bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Mesenchymal Stem Cells on the Viability, Proliferation and Differentiation of B Lymphocytes.
Authors: Tabera Soraya , Perez-Simon Jose A. , Diez-Campelo Maria , Sanchez-Abarca Luis I. , Blanco Belen , Benito Ana , Ocio Enrique , Sanchez-Guijo Fermin M. , Canizo Consuelo , Miguel J.F. San
Published: April 2020

Mesenchymal stem cells (MSC) are multilineage non hematopoietic progenitor cells that play a key role in supporting the lymphohematopoietic system. Their distribution in bone marrow and secondary lymphoid organs allows an intimate interaction with T and B-lymphocytes, which contribute to the normal lymph node development, but this interaction, can not be considered as a simple bi-directional cross-talk and other cell subsets, such as dendritic cells (DC), must be considered. We have analysed the effect of MSC on B-lymphocytes and the pathways involved in these effects. For these propose, we cultured B-cell with or without MSC and analysed different markers involved in the differentiation of B-cells. We found that MSC inhibited proliferation, arresting B lymphocytes in G0G1 phase of cell cycle (figure 1). However, the presence of MSC increased the viability ob B-lymphocytes (double number of viable B-cells: from 13012 to 22835 Annexin-V PE/7 ADD negative events within B-lymphocytes in absence versus presence of MSC). The exposure of B-cells to plasmocytoid DC (pDC) induced B-lymphocytes differentiation increasing both the percentage of CD38++/CD138++ cells as well as the mean fluorescence intensity of both markers (figure 2). Accordingly, different B-cell subpopulations could be identified which represented a continuum in B-cell maturation. While the levels of cytoplasmic immunoglobulin (cIg) were higher among CD38++ cells, the opposite occurred for the expression of surface Ig as well as CD19 and CCR7. Interestingly, the presence of MSC blocked B-cell differentiation. Regarding the pathways involved in these effects, the presence of MSC influenced on ERK 1/2 and p38 pathways, but these effects depended on the culture conditions. Thus, MSC induced phosphorilation of ERK 1/2 MAPK and inhibited phosphorilation of p38 in B-cells cultured with Ig plus CpG (low proliferative conditions) while the contrary occurred in B-cells cultured with TPA (highly proliferative conditions). Therefore we demonstrated that MSC increased viability and blocked cell cycle of B-lymphocytes. Furthermore the plasmocitoid dendritic cells favoured B-lymphocytes differentiation and this process in inhibited in presence with MSC. These effects are at least in part mediated through the ERK 1/2 and p38 pahtways.

Figure Figure
Influence of mTOR Signalling Pathway on the MHC I Immunopeptidome.
Authors: Fortier Marie-Hélène , Caron Etienne , De Verteuil Danielle , Perreault Claude , Thibault Pierre
Published: April 2020

Cell surface major histocompatibility complex (MHC) I molecules are associated with self peptides that are collectively referred to as the self MHC I immunopeptidome (sMII). Despite the tremendous importance of the sMII, very little is known on its genesis and molecular composition. On the other hand, it is well established that the signalling pathway involving mammalian target of rapamycin (mTOR) plays an essential role in the regulation of processes such as ribosome biogenesis and protein translation which are critical for cell growth, proliferation and differentiation. In this work, we studied the influence of mTOR on the sMII for two major reasons:

the tremendous importance of this pathway in oncogenic processes its role in the control of protein synthesis, which is at the origin of the generation of the sMII. To achieve this goal, we developed a novel high-throughput mass spectrometry approach that yields an accurate definition of the nature and relative abundance of unlabeled peptides presented by MHC I molecules.

Starting from EL4 thymoma peptide extracts, more than 200 MHC I-associated peptides were sequenced with high confidence level across more than 5500 peptide clusters reproductibly identified through replicate injections (n = 3). Comparison of the sMII of EL4 thymomas before and after rapamycin stimulation revealed that 13% of the MHC I-associated peptides were significant overexpressed (fold change ≥ 2, p-value ≤ 0.05) after mTOR inhibition. Out of the 27 MHC I peptide candidates showing differential expression, 60% of peptide source proteins were linked to cell development and/or proliferation including ITFIFKSL and NAIKNHWNSTM assigned to FK506 binding protein 12 (mTOR) and myeloblastosis oncogene (c-myb) proteins, respectively. These results indicate that cell signalling events have a major impact on the composition of the sMII that can be monitored by analyses of high-throughput MS-based quantitative profiles.

Figure 1: Relative Quantification of Differentially Expressed MHC I peptides. Volcano Plot representation illustrates MHC I peptides reproductibly detected across replicate injections (n=3). Peptides over-expressed on EL4 cells after rapamycin stimulation are highlighted in blue. Figure 1:. Relative Quantification of Differentially Expressed MHC I peptides. Volcano Plot representation illustrates MHC I peptides reproductibly detected across replicate injections (n=3). Peptides over-expressed on EL4 cells after rapamycin stimulation are highlighted in blue.
Control of Platelet Adhesion by Rigidity Sensing at the Surface of Fibrin Clot.
Authors: Podolnikova Nataly P. , Bowen Benjamin , Lishko Valeryi K. , Podolnikov Andriy V. , Ugarova Tatiana
Published: April 2020

Thrombus formation at sites of vascular injury must occur quickly to reduce blood loss, but is carefully controlled to limit vessel occlusion. Arrest of bleeding is mediated by adhesion and aggregation of platelets and the formation of the fibrin clot. While the interactions responsible for platelet adhesion and thrombus growth have been extensively researched, the mechanisms that limit platelet adhesion are not clear. We have previously demonstrated that plasma fibrinogen is a potent inhibitor of integrin-mediated leukocyte adhesion to fibrin clots and surface-bound fibrinogen, and have provided evidence that fibrinogen reduces cell adhesion by binding to the surface of fibrin rather than blocking leukocyte integrins. Accordingly, cells that engage fibrinogen molecules loosely bound to fibrin (soft substrate) are not able to consolidate their grip on the surface; subsequently, cells detach. Conversely, cells that adhere to the naked fibrin clot (rigid substrate) adhere firmly. Since fibrin and immobilized fibrinogen support platelet adhesion, we examined the effect of soluble fibrinogen on integrin αIIbβ3-mediated adhesion. We show that the anti-adhesive fibrinogen layer formed on the surface of fibrin inhibits platelet adhesion. We also demonstrate that fibrinogen immobilized on plastic at high densities (>20 μg/ml) supports weak platelet adhesion whereas at low concentrations (∼2 μg/ml) it is highly adhesive. An investigation of the mechanism underlying differential platelet adhesion indicates that platelet adhesion to rigid substrates (low-density fibrinogen and naked fibrin gel) induces much stronger phosphorylation of FAK and Syk kinases than that to soft substrates (high-density fibrinogen and fibrin exposed to soluble fibrinogen). Furthermore, the rigid, but not the soft substrates induce recruitment of signaling molecules talin and skelemin to αIIbβ3-containing focal adhesions. Consistent with their limited ability to induce sufficient signaling, soft substrates do not support platelet spreading. These data suggest that circulating fibrinogen prevents stable platelet adhesion by modifying the mechanical properties of the fibrin clot’s surface which results in reduced force generation and insufficient signaling.
Paracrine Suppression of VEGF Secretion by Erythropoietin Inhibits Tumor Growth.
Authors: Smith Carroll R. , Salleng Kenneth J. , Sigounas Vaia Y. , Asch Adam , Sigounas George
Published: April 2020

Several studies have reported that erythropoietin (Epo) is a pleiotropic cytokine with biological properties in addition to its primary function in regulating maturation, growth and survival along the erythroid lineage. Recently, a number of investigators have reported that various neoplastic tissues and human cancer cell lines express Epo and the Epo receptor (EpoR), raising suspicion for the presence of an autocrine-paracrine Epo-EpoR system. It has been shown that inhibition of vascular endothelial growth factor (VEGF) results in an increase of Epo secretion and increased hematocrit in vivo. In this study, we used an in vivo Lewis lung carcinoma model to examine a converse Epo effect on VEGF production and metastasis. Lewis lung carcinoma (LLC) cells were injected subcutaneously into C57BL mice. The plasma levels of VEGF, the tumor vessel formation, the size of the primary tumors and the extent of lung metastatic disease were determined. In addition, intravenously injected LLC cells seeded in the lungs were assessed. Tumor-bearing animals treated with Epo had 23.6% less VEGF in the plasma compared to saline treated mice (p<0.04). There was no correlation between VEGF concentration and hemoglobin levels in either group of animals. Tumor sections indicated that the number of blood vessels was higher (10.7% for inner and 23.8% for outer, respectively) in tumors obtained from animals treated with saline compared to Epo-treated mice (p>0.05). Using non-parametric analysis, we found that there was a statistically significant difference in tumor growth between saline-treated and Epo-treated animals (p<0.05). However, the number of lung metastases derived from primary tumors was similar in both groups. In assessing size of the metastatic tumors, we found that the average volume of lung nodules was 24.2% higher in saline-injected animals compared to Epo-treated mice. The number of tumors seeded in the lungs following intravenous injection of LLC cells was similar in animals treated with a high dosage of Epo, low dosage of Epo or saline. In addition, the average volume of the nodules was reduced by 42% in animals treated with high and low concentrations of Epo compared to the control group (p = 0.03). In conclusion, Epo exerts a paracrine suppressive effect on VEGF secretion resulting in slower tumor growth in this model.
Less Chronic ITP after IVIG for Acute Childhood ITP? A Matched Pairs Analysis from Registry I of the Intercontinental ITP Study Group (ICIS).
Authors: Tamminga Rienk Y.J. , Berchtold Willi , Bruin Marrie C.A. , Buchanan George R. , Kuehne Thomas
Published: April 2020

Introduction: Childhood immune thrombocytopenic purpura (ITP) is a bleeding disorder caused by destruction of platelets with a very small risk of a life threatening bleeding. ICIS Registry I was initiated in 1997 to obtain worldwide prospective data on the natural history of ITP in children. The first analyses of ICIS data showed that 25–47% of the children developed chronic ITP (defined as a platelet count < 150 × 109/l 6 months after diagnosis) depending on age. Previous studies in small numbers of patients suggested that intravenous immunoglobulin (IVIG) treatment at diagnosis might reduce the risk of chronic ITP. Therefore, the ICIS Registry I (with data on >2000 patients) was used to examine prognostic factors related to chronic ITP.

Results: Of the 2605 children with newly diagnosed ITP, between 3 months and 16 years of age, in 1984 the platelet count 6 months after diagnosis was known; 630 (32%) had a platelet count <150 × 109/l. Patient and clinical characteristics associated with a significantly (univariate analysis, p<0.001) higher risk of chronic ITP were: age (<2 yrs: 22%; 2–10: 30%; >10: 49%), platelet count at diagnosis (<10 × 109/l: 25%; 10–20: 37%; 20–50: 40%; >50: 50%) and previous infection (yes: 27%; no 38%). Gender was not of prognostic significance. Investigating the effect of treatment, we performed a matched pairs analysis: two groups were composed, one with platelets ≥150 × 109/l (’controls’) and one with platelets <150 × 109/l (’cases’) at six months from diagnosis, but matched with regard to gender, age, previous infection, platelet count at diagnosis and country of origin. We identified 449 matched pairs. The group with chronic ITP had been more often treated at diagnosis with steroids and less often with IVIG than the controls; the difference between IVIG and steroid treatment was highly significant (p=0.003); patients who were treated with both IVIG and steroids behaved like those with steroids alone; patients without any initial drug treatment behaved in between. Odds ratio (OR) for not having chronic ITP was for IVIG: 1.8 (95% confidence interval: 1.25–2.64) and for steroids 0.60 (0.42–0.87).

Conclusion: Data from the worldwide ICIS Registry I also suggest that IVIG treatment of acute childhood ITP might reduce the risk for chronic ITP while steroids might increase that risk. However, caution is warranted because this study was not a randomized controlled trial, and a considerable number of patients could not be included in the final analyses. A prospective randomised study is needed to confirm these results.
Influences of High-Dose Dexamethasone on Regulatory T Cells, Transforming Growth Factor-β1 and Interleukin-10 of Patients with Chronic ITP.
Authors: Ling Yun , Cao Xiangshan , Yu Ziqiang , Ruan Changgeng
Published: April 2020

Immune thrombocytopenic purpura (ITP) is an autoimmune disorder and high-dose dexamethasone (HD-DXM) has been used as a first-line therapy for patients with ITP. However, little is known about the role of CD4+CD25 + regulatory T (Treg) cells, interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1) in the pathogenesis of chronic ITP and the effects of HD-DXM on them contained Treg cells, IL-10 and TGF-β1. In this study, we investigated the expressions of Treg cells, IL-10 and TGF-β1 in 26 untreated adult patients with chronic ITP. All patients had thrombocytopenia (platelet count <50 × 109/L) for more than 6 months. We also observed short time changes of Treg cells, IL-10 and TGF-β1 after treatment with HD-DXM in these patients. The results showed that a good initial response to HD-DXM occurred in 24 of the 26 patients with chronic ITP (92.3%): the mean platelet count was (84.9±30.4)×109/L [range, (20∼150) ×109/L] one week after the initiation of treatment. The proportion of CD4+CD25+ T cells in the peripheral blood of patients with chronic ITP was significantly higher than that in normal controls(P<0.001); there was no significant difference in the percentage of CD4+CD25high T cells between patients and controls ( P=0.317); but the number of CD4+ FOXP3+ T cells in patients was significantly lower than that in controls (P<0.001). After 4-days treatment with HD-DXM, the numbers of CD4+ CD25+ T cells (P<0.001), CD4+CD25high T cells ( P<0.001), and CD4+FOXP3+ T cells ( P<0.001) in patients were all significantly increased. In the serum of chronic ITP patients, the expression level of TGF-β1 was lower than that of healthy controls (P<0.0001) and HD-DXM could significantly increase it; there was no significant difference in the expression level of IL-10 between patients and controls ( P>0.05) and there was no remarkable change of IL-10 in patients after HD-DXM treatment (P>0.05). The mRNA levels of Foxp3 and TGF-β1 gene in patients were lower than those of controls (P<0.05 and P<0.05); HD-DXM administration significantly increased the expressions of Foxp3 and TGF-β1 gene(P<0.05 and P<0.0001), which were even higher than those of controls(P<0.05 and P<0.05); There was a positive correlation between the Foxp3 mRNA expression and TGF-β1 after treatment with HD-DXM (r =0.403, P=0.041). These results suggest that Foxp3 and TGF-β1 gene are deficient in chronic ITP and the immunosuppressive therapy of glucocorticoids could improve the expression levels of these genes.
Idiopathic Thrombotic Microangiopathies: Antinuclear Antibodies, Platelet Count and Creatinin Level Can Predict a Severe ADAMTS13 Deficiency.
Authors: Coppo Paul , Wynckel Alain , Clabault Karine , Schwarzinger Mickael , Monge Matthieu , Poullin Pascale , Azoulay Elie , Galicier Lionel , Mira Jean-Paul , Bordessoule Dominique , Gorin Norbert C. , Vernant Jean-Paul , Veyradier Agnès
Published: April 2020

Severe ADAMTS13 deficiency in thrombotic microangiopathies (TMA) was reported with a variable frequency, which ranges from 13% to 50%. Whether clinical presentation allows to predict accurately severe ADAMTS13 deficiency remains a matter of controversy. To assess this question, we considered all adult (≥ 18 year-old) patients with idiopathic TMA and ADAMTS13 deficiency (< 15% of normal activity) of our Registry, whose clinical characteristics were compared with those of patients with idiopathic TMA and a detectable (≥ 15% normal activity) ADAMTS13 activity. Inclusion criteria were the association of a microangiopathic hemolytic anemia (< 12 g/dL) with a thrombocytopenia (< 150 × 109/L), and no other identifiable cause for cytopenias (transplantation, cancer and chemotherapy, or HIV infection). Patients were included prospectively from October, 2000, to January, 2007, from 17 National centers. One hundred and thirty-five patients were consecutively included. One hundred and two had a severe ADAMTS13 deficiency (58/96 had a plasma inhibitor), and 33 had a detectable ADAMTS13 activity (mean ADAMTS13 activity: 52.7± 29%). Patients with a severe ADAMTS13 deficiency were younger, displayed a lower platelet count and creatinin level, and presented more frequently antinuclear antibodies (ANA) at diagnosis (39.9± 15 versus 49.6± 17.9 year-old, 20± 19.2 versus 56.6± 42.9 × 109/L, 127± 106 versus 425± 335 μmol/L, and 61.8% versus 24.4%, respectively, p<0.001 in all cases). Chronic renal failure occurred more frequently among patients with a detectable ADAMTS13 activity (19.2% versus 1.1%, p<0.001). In a multivariate logistic analysis, patients with severe ADAMTS13 deficiency were more likely to have ANA (Estimated Odds ratio [OR] 4.81, 95% confidence interval [CI] 1.5–15.4), a lower platelet count (OR 0.98, 95% CI 0.96–1), and a lower creatinin level (OR 0.99, 95% CI 0.98–0.99) than patients with detectable ADAMTS13 activity (p< 0.01, 0.14, and < 0.01, respectively). Positive ANA with a platelet count <31 × 109/L and a creatinin level <194 μmol/L represented a set of criteria that allowed to identify patients with a severe ADAMTS13 deficiency with 52% sensitivity, but with a high specificity, at 97%. These criteria had a high positive predictive value for a severe ADAMTS13 deficiency, at 98.2%, and a negative predictive value of 39.5%. ANA, platelet count and creatinin level can predict accurately a severe acquired ADAMTS13 deficiency in idiopathic TMA. This set of criteria may be helpful in the management of patients that require targeted therapies, in association with plasma exchanges.
Primary Fibrinolysis as the Presenting Sign of Metastatic Prostate Cancer: One Case Report and Literature Review.
Authors: Zhou Rongfu , Ouyang Jian , Chen Bing , Zhou Ming , Xu Yong , Ruan Changgeng
Published: April 2020

Primary fibrinolysis revealing a prostatic adenocarcinoma is rare. Most of the case are limited to biological abnormalities. Here, we describe an unusual case of hematuria and primary fibriolysis as the presenting manifestation of metastatic prostate cancer in a 52-year-old man. The patient consulted for hematuria, ecchymosis and bleeding gums for a month. B-type ultrasound examination showed normal image of prostate. Peripheral blood test showed that the counts of white blood cell, red blood cell and platelet were all in normal range. APTT, prothrombin time and thrombin time were normal. But fibrinogen levels continued to lower than 1.1g/l despite infusions of cryoprecipitate and fresh frozen plasma. Further tests suggested that D-dimer was 9.58 mg/l (normal range: <0.5mg/L), FDP 45 μg/ml, t-PA 90.30 ng/ml (1.00∼12.00 ng/ml), PAI-1 38.3 ng/ml (5.00∼45 ng/ml), α2-PI 118.00% (96.8∼118.8%), PLG 43.30% (57.8∼113.4%), prostate-specific antigen (PSA) 640.2 ng/ml (0∼4 ng/ml), Free PSA 64.2 ng/ml (0∼0.93 ng/ml). PET-CT revealed enhanced metabolizing rate of prostate with enlargement of lymph node in abdomen and multiple bone lesions including rib, vertebration and pelvis. Treatment with a luteinizing hormone-releasing hormone (LHRH) agonist (Diphereline) and a short course of an antiandrogen, led to normalization of all coagulation parameters within 2 weeks, and to clinical improvement and decline in the serum levels of PSA and fPSA. Three months later, the serum levels of PSA and fPSA were normal. We discuss the pathogenesis, differential diagnosis, and association of primary fibrinolysis with prostate cancer along with the management of this condition.
Successful Orthotopic Liver Transplantation in Severe Hemophilia A with High Responding Inhibitor.
Authors: Chen Robert , Paul Seligman , Call Justin , Riske Brenda , Kirschman Ruth Ann , Geraghty Sue , Sally Stabler
Published: April 2020

Orthotopic liver transplantation (OLT) is an effective treatment for both hepatitis C associated cirrhosis, hepatocellular carcinoma, and hemophilia A. Factor VIII activity usually increases into the normal range. Only a few patients with hemophilia complicated by an inhibitor have undergone OLT with both successful outcomes and uncontrolled bleeding being reported. We report early results of OLT in a middle-aged white male severe hemophilia A patient with a history of a high responding inhibitor (historical high - 70 Bethesda units) who had been on immune tolerance for greater than 10 years prior to transplant. A regimen of 40 u/kg of Factor VIII three times per week successfully suppressed inhibitor titers to less than 2 Bethesda units in the previous years. Hand surgery was managed with Factor VII infusions in the year prior to OLT with good results. At the time of transplantation, his inhibitor titer was 0.7 B.U. Due to his history of non-linear kinetics with factor VIII infusion, (5% of a dose remaining at 24 hours), frequent bolus dosing during surgery was employed. He received 10,500 units (116 units/kg) prior to the incision with smaller doses repeated every 2–4 hours. During the operation and the 24 hr immediately post op he required another 27,300 units (300 units/kg) of factor VIII infusion to maintain activity between 61–122%. On post op day 1 he required 46 units/kg to keep activity between 60.2–108%. On post op day 2 he required 35 units/kg to keep activity between 36.8–68.4%. His immunosuppresion included tacrolimus, mycophenolate, and solumedrol taper of 120 mg on day 2, 80 mg on day 3, 40 mg on day 4, and 20 mg day 5. From day 6 to day 8, his total bilirubin increased to 15 and his requirement for Factor VIII also increased to 70 units/kg daily for 3 days to keep his activity between 33.1% to 71.2%. His immunosuppression was increased because of possible acute rejection and solumedrol 500 mg IV was given daily for 3 days. On day 9 his requirement for factor decreased to 11.6 units/kg daily for 4 additional days. Solumedrol was tapered off to prednisone 10 mg po daily. On day 13 post operation, Factor VIII replacement was stopped and his activity was 56.8%, which gradually rose to 81% on day 25. We conclude:

Orthotopic liver transplantation was successful in a hemophilia A inhibitor patient on long term immune tolerance. Factor VIII production by the transplanted liver suppressed the inhibitor and normalized Factor VIII activity up to 4 weeks post transplant. Close follow-up will be required.
Temporal Variation in Intensity of Laboratory Response to DDAVP.
Authors: Tran Minh H. , Hassett Andrea L. Cortese
Published: April 2020

Introduction 1-Desamino-8-D-Arginine Vasopressin (DDAVP) is the mainstay of treatment for mild Type I von Willebrand Disease (vWD). The use of a DDAVP challenge is standard practice in the management of newly diagnosed patients. Pre- and Post- DDAVP administration laboratory studies for vWF: RCoF, vWF: Ag and FVIII:C are expected to rise 2–5 fold from baseline. PTT and Closure Time values would be expected to correct. In a subset of our patients, pre- and post- Platelet Function studies were also tested. We sought to retrospectively study responses in our vWD patient population in terms of time-related and intensity of incremental response to DDAVP.

Results Closure Time prolongation was noted in over 55% of patients with vWD. The Collagen/Epi time appears to be more sensitive for the vWD defect (Collagen/Epi was more frequently prolonged than Collagen/ADP). In vWD patients with Closure Time defects at baseline, 86% were corrected following DDAVP administration, and this correction appears stable over all time points evaluated. Post-DDAVP laboratory responses for vWF: RCoF, vWF: Ag and FVIII:C, respectively: 9.1%, 31.2%, and 16.9% incremented by <50%; 24.7%, 33.8%, and 45.5% incremented by 50–99%; 36.4%, 33.8%, and 28.6% incremented by 100–199%; 18.2%, 0%, and 5.2% incremented by 200–299%; and 11.7%, 1.3% and 3.9% incremented by ≥300%. Peak responses occurred in the 60–89 minute post-DDAVP sampling period (see Figure 1). More than 92% of individuals tested achieved a post-DDAVP vWF: RCoF value of >0.80 U/mL. About 30% and 47.8% of individuals had a prolonged APTT and APTT Mix prior to DDAVP; only 7.8% and 16.7%, respectively, remained prolonged afterwards. There was a trend toward improvement in platelet aggregation variables over time (see Figure 2).

Conclusions Baseline Closure Time abnormalities occurred in over half of the patients and were reliably corrected in most following DDAVP administration. Consistent with established experience, DDAVP produces reliable vWF: RCoF, vWF: Ag and FVIII:C responses in patients with mild Type 1 vWD. Platelet function studies trended toward improvement at greater than 45 minutes post-DDAVP challenge, however, very few patients were studied pre- and post- for platelet function studies and therefore statistical significance was not achieved. Although all patients undergoing DDAVP challenge are asked to avoid antiplatelet drugs, inadvertent use may explain residual Closure Time and Platelet Function test abnormalities.

Post DDAVP Delta Post DDAVP Delta Change in platelet aggregation Following DDAVP Change in platelet aggregation Following DDAVP
Plasma Procofactor Activation by Factor Xla
Authors: Whelihan Matthew F , Mann Kenneth G.
Published: April 2020

The procofactors FV and FVIII are activated by thrombin, FXa and plasmin. During contact pathway-initiated thrombin generation, FXIa activates FIX thus feeding into the coagulation cascade; however, the procofactors FVIII and FV must be activated to achieve a robust level of thrombin generation. We tested the hypothesis that FXIa can activate FV and FVIII. FV (1uM) was subjected to FXIa (100nM) proteolysis. During the reaction the relative activity and integrity were measured at selected time points using a one stage PT clotting assay and SDS-PAGE. Over the 60 minute time course, FV showed a transient 50% activation followed by a reduction in activity to 20%. SDS-PAGE analyses showed that proteolysis of FV by FXIa initially generated fragments with mobilities similar to those produced by α-thrombin. This activation and inactivation pattern suggested that FXIa makes the required activation cleavages at R709, R1018 and R1545 coincidently with inactivating proteolyses. FV was activated with α-thrombin and the reaction quenched by the addition of hirudin prior to FXIa proteolysis. FVa’s cofactor activity was reduced by 50% after 30 min and 80% after 60 min. Analysis of the FXIa cleavage process by SDS-PAGE under reducing conditions showed no intact heavy chain and significant proteolysis of the light chain after 30 minutes. In addition to the Mr = 105000 (HC) and Mr = 75000 (LC), six new products were identified by SDS-PAGE under reducing conditions: Mr = 54000, 50000, 48000, 30000, 22000 and 20000. NH2-terminal sequence analysis indicated a single cleavage in the light chain at R1765 yielding the products Mr = 50000, 48000: (1766à2196) and the Mr = 30000 (1546à1764), also seen with plasmin and FXa. A cleavage in the heavy chain represented by the Mr = 22000, 20000 (R511à709) fragments is also observed. Sequence analysis determined that the Mr = 54000 fragment represented the NH2-terminus of the heavy chain. Western analysis using a heavy chain antibody showed a transient band Mr = 75000 lying under the light chain that is consistent with an initial cleavage R510. A subsequent cleavage, which is coincident with a decrease in the cofactor’s activity, results in a Mr = 30000 product which is consistent with a cleavage somewhere COOH-terminal to R306(R316?). Activity analyses suggest that the initial cleavage in the heavy chain at R510 leads to a FVaXla 1 molecule with similar activity (50%) to that seen in FVa cleaved by APC in the absence of phospholipid. The FVaXla 1 was treated with APC resulting in complete inactivation of the cofactor. We have also observed an analogous FXIa cleavage pattern in FVIII; however sequencing analysis has not yet been attempted. These data suggest that factor XIa may play a role in procofactor activation and inactivation of FV and FVIII in the context of contact pathway-initiated blood coagulation.

Figure Figure
Targeting Autophagy Potentiates Imatinib-Induced Cell Death in Philadelphia Positive Cells Including Primary CML Stem Cells.
Authors: Bellodi Cristian , Lidonnici Maria Rosa , Hamilton Ashley , Helgason Gudmundur V , Soliera Angela R , Ronchetti Mattia , Galavotti Sara , Young Kenneth W , Selmi Tommaso , Van Etten Richard A. , Donato Nicholas , Hunter Ann , Dinsdale David , Tirro’ Elena , Vigneri Paolo , Nicotera Pierluigi , Dyer Martin J.S. , Holyoake Tessa , Calabretta Bruno , Salomoni Paolo
Published: April 2020

Imatinib mesylate (IM), a potent ATP-competitive inhibitor of the BCR/ABL tyrosine kinase, has become standard therapy for patients with chronic myelogenous leukemia (CML). However, the main limitations of IM- and second generation tyrosine kinase inhibitor (TKI)-based therapy are the insurgence of resistance in patients and the intrinsic refractoriness of primitive Philadelphia-positive stem cells. Therefore, there is the need to develop new therapeutic approaches that, in combination with TKI, might be more effective in targeting the stem cell population and preventing the outgrowth of TKI-resistant CML cells. TKI-induced elimination of BCR/ABL-dependent intracellular signals is known to trigger apoptosis, but it is unclear whether this also activates additional cell death and/or survival pathways. We show that IM treatment induces autophagy in CML blast crisis cell lines, CML primary cells and p210BCR/ABL-expressing 32Dcl3 (32D) myeloid precursor cells, but not in 32D cells expressing v-Src or the IM-resistant T315I p210BCR/ABL mutant. IM-induced autophagy does not involve c-Abl, as it is also observed in cells co-expressing p210BCR/ABL and the IM-resistant T315I c-Abl mutant. Induction of autophagy is associated with endoplasmic reticulum-stress and is suppressed by depletion of intracellular calcium. By contrast, ectopic Bcl-2 expression does not block IM-induced autophagy. Suppression of autophagy by pharmacological inhibitors or siRNA-mediated knockdown of essential autophagy genes enhances cell death induced by IM in cell lines and primary CML cells, demonstrating that induction of autophagy has a pro-survival effect. Critically, the combination of TKI with autophagy inhibitors results in near complete elimination of phenotypically (CD34+38−) and functionally (colony forming cells) defined CML stem cells. Together, these findings suggest that autophagy inhibitors may enhance the therapeutic effects of TKI in the treatment of CML.
Bcr-Abl Promotes CML Cell Proliferation through the Suppression of PHLPP Expression.
Authors: Hirano Isao , Nakamura Satoki , Yokota Daisuke , Ono Takaaki , Shigeno Kazuyuki , Fujisawa Shinya , Shinjo Kaori , Ohnishi Kazunori
Published: April 2020

[Background] The proto-oncogene Akt/PKB is activated in many human cancers including leukemias, and regulates the cell survival and apoptosis. Akt/PKB is activated by growth factors or the phosphatidylinositol-3,4,5-triphosphate via PI3K, phosphorylates many substrates for cell survival or proliferation. The tumor suppressor PTEN prevents Akt/ PKB phosphorylation and activation. On the other hand, the PH domain leucine-rich repeat protein phosphatase (PHLPP) dephosphorylates Akt/PKB, induces apoptosis, and suppresses tumor growth. It has been reported that the expression levels of PHLPP are significantly reduced in several colon cancer and glioblastoma cell lines, and Akt/PKB phosphorylation is elevated. However, its function is little known in CML. We found that the expression of PHLPP gene and protein was suppressed in CML cells. In this study, we have investigated the expression and the function of the PHLPP in CML cells.

[Methods] The cells used in this study were human CML cell lines, K562 and Meg01 cells. Primary CML (CP; n=10) cells were obtained from the peripheral blood or bone marrow. Human normal mononuclear cells (MNCs) were isolated from peripheral blood or bone marrow of healthy volunteers after obtaining informed consents. For analysis of PHLPP mRNA expression, quantitative RTPCR was performed in all cell lines treated with Abl kinase inhibitors (STI571, AMN107, and BMS354825). For proliferation analysis and the levels of Akt phosphorylation in CML cells, MTT assays and western blot were performed in all cell lines transfected with Bcr-Abl or PHLPP siRNA, respectively. For colony analysis, the colonies of CFU-GEMM, CFUGM, and BFU-E were counted in CML stem/progenitor cells transfected with PHLPP siRNA or treated with Abl kinase inhibitors.

[Results] In CML cell lines, the expressions of PHLPP mRNA and protein were significantly increased more than normal MNCs by treatment with Abl kinase inhibitors or transfection with Bcr-Abl siRNA. On the other hands, in CML cells transfected with the PHLPP siRNA, it is shown that the phosphorylation levels of Akt were markedly increased compared to the untransfected cells. In CML stem/progenitor cells obtained from patients with CML, the expression of PHLPP gene was suppressed in 10/10 (100 %) of CML. This higher expression was not associated to the counts of blasts of PB (P < 0.001). The transfection with Bcr-Abl siRNA or treatment with Abl kinase inhibitors increased the expression of PHLPP mRNA and protein, and decreased the counts of CFU-GEMM, CFU-GM and BFU-E. [Conclusions] Our results suggest that the Bcr- Abl suppressed the expression of PHLPP mRNA and protein, and the phosphorylation of Akt in CML cells to induce the proliferation of CML cells. Moreover, induction of PHLPP expression might regulate the proliferation of CML stem/progenitor cells.
Long-Term Follow-up Results of IFM9903 and IFM9904 Trials Comparing Non Myeloablative Allotransplantation with Autologous Transplantation in High-Risk De Novo Multiple Myeloma.
Authors: Moreau Philippe , Garban Frederic , Attal Michel , Michallet Mauricette , Marit Gerald , Hulin Cyrille , Benboubker Lotfi , Doyen Chantal , Mohty Mohamad , Yakoub-Agha Ibrahim , Leyvraz Serge , Casassus Philippe , Avet-Loiseau Hervé , Garderet Laurent , Mathiot Claire , Harousseau Jean-Luc
Published: April 2020

The IFM99-03 and IFM99-04 trials were conducted from April 2000 to August 2004. Patients younger than 66 years with high-risk (b2microglobulin > 3 and chromosome 13 deletion by FISH analysis at diagnosis) de novo multiple myeloma (MM) were included and prospectively treated. In both protocols, induction regimen consisted of VAD (4 courses) followed by melphalan 200 mg/m2 (HDM200) plus autologous peripheral blood stem cell transplantation (ASCT). When a HLA-sibling donor was available, ASCT was followed by reduced-intensity conditioning regimen (RIC) allograft (fludarabine, antithymocyte globulin and low dose busulfan): IFM9903 protocol (Garban et al, Blood2006;107:3474–3480). When no donor was available, patients were randomised to receive a second ASCT with HDM220 +/− anti-IL6 monoclonal antibody (BE-8, 250 mg total dose, Diaclone Besançon, France): IFM99-04 protocol (Moreau et al, Blood2006;107:397–403).284 patients met eligibility criteria and received at least one course of VAD. 65 had an available HLA-identical sibling donor and were included in the IFM99-03 trial, and 219 were included in the IFM 99-04 trial. Patients were older in the tandem ASCT trial (median age, 58 vs 54 years; P = .006) and the b2-microglobulin level was also higher in the latter group (median, 4.9 mg/L vs 4.1 mg/L; P = .049). At the reference date of July 1st, 2008, on an intent-to-treat basis, considering the entire population of 284 patients, with a median follow-up of 56 months, the EFS did not significantly differ from tandem ASCT to single autograft followed by allo-RIC (median 22 vs 19 months, P = 0.58). Nevertheless, there was a trend for a superior OS in the double ASCT trial (median 48 vs 34 months, P = 0.07). When considering the comparison of the results of the 166 patients /219 who completed the whole tandem ASCT protocol with those of the 46 patients /65 who underwent the entire auto/allo-RIC program, no difference was observed regarding EFS (median 25 vs 21 months, P = 0.88), but there was again a trend for a superior OS in favour of double ASCT (median OS, 57 vs 41 months, P = 0.08), due to a longer survival after relapse in the tandem ASCT arm. These long-term results indicate that, in a subgroup of high-risk patients with de novo MM, a tandem autologous transplant procedure is at least equivalent or even superior to a combination of autologous followed by RIC allogeneic stem cell transplantation.
Use of a Potassium Channel Blocker to Inhibit Effector-Memory T Cells Isolated from Recipients of Allogeneic BMSCT.
Authors: Cameron Cheryl , Woodliff Jeffrey , McOlash Laura , Hari Parameswaran , Bredeson Christopher , Margolis David , Truitt Robert L.
Published: April 2020

Following allogeneic blood or marrow stem cell transplantation (BMSCT), donor T cells are persistently exposed to host alloantigens and develop into effector-memory (TEM) cells. CD4+ T-EM cells are thought to play a role in perpetuating chronic GVHD (cGVHD) (K. Yamashita et al., BLOOD103:3986, 2004). In contrast to central memory (T-CM) and naïve CD4+ T cells, human CD4+ T-EM cells over-express the voltagegated potassium channel Kv1.3 when activated. As a result, Kv1.3 has emerged as an attractive pharmacological target for the selective suppression of T-EM cells, especially in the setting of autoimmune disease (C. Beeton et al., Mol Pharm67:1369, 2005). We hypothesized that over-expression of Kv1.3 by T-EM cells obtained from allogeneic BMSCT patients with cGVHD would make them preferential targets for Kv1.3-specific potassium channel blockers. To our knowledge, no one has examined the effect of Kv1.3 blockers on T-EM cells from patients with cGVHD. In this study, we isolated T-EM cells as well as central-memory (T-CM) and naïve T cells from fresh (n = 5) and frozen (n = 4) peripheral blood (PB) of patients who had undergone allogeneic BMSCT more than 6 months earlier as well as from normal volunteers by fluorescence-activated cell sorting (FACS). All BMSCT patients had some clinical evidence of cGVHD. T-EM cells were identified by their characteristic immunophenotype: CD4+ CD45RO+/RA- CCR7- CD62L Low. The percentage of CD4+ T-EM cells in 9 patients with cGVHD was 45 ± 21% (range 19%–70%) as compared to approximately 11% in healthy controls. Sorted T-EM, T-CM and naïve CD4+ T cells were polyclonally activated ex vivo using Dynal beads coupled with anti-CD3/CD28 monoclonal antibodies. The proportion of cells secreting gamma interferon (γIFN) was quantified using EliSpot assays to assess functional activity. We first compared the kinetics of γIFN secretion by activated T-EM cells to that of T-CM and naïve T-cells. Only T-EM cells secreted γIFN within 24 hrs of activation; T-CM cells secreted γIFN ≥ 48 hrs after activation, and naïve T cells required ≥ 96 hrs. ShK peptide, a highly specific inhibitor of Kv1.3 channels derived from the sea anemone Stichodactyla helianthus, was added at the initiation of 24-hr cultures. Dose response assays confirmed that T-EM cells in both BMSCT patients and normals were preferential targets of the Kv1.3-specific peptide. On average, T-EM cells were inhibited 26%, 55%, and 69% with ShK concentrations of 1, 10 and 100 nM, respectively. With 100nM ShK, T-EM cells sorted from the peripheral blood of BMSCT patients were inhibited an average of 44 ± 21% (range 17–67%). Our data, using polyclonally activated T-EM cells, support further testing of Kv1.3 blockers against host-alloantigen-specific T-EM cells obtained from BMSCT recipients and as a potential therapeutic agent for cGVHD.
Macrophage Infiltration of Skin Lesions Correlates to Prognosis of Gvhd; A Clue to Refractory Gvhd
Authors: Nishiwaki Satoshi , Terakura Seitaro , Goto Tatsunori , Seto Aika , Watanabe Keisuke , Imahashi Nobuhiko , Tsukamoto Shokichi , Shimba Makoto , Ozawa Yukiyasu , Miyamura Koichi , Ito Masafumi
Published: April 2020

The pathophysiology of acute graft-versus-host disease (aGVHD) is a complex process. Although it is considered an alloimmune attack on host tissues mounted mainly by donor CD8-positive T cells, skin infiltration of T cells is not always the case in biopsy specimens of skin aGVHD, suggesting the involvement of other cellular components. In detailed observation, CD163-positive macrophages which have abilities of antigen presentation and releasing cytokines were frequently observed. We retrospectively reviewed 66 biopsy specimens of skin lesions clinically thought to be aGVHD performed within 100 days after allogeneic stem cell transplantation, and analyzed the relationship between types of infiltrating cells and clinical outcomes. Paraffin-section immunohistochemical analysis was performed using a monoclonal antibody against CD8 and CD163. Counting the total number of CD8-positive T cells and CD163-positive macrophages in 4 fields with 200-fold magnification, median number of CD-8 positive T cells was 78.5 (range; 2–305), and that of CD163-positive macrophages was 149 (range; 38–372). Infiltration of over 100 cells of CD8-positive T cells (CD8ov100) correlated with HLA class I mismatch and grade III–IV a GVHD, while infiltration of over 200 cells of CD163-positive macrophages (CD163ov200) correlated with grade III–IV aGVHD and refractory aGVHD. In logistic analyses, CD163ov200 (Odds 6.29 (95%CI 1.57–25.0); p=0.009) was identified as the only negative predictors of aGHVD outcome. Overall survival of patients with CD163ov200 was significantly lower than that of those with infiltration of under 200 cells of CD163-positive macrophages (63.0% vs. 40.6% at 1 year, respectively; p=0.02). In 23 patients treated with steroid for aGVHD, CD163ov200 was the only significant predictor of refractory aGVHD outcome (Odds 10.9 (95%CI 1.37–83.3); p=0.02), and overall survival of patients with CD163ov200 was significantly lower than that of those with infiltration of under 200 cells of CD163-positive macrophages (63.5% vs. 12.5% at 1 year, respectively; p=0.0004). These results indicate that CD163-positive macrophages may provide a clue for the pathophysiology of refractory GVHD and new treatment strategies.
Recombinant PDGF-BB Enhances Platelet Recovery in Mice with Radiation Induced Thromobocytopenia.
Authors: Ye Jie Yu , Chan Godfrey ChiFung , Chan Shing , Luo Jue Cong , Yang Mo
Published: April 2020

Platelet-derived growth factor (PDGF) involves in the regulation of hematopoietic stem cells. Imatinib mesylate (Gleevec), a PDGF receptor inhibitor, induces thrombocytopenia in 50% of patients with chronic myeloid leukaemia (CML). Our previous studies showed that PDGF enhances megakaryocytopoiesis in vitro via its receptors directly. However, the in vivo effect of recombinant PDGF in thrombocytopenic models has not been reported. In this study, we investigated the in vivo effect of PDGF as well as thrombopoietin (TPO) on hematopoietic stem cells and platelet production in a radiation treated mouse model. We also explored its potential molecular mechanisms on thrombopoiesis. A radiation induced myelosuppression with thrombocytopenic model was established using 4-Gy-irradiated mice. PDGF-BB (1 mg/kg/day) and TPO (1 mg/kg/day) were injected separately after radiotherapy into the mice. Peripheral blood platelets, WBC and RBC from PDGF-BB, TPO and control groups were collected and analyzed on day 0, 7, 14 and 21 respectively. The mice were sacrificed on day 21 and their bone marrows were harvested for CFU assays and histology analysis. PDGF-BB, like TPO, showed a significant promoting effect in platelet production (n=5 p= 0.0078) as well as CFU-MK formation (n=5 p=0.0019) compared with the untreated control group. Histology images also indicated that PDGF-BB increased the number of the megakaryocytic cells and its progenitors in bone marrow of irradiated mice. In order to find out the underlying mechanism, we further studied the in vitro effect of PDGF using megakaryocytic cell line M-07e. Our results demonstrated that PDGF alone (100–200ng/ml) significantly activated the PI-3K/AKT signaling pathway (n=9 p=0.014). PDGF plus PI-3K inhibitor (wortmannin, 100nM) was shown to attenuate the expression of AKT (n=9, p=0.0112). Consistently, imatinib mesylate (Gleevec, 1uM) negatively interfered the expression of AKT when combined with PDGF-BB ((n=4, p= 0.0288). PDGF-BB was found to have a similar anti-apoptotic effect as TPO on M07e cells as showed by Annexin V (n=8, p=0.0281), Mitochondrial Membrane Potential (JC-1) (n=7, p=0.0042), and Caspase-3 (n=5, p=0.0080) assays respectively. In summary, our findings showed that PDGF-BB had an in vivo thromobopoietic effect on the radiation induced myelosuppressive mice models. This effect was likely mediated via PDGF receptor with subsequent activation of PI-3K/AKT pathway, which leads to anti-apoptotic effect on megakaryocytes.
A Myelodysplastic Syndrome Associated with a Clonal GATA1 Mutation.
Authors: Freson Kathleen , De Waele Liesbeth , Thys Chantal , Wittevrongel Christine , Vandenberghe Peter , De Vos Rita , Vyas Paresh , Van Geet Christel
Published: April 2020

Germline exon 4 mutations in the N-terminal zinc finger (N-finger) of transcription factor GATA1 cause a variable degree of X-linked dyserythropoiesis with macrothrombocytopenia. Congenital neutropenia and anemia are described in patients with an exon 2 germline splicing mutation resulting in exclusive expression of the short isoform of GATA1 (GATA1s). Similar but acquired somatic GATA1 exon 2 splicing mutations occur in children with Down Syndrome (DS) and congenital transient myeloproliferative syndrome (TMD) or acute megakaryoblastic leukemia (AMKL). The present study describes a somatic exon 4 GATA1 mutation in a 19-year-old boy with myelodysplastic syndrome (MDS) who has a lifelong history of anemia, neutropenia, severe bone pains and hepatosplenomegaly. Bone marrow biopsy demonstrates bilineage dysplasia with deficient granulopoiesis and dysmegakaryopoiesis. The patient has a normal platelet count but an increased Ivy bleeding time and a prolongation of the PFA100 occlusion time. Platelet ATP secretion and platelet aggregation are decreased. The a to g substitution is present in GATA1 mRNA from platelets and bone marrow mononuclear cells. The mutation is also detected in genomic DNA from purified common myeloid progenitors, but not in common lymphoid progenitors, peripheral blood lymphocytes and skin fibroblasts. This GATA1- E200G mutation is located in a highly conserved residue at the beginning of the N-finger, but does not perturb DNA binding to GATA sites nor reduces its interaction with FOG1. In contrast, the mutation is located at the exonic splice site and is associated with alternative splicing and reduced GATA1 protein levels. In conclusion, this study expands the GATA1 phenotype-genotype spectrum with the development of MDS and platelet dysfunction due to a somatic GATA1 splice mutation.