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Intravenous immunoglobulin (IVIG) is a promising immune-modulatory therapy for limiting harmful inflammation and associated secondary tissue loss in neurotrauma. Here, we show that IVIG therapy attenuates spatial learning and memory deficits following a controlled cortical impact mouse model of traumatic brain injury (TBI). These improvements in cognitive outcomes were associated with increased neuronal survival, an overall reduction in brain tissue loss, and a greater preservation of neural connectivity. Furthermore, we demonstrate that the presence of the main inhibitory FcγRIIB receptor is required for the beneficial effects of IVIG treatment in TBI, with our results simultaneously highlighting the role of this receptor in reducing secondary damage arising from brain injury.

Introduction: Neutrophils are a pivotal cell type in the K/BxN mouse model of rheumatoid arthritis and play an essential role in the progression of the arthritis. They are readily activated by immune complexes (ICs) via their FcγRs to release IL-1β in addition to other cytokines, which are inducing cartilage destruction. Neutrophils also release neutrophil-active chemokines to recruit themselves in an autocrine manner to perpetuate tissue destruction. FcγR-expression on neutrophils is of crucial importance for the recognition of ICs. Methods: In this study, due to its high avidity for binding to FcγRs, we investigated the potential anti-inflammatory effect of a recombinant IgG1 Fc hexamer (rFc-µTP-L309C) on neutrophils in the K/BxN mouse model of endogenously generated chronic arthritis. 200 mg/kg rFc-µTP-L309C and human serum albumin (HSA), used as controls, were administered subcutaneously every other day. Mouse ankle joints were monitored daily to generate a clinical score. Immunohistology was used to evaluate neutrophil infiltration and TUNEL to assess apoptosis. ELISA was used to measure IL-1β. Results: Treatment with rFc-µTP-L309C, but not HSA, was able to significantly ameliorate the arthritis in the K/BxN mice. Significant neutrophil infiltration into the ankle joint was found, but treatment with rFc-µTP-L309C resulted in significantly less neutrophil infiltration. There was no significant influence of rFc-µTP-L309C on neutrophil death or apoptosis. Less neutrophil infiltration could not be correlated to chemokine-mediated migration. Significantly less IL-1β was measured in mice treated with rFc-µTP-L309C. Conclusion: In the endogenous K/BxN mouse model of rheumatoid arthritis, amelioration can be explained in part by inhibition of neutrophil infiltration into the joints as well as inhibition of IL-1β production. Given the observed inhibitory properties on neutrophils, rFc-µTP-L309C may be a potential therapeutic candidate to treat autoimmune and inflammatory conditions in which neutrophils are the predominant cell type involved in pathogenesis.

Recombinant Fc-μTP-L309C is more efficacious than intravenous immunoglobulin (IVIg) at ameliorating antibody-mediated autoimmune diseases through its effects on Fcγ receptors (FcγRs). Fc-μTP-L309C inhibited in-vitro FcγR-mediated phagocytosis 10

Recurrent and persistent airway infections remain prevalent in patients with primary immunodeficiency (PID), despite restoration of serum immunoglobulin levels by intravenous or subcutaneous plasma-derived IgG. We investigated the effectiveness of different human Ig isotype preparations to protect mice against influenza when delivered directly to the respiratory mucosa. Four polyvalent Ig preparations from pooled plasma were compared: IgG, monomeric IgA (mIgA), polymeric IgA-containing IgM (IgAM) and IgAM associated with the secretory component (SIgAM). To evaluate these preparations, a transgenic mouse expressing human FcαRI/CD89 within the myeloid lineage was created. CD89 was expressed on all myeloid cells in the lung and blood except eosinophils, reflecting human CD89 expression. Intranasal administration of IgA-containing preparations was less effective than IgG in reducing pulmonary viral titres after infection of mice with A/California/7/09 (Cal7) or the antigenically distant A/Puerto Rico/8/34 (PR8) viruses. However, IgA reduced weight loss and inflammatory mediator expression. Both IgG and IgA protected mice from a lethal dose of PR8 virus and for mIgA, this effect was partially CD89 dependent. Our data support the beneficial effect of topically applied Ig purified from pooled human plasma for controlling circulating and non-circulating influenza virus infections. This may be important for reducing morbidity in PID patients.

Intravenous immunoglobulin (IVIG) is an effective immunomodulatory treatment for immune dysregulation diseases. However, the mechanisms by which it reduces systemic inflammation are not well understood. NK cell cytotoxicity is decreased by IVIG in women with reduced fertility, but IVIG effects on NK cells in immune dysregulation are less clear. We hypothesized that IVIG modulation of lymphocyte function, especially in NK cells, is important for resolution of inflammation. Our aim was to identify IVIG-induced changes in a cohort of patients with Kawasaki disease (KD) and those that occur broadly in pediatric patients with various immune dysregulatory diseases. Peripheral blood mononuclear cells (PBMCs) of patients with KD or autoimmune/inflammatory diseases were phenotyped pre and post high dose IVIG treatment by flow cytometry. In KD patients, after IVIG infusion Treg cell frequency and the proportion of activated CD25+ immunoregulatory CD56bright NK cells was increased, and multiple lymphocyte subsets showed increased expression of the lymphoid tissue homing receptor CD62L. Importantly, IVIG treatment decreased the frequency of cells expressing the degranulation marker CD107a among cytotoxic CD56dim NK cells, which was reflected in a significant reduction in target cell killing and in decreased production of multiple pro-inflammatory mediators. Interestingly, the activating receptor CD336 was expressed on a higher proportion of CD56bright NK cells after IVIG in both KD and autoimmune/inflammatory patients while other NK receptors were increased differentially in each cohort. In autoimmune/inflammatory patients IVIG induced the proliferation marker CD71 on a higher percentage of CD56dim NK cells, and in contrast to KD patients, CD107a+ cells were increased in this subset. Furthermore, when PBMCs were stimulated ex vivo with IL-2 or Candida antigen in autologous plasma, more of the CD4+ T cells of KD patients expressed CD25 after IVIG therapy but fewer cytotoxic T cells were degranulated based on CD107a expression. In summary, IVIG treatment in patients with immune dysregulation has multiple effects, especially on NK cell subsets and CD4+ T cells, which are compatible with promoting resolution of inflammation. These novel findings provide insight into the immunomodulatory actions of IVIG in autoimmune and inflammatory conditions.

Disruption of the alveolar barrier is one of the main features of Acute Respiratory Distress Syndrome (ARDS), a devastating disease with no pharmacological treatments available at present. Sepsis is also a common risk factor for the development of ARDS, associated with 30% of the cases. During sepsis, hemolysis takes place, leading to the release of haemoglobin and heme in the bloodstream, which have been shown to contritube to cytotoxicity and inflammation. We aimed to develop an in vitro alveolar barrier disruption model that mimics some of the sepsis-induced ARDS main characteristics. For this purpose, epithelial and endothelial co-cultures on an in vitro micro-physiological system (MPS) were exposed to lipopolysaccharide (LPS) and hemin (i.e. the oxidized form of heme) and the effects on barrier disruption and inflammation were investigated. Hemin led to barrier disruption (>90% transbarrier resistance reduction) regardless of the presence of LPS or Peripheral Blood Mononuclear Cells (PBMCs). This was accompanied by an increase in the gene expression of Heme Oxygenase – 1 (> 80-fold) and endothelial damage, indicated by disruption of the cell layers. LPS treatment increased pro-inflammatory cytokine secretion (IL-8, TNF-α and IL1-β) in PBMCs. Hemin combined with LPS also induced pro-inflammatory responses but to a lower extent. In conclusion, our human alveolar barrier disruption model appears to be appropriate to study heme-induced toxicity and inflammation and thus may be a relevant tool for preclinical drug testing.

Therapeutic normal IgG intravenous immunoglobulin (IVIG) is a well-established first-line immunotherapy for many autoimmune and inflammatory diseases. Though several mechanisms have been proposed for the anti-inflammatory actions of IVIG, associated signaling pathways are not well studied. As β-catenin, the central component of the canonical Wnt pathway, plays an important role in imparting tolerogenic properties to dendritic cells (DCs) and in reducing inflammation, we explored whether IVIG induces the β-catenin pathway to exert anti-inflammatory effects. We show that IVIG in an IgG-sialylation independent manner activates β-catenin in human DCs along with upregulation of Wnt5a secretion. Mechanistically, β-catenin activation by IVIG requires intact IgG and LRP5/6 co-receptors, but FcγRIIA and Syk are not implicated. Despite induction of β-catenin, this pathway is dispensable for anti-inflammatory actions of IVIG in vitro and for mediating the protection against experimental autoimmune encephalomyelitis in vivo in mice, and reciprocal regulation of effector Th17/Th1 and regulatory T cells., Karnam et al. show that the β-catenin pathway is dispensable for the anti-inflammatory actions of intravenous immunoglobulin (IVIG) in human dendritic cells and in protecting against experimental autoimmune encephalomyelitis. This study implicates an unknown signaling pathway as a mediator of the anti-inflammatory effects of therapeutic IVIG.

Autophagy plays an important role in the regulation of autoimmune and autoinflammatory responses of the immune cells. Defective autophagy process is associated with various autoimmune and inflammatory diseases. Moreover, in many of these diseases, the therapeutic use of normal immunoglobulin G or intravenous immunoglobulin (IVIG), a pooled normal IgG preparation, is well documented. Therefore, we explored if IVIG immunotherapy exerts therapeutic benefits via induction of autophagy in the immune cells. Here we show that IVIG induces autophagy in peripheral blood mononuclear cells (PBMCs). Further dissection of this process revealed that IVIG-induced autophagy is restricted to inflammatory cells like monocytes, dendritic cells, and M1 macrophages but not in cells associated with Th2 immune response like M2 macrophages. IVIG induces autophagy by activating AMP-dependent protein kinase, beclin-1, class III phosphoinositide 3-kinase and p38 mitogen-activated protein kinase and by inhibiting mammalian target of rapamycin. Mechanistically, IVIG-induced autophagy is F(ab′)2-dependent but sialylation independent, and requires endocytosis of IgG by innate cells. Inhibition of autophagy compromised the ability of IVIG to suppress the inflammatory cytokines in innate immune cells. Moreover, IVIG therapy in inflammatory myopathies such as dermatomyositis, antisynthetase syndrome and immune-mediated necrotizing myopathy induced autophagy in PBMCs and reduced inflammatory cytokines in the circulation, thus validating the translational importance of these results. Our data provide insight on how circulating normal immunoglobulins maintain immune homeostasis and explain in part the mechanism by which IVIG therapy benefits patients with autoimmune and inflammatory diseases.

International audience; Intravenous immunoglobuin (IVIG) exerts protective effects in experimental allergic bronchopulmonary aspergillosis (ABPA) via a sialylation-dependent mechanism. The protection was associated with reduced recruitment of eosinophils, diminished goblet cell hyperplasia, suppressed Th2 and Th17 responses and reciprocally enhanced regulatory T cells and IL-10, and decreased IgE levels in the circulation.

Background High-dose intravenous immunoglobulin (IVIg), and more recently, subcutaneously-delivered Ig (SCIg), are used to treat a variety of autoimmune diseases; however, there are challenges associated with product production, availability, access and efficacy. These challenges have provided incentives to develop a human recombinant Fc as a more potent alternative to IVIg and SCIg for the treatment of autoimmune diseases. Recently, a recombinant human IgG1 Fc hexamer (Fc-μTP-L309C) was shown to be more efficacious than IVIg in a variety of autoimmune mouse models. We have now examined its efficacy compared to IVIg and SCIg in the K/BxN mouse model of endogenous, chronic rheumatoid arthritis (RA). Result Using the serum-transfer K/BxN model and the endogenous autoimmune model, amelioration of the arthritis was achieved. Effective treatment required high and frequent doses of IVIg, SCIg and Fc-μTP-L309C. However, Fc-μTP-L309C was efficacious at 10-fold lower doses that IVIg/SCIg. Also, arthritis could be prevented when Fc-μTP-L309C was given prior to onset of the arthritis in both the endogenous model and in the serum transfer model. Conclusions Our results show that Fc-μTP-L309C is a powerful treatment for the prevention and amelioration of severe, chronic arthritis in a true autoimmune mouse model of RA. Thus, the K/BxN endogenous arthritis model should be useful for testing potential therapeutics for RA. Our findings provide rationale for further examination of the treatment efficacy of immunoglobulin-based therapeutics in rheumatoid arthritis.

Background Recurrent and persistent infections are known to affect airways of patients with Primary Immunodeficiency despite appropriate replacement immunoglobulin serum levels. Interestingly, patients with Chronic Obstructive Pulmonary Disease or with non-CF bronchiectasis also show similar susceptibility to such infections. This may be due to the limited availability of immunoglobulins from the systemic circulation in the conductive airways, resulting in local immunodeficiency. Topical application of nebulized plasma-derived immunoglobulins may represent a means to address this deficiency. In this study, we assessed the feasibility of nebulizing plasma-derived immunoglobulins and delivering them into the airways of rats and non-human primates. Methods Distinct human plasma-derived immunoglobulin isotype preparations were nebulized with an investigational eFlow® nebulizer and analyzed in vitro or deposited into animals. Biochemical and immunohistological analysis of nebulized immunoglobulins were then performed. Lastly, efficacy of topically applied human plasma-derived immunoglobulins was assessed in an acute Streptococcus pneumoniae respiratory infection in mice. Results Characteristics of the resulting aerosols were comparable between preparations, even when using solutions with elevated viscosity. Neither the structural integrity nor the biological function of nebulized immunoglobulins were compromised by the nebulization process. In animal studies, immunoglobulins levels were assessed in plasma, broncho-alveolar lavages (BAL) and on lung sections of rats and non-human primates in samples collected up to 72 h following application. Nebulized immunoglobulins were detectable over 48 h in the BAL samples and up to 72 h on lung sections. Immunoglobulins recovered from BAL fluid up to 24 h after inhalation remained structurally and functionally intact. Importantly, topical application of human plasma-derived immunoglobulin G into the airways of mice offered significant protection against acute pneumococcal pneumonia. Conclusion Taken together our data demonstrate the feasibility of topically applying plasma-derived immunoglobulins into the lungs using a nebulized liquid formulation. Moreover, topically administered human plasma-derived immunoglobulins prevented acute respiratory infection. Electronic supplementary material The online version of this article (10.1186/s12931-019-1057-3) contains supplementary material, which is available to authorized users.

Treatment of autoimmune and inflammatory diseases typically involves immune suppression. In an opposite strategy, we show that administration of the highly inflammatory erythrocyte-specific antibody Ter119 into mice remodels the monocyte cellular landscape, leading to resolution of inflammatory disease. Ter119 with intact Fc function was unexpectedly therapeutic in the K/BxN serum transfer model of arthritis. Similarly, it rapidly reversed clinical disease progression in collagen antibody-induced arthritis (CAIA) and collagen-induced arthritis and completely corrected CAIA-induced increase in monocyte Fcγ receptor II/III expression. Ter119 dose-dependently induced plasma chemokines CCL2, CCL5, CXCL9, CXCL10, and CCL11 with corresponding alterations in monocyte percentages in the blood and liver within 24 hours. Ter119 attenuated chemokine production from the synovial fluid and prevented the accumulation of inflammatory cells and complement components in the synovium. Ter119 could also accelerate the resolution of hypothermia and pulmonary edema in an acute lung injury model. We conclude that this inflammatory anti-erythrocyte antibody simultaneously triggers a highly efficient anti-inflammatory effect with broad therapeutic potential.

Intravenous IgG (IVIG) therapy can be used for immunomodulation. IL-2 is an immunoregulatory cytokine. We evaluated IVIG modulation of human blood lymphocyte response to IL-2 and other cytokines. Neither IVIG nor low concentrations of IL-2 (3-30U/ml) induced lymphocyte proliferation, but in combination they synergistically enhanced proliferation of NK cells. The CD56(bright) cells expanded more than CD56(dim) NK cells, with 90% of NK cells dividing up to 8 generations by day 6, while

Activation of Fc receptors and complement by immune complexes is a common important pathogenic trigger in many autoimmune diseases and so blockade of these innate immune pathways may be an attractive target for treatment of immune complex-mediated pathomechanisms. High-dose IVIG is used to treat autoimmune and inflammatory diseases, and several studies demonstrate that the therapeutic effects of IVIG can be recapitulated with the Fc portion. Further, recent data indicate that recombinant multimerized Fc molecules exhibit potent anti-inflammatory properties. In this study, we investigated the biochemical and biological properties of an rFc hexamer (termed Fc-μTP-L309C) generated by fusion of the IgM μ-tailpiece to the C terminus of human IgG1 Fc. Fc-μTP-L309C bound FcγRs with high avidity and inhibited FcγR-mediated effector functions (Ab-dependent cell-mediated cytotoxicity, phagocytosis, respiratory burst) in vitro. In addition, Fc-μTP-L309C prevented full activation of the classical complement pathway by blocking C2 cleavage, avoiding generation of inflammatory downstream products (C5a or sC5b-9). In vivo, Fc-μTP-L309C suppressed inflammatory arthritis in mice when given therapeutically at approximately a 10-fold lower dose than IVIG, which was associated with reduced inflammatory cytokine production and complement activation. Likewise, administration of Fc-μTP-L309C restored platelet counts in a mouse model of immune thrombocytopenia. Our data demonstrate a potent anti-inflammatory effect of Fc-μTP-L309C in vitro and in vivo, likely mediated by blockade of FcγRs and its unique inhibition of complement activation.

Intravenous immunoglobulin (IVIG) are purified IgG preparations made from the pooled plasma from thousands of healthy donors and are being tested in preclinical mouse models. Inherent challenges, however, are the pluripotency of IVIG and its xenogeneicity in animals. IVIG can alter the viability of human neutrophils via agonistic antibodies to Fas and Siglec-9. In this study, we compared the effects of IVIG on human and mouse neutrophils using different death assays. Different commercial IVIG preparations similarly induced cytokine-dependent death in human neutrophils, whereas they had no effects on the survival of either peripheral blood or bone marrow neutrophils from C57BL/6 or BALB/c mice. F(ab’)2 but not Fc fragments of IVIG induced death of human neutrophils, whereas neither of these IVIG fragments, nor agonistic monoclonal antibodies to human Fas or Siglec-9 affected the viability of mouse neutrophils. Pooled mouse IgG, which exhibited a different immunoprofile compared to IVIG, also had no effect on mouse cells. Together, these observations demonstrate that effects of IVIG on neutrophil survival are not adequately reflected in current mouse models, despite the key role of these cells in human inflammatory and autoimmune diseases.

Several mechanisms account for the beneficial effect of intravenous immunoglobulin (IVIg) in autoimmune and inflammatory diseases. These mechanisms include effects on the cellular compartment and on the humoral compartment. Thus, IVIg impacts on dendritic cells, macrophages, neutrophils, basophils, NK cells, and B and T lymphocytes. Several studies have emphasized that the antiinflammatory effect of IVIg is dependent on α2,6-sialylation of the N-linked glycan on asparagine-297 of the Fc portion of IgG. However, recent reports have questioned the necessity of sialylated Fc and the role of FcγRIIB in IVIg-mediated antiinflammatory effects. In view of the critical role played by Th17 cells in several autoimmune pathologies and the increasing use of IVIg in several of these conditions, by using neuraminidase-treated, desialylated IVIg, we addressed whether the α2,6-sialylation of IgG is essential for the beneficial effect of IVIg in experimental autoimmune encephalomyelitis (EAE), a Th17-driven condition, and for the reciprocal modulation of helper T-cell subsets. We observed no difference in the ability of IVIg to ameliorate EAE irrespective of its sialylation. Our findings thus show that sialylation of IVIg is not necessary for IVIg-mediated amelioration of EAE or for downregulation of Th17 cells and upregulation of regulatory T cells.

In recent years, there has been a surge in the research and development of novel molecules as potential therapeutic alternatives to traditional treatments (such as intravenous immunoglobulins) for autoimmune disorders. The aim of this review is to describe different drug development strategies and evaluate how various molecules have performed in clinical trials to date. Broadly, three main approaches have been pursued. Recombinant fragment crystallisable (rFc) multimers primarily target Fcγ receptors (FcγRs) but may also affect the complement system. These include PF-06755347 (GL-2045), CSL730 (M230), CSL777 and Pan Fc Receptor Interacting Molecule (PRIM). Neonatal Fc receptor (FcRn)-targeting therapeutics block the FcRn receptor and are represented by candidate drugs such as the Fc fragment efgartigimod and the monoclonal antibodies rozanolixizumab (UCB7665), M281 and SYNT001. Finally, Fc and FcγR-targeting therapeutics, comprise molecules that target the Fc of IgG, such as the recombinant soluble FcγIIb receptor valziflocept (SM101/SHP652) and various monoclonal antibodies directed against the receptors. The developmental status of these three classes of molecules ranges from preclinical to ongoing phase 3 clinical studies. Efgartigimod and rozanolixizumab are the most advanced and have demonstrated encouraging results from phase 2 trials in immune thrombocytopenia and myasthenia gravis. Although initial results are promising, further long-term data and a better understanding of the unique mechanisms of action of the different molecules are needed. The efficacy, safety, convenience of administration, duration of effects, and cost will all contribute to determining which of the molecules will be successful in the clinic.

Intravenous immunoglobulin (IVIg) is a widely used treatment for immunodeficiencies and inflammatory autoimmune diseases. Multiple mechanisms of action for IVIg have been proposed, with some involving the Fab region and others related to the Fc region of the immunoglobulin (Ig)G molecule. Changing the glycan at the glycosylation site in the CH2 pocket of the Fc region (for example, via fucosylation), is known to modify the Fc effector function. In a series of publications between 2006 and 2008, it was proposed by Jeffrey Ravetch's group that the addition of a terminal sialic acid to this region was responsible for the anti-inflammatory properties of IVIg 1–3. Based on data generated in the K/BxN arthritis model, the following mechanism of action of IVIg was proposed: the sialylated Fc region first binds to splenic dendritic-cell-specific intercellular adhesion molecule 3 (ICAM-3) grabbing non-integrin (DC-SIGN in humans), which results in production of interleukin (IL)-33, leading to expansion of IL-4-producing basophils and up-regulation the expression of FcγRIIB receptors, thereby taking the inflammatory system into a non-inflammatory state 4. To test this hypothesis, we carried out studies in animal models of idiopathic thrombocytopenic purpura/immune thrombocytopenia (ITP) 5; multiple sclerosis (MS), using the experimental autoimmune encephalomyelitis (EAE) model 6; and rheumatoid arthritis (RA) 7, using the collagen antibody-induced arthritis (CAbIA) and K/BxN models. We assessed the effects of IgG sialylation in these animal models by comparing sialic acid-enriched and -depleted IgG fractions with the untreated product. To achieve the highly sialylated IgG fraction, we used affinity chromatography with sialic acid-specific Sambucus nigra agglutinin (SNA) lectin. Desialylation was achieved using neuraminidase (NAse) to produce the ‘negative’ material. There are two important factors to consider when using the SNA lectin fractionation method 8. First, IVIg fractionated over an SNA column shows increased sialylation mainly in the Fab region, as measured by glycoanalysis with high-performance liquid chromatography (HPLC), or liquid chromatography–mass spectrometry (LC-MS). The findings from both assays indicated that the SNA-binding fraction represents approximately 10% of the IgG molecules in IVIg. However, Fc-sialylation as assessed by LC-MS was increased in one subfraction, representing only 1–2% of the IgG molecule and only by a very small amount 8. Secondly, we have demonstrated that lectin chromatography can result in a skewed antibody pattern of the SNA-binding fraction 8. Antigen specificity of IgG fractions was tested by enzyme-linked immunosorbent assay (ELISA) with a range of pathogen-derived and autoantigens, and demonstrated abnormal antibody concentration patterns in the eluted material compared to the starting material. Our recent study in the antibody-induced K/BxN and CAbIA RA models in mice 7 set out to establish the role of sialylation in antibody-driven inflammation. The therapeutic effect of IVIg and its proposed mechanisms of action were studied using both prophylactic and therapeutic protocols. Prophylactic IVIg protocols have been shown in previous studies to protect against arthritis in the K/BxN model 1,3, and this study demonstrated the efficacy of prophylaxis in the CAbIA model; disease severity was reduced significantly (P

On March 15–17th, 2012 the 2nd international workshop on natural antibodies sponsored by CSL Behring AG took place in Bern, Switzerland. The focus of this workshop centered on new directions and in particular explored the “Future of Immunoglobulin Therapy”. Our international speakers addressed the major themes of understanding the origins of the immunoglobulin (Ig) repertoire and how we use it in our everyday lives to maintain homeostasis and fight infections and diseases. This Ig repertoire is already used as a therapeutic tool in the form of intravenous immunoglobulin (IVIG) for patients suffering from immunodeficiency and autoimmune disorders. The question now arises: what else can we learn and apply for the future of Ig therapy? Are there new directions, new ways of using the repertoire? These questions were discussed in sections covering modified Igs with modified efficacy including catalytic antibodies, the role of Fc receptors and complement inducing autoimmune mediated damage, Ig therapy beyond IgG in particular taking a look at the role of IgA and the therapeutic consequences of the different etiologies of excess amyloid beta in Alzheimer's disease and patients with Downs' syndrome. A major topic addressed the application of biomarkers to define new autoimmune diseases, to refine current therapies with a view to optimising the management of chronic diseases using both genetic and biomarker profiles to forewarn of possible future complications and to monitor fluctuations in disease status. Indeed, some of these topics push the frontiers of autoimmunity beyond its traditional scope and serve the purpose of making us re-evaluate the potential of using the Ig repertoire.

Polyclonal plasma-derived IgG is a mainstay therapeutic of immunodeficiency disorders as well as of various inflammatory autoimmune diseases. In immunodeficiency the primary function of IVIG/SCIG is to replace missing antibody specificities, consequently a diverse Fab-based repertoire is critical for efficacy. Attempts to capture the Ig repertoire and express it as a recombinant IVIG product are currently ongoing. Likewise correction of the defective genes by gene therapy has also been tried. However, both approaches are far from becoming mainstream treatments. In contrast, some of the most important effector mechanisms relevant in therapy of autoimmunity are based on the Fc-portion of IgG; they include scavenging of complement and blockade/modulation of IgG receptors (Fc gamma receptor [FcγR] or the neonatal Fc receptor [FcRn]). These effects might be achieved with appropriately formulated Fc-fragments instead of full-length IgG, as suggested by a pilot study with monomeric plasma-derived Fc in children with ITP and in Kawasaki disease in the 1990s. Since then it has been proposed that structured multimerization of Fc fragments might confer efficacy at much lower doses than with IVIG. Accordingly, various molecular strategies are currently being explored to achieve controlled Fc multimerization, e.g. by fusion of IgG1 Fc to the IgG2 hinge-region or to the IgM tail-piece. Safety considerations will be crucial in the evaluation of these new entities. In a different approach, mutant Fc fragments and monoclonal antibodies have been designed for blockade of the FcRn.

BACKGROUND: Sialylation of the N-linked glycan on asparagine-297 within the Fc region of intravenous gammaglobulins (IVIgs) was shown to be necessary for efficacy of IVIg in the amelioration of experimental inflammatory arthritis. To test the role for Fc sialylation of IVIg in immune modulating therapies beyond the K/BxN arthritis model, we examined the efficacy of sialylated compared to nonsialylated IVIg for the ability to attenuate immune thrombocytopenia (ITP) in a mouse model that approximates the clinical setting of human ITP. STUDY DESIGN AND METHODS: We used a published, passive anti-platelet (PLT) dose-escalation mouse model of ITP that approximates clinical ITP. PLT counts were followed over time before and after IVIg treatment. IVIg from two different manufacturers was used to prepare untreated and neuraminidase-treated IVIg. Sambucus nigra agglutinin (SNA) affinity chromatography was used to obtain sialic acid–enriched and -depleted IVIg. Sialic acid content was determined using Western blot, enzyme-linked immunosorbent assay, and high-performance liquid chromatography. RESULTS: Results were the same using sialylated and desialylated (neuraminidase-treated) IVIg from two different manufacturers. No differences were observed between sialic acid–enriched and -depleted IVIg compared to normal IVIg in their efficacy to alleviate ITP. Using quantitative reverse transcription–polymerase chain reaction, no increase in the spleen FcγRIIB mRNA was detectable, but a pronounced increase of FcγRIIB mRNA in the marrow was seen after IVIg administration. CONCLUSIONS: We conclude that IVIg ameliorates experimental ITP by a mechanism that is independent of sialylation either in the Fc or the Fab region of IVIg.

A highly purified, liquid, 10% immunoglobulin product stabilized with proline, referred to as IgPro10 has recently been developed. IgG was purified from human plasma by cold ethanol fractionation, octanoic acid precipitation and anion-exchange chromatography. The manufacturing process includes two distinctly different partitioning steps and virus filtration, which were also assessed for the removal of prions. Prion removal studies used different spike preparations (brain homogenate, microsomes, purified PrP(sc)) and three different detection methods (bioassay, Western blot, conformation-dependent immunoassay). All of the investigated production steps were shown to reduce significantly all different spike preparations, resulting in an overall reduction of >10log(10). Moreover, the biochemical assays proved equally effective to the bioassay for the demonstration of prion elimination. Four of the manufacturing steps cover three different mechanisms of virus clearance. These are: i) virus inactivation; ii) virus filtration; and iii) partitioning. These mechanisms were assessed for their virus reduction capacity. Virus validation studies demonstrated overall reduction factors of >18log(10) for enveloped and >7log(10) for non-enveloped model viruses. In conclusion, the IgPro10 manufacturing process has a very high reduction potential for prions and for a wide variety of viruses resulting in a state-of-the-art product concerning safety towards known and emerging pathogens.

Enveloped viruses penetrate the host cells by fusion of the viral envelope with a cellular target membrane. One of the best studied viruses with respect to its penetration and uncoating is the alphavirus Semliki Forest virus that is taken up by endocytosis. The alphavirus membrane glycoprotein E1 harbors a so-called fusion peptide, which is responsible for interaction with the endosomal membrane, leading to fusion. Besides this fusion process, cell infection by alphaviruses is accompanied by membrane permeability changes, thus implying some form of pore across the membrane. However, the ability of E1 protein to form ion pores has not been widely accepted. This review provides an overview of studies that confirm earlier results predicting the formation of a proteinaceous pore by the alphavirus spike proteins. Furthermore, different models to explain this pore formation during virus entry are discussed. Viruses are genetic elements that are able to reproduce in a host cell-dependent way. The virus genome, RNA or DNA, is packaged into a protective protein coat; in enveloped viruses this nucleocapsid is surrounded by a lipid bilayer, derived from the host cell membrane during virus budding, which contains viral spike glycoproteins. This packaging protects the virus during the extracellular journey and presents recognition signals for adsorption to the cellular membrane. It provides the means for viral structural conversions upon interaction with the host cell. This leads to successful penetration of the cellular membrane and to the uncovering and release of the nucleic acid for replication (uncoating of the nucleocapsid).

Sodium hydroxide (NaOH) solutions are widely used for the purification of contaminated equipment, as they are known to inactivate a variety of pathogens. However, information about their effect on agents causing transmissible spongiform encephalopathy (TSE) is sparse and contradictory. Scrapie hamster brain homogenate, containing the disease-associated form of the prion protein (PrP(Sc)), was exposed to NaOH. Kinetics studies showed that treatment of brain homogenate with millimolar concentrations of NaOH rapidly abolished the proteinase K-resistant form of the prion protein (PrP(res)). NaOH treatment converted PrP(Sc) into a protease-sensitive form, either in solution or when adsorbed to a metallic surface. If infectivity of TSEs is linked with PrP(res), the results imply that inactivation of TSE occurs more efficiently than currently assumed.

IVIG modulates T cell activation in vitro and inflammatory-autoimmune conditions in vivo. Sialylation of IgG, Fc receptor interactions, modulation of monocyte/macrophage/B cell functions have been implicated in IVIG effects. Subcutaneous IgG (SCIG) therapy is increasingly used for IgG replacement but whether these preparations share the effects of IVIG on T cell modulation is not documented. We compared the potency of SCIG-Hizentra™ (20% IgG preparation) with IVIG-Privigen® (10% IgG) for T cell inhibition, and assessed the involvement of IgG sialylation, monocytes and B cells in this process. Human PBMCs or sorted cells were cultured 3-7 days, and T cells were stimulated with immobilized anti-CD3 mAb or Candida antigen. Thymidine incorporation into DNA was quantitated and cytokines assayed by ELISA/Luminex® assay. IVIG and SCIG both dose-dependently (1-20mg/ml) inhibited (up to >80%) T cell proliferation to anti-CD3 mAb. Response to Candida albicans was comparably inhibited by IVIG and SCIG by 50-80% at 10mg/ml with inhibition even at 3mg/ml (P

Schwab and Nimmerjahn discuss current insights into the immunomodulatory mechanisms of IVIG preparations. The authors promote a recently challenged concept that the sialylation of IgG Fc fragments is likely to be responsible for the therapeutic activity of IVIG, however, they fall short in their discussion of the experimental evidence that does not support this concept. Schwab and Nimmerjahn discuss a hypothetical sialic acid-dependent pathway that involves the binding of Fc-sialylated IgG to mouse SIGNR1 and its human orthologue DC-SIGN (DC-specific ICAM3-grabbing non-integrin) that ultimately leads to the upregulation of the inhibitory Fc receptor (FcR) FcγRIIB on macrophages and dendritic cells (DCs) 1

High-dose i.v. Ig (IVIG) is used to treat various autoimmune and inflammatory diseases; however, the mechanism of action remains unclear. Based on the K/BxN serum transfer arthritis model in mice, IVIG suppression of inflammation has been attributed to a mechanism involving basophils and the binding of highly sialylated IgG Fc to DC-SIGN–expressing myeloid cells. The requirement for sialylation was examined in the collagen Ab-induced arthritis (CAbIA) and K/BxN serum transfer arthritis models in mice. High-dose IVIG (1–2 g/kg body weight) suppressed inflammatory arthritis when given prophylactically. The same doses were also effective in the CAbIA model when given subsequent to disease induction. In this therapeutic CAbIA model, the anti-inflammatory effect of IVIG was dependent on IgG Fc but not F(ab′)2 fragments. Removal of sialic acid residues by neuraminidase had no impact on the anti-inflammatory activity of IVIG or Fc fragments. Treatment of mice with basophil-depleting mAbs did not abrogate the suppression of either CAbIA or K/BxN arthritis by IVIG. Our data confirm the therapeutic benefit of IVIG and IgG Fc in Ab-induced arthritis but fail to support the significance of sialylation and basophil involvement in the mechanism of action of IVIG therapy.

Several mechanisms account for the beneficial effect of intravenous immunoglobulin (IVIg) in autoimmune and inflammatory diseases. These mechanisms include effects on the cellular compartment and on the humoral compartment. Thus, IVIg impacts on dendritic cells, macrophages, neutrophils, basophils, NK cells, and B and T lymphocytes. Several studies have emphasized that the antiinflammatory effect of IVIg is dependent on α2,6-sialylation of the N-linked glycan on asparagine-297 of the Fc portion of IgG. However, recent reports have questioned the necessity of sialylated Fc and the role of FcγRIIB in IVIg-mediated antiinflammatory effects. In view of the critical role played by Th17 cells in several autoimmune pathologies and the increasing use of IVIg in several of these conditions, by using neuraminidase-treated, desialylated IVIg, we addressed whether the α2,6-sialylation of IgG is essential for the beneficial effect of IVIg in experimental autoimmune encephalomyelitis (EAE), a Th17-driven condition, and for the reciprocal modulation of helper T-cell subsets. We observed no difference in the ability of IVIg to ameliorate EAE irrespective of its sialylation. Our findings thus show that sialylation of IVIg is not necessary for IVIg-mediated amelioration of EAE or for downregulation of Th17 cells and upregulation of regulatory T cells.

SUMMARY The development of new virus inactivation procedures has become an area of growing interest mainly due to increased demands concerning the safety of biological products. Photochemical processes represent the most promising methods for the future to inactivate viruses. In these methods, dyes are the most widely used photosensitising reagents. The current article covers a new interesting alternative, namely the use of buckminsterfullerene (C60). The unique properties of this molecule make it a valid candidate for future applications in the inactivation of viruses in biological fluids. ? 1998 John Wiley & Sons, Ltd.

In a process for the inactivation of enveloped viruses in a biological fluid buckminsterfullerene (C60) is used as a photosensitizer. In a photodynamic processes singlet oxygen is generated which is the active agent for the inactivation of viruses present in the fluid. The process comprises the steps of (a) contacting of a solution or dispersion of material derived from the human body or from the bodies of animals with buckminsterfullerene as a photosensitizer, of (b) saturating the solution or dispersion with oxygen; and of (c) irradiating the solution or dispersion with visible or invisible light for activating the oxygen into the singlet state until viruses contained in the solution or dispersion are inactivated. This virus inactivation is specially suitable for protein solutions, e.g. bovine serum albumin, BSA or plasma products of human origin.

Objective Plasmacytoid dendritic cells (PDCs) produce high concentrations of interferon-α (IFNα) following exposure to immune complexes containing nucleic acids. We previously reported that serum from healthy donors inhibits IFNα production by PDCs in response to systemic lupus erythematosus (SLE) immune complexes, and that inhibition is mediated, in part, by IgG. IgG is the major component of intravenous immunoglobulin and is well known to exert antiinflammatory properties. Although suppression of inflammation by the sialylated subfraction of IgG has been implicated in some models, the mechanism of IFNα inhibition by IgG and the importance of sialylation have not been studied. Methods SLE immune complexes or synthetic Toll-like receptor (TLR) agonists were used to stimulate total or individual cell–depleted human mononuclear cell cultures in the presence or absence of IgG, Fc fragments, F(ab′)2 fragments, and their sialylated or unsialylated subfractions. Cytokines were quantified by enzyme-linked immunosorbent assay. Results We identified 2 distinct mechanisms by which IgG inhibits IFNα production. First, IgG Fc fragments inhibited SLE immune complex–stimulated IFNα production via a sialic acid–independent mechanism, by inhibiting immune complex binding to Fcγ receptor IIa on PDCs. In contrast, the F(ab′)2 fragment of the sialylation-enriched fraction of IgG inhibited TLR-7 or TLR-9 agonist–induced IFNα production but did not require the sialic acid residue itself. The inhibitory activity of IgG on TLR agonist–induced IFNα required monocyte production of prostaglandin E2, a potent suppressor of IFNα production by PDCs. Conclusion IgG attenuates IFNα production by PDCs by both cell surface receptor and intracellular pathways, depending on the nature of the inducing stimulus.

Plasmacytoid dendritic cells (PDCs) produce high concentrations of interferon-α (IFNα) following exposure to immune complexes containing nucleic acids. We previously reported that serum from healthy donors inhibits IFNα production by PDCs in response to systemic lupus erythematosus (SLE) immune complexes, and that inhibition is mediated, in part, by IgG. IgG is the major component of intravenous immunoglobulin and is well known to exert antiinflammatory properties. Although suppression of inflammation by the sialylated subfraction of IgG has been implicated in some models, the mechanism of IFNα inhibition by IgG and the importance of sialylation have not been studied.SLE immune complexes or synthetic Toll-like receptor (TLR) agonists were used to stimulate total or individual cell-depleted human mononuclear cell cultures in the presence or absence of IgG, Fc fragments, F(ab')2 fragments, and their sialylated or unsialylated subfractions. Cytokines were quantified by enzyme-linked immunosorbent assay.We identified 2 distinct mechanisms by which IgG inhibits IFNα production. First, IgG Fc fragments inhibited SLE immune complex-stimulated IFNα production via a sialic acid-independent mechanism, by inhibiting immune complex binding to Fcγ receptor IIa on PDCs. In contrast, the F(ab')2 fragment of the sialylation-enriched fraction of IgG inhibited TLR-7 or TLR-9 agonist-induced IFNα production but did not require the sialic acid residue itself. The inhibitory activity of IgG on TLR agonist-induced IFNα required monocyte production of prostaglandin E2, a potent suppressor of IFNα production by PDCs.IgG attenuates IFNα production by PDCs by both cell surface receptor and intracellular pathways, depending on the nature of the inducing stimulus.

Sialylation of the N-linked glycan on asparagine-297 within the Fc region of intravenous gammaglobulins (IVIgs) was shown to be necessary for efficacy of IVIg in the amelioration of experimental inflammatory arthritis. To test the role for Fc sialylation of IVIg in immune modulating therapies beyond the K/BxN arthritis model, we examined the efficacy of sialylated compared to nonsialylated IVIg for the ability to attenuate immune thrombocytopenia (ITP) in a mouse model that approximates the clinical setting of human ITP.We used a published, passive anti-platelet (PLT) dose-escalation mouse model of ITP that approximates clinical ITP. PLT counts were followed over time before and after IVIg treatment. IVIg from two different manufacturers was used to prepare untreated and neuraminidase-treated IVIg. Sambucus nigra agglutinin (SNA) affinity chromatography was used to obtain sialic acid-enriched and -depleted IVIg. Sialic acid content was determined using Western blot, enzyme-linked immunosorbent assay, and high-performance liquid chromatography.Results were the same using sialylated and desialylated (neuraminidase-treated) IVIg from two different manufacturers. No differences were observed between sialic acid-enriched and -depleted IVIg compared to normal IVIg in their efficacy to alleviate ITP. Using quantitative reverse transcription-polymerase chain reaction, no increase in the spleen FcγRIIB mRNA was detectable, but a pronounced increase of FcγRIIB mRNA in the marrow was seen after IVIg administration.We conclude that IVIg ameliorates experimental ITP by a mechanism that is independent of sialylation either in the Fc or the Fab region of IVIg.

When the human body is invaded by foreign organisms it defends itself either through the propagation of a cellular response (cell-mediated immunity) or by the production of soluble proteins. This production of soluble proteins is known as humoral immunity, so-called because it refers to substances found within the humours, or body fluids. While there are a number of components that have been identified as important in the humoral immune response, including complement proteins [1], the key molecules necessary for mediating specific humoral immunity are immunoglobulins (Ig), also known as antibodies (Ab) (see also chapter A3).

Summary 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 α2,6-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.

Virus inactivation by ethyleneimines was first introduced more than 30 years ago. Selective targeting of nucleic acids was reported for oligomeric ethyleneimines. In this study, trimeric ethyleneimine (TEI) was used to inactivate minute virus of mice (MVM; Parvoviridae) and Semliki forest virus (SFV; Togaviridae). The pH-dependency of the inactivation kinetics observed with MVM was different compared to the kinetics reported for other viruses. The higher inactivation rate at higher pH favoured the idea of a mechanism involving protein modifications. Alteration of the isoelectric point and changes in mass could be observed after treatment of soluble proteins with TEI. The uptake of MVM by host cells was reduced or completely blocked by TEI treatment, as shown by monitoring viral internalisation of DNA into target cells. The observed loss of virus infectivity coincided with the inhibition of virus uptake. Thus, virus inactivation by TEI is most likely also a result of chemical modifications of viral surface proteins.

Inactivation of viruses can be induced by singlet oxygen generating agents. The water-insoluble polymeric compound PVNE (poly (1,4-dimethyl-6-vinylnaphthalene-1,4-endoperoxide)) is used as a storage for reactive oxygen and is able to produce thermally generated 1 O 2 in a dark-reaction. Enveloped viruses from two different families, Semliki Forest virus (SFV, Togaviridae) and vesicular stomatitis virus (VSV, Rhabdoviridae) showed a loss of infectivity of up to 8 log 10 /ml (TCID 50 ) when incubated at 37°C with PVNE in buffered solutions. PVNE produces singlet oxygen by thermal decomposition without irradiation. Such chemically generated oxygen excludes reactions involving radicals (type I photoreactions), a problem often encountered in photodynamic processes utilizing dyes as sensitizers. In addition, the water insolubility of the oxygen-carrier allows an easy removal and recycling from aqueous solutions. Therefore, it may prove useful in the inactivation of viruses in biological systems and may be a helpful tool in studies concerning the inactivation mechanism by 1 O 2 .

IVIg is increasingly used for therapy in many autoimmune and inflammatory diseases; however the mechanisms underlying its therapeutic benefit have remained uncertain. We have investigated the mechanisms in the context of modulation of regulatory and pathogenic CD4 T cells in EAE, a murine model of multiple sclerosis. We demonstrate that IVIg delays the onset of EAE by inhibiting encephalitogenic CD4 T cell subsets (Th1 and Th17 cells) and concomitantly expanding foxp3+ regulatory T cells. Furthermore, IVIg renders effector T cells less pathogenic by decreasing the expression of encephalitogenic molecules such as GM-CSF and podoplanin. Intriguingly and contrary to the current arguments, the inhibitory FcγRIIB and sialylation on IgG are dispensable and F(ab’)2 fragments retained this function of IVIg. Additionally, IVIg sequestered effector T cells into the draining lymph nodes leading to decreased infiltration into CNS. IVIg interfered with S1P-S1P1-mTOR pathway to sequester lymphocytes in the draining lymph nodes and reciprocally regulate CD4 T cell subsets. Our study reveals a novel role of immunoglobulins in modulating polarization, pathogenicity and trafficking of T lymphocytes accounting for the observed beneficial effect during IVIg therapy.

Exposure of Aedes albopictus cells infected with Semliki Forest virus (SFV; Togaviridae) to mildly acidic pH (5.6) results in a dramatic increase in the host cell membrane permeability due to pore formation by the virus spike proteins. Identical results were obtained when the cells were infected with two other viruses, Sindbis virus (SIN, Togaviridae) and vesicular stomatitis virus (VSV, Rhabdoviridae). This permeability change could also be observed on isolated virions of SFV, SIN and VSV by measuring the influx of propidium iodide, a nucleic acid-specific fluorescent marker, into the virions. This influx was dependent on the presence of the ectodomains of the viral spikes and could be hampered by zinc ions. Furthermore, haemagglutinin, a membrane protein of influenza A virus (Orthomyxoviridae), expressed in Aedes cells induced a change in membrane permeability identical to that induced by the spike proteins of SFV, SIN and VSV when exposed to low pH. Thus acid-induced membrane permeability changes produced by spike proteins of three different virus families could be demonstrated in infected cells as well as in virions. Therefore, the low pH-induced pore formation by viral spike proteins seems to be more than an event specific for togaviruses and might well be an inherent property of enveloped viruses that use the endocytotic pathway to infect a cell.

Pore formation at mildly acidic pH by SFV spike proteins was investigated using isolated and modified virions. Modification of the virions was performed by limited proteolysis in presence of octylglucoside and resulted in the formation of E1 particles and spikeless particles, respectively. Pore formation was detected by measuring the influx of propidium iodide into the viral particles. The results obtained clearly showed that the presence of E1 alone is sufficient to promote pore formation at mildly acidic pH. Thus E1 represents the pore forming element of the viral spike proteins.

It has been proposed that the anti-inflammatory effects of intravenous immunoglobulin (IVIG) might be due to the small fraction of Fc-sialylated IgG. In this study we biochemically and functionally characterized sialic acid-enriched IgG obtained by Sambucus nigra agglutinin (SNA) lectin fractionation. Two main IgG fractions isolated by elution with lactose (E1) or acidified lactose (E2) were analyzed for total IgG, F(ab')(2) and Fc-specific sialic acid content, their pattern of specific antibodies and anti-inflammatory potential in a human in vitro inflammation system based on LPS- or PHA-stimulated whole blood. HPLC and LC-MS testing revealed an increase of sialylated IgG in E1 and more substantially in the E2 fraction. Significantly, the increased amount of sialic acid residues was primarily found in the Fab region whereas only a minor increase was observed in the Fc region. This indicates preferential binding of the Fab sialic acid to SNA. ELISA analyses of a representative range of pathogen and auto-antigens indicated a skewed antibody pattern of the sialylated IVIG fractions. Finally, the E2 fraction exerted a more profound anti-inflammatory effect compared to E1 or IVIG, evidenced by reduced CD54 expression on monocytes and reduced secretion of MCP-1 (CCL2); again these effects were Fab- but not Fc-dependent. Our results show that SNA fractionation of IVIG yields a minor fraction (approx. 10%) of highly sialylated IgG, wherein the sialic acid is mainly found in the Fab region. The tested anti-inflammatory activity was associated with Fab not Fc sialylation.