Nayer Bagheri

Proinflammatory cytokines regulate FcαR expression by human mesangial cells in vitro

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 (FcαR) by selected cytokines. Binding of 125I‐IgA to quiescent human MC showed 2.55 ×105 sites/cell with an affinity (Ka) of 3.2 ×107 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 pHuFcαR cDNA probe showed time‐dependent increases in mRNA expression in stimulated versus control cells. IL‐6 and tumour necrosis factor‐alpha (TNF‐α) had a biphasic effect on the FcαR mRNA level; at 48 h, IL‐6 increased steady state mRNA levels about six‐fold relative to control, TNF‐α increased mRNA four‐fold, and interferon‐gamma (IFN‐γ) induced FcαR mRNA two‐fold. By reverse transcriptase‐polymerase chain reaction (RT‐PCR), the FcαR expressed on human MC appears highly homologous to that expressed by U937 cells. Altered FcαR expression in response to cytokines may influence the pathogenesis of IgAN by affecting deposition and/or clearance of IgA‐IC in the mesangium.

T cell cytokine polarity as a determinant of immunoglobulin A (IgA) glycosylation and the severity of experimental IgA nephropathy

Immunoglobulin A (IgA) glycosylation, recognized as an important pathogenic factor in IgA nephropathy (IgAN), is apparently controlled by the polarity of T helper (Th) cytokine responses. To examine the role of cytokine polarity in IgAN, inbred mice were immunized by intraperitoneal priming with inactivated Sendai virus (SeV) emulsified in either complete Freunds adjuvant (CFA) or incomplete Freunds adjuvant (IFA), which promote Th1‐ or Th2‐immune response, respectively, and then boosted identically twice orally with aqueous suspensions of inactivated virus. Next, some mice were challenged intranasally with infectious SeV. Mice primed with CFA or IFA had equal reductions in nasal viral titre relative to non‐immune controls, and equally increased serum levels of SeV‐specific IgA antibody. Mice primed with CFA showed higher SeV‐specific IgG than those with IFA. Splenocytes from mice primed with IFA produced copious amounts of interleukin (IL)‐4 and IL‐5, but little interferon‐γ and IL‐2; those primed with CFA had reciprocal cytokine recall responses. Total serum IgA and especially SeV‐specific IgA from mice primed with IFA showed a selective defect in sialylation and galactosylation. Although the frequency and intensity of glomerular deposits and haematuria did not differ, glomerulonephritis in mice primed with IFA and challenged with infectious virus was more severe than in those given CFA, as judged by serum creatinine level. We conclude that the polarity of T cell cytokines controls the pattern of IgA glycosylation and exerts direct or indirect effects on functional glomerular responses to immune complex deposition.

Subcomponent Vaccine Based on CTA1-DD Adjuvant with Incorporated UreB Class II Peptides Stimulates Protective Helicobacter pylori Immunity

A mucosal vaccine against Helicobacter pylori infection could help prevent gastric cancers and peptic ulcers. While previous attempts to develop such a vaccine have largely failed because of the requirement for safe and effective adjuvants or large amounts of well defined antigens, we have taken a unique approach to combining our strong mucosal CTA1-DD adjuvant with selected peptides from urease B (UreB). The protective efficacy of the selected peptides together with cholera toxin (CT) was first confirmed. However, CT is a strong adjuvant that unfortunately is precluded from clinical use because of its toxicity. To circumvent this problem we have developed a derivative of CT, the CTA1-DD adjuvant, that has been found safe in non-human primates and equally effective compared to CT when used intranasally. We genetically fused the selected peptides into the CTA1-DD plasmid and found after intranasal immunizations of Balb/c mice using purified CTA1-DD with 3 copies of an H. pylori urease T cell epitope (CTA1-UreB3T-DD) that significant protection was stimulated against a live challenge infection. Protection was, however, weaker than with the gold standard, bacterial lysate+CT, but considering that we only used a single epitope in nanomolar amounts the results convey optimism. Protection was associated with enhanced Th1 and Th17 immunity, but immunizations in IL-17A-deficient mice revealed that IL-17 may not be essential for protection. Taken together, we have provided evidence for the rational design of an effective mucosal subcomponent vaccine against H. pylori infection based on well selected protective epitopes from relevant antigens incorporated into the CTA1-DD adjuvant platform.

Proteomics analysis of the ezrin interactome in B cells reveals a novel association with Myo18Aα

The molecular regulation of recruitment and assembly of signalosomes near the B cell receptor (BCR) is poorly understood. We have previously demonstrated a role for the ERM family protein ezrin in regulating antigen-dependent lipid raft coalescence in B cells. In this study, we addressed the possibility that ezrin may collaborate with other adaptor proteins to regulate signalosome dynamics at the membrane. Using mass spectrometry-based proteomics analysis, we identified Myo18aα as a novel binding partner of ezrin. Myo18aα is an attractive candidate as it has several protein-protein interaction domains and an intrinsic motor activity. The expression of Myo18aα varied during B cell development in the bone marrow and in mature B cell subsets suggesting functional differences. Interestingly, BCR stimulation increased the association between ezrin and Myo18aα, and induced co-segregation of Myo18aα with the BCR and phosphotyrosine-containing proteins. Our data raise an intriguing possibility that the Myo18aα/ezrin complex may facilitate BCR-mediated signaling by recruiting signaling proteins that are in close proximity of the antigen receptor. Our study is not only significant with respect to understanding the molecular regulation of BCR signaling but also provides a broader basis for understanding the mechanism of action of ezrin in other cellular systems.

Animal models of IgA nephropathy: Formulating therapeutic strategies

Summary: This review seeks to highlight those aspects of ongoing research with animal models that may facilitate development of effective and perhaps specific therapy of IgAN. Oral immunization is typically accompanied by a predominantly Th2 response; defects in the evolution of this typical response, instigated by cyclophosphamide and/or oestradiol, elicit IgAN in mice. The Th2 cytokines that predominate in orally immunized mice, and in patients with IgAN, promote abnormally glycosylated IgA, similar to the IgA in patients. Immune complexes prepared with IgA antibody bearing altered glycosyl residues show enhanced rates of glomerular uptake and altered rates of clearance from the circulation. When immune responses to infectious pathogens are localized to sites remote from a new infection with a serologically related organism, ‘misdirected’ responses permit pathogen replication and antigen production concomitant with strong host antibody responses. The resultant immune complexes elicit IgAN in mice. Similar ‘misdirected’ immune responses may arise in J chain‐deficient subjects. Improved pharmacologic control of B cell trafficking and/or function could rectify these defects. Correction of deficiency in clearance of IgA immune complexes could benefit patients with secondary IgAN. Finally, manipulation of glomerular haemodynamics and/or production or response to intraglomerular cytokines and growth factors might ameliorate disease or forestall progression to glomerulosclerosis. Animal models have served as a conceptual springboard for a wide variety of clinical investigations, and in turn clinical information has guided the design and goal of experimental investigation. Hopefully, this synergy can continue, ultimately resulting in effective and specific therapy.

Development of immune-complex glomerulonephritis in athymic mice: T cells are not required for the genesis of glomerular injury

Chronic injection of dextran into normal mice elicits a glomerulonephritis (GN) that models IgA nephropathy (IgAN) in humans. Since athymic mice lack T cells but nonetheless develop antibodies to polysaccharide antigens such as dextran (DEX), we used athymic mice to study the role of T lymphocytes in the induction of this form of GN, independent of the role of T cells in antibody synthesis. Both mice given injections of diethylaminoethyl (DEAE)-DEX and uninjected mice had circulating IgM and IgA anti-DEX antibodies, which apparently arise as ‘natural antibodies’, but immune complex GN was observed only in the injected mice. All of 15 injected mice exhibited capillary staining for IgA and IgM; none of 12 control mice contained such IgA deposits and only one had capillary staining for IgM (both P<0.001). In addition, IgG and C3 were detected in injected but not control animals. By light microscopy, injected mice exhibited marked expansion of mesangial matrix relative to controls. Electron microscopy showed no glomerular abnormalities in control mice, whereas injected mice showed large organized fibrillar deposits principally in the mesangium. Hematuria and proteinuria were present in all 15 injected mice, but only one of 11 control mice showed hematuria or proteinuria (both P<0.001). These results indicate that chronic injection of DEAE-DEX into athymic mice generates the same clinical and histologic features of GN as in euthymic mice, suggesting that T cells are not necessary to promote GN in this model.