bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Acute Inflammation Induced by Prolonged Exercise on Circulating Stem/Progenitor Cells That Mediate Tissue Repair.
Authors: Papassotiriou Ioannis , Spiropoulos Antonia , Tsironi Maria , Skenderi Katerina , Margeli Alexandra , Graphakos Stelios , Baltopoulos Panayiotis , Goussetis Evgenios
Published: April 2020

The “Spartathlon” ultradistance foot race (246 Km continuous, prolonged, brisk exercise for up to 36 hours) provides a unique model of prolonged duration exercise that reveals dramatic systemic inflammatory changes. Endothelial progenitor cells (EPCs) have been shown to participate in vascular repair and angiogenesis, while circulating bone marrow originated fibrocytes represent multipotent cells mediating tissue repair and remodeling after injury. In this study we investigated the effect of this type of exercise on the number of circulating EPCs and fibrocytes along with molecules of endothelium dysfunction and chemotactic proteins in 10 “Spartathlon” athletes before, at the end and at 48 h post race. The EPCs were obtained by culturing peripheral blood mononuclear cells (PBMC) under endothelial cell conditions (EndoCult) and were measured as colony-forming units (CFUs). Circulating fibrocytes were cultured from PBMCs in IMDM medium supplemented with IL-3 and M-CSF and identified as CD45+CD14+CD34lowCollagen-I+ fibroblastic cells. We also determined the plasma levels of E-, L- and P-selectins, sICAM-1, sVCAM-1, thrombomodulin, lipocalin-2, IL-8 and MCP-1 with appropriate methodology. Circulating EPCs increased by nearly ten-fold in peripheral blood at the end of the “Spartathlon” race (from 48±15 cells/ml to 464±36 cells/ml) and they remained increased (420±28 cells/ml) even at 48h post race. The percentage of the CD45+CD14+CD34lowCollagen-I+ fibrocytes cultured from PBMCs before, at the end, and 48 h post race did not reveal any significant difference (64.5±6.2% vs 70.8±8.5% vs 68±4.8% respectively). Plasma levels of lipocalin-2, IL-8, MCP-1, E-selectin, sICAM, sVCAM and thrombomodulin were increased significantly at the end of the race and returned at the pre race levels 48 h post race, while L- and P-selectins remained unaffected before and at the end of the race, presenting a similar decline at 48 h post race. Our study demonstrates that acute inflammatory tissue damage induced by exhausting exercise increases EPCs but not fibrocytes. Given the ability of EPCs to promote angiogenesis and vascular regeneration and the association of fibrocytes with tissue fibrosis after persistent inflammation, we conclude that this kind of repair cell mobilization may serve as a physiologic repair mechanism in acute inflammatory tissue injury.
Effect of Synthetic Peptides Derived from Human Apo(a) on Cell Proliferation, VEGF Production, and Angiogenesis in Cancer Cells and Vascular Endothelial Cells.
Authors: Bao Bin , Prasad Ananda S. , Dou Dex , Jin Steve , Bao Ginny W.
Published: April 2020

Angiogenesis, the formation of new blood vessels from the existing vascular bed, has been described as one of the hallmarks of cancer, and plays an essential role in tumor growth, invasion, and metastasis. The development of specific anti-angiogenic agents is an attractive therapeutic approach for the treatment of cancer. Apolipoprotein, Apo(a), is a large glycoprotein with multiple kringle structures, which play an important role in regulating the intermolecular reaction in the body. The evidence shows that Apo(a) has anti-angiogenic activity on vascular endothelial cells and inhibits tumor growth in mice. The 38th kringle on Apo(a) has been found to be homologous to the kringle 5 of plasminogen, a known endogenous anti-angiogenic kringle structure. According to our preliminary study, the 38th kringle of Apo(a) had an inhibitory effect on bovine capillary endothelial (BCE) cells. Seven peptides of the 38th kringle on Apo(a) were synthesized for screening of anti-angiogenic activity. Two synthetic peptides (P4 and P5) based on the 38th kringle structure sequence of Apo(a) were found to inhibit the proliferation of BCE cells. In the current study, we examined the effects of P4 and P5 synthetic peptides on cell proliferation by MTT assay, and VEGF production by ELIAS assay in human prostate cancer cells (C4-2B, LNCap, and PC3), and/or vascular endothelial cells such as human aortic endothelial cells (HAEC), human umbilical vein endothelial cell (HUVEC), and mouse microvascular endothelial cells (MVEC) after 2 days of incubation. The results are shown in the Table 1 and 2 below.

Table 1. Effect of synthetic peptides on cell proliferation in cancer and vascular endothelial cells (%) P4 P5 Cell proliferation assay was conducted by MTT methos. The % of cell proliferation in peptide treatment groups was compared to the cells without peptide treatment HAEC 64.7±16.4 62.4±12.0 HUVEC 59.4±3.7 71.7±1.2 MVEC 76.9±6.1 75.8±5.6 C4-2B 64.1±5.1 60.9±4.3 LNCap 77.9±2.9 51.8±1.6 PC3 75.8±3.4 75.4±3.6 Table 2. Effect of synthetic peptides on VEGF production in cancer cells (pg/mL) Control P4 P5 C4-2B 905.0±87.7 694.1±99.1 720.1±106.2 LNCap 1116.5±191.6 717.9±100.1 794.3±119.7 PC3 902.6±52.0 743.2±123.7 721.3±92.7

Our data indicate that these synthetic peptides inhibited the cell proliferation and VEGF production. We also examined the effect of P4 and P5 peptides on angiogenesis in HAEC and MVEC cells after 10h of incubation by Matrigel tube formation assay. The data indicate that angiogenesis declined by 40–50% when P4 or P5 was added to the cell culture. All these data suggest that P4 and P5, synthetic peptides derived from the 38th Kringle of Apo(a) have anti-proliferative and anti-angiogenic effects, which may potentially be applicable for cancer treatment. Further studies are in progress in order to define the mechanisms action of these peptides.
TNF-α-Mediated Tumor Promotion Is Characterized by Enhanced Vasculogenesis and Generation of Myeloid/Endothelial Vascular Leukoctyes.
Authors: Li Bin , Pagni Matthew , Cates Justin , Polk D. Brent , Young Pampee P.
Published: April 2020

Whereas in many contexts myeloid cells are cytotoxic, it is well-established that as yet unknown microenvironment cues instruct the infiltrating tumor associated myeloid cells (TAMs) to drive malignant progression and dissemination. Recently, we and others have characterized a significant subpopulation of tumor associated myeloid cells that co-express endothelial and myeloid markers designated “vascular leukocytes”. Studies suggest that vascular leukocytes play an important role in tumor progression and also demonstrate modest contribution to functional vessels, i.e. vasculogenesis, suggesting that they represent a critical tumor-promoting TAM subpopulation. We have identified TNFα as a key regulator of the vascular transdifferentiation of myeloid progenitors in vitro and within the tumor milieu. TNFα at 40ng/ml significantly increased the numbers of flk-1/VE-cadherin dual positive, early outgrowth EPCs from human CD14+ cells by day 7 (about five fold of the control), starting with increased spindle-shaped population appeared as early as day 3. Consistent with this, we observed increased flk-1 expression by ∼9-fold (p<0.05) in cells treated with 40ng/ml TNFα by real time RT-PCR. Transcripts for VE-cadherin and tie2, both endothelial-enriched, were detected by day 3 in cells exposed to 40ng/ml TNFa but not in its absence (control). TNFα-directed upregulation of endothelial markers in mouse monocytes in vitro was dependent on TNFα receptors as monocytes isolated from mice lacking both TNF receptors displayed significantly delayed endothelial marker upregulation. These data suggested that TNF was a component of the molecular pathway that accelerated, but was not required for, endothelial transdifferentiation of murine and human myeloid cells. Enhanced TNFα expression in both B16 murine melanoma and PyV-mT tumor showed local TNFα significantly promoted tumor growth versus control (>5-fold increase for B16 tumor, p=0.04; >8-fold increase for PyV-mT tumor, p<0.01). Both tumor models indicated that overexpressing TNFα caused higher vascular density over control, while tumor necrosis was significantly reduced. Additionally, we observed increased bone marrow-derived vessels (vasculogenesis) in mouse TNFα-overexpressing tumors, which can be specifically inhibited by an anti-TNFα blocking antibody. A significant increase in association of vascular leukoctyes was detected in tumors overexpressing TNFα by FACs, which was abrogated in the mice lacking TNF receptors. Interestingly, TNF-overexpressing tumors did not recruit greater overall numbers of tumor-associated (myeloid or lymphoid) leukocytes, suggesting a specific role in myeloid to endothelial transdifferentiation in vivo. Our studies suggest that TNFα constitutes part of the microenvironment repertoire that biases recruited myeloid cells towards a proangiogenic/provasculogenic phenotype.
Piperacillin Induced Thrombocytopenia in Critical Care Patients as Compared to Non Critical Patients.
Authors: Naik Seema , Sangal Ashish , Kodali Srinivas , Dumlao Theresa , Kalavar Madhumati , Steier William , He Zili
Published: April 2020

Introduction: Drug-induced thrombocytopenia in the critically ill is documented in the literature with limited frequency. Implicated agents are antiepileptics, anticoagulants, antiarrhythmics, histamine receptor antagonists, and antimicrobials .Piperacillin has only rarely been implicated as a cause of thrombocytopenia, and there is only limited evidence that this complication is caused by immune mechanisms.

Methods: We conducted a retrospective chart review of patients admitted during a 6-month period to determine the frequency of and potential risk factors associated with thrombocytopenia, and the association of acquired thrombocytopenia with the use of piperacillin ICU versus non ICU patients.

Results: Data were collected for 154 patients (M:71, F:83),), admitted to the ICU and Non-ICU setting. 55% of the patients were more than 65 years of age. Platelet drop occurred in 123 patients. Thrombocytopenic patients had a longer ICU stay (11.2 vs 5.6 days) and greater mortality (9% vs 3%) than nonthrombocytopenic patients .The patients developed thrombocytopenia as follows:Mild(100–150k): 22 pts-(15%) ; Moderate(50–100k): 7 pts-(5%) ;and Severe(<50k): 4 pts- (3%). The other drugs during hospitalization were heparin (30%), ceftazidime(11%), famotidine(9%), bactrim(0.3%),esomeprazole (11%), aspirin(18%). Patients received piperacillin for mean duration of 9 days. In patients with thrombocytopenia, the platelet levels dropped by a mean of 15.15% on Day 2 from pretreatment values (range, 2% to 61%) while they were receiving piperacillin. On average, the nadir platelet count was reached 7 days after treatment with piperacillin was initiated (range, 2 to14days). The median time required for platelet recovery was 8.4 days (range 5.6 in ICU to 11.2 in Non-ICU pts).Bleeding was unusual in patients with thrombocytopenia. Platelet transfusions failed to elevate platelet counts.

Discussion: Thrombocytopenia occurs in 23–63% of patients in the intensive care unit (ICU). Factors associated with the disorder in these patients include sepsis, disseminated intravascular coagulation (DIC), blood transfusions, central catheters, and drugs, some of which may have a significant effect on morbidity and mortality. The major mechanisms of drug-induced thrombocytopenia are immune-mediated platelet destruction and concentration-dependent bone marrow suppression. Bone marrow toxicity, mainly neutropenia, has been described as an uncommon secondary effect of most beta-lactams, and it is usually related to large cumulative doses.

Conclusion: Thrombocytopenia is associated with an increased ICU stay and increased mortality. Thrombocytopenic events associated with antimicrobial agents in the ICU may warrant further investigation because of the increased administration of these drugs to combat more virulent bacterial strains.
Salmonella Arizonae Bacteremia Associated with Septic Arthritis Following Consumption of Rattlesnake Capsules in a Patient with Idiopathic Thrombocytopenic Purpura (ITP) on Chronic Steroid Therapy.
Authors: Herrada Juan , Antony Suresh J.
Published: April 2020

Background: Folk medicine plays an important role in some populations in the United States. In particular, the use of rattle snake powder is becoming increasingly popular among the Mexican-American population along the US-Mexico border. Infections associated with the ingestion of this preparation have increasingly been reported in this country. We present a patient who presented with Salmonella Arizonae bacteremia and septic arthritis with underlying ITP on chronic treatment with steroids and surreptitious use of rattle snake powder.

Case Report: A 53 year-old Mexican -American male with a past history of ITP on chronic oral prednisone with intact spleen was admitted with fever, chills, and an acute onset of pain, swelling and tenderness of his left knee. On admission to the hospital, his temperature was 101.5 F, blood pressure was 124/68, and pulse 102/min. The physical findings were significant for swelling and tenderness to the left knee with evidence of synovial effusion and restricted range of motion. The white blood cell count was 18,000cu/mm. Culture of the patient’s left knee synovial fluid aspirate grew Salmonella Arizonae, as did his blood cultures. The pathogen was sensitive to 3rd generation cephalosporins and quinolones, and he was treated with intravenous ceftriaxone for 2 weeks with resolution of the bacteremia and improvement of the septic knee. He subsequently underwent a total knee arthroplasty, and follow up 1 year later revealed no evidence of persistent infection. After the pathogen identification, a careful alternative medicine history revealed the consumption of rattle snake powder preparation intended for treatment of his arthritis and ITP.

Conclusion: Physicians should be aware of Salmonella Arizonae associated illnesses in both immunocompetent as well as immunosuppressed patients. Because the surge of interest in complementary and alternative medicine, a careful history should be elucidated, especially in immunocompromised patients who present with this syndrome.
Heparin-Induced Thrombocytopenia (HIT): A Rural Community Based Experience.
Authors: Mullai N. , Brock Amanda , Harper Shona
Published: April 2020

Background: Heparin-induced thrombocytopenia (HIT) is a known complication of heparin therapy. This study was planned to assess the experience of a community based medical practice with HIT in a rural setting.

Method: A retrospective study was done from medical records of patients suspected clinically of HIT from January 2006 to January 2007. The data were analyzed with regard to test results of patients, especially those who were positive for the HIT antibody and correlated with national statistics.

Result: Fifty-two (52) patients were suspected clinically of having HIT during the study period. All 52 patients received heparin and most of them had cardiac surgery before the onset of thrombocytopenia. Six out of fifty-two (6/52) patients were found to have positive HIT antibody. Two out of six (2/6) also had positive serotonin release assay. Two out of six (2/6) developed heparin-induced thrombocytopenia with thrombosis (HITT). One of the two patients with HITT died of complications. The range of time to obtain test results was 5–7 days. Four out of fifty-two (4/52) patients received thrombin inhibitor lepirudin (Refludan) as alternate anticoagulation.

Conclusion: The overall incidences, time of onset, relation to heparin treatment were similar to that of national averages. The time to obtain diagnostic test results ranged 5–7 days and heparin was withheld in all of them, and more expensive anticoagulation was used for some of them while waiting for the test results. This dilemma in diagnosis and treatment could be avoided if a rapid test that can help to assess the risk early in about 12–24 hrs, is possible. Such a test would be very beneficial especially in small, rural community settings where the availability of expensive testing and medications for HIT are limited.
The Correlation Analysis of Venous Thromboembolism and Acute Myocardial Infarction with the Levels and Polymorphisms of Coagulation and Anticoagulation Factors.
Authors: Yang Linhua , Han Xuanmao , Qiao Zhenhua , Ren Jinfang , Liu Xiue , Hou Lihong , Wang Xuefeng , Ding Qiulan
Published: April 2020

To evaluate levels of coagulation /anticoagulation factors and distribution of their gene polymorphisms in patients with venous thromboembolism(VTE) and acute myocardial infarction(AMI). Of 95 VTE, 95 AMI patients and 95 normal controls were be studied. The results showed that the levels of fibrinogen and D-Dimer, the activies of FII, FV and FVIII were remarkably higher in patient with VTE than those in healthy group, and the activities of FIX, PC and AT were of no significant difference between VTE group and healthy group. The mean levels of fibrinogen, D-Dimer, FV:C and FVIII:C were significantly higher in AMI group than that in controls; while, no significant difference of FII:C, FIX:C, PC:A and AT:A between AMI patients and the controls. In controls, -148CT + -148TT was associated with higher fibrinogen concentrations, the genotypes of other gene polymorphisms were not associated with levels of coagulation /anticoagulation factors. Clear difference were observed for the T allele frequencies of -148C/T and distribution frequencies of FII A19911G were found between VTE group and normal group. there was significant difference of -1476TT genotype distributions and T allele frequencies in -1476A/T between VTE and control subjects. There was significant difference of AC+CC genotype distribution in 1298 A/C polymorphisms between AMI and control, and it was so with the GA genotype in 1793 G/A polymorphisms. But neither genotype frequencies nor allele frequencies of other polymorphisms were found to be no of significant difference between VTE /AMI group and control group. This study proved the importance of combined functions of several coagulant factors in causing VTE and AMI. The carriage of the C148T mutation, FII 20210G/A and -1476A/T polymorphism might be associated with an increased risk of VTE, while the MTHFR 1298A/C and 1793G/A polymorphisms may be related to the development of AMI. Only some of joint occurrence of above polymorphisms might increase the occurrence rate of VTE or AMI.
A Highly Accurate and Linear Clauss Fibrinogen Assay Provides Increased Clinical Value for Patient Reporting throughout the 30 – 1000 mg/dL Medical Decision-Making Range.
Authors: Callahan Janet , Laurant Nathalie , Arcidiacono Sam , Polgar Janos , Mary Doyle , Triscott Mark
Published: April 2020

The availability of accurate and precise Fibrinogen concentration measurements throughout low, normal and elevated ranges are of significant utility in clinical practice. The assessment of elevated fibrinogen levels is valuable as a known marker of coronary heart disease, cerebrovascular disease, cancer, diabetes, hypertension, acute phase reactions, pregnancy, etc. Likewise, the decreased fibrinogen levels associated with liver disease, DIC, thrombolytic therapy, miscarriage, poor wound healing and intracranial hemorrhage support the premise that accurate quantification of very low levels of fibrinogen is needed. The Hemosil TM Fibrinogen-C XC Clauss Fibrinogen (FibC) assay measurement range for Instrumentation Laboratory (IL) ACL TOP family (ACL TOP/CTS) analyzers has been expanded to 30–1000 mg/dL. This assay has the capability of accurately measuring plasma fibrinogen in three mode segments: Near Normal for the 100–500mg/dL range, High Fib for samples 500 to 1000mg/dL and Low Fib for samples less than 100mg/dL. The accuracy of the expanded Fibrinogen assay was assured by calibration with the 2nd International Standard for Fibrinogen calibrator, plasma 98/612 and verification with the SCC/ISTH Secondary Coagulation Standard Lot # 3 as well as a 100 donor normal pool. Precision is consistent across the entire range. Normal, low and high samples demonstrated Total CVs of < 7%. The linearity of values across the expanded Fib C measurement range was established using a statistical evaluation based on CLSI EP6-A: Evaluation of the Linearity of Quantitative Measurement Procedures. Overlapping linearity of the 3 mode segments was demonstrated for Low Fib from 30–150 mg/dL, for Fib C from 85–500 mg/dL, for High Fib from 200 –1000 mg/dL as well as for the overall range of 30–1000 mg/dL. As noted, recovered values from Low Fib to Fib C and from Fib C to High Fib overlap when the tests are evaluated individually, but in practice, this Fib C assay will reflex seamlessly to the appropriate low or high test range. Deviation from Linearity, a statistical measure of goodness of fit, was low, less than 7% for 9 equidistant points between 30 and 1100 mg/dL. Linearity and accuracy were further confirmed in an extensive and statistically rigorous CLSI EP9 method comparison study of 200 samples representing a wide variety of clinical disorders Additional studies investigated interferences and other sample specific conditions. Measurements using reflexive modes to extend the Fibrinogen measurable range deliver timely, precise and accurate information to the practicing clinician over the wide diagnostic range of 30–1000 mg/dL.
Global Analysis of Age-Related Expression Profiles of Mouse Liver Genes: Toward Understanding Age-Related Homeostasis.
Authors: Kurachi Sumiko , Suenaga Emi , Kurachi Kotoku
Published: April 2020

For understanding aging as well as various age-related diseases, it is critical to establish the basic regulatory mechanisms underlying age-related homeostatsis. Through systematic analyses of transgenic mice carrying factor IX and protein C genes, we recently discovered the first molecular mechanism of age-related homeostasis, ASE/AIE-mediated age-related regulatory mechanism of gene expression (Kurachi et. al., Science 1999; Zhang et. al., J. Biol. Chem. 2002; J. Thromb. Haemost. 2005). This laid the foundation for further studies toward an integrated understanding of age-related homeostasis. Here we report global analyses of age-related expression profiles of mouse liver genes. For quantification of expression levels of liver genes of mice at 1, 3, 6, 12, 18 and 24 month of age, Affymetrix GeneChip® Mouse Expression Array 430A (MOE430A with 23,643 probes) were used. These microarray analyses detected 9148 probes as qualified meaningful and the data were subjected to extensive analyses by GeneSpring and IPA network analysis softwares. Real time PCR analyses of representative genes were performed for verifying excellent reproducibility of age-related expression profiles determined by microarray analyses. Age-related stages, particularly of puberty and aging, were identified as the major phases for age-related changes in gene expression. Strong relationships between expression level and ontology, indicating that the highest expression gene group, next highest expression group and lowest level expression gene group are of secreted proteins, mitochondoreal proteins and nuclear regulatory protein, respectively. Most importantly, we successfully identified about a dozen unique and fundamental age-related gene expression patterns including those of age-related stable, gradual increase or decrease, and aging-related dramatic increase or decrease types of expression. These findings led us to a new hypothesis that complex and dynamic age-related regulations of genes are produced by a relatively small number of fundamental regulatory mechanisms. These mechanisms may function independently or in various combinations, thus explaining an essential feature of age-related homeostasis.
Deletion of Pro1648-Leu1650 in VWF73, the Conservative Substrate for ADAMTS13, Causes von Willebrand Disease.
Authors: Su Jian , Bai Xia , Yu Ziqiang , Wang Zhaoyue , Ruan Changgeng
Published: April 2020

The multimer distribution of Von Willebrand factor (VWF) in plasma is regulated by the specific VWF cleaving protease ADAMTS13, which cleaves at the Y1605-M1606 bond in the A2 domain of VWF under the shear stress, plays paramount roles in mediating platelet adhesion to the subendothelium during vascular damage. Quantitative deficiency or qualitative abnormity in VWF caused by the mutations in the VWF gene leads to von Willebrand disease (vWD). There exist three types of vWD. Type 1 vWD is characterized by the partial quantitative deficiency of VWF and normal multimers. Type 3 refers to complete deficiency of VWF. Type 2 vWD refers to the qualitative deficiency of VWF and is subdivided into types of 2A, 2B, 2M, 2N. Meanwhile, the subtype of 2A vWD is also subdivided into two groups regarding ADAMTS13-dependent proteolysis of VWF. Group I includes the mutations G1505R, S1506L, L1540P, V1607D, which hinder the multimer assembly and diminish the secretion of VWF while group II includes R1597W, R1597Q, G1505E, I1628T, E1628K, which make VWF more susceptible to ADAMTS13 -dependent proteolysis. All these published point mutations cluster in the A2 domain of VWF and the corresponding mutation mechanism upon VWF has been elucidated. We have identified a patient with bleeding symptoms and reduced plasma VWF antigen, factor VIII and ristocetin cofactor activity, compatible with clinical von Willebrand disease. Analysis of proband’s plasma VWF multimers in low resolution agarose gels demonstrated similar results compared to the healthy. The patient carried a heterozygous deletion mutation from position 1648 to 1650 resulting in loss of three consecutive amino acids (ProIleLeu) in the pre-pro-VWF. It has been demonstrated that the minimal substrate for ADAMTS13 is intact VWF73, a region from Asp1596 to Arg1668 of von Willebrand factor. The novel deletion mutation in this patient occurred in the intact VWF73 and its mutated effect upon cleavage by ADAMTS13 could be clarified by further experiments such as in vitro recombinant expression of mutated VWF and might strengthen our understanding of the interaction between VWF and ADAMTS13.
Case Report: A Patient with Haemorrhagic Bleeding, an Acquired Factor VIII Inhibitor and Massive Pulmonary Embolism.
Authors: Hildebrandt Martin , Richter Stephan , Schuler Markus , Ludwig Wolf-Dieter
Published: April 2020

A female, 48-year old patient was referred to an emergency ward with dyspnoea, haemoptysis and suspected respiratory infection that had been refractory to prior antimicrobial therapy. The onset of dyspnoea dated back to a febrile episode three months prior to presentation. The patient continued to work in spite of severe shortness of breath. Her history revealed a cervical cancer which had been treated by neoadjuvant radiochemotherapy and extended hysterectomy in 2005. Upon admission, extensive spontaneous haematomas were noted on the upper extremities. Following a diagnostic puncture of the radial artery, severe haemorrhage and compartmentalization developed, and the patient was transferred to our hospital for a surgical emergency intervention and haemostaseological counselling. A decreased factor VIII activity was noted, with a prolonged partial thromboplastin time, a normal von Willebrand Factor activity and concentration, a prolonged bleeding time and a low-titer inhibitor to factor VIII. Additionally, a profound hyponatremia (117 mmol/l) awaited explanation. Haemorrhage was treated with recombinant Factor VIIa, allowing for a successful surgical de-compartmentalization. With regard to the consistent feature of dyspnoea and partial respiratory insufficiency, bronchoscopic examination revealed only minor hemorrhagic suffusion of the bronchial epithelium, but no sign of infection or major pulmonary bleeding. Chest X-ray and laboratory findings showed right ventricular enlargement, bilateral infiltrates and an excessively high NT-pro-BNP concentration in plasma (13027 ng/l). Chest X-ray radiographs performed two months previously for suspected respiratory infection showed pulmonary infiltrates of comparable size, albeit no right ventricular enlargement. Computerized tomography of the chest showed a complete obstruction of the right pulmonary artery and of the lower branch of the left pulmonary artery. Pulmonary arterial pressure was measured at 70 mm Hg. Immunosuppressive therapy was initiated, with concomitant aPTT-adjusted anticoagulation. Upon clinical deterioration with right-ventricular failure, a catheter-based disruption of the emboli was attempted, albeit with no success. A cardio-surgical intervention with thrombendectomy was performed successfully; however, the patient succumbed to sepsis and multiorgan failure ten days later. The autopsy revealed multiple thrombi, a bilateral pulmonary haemorrhagic infarction and a disseminated lymphangiosis and hemangiosis carcinomatosa of the lungs. We conclude that the acquired inhibitor to factor VIII was of paraneoplastic origin and, although detected at a low titre, clinically relevant. Based on previous radiographic findings, a massive pulmonary embolism must be assumed to have occurred months prior to presentation. The anticoagulatory effect of the acquired inhibitor to Factor VIII may have averted the timely detection of the pulmonary embolism.
Amyloidosis Presenting with Platelet Dysfunction and Recurrent Retroperitoneal Hemorrhage (RH).
Authors: Rezazadeh Arash , Laber Damian A. , Sharma Vivek R. , Kloecker Goetz H.
Published: April 2020

CASE REPORT: A 59-year-old white female with history of easy bruising for 2 years and 2 episode of RH in the last 6 month admitted to ICU with her 3rd episode of RH. Her previous w/o included a BM Bx which had shown clonal population of plasma cells kappa chain restrictive (7%) with repeatedly negative SPEP and UPEP. PFA-100 was consistent with severe platelet dysfunction. During the ICU admission platelet transfusion with multiple single donor units did not correct her PFA-100 results, which were confirmed with platelet aggregation results. Both, collagen/epinephrine and collagen/ADP closure times were prolonged (>300 and 128 seconds respectively). The patient however did not receive any medications to explain this apparently intrinsic dysfunction. Coagulation studies including PT, aPTT and factor II, VII, VIII, IX all were within normal limit. Factor X levels were slightly decreased (45%). Patient had selective embolization of the fourth lobar artery of the right kidney. Plasma cells in the marrow showed monoclonal expression for cytoplasmic kappa light chains by both IHC and in situ hybridization. Amorphous pink material was present in the walls of blood vessels. Plasma cells stained positively with CD138. The amyloid material stained positively with Congo red and demonstrated apple green birefringence under polarized light. Skeletal survey demonstrated multiple lytic lesions. Serum free light chain measurement showed high levels of M protein (κ) with κ to λ ration of 4.30. Patient was started on treatment with pulse dose steroid and bortezomib.

DISCUSSION: Amyloidosis can cause hemorrhage with multiple mechanisms including amyloid deposition (amyloid angiopathy), platelet dysfunction, coagulation factor deficiencies, abnormal fibrin polymerization and hyperfibrinolysis. Consumption of factor X by amyloid is often called a culprit for hemorrhage in amyloidosis. The slightly decreased level of factor X in this case provided a valuable clue for diagnosis, but was not likely a cause of bleeding in this patient. Amyloid induced platelet dysfunction along with amyloid angiopathy lead to life threatening hemorrhage in our patient. The mechanism of this platelet dysfunction, which also affected the transfused platelets, is not well explained. This effect on platelets complicates the clinical management. The case also illustrates the value of the free light chain assay, which is more sensitive than SPEP and UPEP. These commonly used tests to diagnose plasma cell dyscrasias are reportedly false negative in 3% of cases.

CONCLUSION: Plasma cell dyscrasia has a wide variety of presentation and should be considered as a part of bleeding work up in complex cases.
Phase II Trial of Oral Panobinostat (LBH589) in Patients with Refractory Cutaneous T-Cell Lymphoma (CTCL).
Authors: Duvic Madeleine , Becker Jürgen C. , Dalle Stephane , Vanaclocha Francisco , Bernengo Maria Grazia , Lebbé Celeste , Dummer Reinhard , Hirawat Samit , Zhang Lei , Marshood Miriam , Laird Glen , Prince H. Miles
Published: April 2020

Background: Panobinostat is a histone deacetylase inhibitor that has shown promising results in cutaneous T-cell lymphoma (CTCL).

Objectives: An open-label, multicenter, Phase II study is being conducted with a primary objective of establishing the efficacy and safety of the pan-deacetylase inhibitor, panobinostat (LBH589), for patients (pts) with relapsed/refractory CTCL with Stage IB–IVA mycosis fungoides (MF) or Sézary syndrome (SS).

Methods: Inclusion criteria include adequate organ function, no clinically significant cardiovascular abnormalities (QTcF ≤ 450 ms, ECOG PS ≤2), failure of ≥2 prior systemic therapies, and no prior HDAC inhibitor treatment. Pts were grouped as having bexarotene therapy (Group [Gr] 1) or bexarotene naïve (Gr 2). Panobinostat was administered at a dose of 20 mg orally on Days 1, 3, 5, weekly, every 28 days until progression or unacceptable toxicity. Response was based on a composite score, including skin assessment with the modified Severity-Weighted Assessment Tool (mSWAT) and systemic disease assessed by CT scan.

Results: To date, 95 pts (Gr 1=62; Gr 2=33) have enrolled with median age of 58 yrs [range 25–88]: 58 male, 37 female; 70 MF, 25 SS. Median prior treatment regimens are 4 and 3 for Gr 1 and 2, respectively. Most pts were ≥Stage IIB at study entry (Gr 1=42; Gr 2=26) and received 1–17+ (median=3) treatment cycles of panobinostat. In Gr 1, 11/62 pts have had confirmed skin responses by SWAT, including 2 complete skin responses. Confirmatory CT scans are pending for 2 patients. In Gr 2, 4/33 pts had confirmed skin and CT scan responses. Common AEs (>20%; all grades, regardless of causality) included diarrhea, thrombocytopenia, nausea, pruritus, fatigue, and asthenia, and Grade 3/4 AEs (>2%, regardless of causality) included thrombocytopenia, neutropenia, pruritus, diarrhea, and hypophosphatemia. Of 4,542 ECGs analyzed, 2 pts have had QTcF >480 ms; 4 had QTcF >60 ms increase from baseline.

Conclusions: Panobinostat continues to demonstrate encouraging clinical activity with a manageable safety profile in pts with CTCL. Per predefined criteria, Group 2 enrollment to Stage 2 is open. Updated efficacy and safety data will be presented.
De-Immunization of Human Factor VIII: Identification of Epitopes in the C2 Domain
Authors: Moise Leonard , Skupsky Jonathan , Tassone Ryan , McMurry Julie A , Martin William D , De Groot Anne S. , Scott David W.
Published: April 2020

Most immune responses to protein antigens are dependent on T-cell recognition of discrete peptide epitopes presented in an MHC groove. The pattern of peptide recognition can be predicted from the primary structure of a given protein based on residues that bind (anchor) to a given HLA phenotype. Algorithms such as EpiMatrix can be applied to identify such epitopes and measure the potential immunogenicity of proteins based on epitope content. One approach to reduce immunogenicity is to generate recombinant proteins whose constituent epitopes have been modified so as to reduce their HLA binding. Modification of the sequence can be performed in silico using algorithms such as OptiMatrix. This “de-immunization” method has been used effectively with a number of therapeutic proteins already in use in clinical trials. It may therefore also be possible to target those residues of fVIII that, while contributing to HLA binding, can be mutated without altering the functional ability of fVIII to initiate clotting. We have begun this de-immunization process with the fVIII, starting with C2 domain because C2 has been confirmed to be a major target of both T cells and inhibitory antibodies. Using EpiMatrix, we selected 10 peptides in human fVIII that would be predicted to bind to eight class II HLA DR molecules that encompass over 95% of the U.S. population. These epitopes were synthesized and eight of the ten were assayed in vitro and found to bind to HLA at IC50 <100μM. Immunization of fVIII knockout mice with whole fVIII or with constituent fVIII peptide epitopes resulted in significant T cell proliferation to the peptide epitopes as measured by thymidine incorporation assays. By contrast, homologous peptide epitopes modified by 1–2 residues elicited significantly lower levels of proliferation. These preliminary results point to the feasibility of generating a de-immunized fVIII molecule with reduced immunogenicity; validation of these results is planned. If such proteins retain even partial activity to initiate clotting, then they would become useful in treating hemophilia A patients to avoid inhibitor formation. (Supported by NIH R43 HL088834-01)
Dual Effect of Platelet Factor Four on the Activities of Factor Xa
Authors: Fiore Martine Marie , Mackie Ian J
Published: April 2020

Platelet Factor 4 (PF4) is a cationic molecule that binds to heparin with high affinity and neutralises the activity of the latter. Our recent studies indicate that heparin can promote an interaction of fXa with PF4 since neutralization of heparin activity by PF4 was dependent on the concentration of protease. To examine the contribution of PF4 in protease function, fXa activity was determined in chromogenic assays. Upon preincubation with fXa and heparin, PF4 (at a concentration of 100 nM) decreased the kcat of S2765 peptide hydrolysis 4-fold and that of prothrombin activation about 2-fold. These results suggested an effect of PF4 on the primary specificity of the protease. In fact, PF4 exerted a mild effect (30 % decrease) on the Na+ dependence of fXa, consistent with linkage between Na+ and S1. PF4 preincubation with fXa also prevented the binding of the S1 probe p-aminobenzamidine (pAB) while simultaneous addition of PF4 and pAB diminished the contribution of PF4. In the presence of excess fVa (relative to fXa), kinetic parameters measuring fXa amidolytic activity in the presence of PF4 were restored to control values in the absence of PF4. Interestingly, high concentrations of PF4 (> 1 μM) totally restored fXa activity toward peptidyl substrate and strongly enhanced prothrombin activation, indicating a dual effect of PF4 on fXa activities. The inhibitory contribution of PF4 during prothrombin activation was due to a three-fold decreased affinity of fXa for fVa while enhancement of prothrombin activation was accompanied by a three-fold increase in fVa-dependent cofactor activity. Thus, the effects of PF4 possibly involved a region of the heparin/fVabinding exosite that is linked to the S1 and Na+ sites. These findings suggest that PF4 is a probe of fVa-dependent changes occurring in the active site of fXa and provide an explanation for the in vivo paradoxical effects of PF4 reported in the literature.
Antisense Transcripts Negatively Regulate Transcription of Multiple Variegated Killer Immunoglobulin-Like Receptor (KIR) Genes
Authors: Cichocki Frank , Lenvik Todd , Anderson Stephen K , Miller Jeffrey S.
Published: April 2020

Natural killer cells are CD3 negative large granular lymphocytes that lyse virally infected and malignantly transformed targets. NK cell functions are regulated by an array of inhibitory and activating receptors, including members of the killer immunoglobulin-like receptor (KIR) family. Human KIR genes are expressed in a variegated and clonally restricted manner on the surface of mature NK cells. While it is well established that individual KIR gene expression is strongly correlated with the DNA methylation status of CpG dinucleotides within the promoter region proximal to the transcriptional start site, the mechanisms that regulate variegated KIR expression are largely unknown. Our goal is to uncover the genetic mechanisms governing KIR transcriptional regulation. We have recently identified an active distal promoter element approximately 1 Kb upstream of the transcriptional start site. This region contains c-MYC binding sites, and KIR expression is increased when MYC is overexpressed in developing NK cells or when c-MYC is physiologically increased by IL-15 activation. Bi-directional promoter activity is found within the previously characterized proximal promoter of all KIR genes. Therefore, double-stranded RNA with homology to the KIR promoter can be generated within this non-coding region. The generation of double-stranded RNA has recently been shown to contribute to promoter DNA methylation of the p15 gene in mammalian cells through a Dicer-independent mechanism. Using quantitative real-time PCR, we found that purified peripheral blood CD56+/KIR3DL1− NK cells express 5-fold more KIR3DL1 antisense transcript than purified CD56+/KIR3DL1+ cells. Therefore, the transcriptional activation of the KIR3DL1 gene correlates with a significant decrease in KIR3DL1 antisense expression. To explore the possibility that KIR antisense transcripts can silence KIR expression, we over-expressed the KIR3DL1 antisense transcript in CD34+ hematopoietic precursor cells and differentiated these cells into NK cells in vitro. After 21 days in culture, we assayed KIR3DL1 mRNA levels using quantitative real-time PCR. KIR3DL1 expression was reduced 4-fold compared to eGFP control cultures, further supporting the hypothesis that antisense transcripts negatively regulate KIR expression. Importantly, expression of the KIR2DL4 gene, which does not contain a promoter with significant homology to KIR3DL1 promoter, was not affected by KIR3DL1 antisense overexpression. This provides the first evidence for gene silencing through double-stranded RNA in the immune system and may be the key mechanism for the establishment of DNA promoter methylation within the KIR locus. Understanding the mechanisms of KIR expression, a requisite for NK cell education/licensing, may allow us to understand the acquisition of effector function and manipulate it for therapeutic benefit.
Beta-catenin Is Essential for Survival of Leukemia Stem Cells Insensitive to Kinase Inhibition in Mice with BCR-ABL Induced Chronic Myeloid Leukemia.
Authors: Li Shaoguang , Hu Yiguo , Chen Yaoyu , Douglas Lori
Published: April 2020

Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) induced by the BCR-ABL oncogene is believed to be developed from leukemic stem cells (LSCs), and we have previously shown in mice that LSCs for CML express the same cell surface markers that are also expressed on normal hematopoietic stem cells. Although the inhibition of BCR-ABL kinase activity by imatinib is highly effective in treating human Ph+ CML in chronic phase, it is difficult to achieve molecular remission of the disease, suggesting that LSCs remain in patients. In this study, we find that following imatinib treatment, LSCs not only remained but also accumulated increasingly in bone marrow of CML mice. This insensitivity of LSCs to imatinib was not due to the lack of BCR-ABL kinase inhibition by imatinib, and proliferating leukemic cells derived from LSCs were still sensitive to growth inhibition by imatinib. These results identify a LSC survival pathway that is not inhibited by imatinib. Furthermore, we show that b-catenin in the Wnt signaling pathway is essential for survival and self-renewal of LSCs, providing a new strategy for targeting these cells.
Arrestin-2 Differentially Regulates PAR4 and P2Y12 Receptor Signaling in Platelets
Authors: Li Dongjun , Woulfe Donna
Published: April 2020

Arrestins play important roles in the function of G Protein-Coupled Receptors (GPCRs) in many cells, but their roles in platelets remain uncharacterized. While the classical role of arrestins is considered to be the internalization and desensitization of GPCRs, more recent studies suggest that arrestins can serve as molecular scaffolds to recruit phosphatidyl inositol-3 kinases (PI3Ks) to GPCRs and promote PI3K-dependent signaling. Due to the multifunctional role of arrestins, we sought to determine whether arrestins regulate Akt activation in platelets and thrombosis in living animals. Co-immunoprecipitation experiments indicate that arrestin-2 associates with PAR4 in thrombin-treated platelets and P2Y12 in ADP-treated platelets, but neither receptor in resting cells. Interestingly, association of arrestin-2 with PAR4 was also stimulated by ADP and PAR4-induced association of arrestin with PAR4 was inhibited by P2Y12 antagonists or apyrase. To determine the functional role of arrestin-2 in platelets, ADP- and thrombin receptor-stimulated Akt phosphorylation was compared in platelets from arrestin-2 knock-out versus WT mice. Akt phosphorylation stimulated by 0.8 mM AYPGKF PAR4 agonist peptide was reduced by an average of 77% in arrestin-2 knock-out platelets compared to WT controls (significantly different, p=0.007, n=3 in each group), but ADP-stimulated Akt phosphorylation was unaffected (p=.38, n=3 each). PAR4-stimulated fibrinogen binding was also reduced in arrestin2−/− platelets (by 58.5% in 1 mM AYPGKF-stimulated platelets compared to WT controls), whereas ADP-stimulated fibrinogen binding was not. Finally, arrestin2 knock-out mice were less sensitive to ferric chloride-induced thrombosis than WT mice: 55% of WT mice (n-=9) formed occlusive thrombi after 2min15sec exposure of the carotid artery to 10% ferric chloride, whereas only 11% of WT mice (n=9) formed occlusive thrombi under the same conditions. In conclusion, arrestin-2 associates with both PAR4 and P2Y12 receptors, but differentially regulates their signaling to Akt and fibrinogen binding and appears to play a net positive role in regulating thrombosis in vivo.
A Comprehensive Lymphocyte Analysis of Dasatinib Treated Chronic Myelogenous Leukemia Patients Reveals T-Cell Oligoclonality.
Authors: Powers John J. , Painter Jeffrey S. , Dubovsky Jason A , Epling-Burnette P.K. , Pinilla Javier
Published: April 2020

On the forefront of targeted cancer therapy are second generation tyrosine kinase inhibitors (TKIs) which impact the outcome of chronic, accelerated, and blast phase chronic myelogenous leukemia (CML). Among these TKIs dasatinib is particularly effective for the treatment of imatinib-resistant Philadelphia chromosome-positive CML; although this drug has been associated with the generation of pleural effusions, fevers, and colitis in some patients. Recent data links these side effects to oligoclonal expansions of large granular lymphocyte (LGL) immunophenotype. To further characterize these events we conducted a comprehensive immunophenotye, T-cell receptor variable region, KIR, and HLA analysis of seven CML patients receiving dasatinib, two of which developed adverse reactions in association with lymphocytosis. Flow cytometric analysis confirmed these proliferative events and elucidated a previously undescribed CD3-CD8+CD56+ population. In addition, we identified multiple patients with decreased CD4 to CD8 T-cell ratios and a shift in CD4+ and CD8+ lymphocyte populations towards an effector and terminal memory phenotype (CD62L-CD45RA-and CD62L-CD45RA+ respectively). Further analysis of the T-cell receptor beta chain showed that all seven patients had oligoclonal populations of either CD4 or CD8 T-cells significantly greater than that of healthy donors. Most of these findings were independent of the occurrence of adverse reactions. All in all our data implicates dasatinib in generating oligoclonal lymphocyte expansions of various phenotypes in all CML patients. It is still unclear as to the underlying molecular mechanisms inducing these expansions, but further exploration could help to prevent or alleviate these reactions and could conceivably provide new tools for future directed immunotherapy.