bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of ADAMTS13 Autoantibodies and Elevated ADAMTS13/fXI Complex in Lupus Anticoagulant Positive Plasmas Suggest a Potential Role for ADAMTS13 in Antiphospholipid Syndrome.
Authors: Guinto Enriqueta R. , Ercolano Ellyn , Grafos Nicholas , Fryer Hugh J.L. , Santulli Clare , Dlott Jeffrey S. , Belter Maryellen , Greenfield Robert S.
Published: April 2020

Reduced ADAMTS13 levels due to congenital deficiency or development of ADAMTS13 autoantibodies has been associated with the severe disorder, thrombotic thrombocytopenic purpura (TTP). ADAMTS13 autoantibodies, reduced activity and elevated level of ADAMTS13/FXI complex have also been reported in TTP and venous thromboembolism (VTE) (1,2). Recently decreased ADAMTS13 activity or high titers of ADAMTS13 autoantibodies were reported in antiphospholipid syndrome (APS) (3), an autoimmune disease associated with VTE, arterial thrombosis and recurrent pregnancy loss. The diagnosis of APS relies upon determination of Lupus anticoagulants (LA) and various antiphospholipid autoantibodies (e.g. anti-CL and anti-beta2GP1). In this study we measured ADAMTS13 autoantibody, antigen, activity and ADAMTS13/fXI complex in plasmas of patients previously evaluated for LA. All of the LA plasmas were STACLOT LA positive and DRVVT negative at the time of diagnosis. In this study we also tested each plasma for dilute Prothrombin time, dPT (ACTICLOT® dPT™ with confirm procedure (4)) and measured anti-CL (IgG and IgM) and anti-beta2GPI (IgG and IgM) antiphospholipid antibodies. ADAMTS13 activity was measured using a new FRET assay, ACTIFLUOR™ ADAMTS13 (1). ADAMTS13 autoantibodies (IgG and IgM) were measured using IMUBIND® ADAMTS13 Autoantibody ELISA and ADAMTS13/fXI complexes using IMUBIND ADAMTS13/fXI ELISA. D-dimer levels were also measured (IMUCLONE® FDP ELISA). In this cohort of LA plasmas, 48/100 tested positive in the dPT screen and 23/48 in dPT confirm. Medium titer of aCL IgM (24/100, 24%) and anti-beta2GPI IgM (11/100, 11%) were predominantly found. ADAMTS13 activity and antigen levels generally fell within the range from normal donors. Anti-ADAMTS13 IgG titer (>14 au/mL) was detected in 11/91 (12%) and anti-ADAMTS13 IgM (>85 au/mL) in 22/96 (23%) plasmas. A subset of thirty- four plasmas was tested for ADAMTS13/fXI complex, 16/34 (47%) had elevated level of ADAMTS13/fXI complex. In the 16 plasmas with elevated ADAMTS13/fXI complex, 13/16 (81%) had elevated aCL IgM and 1/16, elevated beta2GPI IgM. The D-dimer level, a key marker of VTE, was high in 11/16 (68%) of these ADAMTS13/fXI complex -containing plasmas. In a subset of thirty-one plasmas with positive dPT screen and assayed for D-dimer, elevated D-dimer was found in 71% (22/31), of which 50% (11/22) was dPT confirmed.
The Roles of Akt1 and Akt2 in GPIb-IX-Mediated Platelet Activation Signaling.
Authors: Yin Hong , Stojanovic Aleksandra , Hay Nissim , Du Xiaoping
Published: April 2020

The platelet von Willebrand factor (VWF) receptor, glycoprotein Ib-IX (GPIb-IX), mediates platelet adhesion and induces signaling leading to integrin activation. Phosphoinositol 3-kinase (PI3K) is important in GPIb-IX-mediated signaling. PI3K-dependent signaling mechanisms, however, are unclear. To understand the downstream signaling pathway of GPIb-IX signaling, we investigated the roles of PI3K effector kinases, Akt1 and Akt2, in VWF/GPIb-IX-induced platelet activation. VWF/GPIb-IX-induced platelet aggregation was impaired in Akt1- or Akt2-knockout mouse platelets and in human and mouse platelets treated with an Akt inhibitor, SH-6. GPIb-IX-mediated platelet stable adhesion to VWF under shear stress was also inhibited in mouse platelets deficient in Akt1 or Akt2, and in human platelets treated with SH-6. Interestingly, while deficiency of Akt1 or Akt2 caused nearly complete inhibition of stable platelet adhesion to VWF under shear stress, stable platelet adhesion was only partially reduced in platelets treated with both P2Y1 and P2Y12 ADP receptor antagonists, A3P5P and 2MeSAMP or thromboxane A2 pathway inhibitor, aspirin or Syk inhibitor, piceatannol. Therefore, Akt1 and Akt2 are important in early GPIb-IX signaling independent of Syk, ADP or thromboxane A2 (TXA2), in addition to their recognized roles in ADP- and TXA2-dependent secondary amplification pathways. Knockout of either Akt1 or Akt2 diminished platelet spreading on VWF, but not on immobilized fibrinogen. Thus, Akt1 and Akt2 are both required only in the GPIb-IX-mediated integrin activation (inside-out signaling). In contrast, PI3K inhibitors abolished platelet spreading on both VWF and fibrinogen, indicating a role for PI3K in integrin outside-in signaling distinct from that in GPIb-IX-mediated inside-out signaling. Furthermore, Akt1 or Akt2 deficiency diminished VWF-induced cGMP elevation, and their inhibitory effects on GPIb-IX-dependent platelet adhesion were reversed by low concentration of exogenous cGMP, indicating that Akt1 and Akt2 mediate GPIb-IX signaling via the cGMP-dependent signaling pathway. In conclusion, both Akt1 and Akt2 mediate VWF/GPIb-IX-induced signaling pathway leading to platelet activation and the consequent stable platelet adhesion, spreading and aggregation.
Protease-Activated Receptor 3 Negatively Regulates Plasmin-Mediated Protease-Activated Receptor 4 Activation in Platelets.
Authors: Mao Yingying , Jin Jianguo , Kunapuli Satya P.
Published: April 2020

Plasmin, a major extracellular protease, causes intracellular signals to mediate platelet aggregation. Previously, we reported that plasmin-mediated platelet aggregation predominantly occurs through proteolytic cleavage of protease-activated receptor 4 (Quinton et al J. Biol. Chem 2004). We showed that plasmin caused aggregation of mouse platelets more readily than human platelets. In this study, we investigated the mechanism of such a differential sensitivity of mouse platelets to plasmin, using transfected cell lines and platelets. In the platelet system, plasmin caused both human and mouse platelets to shape change and aggregate in a concentration-dependent manner with a different efficiency. Whereas 0.1 U/ml of plasmin causes full aggregation of mouse platelets, that dose only induces shape change in human platelets. In transfected COS7 cells, 1 U/ml plasmin caused a higher intracellular calcium mobilization through mouse PAR4 (mPAR4) than human PAR4 (hPAR4) activation. These results indicate that the mPAR4 is primarily more readily activated by plasmin than hPAR4, possibly due to differences in the primary sequence. Further, mouse and human platelets also differ in terms of the expression of PAR3. It is known that PAR3 acts as a co-receptor for thrombin-induced PAR4 activation. In order to evaluate the contribution of mouse PAR3 to plasmin-mediated mPAR4 activation, we co-expressed mPAR3 and mPAR4 in COS7 cells. Plasmin caused lower mobilization of intracellular calcium when mPAR3 and mPAR4 are co-expressed, compared to the expression of mPAR4 alone. These results indicate that mPAR3, instead of acting as a cofactor to potentiate mPAR4 activation, inhibits plasmin-mediated mPAR4 activation. Consistent with these results, PAR3 null mouse platelets also show a greater plasmin-induced calcium mobilization and aggregation compared to wild-type mouse platelets, which express PAR3 and PAR4. In conclusion, mouse platelets are more readily activated by plasmin than human platelets due to differences in the primary sequence of PAR4. In addition, mPAR3 acts as an inhibitory receptor to plasmin-mediated PAR4 activation, instead of acting as a co-receptor.
P-REX1: A Novel RAC Activing Guanine-Nucleotide Exchange Factor in Human Platelets.
Authors: Morgan-Spencer Alex , Greenberg Daniel
Published: April 2020

A major signaling pathway that regulates platelet shape change and reorganization of the cytoskeleton involves the Rho family of GTPases whose members include Rac, CDC42 and RhoA. These GTPases are converted from their inactive or GDP-loaded state to the active or GTP-loaded state by a class of enzymes called Guanine-nucleotide Exchange Factors (GEFs). GEFs are a family of multi-domain proteins that contain a GDP-GTP exchange domain (DH-PH) as well as other protein interacting domains that are regulated by the activation of receptors present on the platelet surface. We used an affinity binding technique followed by mass spectrometry analysis to identify novel Rac binding GEFs from platelet lysates. Recombinant GST-Rac fusion proteins bound to agarose beads were prepared in the GTP, GDP and nucleotide-free states and incubated with human platelet lysates. Platelet lysate proteins associated with the different GST-Rac preparations (GTP-bound, GDP-bound or nucleotide-free) were eluted and run on SDS-PAGE. Gel slices were then cut out, trypsin digested and analyzed by mass spectrometry. Platelet GEFs were identified by the presence of a DH-PH domain. Using this technique we identified three novel Rac-associated platelet GEFs including P-REX1, a G-βγ protein and phosphatidylinositol (3,4,5)-triphosphate regulated GEF previously found in neutrophils and neurons (Welch, H.C.E et al., Cell108;809–822, 2002). PREX-1 is a 196kDa peptide composed of 1659 amino that specifically activates Rac in neutrophils. In addition to the DH-PH exchange domain, PREX-1 also contains tandem PDZ and DEP domains as well as significant similarity over its C-terminal half to Inositol Polyphosphate 4-Phosphatase. Western analysis with anti-P-REX1 antibody (gift of H.C. Welch, Barbraham Institute, U.K.) showed that PREX-1 is present in purified whole platelet lysates. We have also shown that platelet PREX-1 specifically associates with GST-Rac by affinity pull-down experiments. Functional studies to characterize the exchange activity of platelet PREX-1 are ongoing. PREX-1 is the only known GEF that is directly regulated by G-βγ protein (Hill, K. et al., J Biol Chem280(6):4166–4173, 2005) and represents a novel pathway for platelet G protein coupled receptor signaling through Rac in human platelets.
Removing the Covalent Links between GP Ibα and GP Ibβ Hampers Binding of von Willebrand Factor to GP Ib-IX Complex in CHO Cells.
Authors: Mo Xi , Luo Shi-Zhong , Peng Yuandong , Munday Adam D. , Sun Carol , Berndt Michael C. , Lopez Jose A. , Dong Jing-fei , Li Renhao
Published: April 2020

In the glycoprotein (GP) Ib-IX complex, GP Ibα contains the binding region for its ligand von Willebrand factor (vWF). How the other subunits in the complex, GP Ibβ and GP IX, regulate the GP Ibα-vWF interaction is not clear. Since GP Ibα connects with two GP Ibβ subunits via disulfide bonds, these inter-subunit covalent links may be important to the proper vWF-binding activity of the GP Ib-IX complex. To test this idea, we have obtained Chinese hamster ovary cells stably expressing the mutant GP Ib-IX complex (CHOαSSβIX) in which both residues C484 and C485 of GP Ibα were changed to Ser. As expected, the mutant GP Ibα did not form any disulfide bonds with GP Ibβ in these cells. Nonetheless, co-immunoprecipitation experiments in the digitonin-containing buffer demonstrated that association of GP Ibα with GP Ibβ and GP IX was retained in CHOαSSβIX cells. The expression level of GP Ibα in CHOαSSβIX cells, detected by its binding to WM23, a conformation-insensitive antibody, was comparable to that in the transfected cells stably expressing the wild type GP Ib-IX complex (CHOαβIX). In contrast, binding of CHOαSSβIX cells to AK2, SZ2 or AN51, all of which are GP Ibα-specific conformation-sensitive antibodies, was significantly different from that of CHOαβIX cells. Compared to CHOαβIX cells, ristocetin-induced binding of CHOαSSβIX cells to vWF under static conditions was reduced by about 50% as determined by flow cytometry. Consistently, rolling of CHOαSSβIX cells on the vWF-coated glass slide was significantly faster than CHOαβIX cells under various flow conditions. Thus, in a transfected cell model, removing the disulfide links between GP Ibα and GP Ibβ in the GP Ib-IX complex leads to an alteration in the conformation of the ligand-binding domain in GP Ibα and as a consequence hampers the complex binding to vWF. Our finding suggests that the relative position of GP Ibα and GP Ibβ in the receptor complex is crucial to its vWF-binding activity, which may be exploited to modulate the GP Ibα-vWF interaction.
CPI-17 Contributes to Calcium-Independent ADP-Induced Platelet Shape Change through P2Y1-Gq-PKC Pathways.
Authors: Getz Todd M. , Bhavaraju Kamala , Kunapuli Satya P.
Published: April 2020

The initial event in platelet activation is the reorganization of the cytoskeleton causing the platelets to change from a discoid to a spiculated spheroid shape. Platelet shape change is primarily regulated by the phosphorylation of myosin light chain kinase. We have shown that this process is mediated through both calcium-dependent and calcium-independent Rho kinase pathways. CPI-17, a Protein kinase C (PKC) phosphorylated inhibitory protein of myosin light chain phosphatase, has been shown to have a role in platelet shape change downstream of thrombin-induced platelet activation. CPI-17 is a 17 kDa protein expressed in human platelets shown to inhibit myosin light chain phosphotase activity via PKCs. In this study we examined the role of CPI-17 in ADP-induced shape change and phosphorylation of CPI-17, downstream of the Gq coupled, P2Y1, and the Gi coupled, P2Y12 receptors. CPI-17 phosphorylation occurred upon activation of platelets with 2MeSADP. This phosphorylation was abolished in the presence of the P2Y1 receptor antagonist, MRS-2179. These results indicated that Gq signaling is important for platelet shape change and phosphorylation of CPI-17. In the presence of the calcium chelator, BAPTA, platelets changed shape in response to 2MeSADP; CPI-17 phosphorylation, however, was unaffected by BAPTA treatment under these conditions. However, CPI-17 phosphorylation was inhibited in the presence of the pan PKC inhibitors. These results indicate that CPI-17 phosphorylation occurs downstream of PKC activation. In the presence of BAPTA, treatment with PKC inhibitors decreased platelet shape change possibly due to reduced CPI-17 phosphorylation. The shape change caused by p160ROCK downstream of G12/13 pathways was unaffected by pan PKC inhibitors, but abolished by p160ROCK inhibitors H1152 or Y27632. Platelets incubated with BAPTA, pan PKC inhibitors, and p160ROCK inhibitor H1152, abolished ADP-induced platelet shape change and CPI-17 phosphorylation. In conclusion, ADP-induced platelet shape change occurs through a Gq-mediated, calcium-independent signaling pathway regulated by CPI-17 phosphorylation via PKC activation.
Erythrocytosis Caused by Mutations in the PHD2 and VHL Genes.
Authors: Percy Melanie J. , Furlow Paul W. , Jones Frank G.C. , Lappin Terry R.J. , Lee Frank S. , McMullin Mary Frances
Published: April 2020

Human diseases have provided important insights into the function of fundamental physiological processes, and studying erythrocytosis has increased our understanding of the oxygen sensing pathway. In some cases this rare disorder arises specifically from dysregulation of the erythropoietin (Epo) axis. Epo gene transcription is under the control of a negative feedback mechanism involving the kidneys, which sense tissue hypoxia at the molecular level via the hypoxia inducible factor (HIF) transcription complex. Both subunits of the HIF complex, alpha and beta, are constitutively expressed but only the beta subunit is detectable in the presence of oxygen. Key prolines in the oxygen degradation domain of HIFalpha are hydroxylated by a family of prolyl hydroxylase enzymes, PHD1-3. Of these PHD2 has the most widespread tissue distribution. Upon hydroxylation of HIF, the von Hippel Lindau (VHL) protein, a component of an E3 ligase complex, promotes the ubiquitination of the alpha subunit. HIFalpha is then targeted to the proteasome and the transcription of HIF target genes is prevented. In hypoxia the activity of the PHD enzymes is low and the HIF complex assembles causing upregulation of genes such as Epo. Over the last ten years we have maintained a registry of individuals with erythrocytosis. To date 181 patients have been included with clinical details and consented DNA samples have been collected. There is a preponderance of males with ratio of 1.7 males to each female. The mean age of erythrocytosis individuals on the registry is 37 years. The majority of erythrocytosis patients have inappropriately normal (46%) or raised (26%) serum Epo levels as compared to PV, where the Epo level is often undetectable. Thus it can be inferred that dysregulation of the Epo axis via the oxygen sensing pathway would be a significant cause of erythrocytosis. Consequently, PHD2 and VHL have been investigated. We have now identified 2 different mutations, Pro317Arg and Arg371His, in PHD2. In the tertiary structure of PHD2, Pro317and Arg371 are close to essential iron binding residues and furthermore suggest the location of a HIF binding groove. The Arg200Trp VHL mutation, commonly described as the Chuvash mutation, has been detected in 8 Asian families and their large kindred. Two Western European individuals were found to be compound heterozygous for the Arg200Trp and either the novel Pro192Thr or Gly144Arg variants. Intriguingly 2 further individuals possess one wild type and one mutated allele, Arg200Trp or Leu188Val. Screening other genes of the oxygen sensing pathway has failed to reveal further defects in these individuals. In summary, defects in the oxygen sensing pathway are associated with the development of erythrocytosis. Mutations in VHL are more common than PHD2 and highlight the importance of these proteins in the maintenance of red cell homeostasis.
Autologous Bone Marrow Mononuclear Cell (ABMMC) Transplantation in Type 1 and Type 2 Insulin Dependent Diabetes Mellitus (IDM).
Authors: Carrasco-Yalán Antonio A. , Fernandez-Vina Roberto , Castillo-Aguirre Jorge , Rios Hugo , More L. , Gomez N. , Chirino Samuel , Alvarez Marcos , Aranda M. , Arroyo M. , Rafael J. , Patel A.N. , Tuma-Mubarack Jorge
Published: April 2020

Background: Recent reports have shown that bone marrow-derived stem cell may contribute to islet regeneration. The goal of our study was to evaluate the safety and efficacy of ABMMC transplantation for patients with IDM.

Methods: From June 2005 to January 2007, 28 consecutive patients: 8 Type 1 IDM (T1DM) and 20 Type 2 IDM (T2IDM); who were receiving maximal medical therapy including insulin treatment for 5 years before enrollment. Median time of disease for T2IDM patients was 13 years, without pancreatic islet auto-antibodies. After IRB approval and signed informed consent, bone marrow was harvested and ABMMC were isolated and infused directly into the pancreas via splenic artery using endovascular catheters. Glucose, glycosylated HbA1c and C peptide were measure before and after transplantation. HOMA2 Calculator v2.2 was used to calculated IR and % B (*if Glucose: 3.0 to 25.0 mmol/L and C peptide: 0.2 to 3.5 nmol/L).

Results: There were no study related complications. At 1 year follow-up, mean daily insulin requirement was the same in group T1DM and significantly reduced in group T2IDM, from 42.5 to 4.5 U/d (t=7.94, p<0.001). Ten of the twenty (50%) T2IDM established complete insulin independence. Data in table 1.

Table 1: Median values PRE POST t p T2IDM (n=20) Fasting Glucose (mmol/L) 10.8 6.6 3.98 0.01 Glycosylated HbA1c (%) 9.6 8.1 3.98 0.01 C peptide (nmol/L) 0.5 0.84 5.11 < 0.01 HOMA 2 IR (n=17)* 2.2 2.26 0.94 0.92 HOMA 2 %B (n=17)* 42.4 130.2 4.9 < 0.01 T1DM (n=8) Fasting Glucose (mmol/L) 10.1 11.1 1.382 0.21 Glycosylated HbA1c (%) 8.7 8.7 0.45 0.66 C peptide (nmol/L) 0.17 0.16 1.00 0.35

Conclusions: The use of ABMMC transplantation for T1DM and T2IDM is safe. In this pilot study, only T2IDM patients have significant improvement in pancreatic function demostrated by better glycemic and HbA1c control, and are associated with a significant independence of the insulin. This has formed for a randomized multi-center study which is currently in progress.
Differentiation Hotspots on a Cell Cycle Related Continuum.
Authors: Colvin Gerald A. , Berz David , Dooner Mark S. , Dooner Gerri J. , Johnson Kevin , Aliotta Jason M. , Quesenberry Peter J.
Published: April 2020

Directed differentiation is defined as the ability to program a stem cell at the most primitive level while it still has its reproductive and full proliferative potential. This is in contrast to ex-vivo expansion where the stem cells are forced into specific lineage commitments, limiting the overall therapeutic utility. We have demonstrated differentiation “hotspots” on a cell cycle continuum (Exp Heme35:96, 2007). In this work we showed marked but reversible increases in differentiation potential to megakryocyte and granulocytes at different phases of a single cytokine induced cell cycle passage of highly purified quiescent murine lineagenegative rhodaminelowHoeschtlow (LRH) marrow stem cells. We have reproducibly induced directed stem cell differentiation by capitalizing on inherent changes in sensitivities to inductive cytokine signals in the context of cell cycle position. These cells, when exposed to thrombopoietin, FLT3-ligand and steel factor, synchronously pass through cell cycle. We have found that using a differentiation cytokine cocktail of G-CSF at 0.075ng/ml, GM-CSF at 0.0375ng/ml and steel factor at 50ng/ml, we were able to see enhanced megakaryopoiesis occurring 14-days after culture in those LRH stem cells that were in early to mid S-phase at time of inductive signaling. We have now shown that a megakaryocyte hotspot clusters around 32 hours; the G1/S interface, and that dramatic reversible changes in differentiation potential occur over one hour time intervals. We have confirmed this data by looking at LRH cells through cell cycle transit after initial cell division showing that a megakaryocyte hotspot occurs in two sequential cell cycles and still tied to S-phase at time of inductive signaling of the daughter cells. This hotspot has been demonstrated on a clonal basis, although the kinetics of the hotspot shifts when clonal as opposed to population studies are carried out. An important issue is whether in vitro cytokine exposure, separate from cell cycle status, determines the existence of the hotspot. To address this, we used Hoechst 33342 dye content to assist in separation of different cell cycle fractions (G0–1, early, mid and late components of S, G2/M) of lineage negative Sca-1+ stem cells, a cycling stem/progenitor cell population in which approximately 20% of the cells are in S-phase at isolation. These cells were only exposed to the differentiation cytokines and showed a megakaryocyte hotspot present in only early S-phase cells after 14-days of culture, showing that in vitro cell cycle phase determined the presence of the hotspot, separate from cytokine exposure. These data indicate that differentiation potential of marrow stem cells exists on a cell cycle related continuum and that this potential can be demonstrated on a single cell basis. This suggests a continuum model of stem cell regulation at the stem cell level as opposed to a pure hierarchical model.
Hepatocyte Differentiation by Highly Purified CD34+/CD34− Blast Cells from G-CSF and SCF Primed Bone Marrow from Rhesus Monkey.
Authors: Xia Xiujin , La Russa Vincent F.
Published: April 2020

Hematopoietic stem cells and hepatic stem cells share characteristic markers such as CD34, CD117(c-kit) and CD90 (Thy-1). In this study, we investigated the potential of primitive CD34+/CD34− blast cells to differentiate into hepatocytes in vitro. Rhesus monkeys were administered granulocyte colony stimulating factor, G-CSF, combined with stem cell factor, SCF, the ligand for c-kit receptor, for 3 days. This novel G-CSF and SCF combined cytokine treatment is believed to stimulate the self renewal of primitive blast cells in the bone marrow. Blast cells were purified by a novel technique that combines purging of T-cells and other immune-competent cells that express the Fc receptor. The immuno-depleted mononuclear fraction was labeled with biotin-conjugated SKH6.1 anti-CD34 monoclonal antibody belonging to the class-I epitope. The SKH 6.1 recognizes a heterogeneous blast cell population in rhesus bone marrow that consists mainly of approximately 80% CD34+ and a CD34− small blast cell minor population that also shares low forward and side scatter properties. Blast cells were cultured on Matrigel in a defined medium containing hepatocyte growth factor (HGF), epidermal growth factor (EGF) and fibroblast growth factor-4(FGF-4), and fed every 3–4 days. Conditioned medium was analyzed at weekly intervals for four weeks. ELISA assays were used to detect de-novo synthesis of rhesus monkey specific albumin, and urea production was measured using a quantitative colorimetric assay. Peak production of albumin occurred at 7-days and then began to continuously decrease through week 4, while urea was detected at 7-days but continued to increase through the end of the 4th week. The hepatocyte-like cells were also shown to produce significant intra-cellular glycogen as detected by periodic acid-schiff staining by their 4th week in culture. Hepatocyte function was also characterized by their ability to uptake Dil-Ac-LDL as detected by their strong fluorescence on the 4th week of culture. Our results demonstrated that G-CSF and SCF stimulate the bone marrow by increasing the frequency CD34+ / CD34− blast cells that are able to differentiate into hepatocyte-like cells.
Thrombospondin-1 as a Modulator of ADAMTS13 Activity.
Authors: Palla Roberta , Rossana Lombardi , Zwicker Jeffrey I. , Lawler Jack , De Cristofaro Raimondo , Peyvandi Flora
Published: April 2020

Thrombospondin-1 (TSP-1) is a large homotrimeric glycoprotein of approximatively 450 kDa, synthetised by several cell types, including platelets where it is stored in α-granules. Its multidomain structure enables TSP-1 to interact with many cell-adhesive receptors, integrins, heparan sulfate, as well as with other adhesive glycoproteins, including VWF. In a mouse model, soluble or local platelet released TSP-1 can protect unfolded endothelium-bound and subendothelial VWF from degradation by its plasma protease ADAMTS13. TSP-1 has also been shown to bind to the A3 domain of VWF suggesting a role for competitive inhibition of ADAMTS13-mediated VWF proteolysis. With this background we chose to investigate the inhibition effect of TSP-1 on ADAMTS13 activity by measuring the cleavage of recombinant (r)VWF multimers using rADAMTS13 in absence or presence of TSP-1 at different concentrations. We also assessed whether TSP-1 competes with ADAMTS13 for interaction with the A3 domain of VWF, by comparing cleavage of VWF-A1A2A3 or A1A2 or A2A3 peptides by ADAMTS13 in the presence or absence of TSP-1 by using HPLC analysis.Significant inhibition by TSP1 on ADAMTS13 activity was observed only with TSP-1 plasma concentrations reached after platelet activation (24–160 nM). However, this result was not confirmed at the low concentrations physiologically present in plasma (0.3–0.7 nM). The relative percentage of high molecular weight multimers was proportionally correlated to the TSP-1 concentration. These findings were confirmed using the collagen binding assay to assess VWF activity relative to antigen concentrations. The mean ratio of CBA/Ag was 0.22 in absence of TSP-1 compared to 0.5 and 0.78 in presence of 80 and 160nM of TSP-1. The HPLC analysis of VWF peptides cleavage by ADAMTS13 in presence of increasing concentrations of TSP-1 (0–225 nM) showed that TSP-1 acts as a competitive inhibitor of ADAMTS13 in binding both A1A2A3 and A1A2 peptides. The A1A2A3 peptide demonstrated an apparent affinity for ADAMTS-13 about three-fold higher than that of A1A2 construct (IC50=36.7±4nM and IC50=100.4±25nM, respectively). We also observed a TSP-1 inhibitory effect of ADAMTS13 activity with the A2A3 peptide (IC50=120 nM). These results indicate that the entire A1A2A3 region is necessary to achieve an optimal inhibition of the ADAMTS-13/VWF interaction by TSP-1. The A3 domain plays a central role in this inhibition, although its effect is dependent on the allosteric role played by the A1 domain. Thus, the lower inhibition observed with A2A3 peptide than with the A1A2A3 peptide, could be explained by increased accessibility of ADAMTS13 to A2 domain when the inhibition effect of A1 domain is lacking, as previously described by Nishio et al (PNAS2004;101:10578–83).
Erythropoietin Signaling and Response in Vascular Smooth Muscle Cells.
Authors: Beleslin-Cokic Bojana B. , Cokic Vladan P. , Gojkovic-Bukarica Ljiljana , Noguchi Constance Tom
Published: April 2020

Erythopoietin (EPO) and erythropoietin receptor (EPOR) regulate survival, proliferation, differentiation and viability of erythroid progenitor cells. Beyond erythropoiesis, we have observed that human vascular endothelial cells respond to EPO stimulation by inducing EPOR and endothelial nitric oxide synthase (eNOS) expression, increasing NO production and cGMP, particularly under low oxygen tension. In this study, we investigate the response of vascular smooth muscle (VSM) to EPO stimulation and the contribution of blood vessel to relaxation/contraction. We found that VSM cells express EPOR and that treatment with EPO (5 U/ml) at reduced oxygen tension increased EPOR mRNA by 2 fold. This increased EPO responsiveness at low oxygen tension is accompanied by increased cell proliferation at 2% O2 more than 2 fold. Unlike endothelial cells, EPO did not induce eNOS or NO production in VSM cells. PI-3 kinase was involved in EPO stimulation with no change in MAP kinase. In an isolated blood vessel model system, we checked EPO responsiveness. EPO produced contractions (0.32 ± 0.3 g) of rat renal artery, and pretreatment with LY294002 (10 mM), an inhibitor of PI-3 kinase, statistically significantly inhibited this contraction (58.7 ± 7%). This response was also observed with the endothelium layer removed. In preparations of human internal mammary artery (HIMA) and human saphenous vein (HSV) from patients undergoing coronary artery bypass, EPO did not affect basal vascular tone of HIMA and HSV with the endothelium layer removed, but EPO (5 U/ml) potentated noradrenalin-induced contraction by up to 2 fold in HSV and HIMA. Also, pretreatment with EPO significantly increase angiotensin-evoked contractions of these blood vessels (50 ± 8%, 113 ± 17%, respectively P < 0.01), suggesting that EPO has synergistic effects on angiotensin or noradrenalin-induced [Ca2+] mobilization, particularly on intracellular Ca2+ release. These data suggest that EPO stimulation of vascular smooth muscle may act to modulate or balance the vasodilatory effects of increased NO production by EPO stimulated endothelium. Under oxygen stress vascular smooth muscle can respond to EPO by proliferation and PI-3 kinase activation as a protective effect and in conjunction with Ca2+ release likely contributes to the overall vascular response.
Non-Integrating Gene Transfer To Redirect Specificity of Lymphocytes towards Pediatric CD19+ Malignancies
Authors: Yuen Carrie H. , Andersson Helen , Olivares Simon , Biswal Lisa , Raphael Robert , Killian Tom , Shpall Elizabeth J. , Lee Dean A. , Champlin Richard , Cooper Laurence
Published: April 2020

Pediatric B-lineage malignancies, such as acute lymphoblastic leukemia (B-ALL), generally have a poor prognosis upon relapse of tumor cells exhibiting resistance to conventional chemotherapy. In the absence of new commercially-available therapies for refractory pediatric cancers, it is up to pediatric oncologists to develop and apply new therapeutics. One candidate targeted therapy for B-lineage leukemia/lymphoma, is adoptive transfer of autologous T cells genetically modified to express a chimeric antigen receptor (CAR) and rendered specific for CD19. However, the broad adoption of T-cell gene therapy for children is limited by the expense to produce and release expression vectors and cell products and the risk of insertional mutagenesis. Therefore, we are developing an approach to express the CAR without integration of transgene and therefore without causing genotoxicity. Here we demonstrate that non-viral gene transfer of vector-free in vitro-transcribed messenger ribonucleic acid (mRNA) can efficiently introduce CAR into T cells. We demonstrate, using a commercially available electroporator, that codon-optimized mRNA coding for CD19-specific CAR is expressed on primary human T cells (Figure 1A) that redirects the specificity of CD19+ tumors (Figure 1B). When scaled-up for clinical trials, the electro-transfer of one or more mRNA species into T cells has the advantage of (i) ex vivo manipulation of T cells with preservation of homing receptors for lymph nodes and bone marrow (sites of leukemia/lymphoma), (ii) availability of clinically-meaningful numbers of genetically manipulated T cells within a day of apheresis, (iii) high levels of transgene expression, (iv) reduced cost (preparation of clinical-grade plasmid and mRNA compared with recombinant virus), and (iv) safety (no insertional mutagenesis). The relative ease of gene/mRNA transfer coupled with minimal manipulation of large numbers of T cells allows investigators to give patients multiple doses using existing infrastructure such as already widely exists for processing hematopoietic stem cells and blood banking. We are preparing a multi-institution clinical trial in which pediatric patients with refractory B-ALL receive personalized medication, prepared at each site, in the form of recursive infusions of CAR+ T cells repetitively electroporated with mRNA. This application of transient transfection to adoptive immunotherapy is expected to be of wide appeal for pediatric and adult patients alike with tumors that can be targeted by CAR.

Figure: (A) Expression of CAR in primary human T cells. (B) Specific lysis of CD19+ tumor target by genetically manipulated T cells expressing CAR mRNA Figure:. (A) Expression of CAR in primary human T cells. (B) Specific lysis of CD19+ tumor target by genetically manipulated T cells expressing CAR mRNA
A Heat-Inducible Gene Expression System for a Dominant Negative Version of a TGF-β-Receptor Reveals Antitumor Activity in a Rodent Tumor Model
Authors: Noyan Fatih , Dewey Ricardo A. , Kreipe Hans H. , Fruehauf Joerg , Klein Christoph
Published: April 2020

In an attempt to design novel therapeutic strategies against solid tumors, we hypothesized that spatial and temporal control of transgene expression may allow for the delivery of immunomodulatory molecules at the site of tumor. Here, we assess the potential of the HSP70b promotor to control lentivirus-mediated gene expression in tumor-infiltrating hematopoietic cells. We generated a series of lentiviral vectors and characterized transgene expression in vitro and in vivo. Up to 20fold induction of GFP expression was seen when lentivirus-transduced cells were heated to 43°C in vitro. Only minimal leakiness was seen at 37°C. Next, we analyzed HSP-driven GFP expression in hematopoietic cells upon transplantation of lentivirus-transduced hematopoietic stem cells, using constitutively active promotors and empty vectors as controls. Mice were inoculated with the glioma cell line GL261 and GFP expression was assessed in various organs as well as in tumor-infiltrating hematopoietic cells. Interestingly, HSP70-driven transgene expression was 10–15fold higher in tumor-infiltrating cells when compared to spleen cells, suggesting that the HSP70b promoter was active in the tumor microenvironment. To assess whether confined expression of a dominant negative TGF-β-receptor within the tumor microenvironment would induce anti-tumor immunity, we transplanted mice with hematopoietic stem cells transduced with TGF-β-DNR and injected GL261 cells subcutaneously. The tumor volume was decreased up to 40 fold in mice expressing HSP70b-TGF-β-DNR, comparable to mice transplanted with hematopoietic stem cells that constitutively express TGF-β-DNR. Functional studies suggest a role for perforin, IFN-α and Granzyme B in this TNF-β-DNR-dependent immunomodulatory circuit. In summary, HSP70b-mediated transgene expression in hematopoietic cells may provide a novel experimental strategy for controlled release of immunmodulatory molecules to induce anti-tumor therapies.
Influence of Polymorphisms of Pro-Inflammatory (IL-12, TNFa and LTa) and Anti-Inflammatory Factors (IL-10 and CTLA4) in Autoimmune Hemolytic Anemia.
Authors: Figueiredo Maria S. , D’Abronzo Leandro S. , Oliveira Melca M. , Dreyfuss Juliana L. , Bordin Jose Orlando
Published: April 2020

The Autoimmune Hemolytic Anemia (AIHA) is caused by the destruction of antibody-coated red blood cells, but mechanisms that initiate the production of autoantibodies remains unclear. It had been suggest that decreased production of Th1-type cytokines and production of autoantibodies in AIHA can be secondary to the imbalance between anti- and pro-inflammatory factors. Moreover, the presence of single nucleotide polymorphisms (SNPs) showed association with different production of immunoregulatory factors which may modulate the disease expression in AIHA.

OBJECTIVES: To determine the importance of SNPs of pro- and anti-inflammatory factors in the development of AIHA.

PATIENTS: We studied 17 patients with AIHA who has been followed in the Hematology and Blood Transfusion Unit at the Federal University of Sao Paulo (UNIFESP/EPM), Brazil. The control group was composed by 40 healthy volunteer blood donors.

METHODS: After DNA extraction from peripheral blood samples, the frequency of the SNPs was determinate by PCR-RFLP in patients and healthy individuals. The following SNPs were analyzed: Interleukin 12: IL-12 1188 (A/C), Tumor Necrosis Factor alpha: TNFa-308 (G/A), and Lymphotoxin alpha: Lta +252 (A/G); Interleukin 10: IL-10-592 (C/A), and Cytotoxic T-lymphocyte associated protein 4: CTLA4 exon 1 49 (A/G).

RESULTS: The patient group was composed predominantly by female individuals (14 or 82%) and the median of age was 56 years old (18 to 76 years). The frequency observed for each allele studied in the patient group was: allele A of IL-12 = 0.82; allele G of FNTa = 0.85; allele A of Lta = 0.68; allele C of IL-10 = 0.82; allele G of CTLA4 = 0.59. No differences in allele frequency were found between patient and control groups.

CONCLUSIONS: Our results suggest that these polymorphisms appear not to contribute for the development of the AIHA.
Screening of Pathogenicity-Related Genes in Bone Marrow CD4+ T Cells of Aplastic Anemia Patients.
Authors: Zheng Miao , Liu Wenli , Sun Hanying , Zhou Jianfeng , Zhang Yicheng
Published: April 2020

Recent clinical and experimental data have proved that CD4+ T cells play a crucial role in the pathogenesis of aplastic anemia (AA). However, the mechanisms how CD4+ T cells in AA over-proliferate, activate, infiltrate bone marrow and damage hematopoietic cells have largely remained unknown. Therefore we adopted the suppressive subtractive hybridization (SSH) method so as to screen differentially expressed genes of bone marrow CD4+ T cells between AA patients and normal donors, which may hopefully help clear up the mystery of the pathogenesis of AA. Firstly, we selected a pair of subjects: a typical first-visit AA patient and a healthy donor of the same age and sex. Their bone marrow (BM) mononuclear cells were isolated using lymphocyte separating medium by density gradient centrifugation. After a 24-hour expansion culture, CD4+ T cells were separated with magnetic bead sorting technique. Secondly, with CD4+ T cells of the patient as “tester” and those of the normal donor as “driver”, a cDNA library was established by the SSH method. 110 clones were detected by means of PCR in the established subtractive library, which contained an inserted fragment with 200∼700bp respectively, and the positive detected rate was up to 88%. Then 10 of the resulting subtracted cDNA clones were randomly selected for a respective DNA sequencing and honologic analysis. Among 10 sequenced clones, there were 8 known genes including 2 repeated genes. Lastly, we collected BM samples of 20 AA patients and 20 healthy donors so as to validate the expression levels of differentially expressed genes from SSH library with semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR). We found, compared with normal donors, there were 6 genes among these 8 genes from SSH library up-expressed in BM CD4+ T cells of patients with AA. These genes were transducin (beta) -like 1X-linked receptor 1 (TBL1XR1) (NM024665); ATP-binding cassette, sub-family B (MDR/TAP), member 6 (NM005689); ARP2 actin-related protein 2 homolog (ACTR 2) (NM005722); zinc finger protein 561 (ZNF561) (NM152289); NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 6 (NM002493); eukaryotic translation initiation factor 3, subunit 5(NM003754). These results indicate that some genes are found able to regulate functions of BM CD4+ T cells in AA, concerned with protein synthesis, biology oxidation, signal transduction and cell migration, etc. Therefore, screening and cloning these genes are helpful to elucidate the possible mechanisms of AA.
The Effects of Tcf-1 Gene Silence on Marrow CD4+ T Cells from Aplastic Anemia Patients.
Authors: Zheng Miao , Liu Wenli , Sun Hanying , Zhou Jianfeng , Zhang Yicheng
Published: April 2020

As T-cell special expression gene, T cell factor 1 (Tcf-1) has an HMG-BOX domain be cognized as a transcription factor. We have found from our previous studies that Tcf-1 mRNA expression level in bone marrow (BM) CD4+ T cells from aplastic anemia (AA) patients is markedly higher than that of normal people. It is presumed that up-expression of Tcf-1 gene can be closely related to abnormal functions of BM CD4+ T cells from AA patients. Therefore in the current study, we discussed the effects of Tcf-1 gene silence on the BM CD4+ T cells from AA patients. Three shRNA expression vectors psiRNA1, psiRNA2 and psiRNA3 were constructed with psiRNA-hH1neo G2 plasmid. They were respectively transfected into Jurkat cells in which there was rather high expression level of Tcf-1 gene, then Tcf-1 mRNA of Jurkat cells was assayed by semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR). All three plasmid-derived siRNAs could effectively reduce the expression level of Tcf-1 mRNA in transfected Jurkat cells, but the plasmid-derived siRNA psiRNA2 was found to be the most effective inhibitor of Tcf-1 expression. Next, the selected psiRNA2 were transfected into the BM CD4+ T cells from six AA patients. It was found that the expression level of Tcf-1 mRNA in BM CD4+ T cells from these patients was reduced by 45.0%. At the same time, the expression levels of c-myc and CD44 mRNA in BM CD4+ T cells from these AA patients were also decreased to different extents, 47.7% and 53.7%, respectively. These results indicate that Tcf-1 gene can regulate downstream target genes such as c-myc and CD44 to participate in over-proliferation and activation of BM CD4+ T cells from AA patients. It is likely worthwhile to carry out the in-depth study against Tcf-1 in AA therapy.
Anemia an Old Player or a Cut-Throat in Patients with Coronary Artery Disease or Chronic Heart Failure.
Authors: Corinaldesi Giorgio , Corinaldesi Christian
Published: April 2020

Anemia is associated with an increased number of adverse cardiovascular events (CVD) in particular with coronary artery disease (CAD), and chronic heart failure (CHF), and it is also correlated with gender, aging, renal insufficiency, low BMI. Anemia involves inflammatory cytokines (C-reactive protein, IL-6, MCP-1, TNF-alfa), it reduces marrow response to erythropoietin (EPO) and heme-oxygenases-1(HO-1), it also reduces red cells life span and it may impairs reuse of iron, it mostly reduces, the peak VO-2 (peak aerobic power). The latter appears to be an independent factor that may be associated with an adverse outcome, in fact, for a reduction of one gram of heamoglobin (Hb) the risk of morbility and mortality increase respectively by 32% and 18%. The aim of the study was to determine the clinical implication of anemia in patients with CHF or CAD; we have studied 48 patients (32 male, 16 female) with CHF, and 52 patients (34 male, 18 female) with CAD, with a range of Hb concentration included between 9.4gr/dl and 12.6gr/dl. We have evaluated moreover the tolerance to exercise on a treadmill and six minute walk distance (210+/− 32 m in CAD), (180+/− 28 m in HF), the presence of rest dyspnea, the presence of supraventricular or ventricular arrhythmias (atrial/or ventricular premature beats, sinus tachycardia, or ventricular tachycardia, atrial fibrillation); lower levels of Hb, Fe, TIBC correlate with a greater tendency to develop ventricular arrhytmias instead of supraventricular arrhytmias. Anaemia management included erythropoietin stimulating protein, blood transfusion; we have used darbopoietin 50 mcg every week, and this treatment is associated to a significant improvement in functional class and cardiac and renal function. Epo has a strong cardioprotective effect: reducing left ventricular hypertrophy, infarct size, a higher beta natriuretic peptide level, apoptotic cell death, increase FE and capillary vessels; we have remarked a longer endurance time of exercise testing a greater distance walked (282+/− 64 m in CAD), (248+/− 32m in CHF), a significant increased in the peak oxygen consumption VO2 from 12.6+/− 2.4 to 16.2+/− 2.8 mL/Kg x min. in CAD, 9.8+/− 2.0 to 13.2 +/− 3.4 mL/Kg x min. in CHF. Our data also confirm the link between an increased tendency to develop CVD and a decreased level of Hb.

RISK FACTORS CAD CHF Sex (M/F) 32/16 34/18 Age (Years) 62+/− 6 66+/− 4 Hb (g/dl) 10.8+/− 1.8 10.6+/− 1.2 MCV (fl) 82.6+/− 4.4 77+/− 4.2 Iron (mg/dl) (Fe) 42.8+/− 10.2 36+/− 9.4 Total Iron Binding Capacity (TIBC) 316+/− 68.8 280+/− 62.8
Parvovirus B19 Detection by PCR in Patients with Chronic Renal Failure and Resistance to Erythropoietin: An Interim Report.
Authors: Franco Hector , Escoboza Jose R. Borbolla , Scott Sean P. , Villela Luis M. , Valdez Alejandro , Aviña Ana Elena , Benavides Guillermo
Published: April 2020

Introduction: The human parvovirus B19 (pvB19) is well known as a cause of chronic anemia and red cell aplasia, this due to the high tropism for the erythroid progenitor cells where it replicates. Some reports have suggested that the infection of this virus is related to an erythropoietin resistance in patients with chronic anemia and kidney failure. The most frequent causes of erythropoietin resistance are iron deficiency, chronic inflammation process, vitamin deficiency, hyperparathyroidism and malignancies among others. The aim of this study was to detect the viral DNA of PvB19 and its correlation with hemoglobin levels and human erythropoietin dosage in patients with kidney failure under hemodialysis treatment.

Patients and Methods: All patients had chronic renal failure and were undergoing hemodialysis. The detection of the viral DNA was performed by qualitative polymerase chain reaction (PCR). The PCR reaction was carried out using one specifically designed primer set for PvB19 that encoded for a structural capsid protein (VP1). Erythropoietin resistance (ER) was diagnosed using the European Best practice Guidelines as stated by Hay N (Nephrology, 2002).

Results: A total of 58 samples of peripheral blood were obtained from patients with chronic kidney failure. Mean age was 59.41 (23 to 85 years) and mean Hemoglobin levels were 10.1 g/dL (7–14). Iron was normal in all patients and no patient had active infections. Fifty two patients (89.65%) were receiving recombinant erythropoietin. Among all the samples, 3 cases met ER criteria but only in 1 of them did we find the viral DNA (1.72%). Three cases (5.17%) positive to the viral DNA were found in the non erythropoietin resistance group.

Conclusion: Although ours is a small group and we are still gathering samples, up until now, no relation appears to exist between the presence of PvB19 genetic material and ER.