bluuebunny/crossref_metadata_2025 huggingface dataset:
Synergic Effects of Immunosuppressive Agents on T Lymphocyte Function and Cytokine Expression Pattern.
Authors: Lin Mao Fang , Jiang Yan Ling
Published: October 2019

The regimen including mycophenolate mofetil (MMF) and cyclosporine A (CsA) has been successfully used as an effective method in the prophylaxis of acute graft versus host disease. Herewith we investigated the effect of CsA, mycophenolic acid (MPA) alone or in combination with anti B7–1 mAb on proliferation of T lymphocytes and their possible mechanisms. The T lymphocyte reaction system was established in vitro and was detected by 5-bromo-2-deoxyuridine incorporation method; the expression of IL-2, IFN-γ and IL-10 on mRNA and protein level were detected by RT-PCR and ELISA respectively. Experimental concentration of MPA (50μmol/L) and CsA(0.33μmol/L) inhibited the T lymphocyte proliferation significantly. CsA in combination with anti B7–1 mAb(10mg/L) had a much stronger effect on the inhibition(p<0.01). There was no difference between groups treated either with MPA or with the combination of MPA and anti B7–1 mAb. Both of CsA and MPA could inhibit the protein expression of IL-2 and IFN-γ. The protein expression of IL-2 were 99.70±9.15 pg/ml, 48.19±8.67pg/ml and 42.73±14.64 pg/ml in groups of control, treated with CsA or MPA respectively. The protein expression of IFN-γ in the group treated with CsA or MPA decreased significantly than the control (6.81±5.24 pg/ml, 7.87±4.22 pg/ml vs 82.42±25.55 pg/ml, p<0.05). Compared with that treated with CsA alone, The protein expression of IFN-γ in the group treated with CsA in combination with anti B7–1 mAb decreased significantly (0.30±0.52 pg/ml vs 6.81±5.24 pg/ml, p<0.05). Both of CsA and MPA increased the protein expression of IL-10. Compared with MPA alone, MPA in combination with anti B7–1 mAb increased significantly the expression of IL-10 (941.90±56.61 pg/ml vs 770.95±126.85 pg/ml, p<0.05). The relative expression level of IL-2 mRNA (IL-2/GAPDH) and IFN-γ mRNA (IFN-γ /GAPDH) in the CsA or MPA group was significantly lower than the control (p<0.01). The relative expression level of IFN-γ mRNA in combination group of CsA with anti B7–1 mAb decreased even more significantly (0.38±0.05 vs 0.59±0.02, p<0.01). On the other hand, this combination could up-regulate IL-10 mRNA expression (1.80±0.13 vs 1.38±0.06, p<0.01). Combination of MPA with anti-B7–1 mAb showed a statistically significant increase in IL-10 mRNA expression (1.28±0.06 vs 0.84±0.09, p<0.01) as compared with MPA alone. In conclusion, MPA and CsA induced the changes of cytokine expressive spectrum and the Th1 and Th2 shift might be involved in the immunosuppressive effect. The combination of immunosuppressive agents, especially with anti B7–1 mAb had a synergic effect.
Rerovirus Mediated HSV-TK Gene Transfer Can Influence the Function of Human T-Lymphocyte.
Authors: Cai Zhen , Xue Xingkui , Sun Wenji , Yao Hangping
Published: October 2019

To control graft-versus-host disease (GVHD) following allogeneic hematopoietic stem cell (HSC) transplantation while maintaining the graft antileukemic effects of donor T cells, a suicide gene encoding the herpes simplex virus thymidine kinase (HSV-TK) enzyme was introduced ex vivo via a retroviral vector into donor T cells. Although previous clinical trials showed that it was an efficient tool for controlling GVHD, the gene transduction process of donor T lymphocytes may influence the immunocompetence of the T cells. In the current study, we investigated the impact that the gene manipulation in vitro had on the phenotype and functionality of human donor T lymphocytes. We engineered human T lymphocytes with HSV-TK and puromycin resistance gene with a retrovirus vector and analyzed the phenotype change of gene modified cells with flowcytometry. The secretion of gamma-interferon and interleukin-4 of T lymphocyte were detected with enzyme-linked immunospot(ELIPOT)assays. We also analyzed the lytic activity of T cells against allogeneic targets. The result showed that the number of TK gene transduced T lymphocyte was sufficient for clinical use. There was an inversion of the CD4+/CD8+ ratio in the gene modified T cell (GMC) compared with the fresh peripheral blood lymphocytes (PBLs). In addition, the GMCs showed a higher expression of HLA-DR and had more antigen-experienced cells than did the PBLs. In ELIPOT assays, significantly less gamma-interferon secretion by GMCs was observed compared to PBLs (50 vs. 115 spots/well-3×104cells, p<0.05). The results also showed that IL-4-secreting cell frequencies was similar in GMCs and in PBLs (19.3 vs. 20.3 spots/well-3×104cells, p>0.05). After stimulated by allogeneic PBMCs, the GMCs effectors showed a significant reduction in lytic activity against allogeneic targets in contrast with PBL. In conclusion, HSV-TK gene transduction and selection procedure can change the phenotype and functions of human T lymphocyte. The gene modified T lymphocytes still showed biological activities although lower than those of PBLs.
Comparison of Patient Satisfaction between Medical Staff and Clinical Nurse Specialist Performing Bone Marrow Investigation Procedure.
Authors: Pattison John W. , Neylon Annette J.
Published: October 2019

Bone marrow investigation is an important haematological investigation used to determine a wide range of diverse conditions. Historically the investigation was carried out by a member of the medical haematology team; recently there has been an increasing trend towards non-medical staff taking on the role. At South Tyneside NHS Foundation Trust the Haematology Clinical Nurse Specialist (CNS) was trained and deemed competent by the Consultant Haematologists to carry out this complex procedure. To evaluate the benefits of this change in practice we designed a patient satisfaction survey to determine whether the patient experience is influenced by whether the procedure was carried out by the CNS or a member of the medical team. A simple questionnaire was formulated which was given to patients following bone marrow investigation. The questionnaire was completed and returned anonymously to the audit department for collation of results. Twenty-four patients in total returned questionnaires, twelve from medical staff and twelve from the CNS operators.

There was no variation in explanation of the investigation given to the patient between the two groups. However, all 100% of the patients seen by the CNS were given written information regarding the procedure, whereas only 22% of the medical staff group furnished information to the patient. There was no overall difference between the two groups with respect to opportunity to ask questions relating to the procedure before it was undertaken. Those procedures carried out by the CNS were completed one to two days of the decision to biopsy in 92% of cases, whereas this was only achieved in 44% of those patients seen by a member of the medical team. In addition, there was no difference in the pain experience of either group, with twenty to thirty percent requiring analgesia in both cohorts. The CNS was more likely to supply a follow up appointment to the patient for the test results. In summary, there is no apparent difference in patient satisfaction between medical staff and CNS when bone marrow investigation is required. However, the CNS is more inclined to support verbal information with written documentation and to carry out the procedure within a more rapid time frame. When re-structuring haematological services locally, the role of the CNS in respect to bone marrow investigation should be considered as crucial, particularly regarding significant investigative procedures such as bone marrow biopsy. Importantly, this must be supported by suitable training and competency assessment.
MAP Kinase Signaling Pathway Induced by TPO Is a Negative Regulator of Proplatelet Formation in Human Megakaryocytes.
Authors: de Botton Stephane , Aurade Frederic , Chang Yunhua , Christine Rivat , Loussaief Lamya , Larbret Frederic , Larghero Jerome , Raslova Hana , Vainchenker William , Debili Najet
Published: October 2019

Platelets are formed from mature megakaryocytes (MKs) and arise from the development of cytoplasmic extensions called proplatelets (PPT). Proliferation and full maturation of MKs require TPO, but it is dispensable for platelet shedding. To precisely define the role of different signaling pathway activated by TPO on proplatelet formation (PPF), chemical inhibitors of ERK (PD98059), p38 (SB 203580) and a pan PKC inhibitor (GF109203X) were added to MK derived from human CD34+ cells. As previously reported, the PKC inhibitor leads to a marked decrease in PPF and the p38 inhibitor has no effect on PPF. Addition at day 8 of an inhibitor of the ERK pathway (PD98059) to the purified CD34+CD41+ cultured cells leads to an early PPF associated to a 3-fold increase of PPT. As observed with PD98059, an overexpression of a dominant-negative (DN) form of MEK1 in primary MKs led to a 2-fold increase of PPT. Inhibition of the ERK pathway in the immature MKs surprisingly increased MK mean ploidy, (5.7N in the control versus 10.9N in presence of PD98059). Especially, the highest classes of ploidy (8N, 16N and 32N) were significantly increased. Altogether, those results suggest that the inhibition of MAP/ERK1/2 is an essential step for PPF in mature MKs but also that MAPK/ERK1/2 is a negative regulator of endomitosis. As PPF occurs in the presence of TPO, we investigated whether MAPK activation was down-regulated during MK differentiation. Western blot and flow cytometry analysis were performed and show that ERK activation induced by TPO was dramatically decreased at the late stages of megakaryocytopoiesis. In addition, inhibition of MEK prior to PPF was associated with an activation of caspase 3 in the absence of apoptosis suggesting a direct role of the ERK pathway in the regulation of proplatelet. Altogether, those results show a differential response of MAP kinase pathway to TPO depending on the maturation stage. ERK downregulation occurs when PPF begins and MEK inhibition is associated with increased PPF.
Modulation of Dendritic Cell Chemotaxis by HIV-gp120.
Authors: Bailuguttu Anil P. , Ganju Ramesh K.
Published: October 2019

Dendritic cells are essential antigen-presenting cells involved in the induction of T cell immunity against pathogens, such as Human Immunodeficiency Virus (HIV)-1. During the early stages of HIV infection, immature dendritic cells (iDCs) are the first cellular targets of HIV-1. These HIV-infected iDCs cross mucosal surfaces and facilitate transmission of the virus to nearby CD4+ T cells. To date, the effect of HIV envelope proteins such as gp120 on dendritic cell chemotaxis has not been well characterized. Thus, we studied the effect of recombinant gp120 on the chemotaxis of mature DCs (mDCs) and immature DCs. Immature DCs have been shown to express the CC chemokine receptor, CCR5. In contrast, mature DCs down-regulate CCR5 but upregulate CXCR4 as well as exhibit enhanced chemotaxis towards CXCL12. In our study, we analyzed the effect of soluble gp120 on the chemotaxis of DCs. We have shown that pre-treatment of DCs with M-tropic gp120 significantly inhibited the CCR5-induced chemotaxis of iDCs, whereas pre-treatment of DCs with T-tropic gp120 inhibited the CXCL12-induced chemotaxis of mDCs. In addition, we found that M-tropic gp120 itself induced the chemotaxis of iDCs in a dose-dependent manner, while T-tropic gp120 did not show any effect on iDC chemotaxis. We next analyzed the signaling mechanisms involved in the modulation of iDC chemotaxis by M-tropic gp120 and found that M-tropic gp120 increased the phosphorylation of Src kinase, paxillin, Erk, and Akt in these cells. Moreover, M-tropic gp120 induced an increased expression of Lymphocyte Specific Protein-1 (LSP1), which has been reported to regulate the chemotaxis of various immune cells. Taken together, these results suggest that HIV-gp120 plays an important role in modulating the chemotaxis of mDCs and iDCs. This study may help to elucidate the mechanisms of immune response and contribute to the development of vaccines against HIV.
Notch Agonists: Emerging as a Feasible Therapeutic Approach in AML.
Authors: Sutphin Robert M. , Fang Wendy , Miller Claudia , Zweidler-McKay Patrick A.
Published: October 2019

Introduction: The Notch pathway regulates critical cell-fate decisions affecting the growth and development of human hematopoietic cells. Although Notch1 is a known T cell oncogene, we have discovered that Notch signaling behaves as a tumor suppressor in acute myeloblastic leukemia (AML) inducing growth arrest and apoptosis in both cell lines and patient samples. To characterize the mechanism of this effect we have evaluated the influence of the Notch pathway on key effectors of differentiation, cell cycle, and apoptosis in human AML.

Results: Notch signaling induces rapid growth arrest and apoptosis in a panel of human AML cell lines representing a range of AML FAB subtypes (M2–M6). Specifically, activated Notch1 expression caused a 70–95% reduction in AML cells compared to controls (p<0.001) (Figure 1). Notch-mediated growth arrest occurred in 24–48 hours with cells accumulating in G0/G1. Apoptosis was demonstrated by a 3.8-fold increase in AnnexinV binding (p<0.004) and a 3-fold upregulation of caspase 3 activity (p=0.0002) within 24 hours. The caspase 3 activity was abolished by the caspase 8 inhibitor IETD (p<0.0001) suggesting a potential role for the extrinsic death pathway.

We also found that all four Notch receptors (1–4) are capable of inducing this effect, as is the Notch target gene HES1, suggesting a generalized Notch tumor suppressor effect in AML. Furthermore, Notch signaling through HES1 modulates the expression of key regulators of myeloid differentiation and cell cycle progression including downregulation of CEBPα 2.5-fold (p<0.02) and upregulation of p21WAF1 6-fold (p<0.004) suggesting potential mechanisms.

As a novel therapeutic approach, we synthesized Notch agonists which effectively induce Notch signaling with a >18-fold increase in HES1 expression (p<.0001). Exposure of human AML cell lines and primary patient AML samples to this Notch agonist for 24 hours led to a 3 to 9-fold increase in apoptosis (p<0.017) compared to controls (Figure 2).

Conclusions: We report here that Notch signaling is a novel tumor suppressor pathway in human AML. We demonstrate how Notch agonists can be used to induce growth arrest and apoptosis in human AML cell lines and patient AML samples. As a regulator of cell fate, proliferation and differentiation, Notch effectively disrupts multiple pathways in AML. We propose that Notch agonists represent a novel and feasible therapeutic approach in AML. Pre-clinical evaluation is underway.

Figure.1 Effect of Notch on growth of AML cells Figure.1. Effect of Notch on growth of AML cells Figure.2 Notch Agonist induces apoptosis in AML Figure.2. Notch Agonist induces apoptosis in AML
The KIT D816V-Targeting Drug PKC412 Induces Re-Expression of Bim and Synergizes with Mcl-1 Antisense Oligonucleotides in Producing Growth Inhibition in Neoplastic Human Mast Cells.
Authors: Aichberger Karl J. , Mayerhofer Matthias , Gleixner Karoline V. , Krauth Maria-Theresa , Gruze Alexander , Pickl Winfried F. , Wacheck Volker , Selzer Edgar , Sillaber Christian , Valent Peter
Published: October 2019

Systemic mastocytosis (SM) is a myeloid neoplasm involving uncommitted and mast cell (MC) committed hematopoietic progenitors. In most SM patients, the transforming c-KIT point mutation D816V is detectable. However, so far, little is known about the molecular mechanisms underlying KIT D816V-induced transformation. We examined the expression of two Bcl-2 family members, i.e. anti-apoptotic Mcl-1 and the pro-apoptotic tumor suppressor Bim, in the KIT D816V+ human mast cell line HMC-1. As assessed by RT-PCR, immunocytochemistry, and Western blotting, these cells were found to express Mcl-1 mRNA and the Mcl-1 protein, whereas only trace amounts of Bim were detectable in HMC-1 cells. Exposure of HMC-1 cells to the KIT D816V-targeting drug PKC412 resulted in a substantial increase in expression of Bim and in a marked decrease in expression of Mcl-1. Correspondingly, in Ba/F3 cells with doxycycline-inducible expression of KIT D816V (Ton.Kit.D816V), exposure to doxycycline (induction of KIT D816V) resulted in a significant decrease in expression of Bim, and PKC412 was found to counteract the KIT D816V-induced downregulation in Bim in these cells. Transfection of HMC-1 cells with Mcl-1-specific antisense oligonucleotides (ASO) or an Mcl-1-specific siRNA resulted in reduced survival and in an increased percentage of apoptotic cells compared to cells transfected with scramble control oligonucleotides. In consecutive experiments, combinations of PKC412 and Mcl-1 ASO were applied using varying drug concentrations. In these experiments, both compounds were found to synergize with each other in producing growth inhibition and apoptosis in HMC-1 cells. By contrast, no synergism was observed with combinations of Mcl-1 ASO and imatinib or with Mcl-1 ASO and 2CdA. In summary, our data show that the Bcl-2 family members Mcl-1 and Bim are differentially expressed in neoplastic MC, and that targeting of Mcl-1 is associated with reduced survival of HMC-1 cells. In addition, our data show that Mcl-1 ASO synergize with PKC412 in producing growth inhibition in neoplastic MC. These data support the concept that optimal growth inhibition of neoplastic MC in aggressive variants of SM may be achieved with combinations of targeted drugs.
NPM-ALK Converts JUNB from a Tumor Suppressor to an Oncogene.
Authors: Vesely Paul , Staber Philipp B. , Ott Rene , Pinent Montserrat , Linkesch Werner , Schauer Silvia , Hrzenjak Andelko , Kadin Marshall E. , Sternberg David W. , Hoefler Gerald
Published: October 2019

High expression of the tumor necrosis factor receptor CD30 and the AP-1 transcription factor JunB are the hallmark of anaplastic large cell lymphoma (ALCL). In contrast to the prototypic AP-1 factor c-Jun, JunB exerts an antioncogenic function in most cell types. Its functional role in ALCL remains uncertain. In about 50% of nodal ALCL the balanced chromosomal rearrangement t(2;5)(p23;q35), generating the fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), can be detected. Expression of this fusion kinase induces malignancy in mice and also leads to IL-3 independent outgrowth of the murine hematopoietic cell line Ba/F3.

Using NPM-ALK transduced Ba/F3 cells, we show that NPM-ALK induces JunB expression and activation as verified by quantitative RT-PCR, immunoblot and electro-mobility supershift assay. Interestingly, NPM-ALK transduced Ba/F3 cells also express CD30, which is undetectable in the corresponding wild type cells. Since NPM-ALK induces JunB and CD30 and also leads to growth factor independent proliferation in Ba/F3 cells, these cells mimic conditions present in ALCL.

Knock down of JUNB in NPM-ALK expressing cells using RNA interference leads to downregulation of CD30. Moreover, this partial loss of JunB induces upregulation of p16INK4a and downregulation of CCND1, which directly affect the cell cycle at the G1/S transition. These observations indicate that JunB is an essential factor for CD30 regulation and for neoplastic transformation.

To test if JunB by itself is sufficient to induce CD30 expression and IL-3 independence, we stably transduced Ba/F3 cells with JUNB. In Ba/F3 wild type (WT) cells, JunB expression leads to reverse effects compared to that observed in NPM-ALK transduced Ba/F3. Ba/F3 WT cells do not become IL-3 independent. In addition, compared to vector control, JUNB-transduced Ba/F3 cells show a decrease in proliferation. Furthermore, an induction of p16INK4a and a decrease of CCND1 expression are observed. Moreover, aberrant JunB expression does not trigger CD30 expression in this system.

Taken together, we show that both NPM-ALK and JunB are essential to induce CD30 expression. Furthermore the opposing effects of JunB on p16INK4a and CCND1 in the presence or absence of NPM-ALK indicate that NPM-ALK converts JUNB from a tumor suppressor to an oncogene.
ALOX5AP Gene Variants and Risk of Coronary Artery Disease in Italy. An Angiography-Based Study.
Authors: Girelli Domenico , Martinelli Nicola , Trabetti Elisabetta , Olivieri Oliviero , Cavallari Ugo , Malerba Giovanni , Busti Fabiana , Friso Simonetta , Pizzolo Francesca , Pignatti PierFranco , Corrocher Roberto
Published: October 2019

The gene encoding Arachidonate 5-Lipoxygenase-Activating Protein (ALOX5AP; GeneID: 241; chromosome: 13q12) represents the paradigm of a new class of promising genes identified by powerful genome-wide investigations (Helgadottir A, Nature Genetics 2004), that are currently object of intense studies to confirm their role as possible susceptibility factors for atherothrombosis. Indeed, ALOX5AP encodes the 5-Lipoxygenase-Activating Protein (FLAP; Miller DK, Nature 1990), an essential regulator of the 5-LO/leukotriene pathway, that has been recently implicated in the pathogenesis of atherothrombosis (Funk CD, Nature Review Drug Discovery 2005). We studied a total of 1,431 subjects with or without angiographically documented coronary artery disease (CAD). Seven ALOX5AP single-nucleotide polymorphisms (SG13S25, SG13S377, SG13S114, SG13S89, SG13S32, SG13S41, SG13S35) were examined simultaneously, allowing reconstruction of the at-risk haplotypes “HapA” and “HapB” previously identified in the Icelandic and in the British populations, respectively (Helgadottir A, Nature Genetics 2004). Using a haplotype-based approach, HapA was not associated with either CAD or myocardial infarction (MI). On the other hand, HapB and another haplotype within the same region (that we named “HapC”) were significantly more represented in CAD versus CAD-free subjects, and these associations remained significant after adjustment for traditional cardiovascular risk factors by logistic regression (HapB: OR 1.67, 95% CI 1.04 to 2.67; P=0.032; HapC: OR 2.41, 95% CI 1.09 to 5.32; P=0.030). No difference in haplotype distributions was observed between CAD patients with or without a previous documented MI. Adding our data to current knowledge, some evidence on ALOX5AP as a genetic susceptibility factor for CAD has now emerged in several independent populations (Icelandic, British, Japanese, and Italian). Our study, the first angiography-based to our knowledge, points out a possible role of ALOX5AP in the development of the atheroma rather than in its late complications such as MI.
Double Delayed Intensification (DDI) Is Equivalent to Single DI (SDI) in Children with National Cancer Institute (NCI) Standard-Risk Acute Lymphoblastic Leukemia (SR-ALL) Treated on Children’s Cancer Group (CCG) Clinical Trial 1991 (CCG-1991).
Authors: Matloub Yousif , Angiolillo Anne , Bostrom Bruce , Hunger Stephen P. , Nachman James , Sather Harland , Carroll William L. , Winick Naomi , Gaynon Paul S.
Published: October 2019

Intensification of therapy has improved outcome of children with ALL. CCG 1891 demonstrated that children with intermediate risk ALL (a subset of SR-ALL) had a superior outcome with DDI vs SDI using a modified Berlin-Frankfurt-Muenster (BFM) backbone therapy that included prednisone, in a 3 drug induction (Blood.2002;99:825–33). This benefit was limited to patients with > 5% marrow blasts on Day 7 of induction. CCG-1921 best arm had similar outcome with dexamethasone in induction, and SDI (Blood.2003;101:3809–17). The question remained as to whether DDI would benefit SR patients if they received dexamethasone in induction. Therefore, one hypothesis tested by CCG-1991 was that a regimen with DDI phases would have a superior event free survival (EFS) and overall survival (OS) compared with one that had a SDI phase in children with SR-ALL treated with a modified BFM backbone therapy that included dexamethasone in induction. SR-ALL patients with an unfavorable early response (> 25% marrow blasts at day 14, or >25% marrow blasts at day 7 and >5% marrow blasts at day 14) were not eligible for randomization and were assigned to receive augmented therapy. CCG-1991 used a 2 X 2 factorial design to compare outcome in patients who received SDI vs DDI phases, and oral vs escalating intravenous methotrexate without leucovorin rescue during the interim maintenance phases of therapy. The results of the methotrexate question remain blinded at this time. There were 52 relapses among the 1029 patients randomized to the SDI regimens vs 58 among the 1021 patients randomized to the DDI regimens. Four year EFS was 88.3% (SE = 3.6) for the SDI regimens, and 88.1% (SE = 3.9) for the DDI regimens, p = 0.45, RHR 1.15. The 4 year OS estimate is 96.2% for the SDI, and 94.2% for the DDI; p = 0.25, RHR 1.41. We conclude that there are no benefits to DDI vs SDI in children with SR-ALL and a favorable early marrow response to induction chemotherapy.
Upregulation of Tissue Factor in Polymorphonuclear Leukocytes from Patients with Myeloproliferative Disorders and Inhibition by Hydroxyurea Treatment.
Authors: Maugeri Norma , Cerletti Chiara , Giordano Giulio , Petrilli Maria P. , Fraticelli Vincenzo , de Gaetano Giovanni , Leone Giuseppe , Storti Sergio , Donati Maria B.
Published: October 2019

Polymorphonuclear leukocytes (PMN) from healthy subjects can produce and store tissue factor (TF), which is expressed on PMN surface upon in vitro stimulation with P-selectin. We report here that platelets and PMN from 12 patients with myeloproliferative disorders (MPD) (6 Polycytemia Vera, 6 Essential Thrombocythemia) show up regulation of P-selectin and TF, respectively, in the absence of any in vitro challenge, in comparison to healthy subjects (31±5 vs. 2.8±0.4 percent of P-selectin-positive platelets and 6.4±1.3 vs. 0.8±0.3 percent of TF-positive PMN, both differences P<0.05). The number of circulating mixed platelet-PMN aggregates was also increased (15.1±4.4 percent of platelet-PMN aggregates in MPD vs 2.6±0.5 in healthy, P<0.05). PMN TF expression as well as mixed platelet-PMN aggregates, but not platelet P-selectin, were significantly reduced in six MPD patients after three weeks treatment with hydroxyurea (HU) (6.3±1.7 vs. 1.4±0.2 percent of TF-positive PMN and 25.1±6.3 vs. 4.7±1.1 percent of platelet-PMN aggregates, before and after HU, respectively; both differences P<0.05). In vitro studies performed on PMN separated from healthy donors confirmed HU effects (0–1,400 microM). HU prevented both P-selectin-induced TF expression and mixed cell aggregate formation. The inhibitory effect of HU was specific for P-selectin induced PMN activation, as it did not affect fMLP-induced PMN TF expression. Thus, in MPD patients, platelet P-selectin-mediated TF expression on circulating PMN may play a role in thrombus formation and represents a novel target for the antithrombotic activity of HU.
Cell-Derived Microparticles (C-MP) Are Elevated in Splenectomized Patients and Depressed in Those with Hypersplenism, Suggesting Spleen as Scavenger of C-MP.
Authors: Fontana Vincenzo , Jy Wenche , Dudkiewicz Pamela , Horstman Lawrence L. , Bidot Carlos , Ahn Yeon-Soong
Published: October 2019

BACKGROUND: Cell-derived microparticles (C-MP) are microvesicles released during activation and apoptosis from platelets (PMP), leukocytes (LMP), endothelial cells (EMP) and red cells (RMP). They commonly express phosphatidylserine (PS) judged by binding annexin V (AnV) and carry markers of parent cells. C-MP are involved in thrombosis, inflammation and angiogenesis. Little is known how C-MP are cleared from circulation. To test that the spleen might be involved, we investigated C-MP profiles in patients who underwent splenectomy for various causes (group A), those with hypersplenism (group B), and healthy controls with spleen (group C).

MATERIAL AND METHODS: Group A (Gr A) consisted of 44 patients (18 M and 26 F, mean age 57.2 yr) with splenectomy; group B (Gr B) consisted of 25 patients (12 M and 13 F, mean age 61.6 yr) with hypersplenism. Gr A had 25 ITP, 7 myeloproliferative disorders, 4 lymphoma, 3 hemolytic anemias, 4 trauma related splenectomy, 1 unknown reason. Group B had 19 chronic hepatitis C, 2 alcoholic, 2 idiopathic and 1 autoimmune hepatitis, 1 portal vein thrombosis. Group C (Gr C) were healthy controls with spleen (n=109). Flow cytometry was used to assay C-MP. PMP were identified by CD41+, LMP by CD45+, EMP by CD31+/CD41−, and RMP by glycophorin+. Profile of C-MP and blood counts were compared among the 3 groups.

RESULTS: Table 1 summarizes C-MP data among the 3 groups. Gr A and B were well matched for ages and sex. Mean values of WBC and Hgb were normal in all groups while the platelet count was low in Gr B. Mean values of LMP, EMP and RMP were significantly higher in Gr A compared to Gr B and C, but PMP were not. All species of C-MP (PMP, LMP, EMP, RMP) in Gr B were significantly lower than Gr C (healthy control) and were markedly depressed compared to Gr A (see Table 1). Correlation analysis revealed that PMP correlated with platelet count (p<0.0001) but no correlation between LMP and WBC was seen, or between RMP and Hgb. Accordingly, high LMP and RMP in Gr A cannot explain the relatively high Hgb and WBC levels in this group.

DISCUSSION/COMCLUSIONS: Our data suggest that spleen sequesters and/or clears C-MP of all types (PMP, LMP, EMP, RMP). C-MP express PS (bind AnV), which is known to promote clearance from circulation. Splenectomy is known to be associated with thrombosis but its mechanism in not well elucidated. Elevated C-MP in splenectomized patients may contribute to their increased risk of thrombosis.

Table 1 Gr A (Splntomy) Gr B (Hyperspln) Gr C (Control) p value (A-C) p value (B-C) Patient no. 44 25 109 PMP 13858 7073 15992 n. s. <0.0001 LMP 2043 1180 1385 <0.0001 <0.001 EMP 857 238 436 <0.001 <0.0001 RMP 2964 926 1138 <0.0001 <0.005
Changes in ADAMTS 13 Activity and Von Willebrand Factor Parameters during Acute Dengue Infection.
Authors: Sosothikul Darintr , Seksarn Panya , Pongsawaluk Sureeporn , Lusher Jeanne M.
Published: October 2019

Dengue virus causes febrile illnesses: Dengue Fever (DF) and less frequently a life-threatening illness, Dengue Hemorrhagic Fever (DHF). The pathophysiology of hemostatic defect in dengue infection is thought to relate to the direct effect of virus or cytokines on endothelial activation. To study the state of endothelial activation during dengue infection, we measured plasma levels of von Willebrand factor antigen (vWF:Ag), vWF-collagen binding assay (vWF:CBA), and ADAMTS 13 activity in 42 children (20 with DF and 22 with DHF) during 3 phases of illness: febrile, toxic, and convalescent phase. 38 healthy children comprised as controls. Our data shows that both VWF:Ag and vWF:CBA levels were significantly higher in dengue patients (p ≤ 0.001 in both DF and DHF patients) versus controls. DHF patients had significantly higher of VWF: Ag (p = 0.01, versus DF). ADAMTS 13 activity levels were significantly decreased only in DHF patients during 3 phases of the illness (febrile; mean 78%; p = 0.016, toxic; mean 68%; p<0.001 and convalescent; mean 69%; p<0.001 compared to mean 104% of the controls). Compared to DF patients, DHF patients had significantly lower plasma concentrations of ADAMTS 13 activity during the febrile, toxic and convalescent phase (p = 0.039, p = 0.002 and p =0.003, respectively). Endothelial cell activation is a hallmark of dengue infection especially in DHF patients; indicated by a significant rise in VWF:Ag and vWF:CBA and a reciprocal decline in ADAMTS 13 activity.
Portal Vein Thrombosis Following Splenectomy for Hematologic Diseases: Prevalence and Risk Factors.
Authors: Ciapanna Denis , Nosari Annamaria , Gargantini Livio , Mostarda Giovanni , Nichelatti Michele , Turrini Mauro , Morra Enrica
Published: October 2019

Portal vein thrombosis (PVT) is a potentially fatal complication of splenectomy; the prevalence of PVT remains up till now controversial, such as the related-risk factors. The hematologic patients who underwent splenectomy in Niguarda Hospital, Milan, Italy, between January 1995 and November 2004 were retrospectively reviewed to identify the prevalence of post-splenectomy PVT and risk factors associated with its development. Splenectomy was performed because of malignant (n = 41) or non-malignant (n = 44) hematologic diseases. Indications for splenectomy in the groups with and without PVT were compared with Fisher’s exact test. We tested the reliability of platelet count as a marker of PVT, analysing the receiver operator characteristic (ROC) curve. Cut-off level was chosen for best prediction in term of sensitivity and specificity.

Among 85 patients who underwent splenectomy, the associated non-malignant diseases were immune thrombocytopenic purpura (n = 40), hemolytic anemia (n = 2), Evan’s syndrome (n = 1), cryoglobulinemia (n = 1); malignant diseases were lymphoproliferative (n = 39) and myeloproliferative disorders (n = 2). Four cases of PVT (4,71%) were diagnosed. One of 2 patients with a myeloproliferative disorder had PVT. PVT was also present in 3/39 (7.69%) patients with lymphoproliferative malignancies, all with splenic marginal zone B-cell variety non-Hodgkin’s lymphoma (3/16) . The median splenic weight in patients who developed PVT was 1800 g (range 1500–1980 ) and the median post-surgery platelet count was 661 x 109/liter (range 211–1250). No patient with non-malignant diseases had portal vein thrombosis. The incidence of the event was of 0.0000523 new cases for day-patient, with a 0.005% new incidental case probability. Splenic weight, with a cut-off value of 500 grams for the risk analysis (p=0.029), was significantly correlated to thrombosis. PVT was also significantly correlated to the entity of the post-surgery thrombocytosis (p=0.024), setting the threshold value of thrombosis risk at 850.000/uL (Sensibility 0.50 - Specificity 0.95).

Patients with myeloproliferative and lymphoproliferative disorders seem at higher risk for the development of PVT than those with non-malignant diseases. A splenic weight over 500 grams and a post-surgery thrombocytosis over 850.000/uL are significantly associated with the occurrence of PVT. The presence of these two risk factors should require the prophylactic use of adequate doses of low molecolar weight heparin.
Amplified Expression Profiling of Platelet Transcriptome Reveals Global Activation of Arginine Metabolic Pathways in Patients with Sickle Cell Disease.
Authors: Raghavachari Nalini , Xu Xiuli , Villagra Jose , Kato Greg , Munson Peter J. , Morris Sidney , Gladwin Mark T.
Published: October 2019

In sickle cell disease, ischemia-reperfusion injury and intravascular hemolysis produce endothelial dysfunction and vasculopathy characterized by reduced nitric oxide (NO) and arginine bioavailability. Recent functional studies of platelets in patients with sickle cell disease reveal a basally activated state, suggesting that pathological platelet activation may contribute to sickle cell disease vasculopathy. Studies were therefore undertaken to globally examine transcriptional signaling pathways in platelets that may be dysregulated in sickle cell disease. We demonstrate and validate here the feasibility of comparative platelet transcriptome studies on clinical samples from single donors, by the application of RNA amplification followed by microarray-based analysis of 54,000 probe sets. Data mining for platelet specific or abundant genes identified 118 genes that showed more than a 100-fold increase in transcript expression level compared to all other cells in the database. Most of these genes were clearly annotated as platelet-specific and 84% of these transcripts overlapped with the platelet abundant genes identified in previous gene expression studies. On comparing the platelet gene expression profiles in 18 patients with sickle cell disease in steady state to 12 African American controls, at a 3-fold cut-off and 5% false discovery rate, we identified >100 differentially expressed genes, including multiple genes involved in arginine/NO metabolism and redox homeostasis. Further functional characterization of these pathways using Gene Set Enrichment Analysis, real time PCR, arginase enzymatic assay, and polyamine quantification revealed arginase II-mediated catabolism and diversion of arginine from NO signaling and polyamine synthesis to proline formation. These studies suggest a potential pathogenic role for platelet arginase and ornithine decarboxylase antizyme and provide a novel framework for the study of disease-specific platelet biology.
Symptoms and Signs of Hemochromatosis in HFE C282Y Homozygotes Identified by Screening in Primary Care.
Authors: McLaren Gordon D. , McLaren Christine E. , Adams Paul C. , Barton James C. , Reboussin David M. , Gordeuk Victor R. , Acton Ronald T. , Harris Emily L. , Speechley Mark , Sholinsky Phyliss , Dawkins Fitzroy W. , Snively Beverly M. , Vogt Thomas , Eckfeldt John H.
Published: October 2019

Some patients with hemochromatosis (HC) experience fatigue, heart failure or arrhythmias, diabetes mellitus, liver damage, impotence, or arthritis. We examined self-reported symptoms and clinical conditions in persons homozygous for HFE C282Y, the major HC-associated gene mutation, identified in the Hemochromatosis and Iron Overload Screening (HEIRS) Study, a multi-center, multi-ethnic study in which 101,168 adults were recruited from primary care settings. Non-Hispanic Caucasian C282Y homozygotes were compared to participants without HFE C282Y or H63D alleles (controls) with transferrin saturation (TfS) and serum ferritin (SF) levels in the middle half of gender-specific distributions. Evaluation included a medical history, focused physical examination, and repeat SF. Among 44,082 non-Hispanic Caucasian participants screened at five Field Centers in the United States and Canada, 282 persons homozygous for C282Y were identified, comprising three groups: newly-diagnosed cases with normal (N=64) or elevated (N=131) SF (>200 μg/L in women, >300 μg/L in men), and previously-diagnosed cases (N=87). There were 364 non-Hispanic Caucasian controls. Significant differences were observed for six of 38 outcomes. Previously diagnosed C282Y homozygotes and newly diagnosed homozygotes with elevated SF reported significantly more chronic fatigue than controls (p=0.002). All groups of C282Y homozygotes reported weight loss more often than controls (p<0.001). Excessive thirst was reported more often than controls by newly diagnosed C282Y homozygotes, regardless of SF level (p=0.004), but there was no difference in self-reported history of diabetes. Joint stiffness was more common among newly diagnosed C282Y homozygotes with elevated SF than among control subjects (p<0.001). Swelling or tenderness of the second and third metacarpophalangeal joints and increased pigmentation were also more common among previously diagnosed C282Y homozygotes and newly diagnosed homozygotes with elevated SF than among controls (p=0.001 and p=0.002, respectively). The prevalences of manifestations related to liver or heart disease among C282Y homozygotes were not significantly different from controls. There were no differences among the three groups of C282Y homozygotes in the prevalences of any symptoms or clinical conditions. In summary, some symptoms and conditions associated with HC were more prevalent among C282Y homozygotes than among controls. However, C282Y homozygotes identified by screening in primary care settings did not have a higher prevalence of most symptoms and signs associated with HC than control subjects.
Dietary Iron, Alcohol Consumption and Serum Ferritin Concentrations in African Americans.
Authors: Gordeuk Victor R. , Diaz Sharmin F. , Onojobi Gladys O. , Debebe Zufan , Edossa Amanuel , Nekhai Sergei , Taylor Robert E.
Published: October 2019

We investigated the relationship of dietary iron and alcohol consumption with serum ferritin concentration among 143 inner-city African-Americans from the community. Seventy-one of the participants reported consuming an average of more than four alcoholic drinks per day and 72 less than two alcoholic drinks per week. The mean age was 47 years for the high alcohol group and 49 years for the low alcohol group. Thirteen (18%) of the participants in the high alcohol group were women compared to 27 (38%) in the low alcohol group. Body mass index and rates of positivity for HIV and hepatitis C virus were similar. Typical daily dietary iron and alcohol consumption was calculated based on a dietary questionnaire that has been validated for use among various ethnic groups (University of Hawaii). The relationship of dietary iron content and alcohol consumption with log10 serum ferritin concentration and with log10 ratio of serum ferritin to AST (ferritin/AST) was examined in multivariate linear regression models that adjusted for age, sex, ferroportin Q248H status, caloric intake and serum concentrations of CRP and ALT. Ferritin/AST has been shown to correlate with hepatic iron concentration in the setting of alcoholic liver disease. Both average daily dietary iron from meat, poulty and fish (P = 0.013) and average daily alcohol consumption (P = 0.015) correlated positively with log10 serum ferritin, but average daily non-heme iron content did not (P = 0.9). Similar findings obtained for log10 ferritin:AST. According to this modeling and holding other variables constant, a 70 kg individual with serum ferritin of 100 ng/ml associated with dietary iron from meat, poultry and fish of 3.5 mg/day would have serum ferritin of 135 ng/ml (95% c.i. 107–172) associated with dietary iron from meat, poultry and fish of 7.0 mg/day. Similarly, a 70 kg individual with serum ferritin 100 ng/ml associated with no alcohol intake would have serum ferritin 124 ng/ml (105–148) associated with alcohol consumption of 56 g/day. Our results are consistent with the hypothesis that the amount of dietary heme iron and the degree of alcohol consumption influence the amount of storage iron in the body as reflected by serum ferritin concentration.
Down Regulation of Hepcidin and Haemojuvelin Expression in the Hepatocyte Cell-Line HepG2 Induced by Thalassaemic Sera.
Authors: Weizer-Stern Orly , Adamsky Konstantin , Amariglio Ninette , Levin Carina , Koren Ariel , Breuer William , Rachmilewitz Eliezer , Gardenghi Sara , Rivella Stefano , Cabantchik Yoav , Rechavi Gideon
Published: October 2019

β-thalassaemia represents a group of diseases, in which ineffective erythropoiesis is accompanied by iron overload. In a mouse model of β-thalassaemia we observed that the liver expresses relatively low levels of hepcidin, which is a key factor in the regulation of iron absorption by the gut and of iron recycling by the reticuloendothelial system. We hypothesized that despite the overt iron overload, a putative plasma factor found in β-thalassaemia might suppress liver hepcidin expression. We therefore compared sera from β-thalassaemia and haemochromatosis (C282Y mutation) patients with those of healthy individuals in terms of their capacity to evoke changes in expression of key genes of iron metabolism in human HepG2 hepatoma cells. Sera from β-thalassaemia major patients evoked a major decrease in hepcidin (HAMP) and lipocalin2 (oncogene 24p3) (LCN2) expression, as well as a moderate decrease in haemojuvelin (HFE2) expression, compared to sera from healthy individuals. Significant correlation was found between the degree of downregulation of HAMP and HFE2 evoked by b-thalassaemia major sera (r=0.852, p<0.0009). Decreased HAMP expression was also found in HepG2 cells treated with sera collected from β-thalassaemia intermedia patients. In contrast, the majority of sera from hereditary haemochromatosis patients evoked an increase in HAMP expression, which correlated with their transferrin saturation (r=0.765, p<0.0099). Our results suggest that in β-thalassaemia, serum factors might override the potential effect of iron overload on HAMP expression, thereby providing an explanation for the failure to arrest excessive intestinal iron absorption.
Clathrin-Mediated Endocytosis of Soybean Ferritin by Caco-2 Cells.
Authors: Walter Tomas , Martin Carol San , Theil Elizabeth C. , Pizarro Fernando , Garri Carolina , Nunez Marco T.
Published: October 2019

Background: The mechanisms of intestinal absorption of non-heme and heme iron have been extensively studied. However ferritin, which has been shown to be a good source of bioavailable iron in the diet, could have a different mechanism.

Methods: Here we examined cellular mechanisms by which recombinant soybean ferritin (rSBFn) is absorbed in a cell model for intestinal epithelia. The internalization of mineralized (480 Fe) rSBFn, labeled with radioactive iodine, or the fluorescent probe Oregon Green®, was monitored in cultured, polarized Caco-2 cells. Additionally, internalization of rSBFn iron into the labile iron pool was determined by calcein fluorescence quenching.

Results: Caco-2 cells internalized 131 I-rSBFn from the apical border in a temperature, concentration and time dependent fashion. The apical membrane showed saturable binding sites with a KD = 3.43 ×10−9 M. After internalization, rSBFn was degraded, increasing iron in the cytosolic, labile pool, analyzed as calcein quenching. Horse spleen ferritin also induced calcein quenching. The amount of iron incorporated from 5 nM rSBFn was similar to that contributed by micromolar amounts of heme and non-heme iron. rSBFn endocytosis was inhibited by treatment with short RNAi for the μ 2 sub-unit of the assembly peptide 2 complex, by cytosol acidification and by hyperosmolarity, which all inhibit clathrin-mediated endocytosis. Filipin II, an inhibitor of caveolar endocytosis, had no effect.

Conclusion: These results suggests the presence of a third route of intestinal iron absorption in which ferritin from plant or animal sources is internalized from the apical medium of enterocytes by a clathrin-dependent endocytic process.