bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Activity of Granulocytes Primed In Vivo with Glycosylated Granulocyte Colony-Stimulating Factor (G-CSF) Is Superior To Priming with Non-Glycosylated G-CSF.
Authors: Ribeiro Daniel , Laufs Stephanie , Veldwijk Marlon R. , Zeller Jens W. , Ho Anthony D. , Fruehauf Stefan
Published: April 2020

Recombinant granulocyte colony-stimulating factor (G-CSF) is widely used in the treatment of chemotherapy-induced neutropenia as well as in mobilization of peripheral blood stem cells in context with autologous bone marrow transplantation. Beside recombinant non-glycosylated G-CSF expressed in E.coli (Filgrastim) and glycosylated G-CSF expressed in CHO-cells (Lenograstim), pegylated filgrastim (Pegfilgrastim) has been introduced for single-administration into clinical use. Previous studies suggest a different functional activity of glycosylated vs. non-glycosylated G-CSF. Here we study the effects of these different G-CSF on chemotaxis, oxidative burst and antigen expression of granulocytes including and correlated the results with G-CSF serum levels.

Granulocytes were obtained from 27 patients with hematological malignancies before and after the administration of one of the three G-CSFs (10 Lenograstim, 9 Filgrastim, 8 Pegfilgrastim) and isolated using a polymorphprep density gradient. Chemotactic properties were assessed using a Boyden chamber assay in combination with an under agarose assay, both using fMLP as chemotactic stimulus. Release of superoxide anions served as measure of the oxidative burst after stimulation with PMA using a chemiluminescence assay. The viability and surface antigen expression were assessed by FACS. In addition G-CSF serum levels were determined by ELISA.

Patients receiving Filgrastim showed a significantly impaired chemotaxis contrary to patients receiving Lenograstim (p<0.05), in the Pegfilgrastim group patients showed a strong, yet not significant reduction when compared to Lenograstim. No significant effects could be shown in production of superoxide anions in a chemiluminescence assay.

In all three groups, FACS analysis showed a decrease in expression of CD10, CD11b, CD18 and CD62L and a significant increase of the LPS-receptor CD14 as well as an increased expression of the IgG receptor FcγRI (CD64) following G-CSF treatment. The increase of CD64 in the Pegfilgrastim group was significantly stronger compared to the other groups. Moreover, we observed a significantly stronger increase of CD14 in patients receiving Lenograstim when compared to the Filgrastim group. We did not observe significant differences in G-CSF serum levels between the three groups, the highest mean serum concentrations were found in the Pegfilgrastim group.

Our data argue for an improved functionality of granulocytes primed with glycosylated G-CSF.
Type I Gaucher Disease and Parkinsonism: Prevalence of Parkinson Disease among Ashkenazi Family Members Who Are Carriers of Gaucher Disease.
Authors: Rosenbaum Hanna , Aharon-Peretz Judith , Gershoni-Baruch Ruth , Rowe4 Jacob M.
Published: April 2020

Background: An association between patients suffering from Parkinson Disease (PD) and type I Gaucher disease (GD) was previously described (N Engl J Med, 2004). The goals of the present study were to report the frequency of Parkinson disease among the clinically unaffected family members of Ashkenazi GD patients who are obligate or known glucocerebrosidase (GBA) mutations carriers, and evaluate the clinical course in Type I GD carriers with concomitant manifestations of Parkinson disease (PD).

Methods: A cohort of forty three consecutive unrelated Ashkenazi Type I GD patients followed at the hematology department in the Rambam medical center in Haifa, Israel were evaluated for a family history of PD.

Results: Among forty-three families of Ashkenazi GD patients twelve had one or more relatives with PD. All these subjects were known obligate or identified as (GBA) mutation carriers for one of the common mutations among the Ashkenazi Jewish population namely N370S, 1604 or 84GG. The age of PD onset among GD family members carriers of GBA mutation ranged from 40 to 67 years with a mean of 53 years.

Conclusions: A high rate of relatives with PD was found among GD patient’s families. These results reinforce the association between GD carrier state and PD and suggest that glucocerebrosidase mutations might predispose to PD.

PD in family members of Ashkenazi GD patients Age Genotype PD family history Age onset * GF - Grandfather 18 84GG/1604 mother & 2 GF* 50;55 20 84GG/1226 grandfather 50 59 1226/1226 2 uncles 55 31 1226/1226/no sister 46 18 1226/1226 grandmother 65 27 1226/1226 mother 52 18 1226/1226 father & aunt 70 32 1226/1226 2 aunts (identical twins) 55;67 40 1226/1226/no aunt 65 61 1226/1226 mother 40 8 1226/1226 grandfather 70 13 1226/1226 mother & aunt 60;64
Role of Otx1 in the Differentiation of Myelo-Monocytic Precursors.
Authors: Levantini Elena , Cerisoli Francesco , Bertolotti Francesca , Antonelli Valentina , Galvagno Daniele , Tenen Daniel G. , Hoang Trang , Magli Cristina M.
Published: April 2020

Homeobox-containing genes encode transcription factors implicated in the development and differentiation of multiple cell systems, including hematopoiesis. Several lines of evidence, obtained in our laboratory, indicate that Otx1, a homeobox gene of the paired class strictly required for brain morphogenesis, plays an important role also in blood cell production. It is differentially expressed in hematopoietic progenitors, particularly within the erythroid lineage and loss of Otx1 function results in a cell-autonomous erythroid impairment. Furthermore, Otx1 contributes to the control of erythropoiesis through a direct action on Scl/Tal1, a major hematopoietic regulator. In this study we have investigated whether Otx1 is also implicated in the regulation of myelo-monocytic differentiation. Analysis of Otx1 expression indicated that the gene is transcriptionally active in myelo-monocytic precursors. Moreover, using clonogenic assays we observed that Otx1−/− mice display abnormal frequencies of bone marrow myeloid subpopulations, although the total number of myeloid precursors is normal, as compared to wild type animals. Inactivation of Otx1 leads to a significant decrease of myelo-monocytic (CFU-GM) and monocytic (CFU-M) progenitors, and a concomitant increase of granulocytic precursors (CFU-G). In addition, morphological analysis of Otx1−/− bone marrow cells confirmed a high percentage of cells of the granulocytic lineage. Furthermore, at the molecular level, we detected in null mutant cells the upregulation of the gene encoding the G-CSF receptor and the modulation of other key myeloid regulators. Finally, to understand whether the action of Otx1 in myeloid cells is, like in erythoid cells, mediated by Scl we tested whether expression of a Scl transgene in Otx1−/− mice can re-establish normal ratios between the myeloid subpopulations. Results showed that, in contrast to the findings within the erythroid compartment, constitutive Scl expression does not fully rescue the alterations within the myelo-monocytic lineage. Taken together our data indicate that the brain morphogenetic gene Otx1 is an important regulator of the blood system, as it plays a relevant role in erythropoiesis, and contributes to control also myelo-monocytic differentiation, likely at the level of the commitment events of bipotential progenitor cells.
Immortalized HLA Class I Negative Daudi B Cells Coated with HLA/Peptide Monomers Promote Efficient In Vitro Expansion of Antigen Specific CD8+ T Cells.
Authors: Barber Linda D. , Jordan Susan , Whitelegg Alison M.E. , Madrigal J. Alejandro , Savage Philip
Published: April 2020

Monocyte derived dendritic cells (DCs) have historically been used to stimulate antigen specific T cells in vitro for both experimental and clinical applications. However, this is a time consuming procedure that is restricted by the limited number of DC precursors within peripheral blood. We developed an alternative source of antigen presenting cells (APCs) exploiting the immortalized HLA class I negative human B cell line Daudi. A fusion protein comprising the variable region of an anti CD20 antibody linked to streptavidin was used to target recombinant biotinylated HLA class I peptide complexes to Daudi cells via the cell surface antigen CD20. These alternative APCs were used to induce in vitro proliferation of memory T cells specific for Cytomegalovirus (CMV). CD8+ T cells from an HLA-A*0201 positive, CMV positive individual were stimulated with irradiated Daudi cells coated with HLA-A*0201 monomers presenting a peptide epitope from the CMV coat protein pp65. Staining with fluorescent-labelled HLA class I tetramers showed a 40-fold expansion of pp65 peptide specific CD8+ T cells after 14 days of culture that was comparable to expansion produced by stimulating with peptide antigen presented by DCs. The alternative APCs were also used to induced proliferation of allo-restricted peptide specific CD8+ T cells from an allo-naive individual yielding a population in which 78% bound tetramers representing the HLA-A*0201/peptide combination used for stimulation. Functional specificity of the expanded CD8+ T cells was demonstrated using HLA class I monomer coated Daudi cells as targets in cytolytic killing assays and to stimulate cytokine production that was measured by intracellular staining. Our results show these easy to prepare novel APCs induce efficient in vitro expansion of functional peptide specific CD8+ T cells.
Characterizing Thymoglobulin® Reactivity with Human Myeloma and Plasma Cells.
Authors: Wang Kathy S. , Parmentier Julie , Bendahou Christine , Bloy Christian , Williams John
Published: April 2020

Background. Thymoglobulin®, a preparation of IgG purified from the sera of rabbits immunized with human thymocytes, is FDA approved for the treatment of transplant rejection. The perceived mechanism of action of Thymoglobulin® centers around T cell depletion which enables prolongation of graft survival. However, while Thymoglobulin® clearly effects T cell depletion, it also exhibits a portfolio of immunoregulatory activities that are not based on T cell reactivity yet contribute significantly to its efficacy. Examples include reactivities to adhesion molecules on vascular endothelial cells that inhibit T cell trafficking and to costimulatory determinants on antigen presenting cells that blunts T cell activation respectively (Bonnefoy-Berard et al., Transpl. 1991; Blood 1992; Immunol 1992; J Heart Lung Tx 1996; Rebellato, Transpl 1994; Bourdage Transpl 1995; Michallet Transpl 2003). Aims Bourdage and Hamlin (Transpl 1995) and Zand et al (ASCO, 2004) showed that Thymoglobulin® specifically binds to and induces apoptosis in human B cell lines and myeloma cells. We sought to 1) confirm and extend these findings to non-malignant human B and plasma cells and 2) identify the B cell reactive antibody specificities that reside within Thymoglobulin. Results Thymoglobulin® expresses complement independent, antibody dose dependent cytotoxicity for B cells and myeloma cells at clinically relevant Thymoglobulin® concentrations (effective Thymoglobulin® concentrations in vitro 10 - 100 mg/ml). In addition, Thymoglobulin®, at concentrations 10 – 100 mg/ml, was shown to inhibit human bone marrow derived plasma cell antibody production in vitro in a complement independent manner. Visual observation of Thymoglobulin® containing bone marrow cell cultures showed cell aggregation that was antibody associated. Thymoglobulin® contains antibody reactivities that appear to be B cell selective, as it retains the B cell reactivity even after exhaustive adsorption vs. human T cell antigens. T cell depleted B cell reactive antibodies were shown to significantly inhibit constitutive IgA and IgG production from antibody producing cells within normal human bone marrow cells in vitro. The mechanism by which Thymoglobulin® kills B cells and plasma cells in vitro is unknown, but did not require addition of complement. The nature of the B cell antigens recognized by Thymoglobulin® will be discussed.
Age-Related Induction of CD4+ T-Cell Unresponsiveness Is Reversible and Dependant of the Host’s Environment.
Authors: Horna Pedro , Chavan Rahul , Brayer Jason , Suarez Ildefonso , Sotomayor Eduardo M.
Published: April 2020

A large number of CD4+ T-cells from either aged mice or humans display surface markers associated with an activated/memory phenotype. In spite of these changes however, these T-cells have a markedly decreased ability to proliferate and produce IL-2 in response to antigen stimulation in vitro. The cellular and molecular mechanisms involved in this age-related unresponsiveness of the CD4+ T-cell compartment remain poorly understood. Utilizing a well-established experimental system in which transgenic CD4+ T cells specific for a MHC class II restricted epitope of influenza hemagglutinin (HA) are adoptively transferred into non-transgenic recipients, we have previously elucidated important mechanisms involved in the induction and maintenance of CD4+ T-cell tolerance. Our studies were however limited to the analysis of T-cell function in lymphoma bearing young mice (4 to 10 weeks old). Here, we assessed the influence of the aged microenvironment in determining the phenotype and function of antigen-specific T-cells. CD4+ T-cells from young TCR transgenic mice (2 months old) were adoptively transferred into either old (20–24 months) or young (2 months old) non-transgenic mice. Two weeks later, clonotypic and non-clonotypic CD4+ T-cells were isolated from the spleens of these animals and their phenotype and function were determined in vitro. Reminiscent of the age-related changes observed within the normal CD4+ T-cell repertoire, young transgenic T-cells transferred into aged hosts have acquired an activated/memory phenotype but displayed a significant impairment in antigen-specific proliferation and IL-2 production in response to cognate antigen in vitro. These changes were not due to homeostatic proliferation of the transferred T-cells into the relatively lymphopenic aged host. To determine whether the changes observed in “aged” T-cells were reversible or not, we adoptively transfer old T-cells back into young hosts or into control old mice. While old transgenic T-cells transferred into an old environment remained fully unresponsive, the adoptive transfer of the same old T-cells into a young host restored their ability to proliferate and produce IL-2. Surprisingly, these “old” T-cells were able to produce significantly higher levels of IFN-gamma indicative of their memory/effector phenotype. Furthermore, young animals adoptively transferred with “aged” antigen-specific T-cells were now capable of rejecting A20 B-cell lymphomas expressing HA as a model tumor antigen (A20HA). Taking together, factor(s) present in the aged microenvironment are responsible for limiting the effector function of CD4+ T-cells that seem otherwise well equipped to become fully activated if the proper environment is provided (young microenvironment). The potential role of soluble suppressive factors as well as regulatory T-cells (Tregs) in the unresponsiveness observed in the T-cell compartment of aged hosts will be discussed.
Proteasome Inhibitor Does Not Affect the Function of Human Immune Systems: Effects on Dendritic Cells, T Lymphocytes and NK Cells.
Authors: Bae Jooeun E. , Yu-Tzu Tai , Hidesima Teru , Catley Larence , Li Xianfeng , Prabhala Rao H. , Bachu Ramesh B. , Bertheau Robert , Shammas Masood A. , Samala Sushma , Miller Jeff , Anderson Kenneth C. , Munshi Nikhil C.
Published: April 2020

Bortezomib is the first proteasome inhibitor approved for the therapy of multiple myeloma (MM) based on its in vitro and in vivo activity in myeloma. However, the toxicity and effects of this drug on the human immune function have not been entirely studied. In the present study, we evaluated the effects of Bortezomib on normal human immune cells including dendritic cells (DC), T lymphocytes and NK cells for cell survival, antigen expression, production of cytokines, and other key parameters of immune cell function. In our evaluation of effect of Bortezomib on DC, we did not observe significant change in the expression of cell surface antigens including CD40, CD80, CD83, CD86, HLA-ABC and HLA-DPQR molecules in terms of percentage of cells positive as well as mean fluorescence intensity (MFI). Bortezomib treated immature DC maintained the ability for antigen uptake as measured by uptake of Dextran-FITC (untrt vs. trt = 798 MFI vs. 802 MFI), maintained the expression levels of antigen uptake receptors including mannose (untrt vs. trt = 85% vs. 79%) and DEC-205 (untrt vs. trt = 49% vs. 42%), and the capacity to produce IL-12 (untrt vs. trt = 135 vs. 125 pg/ml). In addition, Bortezomib treated mature DC was able to induce comparable levels of allogenic T cell proliferation to the untreated mature DC as measured by 3[H]-Thymidine incorporation (untrt vs. trt = 212556 cpm vs. 220571 cpm). Furthermore, cell surface antigen expression including CD3, CD4, CD8, CD28, CD154 (CD40L) and TCRab on T lymphocytes were not changed by Bortezomib treatment. The treated T cells also maintained the ability to secrete IFN-g secretion in response to allogenic DC (untrt vs. trt = 85 vs. 88 pg/ml) or Staphylococcal enterotoxin B (untrt vs. trt = 131 vs. 154 pg/ml). The cytolytic activity of the NK cell population was comparable between proteasome inhibitor treated and untreated control cells against the McCAR (untrt vs. trt = 44% vs. 52%) and MM1S (49% vs. 42%) target MM cell lines. This observation was correlated with similar expression levels of CD2, CD11a, CD94, NKp30, NKp44, NKp46, and KARp50.3 activation antigens in treated versus untreated NK cells. These, in vitro results confirm lack of adverse effects of Bortezomib on immune function, and allow us to incorporate of Bortezomib in multimodality therapy that includes immunotherapy.
The Essential of PI3Kγ and PI3Kδ in Thymocyte Survival.
Authors: Diacovo Thomas G. , Vermi William , Montgrain Vivian , Douangpanya Jason , Doggett Teresa , Puri Kamal , Swat Wojciech
Published: April 2020

Class I phosphoinositide 3-kinases (PI3Ks), consisting of PI3Kα, β, γ and δ, are a family of intracellular signalling molecules that play an important role in both innate and adaptive immune responses. In thymocytes, however, their role is less clear as PI3Kγ, but not other class I members, has been postulated to be partially involved in pre-TcR-dependent differentiation without effecting overall T cell numbers. We now report that PI3Kγ in conjunction with its delta counterpart are essential for thymocyte survival and ultimately T cell production. Surprisingly, genetic deletion of the both p110γ and p110δ catalytic subunits resulted in a dramatic reduction in thymic size, cellularity, and lack of cortico-medullary differentiation. Total thymocyte counts in these animals were 27-fold less than observed for wild-type (WT) controls due to a diminished number of DP cells and was associated with T cell depletion in both peripheral blood and secondary lymphoid organs. This phenotype could also be recapitulated in vitro as incubation of thymi harvested from day 14 p110γ−/− embryos with the p110δ specific inhibitor IC87114, but not vehicle control, yielded identical results. Moreover, the observed reduction in the DP population appears to be intrinsic to thymocytes as reconstitution of animals deficient in both p110γ and p110δ catalytic subunits with WT fetal liver cells restored the proportions of DN, DP, SP to that observed in WT controls. The observed defects, however, appear to be due to an excessive amount of apoptosis of the DP thymocyte population, while TCRbeta expression, pre-TCR selection and DP cell production appear to proceed unperturbed. Thus, this study provides evidence that these two distinct class I PI3Ks isoforms work in concert to protect developing thymocytes from apoptotic signals rather than by participating directly in thymocyte differentiation.
Identification of a Potent Inverse Agonist at a Constitutively Active Mutant of Human P2Y12 Receptor.
Authors: Ding Zhongren , Kim Soochong , Kunapuli Satya P.
Published: April 2020

Human platelets express two P2Y receptors: Gq-coupled P2Y1 and Gi-coupled P2Y12. Both P2Y1 and P2Y12 are ADP receptors on human platelets and are essential for ADP-induced platelet aggregation that plays pivotal roles in thrombosis and hemostasis. Numerous constitutively active G protein-coupled receptors have been described in natural or recombinant systems but in the P2Y receptors, to date, no constitutive activity has been reported. In our effort to identify G protein coupling domains of human platelet ADP receptor we constructed a chimeric HA-tagged human P2Y12 receptor with its C-terminus replaced by the corresponding part of human P2Y1 receptor and stably expressed it in CHO-K1 cells. Interestingly, the chimeric P2Y12 mutant exhibited a high level of constitutive activity as evidenced by decreased cAMP levels in the absence of agonists. The constitutive activation of the chimeric P2Y12 mutant was abolished by pertussis toxin, a Gi inhibitor. The constitutively active P2Y12 mutant retained normal responses to 2-MeSADP, with an EC50 of 0.15 ± 0.04 nM. The constitutively active P2Y12 mutant caused Akt phosphorylation that was abolished by the addition of pertussis toxin. Pharmacological evaluation of several P2Y12 antagonists revealed AR-C78511 as a potent P2Y12 inverse agonist whereas AR-C69931MX as a neutral antagonist. In conclusion, this is the first report of a cell line stably expressing a constitutively active mutant of human platelet P2Y12 receptor and the identification of potent inverse agonist.
Redistribution of Activation-Dependent Antigens on the Platelet Surface In Vivo and In Vitro.
Authors: Han Yue , Wu De Pei , Dai Lan , Sheng Wenrong , Ruan Changgeng
Published: April 2020

Platelets play a crucial role in thrombosis, platelet activation induces an alteration of platelet membrane glycoproteins between the platelet surface and surface connected canalicular system (SCCS). In an attempt to better understand this process and assess its significance in the study of thrombosis disease, we used flowcytometry for detecting the redistribution of antigens on the platelet surface following activation. The expression of platelet membrane glycoproteins (GP) Ib, IIIa and P-selectin as well as the platelet agglutination induced by ristocetin were measured in 20 normal subjects, whose platelets were stimulated by the peptide SFLLRNP(TRAP, thrombin receptor activated peptide) at different time points (0~30minute). The same antigens were also analyzed in whole blood in 30 patients with cerebral infarction. As expected, a stable increase of P-selectin was obtained from 2 minute upon TRAP activation, while a reversible reduction of GPIbα and agglutinability by ristocetin were showed at the same time, with a maximum decrease at 2 minute, then followed by a return of GP Ibα to platelet surface. In addition, the platelet membrane GPIIIa was not significantly changed in the initial stage after platelet stimulation and increased by degrees 10 minutes later. Compared with those in normal group, there was an apparent increase of P-selectin and a lower expression of GPIb in the patients with cerebral infarction, while GPIIIa was not significantly changed in all the samples. In summary, TRAP can mediate platelet activation, which leads to the redistribution of GPIb and GPIIIa in a time-dependent manner, together with the release of P-selectin. Meanwhile, the change of activation-dependent antigens on the platelet surface could be an important index reflecting the platelet activation in vivo.
Antiphospholipid Antibodies Impair the Clearance of Apoptotic Platelets.
Authors: Pierangeli Silvia S. , Vega-Ostertag Mariano E.
Published: April 2020

Background: Thrombocytopenia is frequent in patients with the Antiphospholipid Syndrome (APS). The mechanism(s) that lead to abnormal platelet destruction is not understood. Phosphatidylserine (PS) that is exposed in the outer leaflet of the membrane of aged human platelets (AHP) and β2glycoprotein I (β2GPI) mediate their phagocytosis by macrophages without inducing inflammatory or immune responses. We hypothesized that antiphospholipid antibodies (aPL) affect the clearance of AHP and induce immunogenicity of AHP leading to the production of anti-platelet antibodies.

Methods: To examine that question, we studied phagocytosis of AHP by macrophages in the presence of β2GPI, IgG aPL or control IgG (IgG-NHS). AHP labeled with CM-Orange, were incubated with β2GPI and aPL IgG or with IgG-NHS, and added to a monolayer of cultured phagocytes. The fluorescent-positive phagocytes (FPPC) were then tested by fluorescence microscopy. Then the cells were trypsinized and analyzed by flow cytometry. We also examined the immunogenicity (production of anti-platelet antibodies) of AHP treated with IgG aPL and with IgG-NHS by immunizing Balb/c mice with AHP and IgG-aPL or IgG-NHS. The patterns of reactivity of the sera of the immunized mice was examined by immunoblot of human platelet lysates.

Results: aPL IgG produced a significantly lower % FPPC compared to the IgG-NHS-treated cells (15.33 ± 5.31 vs 89.0 ± 7.94, respectively). This was confirmed in the flow cytometric studies: IgG aPL produced significantly lower % of FPPC when compared to IgG-NHS-treated platelets (42.49 ±11.77 vs 78.11± 5.43, respectively).. Mice immunized with AHP and aPL IgG produced significantly higher titers of anti-platelet antibodies (as detected by ELISA) when compared to mice immunized with IgG-NHS (p=0.0033). Furthermore, there was a different pattern of reactivity of the sera with respect to recognition of platelet antigens, when sera of immunized mice were analyzed by immunoblot.

Conclusions: The data indicate that aPL impair the clearance of apoptotic platelets and affect their immunogenicity. This may lead to the production of anti-platelet antibodies and thrombocytopenia in APS.
A Proteomic Approach to the Characterization of the Endothelial Dysfunction in Uremia.
Authors: Carbo Carla , Diaz-Ricart Maribel , Fuste Berta , Cases Aleix , Carrascal Montserrat , Abian Joaquin , Ordinas Antonio , Escolar Gines
Published: April 2020

Deficient hemostasis coexists paradoxically with accelerated atherosclerosis in patients with cronic renal failure (CRF). The high incidence of aterothrombotic events in these patients has been associated with endothelial dysfunction. We have established an in vitro model of cell dysfunction by exposing endothelial cells (EC) to uremic media (Serradell y cols, Thromb Haemost86:1099,2001). However, it is difficult to advance in this research using conventional protein identification techniques. We have applied a proteomic approach to exhaustively characterize the EC dysfunction in uremia. Extracts of confluent EC, grown in the presence of sera from control donors and from patients under hemodialysis, were collected and lysated. Proteins were resolved by molecular weigh (Mr range 10–50kDa) and isoelectric point (pI range 3–10) using two-dimensional gel electrophoresis (2-DE). Spots were visualized by silver staining and further identified by mass spectrometry (MALDI-TOF). A search for significant changes in protein phosphorylation and glycosilation, and overexpression or new presence of proteins was specifically performed in uremic EC. Identification of some of the most prominent proteins revealed molecules related to inflammation (HMGB1, 25148/5.8, Mr/pI; aldose reductase, 35854/6.5; a proteasome component, 29192/5.7) and oxidative stress (superoxide dismutase, 24772/8.3; gluthatione peroxidase, 21899/6.2), both processes associated with CRF. In addition, we observed a significant increase in the protein destrin (18506/8.1), an actin depolymerizing protein. This finding could be related to the previously reported increased detachment of uremic EC when exposed to flowing blood. Therefore, proteomics can be used to compare the proteome of control and uremic EC, helping to reveal information on the molecular basis of the disease. A more exhaustive analysis of 2-DE gels will provide answers and potential therapeutic targets in a short future.
Defibrotide, an Endothelium Protecting and Stabilizing Drug, Has Anti-Angiogenic Potential In Vitro and In Vivo.
Authors: Eissner Guenther , Geissler Edward K. , Iacobelli Massimo , Andreesen Reinhard , Holler Ernst , Koehl Gudrun E.
Published: April 2020

DF is a polydisperse mixture of single-stranded polydeoxyribunucleotides which is successfully used in the treatment of hepatic veno-occlusive disease and other endothelial disorders. Recent pre-clinical evidence suggests that DF might also have anti-neoplastic properties. We addressed the question whether this might be due to the prevention of tumor blood vessel formation (angiogenesis). The anti-angiogeneic potential of DF was tested in vitro (Matrigel™ tube formation and aortic ring assay) and in vivo (dorsal skin-fold chamber model). Our results show that DF quantitatively (100%) blocks tube formation of trans-differentiated human endothelial-like cells (ELC) at concentrations corresponding to pharmacologic DF blood levels (100 μg/mL). Similarly, the sprouting of rat aorta endothelial cells in Matrigel™ was prevented by nearly 100%, when DF was applied on a daily basis. In vivo tumor angiogenesis in a human gastric cancer (TMK-1) grown in skin-fold chambers (nude mice) was also attenuated on day 5 by DF, as measured by microvascular density. Although the exact mechanism of DF action remains to be elucidated, initial Western blotting results show that DF reduces phosphorylation-activation of p70S6 kinase, which is a key target in the PI3K/Akt/mTOR signaling pathway linked to endothelial cell and pericyte proliferation and activation. Taken together, our data suggest that while DF is known for its endothelium-protecting function, it also inhibits tumor blood vessel formation, and thus should be considered for further testing as an anticancer agent.
Efficacy and Safety of a New Intravenous Immunoglobulin in Adult Subjects with Chronic Idiopathic Thrombocytopenic Purpura.
Authors: Leibl Heinz , Varga Gyula , Volkova Zuzana , Gasztonyi Zoltan , Hlusi Antonin , Mayer Jiri , Chojnowski Krzysztof , Wolf Hans-Heinrich , Sharkhawy Marlies , Pavlova Borislava G. , Birthistle Karl , Engl Werner , Walter Simone , Ehrlich Hartmut J.
Published: April 2020

Idiopathic thrombocytopenic purpura (ITP) is a common clinical disorder of immune regulation leading to phagocytic destruction of platelets. Intravenous immunoglobulin (IGIV) has been used successfully to increase platelet count in patients with ITP. Baxter has developed a new IGIV (IGIV,10%), a 10% liquid immunoglobulin preparation with 3 dedicated virus reduction steps integrated into the manufacturing process. The efficacy and safety of this product was assessed in a prospective multi-center study in adult chronic ITP patients with platelet counts of 20x109/L or less. A total of 23 subjects were enrolled and treated for 2 to 5 days with a total dose of 2 g/kg. Of 21 subjects included in the per-protocol analysis data set, 15 responded to treatment (71.4%), achieving a platelet count of at least 50x109/L by Day 8. Fourteen of 15 responders reached this level by Day 5. The median duration of platelet response was 25 days and the highest median platelet count in the responders was 182x109/L. A total of 81 infusions were administered to the 23 subjects in the safety analysis data set. Among the 40 non-serious adverse events related to the use of the study drug, 35 were mild, 3 were moderate, and 2 were severe. The most frequent related adverse events were headache and pyrexia. The results of this study demonstrate that IGIV, 10% TVR Solution is effective in the treatment of adult subjects with chronic ITP and has an excellent safety profile.
Radiometric Quantitation of PAIgG Stratified Treatment Response in ITP Patients.
Authors: Estrada Dexter , Schwartz2 Kenneth A.
Published: April 2020

Because treatment response in ITP is variable, it would be useful to be able to assess which patients are likely to respond to conventional treatment. Toward this goal we correlated the number of platelet surface IgG molecules measured with a quantitative radiometric platelet antibody assay (PAIgG) with treatment related changes in platelet count. We tested the hypothesis that patients’ response to conventional treatment could be stratified based on quantitation of the patients’ PAIgG. Data was obtained via a retrospective chart review. Patients with a diagnosis of idiopathic thrombocytopenic purpura (ITP), age 18 or older with an initial platelet count of <150,000, and quantitation of antiplatelet IgG performed at the time of diagnosis were included in the study. Patients were treated using conventional approaches that included steroids, IVIgG, anti-D antibody, splenectomy, and or cytotoxic medications. Patient response to therapy was evaluated as the change in platelet count, computed as the difference in platelet count obtained at diagnosis and the latest stable platelet count available. There were 64 patients with at least 90 days between initial and follow-up platelet counts. Of the 31 patients whose PAIgG were ≤1,000 molecules/platelet (m/plt), only 1 had an initial platelet count of <50,000. Of the 33 patients who’s PAIgG were >1,000 m/plt 16 had platelet counts of <50,000 (p≤0.001. Patients with PAIgG levels >1000 m/plt exhibited a significantly greater increase in platelet count μ=73,000 ±92,000 as compared to those with PAIgG levels ≤1000 m/plt, μ=18,000 ±43,000 (p=0.03). Although a PAIgG level of 500 m/plt is 3 standard deviations above the mean of normals, a greater response to treatment was noted in patients if their PAIgG level was >1000 m/plt. Patients with PAIgG levels between 500 and 1000 m/plt respond in a similar fashion to those with PAIgG levels <499. In addition, more patients with PAIgG levels >1000 m/plt received conventional treatment (19/33 vs. 10/31; p<0.05). In particular, there were more patients who received cytotoxic treatment in this cohort, (11/33 vs. 3/31; p≤0.025). Similar results were observed in 74 patients with at least 30 days between initial and follow-up platelet counts. This study suggests that measuring the amount of antiplatelet IgG stratifies ITP patients into 2 groups: patients with PAIgG ≤1,000 m/plt with platelet counts over 50,000 and patients with PAIgG >1,000 m/plt platelet counts that are less than 50,000. Those patients whose PAIgG is >1,000 m/plt are more likely to receive treatment and have an increase in platelet count with conventional therapy. We conclude that radiometric quantitation of PAIgG is helpful for predicting a patient’s response to therapy.
Hemolytic Uremic Syndrome Is an Immune Mediated Disease: Role of Anti-CD36 Antibodies.
Authors: Rock Gail , Clark William , Sternbach Marion , Kolajova Maria , McLaine Peter
Published: April 2020

Hemolytic uremic syndrome is a disorder in which platelet microthrombi are formed which have a particular propensity to deposit in the kidney microvasculature resulting in impaired renal function and thrombocytopenia. The mechanism of formation of these microthrombi is not known. In this study, we showed that plasma from five adult and six pediatric cases of HUS caused aggregation and release of adenosine triphosphate from normal platelets. The plasma reacted against platelet lysate in a protein blot and all samples showed reactivity against a band at 88kDa corresponding to the membrane antigen CD36. This was confirmed by probing with Mo91, a monoclonal antibody to CD36. CD36 was also identified in the immune complex formed by incubation of patient plasmas with normal platelet lysate. In other studies, bands of 32kDa and 7.7kDa were obtained when purified verotoxin was protein blotted and probed with either patient plasma or with anti-CD36 antibody Mo91 suggesting structural homologies between CD36 and verotoxin. The data support the concept of an immunological pathogenesis for HUS and suggest that molecular mimicry involving one or both of the homologous domains in membrane-bound CD36 and verotoxin lead to the development of antibodies capable of inducing the pathophysiological events characteristic of HUS.
Treatment of Refractory Chronic Immune Thrombocytopaenic Purpura (ITP) with Rituximab: Report of a Cases Series.
Authors: Garcia-Chavez Jaime , Vela-Ojeda Jorge , Gonzalez-Acosta Jose C. , Ovilla-Martinez Roberto
Published: April 2020

Purpose: To describe the response rates and time to treatment failure in patients with chronic refractory ITP treated with Rituximab.

Methods: Fourteen consecutive patients with chronic refractory ITP, who didn’t achieve a response to first- and second-line treatments (including splenectomy), were recruited between November 2001 and May 2004. Rituximab was administered at 375 mg/m2 iv weekly’ 4 and all patients were pre-medicated with Diphenhydramine (30 mg single dose) and Hydrocortisone (100 mg IV single dose). The first response assessment was performed at week 4, using the following criteria: complete response, platelet count > 100 x 109/L; partial response, platelet count > 50 x 109/L; minimal response, platelet count persisted below 50 x 109/L but without bleeding or need for platelet transfusion; and no response if the platelet count did not change, or the patients required platelet transfusion or remained symptomatic. The response was considered sustained if it persisted for at least 6 months.

Results: The age range was 17–70 years; the baseline platelet count range was 3–37 x 109/L. Five patients achieved a complete response, four patients a partial response, four a minimal response and one no response. The time to response was from 4–32 weeks and 10 patients showed a sustained response. No patients with a response showed bleeding and no patients required the use of other drugs.

Conclusions: These results suggest that Rituximab should have a significant therapeutic effect and an acceptable safety profile in patients with refractory chronic ITP. However, there is a lack of randomized, prospective studies to demonstrate the therapeutic benefit of this treatment.
Reduced Intensity Allogeneic Transplantation for Non-Hodgkin’s Lymphoma: Extended Follow-Up of an Alemtuzumab-Containing Regimen.
Authors: Thomson Kirsty J. , Morris Emma C. , Milligan Don , Cook Gordon , Cavet Jim , Craddock Charles , Hunter Ann , Schey Steve , Parker Anne , Goldstone Anthony H. , Linch David C. , Peggs Karl S. , Mackinnon Stephen
Published: April 2020

Allogeneic transplantation with reduced intensity conditioning is increasingly being used in patients with non-Hodgkin’s Lymphoma (NHL) who fail standard therapy. We report extended follow-up on 121 patients with NHL, who underwent allogeneic transplantation with reduced intensity conditioning at 8 UK centres. Conditioning was with fludarabine 150mg/m2, melphalan 140mg/m2 and alemtuzumab (60–120mg). Cyclosporin A was administered at 3mg/kg from day-1, and stem cell source was bone marrow or PBSC. Diagnoses were in 3 categories: low grade follicular NHL (n=50), mantle cell lymphoma (n=21), and high-grade NHL (n=50, including transformed low grade disease n=15). Donors were HLA-matched siblings in 75 (62%), and unrelated in 46 (38%), of whom 18 were HLA-mismatched at up to 3/10 loci. 48% of patients had failed previous autologous transplantation. Median follow-up was 35 months (1–78). For the group with low grade follicular NHL (n=50), estimated overall survival (OS) was 76% at 1yr and 67% at 4yrs, and non-relapse mortality (NRM) was 16% at 4yrs. Disease relapse or progression occurred in 12 patients, of whom 8 received donor lymphocyte infusions (DLI), with responses in 6. Current progression-free survival (cPFS) is 68% at 4yrs. For the group with mantle cell lymphoma (n=21), estimated OS was 83% at 4yrs, NRM was 11% at 4yrs and relapse or progression occurred in 6 patients. Three patients received DLI, with non-sustained responses in 2. Current PFS is 43% at 4yrs. For high-grade NHL (n=50), estimated OS was 52% at 1yr and 45% at 4yrs. Prior autologous transplantation was common in this group (72%), and NRM was higher at 34% at 1yr and 40% at 4yrs. Progression/relapse occurred in 15 patients, of whom 10 received donor lymphocytes, with responses in 5. Current PFS is 48% at 1yr and 43% at 4yrs. These results, from patients who were often heavily pre-treated, including having failed autologous transplantation, provide encouraging evidence to support the application of reduced intensity allogeneic transplants in NHL. NRM in low grade follicular or mantle cell lymphoma is low, consistent with the use of T cell depletion, and graft-versus-lymphoma effects can be induced with DLI in a subset of cases. The data in follicular lymphoma, in particular, supports consideration of this therapy earlier in the disease. In high-grade disease, NRM appears to be higher, but durable remissions are attainable in a proportion of patients.
Comparison of Rapid Platelet Function Assay with Whole Blood Platelet Impedance Aggregometry for the Definition of Aspirin Resistance.
Authors: Dyszkiewicz-Korpanty Anna M. , Kim Anne , Burner James D. , Frenkel Eugene P. , Sarode Ravindra
Published: April 2020

The reported incidence of aspirin (ASA) resistance ranges from 5 to 30%. Various platelet function assays have been employed to detect aspirin resistance in patients with cardio- and cerebrovascular disease. Such a high proposed incidence of ASA resistance poses a critical need for a rapid point-of -care (POC) platelet function test. Unfortunately, no uniformly accepted definition of ASA resistance exists. Platelet aggregation studies that have been used to define ASA resistance are time consuming and require special technical expertise. The Ultegra Rapid Platelet Function -ASA (RPFA-ASA) has been developed as a POC test that is performed without sample processing. This optical method measures agglutination of fibrinogen-coated beads upon platelet activation with arachidonic acid. In the presence of aspirin effect, however, the agglutination of the beads is inhibited. The described cutoff of ≥ 550 Aspirin Reaction Units (ARU) is termed non-responsiveness to ASA based on a preclinical study and subsequent correlation with epinephrine-induced platelet aggregation in platelet rich plasma. Since RPFA-ASA uses whole blood, we validated its performance characteristics against a classic whole blood platelet aggregation assay (WBA). We studied 50 healthy volunteers, aged 25–75 (24 men, 26 women) with normal CBC, who had not ingested anti-platelet drugs for 14 days prior to the study. Baseline studies included WBA (dual channel aggregometer, Chrono-log Inc., Havertown, PA) using both arachidonic acid (AA -0.5; 0.25 mM) and collagen (1; 2 μg/mL) as well as an RPFA-ASA assay (Accumetrics Inc., San Diego, CA). These studies were repeated after 3 days of ASA (325 mg/d) intake. Based on a review of the literature, we defined an adequate ASA response as a completely inhibited AA-induced platelet aggregation and at least 30% inhibition of collagen-induced aggregation (both concentrations of the agonist). Thus, those with < 30% inhibition of aggregation response to collagen were considered ASA resistant. Eleven subjects were ASA resistant by WBA (20%; 8 females and 3 males (aged 25–63). In contrast, since all 50 subjects achieved ARU values of < 550 ARU, none were recognized as an ASA non-responder by the RPFA-ASA. While the current cutoff of < 550 ARU posed by the Ultegra RPFA-ASA does identify those who have taken ASA, the assay is unable to recognize ASA non-responders. Thus, based on these data, the appropriate cutoff for the recognition of ASA resistance by the RPFA-ASA should be re-adjusted to a significantly lower level to ensure appropriate assay results.
New Approach for Evaluation of Quantitative Risk Factors of Venous Thromboembolism Using Individualized Reference Values.
Authors: Zotz Rainer B. , Gerhardt Andrea , Scharf Rudiger E.
Published: April 2020

The evaluation of quantitative risk factors of venous thromboembolism is characterized by several unsolved problems. The majority of quantitative components of hemostasis are dependent on age, sex, and hormone intake. The cut-off values determined in case-control studies depend on the specific patient/control group and can not be generalized on individuals with other characteristics. Furthermore, the approach does not give reference values dependent on age, sex, and hormone intake which are necessary for risk estimations in clinical practice.

To overcome these disadvantages, we used a multiple regression analysis to create a system of reference values which change continuously depending on age, sex, and hormone intake according to the parameter distribution in healthy controls and calculated the relative risk of hemostatic components (729 patients with first VTE and 675 healthy controls). A significantly increased risk for venous thrombosis was associated with deficiency of protein S activity (odds ratio (OR) 2.8 to 6.1, p=0.0007), free protein S concentration (OR 2.7 to 20.4, p=0.0001), protein C activity (OR 3.2 to 9.4, p=0.0001), antithrombin activity (OR up to 75, p=0.0001), and increased levels of fibrinogen (OR 3.8, p=0.0001), factor VIII:C (OR 3.3, p=0.0001), factor IX (OR 2.3, p=0.0001), factor XI (OR 2.9, p=0.0001), vWF activity (OR 2.4, p=0.0001), and vWF antigen (OR 3.5, p=0.0001).

In contrast to previously published studies, this new approach gives clinically important cut-off values dependent on age, sex, and hormone intake and allows to identify patients at increased risk for venous thrombosis on an individualized basis. Particularly parameters which are highly dependent on age and sex, like protein S activity, can be characterized more precisely using the described procedure. This comprehensive analysis demonstrates that, apart from well-known risk determinants, deficiency of protein S and increased values of FI, FVIII:C, FIX, FXI, vWF, and vWF-Ag are risk determinants predicting venous thrombosis.