bluuebunny/crossref_metadata_2025 huggingface dataset:
MicroRNAs Specific for Immune-Privileged Diffuse Large B-Cell Lymphoma.
Authors: Robertus JanLukas , Harms Geert , Blokzijl Tjasso , de Jong Daphne , Booman Marije , Rosati Stefano , Schuuring Ed , Kluin Philip M. , van den Berg Anke
Published: October 2019

Introduction Primary diffuse large B-cell lymphomas (DLBCL) constitute a heterogeneous group of tumors with a great diversity in both clinical presentation and outcome. Besides nodal DLBCL, about 40% of these tumors present in different extranodal sites, both immune privileged sites such as the testis and the central nervous system (PCNSL) but also non immune privileged sites such as bone, stomach, skin and mediastinum. Remarkably each of these sites show a marked divergence in biological behavior. Recent studies have shown that certain non-coding short RNAs (22nt) called miRNAs may play a role in the pathogenesis of DLBCL. These miRNAs posttrancriptionally down regulate expression of genes. Aim of study To examine the expression levels of selected miRNAs in different DLBCL sites.

Methods and Materials 51 cases of Ann Abour stage I and II primary DLBCL were divided into 4 groups; 19 nodal cases, 11 non-immune privileged site-associated (non-IP) extranodal cases, 9 PCNSL, and 12 testicular DLBCL. Each case was classified GCB or non-GCB using the algorithm as described by Hans et al. 13 miRNAs were selected from current literature on the basis of their involvement in B-cell lymphoma (miR-15, -16, -21, -221, -155, - 181a, -18a, -19, -20a, 17-3p, -17-5p and -92). The expression levels of the mature microRNAs were determined by qRT-PCR using Taqman miRNA assays. Results were compared by using 2−ΔCt and 2−ΔΔCt. Differences in expression levels between the four groups was tested using ANOVA.

Results Of the selected microRNAs, miR-127, miR-17-5p and miR-221 show a significant difference in expression level between the immune privileged site-associated DLBCL (IP-DLBCL) and nodal DLBCL. Both miR-17-5p (p=0.0003) and miR-221 (p=0.0032) show an increased expression level in PCNSL compared to nodal DLBCL and miR-127 shows a significant increased level in testicular DLBCL compared to nodal (p=0.0011) and non-IP (p=0.0393) DLBCL. Also of interest is the fact that miR-17-5p and miR-127 show a significant difference in the level of expression between both PCNSL and testicular DLBCL. MiR-155 shows no significant difference in expression levels between nodal and extranodal DLBCL. Analysis on the basis of GCB and non-GCB classification is currently being undertaken.

Conclusion In PCNSL miR-17-5p and miR-221 are specifically increased whereas miR-127 shows a specifically higher expression in testicular DLBCL when compared to non-IP and nodal DLBCL. Also PCNSL and testicular DLBCL differ in expression of miR-17-5p and miR-127. In contrast to previous reports miR-155 does not show any significant difference between nodal and extranodal groups. This may be caused by the selection of stage I or II primary DLBCL in contrast to previous publications which used a more heterogeneous group of DLBCL.
Intermediate Filaments of Endoskeleton within Human Erythrocytes.
Authors: Terasawa Kazutaka , Taguchi Takehito , Momota Ryusuke , Naito Ichiro , Ohtsuka Aiji
Published: October 2019

The mammalian erythrocytes had been considered to keep their deformable biconcave shape solely by the membrane and the underlying membranous cytoskeleton. However our previous study showed new aspects that human erythrocytes possessed a cytoplasmic endoskeleton apart from the classical membranous cytoskeleton containing beta-actin and neurofilament heavy chain (NF-H) [Terasawa K, et al. Arch. Histol. Cytol2006; 69: 329–340]. Several studies showed that the intermediate filaments such as neurofilament light chain (NF-L), synemin and vimentin were detected in the nucleated erythrocytes [Granger BL, et al. Science.1983; 221: 553–556] and extended from the nuclear envelope to the membranous marginal bands [Granger BL, et al. J. Cell Biol.1982; 92: 299–312]. In the present study, we asked whether the intermediate filaments such as NF-L and vimentin also exist within the newly found endoskeleton of anucleated erythrocytes. NF-L and vimentin were detected within erythrocytes by western blotting and immunofluorescence. The NF-L were detected along the hillside filament group running parallel to the slope between convex periphery and the concave center and vimentin was distributed along several filaments in the whole erythrocytes by the ion-etching/scanning electron microscopy combined with immunocytochemistry. At the hillside region NF-L co-existed with vimentin along the hillside filaments by back scatter emission signals. We concluded that the endoskeletal filaments of the erythrocyte contain intermediate filaments such as NF-L, NF-H and vimentin as those of the nucleated erythrocytes of avian or lower vertebrates. The intermediate filaments of the endoskeleton within the erythrocytes may keep its unique biconcave disc shape against external circumstances.
Pharmacokinetics of Nilotinib in Subjects with Normal or Impaired Hepatic Function.
Authors: Holland Jaymes , Tanaka Chiaki , Zhou Wei , Sethuraman Venkat , Heuman Douglas M. , Schran Horst
Published: October 2019

Nilotinib, a selective and potent inhibitor of Bcr-Abl tyrosine kinase, has demonstrated activity at 400 mg twice daily dosing against imatinib-resistant forms of Bcr-Abl in patients with CML or Ph+ALL in an open label phase I/II study. In this patient population, elevated liver transaminases levels over baseline, which may be related to a combination of past medical history and the hepatotoxic effects of previous medications, are frequently observed but are generally mild and self-limiting. Nilotinib is metabolized in the liver by CYP3A4, and the metabolites are excreted exclusively via the bile into feces. This study was designed to assess pharmacokinetics of nilotinib across a broad spectrum of hepatic impairment in comparison to a control group with normal hepatic function in order to provide guidance for the safe use of the drug in patients with hepatic impairment. Hepatic impairment was categorized using the three Child-Pugh categories (mild, moderate, and severe). The demographics of the impaired and control group were similar with respect to ethnicity, age (years) impaired (47–64) control (48–62), BMI (kg/m2) impaired (23.6–34.7) control (22.8–32.0), and weight (kg) impaired (73.9–103.9) control (73.3–109.5). Serum samples for determination of nilotinib serum concentration by liquid chromatography tandem mass spectrometry were collected up to 120 hours following a single oral 200 mg dose of nilotinib after an overnight fast. Pharmacokinetic parameters were derived from the individual serum concentration-time data by non-compartmental methods. Pre-dose (nilotinib-free) serum samples were obtained to determine ex vivo nilotinib serum protein binding. The results from 14 subjects (6 subjects in each of the mildly and moderately impaired groups and 8 in the control group) in this ongoing study are reported here. Nilotinib exposure at 200 mg dose was similar in patients from a previous clinical study and subjects in the control group of this study. Nilotinib AUC was increased on average by 18% in the mild impairment group, and 23% in the moderate impairment group as compared to the control group. A total of 6 grade 1 and 2 adverse events were reported comprising of back pain, worsening abdominal pain, headache, nausea, heart burn, and indigestion. One adverse event (grade 2 worsening back pain) was reported in the control group. No laboratory or cardiac monitoring findings were considered as adverse events during the treatment period. The currently available results suggest that the pharmacokinetic profile of nilotinib is comparable between subjects with normal hepatic function and subjects with mild or moderate hepatic impairment.
Global Epigenomic Profiling Demonstrates That Myelodysplasia Is Characterized by a Distinct Epigenetic Signature with Aberrant DNA Methylation Changes Involving Various Malignant and Hematopoietic Pathways.
Authors: Zhou L. , Opalinska J. , Sohal D. , Thompson R. , Li Y. , Kotla V. , Pahanish P. , Parmar S. , Kambhampati S. , Figueroa M. , Ye K. , Friedman E. , Wickrema A. , Melnick A. , Greally J. , Verma A.
Published: October 2019

Myelodysplasia (MDS) is a clonal hematopoietic disorder that leads to ineffective hematopoiesis and peripheral cytopenias. DNMT inhibitors such as azacytidine have led to clinical responses in patients, though the genes affected by epigenetic alterations are not well known. Whole genome DNA methylation was analyzed by a recently described novel method, The HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)) that uses differential methylation specific restriction digestion by HpaII and MspI followed by amplification, two color labeling and cohybridization to quantitatively determine individual promoter island methylation. A whole genome human promoter array (Nimblegen) was used to determine the level of methylation of 25626 gene promoters by calculating HpaII/MspI cut fragment intensity ratio. Peripheral blood leucocytes from 13 patients with MDS were compared to 9 age matched normal and anemic controls. Gene expression analysis was performed using 37K oligo maskless arrays on cDNA obtained from the same samples. Analysis showed that whole genome methylation profiling has greater discriminatory power in separating clusters of MDS samples from normal and anemic controls when compared to gene expression analysis. Unsupervised clustering based on epigenetic profiling demonstrated that only two cases of early MDS clustered with normals as compared to absolutely no separation between MDS and normals with clustering based on gene expression patterns. A high correlation (r=0.88–0.96) was observed between global methylation profiles of matched sets of bone marrow and peripheral blood leucocyte samples from selected patients demonstrating that peripheral blood leucocytes can be a valid surrogate for epigenomic analysis. Further analysis showed that genes consistently aberrantly methylated in MDS included Syk kinase, HOXB3, several histone acetyltranferases and others. Functional analysis by Ingenuity showed that cancer and cell signaling pathways were the most affected by epigenetic silencing. Most interestingly, a large proportion of gene promoters were also aberrantly hypomethylated. These included genes from Ras oncogene family, the CDC42 GTPase, various methyl binding proteins and other proteins mainly encoding for cancer and hematopoiesis functional pathways, thus biologically validating our analysis. Therefore, our data demonstrates that MDS is characterized by distinct epigenetic aberrations that are preserved in peripheral blood leucocytes. These can be the basis of future studies on pathogenesis and diagnosis for this disease and can potentially uncover a new set of therapeutic gene targets.
Targeting Myeloproliferative Diseases with JAK2 Inhibitors.
Authors: Markovtsov Vadim , Yu Diane , Gelman Marina , Lang Wayne , Taylor Vanessa C. , Huynh Stacey , Frances Roy , Fang Shuling , McLaughlin John , Bhamidipati Sohasekhar , Clough Jeffrey , Singh Rajinder , Park Gary , Sweeney David , Tonkin Elizabeth , Bahjat Rena , Chang Betty , Pine Polly , Daniel Ruby , Payan Donald G. , Holland Sacha , Hitoshi Yasumichi
Published: October 2019

Limited options provided by the current standard of care for the patients suffering from myeloproliferative diseases (MPDs) prompted an extensive search for the underlying molecular mechanisms of these disorders. Recent discovery of a single activating mutation (V617F) in JAK2 kinase gene associated with the development of the polycythemia vera (PV), essential thrombocythemia (ET) and chronic idiopathic myelofibrosis (CIMF) opened up a possibility to develop highly targeted therapies against these debilitating ailments. To that end, we engineered cytokine-independent Ba/F3 cell line expressing the V617F mutant of JAK2 to screen a focused small molecule library for potential inhibitors of JAK2 V617F -dependent proliferation. We confirmed the ability of hit compounds to inhibit proliferation of JAK2-dependent tumor cell lines using UKE-1 and SET-2 cells carrying the V617F JAK2 mutation. A FACS-based phosphoSTAT5 assay was then used to demonstrate that the hits directly targeted mutant JAK2. JAK3 activity of each compound was evaluated in IL-2-dependent CTLL-2 cell line using phosphoSTAT5 FACS and proliferation assays. To avoid hits with nonspecific antiproliferative activity, the hits were tested in JAK2-independent MOLT4, A549 and H1299 cell lines. Compound hits with the desirable properties were further evaluated for their ability to inhibit JAK2, JAK3 and other kinases in the context of T cell, B cell, or mast cell activation using a variety of cell-based assays as well as in the in vitro biochemical assays. We identified a number of compounds that potently inhibit growth of the two V617F mutant cell lines with EC50s varying from 20 to 500 nM, but do not affect proliferation of control cell lines MOLT4, A549 and H1299 to the same degree. These compounds induce strong and highly specific suppression of STAT5 phosphorylation with IC50s of 10 to 200 nM in SET-2 and V617F JAK2 expressing Ba/F3 cells. One of the hits with the desirable biological and pharmacokinetic profiles was further evaluated in V617F JAK2 Ba/F3 engraftment mouse model where it demonstrated significant extension of survival at 150 and 200 mg/kg bid. Such potent JAK2 inhibitors could become the basis for the next generation of compounds targeting JAK2-dependent myeloproliferative diseases.
Pegfilgrastmin in Children with Severe Aplastic Anemia during Immunosuppressive Therapy.
Authors: Pawelec Katarzyna , Matysiak Michal J.
Published: October 2019

Severe neutropenia and infections connected with leukopenia are same of the main complications in immunosuppressive therapy (IST) of severe aplastic anemia (SAA). Granulocyte-colony-stimulating factors are currently given as supportive therapy in SAA protocol treatment beside antithymocyte globulin and cyclosporine A (CSA). We present four cases of girls aged 6–16 years with SAA without family bone marrow donors, who received pegfilgrastmin in IST. The protocol included: antithymocyte globulin (rabbit) 3.75mg/kg/bw iv for 5 days, CSA 5mg/kg/bw orally for 180 days and pegfilgrastmin (Neulasta-Amgen) 6mg in patients over 45kg and 100μg/kg in children less than 45 kg every 10 days sc until absolute neutrophil count (ANC)reached > 1.5x109/l. Blood was monitored every 7 days during therapy. Two patients received 4 doses of pegfilgrastmin. After this therapy the ANC level was normal without any decreases and we observed slow, gradual rise ANC in this girls In these patients there was a complete remission on day 180 of treatment and both are still in remission month 34. The other two patients received 6 doses of pegfilgrastmin. We observed fairly rapid rise and fall of leukocytes during the first 30 days of therapy. The ANC level became normal on day 70 of treatment. One of the patients is now in partial remission day 320. The other girl did not respond to the first course of IST and she received the second course of IST due to the lack of an unrelated bone marrow donor. Pegfilgrastmin was also administered during this therapy. After 3 doses of pegfilgrastmin leukocyte level rose to 5.3x109/l and ANC level to 2.3x109/l. At present the patient is in partial remission and is still receiving CSA day 210. Pegfilgrastimn administered every 10 days was well tolerated by all four patients and there were no adverse events. We observed that pegfilgrastmin was as effective as filgrastmin which was previously administered. No changes were noted during ANC recovery. However administration of pegfilgrastmin reduced the number of injections given and improved the patient’s well-being.
Vorinostat (SAHA) Inhibits STAT6 Phosphorylation and Transcription, Downregulates Bcl-xL, and Induces Apoptosis in Hodgkin Lymphoma (HL) Cell Lines.
Authors: Buglio Daniela , Georgakis Georgios , Arima Kazuhiko , Liu Yong-Jun , Younes Anas
Published: October 2019

Vorinostat (SAHA) Inhibits STAT6 Phosphorylation and Transcription, Downregulates Bcl-xL, and Induces Apoptosis in Hodgkin Lymphoma (HL) Cell Lines. Although the malignant Hodgkin and Reed Sternberg (HRS) cells of HL are of B-cell origin, they infrequently express B-cell antigens. Recent studies demonstrated that several B-cell specific genes are silenced in HRS cells by epigenetic mechanism, suggesting that this process may be reversible and could be explored therapeutically with deacetylase (DAC) inhibitors or hypomethylating agents. Pan-DAC and isotype-selective DAC inhibitors have shown promising activity in vitro and in vivo in a variety of lymphoid malignancies, including HL. However, the mechanisms of antiproliferative action of DAC inhibitors in HL remain unknown. In this study, we examined the antiproliferative effects of the pan-DAC inhibitor vorinostat (inhibits class I and class II DACs) on HL cell lines and determined its effect on signaling mechanisms that are known to promote HRS cell survival, including STAT3, STAT6, Akt, and ERK pathways. Vorinostat inhibited DACs as evident by the increase in histone-3 acetylation as early as 30 minutes of incubation. Furthermore, vorinostat induced the expression of the cell cycle regulatory protein p21 which was associated with an early increase in the G2M cell cycle fraction in HL cells. Vorinostat had no inhibitory effect on SATA3 or ERK, but inhibited STAT6 phosphorylation and transcription in a dose and time dependant manner. This effect was associated with a decrease in Akt phosphorylation on Ser473 residue. Because STAT6 has been reported to transcriptionally regulate Bcl-XL and Thymus and activation-regulated chemokines (TARC), we examined the effect of vorinostat on these two targets in HL cells. TARC has been shown to play an important role in attracting Th2-type T cells and T-regulatory (T-reg) cells, and to be elevated in sera from patients with HL. Vorinostat downregulated the mRNA expression of TARC in a dose dependent manner, suggesting that it may have a role in regulating chemotaxis of reactive T cells and T-reg cells to HL microenvironment in vivo. Moreover, vorinostat reduced the cellular level of the antiapoptotic protein Bcl-xL which was associated with activation of the caspase pathway, and induction of apoptosis in HL cells. Collectively, these data suggest that DAC inhibition in HL by vorinostat may induce cell death by inhibiting STAT6 and downregulating its antiapoptotic target bcl-xL protein. Furthermore, our data suggest that DAC inhibition may have an added effect in vivo on the cellular component in HL microenvironment by inhibiting TARC.
Intravenous Immunoglobulins Regulation of Human B Cells Is Mediated through Extracellular Signal-Regulated Kinase 1 and 2.
Authors: Ducas Éric , Dussault Nathalie , Racine Claudia , Jacques Annie , Paré Isabelle , Côté Serge , Néron Sonia
Published: October 2019

Intravenous immunoglobulin (IVIg) has become increasingly important as therapy in autoimmune and inflammatory diseases, but its immunomodulatory action is still not fully understood. In the treatment of autoimmune diseases, the large repertoire of human immunoglobulin G (IgG), which constitutes IVIg, is assumed to modulate several immune cells including B cells. As a matter of fact, IVIg can act directly upon B cells in vitro by reducing their expansion and enhancing the differentiation of small populations of both naïve and memory B cells. In the present work, we used a panel of human cell lines representative of B cell major maturation stages and treated them with IVIg in order to identify model cell lines representative of normal B cells that could be used to further investigate IVIg effects on B cell physiology and identify the signal transduction pathways involved. Consistent with the observed effects on B cells, one of these cell lines, the DB cells, which express both FcγRIIB receptor (CD32) and surface IgG (B cell receptor [BCR]), showed reduced proliferation and upregulated secretion in presence of IVIg. As revealed by immunobloting and flow cytometry analysis, DB cells response to IVIg was paralleled by increased phosphorylation of ERK1/2, Gab1 as well as Akt, and addition of ERK1/2 activity inhibitors such as PD98059 to cultures of DB cells abolished the negative IVIg effect on proliferation of this cell line. Furthermore, in contrast to the above described IVIg consequence on ERK1/2 phosphorylation, incubation of DB cells in presence of various amounts of aggregated IgG within intramuscular immunoglobulin (IMIg) or IVIg all prevented ERK1/2 up-regulated phosphorylation. Comparable results were obtained using Pfeiffer cells, which are surface IgG+ as DB cells. Considering that CD32 can induce negative signaling when stimulated by aggregates of monomeric IgG present in IVIg, our results with the DB and Pfeiffer model cell lines thus suggest that the direct effect of IVIg on B cells could involve interaction with their BCR.
Coagulation Factor V-Major Survival Predictor in Patients of Viral Hepatitis Induced Acute Liver Failure.
Authors: Gupta Naveen L. , Sharma Subhadra , Saraya Anoop , Mukhopadhyay Asok K.
Published: October 2019

In acute liver failure, variables favoring survival of patients is based on age, interval between onset of jaundice and encephalopathy, coagulation factor level but all these variables were concluded from the studies that were conducted in Western population where etiology most commonly is paracetamol poisoning and metabolic disorders like Wilson’s disease; where as in Indian subcontinent set up major causative factor is viral hepatitis. Thus we exploited the database collected by observational study to investigate major predictors of survival in viral hepatitis induced acute liver failure. Among 40 patients followed for 1 month(7days) or less if died, there were 12 patients who survived. Tha analysis showed that patients who survived had factor V level on day 3 of diagnosis was in the range of 10%–25% and who died had factor V level below 10% (p<0.004). Thus factor V level on day 3 of diagnosis may help in predicting survival in patients of acute liver failure, thus help in triage for the need of orthotopic liver transplantation which is the only definite curative therapy in patients who have poor survival rates.

t test outcome No. of patients mean Standard deviation p value standard error of mean group statistics Interval died 26 30.6538 10.8552 <0.002 2.1289 survived 14 6.1429 3.3249 0.8886 FactorV day1 died 26 7.5769 2.8309 >0.05 0.5552 survived 14 12.0000 4.3677 1.1673 Factor VIII day1 died 26 225.3396 156.0354 >0.05 30.6011 survived 14 293.3556 206.3757 55.1562 FactorV day3 died 26 5.9615 2.0096 <0.004 0.3941 survived 14 12.9286 4.8272 1.2901 FactorVIII day3 died 26 248.2509 185.7102 >0.05 36.4208 survived 14 274.7226 206.5798 55.2108 Lactate day1 died 26 74.3308 11.5141 >0.05 2.2581 survived 14 59.0929 8.0789 2.1592 Lactate day3 died 26 78.9500 8.8363 >0.05 1.7329 survived 14 62.5000 9.4086 2.5146 Factor VIII:V day1 died 26 29.8545 16.6385 >0.05 3.2631 survived 14 22.9066 8.9435 2.3902 FActorVIII:V day3 died 26 42.4612 31.8050 <0.003 6.2375 survived 14 20.0944 8.7368 2.3350 pH day1 died 26 7.1365 0.1331 >0.05 2.611E-02 survived 14 7.1443 0.1302 2.648E-02 pH day3 died 26 7.0300 0.1479 >0.05 2.901E-02 survived 14 7.2286 0.1075 2.872E-02 pCO2 day1 died 26 41.8462 7.6036 >0.05 1.4912 survived 14 40.7857 7.5261 2.0114 pCO2 day3 died 26 46.3077 10.3760 >0.05 2.0349 survived 14 43.2857 5.8498 1.5634 pO2 day1 died 26 87.3462 4.3169 >0.05 0.8466 survived 14 86.0000 4.3677 1.1673 pO2 day3 died 26 89.3462 4.0194 >0.05 0.7883 survived 14 89.5000 3.6742 0.9820
Safety Profile of Antihemophilic Factor (Recombinant), Plasma/Albumin-Free Method (rAHF-PFM) from Post-Authorization Safety Surveillance (PASS) Program.
Authors: Luu Huong , Spotts Gerald , Gajek Hartwig , Kriukov Alex , Berg Roger , Stephens Darby , Barker Kerry , Ewenstein Bruce
Published: October 2019

ADVATE rAHF-PFM was licensed by the FDA in 2003 and the EMEA in 2004 based on studies of previously treated patients (PTPs) with FVIII levels ≤2%. Subsequent to licensure, a PASS program was launched to document rAHF-PFM experience during the first year of routine therapy in subjects with FVIII baseline levels ≤5%, irrespective of prior FVIII exposure or inhibitor history. As of May 25, 2007, a total of 537 subjects (519 male, 2 female, 16 unreported) were enrolled. Of these 537 subjects, 67 were previously untreated or minimally treated patients (PUPs/MTPs) (≤50 exposure days [EDs]), 444 PTPs (>50 EDs), and 26 subjects had an unknown prior exposure status at study entry. Furthermore, 451 had FVIII ≤2%, 62 FVIII >2% to ≤5%, 5 FVIII >5%, 19 unreported. At enrollment, 421 reported switching from recombinant FVIII therapies (343 first generation FL-rFVIII, 38 second generation FL-rFVIII, and 12 BDD-rFVIII), and 76 from plasma-derived-FVIII (34 VWF-containing pdFVIII). Sixty-four subjects had a history of an inhibitor; 33 underwent immune tolerance induction (ITI). Age cohorts were as follows: 0–2 years (n=29), 2–16 years (n=184), and ≥16 years (n=297); 27 were unreported. A total of 417 subjects completed the one-year observation period; 42 withdrew. Twenty AEs were judged at least possibly related to rAHF-PFM; 11 (mostly inhibitors) were rated as serious. Among 387 PTPs with baseline FVIII ≤2%, 2 recurrent, low-titer (1.4 BU and 1.5 BU) and 1 de novo, low-titer (1.4 BU) inhibitors were reported, providing an interim inhibitor risk estimate of 0.78% (95% CI: 0.16–2.25%). In the remaining PTPs, 1 de novo, high-titer (23 BU) inhibitor was reported in a 60-year-old man (FVIII >5%) following surgery, and 1 de novo, low-titer (4 BU) inhibitor, which disappeared 12 months later, developed in a 4-year-old boy (baseline FVIII=5%). Two high-titer inhibitors were reported in PUPs; 1 successfully eradicated with 5 weeks of ITI using rAHF-PFM (100 IU/kg QD). Other related serious AEs included 2 allergic reactions, 1 ineffective drug response in a subject with an inhibitor and ITI history, and 1 hemarthrosis secondary to trauma. Overall, 2771 new bleeds were recorded. Annual bleed rates were 4.3 and 8.3 among subjects on prophylactic and on-demand therapy, respectively. Of 259 subjects with physician-rated prophylaxis efficacy, 91% had only excellent/good ratings. Of 369 subjects with physician-rated bleed treatment efficacy, 94% had only excellent/good ratings. Ratings were comparable among subjects with an inhibitor history. Interim PASS results indicate rAHF-PFM is safe and effective in the management of hemophilia A patients, including those typically excluded from controlled clinical trials.
Relationship between Procalcitonin Levels and Severity of DIC Induced by Bacterial Infection.
Authors: Kawasugi Kazuo , Shirasaki Ryousuke , Noguti Mizuho , Tashiro Haruko , Shirafuji Naoki
Published: October 2019

 In the past few years, structural and functional studies have demonstrated an increasingly tight interplay between the coagulation and inflammatory systems. These studies support an important role of systemic inflammatory response syndrome (SIRS) in the development of disseminated intravascular coagulation (DIC) in these critically ill patients. Although PCT is important clinical marker, it role in the DIC process is unkown. We measured serum levels of PCT in septic patients with DIC (n=15) and acute promyelocytic leukemia (APL)-induced DIC (n=5). Serum levels of PCT were determined by the EIA (BIOMERIEMX, France).

 The thrombin antithrombin complexes (TAT) levels were higher in both DIC patients as reported by others. ?Levels of PCT elevated in all patients with bacterial infection. In the septic patients with DIC, we found significant elevations of serum PCT levels (97.3±72.3 ng/ml) as compared with septic patients with non overt DIC (30.5±42.4 ng/ml). All the patients that levels of PCT increased than 100 ng/ml died. However, we did not find any difference serum levels of PCT in the APL and EB virus infected patients with DIC.

 These results suggest that assess of thrombin generation capacity may be helpful in the making the diagnosis in septic patients with DIC. It appears that serum levels of PCT may contribute to the severity of DIC induced bacterial infection.
Pharmacokinetics and Pharmacodynamics of Oral Heparin Solid Dosage Form in Healthy Human Subjects.
Authors: Mousa Shaker A. , Zhang Haifeng , Aljada Ahmad , Zhang Fuming , Arbit Ehud , Bender Lewis , Goldberg Michael , Linhardt Robert
Published: October 2019

Heparin is not orally absorbed, presumably because of its size and polyanionic charge and hence is administered parenterally, either by continuous or intermittent infusion or by subcutaneous (SC) injection. However, a formulation that would result in absorption of heparin after oral administration would provide an attractive alternative to parenteral heparin. In that regard several attempts to develop effective non-parenteral heparin formulations have been reported, but they have met with limited success. The present investigation determined the molecular structure, pharmacokinetic (PK), and pharmacodynamic (PD) profiles of oral unfractionated heparin (UFH) containing oral absorption enhancer Sodium N-[8-(2-hydroxybenzoyl) amino] caprylate, salcaprozate sodium (SNAC) and assessed the safety and tolerability of orally dosed heparin solid dosage form versus other routes. Sixteen healthy men were included in this single-dose, three-way crossover, randomized, open-label study. The different arms of the study were as follow: Treatment A- Subjects randomized to Treatment A received two capsules of UFH/SNAC soft gelatin capsules for a total dose of 75,000 U heparin sodium with 150 mL of water; Treatment B: Subjects randomized to Treatment B received 1 mL of heparin sodium USP parenteral 10,000 U/mL via SC injection; Treatment C: Subjects randomized to receive 75,000 U heparin sodium milled powder for oral solution with 150 mL of water; and Treatment D: Subjects randomized to receive 0.5 mL of heparin sodium USP parenteral 5,000 U/mL via IV bolus. Each subject was randomly assigned to receive three of the four treatments so that a total of 12 subjects received each treatment. To characterize the PK of SNAC after oral heparin/SNAC administration and the PD of heparin following the administration of heparin IV, SC, PO alone and PO as heparin/SNAC, 21 blood samples were drawn from all of the subjects receiving treatment A, B, C, and D at the following time points: within 30 minutes pre-dose, at 2, 5, 10, 15, 20, 25, 30, 35, 40, 45 minutes, and at 1,1.5, 2, 2.5, 3, 3.5, 4, 5, 6, and 8 hours post-dose. Blood was drawn into sodium citrate tubes for assessment of anti-factor Xa, anti-factor IIa, aPTT, and SNAC. Disaccharide compositional analysis was performed using capillary high-performance liquid chromatography with electrospray ionization mass spectrometry detection. The PD of heparin was obtained from analysis of plasma anti-factor Xa, anti-factor IIa, aPTT, and total tissue factor pathway inhibitor (TFPI) data. The molecular weight properties and the disaccharide composition of orally administered UFH/SNAC and parenterally administered UFH are identical and consistent with the starting standard UFH administered and achieved anti-factor Xa: anti-factor IIa of 1:1. This is the first true pharmacokinetic study to measure the chemical compositions of heparin administered by different routes.
Direct Interaction of G-CSF-Stimulated-Neutrophils and HUVECs Are Necessary for Production of IL-8 in Endothelial Cells.
Authors: Okamoto Yasuhiro , Watanabe Tsutomu , Onishi Toshihiro , Watanabe Hiroyoshi , Yamaki Yuni , Kodama Yuichi , Nishikawa Takuro , Tanabe Takayuki , Shinkoda Yuichi , Kawano Yoshifumi
Published: October 2019

Background: We previously reported that interleukin (IL)-8 increased up to 207 pg/ml in vivo after administration of granulocyte colony-stimulating factor (G-CSF) at a dose 5μg/kg for five days to mobilize peripheral blood stem cells (PBSC) in hematopoietic stem cell transplantation in cancer patients or healthy donors. Since pre-apheresis serum IL-8 levels correlated with the yield of CD34-positive cells, IL-8 production may be critical to G-CSF induced stem cell mobilization. IL-8 is known to be surged from granules of endothelial cells (EC) by stimulation of GM-CSF, however, precise mechanisms of increased production of IL-8 by G-CSF in vivo is unclear. First, in this study we examined the relationship between G-CSF and IL-8 in vitro.

Method: Peripheral blood was drawn from healthy donor with consent. Neutrophils were separated by density centrifugation method. Human umbilical vein endothelial cells (HUVECs) and G-CSF were purchased. HUVECs were cultured under serum-containing medium and stimulated by G-CSF or HUVECs were co-cultured with neutrophils. IL-8 in culture media was measured by ELISA.

Results: G-CSF alone does not increase IL-8 production in the culture medium either of neutrophils or HUVECs alone, in either short time (6 hours) or long time (up to 4 days) stimulation. However, when HUVECs were co-cultured with neutrophils, IL-8 started to increase 10 hours after starting co-culture and gradually increased up to 3000pg/ml after 4 days culture. IL-8 in co-culture media increased five fold compared to that in the single culture of HUVECs. This increased production of IL-8 was dependent on the number of neutrophils. Further, when neutrophils were pre-incubated by G-CSF at a dose 50ng/ml, IL-8 production in co-culture media increased 70 percent compared to pre-incubation without G-CSF. This increased production of IL-8 was dependent on G-CSF dose. IL-8 production remarkably decreased to 15% when HUVECs and neutrophils were cultured separately by mesh to avoid direct contact.

Conclusion: These data indicates that direct contact between HUVECs and neutrophils is necessary for production of IL-8. Some molecule(s) induced by G-CSF might play an important role in IL-8 production. The effect of stimulation by G-CSF seems less important than that of direct interaction between neutrophils and HUVECs. This correlates well to the fact that high WBC count after G-CSF administration in vivo correlates with the yield of CD34-positive cells in peripheral blood stem cells mobilization. Molecular mechanisms of interaction between HUVECs and neutrophils need to be clarified further.
β2-Glycoprotein I Is a Cofactor for t-PA-Mediated Plasminogen Activation.
Authors: Bu Chunya , Gao Lei , Xie Weidong , Zhang Jianwei , He Yuhong , Cai Guoping , McCrae Keith
Published: October 2019

Regulation of the conversion of plasminogen to plasmin by tissue-type plasminogen activator (t-PA) is critical in the control of fibrin deposition. While several plasminogen activators have been described, soluble plasma cofactors that stimulate fibrinolysis have not been characterized. Here, we report that the abundant plasma glycoprotein, β2-glycoprotein I (β2GPI), stimulates t-PA - dependent plasminogen activation both in the fluid phase and within a fibrin gel. Moreover, depletion of β2GPI from plasma reduced the lysis of fibrin thrombi, and exogenous β2GPI enhanced the activity of t-PA in a rat arteriovenous shunt thrombosis model. β2GPI bound t-PA with high affinity (Kd ∼ 20 nM), and caused a 20-fold increase in the catalytic efficiency (kcat/Km) of t-PA - mediated conversion of Glu-plasminogen to plasmin, primarily by stimulating t-PA amidolytic activity. Structure-function analysis revealed that the region within β2GPI responsible for stimulating t-PA activity is at least partially contained within β2GPI domain V. Finally, stimulation of t-PA - mediated plasminogen activity by β2GPI was inhibited by monoclonal anti-β2GPI antibodies, as well as by anti-β2GPI antibodies from patients with antiphospholipid syndrome (APS). These findings suggest that β2GPI may be an endogenous regulator of fibrinolysis. Impairment of β2GPI-stimulated fibrinolysis by anti-β2GPI antibodies may contribute to the development of thrombosis in patients with APS.
Hepatocytes of Donor Origin in Recipients Liver, after Hematopoietic Stem Cell Transplantation in BetaThalassemia Major.
Authors: Ghavamzadeh Ardeshir , Mirzania Mehrzad , Sedighi Nahid , Yaghmaie Marjan , Kamalian Naser , Alimoghaddam Kamran , Ghaffari Seyed Hamidollah , Azimi Parisima
Published: October 2019

Background Bone marrow and circulating stem cells contains stem cells with the potential to differentiate into mature cells of various organs. We determined whether stem cells transformed to hepathcytes.

Methods Biopsy specimens from the liver were obtained from 11 patients who had undergone transplantation of hematopoietic stem cells from peripheral blood (8 patients) or bone marrow (3 patients). Four female patients had received transplants from a male donor and seven male patients had received transplants from a female donor.All patients had beta thalassemia major and fibrosis in biopsy specimens from the liver before hematopoietic stem-cell transplantation. Hematopoietic stem-cell engraftment was verified by short tandem repeat analysis. The biopsies were studied for the presence of donor-derived hepatocytes with the use of fluorescence in situ hybridization of interphase nuclei and immunohistochemical staining for CD45 (leukocyte common antigen), and a hepatocyte-specific antigen.

Results All 11 recipients of sex-mismatched transplants showed evidence of complete hematopoietic donor chimerism. XY-positive hepatocytes accounted for 4 to 6.7 percent of the cells in histologic sections of the biopsy specimens of female patients. These cells were detected in liver tissue as early as 1 year and as late as 8.5 year after the hematopoietic stem cell transplantation.

Conclusions Bone marrow and circulating stem cells can differentiate into mature hepatocytes in beta thalassemia major patients who undergone hematopoietic stem cell transplantation.
In Vitro Aging of Rat Mesenchymal Stem Cells.
Authors: Zhang Xiaoyan , Li Jianyong , Wu Yujie , Xia Jun , Zhang Yaping , Lu Hua
Published: October 2019

Background Mesenchymal stem cells (MSCs) are now used in repair medicine and transplantation because of its multipotency and immunomodulatory effect. They need in vitro expansion to get adequate number for clinical use. Human MSCs had been observed to enter senescence during in vitro culture. We evaluated that the same phenomenon existed during long term culture of rat MSCs.

Methods Bone marrow from ten male Sprague-Dawley (SD) rats (198±2.12g) were cultured and nonadherent cells were removed three days later. MSCs were identified by osteogenic differentiation and adipocytic differentiation. Cells of each passage were detected for morphology by Wright’s staining, ultrastructure by scanning electron microscope, growth curve by CCK-8 kits detection, osteogenic differentiation and von kossa staining, adipocytic differentiation and oil red staining, βgalactosidase staining, quantitative assay of p16INK4a gene.

Results Rat MSCs were Fusiform shaped or polygon. Cells were becoming flatter and bigger during passaging. More swelling endoplasmic reticulum and demyelinate mitochondrion were observed by scanning electron microscope during passaging. The proliferation of the cells slowed from the 6th passage and stopped at the 8th or 9th passage. The positive rate of βgalactosidase staining and p16INK4a gene increased in cells after 5th passage. Osteogenic and adipocytic potential were attenuated in cells after 6th passage. Conclusions MSCs enter senescence during long term culture. Their potential of proliferation and multipotency dropped.
MicroRNAs 155 and 451 as Possible Key Molecules for Human Erythroid Differentiation.
Authors: Umemura Tsukuru , Masaki Shizuka , Ohtsuka Rie , Abe Yasunobu , Muta Koichiro
Published: October 2019

MicroRNAs (miRNAs) are 18–25-nucleotide noncoding RNAs which play important roles for cell death, proliferation, development and differentiation. MiRNA is an important molecule to regulate genes by suppressing the translation or inducing instability of miRNAs, and is consist of the network system to regulate gene functions in combination with transcription factors. Many recent works demonstrated that some of miRNAs are playing key roles for hematopoiesis and leukemogenesis. In this study, we analyzed the expression of miRNAs(miRNA-155, miRNA-221, miRNA-223, miRNA-451) during differentiation of purified normal human eryhroid progenitors in the liquid culture system. Cells increased almost 500-folds in a number, and differentiated to benzidine-positive mature erythroblasts after days 7 to 9 which were partly red blood cells on days 12 to 14. Since mature erythroid cells loose cellular nucleic acids at the final maturation stages, we measured changes in U6 RNA contents as the internal control for assays of miRNA. Each expression levels of miRNAs were normalized using U6 RNA contents. Analyses of miRNA expressions using quantitative real-time reversetranscriptase polymerase chain reaction have shown that the expression level of miRNA-155 decreased about 200-folds from day 3 to day 12 with almost 87.5% reduction between days 3 and 5. On the other hand, the expression levels of miRNA-451 increased about 270-folds by day 12 in parallel to an increase in benzidine-positive cell numbers. To extend our observation on the up-regulation of miRNA-451 in mature blood cells, we analyzed the miRNA-451 levels in each mature blood cells (red blood cells, granulocytes, lymphocytes and monocytes, platelets) purified from normal human peripheral blood by using a density centrifugation method. miRNA-451 was expressed in red blood cells about 104 folds more than in granulocytes, about 102 folds more than in platelets. Moderate down-regulations of miRNAs 221 and 223 were observed. In conclusion, our observations suggest that the down-regulation of miRNA-155 and the up-regulation of miRNA-451 are key events for normal erythroid differentiation, and that quantitative assays of the two miRNAs may be useful tools for specifying the differentiation stage of each erythroid cells.
GATA-1 Is Functionally Involved in the Human CD11b+ Interstitial/Dermal Dendritic Cell Pathway.
Authors: Gobel Florian , Taschner Sabine , Vaculik Christine , Kumerz Mario , Waltenberger Darina , Elbe-Burger Adelheid , Strobl Herbert
Published: October 2019

CD34+ cord blood progenitors can develop into interstitial/dermal (int) dendritic cells (DC) and Langerhans cells (LC). IL-4 induces common progenitors to differentiate into intDC and suppresses TGF-β1-dependent LC. The transcriptional mechanisms underlying DC versus LC development remain unclear. We found that the erythroid master transcription factor GATA-1 is differentially expressed in DC subtypes: LC were negative for GATA-1. However, GATA-1 was detected in CD34+-derived intDC and the related monocyte derived DC (moDC) with late kinetics and at low expression levels compared to erythroid cells. Induced expression of GATA-1 in DC/LC precursors promoted the formation of intDC and inhibited concomitant monocyte differentiation. Interestingly, GATA-1 and PU.1 are simultaneously expressed during moDC differentiation and both synergise in CD11b expression. Additionally, GATA-1 suppressed vitamin D3 receptor expression, which is a side-effect of moDC development. Taken together, these data indicate the crucial role of GATA-1 in DC development.
Regulation of Chemerin Bioactivity by Plasma Carboxypeptidase N, Carboxypeptidase B (TAFIa) and Platelets.
Authors: Du Xiao-Yan , Zabel Brian A. , Allen Samantha J. , Handel Tracy M. , Lee Peter P. , Butcher Eugene C. , Leung Lawrence L.
Published: October 2019

Chemerin is a potent chemoattractant for cells that express the serpentine receptor CMKLR1 (chemokine-like receptor 1), such as plasmacytoid dendritic cells and tissue macrophages. Chemerin circulates in the blood in a relatively inactive form (prochemerin). Its chemotactic activity is unleashed following proteolytic cleavage of carboxyl-terminal amino acids by serine proteases including plasmin, factor XIIa and VIIa. Recruitment of plasmacytoid dendritic cells and macrophages by chemerin may play a role in local tissue immune and inflammatory responses. The shortest bioactive chemerin peptide NH2-YFPGQFAFS-COOH (9mer) is present in the carboxyl-terminal domain. In this work, we show that plasma carboxypeptidase N (CPN) and B (CPB or activated thrombin-activatable fibrinolysis inhibitor (TAFIa)) (30 nM) remove the C-terminal lysine (K) from YFPGQFAFSK (10mer) (200nM) and enhance the migration of CMKLR1-transfected cells by ∼16-fold. To investigate if sequential proteolysis by plasmin and carboxypeptidases can modulate the activity of chemerin peptides, we generated the carboxyl-terminal 15mer of prochemerin, NH2-YFPGQFAFSKALPRS-COOH. Plasmin cleavage (1 μM) generated a 10mer, which was further processed to 9mer by CPN or CPB (30 nM) cleavage. These sequential cleavages were paralleled by corresponding increases in chemotactic activity. At concentrations as high as 1 μM the 15 mer did not induce chemotaxis; after plasmin cleavage and conversion to 10mer, however, significant chemotactic activity was demonstrated. Treatment with CPN or CPB further enhanced this chemotactic activity. We observed a similar enhancement in bioactivity following sequential plasmin/CPN-or-CPB cleavage of full-length prochemerin. Endogenous plasma CPN supports the activation (∼2.5 fold increase in bioactivity) of plasmin-cleaved prochemerin (0.2 nM). This activation was blocked by incubating plasma with MGTA (DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid), a specific CPN inhibitor. In addition, we show that platelets are a rich source of chemerin (20–30 ng of chemerin/5x108 platelets). Chemerin released from activated platelets triggers CMKLR1-transfectant chemotaxis, which was blocked by anti-chemerin antibodies. Thus, the circulating humoral factors reported here (platelets, serine proteases, and carboxypeptidases) may contribute to the regulation of chemerin bioactivity in vivo and therefore play a critical role in CMKLR1-mediated immune responses.
Hematopoiesis in Bone Marrow Regenerated from Adipose-Derived Stromal Cells.
Authors: Migita Makoto , Fujita Atsushi , Ogawa Rei , Ueda Takahiro , Fukunaga Yoshitaka , Shimada Takashi
Published: October 2019

Background: Adipose-derived stromal cells (ASCs) are highly promising cells for tissue engineering because of their pluripotency and low donor morbidity. We have previously shown that ASCs differentiate into a variety of mesenchymal tissues including bone and bone marrow (BM) like architecture. Osteoblastic cells lining the endosteal surface are a key component of the hematopoietic niche to promote and regulate hematopoietic stem cells (HSCs). In the present study, we investigated whether ASCs derived BM can functionally support hematopoiesis.

Methods: ASCs were isolated from the inguinal fat pads from C57/BL6 mice (Ly5.2) after collagenase treatment and were cultured for three passages in the culture plates. The attached cells were trypsinized and replated onto a hydoxyapatite (HA) scaffold with numerous small pores for 3 days. The scaffold with attached cells was implanted subcutaneously onto the back of C57/BL6 recipient mice. 4.0×105 Lineage negative (Lin-) Ly5.1 BM cells transduced with a lentiviral vector containing the luciferase (Luc) gene were intravenously administered into the recipient mice after lethal irradiation. After 8 weeks, the scaffolds including Luc+ cells were removed and re-transplanted to the lethally irradiated second recipient mice.

Results: Excised HA scaffolds were totally covered with adipose tissues. Hematoxylin-eosin staining showed that microspores were filled with typical bone marrow architecture composed of adipocytes, vasculatures, stroma, and a variety of hematopoietic cells. In vivo bioluminescence imaging showed that Luc+ hematopoitic cells were detected in the scaffolds in the secondary transplanted mice for at least 6 months after transplantation. Bioluminescence signals were also detectable on the whole body including the head, extremities, chest, and abdomen after G-CSF injection.

Conclusions: We demonstrated that BM could be regenerated by ASCs. The ASCs derived BM were capable of maintenance of hematopoietic progenitor cells in vivo. BM regeneration from non-BM cells raises new possibilities to treat various hematopoietic diseases.