bluuebunny/crossref_metadata_2025 huggingface dataset:
Deficiency of 2,3-Dimethoxy-5-Methyl-6-Decaprenyl-1,4-Benzoquinone (Mitoquinone) and Hyperlactic Acidosis in Human Immunodeficiency Virus (HIV) Patients with Head and Neck Squamous Cell Carcinoma (HNSCC) Treated with Nucleoside Reverse Transcriptase Inhibitor (NRTI)-Based HAART and Chemoradiation.
Authors: McPherson Eugene , Mejia E. , Hazel E. , Tassy P.
Published: April 2020

Mitoquinone is a nutrient obtained fromm food and produced in small amounts by the body with a crucial component of oxidative phosphorylation process in the mitochondria generating adenosine triphosphate (ATP). Deficiency of mitoquinone has been described based on failure of biosynthesis caused by gene mutation, inhibition of biosynthesis by HMG coA reductase(statins), compromisal of immune system function in breast cancer and acquired immunodeficiency syndrome (AIDS). Patients with (HNSCC) coinfected with HIV and deficient of mitoquinone, progress more rapidly to AIDS and when treated with statins for dyslipidemia, mitoquinone can become severely depressed and cause serious fatal type B lactic acidosis requiring emergent interventions. Correction of mitoquinone deficiency by supplementing mitoquinone may prevent and reverse the lactic acidosis seen in NRTI-based HAART (Highly Active Antiretroviral Therapy) allowing more effective chemoradiation with less side effects of mucositis, anemia and dermatitis. We present a case of a 44 year old female with HIV disease who developed a right tongue base squamous cell carcinoma (T2N0M0) lesion with trismus, elevated epstein-barr virus DNA (EBVDNA-PCR), dyslipidemia, and renal insufficiency was worked up and staged and treatment started with radiation therapy (XRT). No platinum chemotherapy given due to severe renal disease). Seven weeks of radiation therapy was given with interruption caused by grade II-III mucositis overcome by mitoquinone adjuvant therapy. Immune parameters CD4+ level initially was 71/cumm (10%) with a CD4/CD8 ratio 0f 0.17 and CD16+, CD56+ of 143/cumm (20%). A good response to XRT with less trismus and tumour shrinkage but soon after XRT she developed lactic acidosis with evidence of mitoquinone deficiency, 2.45 mmol/L and 0.4 mg/L respectively. Apolipoprotein A-1 level decreased along with high density lipoprotein-cholesterol (HDL-C). Statin therapy was started and lactic acidosis persisted with no further deteriorations of parameters. Lactic acidosis and mucositis (grade I) improved with mitoquinone supplements. The CD4+ level increased to 132/cumm (20%) and CD16+, CD56+ increased to 298/cumm (26%). CONCLUSIONS:HIV disease patients with HNSCC malignancy and mitoquinone deficiency may be at increase risk for mitochondrial DNA toxicity, chemoradiation side effects and severe lactic acidosis when treated with NRTI-based HAART, statins and XRT. Mitoquinone supplementation may prevent and reverse fatal lactic acidosis, modulate immune status and improve response to chemoradiation therapy, survival and quality of life.
Open-Label Study of Cellular Immunity in Dialysis Patients after Intravenous Ibandronate.
Authors: Bergner Raoul , Weiss Burkhard , Hoffman Martin , Henrich Dirk , Uppenkamp Michael
Published: April 2020

Intravenous infusion of bisphosphonates can cause acute phase reactions which may be caused by changes in the cellular immune system, particularly in lymphocyte subtypes. These immune changes and an acute phase reaction both normally disappear after 48 hours. Previous studies have shown that kidney transplant recipients treated with immunosuppressive therapy and ibandronate for bone protection have a lower rejection rate than those treated with immunosuppressive therapy and placebo.1 This suggests that there may be long-term changes in the immune system occurring with ibandronate treatment. We investigated the long term changes in the cellular immune system during ibandronate treatment in dialysis patients. As these patients are not normally treated with immunosuppressive or cytotoxic agents, which can cause changes in the cellular immune system, they are an eligible population for such a study. In this open-label trial, 16 patients with end-stage renal disease receiving regular hemodialysis were recruited. All patients were treated with ibandronate 2mg every 4 weeks for renal osteopathy; a dose that provides similar AUC and bone exposure as a 6mg dose in patients with normal renal function. The cellular immune system was investigated before first ibandronate application and the measurement was repeated at weeks 2, 4 and 48. The following parameters were measured by blood count differentiation: leucocytes, granulocytes lymphocytes, monocytes. Lymphocyte subtypes were measured by flow cytometry: B-lymphocytes (CD3+/CD19+), T-helper cells (CD3+/CD4+), T-suppressor cells (CD3+/CD8+), natural killer (NK)-cells (CD3+/CD16+56+), helper-inducer cells (CD4+/CD29+), activated T-cells (CD3+/HLA-DR+), activated T-lymphocytes (CD3+/CD25+), naive T-cells (CD3+/CD45RA+) and memory-T-lymphocytes (CD3+/CD45RO+). Twelve patients completed the study and were evaluated. One patient dropped out because of flu-like symptoms with muscle pain after the first ibandronate infusion; however this was well controlled with paracetamol. Three patients died due to concomitant diseases (diabetes and cardiovascular events). There were no statistically significant differences in cellular immunity over time as measured in weeks 0, 2, 4 and 48 (see Figure). In this small-pilot study of dialysis patients receiving ibandronate, no changes in the cellular immune system were observed over time. Changes in different lymphocyte subtypes, which occur in the acute phase reaction after first infusion, were not seen. Reduced rejection rate in transplant recipients after ibandronate infusion cannot be explained by changes in the cellular immune system, and must therefore occur by another mechanism.

Figure Figure
A Shared Gene-Expression Signature in Innate-Like Lymphocytes.
Authors: Yamagata Tetsuya , Benoist Christophe , Mathis Diane
Published: April 2020

Innate and adaptive immunity are the two major arms of the immune system. They rely on very distinct cell-types, primarily distinguished by the source of diversity for non-self recognition, of germline or somatic origin. There exists, however, a subset of lymphocytes whose receptors require rearrangement but result in semi-invariant structures with a high degree of self-specificity. We hypothesized that these innate-like lymphocytes might share a common gene transcription signature. To test this notion, we made pair-wise comparisons of the gene-expression profiles of innate-like lymphocytes and closely paired adaptive system counterparts (NKT vs. CD4T, CD8ααT vs. CD8αβT, B1 vs. B2), and bioinformatically extracted common features and common genes distinguishing innate from adaptive cell-types. A statistically significant “innate signature” was indeed distilled, composed of a small set of genes over- and under-expressed in innate vs. adaptive lymphocytes. Particularly intriguing was the high representation of interferon-inducible GTPases crucial for resistance against intracellular pathogens, and of small G proteins involved in intracellular vacuole maturation and trafficking. Overall, this combined expression pattern can thus be designated as an “innate signature” among lymphocytes.
Integrated Genotyping and mRNA Expression Profiling of Killer Immunoglobulin-Like Receptors.
Authors: Cremer Anja , Heider Ute , Tomiuk Stefan , Arendt Andreas , Schmitz Jurgen , Bosio Andreas , Huppert Volker , Biervert Christian
Published: April 2020

Natural killer (NK) cells belong to a subgroup of lymphocytes (CD3-CD56+) which play an important role in the cellular immune response against virus-infected cells and tumors. The activity of NK cells is regulated by a balance of triggering and inhibitory receptors, including Killer Ig-like Receptor (KIR) molecules which interact with specific HLA class I molecules, predominantly HLA-C, on target cells. The 17 known KIR genes are divided into two classes: activating KIRs and inhibitory KIRs. There is strong evidence that inhibitory KIR mismatch between donor and recipient improves the outcome of haploidentical hematopoietic stem cell transplantation (HSTC) in leukemia patients (Ruggeri et al. 2002). In addition, the KIR-HLA constellation is assumed to have an influence on the severity of graft versus host disease (GvHD). Whether these activities of NK cells are clinically important and to what extent these processes are mediated only by KIR-HLA class I interactions remains to be determined. In human populations, KIR gene haplotypes vary in the number and type of KIR genes they contain. Further diversification is observed by expanded allelic polymorphism at the individual genes. In general, KIR haplotypes contain 7–12 genes plus 2 pseudogenes. Extra KIR heterogeneity is provided at the expression level: different subsets of NK cells within an individual express different KIRs. Recently, it was shown that KIR genotyping alone does not seem to be sufficient for donor KIR assessment because of the lack of gene expression in approximately one-fourth of the individuals for one of the inhibitory KIRs that recognize the three major groups of MHC class I ligands (Leung et al. 2005). KIR phenotyping by flow cytometry using monoclonal antibodies is insufficient due to the lack of specific monoclonal antibodies. For trustworthy analysis, one has to combine KIR genotyping with mRNA expression profiling and flow cytometry. Therefore, we developed a new set of sequence-specific primers (SSP). This primer set can be applied to perform either KIR genotyping or mRNA expression profiling despite the high degree of identity of the genes (80–90%, sometimes more than 95%). The primers of each KIR gene (15 genes and 2 pseudogenes) cover all allelic variants annotated by the IPD KIR Sequence Data Base (status quo July 05). Using this primer set, we genotyped 25 individuals, and compared the results with other sets of KIR primers published elsewhere. Additionally, we show the mRNA expression profile employing the same set of new primers. We confirmed these results on the protein level by flow cytometry.
CD8+ T Cell Responses to the Polycomb Protein EZH2 in Patients with Haemopoietic Malignancies.
Authors: Kalk Emma , Piper Karen , Pratt Guy , Young Lawrence , Steele Jane , Moss Paul
Published: April 2020

Polycomb Group (PcG) genes encode transcriptional repressors that have been implicated in stem cell survival and cell fate determination (Lessard J et al. Blood. 1998). They are the human homologues of the Drosophila Polycomb and Posterior sex comb genes where they maintain segment-specific expression of the homeotic (Hox) genes. Hox gene products are transcription factors involved in cell fate determination and have been implicated in normal haematopoiesis and leukaemic transformation. In mammals, the PcG genes are highly conserved and essential for normal embryonal development and cell cycle control, as well as the regulation of haematopoiesis. Two distinct PcG gene subsets have been identified which demonstrate consistent and often mutually exclusive expression in haemopoietic cells, the patterns correlating with phenotypically distinct populations in the lymph nodes and thymus. BMI-1 and EZH2 are representative examples of these two subsets of PcG genes. BMI-1 expression is essential for stem cell self-renewal whereas EZH2 is required for cell proliferation. Disrupted expression of both of these genes has been demonstrated in a range of haemopoietic and non-haemopoietic malignancies and appears to deliver a proliferative advantage to the cell.

Humoral and T cell immunity to BMI-1 and EZH2 has been observed in patients with hepatocellular and prostate carcinoma, suggesting that these proteins may act as tumour antigens. However, CD8+ T cell responses to peptides derived from the proteins have also been demonstrated in healthy controls indicating that T cell tolerance might not be complete.

Using the cytokine secretion assay and HLA-tetramer staining, HLA A*0201-restricted CD8+ T cell responses to an EZH2-derived peptide have been identified in 7 out of 19 (37%) healthy controls. The frequency of IFNg-secreting cells ranged from 0.001–1.6% of the total CD8+ T cell pool. In the patient cohort, 4 out of 11 donors responded to the peptide (frequency 36%) with a frequency representing between 0.002 and 0.03% of CD8+ T cells. The frequency of the response is therefore comparable between the two groups. The potential role of regulatory CD4+CD25+ T cells in controlling auto-reactive immune responses to PcG proteins is being assessed. Depletion of this population can result in an increased expansion of the tumour-reactive T cell response in vitro.

Thus, although ectopic expression of polycomb proteins is often seen in haemopoietic malignancy there is currently little evidence for a tumour-selective immune response in such patients.
Cas-L/Hef1 Is Required for Marginal Zone B Cell Maintenance and Lymphocyte Trafficking.
Authors: Seo Sachiko , Asai Takashi , Saito Toshiki , Suzuki Takahiro , Ichikawa Motoshi , Ogawa Seishi , Kurokawa Mineo , Chiba Shigeru , Hirai Hisamaru
Published: April 2020

Cas-L (Crk-associated substrate lymphocyte type) which is also known as Hef1 (human enhancer of filamentation 1) was first identified as a protein tyrosine-phosphorylated upon stimulation of b1 integrin. Cas-L possesses a single Src homology (SH) 3 domain and multiple YXXP motifs (substrate domain) as a member of Cas protein family, and is well expressed in peripheral lymphocytes. Previous studies suggest that Cas-L might be involved in Bcr-Abl positive leukemia and adult T cell leukemia. However, the biological function of Cas-L in lymphocytes is little known. We generated Cas-L-deficient mice using a gene targeting strategy. The mice showed a deficit of marginal zone (MZ) B cells and a decrease of cell number in secondary lymphoid organs. To elucidate the mechanism of the MZ B cell defect, the reciprocal bone marrow transfer assays were performed. The results revealed that the defect of MZ B cells in Cas-L-deficient mice is cell autonomous. Next, we analyzed B cell receptor signaling by measurement of intracellular Ca2+ concentration and lymphocyte proliferation. However, we could not find any significant differences between wild type and Cas-L-deficient mice. Cas-L-deficient lymphocytes showed reduced chemotactic response to CXCL12 and CXCL13. The adhesion assay also showed the decreased adhesiveness to VCAM-1 and ICAM-1, which are important for retention of MZ B cells in spleen. Moreover, we found that the lymphocyte trafficking to spleen and lymph nodes was altered in Cas-L-deficient mice. Thus, Cas-L affects homeostasis of MZ B cells and peripheral lymphoid organs, which is considered to be relevant to impaired lymphocyte migration and adhesion.
Standardization of Functional Immune Cell-Based Assays: An Integral Aspect to Vaccine and Biologic Development and Validation.
Authors: Wilkinson Julie , Smith Cecilia , D’Costa Sybil , Rabellino Enrique
Published: April 2020

The utility of the ex-vivo evaluation of immune cell functionality in the context of a) Determining an efficacious vaccine strategy for infectious diseases/cancer, b) Determining a tolerance profile in autoimmunity and transplantation, and c) understanding the basic mechanisms of immune cell responses in disease pathogenesis is well recognized.

However, the benefit of these assays as surrogate markers of immune cell activity in vivo has not been fully realized due to the variable nature of these in vitro assays which is particularly pronounced in T cell functional assays. This variability arises from a variety of factors ranging from choice of assay, source of the cells, the sample processing methodology (isolation, freezing, thawing, and culturing), sample staining protocol for the chosen assay and ultimately data analysis, and data reduction. With a view to reducing variability and standardizing targeted steps of T cell functional assays, an automated methodology for simultaneous staining and analysis of multiple intracellular cytokines and cytotoxicity markers via flow cytometry was developed and validated. A 5-color flow cytometry assay (2–3 surface markers; 2 intracellular markers) was developed to characterize the restricted polyclonal (SEB/CD28) and antigen specific (CEF peptide pool) cytokine and cytotoxic profile response in human PBMCs. A modification to available sample preparation instruments was performed that enabled the automated pipetting, incubation, and staining of intracellular and surface molecules of stimulated human peripheral blood mononuclear cell populations (PBMC) for flow cytometric analysis.

Statistically significant reductions in both inter and intra assay variability was observed in the automated methodology as compared to the manual assay with improvements in CVs for positive cell numbers and mean fluorescence intensity. For example, the inter assay CVs for IFNg cytokine producing CD4+ T cell populations improved from approximately 15 to 5, while the mean fluorescence intensity improved nearly 5 fold with automation. Importantly, the automated methodology furnished comparable responses in percent positive cytokine/cytotoxicity profiles as compared to the manual method while reducing the “handson” sample preparation and analysis time from 2 hours to 20 minutes. With the standardization of functional assays, other sources of variability in assays result can now be addressed specifically e.g. specimen handling, freezing, thawing, culturing, or biological. Standardized multiparametric functional profiling of the cells thus reveals the complex nature of the immune response and lends credence to their use as surrogate markers of efficacy and functionality.
Platelet Apoptosis Events Can Be Triggered by Shear Stresses and Chemical Agonists.
Authors: Leytin Valery , Mykhaylov Sergiy , Allen David J. , Freedman John J.
Published: April 2020

Apoptosis, or programmed cell death, is appreciated as the main physiologic mechanism that regulates cell life-span and serves for controlled deletion of unwanted cells. Since its discovery in 1972, apoptosis was long attributed exclusively to nucleate cells. It took more than 20 years to recognize apoptosis in enucleated cells cytoplasts and anucleate platelets. During the following years, apoptosis has been demonstrated in platelets treated with natural and artificial agonists, in platelet concentrates aged during storage under standard blood banking conditions, and in animal models of suppressed thrombopoiesis and thrombocytopenia. Other studies documented that mechanical forces (shear stresses) stimulate platelet activation and signaling in the absence of exogenous chemical stimuli. We analysed whether shear stresses can trigger platelet apoptosis, a question that has not yet been studied. Using a cone-and-plate viscometer (CAP-2000, Brookfield Engineering Labs, Inc., Middleboro, MA), we exposed human platelet-rich plasma to different shear stresses, ranging from physiologic arterial and arterioles levels (10-44 dynes/cm2) to pathologic high levels (117–388 dynes/cm2) occurring in stenosed coronary, peripheral or cerebral arteries. We found that pathologic shear stresses induce not only platelet activation (P-selectin upregulation and GPIbα downregulation) but also trigger apoptosis events, including mitochondrial transmembrane potential depolarization, caspase 3 activation, phosphatidylserine exposure, and platelet shrinkage and fragmentation into microparticles, whereas physiologic shear stresses are not effective. Platelets subjected to pathologic shear stresses are characterized by impaired platelet function as shown by the absence of ADP-induced platelet aggregation. Apoptosis changes were also induced by the treatment of platelets with calcium ionophore A23187 (10 μM) and thrombin (1 U/mL). Thus, in the present work, we have demonstrated that platelet apoptosis can be induced by chemical stimuli and by mechanical rheological forces (pathologic high shear stresses). Most of shear-induced activation and apoptosis events occur inside of the platelet, including translocation of CD62 from α-granules to the platelet surface, depolarization of mitochondrial inner membrane potential, activation of cytosolic enzyme caspase 3, and translocation of phosphatidylserine from the inner to the outer plasma membrane leaflet. These data suggest that the effects of shear stress on platelet activation and apoptosis are mediated by mechanoreceptor(s) that transmit activation and apoptosis signals to the cell interior. The platelet paradigm of apoptosis induced by chemical agonists and shear stresses suggests that apoptotic cytoplasmic machinery may function without nuclear participation.
SEPT11, a Novel Platelet Septin Partners with the Platelet Septin SEPT5.
Authors: Röseler Sabrina , Bläser Susanne , Bartsch Ingrid , Rempp Hansjörg , Bauer Heike , Lieber Mareike , Busse Anja , Zieger Barbara
Published: April 2020

The septin SEPT11 is a novel member of the highly conserved septin family. Septins are GTP-binding proteins which form heteropolymeric complexes. In non-dividing cells (such as platelets and neurons) septins are implicated in exocytosis. The septins SEPT4, SEPT5 and SEPT8 are expressed in platelets. Platelets from a SEPT5 knockout mouse showed an altered serotonin secretion and platelet aggregation suggesting that SEPT5 is involved in secretion in platelets. Transmission electron microscopy of platelets revealed that SEPT4, SEPT5 and SEPT8 are localized surrounding alpha-granules suggesting that the three septins may be components of the septin complex in platelets and contribute in such a way to platelet biology. Activation of platelets by agonists resulted in the translocation of SEPT4 and SEPT8 to the platelet surface indicating a possible functional role of these proteins in granular secretion. Previously, we had identified the interaction of the septins SEPT5 and SEPT8. The aim of this study was to identify other interaction partners of the human septin SEPT5. Using the yeast two-hybrid system we now demonstrate that SEPT11 partners with SEPT5. Western analysis revealed that SEPT11 is also expressed in platelets. The molecular interaction of SEPT11 with SEPT5 was verified by precipitation of the SEPT5/SEPT11 complex from lysates of human platelets. In addition, using Western analysis and immunofluorescence analysis, the co-expression of SEPT5 and SEPT11 is shown in human endothelial cells (HUVECs). The Western analyses of various mouse tissues showed that the expression pattern of SEPT11 matches with the expression pattern of SEPT5 suggesting that SEPT11 and SEPT5 are components of the corresponding cell specific septin complex. Since SEPT5 and SEPT11 are members of the same septin complex and since SEPT5 has been reported to play an important role in exocytosis, the SEPT5/SEPT11-complex may be involved in exocytosis in platelets and HUVECs.
Properties of Soluble His-Tagged rGPIbα1–483 Wild-Type and Increase-of-Function Mutants.
Authors: Biswas Tapan K. , Miller Jonathan L.
Published: April 2020

The extracellular domain of human platelet GPIbα contains the binding site for von Willebrand factor (vWF) within the amino-terminal 45 kDa region comprising approximately 300 amino acids. In the present studies, we have used CHO cells to synthesize a longer fragment of His1-Phe483, representing nearly the complete extracellular domain of GPIbα, with an additional 8 histidine residues added at the carboxyl end to provide recognition by his-tag reagents. Both wild-type and the increase-of-function mutants Gly233→Val233 and/or Met239→Val239 were employed. For studies of antibody binding, modulator-induced vWF binding, or antibody inhibition of this vWF binding, capture of the recombinant GPIbα could be accomplished by nickel chelate binding of the his-tag. Both botrocetin and ristocetin induced saturable binding of vWF by ELISA, as demonstrated by HRP-conjugated anti-vWF polyclonal antibody. In the case of the different increase-of-function mutants, significant vWF binding was seen even in the absence of added modulator. Inhibition of botrocetin-induced vWF binding was greater for wild-type than for the Met239→Val239 GPIbα mutant with some mabs (e.g., AS-2), whereas the degree of inhibition was essentially equivalent with other mabs (e.g., C-34). When a rabbit polyclonal anti-his antibody was used to immobilize the recombinant proteins instead of the nickel chelate system, binding of vWF and of conformation-dependent mabs was impaired. However, when this same antibody was biotinylated and then captured on a streptavidin plate, excellent capture and subsequent binding characteristics of the recombinant GPIbα were restored. With both this latter system and with the nickel chelate system, a highly sensitive functional vWF activity assay could be established, using botrocetin-induced binding of vWF from human plasma. Linearity of binding was observed between 0.1–0.6% normal plasma, with saturation occurring around 2%. Whereas higher concentrations of plasma actually appeared to displace bound GPIbα to the nickel chelate, the streptavidin-biotin-anti-his attachment method appeared more resistant to such effects. The ELISA-based assay systems developed in this study accordingly provide an and effective approach to study functional interactions of both wild-type and mutant forms of GPIbα with its ligand vWF and also with antibodies directed against different epitopes within the entire extracellular domain of this receptor molecule. The high sensitivity, reproducibility, and rapidity of the assay system also offer the possibility of a useful test for functional assay of plasma vWF.
Expression and Functional Analysis of Disintegrin from Agkistrodon Actus Venom.
Authors: Bai Xia , Fu Jianxin , Su Jian , Dai Lan , Zhang Wei , Ding Kaiyang , Wang Zhaoyue , Ruan Changgeng
Published: April 2020

Disintegrin is a kind of small native peptides derived from the snake venom, containing RGD sequences. Its interaction with integrins, such as aIIbb3, anb3 and a5b1, can inhibit platelet aggregation. hinder the process of angiogenesis and tumor metastasis. In order to further investigate the function of disintegrin in platelet aggregation, cell adhesion and angiogenesis, the sequence encoding the disintegrin domain of agacutin was fused with enhanced green fluorencent protein(eGFP) and inserted in plasmid pQE-30. The recombinant protein was expressed in E.Coli M15 after induction by IPTG, amounting to 38% of the total bacterial protein. The cells expressing integrin such as breast cancer cell line MDA-MB-231 and endothelial cell line EA.hy926 can specifically bind to the recombinant GFP-disintegrin. Flow cytometry showed that the recombinant protein could bind to platelet specfically and could compete with anti-b3 integrin monoclonal antibody CD61. Moreover, the recombinant protein could inhibit platelet aggregation induced by collagen and ADP in a dose-dependent manner, but had no ability to impede the platelet aggregation induced by Ristocetin. Meanwhile, it could inhibit cell (MDA-MB-231 and EA.hy926) adhesion to fibronectin with inhibition rate of 69% and 48%, respectively. Finally, it could induce apoptosis of EA.hy926 endothelial cells and inhibit the angiogenesis on chicken chorioallantoic membrane mode. As a result, it is demonstrated that GFP-disintegrin has the combined quality of its disparate components and can serve as a novel tool for study of tumor and angiogenesis. In addition, the recombinant protein can efficiently inhibit platelet aggregation and angiogenesis, which will be useful for designing the potential drug for anti-thrombosis and anti-tumor metastasis.
Inhibition of Angiogenesis by a Novel Neutralizing Antibody Against Human Vascular Endothelial Growth Factor Receptor 3 (VEGFR-3).
Authors: Chen Hao , Ding Xiu-Yun , Gao Yuan , Ren Fei-Yao , Li Hui , Gao Jian-En , Sun Qi-Hong
Published: April 2020

Vascular endothelial growth factor receptor-3 (VEGFR-3) and its ligands, vascular endothelial growth factor-C (VEGF-C) and D (VEGF-D), are the major molecules involved in the development of the embryonic vascular system and pathological lymphangiogenesis. Throughout embryogenesis, VEGFR-3 is expressed in most endothelial cells, whilst being restricted to lymphatic vessels later in development. This receptor plays a significant role in the normal development of blood and lymphatic vessels. In the present studies, we generated a novel panel of 17 monoclonal antibodies (mAbs) against the human VEGFR-3 and characterized their ability to inhibit the proliferation of human erythroleukemia (HEL) cells and angiogenesis of chick embryo chorioallantoic membrane (CAM). Among these mAbs, BDD073 was demonstrated to inhibit the interaction of soluble VEGFR-3 with VEGF-D and the proliferation of HEL cells. In CAM angiogenesis experiments, the angiogenesis induced by recombinant GST-VEGF-D was decreased in the presence of antibody BDD073. These data indicate that this novel neutralizing antibody against human VEGFR-3 not only might be a potential compounds in blocking the VEGF-D-induced angiogenesis and lymphangiogenesis, but also be a tool for the investigations of biology of VEGFR-3 and analysis of lymphatic vessels in malignant tumors and their metastases.
Von Willebrand Factor Cleaving Protease (ADAMTS-13) Antigen Levels Are Decreased in Patients with Heparin-Induced Thrombocytopenia.
Authors: Hoppensteadt Debra , Cunanan Josephine , Walenga Jeanine M. , Ero Michael P. , Jeske Walter P. , Bick Rodger , Fareed Jawed
Published: April 2020

Heparin-induced thrombocytopenia (HIT) represents a complex pathologic syndrome including all components of the hemostatic system and inflammatory response. ADAMTS-13 is a metalloprotease which mediates the cleavage of von Willebrand factor (vWF) multimers. A deficiency or decreased activity of this protease may result in a TTP-related syndrome manifested by the development of intravascular platelet aggregates, thrombocytopenia, endothelial dysfunction and the deposition of vWF at thrombotic sites. Several recent reports have described decreased functionality of ADAMTS-13 in patients with HIV. This may be due to antibodies than inhibit ADAMTS-13. HIT is characterized by the generation of heparin-induced anti-heparin platelet factor 4 antibodies which are molecularly and functionally heterogeneous. No data is available on the effect of these antibodies on ADAMTS-13. However, increased vWF levels and the generation of ultrahigh molecular weight vWF multimers have been reported in HIT patients. This study was designed to determine the ADAMTS-13 antigen levels in HIT patient plasma samples (n=30) prior to and after treatment with the direct anti-thrombin agent argatroban (ARG 911 study). Plasma samples from normal healthy volunteers (n=30) were used as controls for comparison purposes. ADAMTS-13 antigen levels were quantitated using a newly developed ELISA method from American Diagnostica (Stamford, CT). vWF antigen levels were also measured using an ELISA-based method. The levels of ADAMTS-13 and vWF antigen were reported as percent normal based on the results obtained with the samples from healthy volunteers. The baseline ADAMTS-13 antigen levels varied widely among the HIT patients enrolled in the ARG 911 trial (30–180% NHP) with a mean of 62 ± 24% NHP. On treatment day 1, no significant changes were noted in the ADAMTS-13 levels in HIT patients. However, on the third and 5th-7th day following initiation of argatroban treatment, ADAMTS-13 levels were increased to 81 ± 6% NHP and 86 ± 24% NHP, respectively. The HIT patient group also exhibited higher levels of vWF antigen at baseline (170 ± 27% NHP) which were marginally decreased following treatment with argatroban. During argatroban treatment, a decrease in anti-heparin platelet factor 4 antibody titer and an improvement in platelet count were noted. These results are highly suggestive of a pathogenic role of vWF multimers in HIT syndrome. Furthermore, a decreased ADAMTS-13 antigen level indicates that its regulation in HIT patients may be altered. Additional results on ADAMTS-13 functionality, ADAMTS-13-factor XI complexes and autoantibodies to the metalloprotease may provide additional insights into the pathogenesis of HIT and the pathologic role of vWF related proteins.
Response to Splenectomy Is Durable after a Certain Point in Time in Adult ITP Patients.
Authors: Johansson Eva , Engervall Per , Landgren Ola , Grimfors (deceased) Gunnar , Widell Susanne , Rezai Shahideh , Bjorkholm Magnus
Published: April 2020

Background. Splenectomy is the recommended treatment in adult patients with immune thrombocytopenic purpura (ITP) who fail to respond to corticosteroid treatment or who require unacceptably high doses to maintain a safe platelet level. For these patients splenectomy may produce a good response in 60–80% of patients. However, the observation time after splenectomy has in most published studies been relatively short. Thus, the true long-term response to splenectomy still remains less well defined. The efficacy and safety of splenectomy were assessed in a cohort of adult ITP patients followed for a very long period of time.

Patients and methods. The study included 59 ITP patients (M/F=25/34, median age at splenectomy; 39 yrs, range 14–75 yrs). The median time from diagnosis to splenectomy was 5.5 mos (range 0.4–199 mos). Criteria used to define response to splenectomy were: CR; platelet count >150 x 109/l lasting ≥4 weeks, PR; platelet count ≥50–150 x 109/l lasting ≥4 weeks and, NR; failure to achieve platelet counts ≥50 x 109/l. The criterion for relapse in CR/PR patients was a decrease in platelet counts to <50 x 109/l.

Results. The median follow-up time after splenectomy was 18 yrs (range 2–32 yrs). No serious surgical complications occurred. The overall response rate was 78% with 59% CR and 19% PR. CR and PR patients were younger than NR patients at time of diagnosis (median age: 36 yrs vs 48 yrs, p=0.03) and at splenectomy (median age: 38 yrs vs 51 yrs, p=0.02). Among the 46 responding patients 17 eventually relapsed. A plateau in the progression free survival curve was identified after 12.1 and 7.3 years for patients in CR or PR, respectively.

Figure Figure

At follow-up, 5 of 17 patients relapsing after splenectomy were in CR. Bacteremia was recorded in two patients during a total of 1023 patient years at risk.

Conclusions

We conclude that splenctomy is an effective and safe treatment in adult patients with chronic ITP failing to respond to corticosteroid treatment. The complication rate was low and our findings support the view that response to splenectomy is durable after a certain point in time.
The Prevalence of Pantoprozole Associated Thrombocytopenia in a Community Hospital - A Retrospective Study.
Authors: Katz Randi , Bratcher Jason , Dotan Efrat , Wasserman Carrie , Liebman Michelene , Reddy Sivakumar , Michael Amy , Arastu Aliasad , Panagopoulos Georgia , Spaccavento Colette
Published: April 2020

Proton Pump Inhibitors (PPIs) are widely used in the treatment of peptic ulcer disease, Helicobacter pylori (H pylori) gastritis and gastro-esophageal reflux disease. PPIs are known to be relatively safe medications, with the most common adverse reactions reported as diarrhea, nausea, vomiting, abdominal pain and headache. Thrombocytopenia has not been found to be a significant side effect of PPI use. Furthermore, successful treatment of H pylori associated immune thrombocytopenic purpura (ITP) with PPI therapy has been reported in the literature. In clinical practice, however, PPI use has been thought to be associated with medication-induced thrombocytopenia, although the precise prevalence of this consequence has not been quantified. The purpose of this study is to determine the prevalence of thrombocytopenia in hospitalized patients treated with pantoprazole, a commonly prescribed PPI, compared to non-medicated controls. In this retrospective, case-controlled analysis, we analyzed the platelet counts of 626 patients between 18 and 80 years of age who were admitted to an urban, community-based hospital and were medicated with pantoprazole for at least three days during their hospital stay. Thrombocytopenia was defined as a drop in the platelet count to below 150,000/ml, or a drop of at least 50% from baseline. Patients who had a platelet count below 150,000/ml on admission or were hospitalized for less than 3 days were excluded from the study. A control group of 560 patients with the same inclusion criteria who had not been prescribed pantoprazole was analyzed for comparison. 626 patients were included in the study group and 560 patients in the control group. The median age was 67 years in study group, and 61 years in the control group. The mean platelet count on admission was 260,00/ml in the study group, and 257,000/ml in the control group. Of the subject group, 39 patients (6.2%) met our definition of thrombocytopenia, which was not statistically different from the 35 patients (6.2%) with thrombocytopenia in the control group (p=0.90). Our study compared patients who received PPI therapy with pantoprazole for at least three hospital days to patients without treatment. We found no difference in the rate of thrombocytopenia between hospitalized patients treated with pantoprazole and the non-medicated controls. The rate of thrombocytopenia that we observed is probably an estimate of the overall thrombocytopenia rate in hospitalized patients irrespective of specific medications. While our report did not control for variables such as concurrent medication use, comorbid illnesses, and patient demographics, we are confident that the sample size selected for this study is large enough to provide sufficient evidence that thrombocytopenia is not a significant side effect of pantoprazole therapy.
Evaluation of Qualitative Platelet Disorders in Patients with Hemophilia.
Authors: Kraut Eric H. , Cataland Spero R. , Witkoff Leslie , Jordan Sylvia , Gunning William T.
Published: April 2020

The hemophilias are inherited disorders of the coagulation system with no apparent effects on primary hemostasis. However, bleeding times and platelet aggregation have been reported as abnormal. These observations have been ascribed to undiagnosed Von Willebrand’s Disease (VWD), effects of factor replacement, or drugs. We evaluated three patients with hemophilia including one patient with factor VIII deficiency, one with factor VIII and inhibitor, and one factor IX who had persistent bleeding despite their standard treatment. Initial evaluation of primary hemostasis demonstrated abnormal bleeding time and or platelet function analysis (PFA-100). VWD antigen and ristocetin cofactor were normal. Electron microscopy demonstrated alpha and or delta granule storage pool deficiency. Careful review of all drugs the patients were on did not explain this finding. Repeat evaluation of one patient over time demonstrated persistence of abnormal function. Treatment with DDAVP(Desmopressin) normalized in vitro function in 2 patients. After documentation of these abnormalities, two patients were treated with DDAVP with or without platelets as well as factor replacement or in face of inhibitors FEIBA or FVIIa. Clinical improvement was seen in the 2 patients after this intervention. A prospective study of our hemophilia population has been initiated with the goal of documenting the frequency of storage pool deficiency, the importance of the disorder to bleeding episodes and treatment response, and to determine whether this is inherited or acquired.
Effect of Erythropoietin on Platelet Function and Glycoprotein Expression in Patients with MDS.
Authors: Girtovitis Fotis F.I. , Bougiouklis Dimitrios D. , Makris Michel M.P. , Pithara Elefteria E. , Makris Pantelis P.E.
Published: April 2020

Aim: We decide to study the effect of erythropoietin on the platelet function and glycoproteins expression in patients (pts) with myelodysplastic syndrome (MDS). All patients suffered from primary MDS but none of them received any special treatment. From our study pts that suffered from diseases or they were receiving medication that affect the function of platelets were excluded. Furthermore pts with platelet count<50,000/μl were excluded also because we were unable to check the aggregation of platelets.

Material: 41 subjects were studied, 15 normal subject (10 men and 5 women mean age 66,6±14 years old)and 26 pts (17 men and 9 women with mean age70,9±7,4 years old) suffered from all types of MDS according to FAB criteria (9 with RA, 3 with RARS, 7 RAEB, 4 with CMML and 3 with RAEB-t). We divided them in 2 groups: 1st - 7 Pts receiving human recombinant erythropoietin (rEPO) with mean dose 30.000 iu subcutaneous weekly and 2nd - 19 without rEPO.

Methods.

1- The platelet function was studied in Platelet Ionized Calcium Aggregometer (PICA) using Ristocetin, ADP, Collagen and Adrenalin as stimulators.

2- The expression of platelet glycoproteins (GPIb, IX, IIb, IIIa and P-selectin) was studied using the flow cytometry and special monoclonal antibodies.

This way the percentage of glycoprotein expressed in platelet membrane and MFI were estimated. We performed the statistical analysis of our results using the t-test with common standard deviation..

Results: our results concerning the aggregation test and flow cytometry are presented in tables 1,2 and 3. From the study of our results we can see that

while the decrease of aggregation between the patients under EPO and normal subjects is statistically non significant (p<0.1)the decrease of the corresponding values between the pts that did not received EPO and normal subjects was statistically very significant for all stimulators (p <0,001). pts under EPO show an important increase of platelet expressing GPIIb percentage grater than the expressed percentage of patients without EPO (60% vs. 46,1% correspondingly, p<0.001). The difference of expressed MFI was not statistically significant.

Conclusion: The findings of our study show us that erythropoietin improves the function of platelets in patients with MDS, probably through the increase of platelet percentage which express glycoproteins.

Table 1 Comparison of the mean value of aggregation between the groups Table 2 Comparison of the mean values of the platelets expressing corresponding glycoproteins percentage between the groups Table 3 Comparison of the mean value of MFI between the groups
Hereditary αδ-Storage Pool Deficiency with the Abnormal Structures of the Platelet Microtubules.
Authors: Miyazaki Koji , Kawakami Hayato , Higashihara Masaaki
Published: April 2020

The case is a 27-year-old man, who referred to us presenting the mild bleeding tendency after extraction of a tooth. Bleeding time was 3 minutes by the Duke method, and his platelet count was slightly decreased (7x10 10 /l). His mother also has the similar platelet abnormality. They have no albinism. The peripheral smear demonstrated the abnormal platelet morphology with hypo or agranular and large forms. Almost normal platelets were also present on the smear. A platelet adhesion test showed the impaired collagen adhesion. A platelet aggregation study revealed the abnormally poor responses to adenosine diphosphate, collagen, arachidonate, U-46619, and a relatively retained response to ristocetin. Electron microscopy of the platelets demonstrated 2 populations: one with almost normal distribution of the granules, the other with marked decrease in both α and dense granules, even though the former population had the abnormal granules and the obvious vacuoles in the platelets. Flow cytometry revealed the reduced Mepacrine positive platelets and the decreased surface CD62P induced with phorbor ester in the patient. The patient platelets contained the reduced concentration of both adenosine triphosphate and serotonin, compared with the normal control. The cytoskeleton structures were examined with immunocytochemistry using the specific antibodies. Most platelets of the patient lost the typical coiled marginal bands and showed the yarnball-like structures of the microtubules, although the actin microfilaments were relatively retained. In other words, the platelets even with the almost normal distribution of the granules have a disturbed tubulin organization, which were not apparent in the leukocytes. These results suggested that some platelet-specific proteins, such as β1 tubulin, involved in the formation of the microtubules could be causative for this hereditary storage pool deficiency.
The Comparative Analysis of Serum Proteomes for the Discovery of Biomarkers for Essential Thrombocythemia.
Authors: Kwak Jae-Yong , Lee Na-Ri , Song Eun-Kee , Yim Chang-Yeol , Kwak Yong-Geun
Published: April 2020

Background: Essential thrombocythemia(ET) is a clonal disorder involving a hematopoietic progenitor cell and is manifested by the overproduction of platelets. There is as yet no pathognomonic diagnostic test. In our study, to identify the biomarkers for an initial diagnosis by a lesser invasive method, serum proteins reflecting alteration their proteomes were analysed.

Methods: We compared two-dimensional electrophoresis patterns of human sera of twelve patients with ET with that of normal twelve subjects. The differentially expressed spots were identified by matrix-assisted laser desorption/ionization-time-of flight(MALDI-TOF) and electrospray ionization quadupole time of flight(ESI-Q-TOF)mass spectrometry.

Results: Twenty two spots that expressed differentially in ET group were found. The expression levels of fibrinogen γ, fibrinogen γ-A chain precursor, ALB protein, α-2 macroglobulin, H factor-1, α-2-macroglobulin precursor, ceruloplasmin, α-1-B-glycoprotein, α-2-plasmin inhibitor, α-1-B-glycoprotein and an unknown protein were up-regulated in serum of ET patients, whereas the other proteins including complement fator B preproprotein, afamin precursor, inter-α inhibitor H4, immunoglobulin heavy chain constant region γ 1, immunoglobulin κ light chain and an unknown protein down-regulated.

Conclusion: These results suggest that these proteins can be used as less invasive diagnostic and follow-up biomarkers of ET if further studies were done.

The proteins significantly changed in sera of ET Spot no. Name Accession no. % coverage Mw(KDa)/pI(Measured) Mw(KDa)/pI(database) ET(%) 67 fibrinogen γ 223170 39 50.57/5.6 46.82/5.5 2.54±0.52 69 ” 223170 33 50.57/5.6 46.83/5.5 3.93±1.19 73 fibrinogen γ-A chain precursor 71827 23 48.93/5.6 50.11/5.7 3.89±0.98 74 ” 71827 22 48.93/5.8 50.11/5.7 3.10±0.91 77 ALB protein 27692693 21 50.57/5.9 48.65/6.0 3.77±0.58 89 α-2-macroglobulin 177872 14 263.89/6.1 71.35/5.5 1.95±0.32 100 H factor 1 4504875 20 248.95/5.7 143.79/6.3 5.8±1.78 112 α-2-macroglobulin precursor 4557225 9 151.38/5.7 164.69/6.0 2.18±0.38 125 complement factor B preproprotein 4502397 14 151.38/5.7 86.86/6.6 0.49±0.07 138 afamin precursor 4501987 12 65.01/5.1 85.53/6.3 0.31±0.07 139 ” 4501987 12 65.01/5.1 85.53/6.3 0.36±0.09 140 inter- α inhibitor H4 31542984 12 93.33/5.3 103.57/6.5 0.36±0.08 154 ceruloplasmin 4557485 8 132.89/5.3 123.04/5.4 3.34±0.80 161 α-1-B-glycoprotein 69990 21 84.50/5.0 52.49/5.6 4.23±0.98 162 α-2-plasmin inhibitor 11386143 14 78.58/5.0 54.92/5.8 1.95±0.38 167 α-1-B-glycoprotein 69990 28 80.87/5.3 52.49/5.6 1.73±0.04 210 Not identified - - 38.47/4.6 - 2.77±0.20 249 immunoglobulin heavy chain constant regionγ 1 12054072 15 39.021/8.1 36.65/8.8 0.40±0.09 250 ” 12054072 18 38.79/8.2 36.65/8.8 0.34±0.14 252 ” 12054072 19 38.50/8.3 36.65/8.8 0.37±0.09 262 unknown 15679996 36 24.73/8.2 23.32/8.6 0.30±0.13 265 immunoglobulinκ light chain 2765423 22 25.75/8.8 25.93/9.2 0.28±0.12 Figure Figure