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Plague caused by the Gram-negative bacterium, Yersinia pestis, is still endemic in parts of the world today. Protection against pneumonic plague is essential to prevent the development and spread of epidemics. Despite this, there are currently no licensed plague vaccines in the western world. Here we describe the means of delivering biologically active plague vaccine antigens directly to mucosal sites of plague infection using highly stable microvesicles (outer membrane vesicles; OMVs) that are naturally produced by the abundant and harmless human commensal gut bacterium Bacteroides thetaiotaomicron (Bt). Bt was engineered to express major plague protective antigens in its OMVs, specifically Fraction 1 (F1) in the outer membrane and LcrV (V antigen) in the lumen, for targeted delivery to the gastrointestinal (GI) and respiratory tracts in a non-human primate (NHP) host. Our key findings were that Bt OMVs stably expresses F1 and V plague antigens, particularly the V antigen, in the correct, immunogenic form. When delivered intranasally V-OMVs elicited substantive and specific immune and antibody responses, both in the serum [immunoglobulin (Ig)G] and in the upper and lower respiratory tract (IgA); this included the generation of serum antibodies able to kill plague bacteria. Our results also showed that Bt OMV-based vaccines had many desirable characteristics, including: biosafety and an absence of any adverse effects, pathology or gross alteration of resident microbial communities (microbiotas); high stability and thermo-tolerance; needle-free delivery; intrinsic adjuvanticity; the ability to stimulate both humoral and cell-mediated immune responses; and targeting of primary sites of plague infection., (© 2019 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.)

In coeliac disease (CD), anti-tissue transglutaminase 2 immunoglobulin (Ig)A antibodies (anti-TG2) are produced and deposited in the intestine. PreventCD (www.preventcd.com) is a European multi-centre study, which investigates the influence of infant nutrition and that of genetic, immunological and other environmental factors on the risk of developing CD. The aim of the current study was to evaluate the appearance of intestinal anti-TG2 deposits in very early intestinal biopsies from at-risk infants and their predictive value for villous atrophy. Sixty-five small bowel biopsies, performed in 62 children, were investigated for the presence of intestinal anti-TG2 extracellular IgA deposits by using double immunofluorescence. The biopsies were performed in the presence of elevated serum levels of CD-associated antibodies and/or symptoms suggesting disease. Deposits of anti-TG2 IgA were present in 53 of 53 CD patients and three of three potential CD patients. In potential CD patients, mucosal deposits showed a patchy distribution characterized by some areas completely negative, whereas active CD patients had uniformly present and evident mucosal deposits. Only one of six patients without CD (negative for serum anti-TG2 and with normal mucosa) had intestinal deposits with a patchy distribution and a weak staining. Two of the 53 CD patients received a definitive diagnosis of CD after a second or third biopsy; mucosal deposits of anti-TG2 IgA were evaluated in all samples. Before developing villous atrophy, both patients had anti-TG2 deposits in normal mucosal architecture, antibodies in one patient being absent in serum. We demonstrated that in CD the intestinal deposits of anti-TG2 are a constant presence and appear very early in the natural history of disease., (© 2017 British Society for Immunology.)

The goal of this study was to investigate the glycosylation profile of native immunoglobulin (Ig)G present in serum immune complexes in patients with rheumatoid arthritis (RA). To accomplish this, lectin binding assays, detecting the accessibility of glycans present on IgG-containing immune complexes by biotinylated lectins, were employed. Lectins capturing fucosyl residues (AAL), fucosylated tri-mannose N-glycan core sites (LCA), terminal sialic acid residues (SNA) and O-glycosidically linked galactose/N-acetylgalactosamine (GalNac-L) were used. Patients with recent-onset RA at baseline and after 3-year follow-up were investigated. We found that native IgG was complexed significantly more often with IgM, C1q, C3c and C-reactive protein (CRP) in RA patients, suggesting alterations of the native structure of IgG. The total accessibility of fucose residues on captured immune complexes to the respective lectin was significantly higher in patients with RA. Moreover, fucose accessibility on IgG-containing immune complexes correlated positively with the levels of antibodies to cyclic citrullinated peptides (anti-CCP). We also observed a significantly higher accessibility to sialic acid residues and galactose/GalNAc glyco-epitopes in native complexed IgG of patients with RA at baseline. While sialic acid accessibility increased during treatment, the accessibility of galactose/GalNAc decreased. Hence, successful treatment of RA was associated with an increase in the SNA/GalNAc-L ratio. Interestingly, the SNA/GalNAc-L ratio in particular rises after glucocorticoid treatment. In summary, this study shows the exposure of glycans in native complexed IgG of patients with early RA, revealing particular glycosylation patterns and its changes following pharmaceutical treatment., (© 2017 British Society for Immunology.)

Eosinophils perform numerous tasks. They are involved in inflammatory reactions associated with innate immune defence against parasitic infections and are also involved in pathological processes in response to allergens. Recently, however, it has become clear that eosinophils also play crucial non-inflammatory roles in the generation and maintenance of adaptive immune responses. Eosinophils, being a major source of the plasma cell survival factor APRIL (activation and proliferation-induced ligand), are essential not only for the long-term survival of plasma cells in the bone marrow, but also for the maintenance of these cells in the lamina propria which underlies the gut epithelium. At steady state under non-inflammatory conditions eosinophils are resident cells of the gastrointestinal tract, although only few are present in the major organized lymphoid tissue of the gut - the Peyer's patches (PP). Surprisingly, however, lack of eosinophils abolishes efficient class-switching of B cells to immunoglobulin (Ig)A in the germinal centres of PP. Thus, eosinophils are required to generate and to maintain mucosal IgA plasma cells, and as a consequence their absence leads to a marked reduction of IgA both in serum and in the gut-associated lymphoid tissues (GALT). Eosinophils thus have an essential part in long-term humoral immune protection, as they are crucial for the longevity of antibody-producing plasma cells in the bone marrow and, in addition, for gut immune homeostasis., (© 2015 British Society for Immunology.)

Ataxia-Telangiectasia (A-T) is a genetic condition leading to neurological defects and immune deficiency. The nature of the immune deficiency is highly variable, and in some cases causes significant morbidity and mortality due to recurrent sinopulmonary infections. Although the neurological defects in A-T are progressive, the natural history of the immune deficiency in A-T has not been evaluated formally. In this study we analyse the clinical history and immunological data in 44 patients with A-T who attended the National Ataxia-Telangiectasia clinic in Nottingham between 2001 and 2011. Using patient medical records and Nottingham University Hospitals (NUH) National Health Service Trust medical IT systems, data regarding clinical history, use of immunoglobulin replacement therapy, total immunoglobulin levels, specific antibody levels and lymphocyte subset counts were obtained. T cell receptor spectratyping results in some patients were already available and, where possible, repeat blood samples were collected for analysis. This study shows that subtle quantitative changes in certain immunological parameters such as lymphocyte subset counts may occur in patients with A-T over time. However, in general, for the majority of patients the severity of immune deficiency (both clinically and in terms of immunological blood markers) does not seem to deteriorate significantly with time. This finding serves to inform the long-term management of this cohort of patients because, if recurrent respiratory tract infections present later in life, then other contributory factors (e.g. cough/swallowing difficulties, underlying lung disease) should be investigated aggressively. Our findings also offer some form of reassurance for parents of children with A-T, which is otherwise a progressively severely debilitating condition., (© 2014 British Society for Immunology.)

In coeliac disease, the intake of dietary gluten induces small-bowel mucosal damage and the production of immunoglobulin (Ig)A class autoantibodies against transglutaminase 2 (TG2). We examined the effect of coeliac patient IgA on the apical-to-basal passage of gluten-derived gliadin peptides p31-43 and p57-68 in intestinal epithelial cells. We demonstrate that coeliac IgA enhances the passage of gliadin peptides, which could be abolished by inhibition of TG2 enzymatic activity. Moreover, we also found that both the apical and the basal cell culture media containing the immunogenic gliadin peptides were able to induce the proliferation of deamidation-dependent coeliac patient-derived T cells even in the absence of exogenous TG2. Our results suggest that coeliac patient IgA could play a role in the transepithelial passage of gliadin peptides, a process during which they might be deamidated., (© 2011 The Authors. Clinical and Experimental Immunology © 2011 British Society for Immunology.)

Kawasaki disease (KD) is an acute vasculitis affecting mainly infants and children. Human B cells express Toll-like receptor (TLR)-9, whose natural ligands are unmethylated cytosine-guanine dinucleotide (CpG) motifs characteristic of bacterial DNA. The aim of this study was to clarify the pathogenesis of KD analysing the activation status of peripheral blood mononuclear cells (PBMC), focusing on B lymphocyte activation and functions. Ten patients and 10 age-matched healthy donors were recruited from the Bambino Gesù Hospital of Rome, Italy and enrolled into this study. We determined phenotype profile and immunoglobulin (Ig) production of PBMC from KD patients and age-matched controls. We found that the frequency of CD19(+) B lymphocytes and CD19(+) /CD86(+) activated B lymphocytes from KD patients during the acute phase before therapy was increased significantly. Moreover, B lymphocytes of acute-phase KD patients were more prone to CpG oligodeoxynucleotide (ODN) activation compared with the age-matched controls, as assessed by a significant increase of the number of IgA-secreting cells (SC). In the same patients we found a marked increase of IgM, IgG, interleukin (IL)-6 and tumour necrosis factor (TNF)-α production compared with the control group. In addition, in two convalescent KD patients, conventional treatment with intravenous immunoglobulin (IVIG) restored the normal frequency of CD19(+) B cells, the number of IgA-, IgM- and IgG-SC and the production of IL-6 and TNF-α. Our findings indicate that the percentages of peripheral B lymphocytes of acute-phase KD patients are increased and are prone to bacterial activation in terms of increased numbers of IgA-SC and increased production of IL-6 and TNF-α inflammatory cytokines. Thus, our data support the hypothesis of an infectious triggering in KD., (© 2010 The Authors. Clinical and Experimental Immunology © 2010 British Society for Immunology.)

Infection with Chlamydia pneumoniae (Cp) accounts for around 10% of community acquired bacterial pneumonia and has been associated with other chronic inflammatory conditions. We describe a C57/Bl6 murine model of Cp lung infection characterized by a dose-dependent, resolving neutrophilia followed by lymphocytic infiltration of the lungs. By 21 days post-infection, mice exhibit a T helper type 1 (Th1) polarized serum antibody response with local mucosal antibody secretion and organization of ectopic lymphoid tissue which persisted in the absence of detectable Cp DNA. Macrophage inflammatory protein (MIP)-2/CXCL2, which recruits neutrophils and lymphocytes and is associated with ectopic lymphoid tissue formation, was secreted in the lungs post-infection. In vitro, lung epithelial cells up-regulated MIP-2/CXCL2 in response to both rough lipopolysaccharide (reLPS) and Cp infection. We conclude that Cp infection can have long-term inflammatory effects on tissue that persist after clearance of active infection., (© 2010 The Authors; Clinical and Experimental Immunology © 2010 British Society for Immunology.)

Previous studies have shown the down-regulating in vitro effect of cocoa flavonoids on lymphocyte and macrophage activation. In the present paper, we report the capacity of a long-term rich cocoa diet to modulate macrophage cytokine secretion and lymphocyte function in young rats. Weaned rats received natural cocoa (4% or 10% food intake), containing 32 mg flavonoids/g, for 3 weeks. Spleen immune function was then evaluated through the analysis of lymphocyte composition, their proliferative response and their ability to secrete cytokines and Ig. In addition, the status of activated peritoneal macrophages was established through tumour necrosis factor (TNF)-alpha secretion. The richest cocoa diet (10%) caused a reduction of TNF-alpha secretion by peritoneal macrophages showing anti-inflammatory activity. Similarly, although a 10% cocoa diet increased lymphocyte proliferation rate, it down-regulated T helper 2 (Th2)-related cytokines and decreased Ig secretion. These changes were accompanied by an increase in spleen B cell proportion and a decrease in Th cell percentage. In summary, these results demonstrate the functional activity of a cocoa-high dosage in down-regulating the immune response that might be beneficial in hypersensitivity and autoimmunity.

Recent studies had demonstrated that serum and mesangial immunoglobulin A1 (IgA1) in patients with IgA nephropathy (IgAN) were polymeric and deglycosylated. The current study was to investigate the binding characteristics of monomeric and polymeric normal human IgA1 on mesangial cells and the influence of in vitro deglycosylation of IgA1 molecules. The normal human IgA1 was desialylated and degalactosylated with specific enzymes, respectively. The monomeric IgA1 (mIgA1) and polymeric IgA1 (pIgA1) were separated by Sephacryl S-300 chromatography. The binding capacities of the mIgA1 and pIgA1 to primary human mesangial cells (HMC) were evaluated by classical radioligand assay. Both the native mIgA1 and pIgA1 could bind to HMC in a dose-dependent and saturable manner. The maximal binding capacity of the native pIgA1 were significantly higher than that of the native mIgA1 (P < 0.05). However, the affinity of the native mIgA1 was almost 100 times higher than that of the native pIgA1. After deglycosylation, binding of the two deglycosylated mIgA1 to HMC could not be detected. However, the maximal binding capacities of the two deglycosylated pIgA1 to HMC were increased significantly compared with that of native pIgA1. The affinity of the two deglycosylated pIgA1 was similar to that of native pIgA1 (P > 0.05). The current study suggests differential binding characteristics of native monomeric and polymeric IgA1 on mesangial cells. Glycosylation of IgA1 molecules could significantly affect the binding of IgA1 on HMC.

IgA nephropathy (IgAN) is the most common primary glomerulonephritis, with various pathological phenotypes. Our previous study suggested that aberrant glycosylation of serum IgA1 was associated with different pathological phenotypes of IgAN, and substantial evidence indicated that deglycosylated IgA1 had an increased tendency to form macromolecules. The aim of the current study was to investigate the composition of IgA1-containing macromolecules in different pathological phenotypes of IgAN. Sera from 10 patients with mild mesangial proliferative IgAN (mIgAN), 10 with focal proliferative sclerosing IgAN (psIgAN) and 10 healthy blood donors were collected. The sera were applied and IgA1 binding proteins (IgA1-BP) were eluted from the columns immobilized with desialylated IgA1 (DesIgA1/Sepharose) or desialylated/degalactosylated IgA1 (DesDeGalIgA1/Sepharose), respectively. The amounts of IgA1 and IgG and the glycoform of IgA1 in the IgA1-BP were detected by enzyme-linked immunosorbent assays (ELISAs) and were compared between patients with different pathological phenotypes and normal controls. The amount of IgA1 in IgA1-BP eluted from both columns was significantly higher in patients with both pathological phenotypes of IgAN than in normal controls. In IgA1-BP eluted from DesDeGalIgA1/Sepharose, the desialylation of IgA1 was much more pronounced in patients with both pathological phenotypes of IgAN than in normal controls, while the degalactosylation of IgA1 was much more pronounced only in patients with psIgAN than in normal controls. Furthermore, the amount of IgG in IgA1-BP eluted from DesDeGalIgA1/Sepharose was significantly higher in patients with psIgAN than in normal controls. In patients with psIgAN, the amount of IgG eluted from DesDeGalIgA1/Sepharose was much greater than from DesIgA1/Sepharose. In conclusion, self-aggregated deglycosylated IgA1 with or without IgG were associated with the development of IgAN.

Recent evidence has suggested that IgA1-containing macromolecules and the glycosylation of IgA1 in sera from patients with IgAN might involve the pathogenesis of IgAN. However, whether the different histological phenotypes can be attributed or not to the aberrant glycosylation of macromolecular IgA1 has not yet been elucidated. The aim of the current study is to investigate the glycosylation of IgA1 molecules in serum IgA1-containing macromolecules and their association with pathological phenotypes of IgAN. Sera was collected from 40 patients with IgAN and 20 donors. Twenty patients had mild mesangial proliferative IgAN, the remaining 20 had focal proliferative sclerosing IgAN. Polyethylene glycol 6000 was used to precipitate the macromolecules from sera of patients and controls. Biotinylated lectins were used in an enzyme-linked immunosorbent assay (ELISA) to examine different glycans on IgA1 molecules. The alpha2,6 sialic acid was detected by elderberry bark lectin (SNA) and the exposure of terminal galactose (Gal) and N-acetylgalactosamine (GalNAc) were detected by Arachis hypogaea (PNA) and Vilsa villosa lectin (VVL), respectively. The IgA1 glycans levels corrected by IgA1 concentrations were compared between patients and controls. Reduced terminal alpha2,6 sialic acid of IgA1 (79.89 +/- 25.17 versus 62.12 +/- 24.50, P = 0.034) was demonstrated only in precipitates from sera of patients with focal proliferative sclerosing IgAN, compared with those from controls. Reduced galactosylation of IgA1 molecules in precipitates was demonstrated in patients with both mild mesangial proliferative IgAN and focal proliferative sclerosing IgAN compared with normal controls (24.52 +/- 18.71 versus 76.84 +/- 32.59 P = 0.000 and 33.48 +/- 25.36 versus 76.84 +/- 32.59 P = 0.000). However, no significant difference was found in IgA1 glycosylation in the supernatant between patients and normal controls (P > 0.05). The glycosylation deficiency of IgA1 existed only in serum IgA1-containing macromolecules of patients with IgAN, and was associated with the renal pathological phenotypes. This suggests that aberrant glycosylation of IgA1 in serum macromolecules might be a contributory factor in the pathogenesis of IgAN.

IgA deposition in glomerular mesangium and the interaction with mesangial cells may well be the final common pathway to IgA nephropathy (IgAN). Altered hinge-region O-glycosylation of IgA1 from patients with IgAN may predispose to mesangial deposition and activation of the mesangial cell (MC) by IgA1, via a novel IgA1 receptor, and may be a key event in the pathogensis of IgAN. The aim of this study was to investigate the binding capacity and biological effects of IgA1, from both patients with IgAN and healthy controls, on human mesangial cells (HMC). Serum IgA1 was isolated with jacalin affinity chromatography, heated to aggregated form (aIgA1) and labelled with (125)I. Binding capacity of aIgA1 in vitro to cultured primary HMC was evaluated by a radioligand binding assay and the specificity of binding was determined by a competitive inhibition assay. Intracellular calcium release was studied by confocal analysis and phosphorylation of extracellular signal-regulated kinase (ERK) was determined by Western blot analysis. Change of cell cycles was demonstrated by flow cytometry and HMC proliferation was evaluated by direct cell count. Expression of TGF-beta mRNA and production of supernatant fibronectin were tested by RT-PCR and indirect competitive ELISA, respectively. aIgA1 from both the patients with IgAN and normal controls bound to HMC in a dose-dependent, saturable manner, and was saturated at approximately 500 pmoles per 0.5 ml of aIgA1. aIgA1 from patients with IgAN, however, bound to HMC at a higher speed and Scatchard analysis revealed a Kd of (8.89 +/- 2.1) x 10(-8)m versus (4.3 +/- 1.2) x 10(-7)m for aIgA1 from healthy controls (P = 0.026). The binding was specific because it was only inhibited by unlabelled Mono-IgA1 (mIgA1) and not by serum albumin or IgG. aIgA1 from patients with IgAN could induce release of intracellular calcium, phosphorylation of ERK, DNA synthesis, proliferation of HMC, expression of TGF-betamRNA and secretion of fibronectin in HMC in a similar time-dependent manner as aIgA1 from healthy controls, but the effects were much stronger and the durations were much longer (P < 0.05, respectively). We conclude that aIgA1 from patients with IgAN has a higher binding capacity to HMC and stronger biological effects than aIgA1 from healthy controls. This suggests that direct interaction between IgA1 and HMC and subsequential pathophysiological responses may play an important role in the pathogenesis for IgAN.

We tested whether rat and human MPO have similar antigenic determinants using 36 human MPO-ANCA positive sera, one mouse anti-rat MPO and four mouse anti-human MPO monoclonal reagents. Purified rat and human MPO were used in ELISA, with or without crossinhibition by preincubation with human MPO or irrelevant antigen in the liquid phase. Only one human MPO ANCA positive serum exhibited significant binding in rat MPO ELISA. This binding was poorly inhibited by preincubation with human MPO in the liquid phase, but was conserved after adsorption of non specific anti-rat activity in a chromatography column. Three mouse anti-human MPO IgG monoclonal antibodies did not recognize rat MPO. Only one mouse anti-human MPO IgA monoclonal antibody bound to rat MPO. This binding was poorly inhibited by preincubation with human MPO (35% at 2 micro g/ml). Conversely, the mouse anti-rat MPO monoclonal did not bind human MPO. We have concluded that: (1) Most human MPO-ANCA recognize antigenic determinants on human MPO which are absent on rat MPO. Therefore, human auto-antibodies bind to epitopes which recently appeared after species evolution; (2) Inversely, the mouse anti-rat MPO monoclonal do not bind human MPO. Therefore, rat MPO epitopes have been altered during species evolution; (3) Mice injected with human MPO preferentially develop antibodies against xeno-epitopes which are not present in rodents. Therefore, human MPO may not be the best antigen to raise ANCA in animal models and (4) A comparison of the amino acid sequences of rat and human MPO may help elucidate the major antigenic epitopes.

The human IgA Fc receptor (FcalphaR, CD89) plays an important role in host defence against invading pathogens. To study the properties of the receptor, 12 MoAbs, namely, MIP7c, MIP8a, MIP9a, MIP10c, MIP11c, MIP14b, MIP15b, MIP38c, MIP59c, MIP65c, MIP68b and MIP71a, were generated. The inhibitory effects of the antibodies on FcalphaR functions were tested. Three of the antibodies, MIP7c, MIP8a and MIP59c, were able to block up to 90% of soluble FcalphaR binding to IgA-coated beads and 70-80% of neutrophil phagocytosis of IgA immune complexes (IC). MIP8a could also inhibit IgA IC-induced neutrophil lactoferrin release, while cross-linking of FcalphaR with MIP8a and anti-mouse IgG could elicit neutrophil lactoferrin release. However, IgA IC-induced lactoferrin release required both extracellular calcium and magnesium, whereas MIP8a-induced release did not require extracellular magnesium and only partially required extracellular calcium. In addition, the time course of IgA IC-induced lactoferrin release was slow. Lactoferrin was not detectable if the incubation time was less than 0.5 h. In contrast, MIP8a-induced lactoferrin release was fast. Lactoferrin could be detected within 5 min of incubation. Therefore, neutrophil lactoferrin release induced by IgA IC differed from that induced by cross-linking of FcalphaR with MIP8a.

This study was aimed at verifying whether tissue transglutaminase (tTG) is the sole autoantigen eliciting anti-endomysial antibodies in coeliac disease (CoD) and investigating tTG expression in normal and coeliac mucosa. Twelve anti-endomysial-positive coeliac sera and 12 anti-endomysial-negative control sera (10 microl, diluted 1:5-1:400 in PBS pH 7.3) were preincubated with 10, 20 or 50 microg guinea pig liver tTG at 4 degrees C overnight. Monkey oesophagus tissue slides were then tested with tTG-preincubated and non-preincubated sera to search for IgA anti-endomysial reactivity by indirect immunofluorescence. Moreover, six sections of monkey oesophagus were incubated with an anti-tTG mouse MoAb, six sections with an anti-cytokeratin mouse MoAb and six sections with only 3% bovine serum albumin. Finally, endoscopic duodenal biopsy sections obtained from 12 patients affected by untreated CoD, six patients affected by treated CoD and 10 biopsied controls were immunohistochemically stained with a peroxidase-conjugated anti-tTG MoAb. Our results show that (i) preincubation with tTG abolished endomysial immunofluorescence in most, but not in all, coeliac sera; (ii) the incubation of anti-tTG MoAb with sections of monkey oesophagus resulted in an immunofluorescence staining pattern similar but not identical to that of anti-endomysial-positive coeliac sera; (iii) although tTG expression was present at muscularis mucosae and pericryptal fibroblast in both normal and coeliac mucosa, it was slightly more marked and evident in the latter. Although our absorption experiment was performed with guinea pig liver tTG, we confirm that tTG is the predominant antigen of endomysial antibodies, but we speculate that, at least in some patients, it is not the only one.

Diabetes mellitus is associated with vascular and neurological complications. We have investigated the presence of antibodies to phospholipids and to phospholipid binding plasma proteins in blood samples collected from 68 clinically and biochemically characterized type I and type II diabetic patients and from 252 healthy blood donor controls. Each sample was analysed for antibodies to three phospholipids (cardiolipin, phosphatidylserine and phosphatidylethanolamine), the antibody isotypes (IgA, IgG and IgM), and whether antibody activity was plasma protein-dependent. Patients were considered to have anti-phospholipid antibodies when one or more of these 18 tests was found above predetermined control values. The results of these experiments revealed an increased incidence of anti-phospholipid antibodies in diabetic patients compared with control subjects. The incidence of IgA isotype to phosphatidylethanolamine was higher than the incidence of other isotypes to other phospholipids, and their reactivities were independent of phospholipid-associated proteins. In addition, these antibody findings were studied for associations with prothrombin degradation products, activated factor VII and activated protein C, and with the incidence of diabetic complications. The anti-phosphatidylethanolamine antibody association with proliferative retinopathy was significant.

IgA nephropathy (IgAN) is defined by the predominant deposition of IgA immune complexes (IC) in the glomerular mesangium. Interaction between IgA immune complexes and mesangial cells (MC) could be a linchpin for the genesis of IgAN. We studied the modulation of MC expression of IgA receptors (Fc alphaR) by selected cytokines. Binding of 125I-IgA to quiescent human MC showed 2.55 x 10(5) sites/cell with an affinity (Ka) of 3.2 x 10(7) M(-1). Addition of selected recombinant cytokines had no significant influence on Ka, but increased the number of sites/cell relative to unstimulated cells. Northern hybridization using the pHuFc alphaR cDNA probe showed time-dependent increases in mRNA expression in stimulated versus control cells. IL-6 and tumour necrosis factor-alpha (TNF-alpha) had a biphasic effect on the Fc alphaR mRNA level; at 48 h, IL-6 increased steady state mRNA levels about six-fold relative to control, TNF-alpha increased mRNA four-fold, and interferon-gamma (IFN-gamma) induced Fc alphaR mRNA two-fold. By reverse transcriptase-polymerase chain reaction (RT-PCR), the Fc alphaR expressed on human MC appears highly homologous to that expressed by U937 cells. Altered Fc alphaR expression in response to cytokines may influence the pathogenesis of IgAN by affecting deposition and/or clearance of IgA-IC in the mesangium.

Transfer of 2 x 10(5) congenic or semiallogenic purified TCR alphabeta+ CD4+ T cells to SCID mice leads to an infiltration of the recipient gut lamina propria and epithelium with a donor-derived CD4+ T cell subset which induces a lethal inflammatory bowel disease (IBD) in the recipients. In contrast, IBD was not observed in SCID mice transplanted with unfractionated splenic cells. The earliest detectable pathological changes after CD4+ T cell transfer were proliferation and hypertrophy of the entire colonic epithelial layer, including increased mitotic activity, increased expression of epithelial nuclear proliferation antigen, and elongation of the crypts. Later on, massive mononuclear cell infiltration, hypertrophy of all layers of the colon and occasional epithelial ulcerations were observed. At this stage, accumulations of IgA, IgM and small numbers of IgG1-, IgG2- and IgG3-secreting plasma cells were present in the lamina propria of both the small and large intestine. We conclude that low numbers of intraveneously transferred CD4+ T cells induce IBD in SCID mice. In the late stages of CD4+ T cell-induced IBD, the colonic lamina propria becomes infiltrated with macrophages, neutrophils and plasma cells secreting IgA, IgM, and to a lesser degree IgG antibodies which might play an accessory role in the pathogenesis of IBD.

Immunoglobulin-secreting cells (ISC) in the peripheral blood are active effectors of the human immune defence system on their way to their site of action. We combined immunomagnetic cell separation and ELISPOT to study the expression of maturation markers and homing receptors (HR) on these cells in healthy volunteers. The results revealed that although highly differentiated, peripheral blood ISC are remarkably heterogeneous both with respect to their expression of maturation markers and HR. Moreover, significant differences were demonstrated between the various isotypes. Fewer IgA-secreting cells expressed both markers of early maturation (HLA-DR, HLA-DQ, CD20, and CD21) and of more mature B cells or plasma cells (CD28, CD38, and alpha-syndecan) compared with IgG- and IgM-secreting cells. IgA-secreting cells also showed the lowest proportion of cells positive for the peripheral lymph node HR, L-selectin, or the skin HR, cutaneous lymphocyte antigen (CLA). By contrast, the expression of mucosal HR on IgA-secreting cells did not reveal a more pronounced homing attitude to mucosal tissues than IgG- or IgM-secreting cells. We conclude that peripheral blood ISC are a heterogeneous cell population and that IgA-secreting cells seem to differ from the other isotypes both in respect of expression of HR and the various maturational markers studied.

The mechanism of IgA deposition in the kidneys in IgA nephropathy is unknown. Mesangial IgA is of the IgA1 subclass, and since no consistent antigenic target for the IgA1 has been described, we have investigated the glycosylation of the molecule, as a potential non-immunological abnormality which may contribute to its deposition. IgA1 is rich in carbohydrate, carrying N-linked moieties in common with IgG, but also O-linked sugars, which are rare in serum proteins, and not expressed by IgG or IgA2. Lectin binding assays were designed to examine the expression of terminal galactose on the N-linked carbohydrate chains of purified serum IgG and IgA1, and the O-linked sugars of IgA1 and C1 inhibitor (one of the very few other serum proteins with O-linked glycosylation). No evidence was found for abnormalities of N-linked glycosylation of either isotype in IgA nephropathy compared with matched controls. However, in IgA nephropathy, reduced terminal galactosylation of the hinge region O-linked moieties was demonstrated; this was not seen in C1 inhibitor, which showed normal or increased galactosylation of the O-linked sugars. This abnormality of IgA1 has considerable implications for the pathogenesis of IgA nephropathy, since the O-linked sugars lie in an important functional location within the IgA1 molecule, close to the ligand of Fc receptors. Changes in the carbohydrates in this site may therefore affect interactions with receptors and extracellular proteins, leading to anomalous handling of the IgA1 protein in this condition, including failure of normal clearance mechanisms, and mesangial deposition.

CVID is an acquired disease with low serum immunoglobulins. Patients are classified into three groups on the ability of their B cells to secrete IgM and IgG in vitro in response to IL-2. We have now extended this patient classification to include IgA secretion and to assess whether the patients have IgA+ B cells in the circulation. B cells from almost all of the CVID patients studied were unable to secrete any IgA in vitro, with or without IL-2, although all B cells tested from normal donors showed some spontaneous IgA secretion. Thus, the defect with IgA secretion is more profound than with the secretion of IgM or IgG. Despite this failure to secrete IgA, using flow cytometry techniques all CVID patients tested were found to have IgA+ cells (expressed as a percentage of B cells) within the normal range. This suggests that CVID may involve a defect in the secretion of immunoglobulin isotypes rather than a defect in isotype switching.

In order to gain insight into the production and clinical significance of IgA rheumatoid factor (IgA-RF) in mucosal fluids of patients with rheumatoid arthritis (RA), we examined tear fluid, saliva and serum from 80 patients with RA. Significant correlations were found between IgA-RF levels in tear fluid and saliva (P = 0.002, r = 0.57), saliva and serum (P < 0.001, r = 0.79), and serum and tear fluid (P < 0.001, r = 0.31). No significant correlations were found between total IgA levels in these fluids. Comparison between circulating and mucosal IgA-RF levels after correction for total IgA, revealed that mucosal IgA-RF levels are on average 2.5 times higher than circulating IgA-RF levels. Analysis of IgA-RF specificity showed that lacrimal and salivary IgA-RF reactivity with various IgG subclasses is similar and differs from serum IgA-RF specificity. These results indicate local production of IgA-RF in salivary and lacrimal glands and support the view of a common origin of IgA-RF producing B cells present in mucosal tissues. Mucosal and circulating levels of IgA and IgA-RF were not associated with tests that quantify tear fluid production. This indicates that mucosal and circulating levels of IgA and IgA-RF in patients with RA cannot be regarded as markers for the development of secondary Sjögren's syndrome.

Secretory immunoglobulin A (IgA) was determined by means of an enzyme-linked immunosorbent assay (using as capture antibody an MoAb specific for secretory component) in saliva and serum from 46 patients with IgA mesangial nephritis (IgAGN), 36 with an idiopathic nephrotic syndrome (INS), 30 with an idiopathic membranous nephropathy (MGN) and 40 healthy controls. Secretory IgA levels were elevated in both saliva and serum of patients with primary glomerulonephritis (P < 0.05; Mann-Whitney test) regardless of the histological type of the primary glomerulonephritis. Salivary IgA1 and IgA2 levels were increased in the saliva of patients with IgAGN, INS and MGN (P < 0.05; Mann-Whitney test). The monomeric/total IgA ratio, and interferon-gamma and soluble IL-2 receptor levels, in saliva did not differ between the patients and controls (P > 0.05; Mann-Whitney test). We conclude that the mucosal immune system is activated in forms of glomerulonephritis other than IgAGN.

In the present study we have investigated the clearance kinetics and tissue distribution of monomeric (m) IgG and soluble aggregates of IgG (AIgG) and immune complexes (IC) in normal and Kupffer cell (KC) depleted rats. In normal rats, clearance of mIgG occurred in a biphasic manner with a first half-life (T1/2) (T1) of 36.3 +/- 6.3 min and a second T1/2 (T2) of 168.4 +/- 4.7 min. AIgG composed of 20-27 IgG molecules per aggregate were cleared significantly faster than mIgG with a T1 of 2.5 +/- 0.1 min and a T2 of 32.5 +/- 5.6 min. KC depletion did not have a significant effect on the clearance rate of mIgG (T1: 33.4 +/- 8.9 min; T2; 159.5 +/- 12.5 min), while clearance of AIgG was delayed significantly with T1 4.8 +/- 0.7 min and T2 41.2 +/- 3.2 min. Eight minutes after injection, 77% of AIgG was found in the liver in normal rats while 62% was found in the liver of KC-depleted rats. Double immunofluorescence studies indicated that AIgG in the liver was associated with KC and endothelial cells (EC) in normal rats. In KC-depleted rats, AIgG was strongly associated with EC. A similar staining pattern was observed when IgG-immune IC were administered. The clearance of AIgG in KC-depleted rats was inhibited fully by pre-administration of high concentrations of IgG but not by pretreatment with IgA. asialofetuin (ASFe) or ovalbumin (OVA). Aggregated F(ab')2IgG was cleared with a comparable rate to mIgG from the circulation, again suggesting Fc gamma receptor-mediated elimination of AIgG by EC. There was a reduced degradation of AIgG in rats depleted of KC as compared with normal rats. These data suggest binding and degradation of AIgG by EC in vivo.

Using modified ELISA and spot-ELISA, which permit the parallel determination of heavy chain subclass and the presence of covalently linked J chain, we analysed IgA found in cell culture supernatants or secreted by individual cells from peripheral blood, spleen, bone marrow, gingiva and synovial tissue, with respect to its polymeric or monomeric IgA form (pIgA, mIgA) and IgA1 or IgA2 subclass. The ELISA for determination of J chain in tissue culture supernatants was specific and highly sensitive (detection limit in pg). The results demonstrated that IgA1-producing cells predominated in the tissues examined, and that J chain could be detected in association with the majority of IgA1 and IgA2 secreted by individual cells. With respect to the frequency of cells secreting polymeric, J chain-containing IgA, only 20-30% of cells from the bone marrow were engaged in the synthesis of PIgA. In other tissues the frequency of cells secreting pIgA1 and pIgA2 was considerably higher. Peripheral blood mononuclear cells secreting pIgA2 were easily inducible during stimulation with T cell-dependent pokeweed mitogen, whereas Epstein-Barr virus-transformed cells secreted preferentially mIgA1. When the frequencies of pIgA-, pIgA1- or pIgA2-secreting cells (determined by spot-ELISA technique) from different tissues were correlated with the proportion of pIgA to mIgA (and IgA subclasses) secreted in tissue culture supernatants, data obtained suggest that many individual IgA-producing cells could be engaged in simultaneous secretion of mIgA and pIgA.

Growth and differentiation of cord blood B cells were studied using T cell-depleted populations. In the absence of in vitro activation, cord blood B cells proliferated in response to cytokines including interleukin-2 (IL-2) and interleukin-4 (IL-4); anti-mu-stimulated cord B cells had a lesser response to IL-2 than adult cells. IgM synthesis by cord blood B cells was enhanced by interleukin-6 (IL-6) and decreased by IL-2. In cultures activated by Staphylococcus aureus Cowan I (SAC), cord blood B cells produced lesser increases in IgM than adult B cells regardless of the cytokine added. Cord blood B cells produced no IgG or IgA with any cytokine preparation with or without SAC activation. Supernatants of cord blood T cells pulse-stimulated with phytohaemagglutinin and phorbol myristate acetate contained less IL-2 and IL-6 and had less growth and differentiation activity than adult T cell supernatants. The results confirm a limited cord blood B cell response and also suggest a limitation in production of B cell stimulatory lymphokines by cord blood T cells.

Mucosal ulceration of the oral, and to a lesser extent genital tissues is an essential feature of Behçet's syndrome and is associated with changes in the IgA class of immune responses. Indeed, a significant increase in the proportion of cytophilic IgA1 was found in circulating CD8 and CD4 cells (P less than 0.01), with a corresponding decrease in IgA-Fc receptors on these T cells. Furthermore, 30-40% of the cytophilic IgA1 on T cells may have been of the polymeric secretory type and the rest of the monomeric variety. IgA isotype of B cells was also significantly increased (P less than 0.001), without an overall change in circulating B cells. However, a surprising finding was the significant up-regulation of gamma delta T cell receptor in the CD8 (P less than 0.01) in the absence of a change in the proportion of alpha beta T cell receptor. The results suggest that some common microbial antigen might initiate at the mucosal surface an immune defence reaction characterized by T cells with gamma delta receptors and IgA-specific B cells. However, IgA1 bound to circulating T cells may down-regulate the central T cell function.

Recently we reported on the participation of dietary antigens in the pathogenesis of IgA nephropathy. Particularly, the glomerular deposition of dietary antigens (casein, soybean protein, rice protein) was observed in some cases with IgA nephropathy. It is known that human urinary pepsin (HUP) prepared from the urine of a healthy adult acts as an immune complexase at neutral pH. In this study, the effects of incubation with HUP on the glomerular deposition of dietary antigens was investigated by an immunofluorescence technique in 48 patients with IgA nephropathy, four patients with Henoch-Schönlein purpura nephritis and 69 with other glomerulonephritides. In 33 cases (68.8%) soybean protein and in 12 (25.0%) casein was found in biopsies of patients with IgA nephropathy. After a 3-day incubation with HUP there was no change in the deposition of IgA in 12 cases, a decrease in 22 and complete elimination in 14 cases. The deposits of soybean protein increased in 12 cases, did not change in 20, decreased or was abolished in five and was positively converted in 11. Exceptionally, in five patients treated with HUP, the deposition of soybean protein appeared concomitantly with the disappearance of IgA deposits. Such a concomitance did not occur either in other glomerulonephritides or in studies of other dietary antigens. Thus it appears that soybean protein may be deposited in the glomerular mesangium as an antigen in some patients with IgA nephropathy.

The relative distribution of cells producing subclasses of IgG or IgA was examined by immunohistochemistry in lymph node biopsy specimens from 15 adults with persistent generalized lymphadenopathy due to infection with HIV and from 11 adults with lymph nodes enlarged from other causes. The IgG1 immunocyte fraction was significantly higher and the IgG2 fraction significantly lower associated with HIV infection than in the controls. The proportions of cells producing IgG3, IgG4, IgA I or IgA2 were similar in the two lymph node categories. The altered IgG-subclass distribution of immunocytes in HIV lymphadenopathy concorded with the serum levels that were significantly elevated for IgG1 and lowered for IgG2 compared with a panel of blood donor samples.

Antibodies to 11 different Escherichia coli (E. coli) serotypes were determined by indirect haemagglutination in 53 epileptics treated with diphenylhydantoin (DPH) and in 53 matched controls. Agglutinins were found more often and at higher titres in epileptics (83 . 4%) than in controls (51 . 7%) (P less than 0 . 05). No correlation between the occurrence of E. coli agglutinins and the serum concentration or clearance of DPH, or abnormal biochemical liver tests could be established. E. coli antibodies were predominantly of the IgM classes, and the percentage of positive reactions was slightly, but not significantly, higher in epileptics with elevated serum IgM (88 . 2%) than in those with normal IgM (83 . 1%) (0 . 20 greater than P greater than 0 . 10). The increased incidence of E. coli agglutinins could not be explained by a low serum IgA concentration which may suggest an impaired secretory immune function. The incidences of positive reactions in epileptics with subnormal (less than 37 mg/100 ml), normal (37-285 mg/100 ml) and elevated (greater than 285 mg/100 ml) serum IgA values were 73 . 7, 84 . 7 and 93 . 7% respectively (0 . 01 greater than P greater than 0 . 001). It is suggested that DPH can both suppress and stimulate the humoral immune response in humans, and that genetic or acquired factors may be of importance for the development of modified immune responses.

Most patients with late-onset bypogammaglobulinaemia have normal numbers of B cells. In addition, some of these patients have been shown to have circulating T cells which suppress immunolobulin production in vitro. From our present studies on a large series of patients it appears that these two findings distinguish two separate groups of patients. The majority of the patients have circulating B cells which are 'immature' in the sense that they produce IgM but very little IgG or IgA in vitro. These patients also fail to produce IgA in vivo but their T cells show normal function in vitro and normal T cell markers. Patients with the second form of the disease, including those with associated thymoma, have very few circulating B cells, and relatively preserved IgA production in vivo. In our series it was only this minority of patients whose T cells showed abnormal markers and increased suppressor activity in vitro.

The feasibility of using human cytomegalovirus (CMV)-specific IgA antibody determinations as a signal for early detection of recurrent CMV infections in eight renal transplant recipients was analyzed. Solid phase radioimmunoassay (RIA), enzyme-liked immunosorbent assay (ELISA) and immunoperoxidase assay (IPA) techniques were used for IgA antibody determinations. In parallel, IgG antibodies to CMV were studied by immunoperoxidase assay. A significant rise of CMV-specific IgG antibody titre was observed in all of these patients between 5 and 53 weeks post-transplantation. CMV-specific IgA antibody production was detected close to the time a rise in CMV IgG antibody was observed in seven out of eight patients studied by RIA and ELISA, and in six out of eight patients studied by IPA. In two patients specific CMV IgA antibodies were detected by all three methods before a significant rise of CMV IgG antibody titre was demonstrated. In these patients CMV IgA was detected by RIA earlier than by ELISA and IPA. The potential application of CMV-specific IgA antibody determination for early detection of recurrent CMV infection in renal transplant patients is discussed.

Complement activation may play an important role in renal injury associated with glomerular deposition of IgA immune complexes. The ability of naturally occurring human IgA immune complexes (IgA-IC) and covalently cross-linked human IgA oligomers (X-IgA) to activate complement were examined in vitro and in vivo. Large-sized IgA-IC were isolated from a patient's serum by affinity purification (Jacalin-Sepharose) and gel chromatography. Stable X-IgA were prepared by chemical cross-linking with a heterobifunctional reagent, N-succinimdyl 3-(2-pyridyl-dithio) propionate (SPDP). Treatment of fresh normal human serum with large amounts of either IgA-IC or X-IgA failed to activate C3. The C3 consumption was measured immunochemically by the decrease of the B antigen on the native C3 and by the generation of iC3b. Addition of these complexes to serum did not result in cleavage of factor B. Administration of human IgA-IC or X-IgA to mice, killed after 6 h, resulted in glomerular deposition of IgA. Despite the presence of intense glomerular IgA deposits no C3 was detected. Collectively, these findings suggest that neither soluble nor renal localized human IgA complexes activate complement.

High anti-staphylolysin activity was found in the serum from a patient with multiple myeloma. The activity was confined to the monoclonal IgA protein and was dependent on an intact Fc part of the molecule. An Fc-dependent interaction with protein A was also demonstrated. In addition, a part of the monoclonal IgA was found to be complexed with alpha2-macroglobulin. The results imply an unusual Fc-reactivity of the IgA protein.

Intestinal mucosal immunoglobulin secretion in vitro has been studied by culture of endoscopic biopsy tissues obtained from various sites along the gastrointestinal tract. IgA and IgM were the predominant immunoglobulins produced by intestinal tissues distal to the stomach and secretion of both reached a maximum in the small intestine of normal individuals. In patients with inflammatory bowel disease, characteristic alterations in immunoglobulin production were observed in cultures of large intestinal tissue. In ulcerative colitis (UC), significant reductions in IgA secretion (P less than 0.03) occurred in the rectum during remission and IgM in the colon in active disease (P less than 0.01). However, in active Crohn's disease (CD), rectal IgM secretion was enhanced (P less than 0.004) and IgA diminished (P less than 0.01). IgG secretion was increased throughout the colon in UC especially in distal sites, due largely to significant increases in pre-formed immunoglobulin (P less than 0.02). Similar total increases were observed in colonic tissues from patients with CD, although IgG synthesis in biopsies from rectal sites was normal. These findings suggest that specific abnormalities of intestinal mucosal immunoglobulin synthesis occur in patients with both UC and CD.

A comparative study of membrane expression of immunoglobin (Fc) receptors on human eosinophils and neutrophils has been undertaken using human IgG-1, IgG-2, IgG-3, IgG-4, IgA-1, IgA-2, IgM, IgD and IgE myeloma proteins. Sheep erythrocytes (E) coated with either human IgG-1, IgG-2, IgG-3 or IgG-4 myeloma proteins formed rosettes with human neutrophils and eosinophils. Proportionally more (1 1/2-2 times) rosettes were observed with neutrophils compared to eosinophils. In contrast, E-IgE bound to eosinophils (but not to neutrophils) to a degree that was comparable to E-IgG-1. Although IgG and IgE rosettes were inhibited by aggregates prepared from their corresponding myeloma protein there was no evidence that eosinophils and neutrophils have distinct receptors for IgG subclasses. Cells from four patients with hypereosinophilia were separated on density (metrizamide) gradients. The percentages of E-IgG-1 and E-IgE rosettes with normal- and light-density eosinophils were similar. Neutrophils, but not eosinophils, also bound significantly more E-IgA-1 and E-IgA-2 than the E-human albumin (E-Alb) control. In contrast, neutrophil and eosinophil rosette formation with E-IgM and E-IgD was not significantly different from E-Alb or E alone. These experiments indicate that human neutrophils and eosinophils bind homologous IgG subclass myeloma proteins, eosinophils, but not neutrophils, bind E-IgE with a similar avidity to that observed with E-IgG1, neutrophils, but not eosinophils, readily express demonstrable receptors for IgA-1 and IgA-2 and neither neutrophils nor eosinophils form E-IgM or E-IgD rosettes in greater numbers than the E-Alb controls.

Expression of receptors for IgA (Fc alpha Rs) was investigated on a panel of 35 human B cell lines by labelling with human secretory IgA (0.5 mg/ml) and flow cytometry analysis after staining with fluoresceinated goat anti-human secretory component and/or anti-alpha chain F(ab')2 fragments. Receptors for IgA could be demonstrated on one out of nine Burkitt's lymphoma cell lines, three out of five myeloma cell lines and five out of 21 lymphoblastoid cell lines. The percentage of Fc alpha R-positive cells within the same B cell line varied upon repeated examination. Human dimeric IgA1 lambda myeloma protein revealed the same number of IgA receptor positive cells as did secretory IgA, whereas monomeric IgA did not bind to Fc alpha R. Detection of Fc alpha R was not inhibited when the tests were carried out in the presence of human dimeric IgG, IgM, asialo-orosomucoid, and secretory component but it was abrogated by pre-treatment of the cells with trypsin. The binding characteristics of Fc alpha Rs were studied on the myeloma cell line Esteve, using 125I-labelled human dimeric IgA and secretory IgA. The binding was dose-dependent with rapid kinetics and specific inhibition by unlabelled secretory IgA. Scatchard plot analysis resulted in an equilibrium constant K ranging from 3.2 to 4.7 x 10(6) M/l. No correlation was observed between Fc alpha R expression and differentiation stage, monoclonality, polyclonality of the cell lines, or Ig class produced by the B cells.

Specific surface receptors for the Fc region of human immunoglobulin A (Fc alpha R) were identified on the thymus cells of all 15 patients affected by myasthenia gravis. A large variation of the percentage of Fc alpha R bearing thymus cells was recorded (9 . 5-66 . 0%; mean value 27 . 9 +/- 17 . 8) the high values being associated in most cases with the thymus hyperplasia and the low values with a lipomatosis of the organ. Even the lowest percentage of Fc alpha R bearing cells recorded in myasthenia gravis are considerably higher than those found in normal subjects (0 . 4-4 . 1; mean value 1 . 8) demonstrating that the expression of Fc alpha R on the thymus cells is the consequence of the disease. No attempts were made to identify the cell population bearing Fc alpha R but indirect results indicate that the cells belong to E-rosette forming population, and, in some cases, to a null cell population devoid of the ability to form E and EAC rosettes, Fc alpha R can bind various IgA ligands and the binding is confined to a small fraction of IgA (not more than 5%) considered as cytophilic IgA.

IgA is the prototype of a secretory immunoglobulin and usually is present in external fluids in proportions greater than in serum. Previous studies have suggested that IgE also is a secretory immunoglobulin. We measured IgE, IgA and albumin levels in nasal washings (NW), parotid fluids (PF) and serum in seventeen individuals. In secretions, all of these proteins were measured by radioimmunoassay (RIA). In serum, IgE was measured by RIA, IgA by a turbidimetric method, and albumin by radial immunodiffusion. IgA was detected in NW and PF in all individuals and IgA/total protein ratios in both of these secretions exceeded the corresponding ratios in serum (P less than 0-01). In contrast, IgE was detected only in NW from atopic subjects and IgE/total protein ratios were less than those in serum (P less than 0-02). IgE was not detected in the PF in any of the seventeen subjects. When the proportion (protein/total protein) of IgA, IgE, or albumin in NW was expressed as a percentage of the proportion in serum, IgA and IgE were increased when compared to albumin (P less than 0-01 and P less than 0-03, respectively). These results support the view that IgE is a secretory immunoglobulin in that it is present in NW in proportions greater than expected if it diffused from serum. However, IgE differs from secretory IgA in that ratios of IgE to total protein are greater in serum than in NW. This difference is discussed in terms of local production of IgE and passive diffusion into serum and nasal secretions. Finally, the observation that IgE is present in one external secretion while absent from another indicates that external secretions may be subdivided into two classes on the basis of their content of IgE.

The interaction of cationic proteins with IgG, IgA and IgM were investigated by solid phase radioimmunoassay. All these immunoglobulins showed avid binding, IgM giving the strongest reaction, followed by IgA and then IgG. Fc fragments of IgG gave binding, but F(ab')2 fragments from the three main Ig classes did not, showing that the Fc region is the active part of the molecule. The effects of changes of ionic strength and pH are compatible with the interaction being ionic, and are similar to those seen between immunoglobulins and both Clq and cationic ion exchange gels. The addition of other serum proteins resulted in marked inhibition of the interaction. These phenomena are likely to have fundamental significance for the understanding of interactions of immunoglobulins in vivo and in vitro.

The observation of negatively charged IgA in the mesangium of patients with primary IgA nephropathy (IgA-GN) prompted us to study the charge of serum IgA in IgA-GN, Henoch Schönlein purpura (HSP), alcoholic liver cirrhosis (ALC), membranous nephropathy (MGN) and systemic lupus erythematosus (SLE). Since no abnormal distribution of IgA isoelectric points was detected by isoelectric focusing studies, we developed a sensitive charge-dependent assay using plates coated with either cationized BSA (cBSA) or poly-L-lysine. In 15 IgA-GN sera, the amount of IgA reacting specifically with cBSA (cBSA-IgA) was almost linearly correlated with the poly-L-lysine-binding IgA (r = 0.97, P = 0.0006), suggesting that both assays detect charge-dependent interactions and thus probably measure anionic IgA. Significantly high serum levels of cBSA-IgA were observed in 56% of IgA-GN patients and in 40% of ALC patients. In contrast, normal serum levels of cBSA-IgA were detected in HSP, MGN and SLE. Both, the mono- or polymeric IgA bound to cBSA in a patient's serum studied. Contrasting with the presence of anionic IgA, no increase of cBSA-IgG was observed in IgA-GN. IgA rheumatoid factor (IgA-RF) assay showed high levels in IgA-GN (39%) and in ALC (25%). IgA-RF levels did not correlate with the amount of cBSA-IgA. When 18 patients with IgA-GN were tested after kidney transplantation, increased levels of cBSA-IgA and/or IgA-RF were found to be associated with the recurrence of mesangial IgA deposits in the graft. This suggests that both negatively charged IgA and IgA-RF may play a role in the recurrence of mesangial IgA deposits.

The human liver probably removes circulating polymeric IgA by two routes: (1) secretory component-mediated endocytic transport of polymeric IgA from portal tract blood vessels into bile across biliary epithelial cells and (2) uptake with possible catabolism by hepatocytes and/or sinusoidal phagocytic cells by uncharacterized receptor(s). Failure of either clearance mechanism due to liver disease results in elevated serum polymeric IgA levels. In patients with cholestasis, the raised serum polymeric IgA concentration is due to reflux of biliary secretory immunoglobulin into the blood.

Compared to normal children, pre-natally infected infants had significantly elevated IgM concentrations throughout the first year of life and elevated IgA levels for the first 6 months. In contrast, IgG levels dropped significantly below normal at 3 months post-partum. Infants born without overt disease but with elevated IgM were found to have precocious development of serum IgA when compared to control children. Levels of IgA and IgG in tears were markedly reduced in symptomatic children at 3 months of age. Lymphocyte response in vitro to phytohaemagglutinin and pokeweed mitogen was low in symptomatic infants. These results suggest that intratuerine infection may result in multiple immunological abnormalities.

Several patients with Paget's disease have been reported to have monoclonal IgM proteins in their serum. We have systematically studied 26 patients with Paget's disease severe enough to require diphosphonate therapy. Five of these patients were found to have isolated elevations of serum IgM which were well outside the normal range for their age, sex and race. An additional patient had elevations of both IgG and IgM. These patients seem to represent a subset of Pagetics. They do not differ from the remainder of the group with respect to severity or extent of disease, response to therapy, presence of intercurrent disease or age. They do have a different male to female ratio. These observations suggest that some patients with Paget's disease may have disordered immune regulation or that they may be undergoing a primary response to an agent involved in the aetiology or pathogenesis of the disease.

IgG2 was the predominant specific antibody isotype in mice chronically infected with Y strain Trypanosoma cruzi; IgG1 and IgG3 antibodies were absent or present only at very low levels. Isotype analyses of the acute phase of infection confirmed no early production of IgG1 or IgG3 and no failure in the switch from IgM to IgG. In vivo passive transfer studies of immune serum fractions showed protection to be associated only with the IgG2 isotype. A characteristic specific anti-T. cruzi IgG isotype profile (IgG1, IgG3, greater than IgG2, IgG4) was detected in a majority (39 of 50) of sera from Chagas' disease patients.

A reproducible immunocyto-adherence assay has been developed to study the modulation of Fc receptors for IgA (Fc alpha R), using a murine T cell hybridoma (T2D4), which expresses Fc receptors for all known isotypes of secreted immunoglobulins. By using sheep red blood cells coated with the hapten 2-4-6 trinitrophenyl (TNP), as indicator cells, and a murine monoclonal IgA (MOPC 315) antibody with anti-TNP activity, we were able to study the Fc alpha R on T2D4 cells. We found that: (a) murine Fc alpha R can bind human monoclonal IgA, and this binding is isotype specific since it was inhibited by human monoclonal IgA but not by human monoclonal IgG or IgM; (b) the expression of murine Fc alpha R is unducible by human monoclonal IgA, and this effect is isotype specific since it is not observed with human monoclonal IgM or IgG (c) sera from patients with IgA nephropathy can also induce Fc alpha R expression; by contrast, no induction was observed with normal human sera, (d) in one serum from an IgA-nephropathy patient, the inducer factor was characterized by affinity chromatography on anti-IgA-Sepharose and by gel filtration: high molecular weight IgA, probably IgA aggregates or immune complexes were recognized to be responsible for the induction of murine Fc alpha R expression.

Clearance kinetics and renal deposition of soluble IgA immune complexes (IgA IC) were examined to determine the nephritogenic potential of IgA molecular form in mediating experimental IgA nephropathy. The immune complexes were prepared by mixing purified radiolabelled monomeric (mIgA) or polymeric (pIgA) IgA anti-dinitrophenyl (DNP), derived from MOPC-315 myeloma, with DNP conjugated Ficoll (DNP8-Ficoll). Clearance of IgAIC from the circulation was curve fitted by two exponential components. The first component was similar for both mIgA IC and pIgA IC. The second component was slightly more rapid for mIgA IC than for pIgA IC. Immunofluorescence studies, however, showed that only pIgA IC deposited in the kidneys. Analysis of IgA IC by gradient polyacrylamide gel electrophoresis indicated that mIgA formed only small latticed complexes. The critical role of IgA IC lattice size in renal deposition was confirmed by demonstrating that large latticed mIgA IC, prepared by covalent cross-linking mIgA with a specific affinity labelling antigen, deposited in the kidneys in a pattern similar to pIgA IC. Our results suggest that the monovalency of mIgA is responsible for its inability to form a large latticed nephritogenic complexes.