Anti-FcγRIIB (CD32) Antibodies Differentially Modulate Murine FVIII-Specific Recall Response in vitro.

Fc gamma receptors (FcγRs) for IgG regulate adaptive immune responses by modulating activating and inhibitory signalling pathways within immune cells. Data from a haemophilia A mouse model demonstrate that genetic deletion or blockade of the inhibitory FcγR (CD32) suppresses the formation of antibody-secreting cells (ASCs) in vitro. Mechanisms preventing the FVIII-specific recall response, however, remain unclear. Here, the potential role of CD32 inhibition was studied by differentially modulating receptor activity with selected anti-CD32 monoclonal antibodies (mAbs). Splenocytes from immunized FVIII-/- mice were restimulated with FVIII in the absence or presence of different anti-CD32 mAbs over 6 days. At day 6, cytokine release was quantified from cell culture supernatant and the formation of FVIII-specific ASCs assessed. Binding of FVIII-containing immune complexes (F8-ICs) to bone marrow-derived dendritic cells (BMdDCs) was also investigated. The antagonistic CD32 mAb AT128 suppressed the formation of FVIII-specific ASCs and reduced secretion of IFN-γ and IL-10. In contrast, the agonistic mAbs AT130-2 and AT130-5, and their F(ab')2 fragments, allowed the formation of FVIII-specific ASCs, even though the full IgG of AT130-2 reduced binding of F8-ICs to CD32. Data suggest that an inhibitory signal is transmitted when F8-ICs bind to CD32 and that this signal is required during memory B cell (MBC) activation to support formation of FVIII-specific ASCs. If the inhibitory signal is lacking due to CD32 deletion or blockade with antagonistic anti-CD32 mAbs, FVIII-specific T cell stimulation and ASC formation are suppressed, whereas agonistic stimulation of CD32 restores T cell stimulation and ASC formation.

siRNA mediated knockdown of tissue factor expression in pigs for xenotransplantation.

Acute vascular rejection (AVR), in particular microvascular thrombosis, is an important barrier to successful pig-to-primate xenotransplantation. Here, we report the generation of pigs with decreased tissue factor (TF) levels induced by small interfering (si)RNA-mediated gene silencing. Porcine fibroblasts were transfected with TF-targeting small hairpin (sh)RNA and used for somatic cell nuclear transfer. Offspring were analyzed for siRNA, TF mRNA and TF protein level. Functionality of TF downregulation was investigated by a whole blood clotting test and a flow chamber assay. TF siRNA was expressed in all twelve liveborn piglets. TF mRNA expression was reduced by 94.1 ± 4.7% in TF knockdown (TFkd) fibroblasts compared to wild-type (WT). TF protein expression in PAEC stimulated with 50 ng/mL TNF-α was significantly lower in TFkd pigs (mean fluorescence intensity TFkd: 7136 ± 136 vs. WT: 13 038 ± 1672). TF downregulation significantly increased clotting time (TFkd: 73.3 ± 8.8 min, WT: 45.8 ± 7.7 min, p < 0.0001) and significantly decreased thrombus formation compared to WT (mean thrombus coverage per viewing field in %; WT: 23.5 ± 13.0, TFkd: 2.6 ± 3.7, p < 0.0001). Our data show that a functional knockdown of TF is compatible with normal development and survival of pigs. TF knockdown could be a valuable component in the generation of multi-transgenic pigs for xenotransplantation.

Increased amounts of von Willebrand factor are bound to microparticles after infusion of desmopressin.

Effects of desmopressin (DDAVP) in platelet disorders and primary haemostasis cannot be attributed solely to the increase in FVIII/VWF (von Willebrand factor), as VWF/FVIII concentrates have no effect in these circumstances. Microparticles (MP) can support haemostasis by expression of phospholipids, tissue factor and VWF on their surface. We hypothesized that significant amounts of VWF are bound to MP after DDAVP administration and that consequently depletion of MP should influence VWF:Ag and VWF:RCo plasma levels. Platelet-poor plasma was either obtained well from healthy controls or before and after DDAVP administration from patients with von Willebrand's disease (type 1 or possible type 1) or patients with other bleeding disorders as controls. Concentrations of MP and VWF parameters were determined before and after MP depletion by different methods (magnetic bead selection, plasma microfiltration, ultracentrifugation). Platelet MP and VWF-bearing MP were significantly increased after DDAVP. MP depletion by magnetic bead selection led to a significant reduction in VWF:Ag (-18.0%) and VWF:RCo (-27.7%) plasma levels without changes in VWF multimer composition. As results were similar for DDAVP control subjects, the amount of VWF bound to circulating microparticles was significantly higher after DDAVP administration compared with healthy controls (reduction -11.7%). DDAVP leads to a release of microparticles and increases the amount of VWF bound to microparticles which might explain the clinical efficacy of DDAVP in platelet disorders.

Treatment of lupus-prone NZB/NZW F1 mice with recombinant soluble Fc gamma receptor II (CD32).

OBJECTIVES: Systemic lupus erythematosus (SLE) is a classical autoimmune disorder characterised by the production of IgG autoantibodies against double-stranded DNA (dsDNA). Activation of Fc gamma R-bearing effector cells by immune complexes (ICs) is a key event in SLE pathogenesis as lupus-prone NZB/NZW F(1) hybrids lacking activating Fc gamma receptors (Fc gamma R) are protected against inflammatory kidney damage despite glomerular deposition of ICs. Moreover, soluble Fc gamma Rs inhibit IC-caused Arthus reaction in vivo. Therefore, recombinant human soluble Fc gamma RII (CD32) was evaluated as a novel therapeutic strategy in lupus-like disease in NZB/NZW F(1) hybrids. METHODS: Binding of husCD32 to murine IgG was studied in vitro by binding to IgG-coated erythrocytes and inhibition of phagocytosis of IgG-opsonised murine erythrocytes. In order to examine therapeutic impact of husCD32 in vivo, female NZB/NZW F(1) mice were treated either from week 16 to 20 ("prophylactic", 150 microg/week husCD32) or continuously from week 24 ("therapeutic"; 100 microg/week husCD32) by subcutaneous injections. Controls received buffered saline. RESULTS: In vitro investigations of husCD32 revealed binding to murine erythrocytes coated with murine IgG. Moreover, husCD32 substantially diminished phagocytosis of murine IgG-opsonised murine red blood cells by peritoneal macrophages indicating disruption of IgG-Fc gamma R interaction. There was a therapeutic efficacy of husCD32 to attenuate lupus pathology indicated by significantly delayed onset of proteinuria and weight loss, reduced histopathological findings, delayed development of anaemia and improved survival by prophylactic application. Therapeutic treatment did not reverse nephritis but significantly prolonged survival despite apparent kidney damage. B cell count, concentration of IgG anti-dsDNA autoantibodies and deposition of glomerular ICs was not significantly affected by the application of husCD32. CONCLUSIONS: The results demonstrate binding properties of husCD32 to ICs in vitro and as a proof-of-principle therapeutic efficacy in inhibiting chronic murine lupus pathology in vivo.

CD8alpha+beta(low) effector T cells in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a complex autoimmune disorder characterized by the loss of self-tolerance to nuclear antigens. Aberrant T-cell function plays a central role in lupus pathogenesis. We and others previously demonstrated that peripheral TCRalphabeta+CD3+ T cells express CD8beta either at a high (CD8beta(high)) or low density (CD8beta(low)), thereby defining two functionally distinct subsets. CD8beta(low) T cells express predominantly CD8alphaalpha and less CD8alphabeta as a coreceptor, display a differentiated phenotype and exert effector function. CD8beta(high) T cells appear to be the precursors expressing predominantly the heterodimeric efficient CD8alphabeta coreceptor, exhibiting a naïve phenotype and high proliferative capacity. In the present study, the distribution and functional properties of CD8beta(high) and CD8beta(low) T cells of SLE patients were compared (n = 20) with those of healthy subjects (n = 16). It was found that expansion of CD8beta(low) T-cell subset correlated with disease activity indicating chronic antigenic stimulation leading to a major lack of naïve CD8beta(high) precursor T cells in SLE. Functional characteristics of CD8beta(low) T cells including production of cytokines and cytotoxic granules were not significantly different between patients with SLE and healthy individuals. We speculate that unbalanced CD8beta(high)/CD8beta(low) T-cell relation reflects a skewed homeostasis within the CD8+ T-cell compartment towards fully differentiated effector T cells possibly due to persistent antigen stimulation in SLE.

Thrombelastographic monitoring of recombinant factor VIIa in acquired haemophilia.

Monitoring of the global haemostatic capacity is desired to optimize the treatment with bypassing agents in inhibitor patients. Thrombelastographic methods have been used in ex vivo studies and were suggested useful to evaluate the individual response to bypassing agents. This study aimed at assessing changes in thrombelastographic profiles and their association to clinical outcome in patients treated with recombinant factor VIIa (rFVIIa). Ten patients with acquired haemophilia were treated with rFVIIa for acute bleeding. Thrombelastography was performed after activation with a small amount of tissue factor in samples obtained before and after in vivo administration of rFVIIa. In patients studied before and after a first dose, correction of the thrombelastographic profile was observed but did not predict cessation of bleeding. During steady-state dosing, the median Alpha angle tended to be higher in patients with a good clinical treatment response as compared with patients with a partial or poor response. Similar trends were observed for clotting time and clot formation time. A good clinical treatment response was more frequent in patients with a fully corrected trough-level thrombelastographic profile as compared with patients with an abnormal profile. However, a poor treatment response was observed also in a surgical patient with a normal thrombelastographic profile during steady-state dosing. In conclusion, thrombelastographic monitoring was sensitive to haemostatic changes in response to treatment with rFVIIa. In the limited number of patients studied here, a better clotting profile during steady-state dosing was associated with a better clinical treatment response.

Diagnostic and prognostic value of factor VIII binding antibodies in acquired hemophilia A: data from the GTH-AH 01/2010 study.

UNLABELLED: Essentials Factor VIII (FVIII) binding IgG detected by ELISA could be an alternative to the Bethesda assay. We studied the performance of anti-FVIII IgG ELISA in patients with acquired hemophilia and controls. Anti-FVIII IgG > 99th percentile of controls was highly sensitive and specific. Patients with high anti-FVIII IgG have a lower chance of achieving remission. SUMMARY: Background Acquired hemophilia A is a severe bleeding disorder that requires fast and accurate diagnosis as it occurs often unexpectedly in previously healthy men and women of every age. The Nijmegen-modified Bethesda assay is the diagnostic reference standard for detecting neutralizing autoantibodies against factor VIII (FVIII), but is not widely available, not ideal for quantifying the complex type 2 inhibitors seen in acquired hemophilia, and suffers from high inter-laboratory variability. Objectives To assess the diagnostic and prognostic value of FVIII-binding antibodies as detected by ELISA compared with the Nijmegen Bethesda assay. Methods Samples from the time of first diagnosis and clinical data were available from 102 patients with acquired hemophilia enrolled in the prospective GTH-AH 01/2010 study. Controls (n = 102) were matched for gender and age. Diagnostic cut-offs were determined by receiver-operator curve analysis. The prognostic value was assessed in 92 of the 102 patients by Cox regression analysis of time to partial remission. Results Anti-FVIII IgG above the 99th percentile (> 15 arbitrary units per mL) revealed high sensitivity and specificity (both 0.99; 95% confidence interval, 0.95-1.0) for diagnosing acquired hemophilia. The likelihood of achieving partial remission was related to anti-FVIII IgG concentration (< 300 arbitrary units, 1.0; 300-1050, 0.65; > 1050, 0.39). The Bethesda titer was only associated with the likelihood of partial remission when analyzed in the central laboratory, but not when data from local GTH study sites were used. Conclusion Although the Nijmegen-modified Bethesda assay is the reference standard for demonstrating neutralizing antibodies, the detection of FVIII-binding antibodies by ELISA is similarly sensitive and specific for diagnosing acquired hemophilia. In addition, anti-FVIII IgG may provide prognostic information.

Management of acquired haemophilia A.

Acquired haemophilia A (AHA) is caused by autoantibody inhibitors of coagulation factor VIII (FVIII : C). Recent onset of bleeds and isolated prolongation of the activated partial thromboplastin time (aPTT) are characteristic features of the disorder. Reduced FVIII : C activity and a detectable FVIII : C inhibitor in the Bethesda assay confirm the diagnosis. Patients should be referred to expert centres, whenever possible, and invasive procedures with a high risk of bleeding must be avoided, until haemostasis has been secured by adequate therapy. Bypassing agents capable of inducing sufficient thrombin formation in the presence of FVIII : C inhibitors are treatment of choice, including currently available recombinant factor VIIa (NovoSevenTM) and activated prothrombin complex concentrate (FEIBATM). These agents represent first line therapy to control acute or severe bleeds. To eradicate inhibitors, immunosuppressive treatment (IST) is indicated in patients with AHA. Glucocorticoids, cytotoxic agents and rituximab are most widely used. However, an ideal IST regimen has not been established so far. Adverse events of IST, including infections as the foremost cause death, are frequent complications in AHA.

CMV complications in common variable immunodeficiency.

A patient suffering from common variable immunodeficiency is described, who developed a myelitis under treatment with glucocorticosteroids. Later on, autoimmune complications had to be treated with azathioprine. An exacerbation of the myelitis, retinitis, encephalitis and colitis was observed and a cytomegalovirus infection diagnosed. This infection does not represent a typical complication of common variable immunodeficiency. A functional NK cell defect was detected that may contribute to susceptibility for cytomegalovirus infection in addition to immunosuppressive therapy.

CD8+ T cell populations in common variable immunodeficiency.

In a subgroup of CVID T cell abnormalities have been reported. Peripheral blood T lymphocytes from patients with common variable immunodeficiency (CVID) and blood donors were examined for expression of CD8alpha, CD8beta and CD28. In CVID, three CD8+ T cells could be defined: CD8beta(high)CD28+ (expressing CD8beta at a high median fluorescence intensity), CD8beta(low)CD28+ and CD8beta(low) CD28- cells. The number of CD8beta(low) cells was markedly increased compared to blood donors. After activation, CD8beta(high)CD28+ cells from cord blood differentiated into CD8beta(low) CD28- cells. Therefore, CD8beta(low) cells are induced by activation even in normal donors, but may reflect inflammatory activity in CVID.

Diagnostic workup of patients with acquired von Willebrand syndrome: a retrospective single-centre cohort study.

BACKGROUND: Diagnosis of acquired von Willebrand syndrome (AVWS) remains challenging. Diagnostic algorithms suggest the use of factor VIII (FVIII:C), von Willebrand factor antigen (VWF:Ag), ristocetin cofactor (VWF:RCo), and collagen-binding capacity (VWF:CB), but the sensitivity of these and other laboratory tests for the diagnosis of AVWS is unknown. OBJECTIVES: To analyze the capacity of laboratory tests, including point-of-care testing (POCT), for the identification of patients with AVWS. PATIENTS/METHODS: Thirty-five consecutive patients were enrolled with AVWS diagnosed because of a history of recent onset of bleeding, a negative family history of von Willebrand disease, and abnormal plasma VWF multimers. RESULTS: According to our inclusion criteria, all patients had bleeding symptoms, and the VWF high molecular weight multimers were either decreased or absent. Regarding POCT, PFA-100 was inconclusive, due to anemia or thrombocytopenia, in 29%; the sensitivity was 80% in the remaining patients. The sensitivity of VWF:Ag (23%), VWF:RCo/Ag ratio < 0.7 (26%), VWF:CB/Ag ratio < 0.7 (46%), anti-VWF antibodies (15%) and VWF propeptide/Ag ratio (22%) was too low to rule out the disease. A combination of VWF:Ag < 50 IU dL(-1), VWF:RCo/Ag ratio < 0.7 and VWF:CB/Ag ratio < 0.8 yielded a sensitivity of 86%. Patients diagnosed only because of abnormal VWF multimers showed similar clinical characteristics as other patients. CONCLUSIONS: Early diagnosis of AVWS is difficult, due to lack of sensitivity of the tests used. A substantial number of patients present with normal or increased test results, emphasizing the importance of multimer analysis in all patients with suspected AVWS.

CD8beta/CD28 expression defines functionally distinct populations of peripheral blood T lymphocytes.

Peripheral blood CD8+ T lymphocytes generally express the CD8 coreceptor as an alphabeta heterodimer. On these cells, the CD8beta chain is present either at high (CD8betahigh) or low density (CD8betalow). CD8betahigh cells are CD28+, whereas CD8betalow cells are CD28+ or CD28-. Therefore, three subpopulations of CD8+ T cells can be described: (i) CD8betahighCD28+ (ii) CD8betalowCD28+, and (iii) CD8betalowCD28- cells. Phenotypic and functional characterization of these CD8+ T cell subsets revealed significant differences. CD8betahighCD28+ cells predominantly express CD45RA. In contrast, CD8betalowCD28+ cells frequently express CD45R0 and the activating NK receptor CD161. CD8betalowCD28- cells frequently revert to the CD45RA phenotype. In addition, these cells express CD16, CD56, CD94, and the killer-inhibitory receptors NKB1 and CD158a. Intracellular IL-2 was frequently detected in CD8betahighCD28+ cells and CD8betalowCD28+ cells, but not CD8betalowCD28- cells. CD8betalowCD28+ cells and CD8betalowCD28- cells frequently stained positive for IFN-gamma. In addition, these cells contain intracellular perforin and granzyme A. Expression of Fas (CD95) as well as susceptibility to apoptosis is markedly increased in CD8betalowCD28+ and CD8betalowCD28- cells as compared to CD8betahighCD28+ cells. In vitro activation of peripheral blood lymphocytes triggered expansion of CD8betahighCD28+ cells as well as a development into CD8betalowCD28+ and CD8betalowCD28- cells. Similarly, activation of CD8betahighCD28+ cord blood cells resulted in the appearance of CD8betalowCD28+ and CD8betalowCD28- cells. These data suggest that CD8betahighCD28+ cells can differentiate into CD8betalowCD28+ and CD8betalowCD28- cells upon TCR stimulation. Therefore, the CD8beta/CD28 subsets in peripheral blood may reflect distinct stages of post-thymic CD8+T cell development.