bluuebunny/crossref_metadata_2025 huggingface dataset:
Childhood ALL Treatment and Protocol Compliance Failure at a Single Institute from Most Developed Area of China.
Authors: Tang JingYan , Xue HuiLiang , Gu Long-Jun , Chen Jing , Pan Ci , Ye Hui
Published: October 2019

Purpose To analyze the main reason of failure in children with ALL at a single institute which is at most developed city of developing country China.

Method All the ALL patients who was diagnosed at our hospital from 1998.10 to 2003.12 were analyzed. The date was created from our department tumor registry database. Patient was divided into 3 groups, high, middle and low risk, depends on 1.Age >=10 year, 2. 50×109/L>WBC<100×109/L, 3. chromosome<45,or DNA index <1.16, 4. t(4;11), 5. T-ALL, 6.CNSL and/or TL, 7. WBC>=100×109/L, 8.t(9;22), 9.<1 year or >12 year, 10. Early response (1) Pred.test day 8,pripherial blast >=1,000/μl, (2) Induction day19–21 or day 35 bone marrow blast >=5%. Anyone from item 1 to 5 was middle risk factor, item 6 to 9 was high risk factor. The patients not receiving any therapy after ALL diagnosis were accounted as early protocol compliance failure, receiving therapy less than 15 days were middle protocol compliance failure, giving up therapy or losing follow-up after 15 day with stable disease or CR were accounted as late compliance failure.

Results Total 224 ALL were diagnosed, of them 38 patients went home with no any therapy. That means early protocol compliance failure was 17%. Of the remained 186 patients, 26 patients(12%) belonged to middle protocol compliance failure and 6 (3%) was late compliance failure. So total protocol compliance failure was 31%. The main reason of compliance failure was lacking financial support. Within the 160 patients who received more than 15 days therapy, 50(31%) was high risk, 51(32%) middle risk, and 52(33%) low risk, another 7 at un-know risk group. Of them, 48 patients relapsed(40) or not reached CR(8), 10 died of complication(mainly infection), total treatment failure was 48(30%).

Conclusion Besides the treatment failure, protocol compliance failure is a important reason for childhood ALL survival failure in developing country.
Aclarubicin and Low Dose Arabinoside Cytosine (Ara-C) in Combination with Granulocyte Colony Stimulating Factor (G-CSF) in Treating Acute Myeloid Leukemia (AML) Patients with Relapsed or Refractory Disease and Myelodysplasia Syndrome (MDS): A Multi-Center Study of 112 Chinese Patients.
Authors: Li J.M. , Shen Y. , Wu D.P. , Liang H. , Jin J. , Chen F.Y. , Song Y.P. , Wang J.M. , Qiu X.F. , Hou M. , Qiu Z.C. , Shen Z.X.
Published: October 2019

112 patients with senile acute myelocytic leukemia (AML), refractory or relapsed AML, and MDS-RAEBt entered this study to receive aclarubicin and low dose Arabinoside Cytosine (Ara-C) in combination with Granulocyte Colony Stimulating Factor (G-CSF) for evaluation of efficacy and tolerance of this CGA regimen. Low dose Ara-C was given at the dosage of 10mg/m2/12h, subcutaneously, D1-14 and aclarubicin 14mg/m2/d, intravenously, D1-4 (regimen A) or 7mg/m2 D1-8 (regimen B). Recombinant G-CSF was given at the dosage of 200μg/m2/d, subcutaneously, D1-14. We proved that overall response rate was 19/26 (73.1%) in senile AML, 48/62 (77.4%) in refractory AML, 12/18 (66.7%) in relapsed AML and 10/13 (76.9%) in MDS-RAEBt; and CR rate was at 8/26 (30.8%) in senile patients, 30/62 (48.4%) in refractory AML, 8/18 (44.4%) in relapsed AML and 5/13 (38.5%) in MDS-RAEBt, which were comparable in four groups of patients. 52 patients were followed up. Median progression free survival (PFS) and overall survival (OS) were 9.0±2.2 months and 11.0±1.6 months, respectively. The Kaplan-Meier estimated PFS and OS rate at 12 months were 40.73±8.15% and 42.85±8.23%, respectively. 1 year OS and PFS rate of the patients with CR were 60%±10.8% and 51.3%±13.7%, respectively, compared with the patients with PR, 17.7%±9.3% and 6.4%±6.1%, respectively. The toxic effects were very rare, mainly manifested as hematological changes, neutropenia and thrombocytopenia due to myelosuppression, which was around 70–80% exceeding NCI grade II. And non-hematological toxicities were not observed in this study. CAG regimen seems to be promising for the treatment of various categories of poor-prognosis AML or MDS-RAEBt, with acceptable toxicity.
Identification of a Novel Alternatively Spliced Form of AML1-ETO and Analysis of Its Role in Leukemogenesis.
Authors: Kanbe Eiki , Wang Yang , Boyapati Anita , Yan Ming , Peterson Luke F. , Willman Cheryl L. , Rowley Janet D. , Zhang Don-Er
Published: October 2019

The 8;21(q22;q22) translocation is found in 12% of all AML cases, including 40–50% of AML M2 subtype and a small portion of M0, M1, and M4 subtypes. The translocation brings together the DNA sequence encoding the N-terminus of the AML1 (RUNX1) with nearly all of ETO (MTG8) to generate the AML1-ETO fusion protein. The most commonly known AML1-ETO fusion protein (full length AML1-ETO) is encoded by AML1 exons 1 to 4 and by ETO exons 2 to 11 and has 752 a.a. However, a recently discovered ETO exon 9a can provide a protein translation stop codon after the last amino acid encoded by exon 8. The predicted AML1-ETO protein using exon 9a (AML1-ETO9a) has 575 a.a. and does not include the NHR3 and NHR4 domains at the C-terminus of full length AML1-ETO. To demonstrate the presence of the AML1-ETO9a transcript, we performed RT-PCR using RNA prepared from t(8;21) patient samples. Exon 9a transcript was detected in all patient samples when PCR generated a 595 bp fragment with ETO exon 6 and exon 9 primers and was detected in seven of the 14 samples when PCR generated a 1381 bp fragment with AML1 exon 4 and ETO exon 9a primers, indicating the presence of AML1-ETO9a mRNA in patient samples. The predicted molecular weight of AML1-ETO9a protein with 575 a.a. is 63.8 kDa and a 64 kDa AML1-ETO fusion protein has been reported in both Kasumi-1 cells and t(8;21) leukemic blasts. We compared the effects of AML1-ETO and AML1-ETO9a on proliferation and differentiation of hematopoietic cells using FDCPmix cell line, which is an IL-3 dependent multipotent hematopoietic stem cell line. Both forms of fusion proteins inhibited the myeloid lineage differentiation when cells were cultured in the presence of GM-CSF and G-CSF. However, these fusion proteins showed opposite effects on cell cycle progression. AML1-ETO suppressed cell cycle with prolonged G0/G1; AML1-ETO9a enhanced cell cycle with shortened G0/G1. Expression of these fusion proteins in primary bone marrow cells showed a similar result. Most importantly, only AML1-ETO9a efficiently induced acute myeloid leukemia in mice. Thus, the observation that a shorter form of AML1-ETO is a dominant factor in t(8;21) related leukemogenesis exhibits a new paradigm in the understanding of t(8;21) tumorigenesis.
A FVII Deficiency Induces Cardiac Fibrosis in Mice Resulting in Systolic and Diastolic Dysfunction.
Authors: Xu Haifeng , Sandoval-Cooper Mayra , Donahue Deborah L. , Ploplis Victoria A. , Castellino Francis J.
Published: October 2019

Mice severely deficient in coagulation Factor (F)VII that survive to adulthood have been developed, thus allowing the study of spontaneous bleeding and inflammatory phenotypes under conditions of a deficiency of extrinsic coagulation, especially in organs e.g., the heart, that continually undergo normal mechanical challenge. Mice genetically modified to produce very low levels (∼1% of wild-type) of coagulation FVII (FVIItTA/tTA) spontaneously developed heart abnormalities that were revealed by echocardiography. There were no significant differences between groups at baseline for the hemodynamic and respiratory parameters: temperature and heart rate (HR). The end-systolic functions of FVIItTA/tTA mice heart were comparable to wild-type (WT) controls. However, there were significant changes in the end-diastolic parameters of FVIItTA/tTA hearts. The diameter at end-diastole in FVIItTA/tTA mice was smaller compared to WT mice, which was due to the stiffness of the interventricular wall. When viewed from the parasternal short-axis, these mice had evidence of hypokinesis, indicating alteration of the elastic properties of the left ventricular (LV) wall. Correspondingly, the stroke volume, ejection fraction, fractional shortening, and cardiac output were lower in FVIItTA/tTA mice, compared to WT mice. Mitral valve inflow was compromised in FVIItTA/tTA mice, as demonstrated by the reduced early/late (E/A) filling ratio. We found that the spontaneous impaired cardiac function in FVIItTA/tTA mice was associated with an increased ratio of heart weight to body weight. Decreases in ventricular size, accompanied by reductions in systolic and diastolic functions, suggest a restrictive cardiomyopathy, consistent with an infiltrative myopathic process. Microscopic analysis of mouse hearts showed severe patchy fibrosis in FVIItTA/tTA mice. Hemosiderin deposition in hearts of these mice, along with large increases in inflammatory cells, ultimately resulting in widespread collagen deposition, were found. Significant increases in mRNA levels of TGF-beta, TNF-alpha, and a variety of matrix metalloproteinases, beginning at early ages in FVIItTA/tTA mice, supported the fibrotic pathology. The results of this study demonstrated that hemorrhagic and inflammatory responses to a severe FVII deficiency resulted in the development of cardiac fibrosis, manifest echocardiographically as a restrictive cardiomyopathy, with significantly compromised ventricular diastolic and systolic functions. These data also suggest that a FVII deficiency differentially regulates the expression level of inflammation and extracellular matrix proteins, which result in compromised cardiac function.
Apoptosis Stimulating Protein of p53 (ASPP2) Haploinsufficiency Promotes Irradiation-Induced High Grade Lymphomas in a Mouse Model.
Authors: Kampa Kerstin M. , Akoba Jared D. , Chen Dexi , Beemer Kelly , Gay Joel , Zhu Zhiyi , Padiernos Emerson , Corless Christopher , Baily Alexis , Flemming William H. , Lopez Charles D.
Published: October 2019

Apoptosis Stimulating Protein of p53 (ASPP2) binds the tumor suppressor protein p53 and promotes damage-induced apoptosis in part through stimulation of p53 apoptotic function. We have previously demonstrated that low ASPP2 levels correlate with poor clinical outcome in patients with diffuse large B-cell lymphoma treated with anthracycline-based chemotherapy, which is consistent with its role as a tumor suppressor. We targeted the ASPP2 allele in a mouse model by homologous recombination using a knockout vector that replaced exons 10–17 with a neoR gene. Although ASPP2−/− is an early embryonic lethal event, ASPP2+/– mice appeared developmentally normal and reproduce. Importantly, ASPP2+/– mice developed tumors spontaneously at a higher frequency than sibling ASPP2+/+ controls. We now show that after ionizing irradiation, ASPP2+/– mice develop high grade lymphatic malignancies compared to ASPP2+/+ mice. ASPP2+/+ and +/– mice were irradiated, with either 6 Gy or 10,5 Gy, 8 weeks after birth. Following irradiation, mice were monitored weekly by blood smears and flow cytometry if suspicious cells were observed. Tumor formation was detected as early as 17 weeks post-irradiation. In the 10,5 Gy group, ASPP2+/– mice developed thymic lymphomas (7/31), while no tumors were detected in ASPP2+/+ mice (0/14). Tumor-free survival was statistically significant (p= 0,045, log-rank test). In the 6 Gy dose group 9 out of 28 of the ASPP2+/– mice developed high-grade thymic lymphomas compared to 0/20 ASPP2+/+ mice. Tumor-free survival was statistically significant (p=0,024, log-rank test). Immunophenotyping and flow cytometry on mice with circulating tumor cells from whole unfractionated gated viable bone marrow (Bm) cells revealed that the Bm was typically almost completely replaced by CD4neg, CD5+, CD8+ leukemic/lymphoma cells that are negative for B-cell (B220) and myeloid markers (Mac-1/Gr-1). Preliminary analysis demonstrated that MEFs (mouse embryonic fibroblasts) from ASPP2+/– mice treated with doxorubicin, serum starvation or irradiation had an attenuated G1/G0 checkpoint arrest. Ongoing experiments are being conducted in thymocytes prepared from mice of different ASPP2 and p53 genotypes. Our data demonstrates that ASPP2 plays an important role in suppressing the development of lymphomas after irradiation in a mouse model. Consequently, disruption of the p53-ASPP2 axis may be important in lymphomagenesis and response to therapy. Furthermore, our data provides a rationale for the future study of ASPP2 expression in patients during the formation of secondary hematologic malignancies after therapies with DNA damaging agents.
Rivaroxaban — An Oral, Direct Factor Xa Inhibitor — Prevents Arterial Thrombotic Occlusion in Electrolytically Injured Rat Carotid Arteries.
Authors: Haertlein Barbara , Parry Tom J. , Chen Cailin , Perzborn Elizabeth , Andrade-Gordon Patricia , Damiano Bruce P.
Published: October 2019

Introduction: Rivaroxaban is an oral, direct Factor Xa (FXa) inhibitor in advanced clinical development for the prevention and treatment of thromboembolic disorders, including arterial indications such as acute coronary syndromes (ACS). The aim of this study was to determine whether rivaroxaban can prevent arterial thrombotic occlusion in electrolytically injured rat carotid arteries.

Methods: Anesthetized, male Sprague-Dawley rats were equipped with an intravenous (i.v.) catheter to administer drug or vehicle and an intra-arterial catheter for arterial pressure monitoring and blood sampling. The left carotid artery was exposed and a bipolar electrode placed proximally around it. A pulsed Doppler flow probe was then placed distally around the vessel. Blood was obtained before drug or vehicle administration to determine activated clotting time (ACT), prothrombin time (PT), Russell’s viper venom time (RVVT), activated partial thromboplastin time (aPTT) and thrombin-antithrombin (TAT) complex concentration; baseline carotid blood flow was recorded for 20 minutes. Vehicle (polyethylene glycol:ethanol:water or saline, 0.5 mL/kg/min), rivaroxaban (0.3, 1 and 3 mg/kg; dosing volume 0.5 mL/kg/min), or the low molecular weight heparin enoxaparin (10 mg/kg) were infused i.v. over 30 seconds. After infusion, direct current (3 mA) was applied to the left carotid artery for 5 minutes. Blood flow was monitored for 30 minutes after injury. Blood was sampled again 14.5 and 30 minutes after injury for ACT determination, and after 30 minutes for PT, RVVT, TAT and aPTT determination.

Results: Rivaroxaban dose-dependently increased the median time to arterial thrombotic occlusion (TTO), and was significantly more effective than vehicle at doses of 1 and 3 mg/kg (vehicle, 13.2 minutes; rivaroxaban 1 and 3 mg/kg, >30 minutes; P<0.05 by Kaplan-Meier analysis). Compared with enoxaparin (10 mg/kg), rivaroxaban inhibited TTO at doses of 1 and 3 mg/kg more effectively. Rivaroxaban (0.3–3 mg/kg i.v.) dose-dependently prolonged ACT, PT and RVVT, and substantially decreased the formation of TAT complexes, but did not substantially affect aPTT. Enoxaparin prolonged ACT, aPTT and RVVT, and decreased TAT complex formation, but had no effect on PT.

Conclusions: Rivaroxaban potently inhibited electrolytically induced arterial thrombotic occlusion in rat carotid arteries, resulting in substantial changes in coagulation parameters. The coagulation parameter changes were consistent with inhibition of FXa activity as indexed by the RVVT. These findings suggest that rivaroxaban may be an effective anticoagulant in arterial thrombotic disorders, such as ACS.
Studies of the Affinity and Stoichiometry of Convulxin Binding to Glycoprotein VI: Clues into Its Potent Agonism.
Authors: Herr Andrew B. , Horii Katsunori
Published: October 2019

The primary cell-surface receptor that mediates platelet activation upon exposure to collagen is glycoprotein VI (GPVI). GPVI can be activated by fibrous collagen, soluble collagen mimics, or several snake venom proteins. Upon binding of these agonists, GPVI signaling activates key integrins and triggers secretion of platelet granule contents, leading in turn to activation of neighboring circulating platelets. One of the most potent GPVI agonists from snake venom is convulxin (CVX), an α4β4 C-type lectin-like protein from the South American rattlesnake, Crotalus durissus terrificus. Using analytical ultracentrifugation, we have discovered that CVX exists in solution as a stable dimer of α4β4 rings, creating eight potential binding sites for GPVI. Using a combination of kinetic and equilibrium surface plasmon resonance binding experiments, we show that CVX binds GPVI with high picomolar to low nanomolar affinity at each site, and that all eight sites are likely to be available for binding. Analysis of the previously published crystal structures of CVX suggests that its potency could be due to both high-affinity binding and clustering of GPVI on a single platelet as well as potential bridging of adjacent platelets via the dimeric CVX architecture.
Essential Roles for Mef2c in Lymphoid Commitment and B-Cell Function.
Authors: Stehling-Sun Sandra , Jimenez Rebecca , Hu Andrew , Camargo Fernando D.
Published: October 2019

MEF2 transcription factors are well-established regulators of muscle development. Recently, work in murine models has identified one of these factors, Mef2c, as an important regulator in the pathogenesis and the development of acute myeloid leukemia (AML). However, little is know about the molecular mechanism and physiological role of Mef2c in hematopoiesis. Using conditional gene ablation, we have discovered an unexpected role for MEF2c in hematopoietic stem cells (HSCs), where it is required for pan-lymphoid commitment. Competitive repopulation experiments using Mef2c-null HSCs deleted by means of the Mx1-Cre/poly(IC) approach, revealed completely normal monocytic, granulocytic and erythroid differentiation capacities by mutant cells. Generation and renewal of myeloid progenitors and HSCs was also normal. However, contribution to lymphoid lineages (T-cells, B-cells and natural killer cells) was dramatically reduced. Mef2c-deleted HSCs were able to generate lymphoid primed multipotent progenitors (LMPPs) and expressed normal levels of Flt-3 and the master lymphoid regulator ikaros. However, expression of the interleukin-7 receptor (IL-7R) and the number of phenotypically defined common lymphoid progenitors (CLPs) were substantially reduced. We have found two conserved Mef2c-binding sites in the promoter of the Il-7R gene, indicating that Mef2c could directly regulate Il-7R transcription. This and other potential molecular mechanisms of Mef2c-mediated lymphoid commitment will be discussed. We have also studied the effects of lineage-specific deletion of Mef2c in both myeloid and lymphoid populations. Whereas deletion in myelomonocytic cells using the LysM-Cre strain resulted in no anomalies, B-cell specific ablation with the CD19-Cre line revealed major phenotypical and functional abnormalities. CD19-Cre:Mef2cf/f mice show impaired germinal center formation and reduced antibody production in response to T-cell dependent antigens. In addition Mef2c-null mature B-cells fail to express the mature marker CD23, the low affinity receptor for IgE, which we show is a direct transcriptional target. As a consequence of CD23 reduction, CD19-Cre:Mef2cf/f mice have increased IgE production, thus indicating a potential role of Mef2c in allergic disease. Our work here sheds new light on the molecular mechanisms of lymphopoiesis and identifies MEF2 factors as critical hematopoietic transcriptional regulators.
Effect of Deferasirox on Midazolam Pharmacokinetics.
Authors: Arrowsmith-Bensasson Caroline , Rojkjaer Lisa , Sethuraman Venkat , Skerjanec Andrej
Published: October 2019

Introduction: Deferasirox (Exjade®, ICL670) is an oral iron chelator approved for the treatment of iron overload due to chronic transfusions in more than 80 countries. Deferasirox is mainly eliminated via biliary excretion following glucuronidation. Oxidative metabolism, presumably by CYP1A and CYP2D6 enzymes, accounts for approximately 6% and 2%, respectively. Deferasirox has been shown to be a weak inhibitor of CYP3A4/5 in vitro. It is possible that deferasirox could potentially alter the metabolism of other drugs eliminated via this pathway. Midazolam, a rapid-onset benzodiazepine, is almost exclusively metabolized via CYP3A4/5 making it an ideal probe to investigate the effect of an inhibitor/inducer of CYP3A4/5 in vivo. This drug-drug interaction study was undertaken to investigate the effect of deferasirox on midazolam pharmacokinetics (PK).

Methods: This was a single-center, open-label, one-sequence, cross-over study conducted in healthy volunteers. There were two treatment periods. During Period 1, volunteers received a midazolam 5 mg oral solution and midazolam PK were performed at 0–12 hours. During Period 2, volunteers received doses of deferasirox 30 mg/kg daily for 5 days and on Day 4, a midazolam 5 mg oral solution was given. Midazolam PK were performed at 0–48 hours.

Results: Twenty-two healthy volunteers were enrolled, and 21 completed the study. Mean age was 32 years, 59% of volunteers were male and all were Caucasian. Descriptive statistics for mean plasma concentration of midazolam and its metabolite, 1-hydroxymidazolam, are summarized below:

AUCinf (h.ng/mL) AUClast (h.ng/mL) Cmax (ng/mL) Tmax (h) T½ (h) Midazolam PK Midazolam alone, n=22 Mean (SD) 68.3 (28.4) 63.8 (26.4) 34.4 (17.4) 0.44 (0.17) 3.7 (0.9) Deferasirox + midazolam, n=22 Mean (SD) 61.7 (37.4) 60.3 (37.2) 27.1 (13.6) 0.47 (0.26) 4.4 (1.4) 1-hydroxymidazolam PK Midazolam alone, n=22 Mean (SD) 22.3 (8.8) 20.8 (7.9) 12.8 (6.7) 0.48 (0.15) 3.3 (1.5) Deferasirox + midazolam, n=22 Mean (SD) 25.6 (10.9) 23.8 (10.0) 11.7 (5.3) 0.5 (0.24) 6.1 (3.5)

There was a statistically, but not clinically, significant reduction (17%) in midazolam exposure (AUC) in the presence of deferasirox in comparison with midazolam administered alone. Similarly, Cmax was decreased on average by 23%.

Conclusion: An increase in midazolam exposure, which would have been expected for an inhibitory effect of deferasirox on the CYP3A4-mediated metabolic pathway, was not observed. A decrease in midazolam exposure is suggestive of an induction effect, albeit weak in nature. Drugs that are substrates for CYP3A4 can be co-administered with deferasirox if necessary.
Production of Inflammatory Mediators by Nanoparticle Stimulated Macrophages.
Authors: Hall David J. , Ufkin Melanie , Frear Meghan E.
Published: October 2019

Nanoparticles are receiving attention because of their many potential applications in drug delivery. The ability to treat humans with Polymethylmethacrylate (PMMA) nanospheres and gold nanoparticles is dependent on the biocompatibility of the nanoparticles. One measure of biocompatibility is the interaction between monocyte derived macrophages (MDM) and nanoparticles. Macrophages initiate many immune reactions with a variety of mechanisms, one such reaction is the release of nitric oxide (NO). Our research shows that RAW 264.7 (murine) and THP-1(human) macrophage cell lines, when treated with gold or PMMA nanoparticles, show the production of nitric oxide and degradation of IKB-α, but not reactive oxygen species. In this study, we used primary human MDMs as a more relevant model system. We report that gold nanoparticles and PMMA nanospheres did not cause the production of the same amounts of NO as their cell line counterparts. Western blot analysis showed the degradation of IKB-α during a 60-minute time course in primary human MDM. These studies suggest that the safety concerns over nanoparticle use are not supported for primary human MDM.
Increased Expression of TLR2 and TLR4 in Mononuclear Cells in Patient with Immune Thrombocytopenic Purpura.
Authors: Cheng Yunfeng , Zou Shanhua , Li Feng
Published: October 2019

Immune thrombocytopenic purpura (ITP) is an autoimmune disease characterized by platelet destruction resulting from autoantibodies against self-antigens and T-cell mediated cytotoxicity. Toll-like receptors (TLRs) are pattern recognition receptors important in mediating the immune response and their activation can lead to production of cytokines. Recent data suggest that TLR2 and TLR4 are crucial for the production of inflammatory cytokines and play central role in autoimmune diseases, yet little is known about their roles in ITP. Here we examined the gene expressions of TLR2 and TLR4 in ITP patients. We hypothesize that significant differences will exist between pre-treatment and post-treatment in ITP patients with similar changes reflected in the plasma concentration of cytokines. Total RNA was extracted from mononuclear cells obtained from 12 ITP patients and 15 healthy subjects. TLR2 and TLR4 mRNA expression levels were analyzed using a quantitative real-time PCR method and their protein expressions were validated by western blot. Plasma concentrations of cytokines IL-2, IFN-γ and TNF-α were measured by ELISA. Correlation analyses were carried out between the mRNA expression levels of TLR2 or TLR4 and the plasma levels of IL-2, IFN-γ and TNF-α. The gene expression of TLR2 and TLR4 were significantly increased in ITP patients comparing to healthy control group (p < 0.05 and p < 0.01, respectively). In addition their mRNA expression levels were decreased back into normal range after remission in 8 patients (p > 0.05, compared to healthy control group). Significantly positive correlations were found between the TLR2 mRNA expression level and the plasma concentration of IFN-γ or TNF-α (R = 0.75, p < 0.05; R = 0.83, p < 0.05, respectively). Changes in the gene expression of TLR4 and in the plasma concentration of IFN-γ or TNF-α were also significantly correlated (R = 0.82, p < 0.05; R = 0.88, p < 0.05, respectively). Directional changes in TLR2 / TLR4 and IFN-γ /TNF-α expression were concordant. However, there was no correlation found between TLR2 / TLR4 and IL-2. Differences in TLR2 and TLR4 expression strongly correlated with changes in IFN-γ and TNF-α suggest that the increased gene expressions of TLR2 and TLR4 in ITP patients may contribute to the pathophysiological progression of this disease by increasing the secretion of IFN-γ and TNF-α. Additional studies need to be performed to further clarify the role of TLRs -cytokines pathway in ITP.
Membrane Microparticles from Patients with Steroid-Induced Avascular Necrosis of Femoral Head Increase the Expression of Fas in Endothelial Cells In Vitro.
Authors: Weng Xi-sheng , Wu Zhi-hong , Wei Yi , Qiu Gui-xing
Published: October 2019

High-dose or long-term usage of steroid have been suggested to be at great risk of developing avascular necrosis of femoral head (ANFH). A theory proposed to decipher the mechanism behind the development of steroid-induced ANFH involves vascular compromise and cell death. Membrane microparticles (MMPs) are fragments shed from plasma membrane blebs of virtually all cell types when submitted to a number of stress conditions, including apoptosis. It has been reported that high-dose dexamethasone cause dysfunction and apoptosis of endothelial cells; and MMPs isolated from the plasma of patients with myocardial infarction of preclampsia were found to cause damage in isolated arteries in vitro. We hypothesize that MMPs generated after high-dose or long-term administration of steroid facilitates the apoptosis of endothelial cells initiated by steroid, which contributes to the development of ANFH. MMPs were isolated from the blood of 4 healthy individuals, and 5 patients demonstrated to have ANFH by magnetic resonance imaging (MRI) and X-ray. The mean duration and accumulated dose of steroid administration were 8 months and 2000 mg respectively. The 3rd generation of human umbilical vein endothelial cells (HUVEC) was exposed to MMPs (corresponding to 0.2-fold circulating plasma level) prepared above. After 48 hours, part of cells was lysed to make total cell lysate. And the rest were used to prepare mRNA. The expression level of Fas was detected with reverse-transcript PCR and Western Blot. Our results shown that treatment of HUVEC with MMPs from ANFH patient’s blood significantly increases the transcription and expression of Fas, indicating that MMPs derived from patients with steroid-induced ANFH exacerbates high-dose steroid-induced apoptosis of endothelial cells by enhancing the expression of Fas.
Oral Anticoncipient Induced Angiosarcomatosis and Disseminated Intravascular Coagulation.
Authors: Pfliegler George P. , Kovacs Erzsebet , Nemeth Hajnalka , Szollosi Zoltan , Sapy Peter , Peter Mozes , Koncsek Marta
Published: October 2019

A 50-year old woman presented herself with fever, hemoptoe and cough. On the basis of the clinical signs, elevated d-dimer and suggestive lung-scan, pulmonary embolism was diagnosed and low molecular weight heparin was started. Sonography, however, revealed one giant and two smaller hemangiomas in the liver and CT-scan small angiomas in the lung. Anemia, low platelet count, d-dimer positivity lead to a revision and the diagnosis of dissemniated intravascular coagulation (DIC) was set up. A sudden and severe abdominal pain, preshock, blood in the abdominal cavity indicated the rupture of the hemangioma. The ruptured hemangioma was successfully embolized. While preparing for surgical resection a subsequent bleeding developed but after the addition of rFVIIa it seized, and she became suitable for the removal of the large cavernous hemangioma. In two-month she recovered when bloody sputum reappeared, histological diagnosis from the lung was the same as that of the liver. She recovered, however, the hemangiomas both in lung and the liver were rapidly increasing, therefore a revision of histology was asked. Unfortunately before revision was completed she died from an additional major intrathoracal bleeding. Revised histology proved hemangiosarcoma. This rare and very aggressive disease is usually induced by toxic materials, e.g. vinyl-chloride, thorium dioxide or arsene. In our case her long-term estrogene therapy (from 20 to 44 yrs; subsequently restarted 4 month prior the present complaints due to gynecological bleeding) is probably responsible for the development and transformation of her hemangiomas. In patients with liver angiomas regular control (including sonography) seems to be justified, especially once they are on the pill. Therapeutic options e.g. angiogenesis inhibition by cyclooxygenase II inhibitors or peroxisome proliferator-activated receptor-gamma agonists are discussed.
Compositional Differences in the Oligosaccharide Components of Generic Versions of Enoxaparin and Dalteparin.
Authors: Jeske Walter P. , Brubaker Aleah , Hoppensteadt Debra , Walenga Jeanine M. , Ma Qing , Fareed Jawed
Published: October 2019

Generic versions of the low molecular weight heparins (LMWHs) enoxaparin (Sanofi-Aventis, Paris, France) and dalteparin (Pfizer Inc, NY, USA) have been developed and marketed in India and South America. Regulatory bodies in North America and Europe are evaluating generic versions of enoxaparin and dalteparin. No regulatory approval guidelines exist for these hybrid drugs which are of natural origin and exhibit structural differences due to differences in manufacturing processes. To investigate the oligosaccharide composition of enoxaparin and dalteparin, molecular profiles were compared prior to and after digestion with heparinase-1 (0.1–1.0 U/ml). Functional properties of the pre- and post-heparinase digested agents were profiled in anticoagulant, antiprotease and protease generation assays using whole blood, plasma and serpins such as antithrombin and heparin cofactor II. Additionally, protein binding profiles for both the branded and generic LMWHs were carried out using human serum albumin. At concentrations of heparinase <0.5 U/ml, significant differences in oligosaccharide composition were noted. At higher concentrations, branded and generic LMWHs showed a comparable digestion profile with the exception of differences in salt content and disaccharide formation. The branded and generic products exhibited product-dependent differences in their affinity to heparin cofactor II and antithrombin. The amount of AT affinity components ranged from 20–46% in the nondepolymerized products and was markedly reduced after depolymerization (5–15%). Protamine sulfate and platelet factor 4 titration curves were distinct for the generic versions of enoxaparin. Dalteparin and its generic version showed a stronger inhibition of thrombin generation as measured by amidolytic assay. Protein binding profile also showed measurable differences among these agents. These studies suggest that the manufacturing processes for generic versions of enoxaparin and dalteparin result in products that can be differentiated despite identical anti-Xa unit potencies and molecular profiles. Additional microstructural features such as the presence of anhydromanno groups and other anomeric transformations may also contribute to the chemical and biologic heterogeneity among the generic versions of the branded products. Therefore, besides the molecular profile, additional biologic actions and protein binding profiles can also be used to demonstrate biosimilarities of differentiation among these agents.
The Direct Factor Xa Inhibitor Apixaban Produces Broad Antithrombotic Activity with Limited Effects on Bleeding Time in Rats.
Authors: Schumacher William A. , Bostwick Jeffrey S. , Stewart Anne B. , Steinbacher Thomas E. , Pinto Donald J. , Wong Pancras C.
Published: October 2019

Apixaban is an oral, direct and highly selective factor Xa inhibitor which is in late stage clinical development for the prevention and treatment of thromboembolic diseases. Apixaban is potent against human and rat factor Xa (Ki = 0.08 and 1.3 nM, respectively). The efficacy/safety profile of apixaban was determined in experimental models of thrombosis and hemostasis performed in anesthetized rats. Thrombosis was induced either by topical application of FeCl2 to the vena cava (VT) or carotid artery (AT), tissue factor infusion into the vena cava (TF-VT), or within an extracorporeal arterial-venous shunt (ST). Bleeding time was measured in response to template incision of the renal cortex. Apixaban was administered as a continuous i.v. infusion of 0.1, 0.3, 1 or 3 mg/kg/h starting 60 min before each experimental procedure (n=5 or 6 per dose). These respective doses increased the ex vivo prothrombin time by 1.3, 1.9, 3.0 and 3.9 times control baseline, and achieved plasma concentrations of 0.3, 1.4, 5.0 and 12.2 μM. In comparison to vehicle treatment, the 3 mg/kg/h dose of apixaban decreased thrombus weight by 90 ± 2% in VT, 62 ± 7% in TF-VT, 62 ± 4% in AT, and 79 ± 3% in ST (all p<0.05). A 50% thrombus weight reduction sufficient to prevent vascular occlusion in each model would require projected doses (mg/kg/h) of 0.4 (VT), 1.9 (TF-VT), 0.8 (AT) and 1.1 (ST). The lowest dose level which preserved carotid blood flow at the pre-injury level during thrombus formation was 0.3 mg/kg/h. Apixaban prolonged bleeding time in a dose-dependent manner with no effect detected at 0.1 and 0.3 mg/kg/h, while significant (p<0.05) increases of 1.34 ± 0.12 and 2.13 ± 0.17 times control were observed at 1 and 3 mg/kg/h, respectively. In comparison, aspirin increased bleeding time by 1.42 ± 0.1 times control (n=7, p<0.05) when tested at a 10 mg/kg dose, a dose which produced maximum cyclooxygenase inhibition. These studies predict a wide therapeutic index for apixaban based on the prevention of occlusive thrombosis in a variety of experimental models without excessive bleeding time prolongation. These results are consistent with the safety and efficacy observed with apixaban in phase II clinical trials.
Possible Involvement of Heparin-Binding Protein in Antibody-Mediated Non-Hemolytic Transfusion Reaction.
Authors: Furuta Rika A. , Yasui Kazuta , Matsuyama Nobuki , Fukumori Yasuo , Kimura Takafumi , Hirayama Fumiya , Tani Yoshihiko , Shibata Hirotoshi
Published: October 2019

In antibody-mediated non-hemolytic transfusion reactions, Transfusion-Related Acute Lung Injury (TRALI) tends to occur immediately after a blood transfusion. Leukocyte antibodies or immune complexes have been frequently shown to be associated with the syndrome, although the mechanisms by which they induce TRALI are poorly understood. To explore the mechanism of TRALI, an in vitro whole blood cell culture assay was established in which cells were stimulated by human antibodies and the activation of neutrophils was monitored by a cell surface marker (Mac-1) with FCM. To identify soluble mediators that were released instantly following immune stimulation, we examined the release of soluble factors, including, Perforin, IL-6, TNF-a, and Heparin binding protein (HBP) using ELISAs. In addition, the involvement of two neutrophil FcγRs (FcγRIIIb and FcγRIIa, also known as CD16 and CD32, respectively) in release of HBP was examined during antibody-induced cell activation using anti-FcγR blocking antibodies. Substantial amounts of HBP were released within 30 min upon stimulation by human antibodies, although other soluble mediators were not released within such an eary period. Furthermore, the release of HBP was mediated via signals though both FcγRIIIb and FcγRIIa. HBP that have been known as a multifunctional molecule in early inflammation appears to be a one of the primary effector molecules of antibody-mediated non-hemolytic transfusion reactions including TRALI.
Low ADAMTS13 Activity in Clinical Remission Predicts the Risk of TTP Relapses.
Authors: Wu Haifeng M. , Casper T. Charles , Cataland Spero R. , Lin Shili , Kennedy Melanie , Jin Ming
Published: October 2019

Idiopathic thrombotic thrombocytopenic purpura (TTP) is a devastating disorder characterized by frequent recurrences. A predictive biomarker that allows for the early identification and intervention prior to the overt relapse of disease is needed. In the present study, the assay for ADAMTS13 activity was modified to adequately detect ADAMTS13 activity as low as 0.5%. Serial samples (n=157) collected from 24 TTP patients during periods of sustained clinical remission were then studied. These patients were followed up quarterly for an average of 23 months, during which time 9 episodes of TTP relapses from 6 patients occurred. We measured ADAMTS13 activity in these samples and then employed logistic regression modeling to evaluate the relationship between ADAMTS13 activity levels and the risk of TTP relapses. Our data demonstrated that lower ADAMTS13 activity and younger age are significantly associated with a higher probability of relapse in the 3 months following the date the specimens were taken. In contrast, ADAMTS13 antibody IgG levels are not predictive of TTP relapses. Identification of low ADAMTS13 activity during remission as a key risk factor for TTP relapses provides a new screening strategy to identify patients who may benefit from prophylactic therapy prior to disease relapses.
CD34+ Hematopoietic Progenitor Cells Express CD 184 Antigen and to a Minor Extend MMP9.
Authors: Abendroth Jens , Schmoll Hans J. , Wolf Hans-Heinrich
Published: October 2019

For clinical purposes the CD34 surface antigen characterises hematopoietic progenitor or stem cells (HSC) and will be measured routinely for peripheral hematopoietic stem cell transplantation (PBSCT). Concerning homing mechanisms of HSC not only the number of CD34+ cells could be important. However, there are several other characteristic surface antigens indicating proliferation. For example, the cell surface receptor CD184 has been found on CD34+ HSC and seems to be necessary for stem cell development and migration. CD184 knock-out mice are known to present insufficient hemato- and lymphopoiesis. MMP9, a gelatinase cleaving collagen typ IV, expressed on HSC, is necessary for migration and probably also for HSC engraftment. In leukapheresis products used for PBSCT we were interested in CD184 and MMP9 protease expression on CD34+ cells and their impact on homing.

Methods: 84 leukapheresis products for autologous stem cell transplantation were analysed in 65 patients. The patients suffered from oncologic (n= 45) and haematological (n=20) malignancies. There was no bone marrow infiltration found before starting leukapheresis. Expression of CD184 and MMP9 on CD34+ cells were analysed by flow cytometry. Additionally, the number of CD45+ cells was measured (BD Biosciences). Statistical analysis was run by SPSS using students t-test and Mann-Whitney-U test.

Results: 44,7% (+/− 3,1%) of CD34+ cells presented the CD184+ antigen, but MMP9+ expression was found only in 5% (+/− 1,4%) of CD34+ HSC, respectively. Regarding the patients age, sex or underlying diseases no significant differences were seen.

Discussion: High expression rate of CD184 antigen and MMP9 on CD34+ HSC could be favorable for engraftment and success of hematopoietic stem cell transplantation. Surprisingly, in our study on leukapheresis products less than half of CD34+cells were found to express CD184+ antigen, and only 5% presented MMP9 expression. As time to engraftment after PBSCT of the patients did not differ from mean values either antigen expression could develop on a further step of hematopoietic reconstitution or the patients presented an "alternative pathway" establishing hematopoiesis and immune defense mechanisms. Further studies on proliferation and differentiation mechanisms of HSC are requested.
Fetal Immune Responses Post In Utero Transplantation of Embryonic Stem Cells, Bone Marrow and Fetal Liver Hematopoietic Stem Cells.
Authors: Nedelcu Elena , Moustafa Mohamed , Srivastava Anand , Donahue Jody , Carrier Ewa
Published: October 2019

Introduction: We have previously shown that in utero transplanted ES cells survive and integrate into the fetus development and have established a murine model for the study of the in vivo differentiation of ES cells. The goal of this study was to monitor the fetal immune responses post in utero transplantation of ES cells, bone marrow (BM) and fetal liver (FL) hematopoietic stem cells.

Materials and Methods: Murine (MHC)-mismatched ES cells genetically engineered to express yellow fluorescent protein (YFP-ES cells) were cultured on mitomycin-treated feeder layers for four days prior to in utero transplantation (IUT) in medium containing leukemia inhibitory factor. A cell dose of 5x104 YFP-ES cells ((H-2kb) was injected intraperitoneally in the fetuses of Balb/c (H-2Kd) pregnant mice at E12- E14. BM and FL hematopoietic stem cells (H-2kb) were transplanted in utero at a dosage of 1×109 cells/kg fetal body weight into E12-E14 BALB/c fetuses (H- 2Kd). Fetal immune responses were monitored by in vitro mixed lymphocyte reaction and cytotoxicity assays performed with self (Balb/c), allogeneic ES cells, BM or FL hematopoietic cells, and 3rd party (C3H cells). Cytokine levels (IL-2, IFN-gamma, IL-4 and IL-10) were determined in the cell culture supernatants from cytotoxicity assays. Liver tissue sections were prepared from in utero transplanted fetuses and examined for the presence of lymphocytic infiltration.

Results: In utero transplantation of YFP-ES-cells did not induce tolerance in the fetuses and was associated with increased cytokine production compared with BM and FL groups. Microscopic examination of liver sections of ES cell transplanted group revealed the presence of marked inflammatory infiltrate.

Conclusions: Embryonic stem cells transplanted in utero induce fetal immune responses and increased cytokine production associated with (MHC) upregulation.
Differentiation of Mesenchymal Stem Cells from Rat Bone Marrow into Dopaminergic Neuron-Like Cells In Vitro.
Authors: Chen Li , He Dongmei , Zhang Yuan
Published: October 2019

Mesenchymal stem cells (MSC) from bone marrow cavity are multipotent cells. Their primary function is to support the growth and differentiation of hematologic progenitors. MSCs have been shown to differentiate into a variety of cell types including: bone, adipocytes, cartilage, neuron-like, and muscle-like cells. This project aimed to induce MSCs from rat bone marrow into mature dopamine secreting cells. MSCs were isolated from rat bone marrow, cultured and passaged. After propagating for three generations in vitro culture, MSCs were induced by epidermal growth factor, basic fibroblast growth factor and retinoic acid. After induction, morphologic change was examined by light microscope. NSE,MAP-2a, b and tyrosine hydroxylase (TH) was examined by immunocytochemistry. The related genes of the differentiated neurons, such as Nurr-1, nestin, mash-1,DR2-L,AADC and TH were detected by RT-PCR. After MSCs were inducted for 7 days,14 days and 21 days, dopamine production and release in the extract and medium of dopaminergic-induced cultured cells was assayed by dopamine ELISA. After 14 days of induction, MSC showed neuron-like morphologic changes and expressed NSE, MAP-2a, b and TH. RT-PCR. showed that these induced cells expressed nerves stem cells gene Nestin,Nurr-1 and dopamine nerves gene mash-1,DR2-L,AADC,TH. Most importantly, dopamine ELISA analysis showed the evidence of dopamine release in the extract and medium of dopaminergic-induced clonal MSCs. The results suggest that bone marrow MSCs from rat can be induced to differentiate into dopaminergic neuron-like cells in vitro. Bone marrow MSCs will provide a promising source of neural progenitor cells and may be a favorable candidate for cellular therapy of Parkinson’s disease.
α-Enolase Is the c-myc Promoter-Binding Protein.
Authors: Miller Donald M. , Campbell Hunter , Thomas Shelia D. , Sedoris Kara C.
Published: October 2019

Quadruplex-forming sequences occur disproportionately in the promoters of growth regulatory genes, including c-myc. These sequences appear to play important roles in the regulation of gene expression. The c-myc quadruplex-forming sequence occurs in the c-myc P1 promoter region which is responsible for more than 80% of c-myc expression. We have used proteomic techniques used to identify quadruplex-binding proteins. Two dimensional electrophoretic gels of nuclear extracts were blotted and screened with G-rich (quadruplex forming) and C-rich (control) oligonucleotides. We were surprised to observe that α-enolase binds the G-rich, but not C-rich oligonucleotide. Using electrophoretic mobility shift assays (EMSA), we confirmed that both α-enolase and its alternative translation product, MBP-1, bind quadruplex-forming oligonucleotides. We then tested the hypothesis that both α-enolase and MBP-1 may play important physiologic roles by binding the c-myc quadruplex-forming sequence. Electrophoretic mobility shift assays were used to characterize the binding of α-enolase and MBP-1 to the c-myc quadruplex-forming sequence. Binding of AS1411, a quadruplex forming oligonucleotide, was used as a control. Both α-enolase and MBP-1 demonstrated strong binding to the c-myc P2 promoter sequence (containing the quadruplex-forming sequence). A “supershift” was seen when an anti-α-enolase antibody was included in the incubation although control antibodies did not alter the EMSA pattern. The quadruplex oligonucleotide aptamer, AS1411, competed for α-enolase and MBP-1 binding to the c-myc promoter sequence. However, a nonquadruplex-forming oligonucleotide did not compete. A mutation in the c-myc quadruplex sequence abrogated binding of both α-enolase and MBP-1. In order to test the biological effect of the α-enolase/MBP-1 interaction with the c-myc promoter, we characterized the effect of constitutive overexpression of each α-enolase product, individually. Although, overexpression of MBP-1 by transfected cells resulted in down regulation of c-myc expression and induction of apoptosis, constitutive overexpression of α-enolase did not alter c-myc expression or the rate of cellular proliferation. Cells grown in low glucose medium demonstrate increased MBP-1 levels which results in decreased c-myc expression and inhibition of cellular proliferation. A similar response was seen in hypoxic cells, indicating that enolase/MBP-1 plays an important role in cellular responses to external stress. These results suggest that MBP-1 down regulates c-myc expression by simultaneous binding to the P1 and P2 promoter sequences, while α-enolase binds only the P2 sequence. This suggests a very important regulatory role for α-enolase/MBP-1 in the relationship between cellular metabolism and growth regulation. Enolase/MBP-1 quadruplex-binding likely plays an important role in regulation of c-myc expression.