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To study both the pathophysiologic and the prognostic value of ADAMTS13 in thrombotic microangiopathies (TMAs), we enrolled a cohort of 35 adult patients combining a first acute episode of TMA, an undetectable (below 5%) ADAMTS13 activity in plasma, and no clinical background such as sepsis, cancer, HIV, and transplantation. All patients were treated by steroids and plasma exchange, and an 18-month follow-up was scheduled. Remission was obtained in 32 patients (91.4%), and 3 patients died (8.6%) after the first attack. At presentation, ADAMTS13 antigen was decreased in 32 patients (91.4%), an ADAMTS13 inhibitor was detectable in 31 patients (89%), and an anti-ADAMTS13 IgG/IgM/IgA was present in 33 patients (94%). The 3 decedent patients were characterized by the association of several anti-ADAMTS13 Ig isotypes, including very high IgA titers, while mortality was independent of the ADAMTS13 inhibitor titer. In survivors, ADAMTS13 activity in remission increased to levels above 15% in 19 patients (59%) but remained undetectable in 13 patients (41%). Six patients relapsed either once or twice (19%) during the follow-up. High levels of inhibitory anti-ADAMTS13 IgG at presentation were associated with the persistence of an undetectable ADAMTS13 activity in remission, the latter being predictive for relapses within an 18-month delay. IntroductionThrombotic microangiopathies (TMAs) are defined by the association of acute mechanical hemolytic anemia, thrombocytopenia, and visceral ischemic manifestations related to the formation of platelet thrombi in the microcirculation. 1 Clinically, TMA includes mainly the thrombotic thrombocytopenic purpura (TTP) and the hemolytic uremic syndrome (HUS) characterized by a multivisceral ischemia and a renal ischemia, respectively. 2 Although mechanisms for HUS remain very heterogeneous, pathophysiology for most forms of TTP is related to a severe deficiency of a plasma metalloprotease, ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats). [3][4][5][6] Physiologically, ADAMTS13 is the specific cleaving protease for von Willebrand factor (VWF), a large multimeric glycoprotein crucial for both platelet adhesion and aggregation in the high stress-associated hemodynamic conditions of the microcirculation. 7 A severe enzymatic deficiency of ADAMTS13 causes highly adhesive unusually large multimers of VWF to accumulate in plasma, which may spontaneously bind to platelets and thus induce the formation of platelet thrombi in the microcirculation. In rare cases, clinically relevant ADAMTS13 severe deficiency is related to compound heterozygous or homozygous mutations of the ADAMTS13 gene (Upshaw-Schulman syndrome). [8][9][10] In most cases, severe ADAMTS13 deficiency is secondary to the development of anti-ADAMTS13 autoantibodies (auto-Abs). [11][12] Anti-ADAMTS13 auto-Abs can be detected in vitro either functionally because of their inhibitory effect on ADAMTS13 enzymatic activity [13][14] or, more recently, physically as immunoglobulin G (IgG) or IgM by enzyme-lin...

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The identification of specific genetic loci that contribute to inflammatory and autoimmune diseases has proved difficult due to the contribution of multiple interacting genes, the inherent genetic heterogeneity present in human populations, and a lack of new mouse mutants. By using N-ethyl-N-nitrosourea (ENU) mutagenesis to discover new immune regulators, we identified a point mutation in the murine phospholipase Cg2 (Plcg2) gene that leads to severe spontaneous inflammation and autoimmunity. The disease is composed of an autoimmune component mediated by autoantibody immune complexes and B and T cell independent inflammation. The underlying mechanism is a gain-of-function mutation in Plcg2, which leads to hyperreactive external calcium entry in B cells and expansion of innate inflammatory cells. This mutant identifies Plcg2 as a key regulator in an autoimmune and inflammatory disease mediated by B cells and non-B, non-T haematopoietic cells and emphasizes that by distinct genetic modulation, a single point mutation can lead to a complex immunological phenotype.

Summary.  Background: ADAMTS13‐neutralizing IgG autoantibodies are the major cause of acquired thrombotic thrombocytopenic purpura (TTP). Objective: To analyze the IgG subclass distribution of anti‐ADAMTS13 antibodies and a potential relationship between subclass distribution and disease prognosis. Methodology: An enzyme‐linked immunosorbent assay‐based method was used to quantify the relative amounts of IgG subclasses of anti‐ADAMTS13 antibodies in acquired TTP plasma. Results: IgG4 (52/58, 90%) was the most prevalent IgG subclass in patients with acquired TTP, followed by IgG1 (52%), IgG2 (50%), and IgG3 (33%). IgG4 was found either alone (17/52) or with other IgG subclasses (35/52). IgG4 was not detected in 10% of the patients. There was an inverse correlation between the frequency and abundance of IgG4 and IgG1 antibodies (P < 0.01). Patients with high IgG4 levels and undetectable IgG1 are more prone to relapse than patients with low IgG4 levels and detectable IgG1. Conclusions: All IgG subclasses of anti‐ADAMTS13 antibodies were detected in patients with acquired TTP, with IgG4, followed by IgG1, antibodies dominating the anti‐ADAMTS13 immune response. Levels of IgG4 could be useful for the identification of patients at risk of disease recurrence.

Yeast surface display libraries of human IgG1 Fc regions were prepared in which loop sequences at the C-terminal tip of the CH3 domain were randomized. A high percentage of these library members bound to soluble CD64 and Protein A indicating that the randomization step did not grossly interfere with the overall structure of the displayed Fc. Sorting these libraries by FACS for binders against HER2/neu yielded antigen-specific Fc binders (Fcab; Fc antigen binding) of which one was affinity matured, resulting in Fcab clone H10-03-6 which showed >10-fold improvement in antigen-binding activity versus the parental clone. Pre-equilibrium surface plasmon resonance experiments revealed a K(D) value of 69 nM. H10-03-6 did not react with other members of the HER family and specifically interacted with HER2-positive but not with HER2-negative cells. Importantly, Fcab H10-03-6 elicited potent antibody-dependent cellular cytotoxicity in vitro. Finally, the in vivo half-life in mice was similar to wild-type Fc indicating that the amino acid changes in the CH3 domain did not affect the pharmacokinetic behavior of the recombinant Fc. Our data demonstrate that the Fcab scaffold combines all features of normal antibodies in a small 50 kD homodimeric protein: antigen binding, effector functions and long half-life in vivo.

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine and counterregulator of glucocorticoids, is a potential therapeutic target. MIF is markedly different from other cytokines because it is constitutively expressed, stored in the cytoplasm, and present in the circulation of healthy subjects. Thus, the concept of targeting MIF for therapeutic intervention is challenging because of the need to neutralize a ubiquitous protein. In this article, we report that MIF occurs in two redox-dependent conformational isoforms. We show that one of the two isoforms of MIF, that is, oxidized MIF (oxMIF), is specifically recognized by three mAbs directed against MIF. Surprisingly, oxMIF is selectively expressed in the plasma and on the cell surface of immune cells of patients with different inflammatory diseases. In patients with acute infections or chronic inflammation, oxMIF expression correlated with inflammatory flare-ups. In addition, anti-oxMIF mAbs alleviated disease severity in mouse models of acute and chronic enterocolitis and improved, in synergy with glucocorticoids, renal function in a rat model of crescentic glomerulonephritis. We conclude that oxMIF represents the disease-related isoform of MIF; oxMIF is therefore a new diagnostic marker for inflammation and a relevant target for anti-inflammatory therapy.

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