[Pooled Human Immunoglobulin Preparations as Immunomodulating Drugs].

It is time to celebrate the 125th anniversary of the first successful attempt to develop and use a specific high-titer antitoxic serum for treating diphtheria, a deadly infectious disease. This was followed by major advances in passive immunotherapy 75 years ago (production of pooled human IgG for subcutaneous injection) and 50 years ago (widespread technology for producing immunoglobulin preparations for intravenous administration). More than 200 tons of pooled human IgG are produced per year worldwide. The preparation is used primarily for IgG substitution in patients with primary and secondary immunodeficiencies, as well as for an immunomodulating treatment of a growing number of autoimmune and inflammatory diseases. These preparations contain the pooled IgG antibody repertoire of a large population of healthy plasma donors. This repertoire includes antibodies that neutralize pathogens and their factors of virulence, anti-idiotypic antibodies, and antibodies to other foreign and own proteins, as well as to carbohydrate antigens. Naturally polyspecific antibodies that are present in all healthy individuals play an important role as a first-line defense against bacteria and viruses. After exposure to protein-modifying agents, some IgG molecules can acquire the ability to bind novel structurally unrelated antigens. This phenomenon is referred to as induced polyspecificity. The list of these protein-modifying molecules was shown to include low-pH buffers, free heme, pro-oxidative ferrous ions, reactive oxygen species, etc. Such modified antibody preparations may have a therapeutic potential, since their administration to animals with experimental sepsis or aseptic systemic response syndromes significantly improved survival rates, while the same dose of the native preparation had no effect. We also hypothesize that the aggressive protein-modifying molecules released in sites of inflammation and tissue damage could also modify the antigen-binding behavior of surface immunoglobulin B cell receptors and the structurally related T cell receptors. This "specificity editing" of both types of receptors may play a major role in the body's defense mechanisms.

Targeted silencing of DNA-specific B cells combined with partial plasma cell depletion displays additive effects on delaying disease onset in lupus-prone mice.

Targeting autoreactive B lymphocytes at any stage of their differentiation could yield viable therapeutic strategies for treating autoimmunity. All currently used drugs, including the most recently introduced biological agents, lack target specificity. Selective silencing of double-stranded DNA-specific B cells in animals with spontaneous lupus has been achieved previously by the administration of a chimeric antibody molecule that cross-links their DNA-reactive B cell immunoglobulin receptors with inhibitory FcγIIb (CD32) receptors. However, long-lived plasmacytes are resistant to this chimeric antibody as well as to all conventional treatments. Bortezomib (a proteasome inhibitor) depletes most plasma cells and has been shown recently to suppress disease activity in lupus mice. We hypothesized that the co-administration of non-toxic doses of bortezomib, that partially purge long-lived plasma cells, together with an agent that selectively silences DNA-specific B cells, should have additive effects in an autoantibody-mediated disease. Indeed, our data show that the simultaneous treatment of lupus-prone MRL/lpr mice with suboptimal doses of bortezomib plus the chimeric antibody resulted in the prevention or the delayed appearance of the disease manifestations as well as in a prolonged survival. The effect of the combination therapy was significantly stronger than that of the respective monotherapies and was comparable to that observed after cyclophosphamide administration.

Inflammation-induced enhancement of IgG immunoreactivity.

Natural polyreactive IgG antibodies are found in the sera of all healthy individuals. The in vitro exposure of pooled human IgG to protein-destabilizing chemical or physical factors has been previously shown to result in the exposure of their "hidden" polyspecificity. We hypothesize that such an enhancement of their pre-existing immunoreactivity may occur in vivo in the aggressive microenvironment of inflammation sites. An increase in the antigen binding intensity as well as of the number of recognized antigens was observed in the sera of IgG-infused immunodeficient SCID mice with induced local inflammation. The expansion of the IgG pathogen-binding repertoire may have important biological consequences.

Iron ions and haeme modulate the binding properties of complement subcomponent C1q and of immunoglobulins.

The complement system and circulating antibodies play a major role in the defence against infection. They act at the sites of inflammation, where the harsh microenvironment and the oxidative stress lead to the release of free iron ions and haeme. The aim of this study was to analyse the consequences of the exposure of C1q and immunoglobulins to iron ions or haeme. The changes in target recognition by C1q and in the rheumatoid factor activity of the immunoglobulins were investigated. The exposure of C1q to ferrous ions increased its binding to IgG and to IgM. In contrast, haeme inhibited C1q binding to all studied targets, especially to IgG1 and C-reactive protein. Thus, the haeme released as a result of tissue damage and oxidative stress may act as a negative feedback regulator of an inappropriate complement triggering as seen in ischaemia-reperfusion tissue injury. The results also show that iron ions and haeme were able to reveal rheumatoid factor activity of IgG. The modulation of the C1q-target binding as well as the revealing of rheumatoid factor activity of IgG by exposure to redox-active agents released at the sites of inflammation may have important consequences for the understanding of the immunopathological mechanisms of inflammatory and autoimmune diseases.

IgM-enriched human intravenous immunoglobulin suppresses T lymphocyte functions in vitro and delays the activation of T lymphocytes in hu-SCID mice.

Previous studies of an experimental human immunoglobulin preparation for intravenous use, containing normal pooled IgM (IVIgM), have shown its beneficial therapeutic effect in experimental autoimmune diseases. The mechanisms of its immunomodulatory activity remain however, poorly understood. In the experiments reported here, IVIgM inhibited the proliferation of various autonomously growing human lymphoid cell lines in vitro, as well as of MLR- and of PHA-stimulated human T-lymphocytes. These effects of IVIgM were observed at non-apoptotic concentrations and were stronger on a molar basis than those of normal pooled IgG for intravenous use (IVIg). Both preparations, when administered to SCID mice, repopulated with human peripheral blood mononuclear cells, delayed the expression of the early activation marker CD69 on both human CD4+ and CD8+ T-lymphocytes, activated by the mouse antigenic environment. The data obtained show that normal pooled human IgM exerts a powerful antiproliferative effect on T-cells that is qualitatively similar but quantitatively superior to that of therapeutic IVIg. Our results suggest that infusions with IVIgM might have a significant beneficial immunomodulating activity in patients with selected autoimmune diseases.

Breaking of tolerance to native DNA in nonautoimmune mice by immunization with natural protein/DNA complexes.

A major event in the pathogenesis of systemic lupus erythematosus (SLE) is the breaking of tolerance to native DNA and the appearance of IgG anti-double-stranded (ds) DNA antibodies. The mechanisms of the losing of tolerance are not well understood. Continuous efforts have been made in the past to induce anti-native DNA IgG autoantibodies in non-autoimmune animals but the relevance of the approaches used to what happens in spontaneous disease is unclear. We succeeded in breaking tolerance to native DNA in nonautoimmune-prone BALB/c mice by immunization with natural DNA/protein complexes. These complexes included nucleosomes, crude commercial histone and nucleohistone preparations. The anti-dsDNA IgG response lasted for more than an year. IgG deposition in the kidneys of the animals was repeatedly shown. As DNA-specific B cells behave in many ways as non-autoreactive B cells, we suggest that the activity of the self-reactive B lymphocytes could be selectively inhibited in a way that mimics a physiological mechanism controlling the magnitude and duration of the IgG antibody response to foreign antigens.

The autoreactivity of therapeutic intravenous immunoglobulin (IVIG) preparations depends on the fractionation methods used.

Natural immunoglobulin G (IgG) autoantibodies are present in the plasma of healthy individuals and, as a result, in pooled therapeutic intravenous immunoglobulin (IVIg) preparations. The production processes of commercial IVIg preparations involve different fractionation and virus-inactivation steps that include in some cases treatments at extreme conditions. Different physical and chemical treatments are known to augment greatly the reactivity of natural autoantibodies to self-antigens. It is not clear to what extent the self-reactivity of IVIg preparations is due to the presence of natural IgG antibodies in the plasma pools used for fractionation, and to what extent it is due to the treatments that the IgG molecules have been subjected to during the fractionation process. We compared the binding of seven different commercial IVIg preparations to human liver antigens. All studied IVIg's could be clearly separated into two distinct groups: those that possess significant self-reactivity and those with low binding to self-antigens. Increased self-binding was seen in the preparations produced using a fractionation step at low pH. The treatment of IVIg at low pH resulted in increasing the inhibitory effect of the pooled IgG on PHA-induced proliferation of human peripheral blood mononuclear cells. IVIg's with high and low self-binding may have different immunomodulating activities when infused to autoimmune patients.

Rhodococcus equi infection in transplant recipients: a case of mistaken identity and review of the literature.

The incidence of Rhodococcus equi infection in solid-organ transplant recipients continues to rise throughout the world. Unfortunately, this opportunistic pathogen is still underestimated and potentially disregarded by physicians and microbiology laboratories due to its morphology on Gram staining. Pulmonary involvement is the most common finding in the immunocompromised host. We report a case of a 63-year-old heart-transplant recipient who presented with increasing fatigue and nonproductive cough for 3 weeks. After full evaluation, a lung abscess was demonstrated by thoracic computerized tomography (CT). Blood and sputum cultures were remarkable for heavy "diphtheroids." Although the Gram-stain result was initially interpreted as a contaminant, a clinical suspicion for Rhodococcus assisted in further investigation. Broncheoalveolar lavage and CT-guided biopsy of the lung abscess revealed heavy growth of diphtheroids. However, further evaluation by a reference laboratory demonstrated mycolic acid staining consistent with R. equi. Surgical drainage and prolonged antibiotic therapy resulted in complete remission of the pneumonia and abscess. This represents the fourth reported case of R. equi infection in a heart transplant recipient. It is imperative that all physicians and laboratory staff consider R. equi when an immunocompromised patient has any type of pneumonia, especially with abscess formation.

Normal human immunoglobulin suppresses experimental myasthenia gravis in SCID mice.

Serum IgM has been shown to participate in the control of IgG autoreactivity in healthy subjects. We have recently shown that an immunoglobulin preparation of pooled normal human IgM (IVIgM) contains anti-idiotypic antibodies against disease-associated IgG autoantibodies in autoimmune patients and protects rats from experimental autoimmunity. The aim of the present study was to asses the in vitro and in vivo immunomodulatory effects of IVIgM in comparison with IgG, in SCID mice reconstituted with thymic cells from a myasthenia gravis patient. Non-leaky SCID mice were injected i.p. with 60 x 10(6) thymic cells from a patient with myasthenia gravis and 1 day later boosted with 10(6) irradiated acetylcholine receptor (AchR)-expressing TE671 cells. On days 14, 21 and 28, mice were treated with IVIgM or with equimolar amounts of human serum albumin. The level of anti-AchR antibodies in the sera of three out of four IgM-treated animals was less than 1 nM. Further, there was a significant decrease in the loss of endplate AchR on the diaphragms of IgM-treated SCID mice. These findings indicate that pooled normal IgM exerts an immunoregulatory role in experimental myasthenia gravis, and suggests that IgM may be considered as an alternative approach in the therapy of autommune diseases.

Inhibition of cell adhesion by antibodies to Arg-Gly-Asp (RGD) in normal immunoglobulin for therapeutic use (intravenous immunoglobulin, IVIg).

Intravenous immunoglobulin (IVIg) therapy is associated with a broad range of immunomodulatory activities. Several of the postulated mechanisms of IVIg action relate to the presence of antibodies to molecules relevant for regulation of the immune response. This article reports that IVIg contains antibodies to the Arg-Gly-Asp (RGD) sequence, and the attachment site of a number of adhesive extracellular matrix proteins, including ligands for beta1, beta3, and beta5 integrins. Anti-RGD antibodies were identified in IVIg by enzyme-linked immunosorbent assay and by using the BIAcore (BIAcore, Uppsala, Sweden) technology. The affinity of anti-RGD antibodies to a synthetic RGD-containing peptide and to fibronectin (Fn) was found to be in the micromolar range. F(ab')2 fragments specific for RGD were purified from IVIg by affinity chromatography. Anti-RGD F(ab')2 antibodies inhibited adenosine diphosphate induced alphaIIb/beta3 integrin-mediated platelet aggregation and the adhesion of activated alpha4beta1 integrin-expressing B cells to Fn. Adhesion of unstimulated platelets to fibrinogen (Fg) involving both the gamma-chain dodecapeptide sequence and the RGD sequence was inhibited by anti-RGD antibodies. In addition, adhesion of thrombin-stimulated platelets to von Willebrand factor or Fg was completely inhibited by affinity-purified anti-RGD antibodies. Our results suggest that the presence of natural IgG antibodies to the RGD motif may contribute to the immunomodulatory and anti-inflammatory effects of therapeutic preparations of normal IgG.

Pooled normal human polyspecific IgM contains neutralizing anti-idiotypes to IgG autoantibodies of autoimmune patients and protects from experimental autoimmune disease.

Normal human serum contains IgM antibodies that regulate the natural autoantibody activity of IgG in autologous serum. In the present study, we show that pooled normal human IgM (IVIgM) purified from plasma of more than 2,500 healthy donors and processed in a similar fashion to that of therapeutic preparations of pooled normal human IgG (IVIg) suppresses activity of IgG autoantibodies purified from the serum of patients with autoimmune diseases in vitro. The inhibitory effect of IVIgM was greater or equivalent to that of IVIg on a molar basis. We show that IVIgM contains anti-idiotypic antibodies directed against idiotypic determinants of autoantibodies, in particular by showing that Sepharose-bound IVIgM selectively retained F(ab')2 fragments of IgG autoantibodies. The infusion of (Lewis x Brown-Norway) F1 rats with IVIgM protected the animals against experimental autoimmune uveitis induced by immunization with the soluble retinal S antigen, as evidenced by clinical scoring and histopathological analysis. The present findings provide a rationale for considering pooled IgM for immunomodulation of autoimmune disease.

An IgA, IgG and IgM-containing human immunoglobulin for oral administration to high-risk bottle-fed newborns. I. Immunochemical characterisation and antibody activity.

Antibodies to a wide spectrum of infectious agents belonging to the IgA, IgM and IgG isotypes are thought to be one of the protective factors in human milk. Cow milk-fed newborns are at an increased risk of infections as well as of allergic diseases and of necrotising enterocolitis. A reasonable approach would be to add to the milk formula fed to them the immunoglobulins present in human milk. We developed a pasteurised immunoglobulin preparation from pooled donor plasma ('Orabulin') containing 75% IgG, 18% IgA and 6% IgM for feeding to high-risk bottle-fed babies. Its molecular composition was studied by HPLC and by SDS-PAGE. The levels of IgA, IgG and IgM antibodies in Orabulin were compared to these in the immunoglobulin fraction of human colostrum and an enrichment was found. It is suggested that the presence of a standardised amount of human IgM in an immunoglobulin preparation intended for feeding to newborns may bring an additional advantage because of the high opsonising and virus-neutralising activity of the antibodies of this isotype.

Modulation of autoimmune responses by intravenous immunoglobulin (IVIg).

Significant progress has been made in understanding the mechanisms by which intravenous immunoglobulins (IVIg) exert immunomodulatory effects in the treatment of autoimmune diseases. A unique property of immunoglobulins is the diversity of variable (V) regions. The evidence discussed in this communication supports our notion that the diversity of V regions in IVIg preparations is a determining factor for the anti-inflammatory substitutive and immunomodulatory functions of IVIg therapy. We have demonstrated the presence in IVIg, of anti-idiotypic antibodies directed against various autoantibodies. The ability of IVIg to interact through V regions with complementary V regions of antibodies and antigen receptors as well as with relevant soluble and surface molecules provides the basis for inducing the selection of immune repertoires. The study of the mechanisms by which IVIg mediates selection of autoreactive repertoires is essential for our understanding of the mechanisms underlying the emergence of pathological autoimmunity and of the physiological role of natural antibodies in the establishment and maintenance of tolerance to self and homeostasis of autoreactivity in healthy individuals.

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

Pooled therapeutic intravenous immunoglobulin (IVIg) is increasingly used for the treatment of autoimmune and systemic inflammatory diseases. The lack of a clear understanding of the mode of action of IVIg has been an obstacle to a more rational use and schedule of administration of IVIg and to the improvement of the existing IVIg preparations. Several nonmutually exclusive mechanisms have been proposed to account for the beneficial immunomodulatory effects of IVIg. These include the functional blockade of Fc receptors on phagocytes, inhibition of the deposition of activated complement components on target cells, modulation of the secretion of cytokines and cytokine antagonists, interference with T and B cell proliferation, neutralization of pathological autoantibodies, and long-term selection of immune repertoires.

Natural polyreactive IgA and IgM autoantibodies in human colostrum.

Secretory antibodies against bacteria and viruses in human colostrum and milk are known to be important protective factors for the breast-fed infant. The authors have shown by enzyme immunoassay that colostrum contains IgA and IgM antibodies to a number of autoantigens: native DNA, actin, myosin, myoglobin, laminin, transferrin and thyroglobulin. These antibodies were polyspecific-those with anti-DNA reactivity immunopurified on a DNA-cellulose affinity column bound to a panel of self- and environmental antigens. The levels of natural autoantibodies in the immunoglobulin fraction of human colostrum were 3-10 times lower (when presented as antibody activity per microgram of immunoglobulin) than in the immunoglobulin fraction of serum. The biological significance of the presence of B cells with autoantibody specificity in the mammary gland and of natural autoantibodies in colostrum and milk is not clear. It has been suggested that self-reacting autoantibodies in serum play a major role in the selection of the pre-immune B-cell repertoire and in the maintenance of the immune homeostasis. The authors hypothesize that the natural autoantibodies in colostrum and milk may contribute to the selection process of physiological repertoire during the early postnatal period in breast-fed infants. This could explain the lower frequency of allergic, inflammatory and autoimmune diseases and lymphomas which is seen in their later life when compared with that observed in children who have been formula-fed after birth.

Antibodies to a conserved region of HLA class I molecules, capable of modulating CD8 T cell-mediated function, are present in pooled normal immunoglobulin for therapeutic use.

Intravenous immunoglobulin (IVIg) is increasingly used for the treatment of autoimmune diseases and the prevention of infections and of graft versus host reactions in recipients of allogeneic bone marrow transplants. The immunomodulatory effects of IVIg are largely dependent on their ability to interact with membrane molecules of lymphocytes. We report here that IVIg recognizes the B07.75-84 peptide, corresponding to a conserved region of the alpha I helix of the first domain of HLA-B7 01, which represents a nonpolymorphic determinant of HLA class I molecules. Intact IVIg and its F(ab')2 fragments bound to the peptide as well as to purified soluble HLA and to HLA on a human T cell line. Binding of IVIg to HLA was assessed by ELISA, immunofluorescence, and real-time analysis of the interaction using the BIAlite system. The binding of antipeptide antibodies to HLA was inhibited by free peptide. Antipeptide antibodies isolated from IVIg by affinity chromatography inhibited CD8 cell-mediated cytotoxicity of an influenza virus-specific human T cell line. The presence in IVIg of antibodies to critical regions of HLA class 1 molecules suggests a possible role for IVIg in modulation of class-I-restricted cellular interactions in the immune response.

Variable region-connected, dimeric fraction of intravenous immunoglobulin enriched in natural autoantibodies.

The beneficial effect of intravenous immunoglobulin (IVIg) therapy in patients with autoimmune diseases is at least partially dependent on the content in IVIg of antibodies capable of interacting with variable regions (idiotypes) of autoantibodies. In the present study, we have evaluated the antibody activity to a panel of self and environmental antigens of IVIg preparations and their dimer-enriched fractions. Dimers were either obtained by affinity chromatography of IVIg on Sepharose-bound F(ab')2 fragments of IVIg or by size exclusion gel filtration chromatography of IVIg. Enrichment of IVIg in dimers was found to be associated with an increase in the antibody activity against self-antigens as compared with unchromatographed IVIg. Our findings extend previous observations on enhanced autoantibody content of the affinity chromatography-separated 'connected' fraction of IVIg and suggest that therapeutic preparations of IVIg enriched in dimers may be obtained by size exclusion chromatography. Separation by size increases the feasibility of industrial-scale preparation of IVIg with high dimer content that are endowed with high potential immunomodulatory activity in vivo.

Antibodies to the CD5 molecule in normal human immunoglobulins for therapeutic use (intravenous immunoglobulins, IVIg).

IVIg are increasingly used for the treatment of autoimmune diseases. In the present study, we show that IVIg contain antibodies directed against CD5, a cell surface molecule of T cells which is also a marker of the autoantibody-producing CD20+ ('B-1') subset of B lymphocytes. Antibodies to the CD5 molecule were demonstrated in IVIg by the ability of therapeutic preparations of IVIg to inhibit the binding of labelled CD5 MoAb to the CD5-expressing human T cell line H9. Preincubation of H9 cells with IVIg or with F(ab')2 fragments prepared from IVIg resulted in dose-dependent inhibition of the binding of CD5 antibody. The presence in IVIg of antibodies to the CD5 molecule was further confirmed by the binding of IVIg to mouse L cells that expressed human CD5 molecules following a stable transfection with CD5 cDNA. Human CD5 antibodies in IVIg provide therapeutic immunoglobulin preparations with the potential of modulating T cell functions through CD5, and of regulating the expression of B cell subsets expressing CD5. This may have implications for the treatment of autoimmune diseases.

Shigella flexneri 2a and sonnei I vaccine with two attenuating markers: construction, tolerability and immunogenicity in 143 children aged 3-17 years.

The vaccine candidates were constructed through stepwise incorporation of weakly attenuated purine auxotrophy with subsequent rifampicin resistance (RNA polymerase) mutation to yield optimal attenuation. These strains showed a maintained invasiveness for conjunctival epithelia. Therefore, while not causing keratoconjunctivitis, they were excreted for a short but marked period and provided partial protection in the Sereny test. Children tolerated the maximum dose of 1-3 x 10(9) colony-forming units (c.f.u.) of lyophilized vaccine without side effects. This total dose distributed among three oral inoculations, induced coproantibodies in 70% of recipients during the first three months. However, the single application of this total dose for primary immunization or booster proved to be more efficient: 90% of the subjects had markedly elevated titres for not less than six months.

IgG subclasses distribution in immunoglobulin preparations for intravenous use.

The levels of IgG subclasses were determined, by radial immunodiffusion using monoclonal antibodies, in 16 production batches of two human immunoglobulin preparations for intravenous use--Immunovenin and Immunovenin-intact. While the partially degraded preparation--Immunovenin contained exclusively IgG1, the subclass distribution in Immunovenin-intact was close to that observed in normal human serum.