IVIg increases interleukin-11 levels, which in turn contribute to increased platelets, VWF and FVIII in mice and humans.

The mechanisms of action of intravenous immunoglobulins (IVIg) in autoimmune diseases are not fully understood. The fixed duration of efficacy and noncumulative effects of IVIg in immune thrombocytopenia (ITP) and acquired von Willebrand disease (AVWD) suggest other mechanisms besides immunological ones. Additionally to the peripheral destruction of platelets in ITP, their medullary hypoproduction emerged as a new paradigm with rescue of thrombopoietin receptor agonists (TPO-RA). In an ITP mouse model, interleukin (IL)-11 blood levels increase following IVIg. IL-11 stimulates the production of platelets and other haemostasis factors; recombinant IL-11 (rIL-11) is thus used as a growth factor in post-chemotherapy thrombocytopenia. We therefore hypothesized that IVIg induces IL-11 over-production, which increases platelets, VWF and factor VIII (FVIII) levels in humans and mice. First, in an ITP mouse model, we show that IVIg or rIL-11 induces a rapid increase (72 h) in platelets, FVIII and VWF levels, whereas anti-IL-11 antibody greatly decreased this effect. Secondly, we quantify for the first time in patients with ITP, AVWD, inflammatory myopathies or Guillain-Barré syndrome the dramatic IL-11 increase following IVIg, regardless of the disease. As observed in mice, platelets, VWF and FVIII levels increased following IVIg. The late evolution (4 weeks) of post-IVIg IL-11 levels overlapped with those of VWF and platelets. These data may explain thrombotic events following IVIg and open perspectives to monitor post-IVIg IL-11/thrombopoietin ratios, and to assess rIL-11 use with or without TPO-RA as megakaryopoiesis co-stimulating factors to overcome the relative hypoproduction of platelets or VWF in corresponding autoimmune diseases, besides immunosuppressant.

Therapeutic factor VIII does not trigger TLR1.2 and TLR2.6 signalling in vitro.

The administration of therapeutic factor VIII (FVIII) to patients with haemophilia A induces the development of inhibitory anti-FVIII IgG in a substantial number of patients. For an antigen-specific immune response to develop, antigen-presenting cells (APCs) need to mature and procure appropriate co-stimulatory signals to T cells at the time of presentation of the endocytosed antigen. The nature of the danger signals that induce APC maturation, thus initiating the anti-FVIII immune response, are yet ill-characterized. Contradictory reports on a direct effect of therapeutic FVIII on APC maturation have been released. Here, we investigated whether FVIII directly triggers Toll-like receptor 2 (TLR2) signalling. The capacity of human recombinant FVIII to promote the maturation of a mouse bone marrow macrophage cell line (BMA) was investigated by flow cytometry. In parallel, the triggering of TLR1.2 or TLR2.6-expressing HEK293 cells by FVIII was analysed following transfection of the cells with a reporter construct for NFκB activity. In contrast, to zymosan, a known TLR2 agonist, human recombinant FVIII did not induce the maturation of mouse BMA macrophages, as analysed by the levels of expression of CD80, CD86, CD40 and I-Ab at the cell surface. Furthermore, incubation of FVIII with cells expressing TLR2 paired with TLR1 or TLR6, failed to activate NFκB, whereas NKκB activity was triggered in the presence of zymosan. Our results confirm that FVIII alone is insufficient to trigger the maturation of APCs that is required to initiate an immune response.

Intravenous immunoglobulins in immunodeficiencies: more than mere replacement therapy.

Intravenous immunoglobulin (IVIG) is a therapeutic compound prepared from pools of plasma obtained from several thousand healthy blood donors. For more than 20 years, IVIG has been used in the treatment of a wide range of primary and secondary immunodeficiencies. IVIG now represents a standard therapeutic option for most antibody deficiencies. Routinely, IVIG is used in patients with X-linked agammaglobulinaemia (XLA), common variable immunodeficiency (CVID), X-linked hyper-IgM, severe combined immunodeficiency, Wiskott-Aldrich syndrome, and selective IgG class deficiency. In addition, IVIG is used extensively in the treatment of a wide variety of autoimmune disorders. IVIG is administered at distinct doses in the two clinical settings: whereas immunodeficient patients are treated with replacement levels of IVIG, patients with autoimmune and inflammatory diseases are administered with very high doses of IVIG. Several lines of experimental evidence gathered in the recent years suggest that the therapeutic beneficial effect of IVIG in immunodeficiencies reflects an active role for IVIG, rather than a mere passive transfer of antibodies.

Bortezomib delays the onset of factor VIII inhibitors in experimental hemophilia A, but fails to eliminate established anti-factor VIII IgG-producing cells.

BACKGROUND: Replacement therapy with exogenous factor VIII to treat hemorrhages induces inhibitory anti-FVIII antibodies in up to 30% of patients with hemophilia A. Current approaches to eradicate FVIII inhibitors using high-dose FVIII injection protocols (immune tolerance induction) or anti-CD20 depleting antibodies (Rituximab) demonstrate limited efficacy; they are extremely expensive and/or require stringent compliance from the patients. OBJECTIVES: To investigate whether the proteasome inhibitor bortezomib, which depletes plasmocytes, modulates the anti-FVIII immune response in FVIII-deficient mice. METHODS AND RESULTS: Preventive 4-week treatment of naïve mice with bortezomib at the time of FVIII administration delayed the development of inhibitory anti-FVIII IgG, and depleted plasma cells as well as different lymphoid cell subsets. Conversely, curative treatment of inhibitor-positive mice for 10 weeks, along with FVIII administration, failed to eradicate FVIII inhibitors to extents that would be clinically relevant if achieved in patients. Accordingly, bortezomib did not eradicate anti-FVIII IgG-secreting plasmocytes that had homed to survival niches in the bone marrow, despite significant elimination of total plasma cells. CONCLUSIONS: The data suggest that strategies for the efficient reduction of anti-FVIII IgG titers in patients with hemophilia A should rely on competition for survival niches for plasmocytes in the bone marrow rather than the mere use of proteasome inhibitors.

6th International Immunoglobulin Symposium: poster presentations.

The posters presented at the 6th International Immunoglobulin Symposium covered a wide range of fields and included both basic science and clinical research. From the abstracts accepted for poster presentation, 12 abstracts were selected for oral presentations in three parallel sessions on immunodeficiencies, autoimmunity and basic research. The immunodeficiency presentations dealt with novel, rare class-switch recombination (CSR) deficiencies, attenuation of adverse events following IVIg treatment, association of immunoglobulin (Ig)G trough levels and protection against acute infection in patients with X-linked agammaglobulinaemia (XLA) and common variable immunodeficiency (CVID), and the reduction of class-switched memory B cells in patients with specific antibody deficiency (SAD). The impact of intravenous immunoglobulin on fetal alloimmune thrombocytopenia, pregnancy and postpartum-related relapses in multiple sclerosis and refractory myositis, as well as experiences with subcutaneous immunoglobulin in patients with multi-focal motor neuropathy, were the topics presented in the autoimmunity session. The interaction of dendritic cell (DC)-SIGN and alpha2,6-sialylated IgG Fc and its impact on human DCs, the enrichment of sialylated IgG in plasma-derived IgG, as wells as prion surveillance and monitoring of anti-measles titres in immunoglobulin products, were covered in the basic science session. In summary, the presentations illustrated the breadth of immunoglobulin therapy usage and highlighted the progress that is being made in diverse areas of basic and clinical research, extending our understanding of the mechanisms of immunoglobulin action and contributing to improved patient care.

Splenic marginal zone antigen-presenting cells are critical for the primary allo-immune response to therapeutic factor VIII in hemophilia A.

BACKGROUND: Alloimmune responses to intravenously administered protein therapeutics are the most common cause of failure of replacement therapy in patients with defective levels of endogenous proteins. Such a situation is encountered in some patients with hemophilia A, who develop inhibitory anti-factor (F)VIII alloantibodies after administration of FVIII to treat hemorrhages. OBJECTIVES: The nature of the secondary lymphoid organs involved in the initiation of immune responses to human therapeutic has not been studied. We therefore investigated this in the case of FVIII, a self-derived exogenous protein therapeutic. METHODS: The distribution of intravenously administered FVIII was followed after FVIII-deficient mice were injected with radiolabeled FVIII and using immunohistochemistry. The role of the spleen and antigen-presenting cells (APC) in the onset of the anti-FVIII immune response was analyzed upon splenectomy or treatment of the mice with APC-depleting compounds. RESULTS: FVIII preferentially accumulated in the spleen at the level of metallophilic macrophages in the marginal zone (MZ). Surgical removal of the spleen or selective in vivo depletion of macrophages and CD11c-positive CD8 alpha-negative dendritic cells resulted in a drastic reduction in anti-FVIII immune responses. CONCLUSIONS: Using FVIII-deficient mice as a model for patients with hemophilia A, and human pro-coagulant FVIII as a model for immunogenic self-derived protein therapeutics, our results highlight the importance of the spleen and MZ APCs in the initiation of immune responses to protein therapeutics. Identification of the receptors implicated in retention of protein therapeutics in the MZ may pave the way towards novel strategies aimed at reducing their immunogenicity.

Mechanisms of action of intravenous immunoglobulin in autoimmune and inflammatory diseases.

Intravenous immunoglobulins (IVIg) exert a broad range of immunoregulatory functions that provide a basis for the beneficial effects of IVIg in autoimmune and systemic inflammatory disorders. This review focuses on the effects f IVIg on humoral and cellular immunity that may be of relevance for the treatment of inflammatory neurological diseases.

Natural autoantibodies as tools to predict the outcome of immune response?

Natural autoantibodies (NAbs), produced by B-1 B-cells, are directed against autoantigens and pathogens. NAbs can capture and present antigen to T helper cells thereby initiating adaptive immunities. It is proposed that screening for NAbs against pathogens will predict the strength of an antigen-induced immune response and could be used as a tool for vaccine development.

Mechanisms of action of intravenous immunoglobulin in autoimmune and inflammatory diseases.

Therapeutic polyclonal intravenous immunoglobulin (IVIg) consists of normal IgG obtained from the pools of plasma of several thousand healthy blood donors. IVIg is used as substitutive treatment of primary and secondary immunodeficiences. Since the first study of Paul Imbach who demonstrated the beneficial effect in idiopathic thrombocytopenic purpura, IVIg is also used in a number of autoimmune and inflammatory diseases. The immunoregulatory effects of IVIg in autoimmune diseases depend on the interaction of Fc portion of immunoglobulins with Fc receptors and on the selection of lymphocyte repertoires of patients through variable regions of infused immunoglobulins. IVIg modulates the activation and effector functions of B and T lymphocytes, neutralizes pathogenic autoantibodies, interferes with antigen presentation and has a strong anti-inflammatory effect which depends on its interaction with the complement system, cytokines and endothelial cells. The immunomodulatory potential of IVIg in patients is thus a result of a variety of complex mechanisms that act in a synergy.

Immune responses of goats against foot-and-mouth disease quadrivalent vaccine: comparison of double oil emulsion and aluminium hydroxide gel vaccines in eliciting immunity.

The epidemiological role of small ruminants in foot-and-mouth disease (FMD) outbreaks has been generally neglected. Although, the disease in these species is sub-clinical in nature, their role as virus carriers represents a reservoir for further infection and spread of disease. Data on the usefulness of polyvalent FMD vaccine (FMDV) in goats is scant. Thus, the present study was undertaken to evaluate the benefits of a highly potent polyvalent FMDV in goats. In the present investigations, FMDV quadrivalent double oil emulsion (Montanide ISA 206) vaccines were tested in goats at reduced doses of 2 ml per animal (antigen payload 3.5 microg per serotype per dose). The oil adjuvant elicited superior immune response at any given period than aluminium hydroxide gel (AGS) vaccine and the rapidity of development of response was quicker. The duration of immunity also appeared to be maintained for long period. The differences in immune response between two adjuvant groups were statistically significant (P<0.05). The differences were apparent even in kinetics of immune response. Unlike cattle, goats were found to be late responders for oil-adjuvanted vaccine. Our results indicate possible universal usage of double oil emulsion vaccines for disease control programs irrespective of species of animals.

Early antibody responses of cattle for foot-and-mouth disease quadrivalent double oil emulsion vaccine.

Foot-and-mouth disease (FMD) is an economically important disease of cloven-hoofed animals. The multiplicity of FMDV serotypes in animals poses a central problem in the policy of vaccination and is of much concern to health authorities. Hence it is the practice of vaccination with polyvalent vaccine for prophylactic measure. In the present report, we analysed the early antibody responses elicited by FMDV quadrivalent (FMDV O, A, C and Asia 1 serotypes) double emulsion (Montanide ISA 206) vaccines in cattle. We observed variations between various viral serotypes in eliciting early antibody response although neutralizing antibody response against all the four serotypes were detected as early as fourth day following vaccination. The duration of immunity also appeared to maintain for long period. The neutralizing antibody titres were maintained well above 2log(10) even after 6 months of vaccination irrespective of serotypes. Thus, allows the possibilities of two vaccinations per year for the maintenance of herd immunity.

Pathophysiology of inhibitors to factor VIII in patients with haemophilia A.

The occurrence of factor VIII (FVIII) inhibitors is one of the major complications of the treatment of haemophilia A. We present this review as a description of the major players of the antiFVIII immune response, with particular emphasis on the nature and properties of the different antiFVIII antibodies, their mechanisms of action in inhibiting FVIII activity, their potential neutralization by anti-idiotypic antibodies, and the importance of the T cell in participating in the induction of FVIII inhibitors. We briefly conclude on the avenues that remain to be explored in order to establish efficient therapeutic approaches aimed at eliminating FVIII inhibitors in patients with haemophilia A.

The concept of idiotypic vaccination against factor VIII inhibitors in haemophilia A.

Idiotypic vaccination has proven successful in several animal models and human trials. Here we suggest that the expression of cross-reactive idiotypes on factor VIII (FVIII) inhibitors of patients with haemophilia A, patients with anti-FVIII autoimmune disease and natural anti-FVIII antibodies of healthy individuals, together with the ability of anti-idiotypic reagents to neutralize anti-FVIII antibodies, provides a rationale for designing a vaccine strategy aimed at preventing the occurrence of or suppressing inhibitors, based on the induction of protective anti-idiotypes. Here we discuss the rationale supporting the concept of using idiotypic vaccination to prevent the occurrence of FVIII inhibitors in patients with haemophilia A.

Protective immune response to 16 kDa immunoreactive recombinant protein encoding the C-terminal VP1 portion of Foot and Mouth Disease Virus type Asia 1.

Recombinant protein of Foot and Mouth Disease Virus (FMDV) type Asia 1 corresponding to the C-terminal half of VP1 was expressed in Escherichia coli. As an alternative to the synthetic peptide, this selected C-terminal region was used as a protein vaccine in guinea pigs in order to study the immune response with various adjuvant formulations: immune stimulatory complexes (ISCOMs), Montanide ISA 206, Freund's incomplete adjuvant (FIA), lipopolysaccharide (LPS) and cytokine mixture. A primary dose of 40 microg/animal followed by a booster of the same dose was injected after a 21-day interval. The sera were collected at intervals of 21, 42 and 63 days after the booster. The humoral response to vaccine was monitored by sandwich enzyme-linked immunosorbent assay (ELISA) and a serum neutralization test (SNT). The guinea pig sera showed high titers both in ELISA and SNT, which could be protective. Further, irrespective of the adjuvant preparation used, the vaccine conferred protection against the challenge virus 105 days post-vaccination in 13 of 15 animals (86%). The results indicated that a combination of recombinant protein ISCOMs and Montanide ISA 206 would be a better choice for achieving early protective titers and longer lasting immunity and that the C-terminal half of the VP1 protein may be tried as a safe vaccine for secondary immunization.

Immuno affinity purification of foot and mouth disease virus type specific antibodies using recombinant protein adsorbed to polystyrene wells.

The specificity of foot and mouth disease virus (FMDV) serological tests depends largely on the quality and purity of the antibodies used. Such type specific antibodies can be generated by hybridoma technology. Alternatively, the specific antibodies can be selected from polyclonal serum by immunoaffinity chromatography using recombinant protein/peptide bound affinity matrices. Based on this approach, we purified selectively antibodies against the major epitopes of VP 1 of FMDV serotype Asia 1 using recombinant protein adsorbed to polystyrene wells. Optimum buffer conditions were standardised for efficient elution. Buffer consisting of 4 M MgCl2 with 75 mM HEPES pH 6.5 was found to be optimum with respect to elution efficiency of bound antibodies and integrity of antigen. The specific reactivity of eluted antibodies was confirmed by dot-enzyme linked immunosorbent assay (dot-ELISA) and antigen capture reverse transcription polymerase chain reaction (Ag/RT-PCR). The effect of temperature and repeated elution on the stability of coated protein were studied.