Systemic Activation of Neutrophils by Immune Complexes Is Critical to IgA Vasculitis.

In IgA vasculitis (IgAV) perivascular deposition of IgA1 immune complexes (IgA-ICs) is traditionally considered the fundamental trigger for polymorphonuclear neutrophil (PMN)-mediated damage. We propose that IgA-IC deposition, although mandatory, is not sufficient alone for IgAV. Serum IgA-IC levels and IgA-IC binding to PMNs were quantified in IgAV patients and controls. Activation of PMNs was evaluated by neutrophil extracellular trap (NET) release, adherence, and cytotoxicity assays and in a flow system to mirror conditions at postcapillary venules. In vitro results were related to findings in biopsies and a mouse vasculitis model. During acute IgAV flares we observed elevated serum levels of IgA-ICs and increased IgA-IC binding to circulating PMNs. This IgA-IC binding primed PMNs with consequent lowering of the threshold for NETosis, demonstrated by significantly higher release of NETs from PMNs activated in vitro and PMNs from IgAV patients with flares compared with surface IgA-negative PMNs after flares. Blocking of FcαRI abolished these effects, and complement was not essential. In the flow system, marked NETosis only occurred after PMNs had adhered to activated endothelial cells. IgA-IC binding enhanced this PMN tethering and consequent NET-mediated endothelial cell injury. Reflecting these in vitro findings, we visualized NETs in close proximity to endothelial cells and IgA-coated PMNs in tissue sections of IgAV patients. Inhibition of NET formation and knockout of myeloperoxidase in a murine model of IC vasculitis significantly reduced vessel damage in vivo. Binding of IgA-ICs during active IgAV primes PMNs and promotes vessel injury through increased adhesion of PMNs to the endothelium and enhanced NETosis.

Recurrence of IgA nephropathy after kidney transplantation: experience from the Swiss transplant cohort study.

BACKGROUND: Recurrence of IgA nephropathy (IgAN) after kidney transplantation occurs in about 30% of patients. The relevance of recurrence for the long-term graft survival is expected to increase, since graft survival continues to improve. METHODS: In a nested study within the Swiss Transplant Cohort Study the incidence of IgAN recurrence, predictive factors, graft function and graft and patient survival were evaluated. Serum concentration of total IgA, total IgG, Gd-IgA1 and IgA-IgG immune complex were measured using ELISA-based immunologic assays. RESULTS: Between May 2008 and December 2016, 28 women and 133 men received their kidney allograft for end-stage kidney disease due to IgAN in Switzerland. Over a median follow-up time of 7 years after transplantation, 43 out of 161 patients (26.7%) developed an IgAN recurrence, of which six (13.9%) had an allograft failure afterwards and further four patients (9.3%) died. During the same follow-up period, 6 out of 118 patients (5%) each experienced allograft failure or died without prior IgAN recurrence. After 11 years the risk for IgAN recurrence was 27.7% (95%-CI: 20.6-35.3%). Renal function was similar in patients with and without recurrence up to 7 years after transplantation, but worsened thereafter in patients with recurrence (eGFR median (interquartile range) at 8 years: 49 ml/min/1.73m2 (29-68) vs. 60 ml/min/1.73m2 (38-78)). Serum concentration of total IgA, total IgG, Gd-IgA1 and IgA-IgG immune complex within the first year posttransplant showed no significant effect on the recurrence of IgAN. Younger recipients and women had a higher risk of recurrence, but the latter only in the short term. CONCLUSIONS: Our study showed a recurrence risk of 28% at 11 years after transplantation, which is consistent with previous literature. However, the predictive value of known biomarkers, such as serum Gd-IgA1 and IgA-IgG IC, for IgAN recurrence could not be confirmed.

Differential expression of microRNA miR-150-5p in IgA nephropathy as a potential mediator and marker of disease progression.

Understanding why certain patients with IgA nephropathy progress to kidney failure while others maintain normal kidney function remains a major unanswered question. To help answer this, we performed miRNome profiling by next generation sequencing of kidney biopsies in order to identify microRNAs specifically associated with the risk of IgA nephropathy progression. Following sequencing and validation in independent cohorts, four microRNAs (-150-5p, -155-5p, -146b-5p, -135a-5p) were found to be differentially expressed in IgA nephropathy progressors compared to non-progressors, and patients with thin membrane nephropathy, lupus nephritis and membranous nephropathy, and correlated with estimated glomerular filtration rate, proteinuria, and the Oxford MEST-C scores (five histological features that are independent predictors of clinical outcome). Each individual microRNA increased the discrimination score of the International IgAN Prediction Tool, although due to the small number of samples the results did not reach statistical significance. miR-150-5p exhibited the largest amplitude of expression between cohorts and displayed the best discrimination between IgA nephropathy progressors and non-progressors by receiver operating curve analysis (AUC: 0.8). However, expression was similarly upregulated in kidneys with established fibrosis and low estimated glomerular filtration rates at the time of biopsy. Consistent with a more generic role in kidney fibrosis, in situ hybridization revealed that miR-150-5p was found in lymphoid infiltrates, and areas of proliferation and fibrosis consistent with the known drivers of progression. Thus, miR-150-5p may be a potential functional mediator of kidney fibrosis that may add value in predicting risk of progression in IgA nephropathy and other kidney diseases.

Macrophage interactions with collecting duct epithelial cells are capable of driving tubulointerstitial inflammation and fibrosis in immunoglobulin A nephropathy.

BACKGROUND: Tubulointerstitial fibrosis is a powerful predictor of future progression inimmunoglobulin A (IgA) nephropathy (IgAN). Proximal tubular epithelial cells (PTECs), in concert with infiltrating macrophages, are regarded as the agents provocateurs for driving this fibrotic process. However, evidence is now emerging for a contributory role of the distal nephron. The aim of this study was to examine the potential influence of macrophages on collecting duct epithelial cells (CDECs) and their combined role in the progression of IgAN. METHODS: CDECs were cultured with macrophage-conditioned media (MCM) generated from human monocyte cell lines U937 and THP-1 stimulated with or without 100 µg/mL galactose-deficient IgA1. CDECs were analysed for evidence of inflammation and fibrosis. RESULTS: Staining of IgAN biopsies for CD68+ macrophages revealed the presence of macrophages juxtaposed to collecting ducts and within their lumina. CDEC exposed to MCM from IgA1-stimulated THP-1 cells (THP-1-IgA-MCM) exhibited markedly increased expression of neutrophil-associated gelatinase (NGAL) and proinflammatory cytokinesinterleukin (IL)-1ß, tumour necrosis factor-α, IL-6 and IL-8 compared with MCM from non-IgA-stimulated THP-1 cells (THP-1-MCM). U937-IgA-MCM increased fibronectin levels and reduced E-cadherinmRNA expression. THP-1-IgA-MCM-derived exosomes induced similar increases in NGAL and cytokine expression while in cross-over experiments exosomes extracted from IL-1ß-exposed CDEC induced IL-1ß and IL-6 mRNA expression in both sets of macrophages. MiRnome analysis revealed that microRNA (miR)-146a, -155 and -200b exhibited a >2-fold increase in expression in CDEC treated with THP-1-IgA-MCM compared with THP-1-MCM. Enforced miR-146a suppression further enhanced NGAL expression, while ectopic miR-146a over-expression downregulated it. NGAL mRNA and miR-146a were upregulated in the biopsies of patients with progressive IgAN compared with non-progressive IgAN. CONCLUSIONS: Taken together, these data suggest that CDEC-macrophage interactions potentially contribute to the tubulointerstitial fibrosis characteristic of progressive IgAN.

Galactose-deficient IgA1 in skin and serum from patients with skin-limited and systemic IgA vasculitis.

BACKGROUND: IgA vasculitis (IgAV) encompasses a systemic form involving kidneys, gut, skin, or joints, and a skin-limited form. One characteristic feature of systemic IgAV is deposition of galactose-deficient IgA1 (GD-IgA1) in kidneys (as in IgA nephropathy). The relevance of GD-IgA1 for cutaneous vasculitis is unknown. OBJECTIVE: We investigated whether GD-IgA1 is deposited perivascularly in systemic and also skin-limited IgAV and whether its serum levels differ between both forms. METHODS: In a case-control study, deposition of GD-IgA1 was analyzed immunohistochemically by KM55 antibody in skin biopsy specimens from 12 patients with skin-limited IgAV and 4 with systemic IgAV. GD-IgA1 levels were compared by enzyme-linked immunosorbent assay in sera from 15 patients each with skin-limited and systemic IgAV and from 11 healthy individuals. RESULTS: All biopsy samples from systemic IgAV, and also from skin-limited IgAV, revealed perivascular GD-IgA1 deposition. The average GD-IgA1 concentration in serum was significantly higher in systemic IgAV than in skin-limited IgAV, despite overlap between the groups. LIMITATIONS: Although high GD-IgA1 levels may be predictive of systemic IgAV, patient numbers were too low to determine cutoff values for systemic versus skin-limited IgAV. CONCLUSION: Perivascular GD-IgA1 deposition is a prerequisite for systemic and skin-limited IgAV; however, high GD-IgA1 levels in some patients with systemic IgAV suggest a dose-dependent effect of GD-IgA1 in IgAV.

Galactosylation of IgA1 Is Associated with Common Variation in C1GALT1.

IgA nephropathy (IgAN), an important cause of kidney failure, is characterized by glomerular IgA deposition and is associated with changes in O-glycosylation of the IgA1 molecule. Here, we sought to identify genetic factors contributing to levels of galactose-deficient IgA1 (Gd-IgA1) in white and Chinese populations. Gd-IgA1 levels were elevated in IgAN patients compared with ethnically matched healthy subjects and correlated with evidence of disease progression. White patients with IgAN exhibited significantly higher Gd-IgA1 levels than did Chinese patients. Among individuals without IgAN, Gd-IgA1 levels did not correlate with kidney function. Gd-IgA1 level heritability (h2), estimated by comparing midparental and offspring Gd-IgA1 levels, was 0.39. Genome-wide association analysis by linear regression identified alleles at a single locus spanning the C1GALT1 gene that strongly associated with Gd-IgA1 level (ß=0.26; P=2.35×10-9). This association was replicated in a genome-wide association study of separate cohorts comprising 308 patients with membranous GN from the UK (P<1.00×10-6) and 622 controls with normal kidney function from the UK (P<1.00×10-10), and in a candidate gene study of 704 Chinese patients with IgAN (P<1.00×10-5). The same extended haplotype associated with elevated Gd-IgA1 levels in all cohorts studied. C1GALT1 encodes a galactosyltransferase enzyme that is important in O-galactosylation of glycoproteins. These findings demonstrate that common variation at C1GALT1 influences Gd-IgA1 level in the population, which independently associates with risk of progressive IgAN, and that the pathogenic importance of changes in IgA1 O-glycosylation may vary between white and Chinese patients with IgAN.

ß1,4-galactosyltransferase 1 is a novel receptor for IgA in human mesangial cells.

IgA nephropathy is characterized by mesangial deposition of IgA, mesangial cell proliferation, and extracellular matrix production. Mesangial cells bind IgA, but the identity of all potential receptors involved remains incomplete. The transferrin receptor (CD71) acts as a mesangial cell IgA receptor and its expression is upregulated in many forms of glomerulonephritis, including IgA nephropathy. CD71 is not expressed in healthy glomeruli and blocking CD71 does not completely abrogate mesangial cell IgA binding. Previously we showed that mesangial cells express a receptor that binds the Fc portion of IgA and now report that this receptor is an isoform of ß-1,4-galactosyltransferase. A human mesangial cell cDNA library was screened for IgA binding proteins and ß-1,4-galactosyltransferase identified. Cell surface expression of the long isoform of ß-1,4-galactosyltransferase was shown by flow cytometry and confocal microscopy and confirmed by immunoblotting. Glomerular ß-1,4-galactosyltransferase expression was increased in IgA nephropathy. IgA binding and IgA-induced mesangial cell phosphorylation of spleen tyrosine kinase and IL-6 synthesis were inhibited by a panel of ß-1,4-galactosyltransferase-specific antibodies, suggesting IgA binds to the catalytic domain of ß-1,4-galactosyltransferase. Thus, ß-1,4-galactosyltransferase is a constitutively expressed mesangial cell IgA receptor with an important role in both mesangial IgA clearance and the initial response to IgA deposition.

The solution structures of native and patient monomeric human IgA1 reveal asymmetric extended structures: implications for function and IgAN disease.

Native IgA1, for which no crystal structure is known, contains an O-galactosylated 23-residue hinge region that joins its Fab and Fc regions. IgA nephropathy (IgAN) is a leading cause of chronic kidney disease in developed countries. Because IgA1 in IgAN often has a poorly O-galactosylated hinge region, the solution structures of monomeric IgA1 from a healthy subject and three IgAN patients with four different O-galactosylation levels were studied. Analytical ultracentrifugation showed that all four IgA1 samples were monomeric with similar sedimentation coefficients, s(0)20,w. X-ray scattering showed that the radius of gyration (Rg) slightly increased with IgA1 concentration, indicating self-association, although their distance distribution curves, P(r), were unchanged with concentration. Neutron scattering indicated similar Rg values and P(r) curves, although IgA1 showed a propensity to aggregate in heavy water buffer. A new atomistic modelling procedure based on comparisons with 177000 conformationally-randomized IgA1 structures with the individual experimental scattering curves revealed similar extended Y-shaped solution structures for all four differentially-glycosylated IgA1 molecules. The final models indicated that the N-glycans at Asn(263) were folded back against the Fc surface, the C-terminal tailpiece conformations were undefined and hinge O-galactosylation had little effect on the solution structure. The solution structures for full-length IgA1 showed extended hinges and the Fab and Fc regions were positioned asymmetrically to provide ample space for the functionally-important binding of two FcαR receptors to its Fc region. Whereas no link between O-galactosylation and the IgA1 solution structure was detected, an increase in IgA1 aggregation with reduced O-galactosylation may relate to IgAN.

Spleen tyrosine kinase is important in the production of proinflammatory cytokines and cell proliferation in human mesangial cells following stimulation with IgA1 isolated from IgA nephropathy patients.

IgA immune complexes are capable of inducing human mesangial cell (HMC) activation, resulting in release of proinflammatory and profibrogenic mediators. The subsequent inflammation, cellular proliferation, and synthesis of extracellular matrix lead to the progression of IgA nephropathy (IgAN). Spleen tyrosine kinase (SYK) is an intracellular protein tyrosine kinase involved in cell signaling downstream of immunoreceptors. In this study, we determined whether SYK is involved in the downstream signaling of IgA1 stimulation in HMC, leading to production of proinflammatory cytokines/chemokines and cell proliferation. Incubation of HMC with IgA1 purified from IgAN patients significantly increased the synthesis of MCP-1 in a dose-dependent manner. There was also significantly increased production of IL-6, IL-8, IFN-γ-inducible protein-10, RANTES, and platelet-derived growth factor-BB. Stimulation of HMC with heat-aggregated IgA1 purified from IgAN patients induced significantly increased HMC proliferation. Both pharmacological inhibition of SYK and knockdown of SYK by small interfering RNA significantly reduced the synthesis of these mediators and inhibited HMC proliferation. Moreover, positive immunostaining for total and phospho-SYK in glomeruli of kidney biopsies from IgAN patients strongly suggests the involvement of SYK in the pathogenesis of IgAN. To our knowledge, we demonstrate, for the first time, the involvement of SYK in the downstream signaling of IgA1 stimulation in HMC and in the pathogenesis of IgAN. Hence, SYK represents a potential therapeutic target for IgAN.

Combined walking exercise and alkali therapy in patients with CKD4-5 regulates intramuscular free amino acid pools and ubiquitin E3 ligase expression.

Muscle-wasting in chronic kidney disease (CKD) arises from several factors including sedentary behaviour and metabolic acidosis. Exercise is potentially beneficial but might worsen acidosis through exercise-induced lactic acidosis. We studied the chronic effects of exercise in CKD stage 4-5 patients (brisk walking, 30 min, 5 times/week), and non-exercising controls; each group receiving standard oral bicarbonate (STD), or additional bicarbonate (XS) (Total n = 26; Exercising + STD n = 9; Exercising +XS n = 6; Control + STD n = 8; Control + XS n = 3). Blood and vastus lateralis biopsies were drawn at baseline and 6 months. The rise in blood lactate in submaximal treadmill tests was suppressed in the Exercising + XS group. After 6 months, intramuscular free amino acids (including the branched chain amino acids) in the Exercising + STD group showed a striking chronic depletion. This did not occur in the Exercising + XS group. The effect in Exercising + XS patients was accompanied by reduced transcription of ubiquitin E3-ligase MuRF1 which activates proteolysis via the ubiquitin-proteasome pathway. Other anabolic indicators (Akt activation and suppression of the 14 kDa actin catabolic marker) were unaffected in Exercising + XS patients. Possibly because of this, overall suppression of myofibrillar proteolysis (3-methylhistidine output) was not observed. It is suggested that alkali effects in exercisers arose by countering exercise-induced acidosis. Whether further anabolic effects are attainable on combining alkali with enhanced exercise (e.g. resistance exercise) merits further investigation.

An update on the pathogenesis and treatment of IgA nephropathy.

Over the past two decades significant progress has been made in unravelling the complex pathogenesis of immunoglobulin A nephropathy (IgAN). Excess amounts of poorly galactosylated immunoglobulin (Ig)A1 in the serum appear to be the trigger for generation of glycan-specific IgG and IgA autoantibodies, resulting in the formation of circulating IgA immune complexes, which are pivotal to the development of nephritis. It remains unclear why there is an increase in poorly galactosylated IgA1 molecules in the serum in IgAN. One intriguing possibility is that this IgA is derived from displaced mucosal B cells, which have mis-homed from their mucosal induction sites to systemic sites, where they secrete polymeric, poorly galactosylated IgA directly into the circulation rather than onto mucosal surfaces. Lack of a clear appreciation of the origins of poorly galactosylated IgA1 and an incomplete understanding of immune complex formation have hampered development of specific therapeutic strategies to prevent mesangial IgA deposition. Clinicians have therefore been left to manage patients with generic therapies, mainly by control of blood pressure and renin-angiotensin blockade. A paucity of high-quality clinical trials has meant that evaluation of additional therapies, particularly immunosuppressive regimens, has been difficult and there remains a great deal of confusion over the optimum treatment of patients at high risk of progressive chronic kidney disease.

New Insights into the Pathogenesis and Treatment Strategies in IgA Nephropathy.

Background: Immunoglobulin A nephropathy (IgAN) is the most common form of primary glomerulonephritis worldwide. It is defined by mesangial IgA deposition, with consequent mesangial cell proliferation, inflammation, and tubulointerstitial fibrosis. Summary: Approximately 30% of affected patients will progress to end-stage kidney disease within 20 years of diagnosis. Currently, there is no disease-specific treatment available and management recommendations are, in general, limited to optimization of lifestyle measures and use of renin-angiotensin-aldosterone system blockers. More recently, advances in the understanding of the pathogenesis of IgAN have informed the development of novel therapeutic strategies that are now being tested in clinical trials. These have focused on different areas that include modulating the production of poorly galactosylated IgA1, which is central to the development of IgAN, and inhibiting the downstream signaling pathways and complement activation that are triggered following mesangial IgA1 deposition. In this review, we will summarize important pathogenic mechanisms in IgAN and highlight important areas of interest where treatment strategies are being developed. Key messages: IgAN is a common form of primary glomerulonephritis for which there is no current approved specific therapy. Recent advances in the understanding of its pathogenesis have led to the development of novel therapies, with the hope that new treatment options will be available soon to treat this condition.

Higher serum galactose-deficient immunoglobulin A1 concentration is associated with stronger mesangial cellular inflammatory response and more severe histologic findings in immunoglobulin A nephropathy.

BACKGROUND: Galactose-deficient immunoglobulin A1 (Gd-IgA1) is known to play a key role in the pathogenesis of IgA nephropathy (IgAN). We aimed to evaluate whether serum Gd-IgA1 is associated with in vitro activation of mesangial cells in individual patients and how this affects the clinical and histologic parameters. METHODS: Serum samples and clinical and histologic data were collected in the University Hospital Basel and Hammersmith Hospital, London. Serum levels of IgA1 and Gd-IgA1 were measured by enzyme-linked immunosorbent assay (ELISA) and lectin-binding assay using lectin Helix aspersa (HA). Primary human mesangial cells were stimulated with IgA1 isolated from serum from individual patients and the concentrations of monocyte chemoattractant protein-1 and interleukin-6 were measured in cell culture supernatant by ELISA. RESULTS: Thirty-three patients were enrolled. A significant correlation was observed between serum Gd-IgA1 levels and the concentration of MCP-1 in the culture supernatant in individual patients (Spearman r = 0.5969, P = 0.0002). There was no significant correlation between serum Gd-IgA1 levels and proteinuria or estimated glomerular filtration rate at diagnosis. However, the serum Gd-IgA1 level was significantly higher in patients with segmental glomerulosclerosis (S0 versus S1, P = 0.0245) and tubular atrophy/interstitial fibrosis (T0 versus T1 and T2, P = 0.0336; T0 versus T2, P = 0.0225). CONCLUSIONS: Higher serum Gd-IgA1 concentration is associated with stronger mesangial cell inflammatory response with production of a greater amount of MCP-1 in vitro. This in turn is associated with severe histologic changes. The disease progression with worse renal outcome in patients with higher serum Gd-IgA1 may be therefore mediated by more pronounced mesangial cell inflammatory response leading to more severe histologic changes.

Effect of Immunosuppressive Drugs on the Changes of Serum Galactose-Deficient IgA1 in Patients with IgA Nephropathy.

Galactose-deficient IgA1 (Gd-IgA1) and IgA-IgG complexes are known to play an important role in the pathogenesis of IgA nephropathy (IgAN). We aimed therefore to determine the impact of immunosuppression on the serum levels of Gd-IgA1, total IgA1 and IgA-IgG complexes in IgAN patients. In a retrospective study, serum samples from IgAN patients collected before transplantation (t0) and at 3- and 6-month posttransplant (t3 & t6) were used to measure the levels of Gd-IgA1, total IgA1 and IgA-IgG complexes. The area under the curves (AUC) of immunosuppressants was calculated by the plot of plasma trough level or dosage of each immunosuppressant versus time and was interpreted as the extent of drug exposure. Thirty-six out of 64 IgAN patients, who underwent kidney transplantation between 2005 and 2012, were enrolled. From t0 to t3, serum Gd-IgA1 and total IgA1 decreased significantly (24.7 AU (18.6-36.1) to 17.2 (13.1-29.5) (p<0.0001); 4.1 mg/ml (3.6-5.1) to 3.4 (3.0-4.1) (p = 0.0005)), whereas IgA-IgG complexes remained similar. From t3 to t6, Gd-IgA1 and IgA-IgG complexes significantly increased (17.2 AU (13.1-29.5) to 23.9 (16.8-32.0) (p = 0.0143); OD 0.16 (0.06-0.31) to 0.26 (0.14-0.35) (p = 0.0242)), while total IgA1 remained similar. According to median regression analysis, AUC of prednisone t0-6 was significantly associated with the decrease of Gd-IgA1 t0-6 (P = 0.01) and IgA1 t0-6 (p = 0.002), whereas AUC of tacrolimus t0-6 was associated with the decrease of IgA1 t0-6 (p = 0.02). AUC of prednisone t0-3 was associated with the decrease of IgA-IgG complexes t0-3 (p = 0.0036). The association of AUC prednisone t0-6 with Gd-IgA1 t0-6 remained highly significant after adjustment for other immunosuppressants (p = 0.0036). Serum levels of Gd-IgA1, total IgA1 and IgA-IgG in patients with IgAN vary according to the changing degrees of immunosuppression. The exposure to prednisone most clearly influenced the serum levels of Gd-IgA1.

Lymphocyte responses to influenza and tetanus toxoid in vitro following intensive exercise and carbohydrate ingestion on consecutive days.

The effect of carbohydrate (CHO) ingestion on antigen- (rather than mitogen-) stimulated T-cell responses to prolonged, intensive exercise may give a more realistic insight into the effect of CHO on T-cell functional capacity and subsequent infection risk. This study investigated the effect of CHO ingestion during prolonged, intensive exercise on influenza- and tetanus toxoid-stimulated T-cell cytokine mRNA expression and proliferation. Mitogen- [phytohemagglutinin (PHA)] stimulated proliferation was assessed for comparison. Responses were assessed following exercise on consecutive mornings to determine any carryover effect. Fifteen male games players performed two exercise trials in a double-blind, randomized, crossover design. Each trial comprised 90 min of intensive, intermittent running on consecutive mornings, with either CHO (6.4% wt/vol) or placebo (PLA) beverage ingestion before, during, and after each bout of exercise. Postexercise CD3(+) cell counts were higher in PLA than CHO on both days (P < 0.05). Antigen-stimulated T-cell cytokine mRNA expression was unaffected by exercise or CHO ingestion. Before exercise on day 2, T-cell proliferative responses to PHA, influenza, and tetanus toxoid were higher in CHO than PLA by 99, 80, and 58%, respectively (P < 0.01 for PHA, P < 0.05 for influenza and tetanus toxoid). At 1 h postexercise on day 2, PHA-induced proliferation was 70% higher in CHO than PLA (P < 0.05), yet there were no differences between trials for antigen-induced proliferative responses. Therefore, mitogen-induced T-cell proliferation following strenuous exercise and CHO does not necessarily reflect responses to specific antigens and, consequently, may not provide a good model for the situation in vivo.

Abnormal IgD and IgA1 O-glycosylation in hyperimmunoglobulinaemia D and periodic fever syndrome.

In order to determine the glycosylation pattern for IgD, and to examine whether there are changes in the pattern of IgD and IgA1 O-glycosylation in patients with hyperimmunoglobulinaemia D and periodic fever syndrome (HIDS) during acute febrile attacks and during periods of quiescence, serum was obtained from 20 patients with HIDS and 20 control subjects. In the HIDS group, serum was obtained either during an acute febrile episode (n = 9) or during a period of quiescence (n = 11). The O-glycosylation profiles of native and desialylated IgA1 and IgD were measured in an ELISA-type system using the lectins Helix aspersa and peanut agglutinin, which bind to alternative forms of O-glycan moieties. IgD is more heavily O-galactosylated and less O-sialylated than IgA1 in healthy subjects. HIDS is associated with more extensive O-galactosylation of IgD and a reduction in O-sialylation of both IgD and IgA1. These changes are present both during acute febrile attacks and periods of quiescence. The T cell IgD receptor is a lectin with binding affinity for the O-glycans of both IgD and IgA1. The observed changes in IgD and IgA1 O-glycosylation are likely to have a significant effect on IgD/IgA1-T cell IgD receptor interactions including basal immunoglobulin synthesis, and possibly myeloid IgD receptor-mediated cytokine release.

Immunopathogenesis of IgAN.

The defining hallmark of IgA nephropathy (IgAN) is deposition of polymeric IgA1 in the glomerular mesangium accompanied by a mesangial proliferative glomerulonephritis. The mechanisms involved in mesangial polymeric IgA1 deposition and the initiation of inflammatory glomerular injury remain unclear. This lack of a complete understanding of the pathogenesis of IgAN has meant that there is still no treatment known to modify mesangial deposition of IgA. Increasing evidence, however, supports the importance of IgA-containing immune complex formation as a pivotal factor driving mesangial IgA deposition and triggering of glomerular injury. A number of potentially important changes to the IgA1 molecule have been identified in IgAN, which may contribute to immune complex formation. These changes suggest that the polymeric IgA1 that deposits in IgA nephropathy is derived from mucosally primed plasma cells. The presence of this IgA in the circulation reflects displacement of mucosal B lineage cells to systemic sites and may be the result of mishoming of lymphocytes trafficking along the mucosa-bone marrow axis.

O-glycosylation of serum IgA1 antibodies against mucosal and systemic antigens in IgA nephropathy.

In IgA nephropathy (IgAN), serum IgA1 with abnormal O-glycosylation deposits in the glomerular mesangium. The underlying mechanism of this IgA1 O-glycosylation abnormality is poorly understood, but recent evidence argues against a generic defect in B cell glycosyltransferases, suggesting that only a subpopulation of IgA1-committed B cells are affected. For investigation of whether the site of antigen encounter influences IgA1 O-glycosylation, the O-glycosylation of serum IgA1 antibodies against a systemic antigen, tetanus toxoid (TT), and a mucosal antigen, Helicobacter pylori (HP), was studied in patients with IgAN and control subjects. Serum IgA1 was purified from cohorts of patients with IgAN and control subjects with HP infection and after systemic TT immunization. The IgA1 samples were applied to HP- and TT-coated immunoplates to immobilize specific antibodies, and IgA1 O-glycosylation profiles were assessed by binding of the O-glycan-specific lectin Vicia villosa using a modified ELISA technique. Although total serum IgA1 had raised lectin binding in IgAN, the O-glycosylation of the specific IgA1 antibodies to TT and HP did not differ between patients and control subjects. In both groups, IgA1 anti-HP had higher lectin binding than IgA1 anti-TT. This study demonstrates that IgA1 O-glycosylation normally varies in different immune responses and that patients produce the full spectrum of IgA1 O-glycoforms. IgA1 with high lectin binding was produced in response to mucosal HP infection in all subjects. The raised circulating level of this type of IgA1 in IgAN is likely to be a consequence of abnormal systemic responses to mucosally encountered antigens rather than a fundamental defect in B cell O-glycosylation pathways.