14 research outputs found
Detection of False-Negative Results in Nested Primer PCR of Proviral HIV-1 DNA
No full textA control template for a competitive nested primer PCR of the HIV-1 gag region was constructed. This construct shares the primer recognition sequences with the wildtype template and yields a 97-bp fragment after amplification (wild-type: 115 bp). To provide an internal control for the individual PCR runs, six copies of this nested primer control plasmid were introduced into a reaction tube containing the specific sample (under the PCR conditions used, this copy number reproducibly gave a positive PCR signal). The results of our study show the feasibility of this concept by analyzing a plasmid (pBH10) containing HIV-1 wildtype sequences, and examination of samples from a cohort of HIV-1-seropositive subjects demonstrated the clinical usefulness of this test. The control plasmid was detectable in all of the samples but one, which without the use of the control template would have yielded a false-negative result
Quantitative Multiple Competitive PCR of HIV-1 DNA in a Single Reaction Tube
No full textA quantitative multiple competitive PCR (QMC-PCR) for determination of DNA copy numbers is described. Four competitive DNA templates for the env region of HIV-1 were constructed with sizes longer (187 and 163 bp) or shorter (122 and 105 bp) than the 142 bp of the wild-type PCR product. Varying amounts of each of these competitors are introduced together with the sample into a single reaction tube. Since competitors and wild-type fragments share the same primer recognition sequence (SK68/SK69), amplification occurs according to the rate of the introduced copy numbers. The PCR products are run on an agarose gel, and the copy number of the sample is determined by analyzing the bands with a video densitometer and calculating the equivalence point in a linear regression plot
Evidence of protective effects of recombinant ADAMTS13 in a humanized model of sickle cell disease
No full textSickle cell disease (SCD) is an inherited red blood cell disorder that occurs worldwide. Acute vaso-occlusive crisis is the main cause of hospitalization in patients with SCD. There is growing evidence that inflammatory vasculopathy plays a key role in both acute and chronic SCD-related clinical manifestations. In a humanized mouse model of SCD, we found an increase of von Willebrand factor activity and a reduction in the ratio of a disintegrin and metalloproteinase with thrombospondin type 1 motif, number 13 (ADAMTS13) to von Willebrand factor activity similar to that observed in the human counterpart. Recombinant ADAMTS13 was administered to humanized SCD mice before they were subjected to hypoxia/re-oxygenation (H/R) stress as a model of vaso-occlusive crisis. In SCD mice, recombinant ADAMTS13 reduced H/R-induced hemolysis and systemic and local inflammation in lungs and kidneys. It also diminished H/R-induced worsening of inflammatory vasculopathy, reducing local nitric oxidase synthase expression. Collectively, our data provide for the first-time evidence that pharmacological treatment with recombinant ADAMTS13 (TAK-755) diminished H/R-induced sickle cell-related organ damage. Thus, recombinant ADAMTS13 might be considered as a potential effective disease-modifying treatment option for sickle cell-related acute events
Persistence of circulating ADAMTS13-specific immune complexes in patients with acquired thrombotic thrombocytopenic purpura
No full textAnti-ADAMTS13 autoantibodies are the main cause of acquired thrombotic thrombocytopenic purpura. Binding of these antibodies to ADAMTS13 eventually results in the formation of antigen-antibody immune complexes. Circulating ADAMTS13-specific immune complexes have been described in acquired thrombotic thrombocytopenic purpura patients, however, the prevalence and persistence of these immune complexes over time has hitherto remained elusive. Here, we analyzed a large cohort of patients with acquired thrombotic thrombocytopenic purpura for the presence of free and complexed anti-ADAMTS13 antibodies. In the acute phase (n=68), 100% of patients had free IgG antibodies and 97% had ADAMTS13-specific immune complexes. In remission (n=28), 75% of patients had free antibodies (mainly IgG) and 93% had ADAMTS13-specific immune complexes. Free antibodies were mainly of subclasses IgG1 and IgG4, whereas IgG4 was by far the most prevalent in ADAMTS13-specific immune complexes. Comparison of ADAMTS13 inhibitor and anti-ADAMTS13 IgG (total and subclasses) antibody titers in acute phase and in remission samples showed a statistically significant decrease in all parameters in remission. Although non-significant, a trend towards reduced or undetectable titers in remission was also observed for ADAMTS13-specific immune complexes of subclasses IgG1, IgG2 and IgG3. For IgG4, no such trend was discernible; IgG4 immune complexes persisted over years, even in patients who had been treated with rituximab and who showed no features suggesting relapse
Subcellular distribution of enzymes involved in the biosynthesis of cyanelle murein in the protist Cyanophora paradoxa
No full textAbstractCyanelle containing organisms, notably Cyanophora paradoxa, best studied among them, are unique with respect to the occurrence of peptidoglycan (murein) within an eukaryotic cell. Enzyme activities involved in the biosynthesis of UDP-N-acetyl-muramylpentapeptide could be localized within the cyanelle compartment. Some of the enzymes performing later steps of murcin biosynthesis were detected in the postcyanelle supernatant rather than in the cyanelle lysate. This is taken to reflect a ‘periplasmic’ location of these enzymes that are partially liberated upon rupture of the cyanelle outer membrane
Potential for Recombinant ADAMTS13 as an Effective Therapy for Acquired Thrombotic Thrombocytopenic Purpura
No full textThe metalloprotease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) regulates the size of von Willebrand factor multimers. A deficiency in ADAMTS13 activity is associated with the life-threatening disease thrombotic thrombocytopenic purpura (TTP). The vast majority of patients have acquired TTP, where circulating anti-ADAMTS13 autoantibodies are causative for the decreased ADAMTS13 activity. Current treatment consists of plasma exchange, but improved therapies are highly warranted.sponsorship: C. Tersteeg is a Postdoctoral Fellow supported by the Research Foundation-Flanders (FWO), Belgium (12N0715N). This study was funded by the FWO G.0584.11 N, the KU Leuven OT grant OT/14/71, and KU Leuven program financing PF/10/014. (Research Foundation-Flanders (FWO), Belgium|12N0715N, FWO|G.0584.11 N, KU Leuven OT grant|OT/14/71, KU Leuven program|PF/10/014)status: Publishe
後天性血栓性血小板減少性紫斑病治療における血漿交換不応例は、ADAMTS13インヒピター力価の急上昇によって発生する : 治療中の患者血漿中ADAMTS13抗原抗体複合体の動態可視化
No full textBACKGROUND: Plasma exchange (PE) is the first-line treatment for primary acquired thrombotic thrombocytopenic purpura (aTTP) with severe deficiency of ADAMTS13 activity (ADAMTS13:AC). Some patients are poor responders to PE, raising concern over multiple pathogenetic pathways. STUDY DESIGN AND METHODS: Based on 52 aTTP patients in our national cohort study, we monitored plasma levels of ADAMTS13, clinical and laboratory findings, and outcomes. In a representative poor responder to PE, we examined an ADAMTS13 inhibitor (ADAMTS13:INH) complex in plasma milieu, by means of a large-pore isoelectric focusing (IEF) analysis. RESULTS: Of 52 aTTP patients, 20 were good responders and 32 were poor responders. In the latter group, plasma ADAMTS13:AC levels never increased to more than 10% of normal during 14 days after PE initiation. Mean (±SD) plasma ADAMTS13:INH titers (Bethesda unit/mL) were 5.7 (±4.5) before PE, but decreased to 1.4 (±0.8) on the fourth PE day and then remarkably increased to 14.8 (±10.0) on the 10th PE day, termed "inhibitor boosting," and then slowly decreased to undetectable level over 1 month. On admission, none of the routinely available clinical and laboratory markers differentiated these two groups. However, elevated pre-PE levels of ADAMTS13:INH were correlated with a poor response. We visualized an ADAMTS13:INH (immunoglobulin G) complex in a patient plasma by an IEF analysis and found proteolytic fragment of ADAMTS13 antigen by a two-dimensional IEF and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. CONCLUSION: Findings from this cohort of aTTP patients demonstrated that inhibitor boosting often occurs in aTTP patients in Japan. Poor responders could be predicted by elevated pre-PE ADAMTS13:INH levels on admission, but not by routinely collected clinical or laboratory data.博士（医学）・乙第1412号・平成29年11月24日© 2015 AABB(American Association Of Blood Banks)Copyright © 1999 - 2017 John Wiley & Sons, Inc. All Rights ReservedThis is the pre-peer reviewed version of the following article: [https://onlinelibrary.wiley.com/doi/full/10.1111/trf.13182], which has been published in final form at [http://dx.doi.org/10.1111/trf.13182]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions
Epitope mapping of ADAMTS13 autoantibodies in acquired thrombotic thrombocytopenic purpura
No full textPoor responder to plasma exchange therapy in acquired thrombotic thrombocytopenic purpura is associated with ADAMTS13 inhibitor boosting: visualization of an ADAMTS13 inhibitor complex and its proteolytic clearance from plasma.
No full textBACKGROUND: Plasma exchange (PE) is the first-line treatment for primary acquired thrombotic thrombocytopenic purpura (aTTP) with severe deficiency of ADAMTS13 activity (ADAMTS13:AC). Some patients are poor responders to PE, raising concern over multiple pathogenetic pathways. STUDY DESIGN AND METHODS: Based on 52 aTTP patients in our national cohort study, we monitored plasma levels of ADAMTS13, clinical and laboratory findings, and outcomes. In a representative poor responder to PE, we examined an ADAMTS13 inhibitor (ADAMTS13:INH) complex in plasma milieu, by means of a large-pore isoelectric focusing (IEF) analysis. RESULTS: Of 52 aTTP patients, 20 were good responders and 32 were poor responders. In the latter group, plasma ADAMTS13:AC levels never increased to more than 10% of normal during 14 days after PE initiation. Mean (±SD) plasma ADAMTS13:INH titers (Bethesda unit/mL) were 5.7 (±4.5) before PE, but decreased to 1.4 (±0.8) on the fourth PE day and then remarkably increased to 14.8 (±10.0) on the 10th PE day, termed "inhibitor boosting," and then slowly decreased to undetectable level over 1 month. On admission, none of the routinely available clinical and laboratory markers differentiated these two groups. However, elevated pre-PE levels of ADAMTS13:INH were correlated with a poor response. We visualized an ADAMTS13:INH (immunoglobulin G) complex in a patient plasma by an IEF analysis and found proteolytic fragment of ADAMTS13 antigen by a two-dimensional IEF and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. CONCLUSION: Findings from this cohort of aTTP patients demonstrated that inhibitor boosting often occurs in aTTP patients in Japan. Poor responders could be predicted by elevated pre-PE ADAMTS13:INH levels on admission, but not by routinely collected clinical or laboratory data.博士（医学）・乙第1412号・平成29年11月24日© 2015 AABB(American Association Of Blood Banks)Copyright © 1999 - 2017 John Wiley & Sons, Inc. All Rights ReservedThis is the pre-peer reviewed version of the following article: [https://onlinelibrary.wiley.com/doi/full/10.1111/trf.13182], which has been published in final form at [http://dx.doi.org/10.1111/trf.13182]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.identifier:Transfusion Vol.55 No.10 p.2321-2330 (2015 Oct)identifier:00411132identifier:http://ginmu.naramed-u.ac.jp/dspace/handle/10564/3393identifier:Transfusion, 55(10): 2321-233
ADAMTS13 gene defects in two brothers with constitutional thrombotic thrombocytopenic purpura and normalization of von Willebrand factor-cleaving protease activity by recombinant human ADAMTS13
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