A multi‐centre,single‐arm,open‐label study evaluating the safety and efficacy of fixed dose rituximab in patients with refractory,relapsed or chronic idiopathic thrombocytopenic purpura (R‐ITP1000 study)

The efficacy of a fixed‐dose rituximab schedule was prospectively explored in primary/acute refractory, relapsed or chronic (platelet count >10 × 10⁹/l and ≤50 × 10⁹/l) idiopathic thrombocytopenic purpura (ITP). Patients received two doses of rituximab (1000 mg) on days 1 and 15 and were followed‐up on weeks 1–8, 12, 26, 39 and 52. A total of 122 patients were included in the safety population; efficacy was analysed in 108 patients. Overall response rate (ORR) at week 8, defined as the proportion of patients achieving complete response (CR; platelet count >150 × 10⁹/l) or partial response (PR; platelet count >50 × 10⁹/l) was 44%. Therapeutic response, defined as achieving a response at week 8, with at least a minor response (MR; platelet count >30 × 10⁹/l), sustained up to weeks 26 and 52 and accompanied by a reduction in ITP medications, was achieved in 44% (week 26) and 35% (week 52) of patients, respectively. Treatment was well tolerated with no safety concerns. While this study failed to meet its primary endpoint of an ORR of 50%, the efficacy of two fixed doses of rituximab appear to provide similar efficacy to the standard 375 mg/m² four‐dose schedule in relapsed/chronic ITP.     

Thrombotic microangiopathies

Objective

To review the clinical features and pathophysiologic mechanisms of the thrombotic microangiopathies (TMAs) including acquired and congenital thrombotic thrombocytopenic purpura (TTP), Shiga toxin-induced and atypical (non-Shiga toxin-induced) hemolytic uremic syndrome (HUS), and the TMAs associated with pregnancy, drugs, and organ transplantation.

Methods

PubMed Medline was used to identify articles published from 2000 to July 2013 using the following key words: thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, Shiga toxin, ADAMTS13, and eculizumab. Articles in languages other than English, papers available in abstract form only, and nearly all single case reports were excluded. Small series, reports from registries and study groups, reviews, guidelines, and articles concerning pathophysiology and therapy were preferentially considered.

Results

Impaired post-secretion processing of unusually large von Willebrand multimers due to deficiency of ADAMTS13 (IgG antibodies or congenital), dysregulation of the alternative complement pathway (mutations and/or specific antibodies), and endothelial injury are pathophysiologic mechanisms involved in the TMAs. Acquired and congenital TTP are due primarily to severe ADAMTS13 deficiency, atypical HUS is commonly associated with complement dysregulation, and Shiga toxin, drugs, immune complexes, and others likely damage endothelium. However, there is considerable mechanistic overlap, and the TMAs often have multifactorial causation. Plasma procedures, complement pathway inhibition, immunosuppression, and general supportive care are the principal therapies.

Conclusions

The TMAs are very important conditions because of their associated organ damage and mortality rates. Prompt recognition and categorization by both clinical presentation and pathophysiologic mechanisms should become routine as they are crucial to an optimal treatment plan. Treatment advances have substantially reduced the morbidity of these disorders. Investigational therapies are promising.     

Evaluation of a chromogenic commercial assay using VWF-73 peptide for ADAMTS13 activity measurement

Introduction

Thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy (TMA), related to a severe functional deficiency of ADAMTS13 activity (< 10% of normal). ADAMTS13 activity is thus crucial to confirm the clinical suspicion of TTP, to distinguish it from other TMAs, and to perform the follow-up of TTP patients.

Material and methods

We compared the performance of the commercial chromogenic assay Technozym® ADAMTS13 Activity ELISA (chromogenic VWF73 substrate, Chr-VWF73, Technoclone, Vienna, Austria), to that of our in-house FRETS-VWF73 used as reference method. A large group of 247 subjects (30 healthy volunteers and 217 patients with miscellaneaous TMAs) was studied.

Results

The lower limit of detection of the Chr-VWF73 was 3%, which is well adapted to the clinically relevant threshold for TTP diagnosis (10%). Our results showed a reasonable agreement between FRETS-VWF73 and Chr-VWF73 assays to distinguish samples with an ADAMTS13 activity < 10% from those with an ADAMTS13 activity > 10%. However, Chr-VWF73 assay provided false negative results in ~ 12% of acute TTP patients. Inversely, the Chr-VWF73 assay globally underestimated ADAMTS13 activity in detectable values ranging from 11 to 100% (with a great variability compared to FRETS-VWF73), which may be a concern for the follow-up of TTP patients in remission.

Conclusion

In-house assays developed and performed by expert laboratories remain the reference methods that should be used without limitation to control values provided by commercial assays when needed. Also, the development of an international reference preparation will be crucial to improve standardization.     

Evidence for a role of anti-ADAMTS13 autoantibodies despite normal ADAMTS13 activity in recurrent thrombotic thrombocytopenic purpura

Background

Severe ADAMTS13 deficiency is a critical component of the pathogenesis of idiopathic thrombotic thrombocytopenic purpura but is found only in about 60% of patients clinically diagnosed with this disease.

Design and Methods

Over a period of 8 years and six episodes of thrombotic thrombocytopenic purpura we studied the evolution of the anti-ADAMTS13 antibody response in a patient using different ADAMTS13 assays and epitope mapping.

Results

Anti-ADAMTS13 autoantibodies were found in all episodes but were inhibitory only in the last two episodes. In a flow-based assay, normal ADAMTS13 activity was found only during the first disease episode, while ADAMTS13 activity was normal using a static assay in episodes 1 and 3, and severely deficient in the last two episodes. Fluorescence evolution in a modified fluorescence resonance energy transfer assay using a von Willebrand factor A2 domain peptide substrate was linear in episodes 1, 5 and 6, but increased exponentially in episodes 3 and 4. Despite the variable functional characteristics of the anti-ADAMTS13 autoantibodies, their principal epitope was the ADAMTS13 spacer domain in all episodes.

Conclusions

The patient is unique as he displayed features of maturation or shaping of the anti-ADAMTS13 autoantibody response during the course of multiple episodes of thrombotic thrombocytopenic purpura. Anti-ADAMTS13 autoantibodies may be important in vivo despite normal ADAMTS13 activity in routine assays. Consequently, treatment decisions should not be based solely on activity assay results.     

Autoantibody-mediated complement activation on platelets is a common finding in patients with immune thrombocytopenic purpura (ITP)

Background: It is commonly accepted that antibody‐mediated removal of platelets represents a major mechanism of platelet destruction in immune thrombocytopenic purpura (ITP). Although complement activation may participate in platelet clearance, frequency and specificity of complement activation have not yet been studied systematically in ITP. Patients and methods: We examined blood samples from 240 patients with ITP. Samples were assessed for the presence of free and bound platelet autoantibodies by a standard glycoprotein‐specific assay (monoclonal antibody‐specific immobilization of platelet antigens). The ability of all sera to fix complement to a panel of human platelets was investigated in a complement fixation (CF) assay. Fixation of C1q to isolated GP IIb/IIIa was assessed by flow cytometry. Results: Glycoprotein‐specific autoantibodies were detected as platelet‐bound antibodies in 129 (54%) and as additional free antibodies in 26 (11%) and were undetectable in 111 (46%) patients. Assessing these subgroups for CF, 103 (65%), 21 (81%), and 33 (30%) sera gave positive results. If GP IIb/IIIa was absent from the test platelets, 81 (67%) lost their ability to fix complement; if GP Ib/IX was absent, 37 (30%) lost their ability to fix complement. C1q fixation to immunobeads coated with GP IIb/IIIa was observed in 50% of sera containing anti‐GP IIb/IIIa antibodies. Conclusions: In a significant number of patients with chronic ITP, platelet autoantibodies are capable of activating the classical complement pathway. CF is even present in ITP sera without detectable autoantibodies, indicating that current techniques for autoantibody detection may be insufficient. The major targets for complement‐fixing autoantibodies in ITP are GP IIb/IIIa and GP Ib/IX.     

TICLOPIDINE-INDUCED THROMBOTIC THROMBOCYTOPENIC PURPURA: TWO CASE REPORTS TREATED WITH PLASMA EXCHANGE PLUS STEROIDS

Thrombotic thrombocytopenic purpura (TTP) has a high mortality rate if left untreated with plasma exchange promptly. We report two cases of ticlopidine-induced TTP, which lesser dosages of ticlopidine (200–250 mg/day) were prescribed and were treated with plasma exchange (PE) plus steroids. The first case was treated successfully, but the second case did not respond to our treatment and died of a progressive disease complicated with pneumonia. In sum, we recommend careful use of ticlopidine, regardless of the dosages prescribe and regardless of how long the drug is used. Moreover, the adverse effect of ticlopidine should be closely monitored.     

Simvastatin Increases ADAMTS13 Expression in Podocytes

Introduction

ADAMTS13 is a specific von Willebrand factor–cleaving protease. Severe deficiency of ADAMTS13 is the main cause of thrombotic thrombocytopenic purpura. ADAMTS13 is mainly synthesized and released from hepatic stellate cells and endothelial cells, but is also expressed in other cells, including kidney podocytes. Simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, has a beneficial effect on atherosclerosis and also has anti-inflammatory and antithrombotic properties. A recent study indicates that ADAMTS13 reduces inflammatory plaque formation during early atherosclerosis in mice. In our study, we investigated the effects of simvastatin on inflammatory cytokines–induced ADAMTS13 expression in podocytes.

Materials and Methods

A conditionally immortalized mouse podocyte cell line was utilized to study the expression of ADAMTS13 in podocytes. The influence of TNF-α, IL-4, IL-6 and simvastatin on ADAMTS13 was investigated. ADAMTS13 mRNA levels in podocytes were measured by using real-time PCR and protein levels were detected by Western blotting.

Results

Simvastatin significantly up-regulated the expression levels of ADAMTS13 mRNA and protein in podocytes. IL-6 decreased ADAMTS13 expression, and TNF-α had no significant effects on ADAMTS13 expression in podocytes. IL-4 reduced ADAMTS13 mRNA expression but not its protein level. Simvastatin was able also reversed the inhibitory effect of IL-6.

Conclusions

We demonstrate that simvastatin increases the expression of ADAMTS13 in a dose-dependent manner in podocytes, which likely contributes to the antithrombotic property of statin. Different inflammatory cytokines have different effects on the levels of ADAMTS13 mRNA expression and protein within podocytes.     

Enhancing global vaccine pharmacovigilance: Proof-of-concept study on aseptic meningitis and immune thrombocytopenic purpura following measles-mumps containing vaccination

New vaccines designed to prevent diseases endemic in low and middle-income countries (LMICs) are now being introduced without prior record of utilization in countries with robust pharmacovigilance systems. To address this deficit, our objective was to demonstrate feasibility of an international hospital-based network for the assessment of potential epidemiological associations between serious and rare adverse events and vaccines in any setting. This was done through a proof-of-concept evaluation of the risk of immune thrombocytopenic purpura (ITP) and aseptic meningitis (AM) following administration of the first dose of measles-mumps-containing vaccines using the self-controlled risk interval method in the primary analysis. The World Health Organization (WHO) selected 26 sentinel sites (49 hospitals) distributed in 16 countries of the six WHO regions. Incidence rate ratios (IRR) of 5.0 (95% CI: 2.5–9.7) for ITP following first dose of measles-containing vaccinations, and of 10.9 (95% CI: 4.2–27.8) for AM following mumps-containing vaccinations were found. The strain-specific analyses showed significantly elevated ITP risk for measles vaccines containing Schwarz (IRR: 20.7; 95% CI: 2.7–157.6), Edmonston-Zagreb (IRR: 11.1; 95% CI: 1.4–90.3), and Enders’Edmonston (IRR: 8.5; 95% CI: 1.9–38.1) strains. A significantly elevated AM risk for vaccines containing the Leningrad-Zagreb mumps strain (IRR: 10.8; 95% CI: 1.3–87.4) was also found. This proof-of-concept study has shown, for the first time, that an international hospital-based network for the investigation of rare vaccine adverse events, using common standardized procedures and with high participation of LMICs, is feasible, can produce reliable results, and has the potential to characterize differences in risk between vaccine strains. The completion of this network by adding large reference hospitals, particularly from tropical countries, and the systematic WHO-led implementation of this approach, should permit the rapid post-marketing evaluation of safety signals for serious and rare adverse events for new and existing vaccines in all settings, including LMICs.     

An approach to evidence-based therapeutic apheresis

This review is derived from a memorial lecture honoring Dr. Francis Morrison, a former President of the American Society For Apheresis (ASFA). The author had numerous professional contacts with Dr. Morrison through ASFA in the early 1990s, having served with him on the Board of Directors and followed him as President, and also came to know him well on a personal level. Professionally, Dr. Morrison stood out as a courtly gentleman with a marvelous baritone voice whose ability to facilitate organization contributed to a growing sense of dignity and purpose in the affairs of the society. On the personal side, however, there wasn't an ounce of pretension in him. He was accessible and down-to-earth; a genuine character with an active and appealing sense of humor. Not surprisingly, he seemed to have a wealth of insight and "common sense," and since the topic of this study is a kind of common sense approach to assessing the effectiveness of therapeutic apheresis, it seems a fitting way to honor his memory.     

特发性血小板减少性紫癜的细胞免疫异常研究新进展

长期以来认为特发性血小板减少性紫癜的病因主要是抗体介导的血小板破坏增多，但是体液免疫异常机制并不能解释发病中的所有现象。越来越多的研究表明细胞免疫异常参与了血小板破坏机制。本综述就T细胞耐受、T细胞凋亡异常、T细胞的异常激活、T细胞亚群及其功能改变、T细胞的细胞毒作用等作了概述。