Hypersensitivity to intravenous iron: classification, terminology, mechanisms and management.

Intravenous (IV) iron therapy is widely used in iron deficiency anaemias when oral iron is not tolerated or ineffective. Administration of IV-iron is considered a safe procedure, but severe hypersensitivity reactions (HSRs) can occur at a very low frequency. Recently, new guidelines have been published by the European Medicines Agency with the intention of making IV-iron therapy safer; however, the current protocols are still non-specific, non-evidence-based empirical measures which neglect the fact that the majority of IV-iron reactions are not IgE-mediated anaphylactic reactions. The field would benefit from new specific and effective methods for the prevention and treatment of these HSRs, and the main goal of this review was to highlight a possible new approach based on the assumption that IV-iron reactions represent complement activation-related pseudo-allergy (CARPA), at least in part. The review compares the features of IV-iron reactions to those of immune and non-immune HSRs caused by a variety of other infused drugs and thus make indirect inferences on IV-iron reactions. The process of comparison highlights many unresolved issues in allergy research, such as the unsettled terminology, multiple redundant classifications and a lack of validated animal models and lege artis clinical studies. Facts and arguments are listed in support of the involvement of CARPA in IV-iron reactions, and the review addresses the mechanism of low reactogenic administration protocols (LRPs) based on slow infusion. It is suggested that consideration of CARPA and the use of LRPs might lead to useful new additions to the management of high-risk IV-iron patients.

Enhancing effect of autologous human erythrocytes on generation of C3 cleavage products beyond iC3b.

The in vitro formation of C3d and C3c in fresh normal human serum (NHS) after addition of five different activators of the complement (C) system was studied. Following C-activation in NHS (n = 53) by Sephadex G-200 beads, the conversion of C3 was found to proceed to iC3b with a variable but restricted generation of C3d. Similar results were obtained by use of heat-aggregated IgG, Escherichia coli, zymosan, and cobra venom factor. However, comparing the C3d concentration following activation in the presence and absence of autologous red blood cells (RBC) at 37 degrees C the generated C3d was found to be 2- to 3-fold higher in the presence of RBC after 30, 60, and 210 min. Preincubation of RBC with polyclonal anti-CR1 antibodies resulted in a dose-dependent reduction of the amount of C3d generated. C-activation induced by Sephadex G-200 beads, in the absence of RBC, generated iC3b without a significant production of C3d. After removal of the activator beads, addition of RBC resulted in a decrease of iC3b and a clear increase in the C3c and C3d concentration within 3 h. Western blotting analysis of the C3d produced in the presence of RBC showed that the molecular weight (36 kilodaltons) was similar to that of C3d formed in vivo.

Heterogeneity of C4d and C3d and their complex formation with serum albumin.

High molecular weight (Mr around 175 KD) forms of C4d and C3d as well as free C4d and C3d (Mr about 40 KD) were demonstrable in normal human serum (NHS). Following in vitro C activation in NHS by delta IgG, the 40 KD C4d component increased markedly. By immunofixation it was shown that the high molecular forms of C4d and C3d reacted with biotinylated anti-human albumin IgG, whereas the 40 KD-free C4d and C3d fragments did not. Furthermore, the incorporation of anti-albumin IgG in the first dimensional gel in crossed immunoelectrophoresis caused retention of the 175 KD C4d component but not of free C4d. The 175 KD C4d had a distinctly higher electrophoretic migration velocity (post-albumin region) than the 40 KD C4d fragment. The C3d-, C4d-serum albumin complexes could not be dissociated by reducing agents (DTT, mercaptoethanol), a non-ionic detergent, or exposure to high and low ionic strength.

Comparison of different antibody preparations against pregnancy-associated plasma protein-A (PAPP-A) for use in localization and immunoassay studies.

Four antibody preparations against pregnancy-associated plasma protein (PAPP-A) were compared in order to find an explanation for the contradictory results published on tissue localization, clinical usefulness and biological function of PAPP-A. One of the preparations studied was a rabbit anti-PAPP-A antiserum which has been offered for general scientific use (Bischof et al. 1979). Only the IgG fraction of anti-PAPP-A antisera which appeared to be monospecific and had been further absorbed with fetal connective tissue gave specific uniform staining of the cytoplasm of the syncytiotrophoblast exclusively. Circulating PAPP-A could not be detected by RIA employing this IgG preparation in the non-pregnant state, or before 18 days after conception. Circulating PAPP-A could be detected in all seven pregnant women studied within 4 weeks after conception. Identical results were obtained with a commercially available IgG fraction against PAPP-A.

Complement activation, circulating protease inhibitors and pregnancy-associated proteins in severe pre-eclampsia.

Circulating protease inhibitors, pregnancy-associated proteins and the split product of complement factor 3 (C3d) were measured in 14 women with severe pre-eclampsia and their matched controls. Only the mean levels of antithrombin III were observed to be significantly lower in pre-eclampsia (P less than 0.02).

Immunohistochemical aspects of immunological cross-reaction and masking of epitopes for localization studies on pregnancy-associated plasma protein A.

The influence of antibody absorption procedures and proteolytic pre-treatment of formaldehyde-fixed placental tissue on the localization of pregnancy-associated plasma protein A by immunoperoxidase technique was examined. Apparently monospecific IgG fraction of the anti-plasma protein applied directly on fixed tissue resulted in staining of connective tissue and a thin apical rim of the syncytiotrophoblast. Further absorption of the antibody with foetal connective tissue abolished this staining reaction. Pre-treatment of the fixed placental tissue with trypsin prior to application of the antibody, which had been absorbed with connective tissue, resulted in staining within the cytoplasm of the syncytiotrophoblast exclusively. Identical staining was seen when this IgG preparation was used directly on frozen placental tissue. The results point to the importance of the specificity of the antibody preparations and of proteolytic unmasking of epitopes when fixed tissues are used for localization studies of pregnancy-associated plasma protein A by immunoperoxidase technique.

Characterization of human pregnancy zone protein. Comparison with human alpha 2-macroglobulin.

Native human pregnancy zone protein (PZP), a close homolog of alpha 2-macroglobulin (alpha 2M), can be obtained in approximately 20% yield from pooled late pregnancy plasma or serum by a combination of polyethylene glycol precipitation, euglobulin precipitation, DEAE-Sephacel chromatography, zinc-chelate affinity chromatography, and negative affinity chromatography on insolubilized antibodies against human serum proteins. Both proteins are similarly organized as disulfide-bridged dimers of 360 kDa containing 180-kDa subunits. These dimers constitute the proteinase-binding units of PZP, and in contrast to alpha 2M, they appear to be only loosely associated, indicating a subtle difference in the quaternary structure of these alpha-macroglobulins. The preparations contain functionally intact beta-cysteinyl-gamma-glutamyl thiol esters, located in the same nonapeptide sequence as found in alpha 2M, and form complexes with a variety of proteinases in which a large fraction of the proteinase is bound covalently. Proteinases bound to PZP are still active and poorly accessible to reaction with large inhibitors like alpha 1-proteinase inhibitor. The structural and functional features of PZP indicate that PZP and alpha 2M, although extremely similar, may have different yet overlapping sets of proteinases as targets. It is possible that PZP mainly controls the activity of cellular proteinases released under conditions of increased cellular turnover and that PZP could be the human equivalent to the acute phase alpha-macroglobulins known in other species.

Antigenic determinants of pregnancy-associated alpha 2-glycoprotein and alpha 2-macroglobulin defined by poly- and monoclonal antibodies.

Human alpha 2-macroglobulin and pregnancy-associated alpha 2-glycoprotein (PA alpha 2G) share several physicochemical characteristics. By the use of unabsorbed or absorbed polyclonal antibodies to these antigens, the existence of common epitopes in these molecules were demonstrated in crossed immunoelectrophoresis. Two monoclonal antibodies out of 9 raised against purified PA alpha 2G were demonstrated to react with both antigens, indicating close immunochemical relatedness between these macroglobulins. The findings might have functional implications.

Characterization of tryptic fragments of human complement factor C3.

C3c and C3d fragments were prepared in pure form from trypsin-digested human C3, and the individual chains of tryptic C3c were isolated by gel filtration on Sepharose 4B in 6M guanidinium hydrochloride. No low mol. wt (Mr) fragments were identified. The polypeptide chains were characterized with regard to Mr, amino acid composition and N-terminal amino acid sequence. Tryptic C3c consisted of one fragment from the beta-chain (Mr 64,000) and two from the alpha'-chain (Mr 40,000 and 23,000). The beta-chain fragment was derived from the C-terminal part of the chain, and the 23,000-Mr component constituted the amino terminal end of the alpha-chain. The 40,000-Mr fragment emanated from the C-terminal end of the alpha-chain. Tryptic C3d displayed microheterogeneity on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, but possessed a homogeneous N-terminal, identical to that described by Tack et al. (1980) (Proc. natn. Acad. Sci. U.S.A. 77, 5764-5768). By utilization of antisera against subunits of C3 and C3c in immunoblotting a degradation scheme for C3 by trypsin was proposed and the positions of the fragments in the intact molecule indicated.

Application of electroblotting technique to studies of the intestinal antibody response to extractable fecal antigens.

Water-soluble and sodium dodecyl sulfate (SDS)-extractable fractions were prepared from feces of four patients with inflammatory and six with non-inflammatory bowel disease. Both types of fractions were run on polyacrylamide gel electrophoresis, followed by electroblotting on nitrocellulose sheets of the separated components. The antibody reactivities in serum and in the intestinal wall against the extracted and separated fecal components were investigated. A striking lack of reactivity was observed when serum was used as antibody source against the fecal extracts both in patients with inflamed and in those with non-inflamed intestinal mucosa. The locally produced gut-associated IgG reacted more intensely with SDS-extractable fecal components than with water-soluble components. A strong reaction between intestinal-wall IgG extracts and a water-soluble antigen of 46-48 kD in the feces of two colitis patients was, however, observed. No clear correlation between the single bands and the occurrence of inflammatory bowel disease could be established because of the small number of patients.

Preparation of antibodies against the fourth complement component (C4) and development of a direct electroimmunoassay for quantification of C4d.

The specificity of several preparations of antihuman C4 antibodies were examined by crossed immunoelectrophoresis. Two antibody preparations with anti-C4c and anti-"total" C4 reactivity respectively were prepared by immunoadsorption procedures and defined by comparison with reference antibodies of known specificity. These two antibody preparations were used in the development of a rocket immunoelectrophoresis with an intermediate gel for specific and direct quantification of C4d. This method permits the selective quantification of activation of the classical complement pathway as opposed to the alternative pathway activation.

The selection of antibodies with defined desorption properties from precipitated immune complexes for use in immunoadsorption procedures.

A general method for preparing immunosorbents with preselected antibody avidity is described. The method, which is a modification of a method described previously, also includes immunospecific purification of the ligand prior to coupling on the gel matrix. Polyclonal anti-alpha-1-fetoprotein antibodies in precipitated immune complexes were separated according to their avidity (low, intermediate and high) by dissociation with agents of increasing efficiency. After solid-phase coupling the antigen binding activity of the separated antibody preparations was examined according to recovery, capacity and binding strength. Antibodies of intermediate avidity derived from the immune complexes demonstrated optimal properties for preparative affinity chromatography.

Immunoelectrophoretic analysis of C4 split products expressing D but not C epitopes: influence of storage, Ca2+ and Ca2+-chelating agents.

Based on immunoelectrophoretic methods a heterogeneity in the electrophoretic mobility of C4d was observed. C4d was defined immunochemically as C4 molecules expressing D but lacking C epitopes. A beta-mobile form was observed when EDTA or heparin was not added to the sample prior to electrophoretic analysis. This component was generated during electrophoresis. Another C4d component migrating to the post-albumin region probably represented an in vivo generated split product. However, this C4d form was also produced during storage of serum or plasma at room temperature and its formation was enhanced in the presence of EDTA. Based on these findings standard conditions for collection and storage of clinical samples for quantification of C4d by electroimmunoassay are suggested.

Partial primary structure of human pregnancy zone protein: extensive sequence homology with human alpha 2-macroglobulin.

Human pregnancy zone protein (PZP) is a major pregnancy-associated protein. Its quaternary structure (two covalently bound 180-kDa subunits, which are further non-covalently assembled into a tetramer of 720 kDa) is similar to that of human alpha 2-macroglobulin (alpha 2M). Here we show, from the results of complete or partial sequence determination of a random selection of 38 tryptic peptides covering 685 residues of the subunit of PZP, that PZP and alpha 2M indeed are extensively homologous. In the stretches of PZP sequenced so far, the degree of identically placed residues in the two proteins is 68%, indicating a close evolutionary relationship between PZP and alpha 2M. Although the function of PZP in pregnancy is largely unknown, its close structural relationship to alpha 2M suggests analogous proteinase binding properties and a potential for being taken up in cells by receptor-mediated endocytosis. In this regard our studies indicate a bait region in PZP significantly different from that present in alpha 2M. PZP could be the human equivalent of the acute-phase alpha-macroglobulins (e.g., rat alpha 2M and rabbit alpha 1M) described earlier.

Factor I deficiency and C3 nephritic factor: immunochemical findings and association with Neisseria meningitidis infection in two patients.

The complement system was examined in two patients with systemic Neisseria meningitidis infections, both of whom had reduced or nondetectable CH50 as analysed by both pathways. C3 measured by conventional technique revealed 19% anti-C3c-reactive protein in the plasma of patient 1 and 3% in patient 2. Patient 1 had circulating C3b but no detectable C3c, C3d, or C4d, whereas patient 2 had normal levels of C3c and C4d and strongly elevated levels of C3d. Factor B analysis revealed no demonstrable native factor B and small amounts of Bb in patient 1 and normal concentration of native factor B plus trace amounts of Bb in patient 2. The depletion of C3 in both patients was due to uncontrolled activation caused by complete factor I deficiency (patient 1) and circulating C3 nephritic factor (patient 2). Both parents of patient 1 had factor I concentrations below (mean-2 SD) that seen in normal healthy individuals (n = 20). Circulating immune complexes (IC) were demonstrated in patient 1 only, whereas serum from both patients had strongly reduced capacity to solubilize preformed IC.

Immunohistochemical demonstration of pregnancy-associated plasma protein A (PAPP-A) in the syncytiotrophoblast of the normal placenta at different gestational ages.

The immunoperoxidase technique was used to study the localization of pregnancy-associated plasma protein A (PAPP-A) in formaldehyde-fixed paraffin-embedded tissue from normal human placentae at the gestational age of 8, 15 and 40 weeks. Sections of formaldehyde-fixed tissue treated with a proteolytic enzyme and incubated in antiserum against PAPP-A either raised in goats or rabbits showed that PAPP-A was distributed in the cytoplasm of the syncytiotrophoblast. The protein was not found in the cytotrophoblast. Sections without pretreatment with trypsin and incubation in goat anti-PAPP-A showed no staining reaction, whereas incubation in rabbit anti-PAPP-A revealed a staining of the syncytiotrophoblast surface. The results indicate that PAPP-A is probably synthesized in the syncytiotrophoblast.

Charge and size heterogeneity of C3d following in vivo and in vitro activation of the complement system.

Split products of the third complement factor (C3) expressing D but not C epitopes (C3d) were analyzed by crossed immunoelectrophoresis and size chromatography. Four molecular forms (termed 1, 2, 3, and 4 from the anodic side) were identified. The precipitation pattern of C3d in serum following acute in vivo activation was similar to the patterns observed using in vitro activation by MgCl2, zymosan, Escherichia coli, and delta IgG. These patterns were different from those observed in normal human serum and serum from a patient suffering from systemic lupus erythematosus. The molecular weights of forms 1 and 4 were approximately 170,000 daltons and those of forms 2 and 3 approximately 40,000 daltons.

Production of antibody against C3c epitopes which are inaccessible on native C3.

Anti-C3c antiserum was induced by immunizing rabbits with autologous erythrocytes coated with human C3b/C3bi. By absorption with human plasma this antiserum was rendered specific for C3c epitopes which are not expressed on native C3. This specificity may be analogous to that of immunoconglutinin.