bluuebunny/crossref_metadata_2025 huggingface dataset:
TPO Exerts a Protective Effect on Iron-Induced Apoptosis Via Erk1/2 Signaling in Human Mesenchymal Stem Cells.
Authors: Zhang Wei , Yang Mo , Chan Shing , Chan Godfrey ChiFung
Published: April 2020

Osteoporosis is a very common problem among adolescent and adult patients with thalassaemia major (TM). The pathogenesis of osteoporosis in TM is related to several factors including iron overload. Bone is derived from osteoblasts. And osteoblasts are differentiated from mesenchymal stem cells (MSC). Therefore, iron-overload induced MSC damage may contribute to osteopenia and osteoporosis. The effect of iron-overload on MSC has not been investigated previously. We hypothesize that iron-overload may induce apoptosis in MSC by caspase-dependent pathway, and haematopoietic growth factor thrombopoietin (TPO) and calcium channel blocker amlodipine may have a protective effect on iron-induced apoptosis in these cells. We have shown that iron (FeCl3) reduced hMSCs viability in a dose-dependent manner (0–0.6 mM) (n=5). By annexin V and PI staining, apoptotic cells were found to be significantly increased after iron treatment (0.3 mM) for 72 hrs (n=4). The expression of active caspase-3 was significantly increased in iron-treated cells (0.15mM, 0.3mM) (n=5). Iron treatment also increased the proportion of cells containing JC-1 monomers, indicating a trend in the drop of mitochondrial membrane potential. TPO exerted protective effect on iron-induced apoptosis in hMSCs. Human MSCs were cultured in the presence of iron (0.3 mM) with or without TPO (50 ng/ml) for 72 hrs (n=4). The cell viability was significantly increased with the treatment of TPO. Dot-plot analysis of annexin V/PI staining demonstrated that TPO significantly reduced the population of apoptotic cells. Incubation with TPO also decreased the iron-induced caspase-3 expression. Flow cytometric dot-plot analysis of hMSCs also showed trends of amelioration of the increase in JC-1 monomers in the iron plus TPO. The population of phospho-Erk1/2 was also significantly increased in TPO-treatment, and the increased phospho-Erk was significantly reversed by the upstream signaling inhibitor PD098059. Calcium channel blocker amlodipine (10−9M) also had a protective effect on iron-induced apoptosis in these cells. Our findings suggest that iron-overload induces apoptosis in hMSCs via the caspase-dependent pathway and that TPO and amlodipine might exert a protective effect on iron-induced apoptosis via the activation of Erk1/2 signaling. The use of either haematopoietic growth factor or calcium channel blocker for the protection of hMSCs from iron induced toxicity is a novel concept. Our study has the potential in minimizing the bone damage induced by iron-overload in patients with thalassaemia major.
Taxanes Induce a Rapid Mobilization of Different Populations of Circulating Endothelial Progenitors by SDF-1 Modulation in Cancer Patients.
Authors: Bono Anna , Quarna Jessica , Marighetti Paola , Calleri Angelica , Antoniotti Pierluigi , Mancuso Patrizia , Voest Emile E , Langenberg Marlies , Roodhart Jeanine , Shaked Yuval , Kerbel Robert S , Bertolini Francesco
Published: April 2020

There is increasing clinical evidence that the addition of certain anti-angiogenic drugs can enhance the anti-cancer activity of many different chemotherapeutic drugs. Hypotheses to explain how anti-angiogenic drugs act as chemosensitizing agents include the “cancer vessel normalization” scenario and the ability of anti-angiogenic drugs to slow down tumor regrowth and thus increase the degree and durability of the tumor response. We have previously observed in preclinical studies a mobilization of circulating endothelial progenitors (CEPs) several days after the administration of maximum tolerable dose (MTD) cyclophosphamide (Bertolini et al, 2003), and suggested that such a CEP mobilization may facilitate tumor cell repopulation during the subsequent drug-free break that is necessary to allow recovery from the toxic side effects of such therapy. We report here preclinical and clinical evidence that several chemotherapeutics, and in particular taxanes, induce a rapid CEP increase. In previous mouse studies (Shaked et al, Cancer Cell, in press 2008), 4–24 hours after MTD administration of taxanes we observed a 3–8 fold CEP increase (depending on the mice strain, drug and dose administered). A similarly rapid CEP mobilization was observed (albeit at a less relevant level) after 5-FU administration, but not after the administration of drugs such as gemcitabine, anthracycline derivates or topo-isomerase inhibitors. The rapid mobilization induced by taxanes resulted in a colonization of treated tumors by CEPs, that was abrogated by the previous administration of anti-angiogenic drugs blocking VEGF or VEGFR2. A rapid increase in circulating SDF-1 levels was similarly observed after taxane administration, and inhibition of the CXCR4-SDF-1 axis by monoclonal antibodies blocked CEP mobilization and significantly enhanced the anti-tumor activity of taxanes. In clinical studies in cancer patients in Milan and Utrecht (n=54), we observed that MTD taxanes induced a rapid (by 4 hours) increase in circulating SDF-1 levels and a parallel 2–4 fold increase in a variety of different CEP populations (evaluated by six-colour flow cytometry as viable CD45- CD34+VEGFR2+ cells, CD133+ cells, CD45-CD133+ cells, CD45-CD34+CD133+ cells and CD45-CD34+CD133+CXCR4+ cells). Circulating levels of G-CSF, VEGF, VEGFR1 and VEGFR2 were not significantly modulated 4–24 hours after taxane administration. Taken together, our data indicate a novel SDF-1-related mechanism explaining the chemosensitizing activity of anti-angiogenic drugs and suggest that VEGF/VEGFR and SDF-1 inhibition can increase the anti-cancer efficacy of taxanes. Further investigations about chemotherapy-induced CEP mobilization might have a clinical impact in cancer and vascular medicine.
Activation of Endothelial Cells by Cocaine.
Authors: Hobbs William , Chen Junmei , Lopez Jose A.
Published: April 2020

Cocaine use is associated with cardiac ischemia, sudden death, and stroke in patients with few additional risk factors for cardiovascular disease. Despite the very short halflife of cocaine in circulation (less than 1 hr), the risk of these cardiovascular events remains elevated for up to one week after cocaine ingestion. Pathologic findings following cocaine-associated cardiac events include platelet-rich microthrombi and accelerated atherosclerosis. In addition, observational studies have identified increased levels of von Willebrand Factor (VWF) at steady state in plasma of chronic cocaine abusers, which increase following cocaine challenge. Plasma VWF is derived primarily from endothelial cells, with a smaller contribution from platelets. Thus, cocaine may activate platelets and/or endothelial cells contributing to tissue ischemia, in addition to its well-known effects of vasoconstriction and increased tissue metabolic demand. When first released, VWF is hyperadhesive and capable of spontaneously binding platelets. This form, termed ultralarge VWF (ULVWF) because some of the multimers are extremely large, is normally rapidly processed by the metalloprotease ADAMTS13 into the smaller, less reactive forms usually found in plasma. Failure of this process, due to congenital or acquired deficiency of ADAMTS13, leads to accumulation of ULVWF, and in its most severe form produces the devastating microthrombotic disease, thrombotic thrombocytopenic purpura (TTP). We hypothesized that cocaine could activate the endothelium to secrete ULVWF, and thus promote thrombosis and/or vaso-occlusion. To test this possibility, we exposed cultured human umbilical vein endothelial cells (HUVEC) to various concentrations of cocaine. We found that 0.5 mg/ml cocaine, a concentration comparable to peak blood levels detectable in cocaine abusers, induced secretion of ULVWF almost as efficiently as did 25 μM histamine, a potent ULVWF secretogogue. If platelets were then perfused over the endothelium in a parallel plate flow chamber at 2.5 dyne/cm2, they spontaneously formed beads-on-a-string structures on the attached ULVWF strands. We also evaluated the ability of cocaine metabolites to induce ULVWF release. Neither norcocaine nor ecoginine methyl ester (both minor metabolites) stimulated ULVWF release. However, benzoylecgonine, the major cocaine metabolite (detectable in plasma up to 6–8 days after cocaine use) potently induced ULVWF release, as detected by the formation of ULVWF strings and accumulation of higher molecular weight VWF multimers in the HUVEC supernatant on non-reducing agarose gels. These effects of cocaine are likely to contribute to cardiovascular syndromes associated with cocaine use, including the triggering of vasoocclusive crises in sickle cell anemia, and may explain the observed association of cocaine use with TTP. Furthermore, these results suggest that clinical management of cocaineinduced ischemia or vaso-occlusion may benefit from therapies aimed at disrupting ULVWF.
Vascular Branching Morphogenesis of the Placenta Requires Expression of Ott1(Rbm15) in the Trophoblast Compartment.
Authors: Raffel Glen D. , Chu Gerry C , Cullen Dana E. , Bronson Roderick T , Bernard Olivier A , Gilliland D. Gary
Published: April 2020

OTT1(RBM15) is a transcriptional activator/repressor known to be a fusion partner in t(1;22) infant acute megakaryocytic leukemia. Conditional deletion of Ott1 in adult mice resulted in expansions of the megakaryocytic, myeloid and pluripotent Lin−Sca-1+c-Kit+ populations and a block in pre B lymphoid development, suggesting a global regulatory role in hematopoiesis. Germline deletion of Ott1 is homozygous lethal after E9.5, producing developmentally delayed embryos with open notochords. Further investigation into the cause of embryonic death revealed a placental defect characterized by arrested vascular branching morphogenesis of the invading fetal vessels, leading to embryonic demise through inadequate oxygenation. Histopathology and in situ hybridization of Ott1-deficient placentas identified a dramatic reduction in the spongiotrophoblast and syncytiotrophoblast layers. Expression of Vegf, a critical element of placental vasculogenesis, was still observed, thus excluding the requirement for Ott1 in its induction. By using Sox2-cre with a conditional allele of Ott1, complete excision of Ott1 was achieved within the embryo yet the placental trophoblast compartment was left undeleted. These embryos were now able to form normal placentas with intact vascular branching morphogenesis and survive to term. This result demonstrates that Ott1-mediated vascular branching morphogenesis is regulated by the trophoblast compartment rather than the invading fetal vasculature. Additionally, the Sox2-cre-deleted pups manifested cardiac ventricular septal defects and hyposplenia and succumbed during the perinatal period. Hox, Notch and hypoxia-related pathways are important for both cardiac and splenic development, however downstream target gene expression was not perturbed. In summary, Ott1-dependent pathways, in addition to their implicit involvement in a congenital leukemia, are essential for vascular branching morphogenesis in the placenta and organogenesis of the mesoderm-derived heart, spleen and blood. Mutation of OTT1 or alterations in its regulation may therefore underlie human disorders such as placental insufficiency, asplenia or congenital cardiac malformations.
Carcinoembryonic Antigen (CEA) Regulates Tumor-Angiogenesis in Colon Carcinomas.
Authors: Braemswig Kira , Poettler Marina , Kalinowska Wazlawa , Zielinski Christoph , Prager Gerald W
Published: April 2020

Human carcinoembryonic antigen (CEA) is a cell surface adhesion molecule member of the Immunoglobulin Superfamily (IgSF). Aberrant upregulation and secretion of soluble CEA is a common feature found in a wide variety of human cancers such as colon, breast and lung. Previous in vitro and in vivo results have demonstrated that CEA can affect tumor cell behavior including the inhibition of cell differentiation and apoptosis. However, any functional effects on angiogenic endothelial cell behavior are so far unknown. In the present work we found that in endothelial cells exogenous CEA led to a time and dose dependent increase in ERK phosphorylation, which was inhibited by the specific MEK inhibitor U0126. Thereby, the observed CEA effect was comparable in time and intense with the canonical angiogenic growth factor VEGF. The CEA-induced ERK phosphorylation was not affected by the blockage of VEGFR-2 / flk-1 using a specific inhibiting peptide (CBO-P11), which indicates a VEGF-independent mechanism. Furthermore, co-stimulation of endothelial cells with VEGF and CEA shows synergistic effects on ERK phosphorylation. While in endothelial cells no endogenous expression of CEA is detected, its putative receptor, the CEA receptor (CEAR), is highly expressed as shown by immunohistochemical staining of paraffin-embedded colon carcinoma sections as well as in biochemical analyses. When an activating antibody against CEAR was used, CEA-induced ERK phosphorylation was mimicked, while downregulation of CEAR by siRNA diminished CEA-induced signal transduction, significantly. To test a biological relevance of our findings, we first measured endothelial cell proliferation: CEA led to a dose dependent increase in endothelial cell proliferation in vitro, which again revealed a synergistic effect with VEGF. Thereby, CEA-induced endothelial cell proliferation was again independent of VEGFR-2 / flk-1. A biological role of CEA in tumor-angiogenesis was reflected by an in vivo model using CEA Mimotope immunized BALB/c mice, which were transplanted with MethA/CEA overexpressing tumor cells. Immunohistological analyses of these tumors revealed a significantly reduced vascular density, which was accompanied with diminished tumor growth. Our data provide first evidence of CEA as a novel pro-angiogenic activator of endothelial cells, which results in an increase in endothelial cell proliferation, independent of VEGFR-2. Furthermore, by targeting CEA in an in vivo mouse model, tumor-angiogenesis was markley reduced, indicating a potential therapeutic target in cancer.
Genetic Polymorphisms of β-Cell Differentiation Confer Risk of Alterations of Glucose Metabolism in Survivors of Childhood Acute Lymphoblastic Leukemia.
Authors: Surapolchai Pacharapan , Hongeng Suradej , Mahachoklertwattana Pat , Pakakasama Samart , Winaichatsak Angkana , Wisanuyothin Nittaya , Pasomsub Ekawat , Mahasirimongkol Surakameth , Sirachainan Nongnuch
Published: April 2020

Purpose: Long-term survivors of acute lymphoblastic leukemia (ALL) seem to be at risk of insulin resistance, impaired glucose tolerance and may develop typical signs of metabolic syndrome with disturbances in lipid metabolism. The aim of the present study was to study the prevalence of alterations in glucose metabolism and the predisposing factors of these disturbances in survivors of childhood ALL. Furthermore, we intended to identify the role of such genetic polymorphisms affecting β-cell function and glucose metabolism and possibly to develop them as markers for monitoring β-cell dysfunction following treatment for ALL in the future.

Patients and methods: In 131 ALL survivors after cessation of therapy, an oral glucose tolerance test was performed to determine β-cell function/insulin sensitivity. Six single-nucleotide polymorphisms of PAX4 and TCF7L2 genes were genotyped in order to evaluate the association between these polymorphisms and β-cell function/insulin sensitivity.

Results: Ten out of 131 (7.6%) survivors had impaired glucose tolerance (IGT) while 40 out of 131 (30.5%) had insulin resistance (IR) and demonstrated characteristics of metabolic syndrome (hyperinsulinemia, hypertriglyceridemia, low HDL-C). In the logistic regression analysis, the most important factor predicting IGT and IR was older current age (p= 0.010 and <0.001, respectively) while the PAX4 R192H mutation (rs2233580) was significant associated only to IGT after adjusted for age (p=0.043) (adjusted OR 5.28, 95%CI 1.06–26.40).

Conclusion: Our findings suggest that older age of childhood ALL survivors is the most important risk factor of IGT and IR, underlining the need to continue regular and long-term screening till adulthood. Moreover, after adjusted for age, the PAX4 R192H variant (rs2233580) seemed to be associated with IGT. If this association is confirmed, institution of lifestyle and therapeutic measures may be needed to reduce metabolic risk and diabetes.
Molecular Response in Core Binding Factor Acute Myelogenous Leukemia with Fludarbine, Cytarabine, G-CSF and Gemtuzumab Ozogamicin.
Authors: Borthakur Gautam , Faderl Stefan , Verstovsek Srdan , Jones Dan , Estrov Zeev , O’Brien Susan , Bass Joyce , Kantarjian Hagop
Published: April 2020

Background: Core binding factor associated acute myelogenous leukemias (CBF AML) are characterized by unique fusion transcripts that are amenable to molecular monitoring. Multiple log reduction in the fusion transcript level has been reported to be associated with lower relapse rates in CBF AML (Liu-Yin JA et al. ASH 2007# 543). Addition of gemtuzumab ozogamicin (GO) to induction and consolidation has resulted in improved disease free survival (DFS) in patients with AML and “standardrisk” or “favorable cytogenetics” (Burnett,A. et al. ASH 2006#13). Historical data from M.D. Anderson Cancer Center indicates that induction/consolidation therapy with fludarabine, cytarabine and granulocyte colony stimulating factor (GCSF) (FLAG) results in improved event free survival (EFS) (median EFS not reached after median followup of 118 weeks) in patients with CBF AML (Borthakur et al. 2008, Cancer in press).

Method: In an attempt to improve upon these results we have initiated a study incorporating GO with FLAG (GO-FLAG) in induction/consolidation for treating patients with newly diagnosed CBF AML. We report on molecular responses in patients enrolled in this study. Extracted RNA is reverse transcribed and analyzed by real-time quantitative PCR for the fusion transcript associated with CBF leukemias. Values are expressed as a percentage of fusion transcript to normalizing ABL transcripts. The sensitivity of detection is approximately 1 in 100,000 for AML1-ETO and 1 in 10,000 for CBFb-MYH11.

Results: Seventeen (Inv 16=6/t 8;21=11) patients (male=15)[median age 49 years (range, 29–73)]have been treated to date with a median follow-up of 10 months (range,1–16). Patient characteristics at presentation include median WBC 10.4 (range, 1.5–41.5) x 106/ml, hemoglobin 9.3 (range, 7.8–11.9) gm/dL, platelet count 22 (range, 6–365) x 109/ml. Additional cytogenetic abnormalities were present in 10 patients; +8 and –Y being most frequent. Two patients had kit mutations. All patients achieved complete remission (CR) except one (partial remission). Two patients died in CR from infectious complications. There has been no relapse. Median pre-treatment value for fusion transcript to Abl ratio by Q-PCR is 100 (range, 100–857). After induction the median Q-OCR value dropped to 0.04 (range, 0–9.17) and after three cycles of therapy the median value is 0.01 (range, 0–0.12).

Conclusion: Induction/consolidation therapy with GO-FLAG regimen in CBF AML results in excellent molecular response. Further patient accrual and follow-up will generate information about impact on overall and disease/event free survival.
High Rate of Second Malignancies after Reduced Intensity Double Cord Blood Transplants in Adults.
Authors: Ballen Karen K , Cutler Corey , Yeap Beow Y , McAfee Steven L , Dey Bimalangshu R , Attar Eyal C , Haspel Richard L , Liney Deborah , Koreth John , Ho Vincent , Alyea Edwin P , Soiffer Robert J , Spitzer Thomas R , Antin Joseph H
Published: April 2020

Reduced intensity conditioning (RIC) double cord blood transplantation is an effective strategy in adults to provide allogeneic donors for patients with uncommon HLA types. However, the risk of second cancers has not been established. We treated 53 patients with hematologic malignancies using a RIC regimen of fludarabine 30 mg/m2/day Days −8 through −3 (total dose 180 mg/m2), melphalan 100 mg/m2 Day −2 and rabbit antithymocyte globulin 1.5 mg/kg Days −7, -5, -3, -1 (total dose 6 mg/kg). Cord blood units were a 4/6 or higher HLA A, B, DR allele match with the patient and each other and achieved a minimum precryopreservation cell dose of 3.7 x 107 NC/kg. Twenty-one patients received GVHD prophylaxis with cyclosporine and mycophenolate mofetil and 32 patients received tacrolimus and sirolimus. Median age was 49 (range 19–67) years and the majority of patients had acute leukemia or relapsed lymphoma. Median follow-up was 27 months among the 29 patients still alive. Nine patients developed a second malignancy. Seven patients had lymphoma and two had MDS/MPD. The second cancers occurred at a median of 141 days (range 39 days-26 months) after transplant. Five second cancers (both MDS/MPD and 3 of 7 lymphomas) were donor derived; the origin of the others was not determined. Patients with Epstein Barr Virus lymphoproliferative disorder (EBV-LPD) had a median EBV viral load of 445,000 copies/ml (range 3300–5.4 million). Six of seven patients with lymphoma had wide spread organ involvement; 6 patients were treated with Rituxan but only one of them had a sustained response; the rest died of EBV-LPD. Both cases of MDS/MPD were donor derived and fatal. One occurred in a patient transplanted for CLL; the other patient was transplanted for AML. The overall survival for the entire cohort of 53 patients was 55% at two years. The actuarial rate of second malignancy was 19% at 26 months post-transplant (2 year rate of 16%). The rate of donor-derived myeloid disease was 6%. Second malignancy was the leading cause of death, with relapse the second leading cause of death. Neither age, GVHD prophylaxis, HLA matching, nor total nucleated cell dose predicted for a higher rate of second malignancy. There was a trend to a higher risk of second cancer in patients who received an infused CD34+ cell dose of ≤ 1.9 x 105 cells/kg)—(30% vs. 7%, P=0.070). In summary, 1) Second malignancies occur at a high rate after RIC cord blood transplantation; 2) A low infused CD34+ cell dose/kg may be associated with a higher risk of second cancer; 3) Strategies to reduce thymoglobulin dose may be important to reduce the incidence of EBV-LPD; 4) It is important to assess the genetic origin of post-transplant myeloid diseases to establish the risk of donor-derived leukemia.

723: Recurrence, Secondary Neoplasia, and Late Complications
Engraftment Syndrome Following Unrelated Cord Blood Transplant in Pediatric Patients.
Authors: Domm Jennifer , Wang Li , Calder Cassie , Manes Becky , Frangoul Haydar
Published: April 2020

Engraftment syndrome or hyper-acute graft versus host disease like syndrome describes a constellation of symptoms that occur prior or during neutrophil recovery after both allogeneic and autologous stem cell transplant. The usual symptoms are high grade fever and diffuse erythematous rash. There are no prior published reports describing the incidence and clinical implications of engraftment syndrome in recipients of unrelated cord blood transplant. There were 326 patients ≤ 18 years of age with malignant (N=229) or non-malignant (N=97) diseases enrolled on a prospective National Heart Lung Blood Institute (NHLBI) sponsored cord blood transplant study between 1999 and 2003. Dataset was abstained after signed agreement with the NHLBI and local IRB approval. All patients received myeloablative preparative regimen with either total body irradiation or busulfan based regimens with cyclosporine and prednisone GVHD prophylaxis. All patients received anti-thymocyte globulin as part of their conditioning regimen. ES was defined as high grade fever and diffuse erythematous rash without documented infections. The median age was 4.65 years (range 0.04 – 17.90) with 127 (60%) male, 264 (81%) had a performance status of ≥ 90. Two hundred twenty eight (70%) of the patients had malignant diseases and 179 (55%) received a cord blood unit matched at 3/6 or 4/6 HLA antigens and 147 (45%) received a cord blood unit matched at 5/6 or 6/6 HLA antigens. The median total nucleated cell dose per kg infuses was 6.94 x 107/kg (range 0.08–809.4 x 107/kg). Sixty-four patients (20%) developed engraftment syndrome at a median of 10 days (range 5–24) post transplant. All patients developed engraftment syndrome prior to engraftment and were treated with high dose steroids. Patients who developed engraftment syndrome had a median age of 2.47vs. 5.57 years, 48% had nonmalignant disease versus 25%, compared to those who did not develop engraftment syndrome respectively. The median pre-cyopreserved total nucleated cells infused was 8.58x107/kg (range 1.54x107 to 27.49 x107) in those who developed engraftment syndrome compared to 6.68x107/kg (range 0.08 x107 to 80.91x107) to those who did not. A multivariable analysis using Cox regression analysis using engraftment syndrome as a time dependent co-variable was performed including patients age, sex, performance status (<90 versus ≥ 90), disease (malignant versus non malignant), HLA match (3–4/6 versus 5–6/6 HLA match), and total nucleated cell count per kg infused. Overall survival was significantly worse in female recipients (HR 1.78, CI 1.31–2.41, p=0.0002), those with malignant diseases (HR 1.75, CI 1.15–2.66, p=0.009), patients with performance status <90 (HR 1.66, CI 1.15–2.40, p=0.007) and those who developed engraftment syndrome (HR 1.61, CI 1.15–2.28, p=0.006). Engraftment syndrome is a common complication following unrelated cord blood transplant in pediatric patients that increases the risk of overall mortality.
Acute Treatments and Early Outcomes of Childhood Arterial Ischemic Stroke: First Analysis of the International Pediatric Stroke Study.
Authors: Goldenberg Neil A , Bernard Timothy J. , Gordon Anne , Fullerton Heather J. , deVeber Gabrielle A.
Published: April 2020

Background: In pediatric arterial ischemic stroke (AIS) clinical treatment trials are lacking and treatment practices variable. Factors determining treatment selection and outcomes are important to delineate.

Objective: We aimed to (1) describe acute treatments and outcomes in childhood AIS, and (2) test putative variables for treatment selection, and outcome in a prospective-retrospective multicenter international study.

Methods: We evaluated treatments and early outcomes of children enrolled in the International Pediatric Stroke Study with AIS diagnosed at >28 days and <18 years of age from 2003 to 2007. Putative predictor variables for antithrombotic treatment selection included age, clinical AIS subtypes, geographic region (Asia, Australia, Canada, Europe, South America, and U.S.), and diagnosis pre- versus post-2004 (when pediatric AIS guidelines published).

Results: Among 676 children with acute AIS, anticonvulsants and antibiotics were administered acutely in 57% and 40%, respectively; use of each decreased with age (P<0.001). Acute anticoagulants (AAC; with/without concomitant anti-platelet therapy) were selected more frequently than either acute anti-platelet agents (AAP) alone or no acute antithrombotic treatment (NAAT) (43% AAC vs. 28% AAP alone and 29% NAAT). NAAT decreased with increasing age. AAC was most frequent in cerebral/cervical arterial dissection (n=52; 75% AAC vs. 6% AAP) and least frequent with moyamoya syndrome (n=72; 31% AC vs. 43% AAP). AAC was most common in Europe and Canada, AAC vs. AAP relatively balanced in the U.S., and AAP most common in Asia and South America. AAC use was similar pre- versus post-2004. At hospital discharge 71% had neurological deficits independent of age, subtype, or geographical region. Mortality at discharge was 3%.

Conclusions: Acute anticoagulation is frequently but not uniformly employed in childhood AIS. With current treatment, the prevalence of neurological deficit at hospital discharge is high. These findings reflect disparity in published guidelines and highlight the need for clinical trials to reduce adverse outcomes.

Figure 1. Acute antithrombotic therapy in childhood AIS, by geographic region. Figure 1. Acute antithrombotic therapy in childhood AIS, by geographic region.
Platelet Responsiveness to Aspirin Loading in Patients with ST Elevation MI Undergoing Primary Percutaneous Intervention Is Associated with Myocardial Reperfusion and Clinical Outcome.
Authors: Varon Davi d , Matetzky Shlomi , Fefer Paul , Shenkman Boris , Shechter Michael , Levi Nitza , Savion Naphtali , Hod Hanoch
Published: April 2020

Background: In patients with stable coronary artery disease, and patients undergoing elective percutaneous coronary intervention (PCI), laboratory resistance to aspirin is associated with a higher incidence of adverse events. Nevertheless, the responsiveness to aspirin in acute myocardial infarction (AMI) and its implications have not yet been investigated.

Methods: The study comprised 76 aspirin naïve patients who underwent primary PCI (PPCI) for ST-elevation MI (STEMI). Platelet reactivity was assessed 30–60 mins after a loading dose of 300mg chewable aspirin, by conventional aggregometry and Impact R, where platelet reactivity to arachidonic acid (AA) was expressed by platelet deposition under flow conditions.

Results: Patients were stratified using the median value of AA-induced platelet aggregation (PA) (49%) to good responders to aspirin (n=38), who had a median AA-induced PA of 33% (25–41), and poor responders to aspirin (n=38), who had a median AA-induced PA of 77% (70–84). Similarly, good compared with poor responders had higher surface coverage by Impact R (3.9±2.6 vs. 2.2±1.3, p=0.003). Good versus poor responders were similar regarding baseline demographic, clinical and angiographic characteristics. However, good responders were more likely to demonstrate early ST-segment resolution ≥ 70% after PPCI (84% vs 54%, p <0.01), suggestive of better myocardial reperfusion. Good compared to poor responders had a lower incidence of adverse cardiovascular events (re-infarction, need for re-intervention, congestive heart failure and/or death) throughout a 6-month follow-up (11% vs 24%, respectively; p<0.05).

Conclusions: Ex vivo poor platelet responsiveness to aspirin loading in STEMI patients is associated with a worse prognosis in patients
Predictive Factors for Poor Outcome in Danaparoid-Treated HIT-Patients: A French Multicentre Case-Control Study.
Authors: Elalamy IsmaïL. , Tardy-Poncet B. , Mulot A. , DeMaistre E. , Pouplard Claire , Nguyen Philippe , Trossaert M. , Blanloeil Y. , Cleret B. , Gruel Yves , Lecompte T. , Tardy B.
Published: April 2020

Objective: to identify predictive factors of complications and mortality in danaparoid-treated HIT-patients.

Patients and Methods: case-control study involving HIT-patients defined by a relative decrease in platelet count of over 40% with or without thrombosis at presentation and positive laboratory testing (functional assay +/− ELISA). A patient presenting a complication 48 hours after heparin replacement with danaparoid (thrombosis, DIC, amputation, haemorrhage, death) was considered as a case.

Results: 49 cases and 65 controls were included (mean age 67±14 y; 51% women), from cardiovascular (53%) or orthopaedic (5%) surgery, intensive care (6%) or medicine (8%) units. Mean duration to complication was 7 days (D): death (n=26); thromboses (n=23); DIC (n=6); amputations (n=4); and haemorrhages (n=8).

In univaried analysis, compared to controls, cases presented:

an earlier HIT (7.9±3.5 versus 10.1±7.1 D; p=0.03) a lower platelet nadir (53±37 versus 70±37 G/L ; p=0.01), a more frequent thrombosis at HIT diagnosis (59% versus 32%, p=0.004), a later heparin withdrawal after HIT diagnosis (2.8 versus 1.3 d; p=0.01), a delayed heparin replacement (3.7 versus 1.9 d ; (p=0.001), an inadequate dosage of replacement therapy (44% versus 19% ; p=0.004).

In multivaried analysis, predictive factors leading for poor outcome were:

an initial thrombosis at HIT diagnosis (OR=3.2 [1.3–8]), a delay greater than 48 h for replacement therapy with danaparoid (OR=6.6 [2.5–17.3]), insufficient doses of danaparoid (OR=4.3 [1.5–12]).

Conclusion: this study confirms the mandatory need for a prompt replacement therapy and strict adherence to recommended doses for HIT-patients.
Prothrombin Complex Concentrate for Warfarin Reversal: A Single Dose Is Effective for All INRs >4.5.
Authors: Gatt Alexander , Riddell Anne , van Veen Joost J , Kitchen Steve , Tuddenham Edward , Makris Michael
Published: April 2020

Introduction: 1% of patients of warfarin experience life-threatening bleeding per year and require emergency reversal of their anticoagulation. This is usually done by using a combination of vitamin K and prothrombin complex concentrate (PCC), FVIIa or FFP. Algorithms for warfarin reversal commonly use INR thresholds to guide the PCC dose. However, the INR system has only been validated for values up to 4.5.

Aim: To compare the ability of PCC, FVIIa and FFP to reverse the warfarin effects on the INR and thrombin generation (TG) as measured by the calibrated automated thrombography (CAT). We hypothesised that INRs greater than 4.5 would not correlate with TG and in which case one concentration of reversal agents would be suitable to reverse warfarin in all these patients.

Methods and Results: A pool of plasma from patients on warfarin (final INR 4.8) was spiked with Beriplex P/N (25Units/kg), FVIIa (~140μg/kg) and FFP (~20ml/kg). The INR normalised with Beriplex and FVIIa and was reduced to 1.4 with FFP. However, only Beriplex completely normalised TG parameters. In view of this Beriplex was used for subsequent reversal experiments. We analysed 48 plasma samples with INR >4.5 (median 5.5; range 4.6–8.0) and measured TG at 5pM tissue factor. We found a significant moderate correlation between the INR and the endogenous thrombin potential (ETP) which is the maximum amount of thrombin generated (r=−0.59; P= <0.0001). The mean ETP was 279.4nM.min (95%CI 240 – 318.9; SD 135.9). 2 plasma samples with INR >8.0 were spiked with 5 concentrations of Beriplex P/N (7.5, 15, 25, 35 & 50Units/kg) in order to decide which is the ideal concentration of this PCC to reverse warfarin effects on the CAT. Both samples showed that a concentration between 25 and 35Units/kg could normalise TG without producing hypercoagulability. Hence we chose 30Units/kg to spike another 10 samples with INR >4.5 (mean 6.4; range 4.7–8.0). The INR, TG and FII, VII, IX and X were tested after the addition of Beriplex P/N. The INR normalised in all samples (median 1.2; range 1.08–1.3) as did the ETP (median 1942nM.min; range 1546–2753nM.min), FII (mean 114U/dl; range 76–126U/dl), FIX (mean 127U/dl; range 106–154U/dl) and FX (mean 131U/dl; range 120–140U/dl). FVII increased to a mean 52U/dl (range 20–68U/dl). A lower concentration of Beriplex (25U/ kg) did not satisfactorily normalise the INR and TG in all samples tested.

Conclusion: PCCs fully reverse the effects of warfarin when compared to standard doses of FFP and FVIIa. INRs >4.5 still correlate with TG but the amount of thrombin generated was comparatively low making the use of one dose of PCC (30U/kg) suitable for reversing the effect of all INRs >4.5. This not only simplifies the reversal algorithms but reduces cost since doses up to 50U/kg are sometimes advocated for INRs >6.0.
High Sensitivity Detection of Bacillus Cereus in Plasma Samples.
Authors: Rao Shilpakala Sainath , Mohan Ketha V. K. , Atreya Chintamani D
Published: April 2020

Microbial contamination of blood and blood products remains a major concern in transfusion medicine. Detection of bacterial contamination has been traditionally carried out by inoculating the contaminated blood/blood products into a liquid or solid nutrient medium followed by 18–24 h incubation period to observe any bacterial colony formation (growth). Although this technique is highly sensitive and specific, the test requires at least 24 hours for accurate diagnosis, which is a limitation Nucleic acid-based and antigen-based assays that have been recently in use also have limitations either with regard to time, sensitivity or specificity. In the present study we utilized Bacillus cereus and bacteriolysin (PlyG) derived from its g-phage virus as a model system for high sensitivity detection of this B. cereus in plasma. Six overlapping synthetic peptides in the bacterial binding C-terminal region of PlyG were tested in the study. The length of the synthetic peptides ranged from 20 to 10-mer and were biotinylated for subsequent detection. Peptide binding to bacteria was detected using either Streptavidin-conjugated horse radish peroxidase (HRP) on a nitrocellulose membrane or by using streptavidin-conjugated fluorescent Q-dots (liquid nano crystals) and analyzing under a fluorescence microscope. Various dilutions of log-phase cultures of B. cereus were spiked into plasma samples and incubated with the peptides for 1 hour followed by detection using either the streptavidin-HRP or streptavidin-Q-dots. Results revealed that two out of six peptides bound strongly to Bacillus in both the assays, while the rest of the peptides were either non-binders or weakly-binding. The membrane-based assay demonstrated an assay sensitivity of 102 colony forming units (CFU)/ml whereas the Q dots-based fluorescence assay was able to detect even a single bacterium. Overall, our findings suggest that both these assays are rapid and sensitive and results could be achieved within 2–3 hours. Quantitation of the fluorescent signal by fluorometry in a 96-well plate format is in progress.

The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.
Front-Line Chemo-Immunotherapy Rituximab-FC + Rituximab Maintenance in Patients with Untreated Advanced Stage Non Follicular Lymphoma (INFL): Preliminary Results of a Prospective Study of the Intergruppo Italiano Linfomi and Roche (ML18324).
Authors: Baldini Luca , Pulsoni Alessandro , Rossi Giuseppe , Vitolo Umberto , Morra Enrica , Olivero Barbara , Cavalieri Elena , Merli Francesco , Rigacci Luigi , Nobile Francesco , Orsucci Lorella , Brugiatelli Maura , Musto Pellegrino , Deliliers Giorgio Lambertenghi , Gamba Enrica
Published: April 2020

Fludarabine in combination with cyclophosphamide (FC) plus rituximab (R) is an effective treatment for newly diagnosed as well as relapsed low-grade non-Hodgin’s lymphoma (NHL), including chronic lymphocytic leukemia (TAM 2006 and 2008, Keating 2005, Sacchi 2007). Maintenance treatment with R improves overall and progression-free survival in patients both with and without rituximab during induction, with relapsed/resistant follicular NHL (Van Oers 2006). We investigated efficacy and safety of the chemo-immunotherapy FCR followed by rituximab maintenance treatment in patients with advanced untreated indolent B-cell non follicular lymphomas (INFL). Patients and methods: from July 2005 to May 2007, 47 pts whit untreated advanced stage INFL (23 lymphocytic, 20 limphoplasmacytic and 4 nodal marginal zone NHL) were enrolled in an open label, single arm, multicenter phase II study, consisting with 6 courses of FC (Fludarabine, 25 mg/m2 i.v. plus Cyclophosphamide, 250 mg/m2, days 2–4) plus 8 doses of R (375 mg/m2, day 1 every FC cycle and day 14 of cycles 4 and 5) followed by R maintenance (375 mg/m2 every two months for 4 doses). Prophylactic antibiotic treatment with cotrimoxazole (two tablets three times a week) and antifungal profilaxis with itraconazole was planned from the beginning of chemotherapy to three months later or until normalization of CD4 count. The primary endpoint of this study is the percentage of failure free patients after two years from the treatment start. Results: 46/47 pts were evaluable for therapeutic response: median age 59 years (31–68), M/F ratio 28/18; stage III/ IV 44; B symptoms and splenomegaly 11 and 14 pts respectively; FLIPI scores were: 0–1, 16 pts (34.8%), score 2, 19 pts (41.3%) and score ≥ 3, 11 pts (23.9%). Forty-five patients received all the 6 courses of treatment and 1 patient 3 courses only. The ORR was 96% (44/46 pts) with 28 complete (61%) and 16 (35%) partial responses; 2 pts were in MR/SD. A total of 274 courses of FRC were given to 47 patients. The most frequent serious toxicity (NCI-CTC grade 3–4) was neutropenia that was reported in 55% of the courses. Three cases of Herpes zoster infection were reported. All the patients are still alive. Conclusions: in a series of INFL at diagnosis, FCR regimen is safe and effective with a very high CR rate. The whole study will provide insights on the role of R maintenance after R-chemotherapy in a particular subset of low-grade NHLs.
The Role of the P1 Residue of Protease Nexin 2 and Basic Pancreatic Trypsin Inhibitor in the Mechanism of Factor XIa Inhibition.
Authors: Navaneetham Duraiswamy , Sinha Dipali , Walsh Peter N.
Published: April 2020

Factor XIa (FXIa), a plasma serine protease that activates FIX, is regulated by protease nexin 2 (PN2), a Kunitz-type protease inhibitor (KPI) secreted on platelet activation. The Kunitz protease inhibitor domain of protease nexin 2 (PN2KPI) is highly homologous (45% primary sequence identity) to basic pancreatic trypsin inhibitor (BPTI, another Kunitz-type protease inhibitor) and their backbone tertiary structures are nearly identical (J Biol Chem280: 36165, 2005; J Mol Biol230: 919, 2005). However, PN2KPI (Ki 1 nM) is 600-fold more potent as a FXIa inhibitor than bovine basic pancreatic trypsin inhibitor (BPTI; Ki 630 nM). The present study is aimed at examining why these two structurally similar inhibitors are so different in their affinities and specificities in FXIa inhibition and analyzing the mechanisms of FXIa inhibition by these two inhibitors. Reasoning that the P1 residue (Arg15 in PN2KPI and Lys15 in BPTI) might play a crucial role in determining the affinity and specificity for FXIa we expressed BPTI-K15R (mimicking PN2KPI at the P1 site) and PN2KPI-R15K (mimicking BPTI at the P1 site) and examined their inhibitory properties. BPTI-K15R was found to inhibit FXIa with a Ki (10 nM) ~60-fold tighter than BPTI (Ki 630 nM), whereas PN2KPI-R15K inhibited FXIa with a Ki (32 nM) that was 32- fold impaired compared with PN2KPI (Ki 1 nM). Progress curves of peptidyl fluorogenic substrate (Boc-Glu-Ala-Arg-AMC) hydrolysis by FXIa in the presence of all the inhibitors except BPTI clearly demonstrated higher initial rates that continuously decreased until reaching a steady state, typical of slow inhibition. Progress curves obtained using BPTI, on the other hand, were found to be linear at all concentrations of the inhibitor. Analysis using steady state kinetics clearly demonstrated that BPTI is a competitive inhibitor in FXIa hydrolysis of the peptidyl chromogenic substrate, pyro-Glu-Pro-Arg-pNA. Analysis of progress curves using kinetic equations for slow, tight-binding inhibitors confirmed that the formation of the FXIa:PN2KPI complex occurs in a single step that is slow, whereas formation of FXIa:PN2KPI-R15K, FXIa:BPTI and FXIa:BPTI-K15R complexes do not conform to the same kinetic mechanism. Thus PN2KPI and BPTI inhibit FXIa by distinctly different mechanisms and the Arg at the P1 site of PN2KPI confers specificity and high affinity for FXIa inhibition and in part determines the mechanism of PN2KPI as a slow, tight binding inhibitor.
Characteristics and Clinical Outcomes of Adult Aplastic Anemia with Abnormal Cytogenetics at Diagnosis.
Authors: Kim Sung-Yong , Kim Myungshin , Han Kyungja , Lee Seok , Lee Mark Hong , Lee Jong-Wook , Min Woo-Sung , Kim Chun-Choo
Published: April 2020

Background and methods Occasionally, patients with acquired aplastic anemia (AA) present with abnormal cytogenetics in bone marrow cells at diagnosis. The diagnosis and treatment of such patients have not been established and have been center-dependent. We have treated adult AA patients with abnormal cytogenetics in a same way as those with normal cytogenetics. Presently, the characteristics and clinical outcomes of 600 adult AA patients who had successful cytogenetics at diagnosis were retrospectively evaluated. Our aim was to determine the characteristics and clinical courses of AA patients with cytogenetic abnormalities at diagnosis who were treated as those with normal cytogenetics.

Results Characteristics : Of the evaluable patients, 572 (95.3 %) had normal cytogenetics and 28 (4.7 %) had abnormal cytogenetics at diagnosis. The most frequent abnormality was trisomy 8 (n=15) followed by monosomy 7/deletion 7q (n=5) and deletion 1q (n=5). Other chromosome abnormalities were isochromosome 17q (n=1), trisomy 15 (n=1) and monosomy 21 (n=1). There was no significant statistical difference in gender (P=0.562), Hepatitis B or C infection (P=0.402), paroxysmal nocturnal hemoglobinuria (P=0.709) and severity of AA (P=0.325) between patients displaying normal cytogenetics and abnormal cytogenetics. The age of abnormal cytogenetics patients was significantly lower than normal cytogenetics patients (P<0.001).

Immunosuppressive therapy : A total of 334 patients received immunosuppressive therapy using antithymocyte globulin and cyclosporine. Six months after commencement of therapy, 165 (50.9 %) patients responded partially or completely. Multivariate analysis revealed abnormal cytogenetics (HR=0.250; 95% CI=0.077–0.808; P=0.021), absence of paroxysmal nocturnal hemoglobinuria and age (≥ 67) as independent predictors for the poor response to immunosuppressive therapy.

Leukemic transformation : Kaplan-Meier modeling revealed that abnormal cytogenetics was also associated with higher cumulative leukemic transformation rate (P<0.001) and lower leukemia-free survival (P=0.021). Of note, the cause of all deaths in non-severe AA patients with abnormal cytogenetics was leukemic transformation.

Conclusion: Patients with abnormal cytogenetics at diagnosis of AA tend to respond poorly to immunosuppressive therapy and present with a high leukemic transformation risk, which suggests that cytogenetic abnormalities should be emphasized more than morphological features in diagnosis and treatment decision in AA.