclerosis (pooled 1-year EDSS change −0.46, 95% CI [−0.92,0.01] in favor of IVIG; pooled change in annual exacerbation rate −0.82, 95% CI [−1.54, −0.11] in favor of IVIG; pooled odds of remaining exacerbation free 0.24, 95% CI [0.12, 0.50] in favor of IVIG ) and idiopathic chronic inflammatory demyelinating polyneuropathy (pooled change in disability score −0.67, 95% CI [−1.04, −0.30] in favor of IVIG; pooled odds of achieving treatment response 4.43, 95% CI [2.20, 8.91] in favor of IVIG). There was insufficient evidence to determine whether this therapy was more effective for Guillain-Barré Syndrome than plasma exchange (pooled change in disability score −0.11, 95% CI [−0.37,0.14]; pooled odds of improving by 1 or more grades on the Hughes disability scale 1.91, 95% CI [1.11, 3.28] in favor of IVIG; pooled odds of mortality 0.84, 95% CI [0.31, 2.29]). There was also insufficient evidence regarding paraprotein-associated chronic inflammatory demyelinating polyneuropathy and multifocal motor neuropathy. No evidence of benefit was observed for inclusion body myositis and myasthenia gravis. Based on evidence from lone randomised trials, there also exists evidence indicating that IVIG may be useful for treatment of dermatomyositis, stiff person syndrome and Lambert-Eaton Syndrome.
A VWF Fragment Spanning the A1, A2, and A3 Domains Supports Platelet Adhesion Better Than the Isolated A1 Domain and Is Considerably More Resistant to Cleavage by Adamts-13 Than the Isolated A2 Domain.
Authors: Padilla Arnoldo A. , Lopez Jose A. , Cruz Miguel A.
Published: October 2019

von Willebrand factor (VWF) is a large multimeric protein whose monomers are of the domain structure D’-D3-A1-A2-A3-D4-B1-B2-B3C1-C2. The three tandem A domains mediate several important functions of the protein including supporting platelet adhesion, through an interaction of A1 with the platelet GP Ib-IX-V complex, and binding collagen, through A3 and A1. Further, the A2 domain contains the cleavage site for the plasma metalloprotease ADAMTS-13, which processes the newly synthesized and extremely adhesive ultra-large forms of VWF (ULVWF) to the forms normally found in plasma. Our goal was to compare the functions of the individual A domains as isolated domains to their function in the context of the A1-A2-A3 fragment. All recombinant domains were produced in bacteria. The A1-A2-A3 fragment contained an N-terminal thioredoxin-tag and a C-terminal 6Xhis tag and was purified in 2 steps using nickel and heparin columns. The recombinant protein was correctly folded, as indicated by the fact that it was recognized by a number of conformation-specific antibodies. Using ELISA, we examined the interaction of the fragment with both glycocalicin (the soluble extracellular domain of GP Iba) and collagen. At equivalent concentrations, A1-A2-A3 bound collagen better than did the isolated A1 and A3 domains, but the binding to glycocalicin was approximately 7.5 times lower than the A1 domain. We then tested the ability of the fragment to support platelet adhesion under flow, by coating the fragment onto a coverslip that made up the bottom of a parallel-plate flow chamber. In contrast to the findings with immobilized glycocalicin, immobilized A1-A2-A3 supported the attachment, rolling, and firm adhesion of the platelets to the surface, whereas A1 did not support firm adhesion. Finally, we tested cleavage of the fragment by plasma ADAMTS-13, using both ELISA and western blotting to detect cleavage. The rate of cleavage of the A1-A2-A3 fragment was much slower than that of the recombinant A2 domain, needing a longer incubation time to achieve a similar percentage of cleavage in a 15 fold higher concentration of plasma. These results show clearly that within the triplicate A domains of VWF, adjacent A domains influence each other’s functions, possibly by binding each other or by conformational modulation. The fact that the fragment is able to support firm platelet adhesion in isolation suggests that it may contain a second, previously unidentified binding site for platelet receptors.
Three New Mutations of Glucose-6-Phosphate Isomerase Associated with Chronic Hemolytic Anemia.
Authors: Vercellati Cristina , Fermo Elisa , Marcello Anna , Mariani Mariagabriella , Bianchi Paola , Barcellini Wilma , Ladogana Saverio , De Fanti Alessandro , Zanella Alberto
Published: October 2019

Glucose-6-phosphate isomerase (GPI, EC 5.3.1.9) is a dimeric enzyme that catalyses the reversible isomerisation of glucose-6-phosphate (G6P) to fructose-6-phosphate (F6P), the second reaction step of glycolysis. GPI deficiency is the second most common erythro-enzymopathy of anaerobic glycolysis, after pyruvate kinase. The gene locus encoding GPI is located on chromosome 19q13.1 and contains 18 exons. So far about 50 cases of GPI deficiency have been described, and 29 mutations have been reported at the nucleotide level: 25 missense, 2 nonsense and 2 splice site. The aim of this study is to describe the hematological, biochemical and molecular characteristics of two new families affected by GPI deficiency with chronic hemolytic anemia.

Case1: The propositus was a 1 yrs old boy of Northern Italian origin with chronic hemolytic anemia and need for blood transfusion in concomitance of infectious episodes. At the time of the study Hb was 7.6 g/dL, reticulocytes 231x109/L, total bilirubin 1.4 mg/dL. The study of the most important red cell enzymes revealed a very low GPI activity (12.5% of normal). The parents were not consanguineous, and displayed intermediate GPI activity (66% of normal in both). The sequence of complete erythrocyte GPI gene revealed the presence of two new missense mutations: G145C (Gly49Arg) and C921A (Phe307Leu). Since both these aminoacids are conserved in eukaryotic GPI enzymes, the mutations G145C and C921A are likely to impair the enzyme function or stability.

Case2:The propositus was a 8 yrs. old girl of Southern Italian origin with a history of chronic hemolytic anemia and occasional need for blood transfusion. At the age of 7 yrs. she was splenectomised reducing the need for blood transfusion. At the time of the study Hb was 9.4 g/dL, reticulocytes 364x109/L, total bilirubin 12.3 mg/dL. The propositus was homozygote for Gilbert’s syndrome. GPI activity was 20% of normal. The parents were not consanguineous, and displayed intermediate GPI activity (55% and 61% of normal). The sequence of complete erythrocyte GPI gene revealed the presence of two missense mutations, G301A (already described in an Italian patient by Baronciani et al Blood 88:2306,1996) and the new variant G1009A, which result in the aminoacidic substitutions Val101Met and Ala337Thr respectively.
Changes of Subsets of DC 1 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 measure the changes of subsets of dendritic cells 1 (DC1) in the bone marrow of severe aplastic anemia (SAA) patients and evaluate the relationships between the CD11c+CD83+cells and Th1 cells, CD3+CD8+ cells or hematopoietic function.

Methods By FACS, the quantity and ratio of CD11c+CD1a+ cells, CD11c+CD83+ cells, Th1 cells, and CD3+CD8+ cells in the bone marrow of SAA patients and normal controls were detected respectively. The relationships between CD3+CD8+ cells and Ret or ANC, between Th1 cells and CD3+CD8+ cells, Ret or ANC, between CD11c+CD83+ cells, and Th1 cells, CD3+CD8+ cells, Ret or ANC were evaluated.

Results In normal control’s bone marrow, the percentages of Th1 cells, CD11c+CD1a+ cells, CD11c+CD83+ cells and ratio of CD11c+CD83+ /CD11c+CD1a+ was 0.42±0.30%, 0.38±0.29%, 0.37±0.32% and 1.07±0.10 respectively. In the untreated SAA patient’s bone marrow, they were 4.87±0.54%, 1.73±0.24%, 3.38±0.56% and 2.21±0.32 respectively, and increased markedly(p<0.01). In recovering SAA patient’s bone marrow, the percentages of Th1 cells, CD11c+CD1a+ cells and CD11c+CD83+ cells decreased significantly[0.53±0.22%, 0.61±0.23%, 0.65±0.22%, respectively (p<0.01)]. The ratio of CD11c+CD83+/CD11c+ CD1a+ of recovering SAA patients was 1.37±0.25 which was similar to that of normal controls (p>0.05). The percentages of CD3+CD8+ cells of untreated SAA patients was 32.32±10.22%, and that of recovering SAA patients decreased to 13.67%±5.24 significantly (p<0.01). The percentages of CD3+CD8+ cells of SAA patients were correlated to their Ret and ANC (P<0.05) negatively. Their Th1 cell percentages were correlated to their CD3+CD8+ cells positively (P<0.01), but to their Ret and ANC negatively(p<0.01). SAA patient’s CD11c+CD83+ cell percentages were correlated to their Th1 cell and CD3+CD8+ cells positively (P<0.01, P<0.05), but to their Ret and ANC negatively(p<0.01).

Conclusion Both immature DC1 and activated DC1 increased in the bone marrow of SAA patients, and the balance of subsets of DC1 shifted from stable form to active one, which might promote Th0 cells to polarize to Th1 cells, then cause the over-function of T lymphocytes and hematopoietic failure in SAA.
Development of an In Vivo Model To Assess Factors That May Stimulate the Generation of an Immune Reaction to Epoetin Alfa.
Authors: Ryan Mary H. , Shanahan Mary F. , McMahon Frank , Jolliffe Linda , Brigham-Burke Mike , Heavner George A. , Farrell Francis X.
Published: October 2019

The incidence of pure red cell aplasia (PRCA) in patients with chronic kidney disease associated with the subcutaneous (sc) administration of epoetin alfa (EPREX®) began to increase in 1998. As part of an intensive investigation into the reasons for this increase, an in vivo model was developed to assess the ability of potential causative factors to stimulate an immune response to recombinant human erythropoietin (rHuEPO). Due to species differences in protein sequence and as well as antigen processing and/or presentation, animal models cannot fully predict immunogenicity in humans. However, changes in relative immunogenicity have been readily detected in animal models. Moreover, transgenic animals have been utilized to negate the effects due to sequence differences between species. The EPO sequence is highly homologous between mouse and man. We found it difficult to generate anti-EPO antibodies in both human EPO transgenic mice and two normal mouse strains (Balb/c and BD-F1). To mitigate the complicating factors associated with the transgenic model (e.g. difficulty breeding, improper regulation of EPO production), normal mouse strains were selected for the development of animal models. Mice were injected sc with rHuEPO weekly for 4–6 weeks. Serum samples were analyzed 1–2 weeks following the last injection to ensure complete clearance of the injected protein. Serum samples were screened by an ELISA and/or by a surface plasmon resonance (BIAcore) method. In animals injected with rHuEPO alone, anti-EPO antibodies were either absent or present at very low levels. The addition of an adjuvant to the immunization protocol was able to boost both the rate of occurrence and titer of the immune response and resulted in the generation of anti-EPO antibodies that, in most cases, recognized both human and mouse EPO. Some of the mice exhibited a dramatic reduction in hematocrit, suggesting that the anti-EPO antibodies neutralized endogenous EPO. The development of an anti-EPO antibody response in humans is an extremely rare event that cannot be reproduced in an animal model without significant manipulation of the system (e.g. use of an adjuvant with doses of EPO considerably higher than therapeutic levels). Nevertheless, the fact that anti-EPO antibodies can develop with an appropriate immunization protocol allows for this model to be used to evaluate factors that may potentiate an immune response to rHuEPO.
L-Glutamine Therapy Increases Exercise Endurance of Sickle Cell Anemia Patients.
Authors: Macan Henry R. , Casaburi Richard , Tsurugaya Hideki , Porszasz Janos , Koh Han , Tanaka Kouichi R. , Niihara Yutaka
Published: October 2019

We have demonstrated previously that oral L-glutamine administration can improve NAD redox potential in sickle red blood cells (RBC) and decrease the endothelial adhesion rate of sickle RBC. Clinically, there was subjective reporting of increased energy level. To confirm the reporting of increased energy level objectively, a study incorporating exercise tolerance testing was designed. After proper consent, 7 homozygous sickle cell anemia volunteers participated in the study, which was composed of two parts. The first is incremental work rate test where the work rate increased incrementally. The second part is the constant work rate test. In this test the 80% maximum work rate for the incremental test is utilized and the patients are tested for duration of exercise. These tests were conducted at baseline and after 8 to 12 weeks of therapy with oral L-glutamine at 30 grams a day. Two of the patients withdrew prior to completion of the follow up testing. There were 6 patients who completed the incremental work rate test and 4 patients who completed constant work rate test, respectively, at baseline and after treatment. Two of the 6 patients who completed the incremental work rate test at baseline declined to have the constant work rate test, resulting in 4 patients completing the constant work rate test. The results were as following: The work rates were 88 watts at baseline and 91.5 watts after treatment, with p = 0.18, for the incremental work protocol and 5.87 minutes at baseline and 7.27 minutes after treatment, with p = 0.02, for constant work protocol. For the incremental work rate, the data show a trend for increase with L-glutamine treatment. For the constant work protocol, there was a significant improvement in the work rate after treatment with L-glutamine. The data are consistent with the subjective reporting from a previous study suggesting that L-glutamine therapy improved the exercise endurance of sickle cell anemia patients

Test Incremental Constant Mean value Peak work rate SD± Duration SD± Pre- 88 watts 25.58± 5.87 minutes 1.65± Post- 91.5 watts 21.69± 7.27 minutes 1.14± Significance P value One-tail test 0.18 0.02
Outcome of Transitioning Pediatric Patients with Sickle Cell Disease to Adult Programs.
Authors: Ballas Samir K. , Dampier Carlton
Published: October 2019

The transition of medical care of patients with sickle cell disease (SCD) from pediatric to adult providers represents a milestone in their lives. Major concerns among adolescents and young adults about transition include taking responsibility for self, making own decisions, cost of medical care, fear of suboptimal pain management, and reluctance to leave known providers. In this study we present our experience in the process of transition to adult care and its outcome over the last ten years. Adolescents and young adults were given information about the nature of medical care provided by adult internists and hematologists. The sickle cell programs available in the city were described. Moreover, site visits to the hospitals where adult care was to be provided were arranged. During these visits, adolescents and young adults had the chance to meet the hematologist and other potential providers and ask questions, visit the emergency room, the clinic, and the sickle day unit if applicable. Patients were empowered to choose the program to which they wished to be transitioned. During the last 10 years, 90 adolescents and young adults (See Table) with SCD (Sickle Cell Anemia [SS], Hemoglobin SC Disease, and Sickle Thalassemia [ST]) were transitioned to the adult sickle cell program of Thomas Jefferson University. Age of transition varied between 18 and 25 years. Eighteen patients (20%) died. Age at death was 24.9 ± 2.95 years and the male/female ratio was 10:8. Complications of sickle cell disease after transition included leg ulcers, stroke, avascular necrosis, anxiety, depression, and priapism. Nineteen patients (10 males, 9 females) were employed. Twenty-nine (32%) patients developed chronic pain syndrome and its sequelae. Many patients failed to achieve their childhood goals. The data show that a significant number of patients die within 10 years after transition. The quality of life of survivors is suboptimal and drifts into issues of chronic pain management in the adult environment. Identifying these issues may provide predictors that identify children at risk to have undesirable outcomes after transition. Aggressive management and refining the process of transition should improve the outcome after transition.

Distribution of the Transitioned Patients

SS SC ST Total Male 31 8 4 43 Female 34 8 5 47 Total 65 16 9 90
Finding of 8 New Therapeutic Agents by the Use of New High-Throughput Drug Screening Methods Established by the Nhlbi Sicke Cell Disease Reference Laboratory.
Authors: Asakura Toshio , Abdulmalik Osheiza , Iyamu Efe , Chen Qiukan , Yang Jisheng , Brugnara Carlo , Evans Greg
Published: October 2019

The NHLBI Sickle Cell Disease (SCD) Reference Laboratory (Ref Lab) was established in 1997 to facilitate rapid, efficient, accurate and economical evaluation of agents or therapies that may be beneficial for the treatment of patients with sickle cell disease. On the basis of the assumption that the unsuccessful finding of antisickling agents in the past was attributed to insufficient pre-clinical drug evaluation, the SCD Ref Lab proposed to screen drugs using more than 10 in vitro tests to study if a drug inhibits or promotes sickling, hydrates or dehydrates SS cells, prolongs or shortens the delay time prior to deoxy-Hb S polymerization, induces Hb F, increases or decreases the solubility of deoxy-Hb S; Hb oxygen affinity; membrane ion transport, adhesion of SS cells to endothelial cells; met-Hb S formation; hemolysis and the amount of intracellular denatured Hb S. In addition, drugs that showed a beneficial effect without significant adverse effects in these tests were further studied using transgenic mice that produce human sickle hemoglobin. To perform many in vitro tests efficiently, the SCD Ref Lab established a new high-throughput drug evaluation protocol by which many tests are conducted simultaneously using a small volume of SS blood samples or Hb S. To date, we have found 8 new antisickling agents. They are NS3623 (Blood97:1451–7, 2001); MX-1520 (Brit. J. Haematol.125:788–795,2004); NIPRISAN [Brit. J. Haematol.118: 337–343, 2002 and 122: 1001–8 (2003)]; FLOCOR (manuscript in preparation); and 5HMF which forms a Schiff base adduct with Hb S. In vivo studies using transgenic sickle mice showed that pretreatment with each of these drugs prevented the formation of sickled cells in the blood and prolonged the survival time under severe hypoxic conditions. We also found that Amidox, Didox and Trimidox increased the synthesis of Hb F in K562 cells (Am J. Hematol.63: 176–183, 2000).
Prevalence of Respiratory Symptoms in Children with Sickle Cell Disease Compared to Children with Other Hematological Diseases.
Authors: Mueller Brigitta Ursula , Acosta Monica , Dinu Marlen , Sockrider Marianna , Shardonofski Felix , Smith E. O’Brian
Published: October 2019

Acute chest syndrome (ACS) and pulmonary hypertension are complications of sickle cell disease (SCD). The effects of pre-existing respiratory symptoms on the frequency of complications of SCD, such as vaso-occlusive crises and ACS, have not been fully explored. Many therapeutic options exist for patients with reactive airway disease or other recurrent respiratory problems. Control of associated symptoms may help prevent the occurrence of ACS and other sickle cell-related problems. We compared the prevalence of respiratory symptoms in SCD patients compared to patients with other hematological disorders.

Methods: Two questionnaires (one for patients with SCD, one for patients with other hematological disorders) were developed, which included questions regarding respiratory symptoms (especially wheezing and cough, frequency of these symptoms, previous diagnosis of asthma), personal history and environmental exposure. The study was approved by the Institutional Review Board, and patients and their caregivers were interviewed during a routine visit to the outpatient hematology clinic after giving informed consent.

Results: To date, 124 subjects have been interviewed (62 males and 58 females, ages 3–18 years, mean age 10.1yrs), and 120 questionnaires were evaluable, including 59 from patients with SCD and 61 from the comparison group. Respiratory symptoms (wheezing and cough) were more frequent in SCD patients compared to patients with other hematological diseases. Wheezing was present in 34 (58%) SCD patients and in 20 (33%) patients in the other group (p<0.01). Twelve patients (20%) in the SCD group missed school due to respiratory problems (over half of them missed more than 7 days of school) compared to 4 (6%) patients (only 1 missing school for more than 7 days) in the other group (p<0.04). Hospitalization due to respiratory problems before the age of 2 years was more common in SCD patients: 23 (39%) compared to 8 (13%) in other hematological patients (p<0.003).

Conclusions: The prevalence of pulmonary symptoms is higher in patients with SCD compared to patients with other hematological disorders and associated with frequent hospitalizations and school absences.
Nitrated Lipids in a Murine Model of Sickle Cell Disease at Baseline.
Authors: Schwartz Jeffrey , Baker Paul R.S. , Schopfer Francisco J. , Freeman Bruce A.
Published: October 2019

Introduction

Sickle cell disease (SCD) is increasingly recognized as a disorder of inflammatory homeostasis. One major focus in recent years has been understanding the role of Nitric Oxide (NO) in the pathophysiology of SCD. NO is a critical mediator of inflammatory pathways and current evidence supports the precept that NO bioavailability is decreased in SCD, resulting in normal concentrations of NO at baseline but an inability to increase NO during stress. Nitrated fatty acids, such as Nitrolinoleate (LNO2), have recently been reported as potent and abundant anti-inflammatory signaling mediators with the ability to cause vasorelaxation and inhibition of platelet and neutrophil activation. Evidence supports their anti-inflammatory signaling is mediated through the release of NO and NO-related products. LNO2 has not previously been described in patients with SCD and our objective was to quantify LNO2 in a murine model of SCD at baseline.

Methods

Whole blood was obtained from transgenic sickle cell and wild type mice (n = 5 and 6, respectively). Blood was centrifuged and separated into plasma and packed red blood cells (RBCs). These biological samples were prepared for lipid analysis by the method of Bligh and Dyer; care was taken so that the pH of the extraction milieu was consistently maintained at 7 so as to avoid artifactual nitration. Samples were analyzed for free LNO2 content by electrospray ionization tandem mass spectrometry. Using a hybrid triple quadrupole ion trap mass spectrometer, MRM transitions were monitored that specifically identified nitrated linoleic acid species; these species were concomitantly confirmed by the qualitative analytical abilities of the ion trap. The presence of nitrated linoleic acid was confirmed by HPLC chromatographic retention times, MS/MS “fingerprints” and was quantitated by the inclusion of a known quantity of 13C-labeled LNO2.

Results

LNO2 concentration was calculated as a function of the ratio of analyte to internal standard peak areas by using an internal standard curve linear over five orders of magnitude. Free LNO2 in the RBCs and plasma of 5 transgenic sickle cell mice were 3.97 ± 2.56 nM and 12.37 ± 9.83 nM, respectively. Free LNO2 in the RBCs and plasma of 6 wild type mice were 9.49 ± 8.32 nM and 14.91 ± 10.08 nM, respectively. There were no significant differences in LNO2 concentration between any of the groups.

Conclusions

LNO2 is present in both transgenic sickle cell mice and wild type mice in comparable concentrations at baseline. As a mediator of NO anti-inflammatory signaling, this is consistent with human studies showing comparable concentrations of NO metabolites at baseline between sickle cell patients and healthy controls. Further study of LNO2 in sickle cell disease is warranted to better understand its role in the inflammatory process associated with acute stress, such as vaso-occlusive pain crisis and acute chest syndrome, when NO bioavailability is decreased.
The Outcomes of Preimplantation Genetic Diagnosis Therapy in Treatment of β Thalassemia - a Retrospective Analysis.
Authors: Qureshi Naveen , Foote Drucilla , Madore Rebecca , Walters Mark C. , Singer Sylvia Titi , Quirolo Keith C. , Vichinsky Elliott P.
Published: October 2019

BACKGROUND: β thalassemia is one of the most common single gene disorders. Hematopoietic stem cell transplantation (HCT) from a human leukocyte antigen (HLA)-identical sibling donor is a curative option that minimizes the risk of graft-versus-host disease, compared to alternative donor HCT. In families that have an affected child, preimplantation genetic diagnosis (PGD) can be utilized to select an unaffected embryo that is HLA-identical. Briefly, this procedure requires in vitro fertilization, oocyte retrieval, fertilization and blastomere biopsy for preimplantation analysis and identification of unaffected HLA-identical embryos. After delivery, umbilical cord blood from the sibling donor is collected for HCT. In our institution, PGD has been pursued as a therapeutic option by families with thalassemia. The estimated cost of this uninsured procedure is $20,000 per cycle.

METHODS: Families affected with β thalassemia who attempted PGD were identified and reviewed for indication, attempted cycles, successful pregnancy and transplantation outcome.

RESULTS: Eight identified families affected by thalassemia underwent PGD. The diagnosis of their affected children included: 6 cases of β thalassemia major and 2 cases of transfusion dependent E β thalassemia patients. A total of 14 cycles of PGD were attempted, ranging from 1–4 attempts per family. Following successful identification of HLA-identical cells, 2 pregnancies occurred (1 early miscarriage, 1 successful delivery). This pregnancy resulted in the engraftment of a β thalassemia child.

CONCLUSION: PGD including selection of HLA-identical sibling embryos is a novel, therapeutic approach for patients with β thalassemia. While this offers the possibility of recruiting a suitable donor for HCT, it is limited by significant financial and emotional burdens that it places on families affected with β thalassemia. Improvements in its efficiency and cost will make this a more viable option for affected families.
Quality of Life in Patients with Thalassemia.
Authors: Pakbaz Zahra , Treadwell Marsha , Yamashita2 Robert , Foote Drucilla , Vichinsky Elliott
Published: October 2019

Introduction: Thalassemia patients have experienced a dramatic increase in lifespan secondary to improved and more complicated therapy. The impact of the disease and its treatment on quality of life is largely unknown. Determining the degree of health impairment as perceived by the patient is essential information needed to recommend suitable therapy. Factors that affect the quality of life of thalassemia major patients may differ from thalassemia intermedia patients. Identifying the specific causes for negative patient assessment of overall health status can result in successful intervention. The objective of this study is to determine the factors that affect quality of life in both transfused and non-transfused thalassemia patients.

Methods: The Dartmouth Care Cooperative Chart System (COOP) questionnaire is a standardized, validated, visual screening tool that screens the major domains affecting quality of life. Patients rate quality of life from excellent (1) to poor (5). Scores of 1 and 2 are normal. A Score of 3 are mild to moderate impairment, and 4 and 5 are severe abnormalities. 50 patients were randomly selected during a comprehensive visit to complete a COOP questionnaire. These results were augmented by a brief medical history and chart review.

Results: Forty-eight thalassemia patients, including 25 transfusion dependent (Tx) and 23 non-transfused (Non-Tx) patients (50% female) were randomly selected and completed the COOP questionnaire. The mean age of the total population was 19 years (0.9–37.9 yrs) and was similar in both groups. Half of both groups had scores of 3 or less indicating no severe impairment. However, 56% of patients had ratings of 3 indicating mild to moderate impairment of major domains. Four patients (2 Tx, 2 Non-Tx) had repeated 4–5 ratings in multiple domains indicating severe impairment of quality of life. The most commonly reported affected domains were feelings, such as anxiety, depression, and concern of overall health status or indications of recent deterioration in health.

Conclusion: At least 50% of transfusion and non-transfusion dependent thalassemia patients demonstrate some impairment in quality of life. Forty-four percent of the population had a severe impairment in at least one domain. In particular, mental health issues are widespread. In contrast to previous beliefs, non-transfusion dependent patients also suffer serious impairment in quality of life. This data suggests all patients should undergo quality of life assessments and intervention, which focuses on affected domains. Studies to determine if quality of life affects patients’ adherence to chelation therapy are needed.
Plasma Gelatinolytic Activity in Leukemias.
Authors: Wrzyszcz Aneta , Urbaniak Joanna , Wrobel Tomasz , Mazur Grzegorz , Tomaszewska-Toporska Beata , Kuliczkowski Kazimierz , Wozniak Mieczyslaw
Published: October 2019

Gelatinase A (Matrix Metalloproteinase 2, MMP-2) and B (Matrix Metalloproteinase 9, MMP-9) are secreted by connective tissue, endothelial, epithelial and hematopoietic cells. Elastase and gelatinases produced by granulocytes are released in whole blood in response to varied stimuli. They induce breakdown of connective tissue and play an important role in processes of cell transmigration and tissue remodeling. The aims of this study were to determine and compare the plasma concentration of PMN elastase and the gelatinase activity in patients with chronic myelogenous leukemia (CML, n=16), acute myeloblastic leukemia, AML, n=53 and acute lymphoblastic leukemia, ALL, n=18) and in healthy persons (n=22). We measured activity of MMP-2 and MMP-9 using gelatine zymography and concentration of elastase from polymorphonuclear leukocytes (PMN) in complex with alpha1-proteinase inhibitor using ELISA. The MMP-9 activity in plasma from patients with CML was higher than that from AML, ALL and reference groups. The MMP-9 activity in AML and ALL group was lower than in healthy persons. There was no significant difference in the MMP-2 levels between the plasma samples from patients with CML or ALL and those from healthy subjects, but MMP-2 activity was lower in the reference group than in AML. PMN elastase concentration in plasma from the healthy reference group was lower than that from CML and AML patients. Furthermore, PMN elastase concentration in plasma from CML patients was higher than that from AML and ALL. We found significant correlation between MMP-9 activity and white blood cells count in the reference group (r=0,56), CML group (r=0,70) and AML group (r=0,44). The PMN elastase concentration increased proportionally to the white blood-cell count (r=0,51) in plasma from patients with CML only. There was significant relationship between the count of granulocytes in different stages of maturation with PMN elastase and MMP-9 activity: promyelocytes (r=0,73 for elastase and r=0,75 for MMP-9), myelocytes (r=0,63 and r=0,79), metamyelocytes (r=0,56 and r=0,83), band neutrophils (r=0,64 and r=0,74), segmented neutrophils (r=0,73 for MMP-9) and eosinophils (r=0,64 for MMP-9) in blood from CML patients. Increased MMP-9 activity was correlated with increased MMP-2 activity in the reference group (r=0,58) and AML group (r=0,50), however it was correlated with decreased MMP-2 activity in the CML group (r=−0,59). In the CML group it was observed that decreased MMP-2 activity correlated with an increased count of: blasts (r=−0,64), promyelocytes (r=−0,77), myelocytes (r=−0,63), metamyelocytes (r=−0,64) and band neutrophils (r=−0,60). Gelatin zymography can be employed to display differences of plasma MMP-2 and MMP-9 activity between patients with chronic myelogenous leukemia, acute leukemia and healthy persons.
Evaluation of the Usefulness of Detected Platelet Antibodies for the Prognosis of Treatment Results in Patients with Thrombocytopenic Purpura.
Authors: Sikorska Anna , Maslanka Krystyna , Konopka Lech , Zupanska Barbara
Published: October 2019

Platelet antibodies are the main cause of platelet destruction in immunological thrombocytopenic purpura. However, the significance of their detection for prognosis of treatment results in ITP patients (idiopathic immune thrombocytopenic purpura) has not yet been determined.

The aim of this work was to evaluate the effectiveness of treatment with corticosteroids, splenectomy and immnunosupressive drugs of 409 ITP patients in relation to the detection of platelet antibodies.

Platelet antibodies were determined in the ITP patients under observation:

in connection with autologous platelets - with platelet immunofluorescence test (PIFT),

in serum - with immunoenzimatic test using monoclonal antibodies directed at GPII/IIIa, GPIb, GPIa/IIa (MAIPA - monoclonal antibody immobilization of platelet antigens).

In treatment effectiveness prognosis the multifactor statistical analysis was used involving not only platelet antibodies but also other parameters such as age, sex, course of disease, megacariocyte count in bone marrow. In patients after splenectomy -the spleen-liver index and the time lapse between ITP diagnosis and surgical procedure were additionally taken into account.

The statistical analysis revealed that platelet antibodies had no significant effect on the treatment results prognosis in ITP patients. However, while analysing groups of patients with different treatment administered, it was found that the effect of platelet antibodies on positive prognosis treatment was significant though not very big effect in the case of patients treated with corticosteroids and immunosuppressive drugs while nonsignificant in the case of patients after splenectomy.

Additional factors significantly effecting positive treatment prognosis in all patients were: severe/acute course of disease, increased/normal megacariocyte count, young age, female sex. In splenectomy patients the spleen-liver index (up to 5.7:1) seems to be a significantly beneficial factor as well as the time lapse between diagnosis and procedure (up to 9 years since diagnosis).
Functions of Thrombin Receptors in the Reversible Distribution of Platelet Glycoprotein Ib within Activated Platelets.
Authors: Yue Han , Pasquet Jean-Max , Nurden Alan , Changgeng Ruan
Published: October 2019

Platelets are critical in normal haemostasis and arterial thrombosis. As a potent activator, thrombin activates human platelets by cleavage of the protease-activated receptors PARs, exposing new amino terminus which serve as tethered peptide ligands, binding to the receptors induce transmembrane signaling. Thrombin also binds to platelet glycoprotein Ib, whose redistribution within platelet was showed to be reversible upon to thrombin or to PAR1 and PAR4 peptides. In an attempt to understand the reversible expression of platelet GPIb and cytoskeleton reorganization during platelet activation, then determine functions of thrombin receptors in GPIb redistribution. we used peptide SFLLRNPNDKYEPF (PAR1-AP, trap) and AYPGKF (PAR4-AP) for stimulating platelet at different time points (0~60minute), detected the platelet surface GPIbα and P-selectin with flowcytometry, and compared the alteration of GPIbα, actin and myosin in cytoskeleton by western-blot, then analyzed the membrane cytoskeleton followed by GPIbα immunoprecipitation.

As expected, a reversible interalisation of GPIbα was obtained by PAR1 or PAR4 activation, and a transient change of actin, myosin and GPIbα /myosin GPIbα /actin association were also found in this course. These transient internalisations were apparently blocked by Cytochalasin D (inhibitor of actin polymerisation) or BAPTA/AM (calcium chelator). At the same time, ApyraseVII had weak effect on GPIbα interalisation, although the return of GPIbα to platelet surface was accelerated by this low ATP/ADPase, which quickened GPIbα dissappearance in cytoskeleton and the dissociation of GPIb/myosin or GPIb/actin during PAR1-AP activation.100nM and 10μM wortmannin were used to inhibit phosphatidylinositol 3-kinase (PI3-K) and/or myosin light chain kinase (MLCK) in our experiments. In response to both PARs activation, GPIbα interalisation was partly inhibited by 100nM wortmannin but little change for 10μM wortmannin, and a delayed restoration of surface GPIbα was observed in the presence of 10μM wortmannin upon PAR1-AP activation.. Apart from the above reagents, Ro-31-2220 (inhibitor of protein kinase C) induced a decreased GPIbα centralisation in response to PAR1 activation, and blocked the pool of GPIbα inside platelet in the latter course of PAR4 activation (p<0.05 at 10,30 min). Globally, Our study confirmed that thrombin receptors are important for platelet signal transmission, Stimulation of either receptor is sufficient to trigger platelet activation and induce GP Ib redistribution, which is correlated with cytoskeleton reorganisation, depending on the actin polymerisation and calcium mobilisation and implicate both myosin and actin. Our results also suggested critical roles of ADP, PI3-K or protein kinase C (PKC) in the GPIb redistribution.