bluuebunny/crossref_metadata_2025 huggingface dataset:
Compound Heterozygeous RAG-1 Mutations in 2 Patients Presenting with Chronic Disfiguring Granulomatous Skin Lesions.
Authors: Schuetz Catharina , Gudowius Sonja , Niehues Tim , Schulz Ansgar , Schwarz Klaus , Debatin Klaus-Michael , Friedrich Wilhelm
Published: April 2020

Introduction

Mutations of the recombinase activating genes (RAG) cause a severe combined immunodeficiency (SCID) characterized by absent or low T and B cells due to defective T/B cell receptor rearrangement and failure of lymphocyte maturation. Presenting clinical features commonly are failure to thrive and opportunistic infections in early infancy. We observed 2 patients with RAG mutations where partial T- and B-cell immunity is preserved. These patients did not suffer from severe infections but presented with unusual symptoms and signs, e.g. unexplained lymphogranulomatous skin lesions mimicking malignant lymphoma and lupus vulgaris.

Case presentation

Both patients, who are offsprings of unrelated German parents, underwent genetic screening because of unclear primary immunodeficiencies and were found to have compound heterozygeous RAG-1 mutations (Patient 1: R507W, R737H; R314W and patient 2: R778Q; R975W). Both have no evidence of thymic tissue on ultrasound and have persistant lymphopenia with low T-cell numbers, but are capable of mounting positive antibody responses to tetanus toxoid. In both patients unusual skin lesions were leading clinical abnormalities.

Patient 1, a 6 year-old girl, has been suffering from progressive papulonodular skin lesions of extremities and face since the age of 1 year. Biopsy showed highly monomorphous T-cell infiltrates leading initially to a diagnosis of cutaneous T-cell lymphoma treated with chemotherapy. As progression occurred chemotherapy was stopped and the lesions were reinterpreted as poorly organised sarcoid-like granulomas of unknown etiology. At 5 years of age the girl developed an EBV lymphoma of her right tonsil treated with rituximab. The patient is undergoing at present hematopoetic stem cell transplantation (HSCT).

Patient 2 is an 8 year-old girl, who at the age of 2 years developed persistant ulcerative skin lesions on her face and extremities. Granulomatous epitheloid tissue was found in skin biopsies, and later also in lung and tongue biopsies. No pathogens including mycobacteria were isolated. No improvement was seen on immunosuppression. Skin grafting was necessary in her lower extremity twice after partial destruction of the Achilles tendon by progressive ulceration. HSCT is planned in the near future.

Conclusion

RAG mutations may be associated with a combined immunodeficiency where uncontrolled granuloma formations of the skin, but not opportunistic infections, are leading clinical findings. It is unclear whether these disfiguring lesions are caused by pathogens such as mycobacteria which are difficult to culture or whether they represent reactive sarcoid-like granulomas as seen in other primary IDs, e.g. common variable immunodeficiency.
Specific Cytotoxicity and Clonal Expansion of TCR Vβ Subfamily T Cells Induced by PML-RARα Peptide.
Authors: Li Yangqiu , Tang Ji , Yang Lijian , Chen Shaohua , Zhou Yubing
Published: April 2020

The analysis of T cell receptor (TCR) Vβ repertoire is one of the sensitive methods to identify the clonal expansion T cells which response to tumor associated antigens. Understanding the clonality and restricted usage of TCR Vβ repertoire of expanded T-cells induced by PML-RARα peptide may be useful in helping design the new immunotherapeutic strategy specifically for acute promyelocytic leukemia (APL).The aim of the present study was to investigate the specific cytotoxicity and clonality of TCR Vβ repertoire in cord blood T cells induced by PML-RARα peptide (LSSCITQGKAIETQSSSSEE) in vitro. Cord blood mononuclear cells were amplified by IL-2, anti-CD3 and anti-CD28 antibody with different concentration (16.7μg /ml, 33.3μg /ml or 50μg /ml respectively) of synthetic PML-RARα peptide. The induced T cells were collected at different time points after culture (3, 6, 9, 10, 12 or 15 days). The expression and clonality of TCR Vβ subfamilies within induced T cells were analyzed by using RT-PCR and genescan technique. The cytotoxicity of induced T cells was detected by LDH release assay. The results showed that the best condition for T cells induction and amplication was at a concentration with 16.7μg /ml of PML-RARα peptide and at a culture duration with 10 to 15 day. TCR Vβ repertoire analysis showed that restricted expression and cloanl expansion of TCR Vβ subfamily cord blood T cells could be identified after induction by PML-RARα peptide. Clonal expanded T cells were found in Vβ13, Vβ14 and Vβ16 subfamlies respectively. The induced T cells were showed to have the specific cytotoxicity for NB4 cell line (effector cells: tagerted cells=20:1), the cytotoxicity rates were 49.65±6.7% (p<0.05) at day 10th and 73.13±8.42% (p<0.01) at day 15th after culture, which show statistical significance in compare to the control group (without PML-RARα induction). In conclusions, the PML-RARα peptide could induce the clonal expansion T cells from cord blood in vitro, which may have specific cytotoxicity for PML-RARα+ cells.
CD5+B Lymphocytes in the Bone Marrow of Immunic Hemocytopenia Patients and Its Clinical Significancy.
Authors: Shao Zonghong , Xing Limin , Fu Rong , Liu Hong , Shi Jun , Bai Jie , Tu Meifeng , Wang Huaquan , Cui Zhenzhu , Jia Hairong , Sun Juan , Yang Chongli
Published: April 2020

Background A subpopulation of B lymphocytes expressing CD5 antigen is involved in several autoimmune diseases through the release of autoantibodies. We investigate the quantitative of CD5+ B lymphocytes in the bone marrow of the patients with autoimmunic hemocytopenia and study the relationship between quantity of CD5+B cells and clinical or laboratorial parameters of these patients.Methods Quantities of CD5+B lymphocytes in the bone marrow(BM)of 14 patients with autoimmune hemolytic anemia(AIHA) or Evans syndrome, 22 immunorelated pancytopenia (IRP) patients and 10 normal controls were assayed by FACS. The correlation between their clinical or laboratory parameters and CD5+B lymphocytes was analyzed.

Results The qutantity of CD5+B lymphocytes of AIHA/Evans syndrome ((34.64±19.81)% or IRP((35.81±16.83)%) patients was significantly higher than that of normal controls((12.0±1.97)%)(p<0.05). There was no significant difference between those of AIHA/Evans syndrome and IRP patients(p>0.05). The quantity of CD5+B lymphocytes in the bone marrow of all cytopenic patients showed negative correlation with complement C3(r=−0.416, p<0.05). In AIHA/Evans syndrome patients, the quantity of BM CD5+ B lymphocytes showed positive correlation with indirect bilirubin(IBIL) (r=1.00, p<0.05); In Evans syndrome patients, the quantity of CD5+B lymphocytes in their bone marrow showed positive correlation with PAIgG (r=0.761, p<0.05) and PAIgM(r=0.925, p<0.05). The qutantity of CD5+B lymphocytes in the bone marrow of all cytopenic patients showed negative correlation with treatment response (correlation coefficient tau-b=−0.289, p<0.05), but no relation with the yields of CFU-E, BFU-E,CFU-F and CFU-GM BM in vitro.

Conclusion BM CD5+B lymphocytes of the patients with autoimmunic hemocytopenias significantly increased and was related to the disease severity and clinical response. It was suggested that CD5+B lymphocytes might play an important role in the pathogenesis of autoimmunic hemocytopenia.
Gene Expression Profiling of Natural Killer Cell Subsets Isolated Using Novel MACS Methods.
Authors: Schmitz Jurgen
Published: April 2020

Natural Killer cells are now regarded as an important (Hokland, Mol Immunol 2005) but heterogenous (Cooper, Blood 2001) lymphocyte subset. Different Natural Killer cell subsets have different functions (Cooper, Blood 2001; Mavilio, PNAS 2005; Trotta, Blood 2005) and further research will be required to identify the role of those NK cell subsets in immune responses. We have developed novel methods, based on magnetic cell sorting, for purification of several NK cell subsets and have evaluated the separation performance of these kits using peripheral blood mononuclear cells from 10 donors. More than 1x105 CD56brightCD16− cells from 1x108 PBMC can be isolated with >70% purity (>85% CD56+CD16−), and more than 1x106 CD56dimCD16+ cells with >94% purity. Additional subsets have been identified based on CD8 expression: >2x106 CD8+ NK cells can be purified with >90% purity.

Only limited data has previously been published on gene expression profiling of NK cell subsets using DNA microarrays (Koopman, J Exp Med 2003). We thus have used these MACS sorted NK cell subsets from 3 donors for an immune system specific microarray analysis (“PIQOR Immunology”). Usually gene expression profiling experiments are combined with flow cytometry characterization and functional tests. We used a novel amplification method to perform microarray analysis from mRNA isolated from as little as 1000 cells. Genes for surface molecules known to be differently expressed on NK cell subsets and immune effector molecules produced preferentially by single subsets served as control for the combination of amplification and microarray analysis (CD56, CD16, CD8, CD62L, CD122, Perforin, Granzyme B). Results of the analysis will be shown.
Decreased TCR ζ-Chain Expression in T Cells from Patients with Aplastic Anemia.
Authors: Solomou Elena E. , Keyvanfar Keyvan , Young Neal S.
Published: April 2020

The ζ-chain is a unique component of the T cell receptor (TCR) with no sequence homology to other CD3 molecules. The ζ-chain acts to stabilize the TCR on the cell surface and to transmit signals to different transduction pathways in the cell. T cells from patients with autoimmune diseases, with chronic inflammation, and suffering malignant tumors are functionally impaired in that they express decreased ζ-chain. Acquired aplastic anemia (AA) is a bone marrow failure syndrome characterized by T-cell immune mediated destruction of hematopoietic stem cells. Oligoclonal expansion of T cells correlates with disease activity. In this study, we examined TCR ζ-chain protein levels in aplastic anemia patients. Twelve out of sixteen patients (75%) examined displayed decreased ζ-chain protein levels compared to normal controls (p=0.008). As a control, no differences were detected in expression of the TCR ε-chain. Patients and healthy donors displayed comparable amounts of Zap-70, the kinase responsible for full phosphorylation and activation of the ζ-chain. There was no correlation with disease status or medical treatment at the time of sampling. Four patients were re-examined for ζ-chain protein levels at 3 month intervals, but there was no change in their ζ-chain expression pattern despite hematologic improvement. We hypothesized correlation between ζ-chain and IFN-g levels in T cells from patients with AA. When we estimated IFN-g levels by flow cytometry, all 5 patients examined had increased IFN-g levels, of whom 4 had decreased ζ-chain levels: intracellular IFN-g was reciprocally correlated with ζ-chain protein levels. In summary, our data indicate that TCR ζ-chain expression is decreased in the majority of patients with AA, regardless of disease activity or treatment status. ζ-chain-deficient T cells from AA patients also overproduce IFN-g. Is ζ-chain down-regulation the primary event leading to IFN-g over-production, or is IFN-g responsible for the ζ-chain down-regulation? One possible explanation is an intrinsic defect in the TCR leading to alterations in kinases and transcription factors downstream of the ζ-chain-Zap-70 association. Whether ζ-chain expression correlates with expanded Vb clones observed is to be established. The seqeunce of molecular events described in the current work may lead to the Th1 shift observed in aplastic anemia, but further studies are needed to explore the possible role of ζ-chain downregulation to the pathogenesis of AA.
Galectin-1 Purified from Human Platelets.
Authors: Fink Nilda Ester , Gonzalez Mariana M.
Published: April 2020

Galectins are a family of lectins which exhibit β-galactoside-binding activity. They are Ca++-independent and have a common consensus sequence in the carbohydrate binding domain. Fourteen mammalian members of this family have been sequenced and well-characterised in different species, although galectins-1 and -3 have been the most thoroughly studied. They have biological functions in the regulation of the immune system such as allergic inflammation and host defence (activation of mast cells, neutrophils and eosinophils, stimulation of interleukins production and in the generation of oxidizing radicals), but scarse information is avalaible on biological functions in platelet function and hemostasia. The objective of this work was to detect and purified galectins from human platelets. Platelet-rich plasma obtained from blood bank, with negative serology for HIV, HBsAg and HVC, was filtered by a Pall Purecell PL filter (USA) to produce leucoreduction. Later, platelets were centrifuged and the pellet was homogenized in MEPBS (mercaptoethanol-EDTA-phosphate buffer saline)-200 mM lactose. The supernatant was treated by anion-exchange and affinity chromatography (lactose-agarose) followed by size exclusion in FPLC. We determined that an amount of 6-10 fg was present in a single platelet. The molecule was a dimer with a subunit of 14 kDa. Purified protein was isolated by PAGE-SDS and electroblotted onto nitrocellulose sheets. Identity was revealed employing polyclonal and monoclonal anti-galectin antibodies. Positive and negative controls were running simultaneously. Amino acid sequencing of galectin peptides performed indicated that it was a galectin-1. We conclude that galectin-1 is present in human platelets. Potential roles of galectin-1 in platelet function and hemostasia are under study.
New Insights from the Structure Function of the Catalytic Region of Human Platelet PDE3A: The Role of the Unique 44 Amino Acid Insert.
Authors: Hung Su-Hwi , Zhang Wei , Tao Jing , Kim Carolyn , Noone Rachel , Ezon David , Colman Roberta F. , Colman Robert W.
Published: April 2020

Human platelet PDE3A degrades cAMP, the major intracellular inhibitor of platelet function, and thus potentiates platelet activation. PDE3A is irreversibly inactivated by the affinity label Sp-cAMPS-BDB. The inactivation is prevented by Sp-cAMPS indicating that the affinity label is targeted at the cAMP binding site. We now use Sp-cAMPS-BDB with the aim of identifying nonconserved amino acids in substrate binding. After incubating Sp-cAMPS-BDB with PDE3A followed by reduction with [3H]NaBH4, the incorporation was 1.1 mol/mol. HPLC analysis of the tryptic digest yielded a radioactive octapeptide T806YNVTDDK813 in the 44-amino acid insert of PDE3A. Molecular modeling of PDE3A based on the PDE3B structure suggests the insert is a flexible loop. Incorporation of Sp-cAMPS-BDB indicates loop interaction with the substrate. Since Sp-cAMPS-BDB reacts with the nucleophilic residues, Y807, D811 and D812 were each mutated to alanine. Sp-cAMPS-BDB inactivates D811A and D812A but not Y807A, suggesting Y807 is the residue modified by Sp-cAMPS-BDB. Y807A affects the Km but not kcat, suggesting its involvement in cAMP binding. Kinetic analyses of 11 loop mutants reveal that H782A, T810A, Y814A and C816S each affects the kcat but not Km, indicating that catalysis is modulated. We conclude that binding of cAMP to the flexible loop of platelet PDE3A induces a conformational change which allows interaction with essential catalytic residues. These findings provide a new strategy for developing antiplatelet agents to treat patients with reocclusion of coronary arteries who are resistant to aspirin or whose chronic congestive heart failure prevents utilizing cilostazol.
Proteomic Analysis Reveals Intrinsic Differences between Phenotypically Identical Mesenchymal Stem Cells.
Authors: Mazhari Sam , Desai Jyoti , Chamberlain Jason , Porada Chris , Zanjani Esmail D. , Almeida-Porada Graca
Published: April 2020

We have previously reported the isolation of Stro-1+, CD45-, GlyA-human MSC from human bone marrow (BM), liver (LV), and brain (BR). Despite their similar morphology and phenotype, these cells, upon in vivo transplantation into the pre-immune fetal sheep model, seemed to differentiate more efficiently into cells of their tissue of origin. For instance, all 3 sources of MSC gave rise to blood, with average levels of human CD45+ cells in chimeric animals of 8.7% for LVMSC, 3.3% for BRMSC, and 4.5%for BMMSC. Human neural cells were also detected in the brain of transplanted animals with the highest levels obtained with BRMSC (1.33%) and lowest with LVMSC (0.4%). However, BRMSC generated very few human hepatocytes (2–10 hepatocytes/section), while both LVMSC and BMMSC gave rise to much higher levels of hepatocytes. This led us to hypothesize that despite the similar phenotype, the proteome makeup of MSC from different tissues may be distinct. To test this hypothesis, we examined the commonalities and differences in the protein repertoire of cultured MSC from these 3 different sources. The eighty most prominent protein spots from the 2-D gel of each cell population were removed and identified by MOLDI-TOF/TOF mass spectroscopy after Trypsin digestion. Our results demonstrate that there are a large number of common proteins between the LV, BM and BR MSC (> 75%). Besides the cytoskeletal and associative proteins such as Vimentin, β-actin, cofilin, Tropomyosin and Lamin A/C, the cells contained a large quantity of proteins for protein processing such as Cyclophilin A, Endoplasmic reticulum protein ERp29, Disulfide-isomerase ER60 and Glucose-regulated proteins 75 and 78, which may be important for the broad differentiative potential of MSC. Despite these similarities, forty percent of the identified proteins showed significantly different expression levels between the three types of cells (> 300%), the majority of which were not proteins previously shown to be BM-, Liver or Brain-specific. Interestingly, however, LV-derived cells contained a 5-fold greater concentration of Albumin than BM or Brain cells, which may explain their greater tendency to form liver tissue in vivo. Likewise, since BMMSC were enriched in smooth muscle associated proteins, potentially they may constitute a better source to be used for muscle repair. Thus, the definition of the different MSC proteomes can lead not only to the discover of critical differences involved in differentiation/plasticit into different cell fates, but may also help us to identify distinct cell populations that are ideally suited for cell therapies for specific organs.
Identification of Inherited Macrothrombocytopenias Based on Mean Platelet Volume among Patients Diagnosed with Idiopathic Thrombocytopenia.
Authors: Gohda Fumito , Uchiumi Hideki , Handa Hiroshi , Takafumi Matsushima , Tsukamoto Norifumi , Morita Kimio , Amagai Hatsuo , Murakami Masami , Murakami Hirokazu , Nojima Yoshihisa , Karasawa Masamitsu
Published: April 2020

Inherited macrothrombocytopenia is a rare illness that is often misdiagnosed as idiopathic thrombocytopenia (ITP), a more widespread acquired disease. The hallmark of a diagnosis of these diseases is the presence of giant platelets. Automated blood cell counters in routine clinical use usually miss giant platelets and underestimate mean platelet volume (MPV). Incorrect diagnoses might expose patients to a risk of unnecessary treatment. The ADVIA 120 hematology counter efficiently detects large platelets based on two-dimensional laser light scatter. This two-dimensional approach provides a more accurate estimation of platelet volume than conventional one-dimensional analysis. The present study measures and re-evaluates MPV using the ADVIA 120 in 112 patients who had initially been diagnosed with ITP.

We identified 11 unrelated patients as having probable macrothrombocytopenia (average MPV of 19.2 ± 3.8 fL; normal range 7.8–10.2). A diagnosis of inherited macrothrombocytopenia was immediately ruled out in 5 of the 11 patients, because their medical records indicated that their platelet counts had once been normal or became normalized (>100x199/L) in response to steroid therapy. Of the remaining 6 patients, platelets aggregated abnormally in response to ristocetin in 3 patients and MYH9-RD was suspected in one patient since Döhle-like bodies were apparent in granulocytes on blood films. DNA analyses confirmed that 3 of these patients had Bernard-Soulier syndrome and 1 had MYH9-related disease, both of which are the most common forms of inherited macrothrombocytopenia. We detected 3 homozygous mutations in the 3 patients with Bernard-Soulier syndrome: A3171T and 4444insT in the GPIbα gene that created new premature termination codons, and the missense mutation, G1913A in the GPIX gene, that caused the loss of GPIX surface expression. In one patient with MYH9-related disease, we found a heterozygous missense mutation of C5797T in exon 40 of the MYH9 gene. We stress that all four patients had received high-dose steroid therapy and/or splenectomy before this study according to a diagnosis of ITP.

Checking MPV using the ADVIA 120 in thrombocytopenic patients is a useful method of correctly diagnosing inherited macrothrombocytopenia, and thus avoiding patient exposure to unnecessary and sometimes toxic treatment.
Interaction of Recombinant Factor VIIa with Rehydrated, Lyophilized Platelets.
Authors: Fischer Thomas H. , Wolberg Alisa S. , Bode Arthur P. , Ramer Kevin J. , Nichols Timothy C.
Published: April 2020

The experiments presented here were undertaken to determine if factor VIIa (rFVIIa, the Novo Nordisk product NovoSeven™) will directly bind to rehydrated, lyophilized (RL) platelets (Stasix™ platelets, Entegrion, Inc. trade) for the formation of a catalytic surface with an enhanced ability to generate thrombin. The relationship of rFVIIa to the RL platelet surface was examined by measuring equilibrium and non-equilibrium binding of the coagulation factor to the cells, by studying the subcellular localization of the coagulation factor on RL platelets, and by following the effects of the surface modification on the kinetics of thrombin generation. The association of rFVIIa with RL platelets occurred with an on rate of 3.6x103 sec−1moles−1. Saturation occurred in minutes and was calcium dependent. Disassociation (in plasma or citrated saline) was slow, with over half of the coagulation factor remaining bound after two hours (with slow and fast rate constants of 5.0x10−5 and 4.1x10−4 sec−5 respectively). These results define a binding site with an apparent equilibrium constants of 110 nM. Equilibrium binding of rFVIIa to RL platelets was analyzed with flow cytometry and Western analysis. The rFVIIa was bound to RL platelets in a dose-dependent manner when incubated at concentrations of 0.3 to 10.0 uM rFVIIa and 3x104 to 106 RL platelets/ul in citrated saline. When high concentrations of rFVIIa were bound to RL platelets densities of over one million molecules of rFVIIa per RL platelet was obtained. Fluorescent microscopy analysis revealed that the rFVIIa was localized to the surface membrane and that some rFVIIa localized internally to the outer surface of the surface connected open canalicular system and/or sites of internal trafficking. Flow cytometric analysis with annexin V demonstrated that considerable quantities of phosphatidylserine were present on the external surface of the RL platelet membrane for potential facilitation of rFVIIa binding. The effect of RL platelet surface modification by rFVIIa on thrombin generation was investigated by following the hydrolysis of the thrombin-specific fluorogenic substate D-phe-pro-arg-ANSNHin plasma. rFVIIa and RL platelets accelerated thrombin generation in this system with rFVIIa being approximately twice as effective (per molecule of the recombinant protein) when added to the assay system pre-bound to RL platelets as compared to being initially free in the plasma. Similar results were obtained when free and RL platelet bound rFVIIa were tested in factor IX-deficient plasma. These experiments show that rFVIIa retains activity when super-saturated on the RL platelet membrane. The results of the studies presented here suggest that RL platelets can be used to concentrate rFVIIa at sites of vascular injury.
Diagnostic Criteria for Hematopoietic Stem Cell Transplantation-Associated Microangiopathy (TAM): Results of a Consensus Process by an International Working Group.
Authors: Ruutu Tapani , Barosi G. , Benjamin R. J. , Clark R. E. , George J. N. , Gratwohl A. , Holler E. , Iacobelli M. , Kentouche K. , Lämmle B. , Moake J. L. , Richardson P. , Socié G. , Zeigler Z. , Niederwieser D. , Barbui T.
Published: April 2020

The pathogenesis of microangiopathy following stem cell transplantation is poorly understood. Endothelial injury is probably an important factor. The spectrum of transplantation-associated microangiopathy (TAM) extends from minor laboratory findings to life-threatening clinical complications. TAM has not been properly defined, a large number of definitions have been used, and the reported incidences and outcomes of this complication have varied greatly. For purposes of clinical study, and to hasten the development of prophylaxis and therapy for this complication, widely accepted and uniform criteria for TAM are essential. An International Working Group was, therefore, formed with the goal of developing a consensus formulation of the criteria necessary for the diagnosis of TAM. Using a mail-only process, 14 experts in microangiopathic disorders and/or transplantation were asked to propose a list of candidate diagnostic criteria, to select those considered necessary, and to rank those considered optional in order to identify a core set of criteria. The three necessary criteria in the core set consisted of: 1)”increased percentage (>4%) of schistocytes in the blood”; 2)”de novo, prolonged, or progressive thrombocytopenia (platelet count ≤50x109/l or a 50% or greater reduction from previous counts)”; and 3)”sudden and persistent increase in LDH”. The four most highly ranked optional criteria were: “decrease in Hb concentration or increased red cell transfusion requirement”, “decrease in serum haptoglobin”, “sudden and persistent increase in BUN or creatinine”, and “neurological symptoms”. In an appropriateness panel process, the experts were then asked to score the diagnosis of 16 patient profiles as appropriate or not appropriate for TAM. Using the experts’ consensus, the performance (sensitivity and specificity) of 24 possible definitions of the disorder obtained from the core set of criteria was evaluated. The definition of TAM with the highest final score was: the three necessary criteria, plus decrease in Hb concentration or increased red cell transfusion requirement plus decrease in serum haptoglobin. The Working Group, consequently, proposes that the diagnosis of TAM requires fulfilment of all of the following criteria: 1) increased percentage (>4%) of schistocytes in the blood; 2) de novo, prolonged, or progressive thrombocytopenia (platelet count ≤50x109/l or 50% or greater reduction from previous counts); 3) sudden and persistent increase in LDH; 4) decrease in Hb concentration or increased red blood cell transfusion requirement; and 5) decrease in serum haptoglobin. This definition has >80% sensitivity and specificity.
The Clinical Efficacy of Helicobacter pylori Eradication in Patients with Helicobacter pylori-Positive Chronic Thrombocytopenic Purpura.
Authors: Nam Seung-Hyun , Kim Bong-Seog , Lee Jae Hoon , Song Hong Suk , Bae Sung-Hwa , Hyun Myung Soo , Yuh Young Jin , Lee Sang-Jae , Park Seonyang
Published: April 2020

Background: Helicobacter pylori (H pylori) has been implicated in the pathogenesis of some autoimmune diseases including idiopathic thrombocytopenic purpura (ITP). Several studies recently showed a high prevalence of H pylori infection in patients with ITP and reported a platelet recovery after bacterial eradication therapy. The prevalence of H pylori infection and effect of its eradication in Korea patients with chronic ITP were investigated.

Methods: The study included among 35 patients with chronic ITP from eight hospitals. H pylori infection was assessed by urea breath test, rapid urease test or microbial culture. H pylori eradication was performed with amoxicillin, clarithromycin and omeprazol regimen for 7 days or bismuth, metronidazol and tetracycline regimen for 10 days. Eradication was assessed by urea breath test 4 weeks after treatment. Platelet counts were monitored serially after the end of treatment.

Results: Thirty five patients with chronic ITP were evaluated, including 12 males and 23 females. Median age was 57 years (range 30–79). The median platelet count before eradication was 23,000/uL (range 4,000–66,000/uL). Sixteen patients were performed the splenectomy previously. H pylori infection was found in 23 (65%) of 35 patients. In 21 patients performed the eradication, 6 patients (28.5%) had a significant increase in platelet count after 2 weeks and 2 months. The response duration was 1–27 (median 7.6) months.

Conclusions: This study confirmed the efficacy of H pylori eradication in increasing platelet in adult chronic ITP patients. We must consider the investigation and eradication of H pylori infection in ITP patients as a simple, inexpensive tool in management of the chronic disease.
Correlation between Helicobacter Pylori Eradication and Platelet Count in Immune Thrombocytopenic Purpura (ITP).
Authors: Jang Jun Ho , Kim Hyeoung Il , Lee Hyun Woo , Kang Seok Yun , Park Joon Seong , Choi Jin Hyuk , Lim Ho Yeong , Kim Hugh C.
Published: April 2020

BACKGROUND: Helicobacter pylori has clearly been implicated in the pathogenesis of gastric and duodenal ulcers, gastritis, and gastric malignancy. Remarkably, eradication of H. pylori from the gastric mucosa has been associated with improvement of systemic disease, including Sjögren’s syndrome, rheumatoid arthritis, autoimmune thyroid disease, and immune thrombocytopenic purpura (ITP).

PURPOSE: To investigate the relationship between Helicobacter pylori infection and the clinical features of idiopathic thrombocytopenic purpura (ITP), and to examine the effects of H. pylori eradication on platelet counts.

METHOD: A 13C urea breath test (UBT) for H. pylori infection was performed in a 25 consecutive patients with ITP at Ajou University School of Medicine, Suwon, Korea. Patients who tested positive for H. pylori received standard eradication therapy if their platelet count was < 50 x 109/L.

RESULTS: H. pylori infection was detected in 18 patients (72%) and eradication therapy was successfully administered to all infected patients. H. pylori infection was not associated with dyspepsia or other clinical or laboratory features. Platelet responses were observed in 6 (33%) of these patients, which lasted for more than 4 months in 4 patients. Platelet associated antibody and anti platelet antibody were negative to all patients.

CONCLUSION: H. pylori eradication may improve the platelet counts in some of adults (33%) in whom the ITP is of recent onset.
Aspirin Resistance - A Comparison of Platelet Functions and 11-Dehydro Thromboxane B2 and 8-epi Prostaglandin F2α Plasma Concentration.
Authors: Zawilska Krystyna , Zytkiewicz Marcin , Hanszke Ewelina , Gielwanowska Liwia , Psuja Piotr , Turowiecka Zofia , Gaik Irmina , Zozulinska Maria , Duszynska Malgorzata
Published: April 2020

Introduction. Laboratory tests do not reveal any changes due to administration of aspirin (75 – 150 mg/d) in about 25% of patients. The definition of aspirin resistance and parameters of different laboratory tests for its identification have not yet been established.

Aim. The aim of our study was to compare the results of platelet aggregation studies, the closure times in PFA-100, the plasma concentrations of 11-dehydro thromboxane B2 (11-d TxB2) and 8-epi prostaglandin F2α (8-epi PgF2α) in patients receiving aspirin chronically.

Material. The study group consisted of 22 patients taking aspirin for the secondary prevention of ischemic stroke (IS). All patients were at least 6 months after the acute onset, and had a diminished intraplatelet concentration of malonyldialdehyde due to aspirin ingestion.

Methods. Following parameters have been evaluated:

Platelet aggregation induced by either ADP (3,5 and 5,0 μM), collagen (2 μg/ml) or arachidonic acid (AA) (0,6 mM); Closure time in PFA-100 (collagen/epinephrine and collagen/ADP cartridges); Plasma 11-d TxB2 and 8-epi PgF2α concentration measured by ELISA method (Cayman Chemicals).

Aspirin resistance has been determined by the following criteria: the intensity of ADP induced platelet aggregation ≥ 60%, collagen induced aggregation ≥ 70%, AA induced aggregation ≥ 20%, PFA-100 closure time ≤ 165 s, 11-d TxB2 concentration ≥ mean of the control group ± 2 SD.

Results.

The frequency of aspirin resistance in patients after ischaemic stroke Aggregation ADP 3,5 μM ADP 5,0 μM Collagen Arachidonic acid PFA-100 11-d TxB2 63,6% 45,5% 18,2% 13,6% 54,5% 50,0%

Statistically significant correlations have been found between the plasma concentration of 11-d TxB2 and PFA-100 (Col/Epi) closure times and between plasma concentration of 8-epi PgF2α and PFA-100 (Col/Epi) closure times. There was no difference in mean plasma concentration of 8-epi PgF2α between the group of patients and controls.

Conclusions.

The most valid laboratory method of aspirin resistance identification in ischemic stroke patients (besides a mesurement of 11-dehydro thromboxane B2) seems to be PFA-100 closure time. Our PFA-100 studies reveal a much higher percentage of aspirin resistance as compared to former studies. The plasma concentration of 8-epi prostaglandin F2α remains in the normal range in patients taking aspirin for the secondary prevention of ischemic stroke.
Rituximab for the Treatment of Recurrent Thrombotic Thrombocytopenic Purpura.
Authors: Bortolheiro Teresa C. , Castro N. S. , Rodolfo Cançado D. , Carlos Chiattone
Published: April 2020

Background: Thrombotic thrombocytopenic purpura (TTP) is a rare and severe disease, characterized by thrombocytopenia, microangiopathic anaemia, fever, renal failure, and neurological manifestation caused by deposition of von Willebrand factor - platelet rich hyaline thrombi in the arterioles and capillaries. Plasma exchange has decreased the mortality in TTP from almost 100% to 10–30%. However, relapses occur in more than one-third of patients, a subset of whom develop multiple relapses or chronic disease requiring numerous sessions of plasma exchange. This treatment is costly and associated with many adverse reactions. Case

Report: We describe a female patient - diagnosed with TTP in 1996 - who did not achieve a sustained remission with plasma exchange, steroids and vincristine, but who remained in remission after a splenectomy performed in 1997. The patient underwent relapse in 2003 and restarted plasma exchange, with progressive improvement of cytopenias. Plasma exchange was reduced in frequency from daily sessions to every other day. There was a lowering of platelet levels and an increase in LDH levels - steroids and vincristine were re-initiated, but without response. The patient was treated with four, weekly doses (375 mg/m2) of rituximab. Plasma exchange was only required during the first 2 weeks of rituximab treatment, and was discontinued after the second dose of rituximab. The patient remained in complete remission for 9 months, but was then admitted to hospital with femoral thrombosis, and normal LDH and platelet levels. After 40 days of thrombosis therapy, the patient had a further relapse and refused plasma exchange. Treatment with rituximab was restarted as previously, and the patient achieved a complete remission within 2 weeks - now sustained for 11 months.

Conclusion: In comparing this patient with eight previous cases not treated with rituximab, we conclude that rituximab is a very effective treatment for TTP, which is not associated with clinically significant toxicity. Randomized clinical trials are required to confirm these findings.
Expression of Vascular Endothelial Growth Factor (VEGF) Associated with Its Receptor (VEGFR) in Relation to Tissue Factor (TF) in Human Breast Cancer Tissue.
Authors: Wojtukiewicz Marek , Sierko Ewa , Zimnoch Lech , Thorpe Philip , Brekken Ralf A. , Kisiel Walter
Published: April 2020

New blood vessel formation is an important process in tumor growth and dissemination. The relationship between vessel density and survival of patients was documented in breast cancer patients. Vascular endothelial growth factor (VEGF) is of paramount importance in new blood vessel development. VEGF exerts its biological function through binding to specific receptors (VEGFR-1/Flt-1 and VEGFR-2/KDR/Flk-1). Recent studies revealed that tissue factor (TF), apart from being the main procoagulant in cancer patients, is endowed with biological activities that do not relate to its hemostatic function. There exists a large body of evidence that TF is one of the key regulators of angiogenesis. In vitro experiments demonstrated that TF stimulates the synthesis of VEGF in different cell types. The purpose of the study was to assess the expression of VEGF and VEGF coupled to its receptor in relation to the expression of TF. Studies were performed on breast cancer fragments and metastatic axillary lymph node tissues obtained during surgical treatment of previously untreated patients. The tissues were fixed according to AMeX method and embedded in paraffin. Immunohistochemical staining procedures according to the ABC method and Envision Double Staining System (DAKO) were employed using polyclonal antibodies directed to TF and VEGF and a novel monoclonal antibody that binds to VEGF only when it is associated with its receptor - VEGFR. In breast cancer and lymph node tissues the co-localization of TF and VEGF in cancer cells was observed. Of particular interest was the co-expression of TF and VEGF in association with its receptor - VEGFR in cancer cells and endothelial cells of small blood vessels. Furthermore, VEGF coupled to its receptor was localized in dendritic cells in metastatic lymph nodes, which may indicate its influence on the host immune response system. The observed co-localization of TF and VEGF (including VEGF in association with VEGFR) suggests a functional interrelationship that exists between TF and VEGF in breast cancer in vivo.
Factor VIII A2 Domain Residues 497–510 and 584–593 Comprise the R8B12 Epitope.
Authors: Ansong Charles , Miles Stephen , Fay Philip J.
Published: April 2020

The murine monoclonal antibody R8B12 recognizes the C-terminal region (residues 563–740) of the A2 subunit of factor VIIIa (Fay et al. J. Biol. Chem. 266:20139, 1991), as judged by western blotting. The location of the R8B12 epitope within the A2 subunit is not known. In the present study we used affinity-directed mass spectrometry (Zhao et al. Proc. Natl. Acad. Sci. USA.93:4020, 1996) to map the R8B12 epitope. Purified A2 subunit was subjected to proteolytic digestion with trypsin. The digest was then subjected to immunoprecipitation (IP) using R8B12 IgG, in which peptide(s) are selected that bind the antibody. Subsequently the masses of the affinity-selected peptides were determined directly from the immune complex by MALDI-TOF mass spectrometry. Proteolysis of A2 with trypsin generated a pre-IP peptide fingerprint that covered ~40% of the A2 domain sequence (Figure, left). Analysis of the post-IP peptide fingerprint showed two masses, 1309 and 1654, as representing affinity-selected peptide fragments (Figure, right). A theoretical database search identified the 1309 mass peak as A2 domain residues 584–593 and the 1654 mass peak as A2 domain residues 497–510. Direct sequencing of both mass peaks confirmed the results of the theoretical database search. These residues mapped to regions on the A2 domain of the factor VIII A domain homology model that are surface exposed and proximal to each other. Taken together, the above results suggest that A2 domain residues 497–510 and 584–593 represent a discontinous epitope for R8B12. Furthermore, based upon blotting specificity of R8B12 to the C-terminal portion of A2, we speculate that the latter sequence that forms this epitope makes a substantially greater contribution to the binding energy.

Figure Figure
The Role of Erythropoietin in Vascular Endothelial Cells and Thrombosis.
Authors: LaMontagne Kenneth R. , Bdeir Khalil , Carinato Maria E. , Butler Jeannene , Cines Douglas B. , Farrell Francis X.
Published: April 2020

Erythropoietin (EPO) is an essential glycoprotein hormone that regulates erythrocyte production in hematopoietic tissues by stimulating growth, preventing apoptosis, and inducing the differentiation of red blood cell precursors. Clinically, EPO has been extensively used to increase levels of hemoglobin in the treatment of patients with anemia due to chronic renal failure, cancer chemotherapy, and HIV therapy. Recent studies suggest that the function of EPO and its cognate receptor, EPOR, are not strictly limited to erythroid lineages. EPOR expression has been identified in nonhematopoietic cells and tissues including endothelial, neuronal, and tumor cells. Epoetins have shown benefit in correcting anemia in chronic kidney disease and in chemotherapy-associated anemia in cancer patients. However, the incidence of thrombosis appears to be higher in patients administered EPO versus controls transfused to comparable hemoglobin levels. In particular, incidences seem higher in oncological patients treated with EPO and receiving combined radiation and chemotherapy than in those receiving chemotherapy alone. To address the potential interaction and responsiveness of the vasculature to EPO, we evaluated EPO using vascular endothelial cell assays. Biochemical analysis (eg, receptor expression, ligand-receptor binding, and activation of signaling cascades) as well as cellular analysis (eg, proliferation, migration, and differentiation) will be discussed. To evaluate the effects of EPO on thrombosis we used the in vivo pulmonary micoemboli (ME) model. In this fibrinolysis model, a preformed 125I-fibrin microemboli with a defined particle size is infused into mice intravenously. Microemboli lodge primarily in the lungs and the rate of radioactivity lost is measured over time. Defects in fibrinolysis have been identified in mice lacking either tPA or uPA. Mice were exposed acutely or chronically to EPO and the dissolution of ME was tested. Mice were injected subcutaneously with either a single dose of EPO (2500 IU/kg) and for the chronic studies EPO (1200 IU/kg) or control buffer three times a week for three weeks. The residual radioactivity in the lungs was measured after 10 and 90 minutes. Red blood cell count, hemoglobin, and hematocrit were analyzed weekly to confirm EPO activity. Our results indicate that neither a single (acute) dose of EPO nor chronic treatment using EPO affected the rate of pulmonary fibrinolysis. This suggests that EPO treatment does not significantly impair the function of the pulmonary fibrinolytic system proteins (eg, uPA, tPA, plasminogen, and PAI-1) using the ME model. We cannot exclude EPO’s potential influence on other aspects of coagulation and fibrinolysis.
Biphasic Waveform of the Activated Partial Thromboplastin Time (A2 Flag) Is Associated with Adverse Events in the Coronary Care Unit.
Authors: Bane Christopher , Leya Ferdinand S. , Lewis Bruce E. , Walenga Jeanine M.
Published: April 2020

Objective: An abnormality of the optical transmission waveform obtained during measurement of the activated partial thromboplastin time (aPTT) has been described in association with DIC and sepsis. This biphasic waveform (BWF) is caused by the in vitro formation of Ca2+-induced complexes between VLDL and CRP. A review of the literature revealed no studies on the BWF in cardiac patients. The purpose of this study was to determine whether the BWF is associated with adverse clinical outcomes among patients in a Coronary Care Unit (CCU).

Patients: Consecutive patients admitted to the CCU over a two-month period (n=83) were retrospectively evaluated. Standard of clinical care was not altered.

Methods: Laboratory results including aPTT, aPTT waveform analysis, troponin, beta-natriuretic peptide (BNP), complete blood count (CBC), basic metabolic panel (BMP), and high sensitivity C-reactive protein (hsCRP) were analyzed when available. BWF values beyond a pre-set threshold [5 SD’s lower than the mean value of a normal population (n=40)] were signaled by the A2 Flag. Hospital charts were reviewed to determine coronary artery disease (CAD), hypertension (HTN), diabetes mellitus (DM), hyperlipidemia, and renal dysfunction. Hospital course including invasive and surgical procedures and medical interventions were recorded.

Results: The A2 Flag was detected in 23 of 83 patients (27.7%) during the entire course of CCU treatment. Using a lower Slope_1 threshold value (as had been used in the DIC/sepsis studies) produced an A2 flag in >90% of the patients. Patients in the A2 Flag and no-Flag groups were comparable for age, gender, HTN, DM and hyperlipidemia. Of the patients with an A2 Flag, 73.9% had acute coronary syndrome (encompassing the spectrum of clinical conditions ranging from unstable angina to non-ST-segment elevation MI to ST-segment elevation MI) vs 59.6% in the no-Flag group, 95.6% had CAD vs 83.8%, 52.1% had arrhythmia vs 32.2% (p=0.020), 60.8% had renal dysfunction (creatinine >1.5) vs 32.2% (p=0.002), 47.8% had elevated BNP vs 22.5% (p=0.037), and 73.9% had elevated troponin vs 50.0%. Length of stay was longer in the A2 Flag group (16.2 vs 7.2 days; p<0.001). Six of seven patients that expired had an A2 Flag. One patient with an A2 Flag expired during a subsequent readmission shortly after the course of this study.

Conclusion: The A2 Flag result is reported with the aPTT and thus is a rapid test available at no additional cost. As an adjunct to routine lab testing, aPTT waveform analysis appears to be a novel means to identify a select population of CCU patients that are at higher risk for adverse cardiac events and warrants further investigation. This pilot study is being used to develop a larger prospective study to determine if the A2 Flag can improve patient care through earlier diagnosis and treatment intervention.