Ahmed Boumediene

Transglutaminase is essential for IgA nephropathy development acting through IgA receptors

Transglutaminase 2 is required for the development of IgA nephropathy.

Use of both CD63 up regulation and IgE down regulation for the flow cytometric analysis of allergen induced basophil activation. Definition of an activation index.

Abstract.Objective and design:The aim of this study was to compare the use of a late (CD63) and an early (IgE) marker of basophil activation in the flow cytometric diagnosis of beta-lactam induced allergic hypersensitivity reactions.Subjects:Twelve patients who had had a clear cut betalactam induced immediate reaction and 16 controls were selected, as well as 11 patients who had had an immediate reaction to bee or wasp stings.Methods:Leukocyte suspensions were incubated with allergen dilutions as well as 2 positive controls (anti-IgE and NFormyl- Methionyl-Leucyl-Phenylalanine (fMLP)). Basophils were labelled with an anti-IgE FITC (fluorescein isothiocyanate) and an anti-CD63 PE (phycoerythrin). Results were expressed as percentage CD63 expression and index calculated according to a specific algorithm including the two activation markers.Results:Significant CD63 expression (>5 %) was observed in 3/12 cases for the beta-lactam sensitized population, in 0/16 cases for the controls and in 11/11 cases for the venom sensitized population. A significant index (determined by a ROC analysis) was observed in 11/12 beta-lactam sensitized patients and in 0/16 controls.Conclusion:These results show that IgE (an early activation marker) is more sensitive than CD63 (a later activation marker) in the diagnosis of beta-lactam allergy.

Replacement of Iγ3 germ-line promoter by Iγ1 inhibits class-switch recombination to IgG3

Class-switch recombination (CSR) enables IgM-producing B cells to switch to the production of IgG, IgE, and IgA. The process requires germ-line (GL) transcription that initiates from promoters upstream of switch (S) sequences and is regulated by the 3′ regulatory region (3′RR) located downstream of the Ig heavy chain (IgH) locus. How the 3′RR effect its long-range activation is presently unclear. We generated a mouse line in which Iγ3 GL promoter was replaced by Iγ1. We found that GL transcription could initiate from the inserted Iγ1 promoter and was induced by increased concentrations of IL-4 and that the transcripts were normally spliced. However, when compared with GL transcripts derived from the endogenous Iγ1 promoter in the same stimulation conditions, those from the inserted Iγ1 promoter were less abundant. CSR to Cγ3 was abrogated both in vivo and in vitro. The results strongly suggest that the endogenous Iγ1 promoter insulates the inserted Iγ1 from the long-range activating effect of the 3′RR. The implications of our findings are discussed in light of the prominent models of long-distance activation in complex loci.

Analysis of IgE down regulation induced by basophil activation. Application to the diagnosis of muscle relaxant allergic hypersensitivity by flow cytometry.

No Abstract..

Tacrolimus Pharmacodynamics and Pharmacogenetics along the Calcineurin Pathway in Human Lymphocytes

BACKGROUND Although therapeutic drug monitoring has improved the clinical use of immunosuppressive drugs, there is still interpatient variability in efficacy and toxicity that pharmacodynamic monitoring may help to reduce. To select the best biomarkers of tacrolimus pharmacodynamics, we explored the strength and variability of signal transduction and the influence of polymorphisms along the calcineurin pathway. METHODS Peripheral blood mononuclear cells from 35 healthy volunteers were incubated with tacrolimus (0.1-50 ng/mL) and stimulated ex vivo. Inhibition of NFAT1 (nuclear factor of activated T cells 1) translocation to the nucleus and intracellular expression of interleukin-2 in CD4(+) and CD8(+) T cells and the surface activation marker CD25 in CD3(+) cells were measured by flow cytometry. We sequenced the promoter regions of immunophilins and calcineurin subunits and characterized selected single nucleotide polymorphisms in the genes of the calcineurin pathway with allelic discrimination assays. RESULTS All responses closely fitted an I/Imax sigmoid model. Large interindividual variability (n = 30) in I0 and IC50 was found for all biomarkers. Moreover, strong and statistically significant associations were found between tacrolimus pharmacodynamic parameters and polymorphisms in the genes coding cyclophilin A, the calcineurin catalytic subunit α isoenzyme, and CD25. CONCLUSIONS This study demonstrates the consistency and large interindividual variability of signal transduction along the calcineurin pathway, as well as the strong influence of pharmacogenetic polymorphisms in the calcineurin cascade on both the physiological activity of this route and tacrolimus pharmacodynamics.

IgA Structure Variations Associate with Immune Stimulations and IgA Mesangial Deposition

IgA1 mesangial deposition is the hallmark of IgA nephropathy and Henoch-Schönlein purpura, the onset of which often follows infections. Deposited IgA has been reported as polymeric, J chain associated, and often, hypogalactosylated but with no information concerning the influence of the IgA repertoire or the link between immune stimuli and IgA structure. We explored these issues in the α1KI mouse model, which produces polyclonal human IgA1 prone to mesangial deposition. Compared with mice challenged by a conventional environment, mice in a specific pathogen-free environment had less IgA deposition. However, serum IgA of specific pathogen-free mice showed more galactosylation and much lower polymerization. Notably, wild-type, α1KI, and even J chain-deficient mice showed increased polymeric serum IgA on exposure to pathogens. Strict germfree conditions delayed but did not completely prevent deposition; mice housed in these conditions had very low serum IgA levels and produced essentially monomeric IgA. Finally, comparing monoclonal IgA1 that had different variable regions and mesangial deposition patterns indicated that, independently of glycosylation and polymerization, deposition might also depend on IgA carrying specific variable domains. Together with IgA quantities and constant region post-translational modifications, repertoire changes during immune responses might, thus, modulate IgA propensity to deposition. These IgA features are not associated with circulating immune complexes and C3 deposition and are more pertinent to an initial IgA deposition step preceding overt clinical symptoms in patients.

Gammopathy with IgA mesangial deposition provides a monoclonal model of IgA nephritogenicity and offers new insights into its molecular mechanisms

BACKGROUND Henoch-Schönlein purpura (HSP) and IgA nephropathy (IgAN) are characterized by mesangial deposition of polyclonal IgA eventually showing aberrant glycosylation, affinity for mesangial cells and/or co-precipitation with antigen, bacterial peptides, autoantibodies or soluble receptors. IgA were also suggested to be negatively charged and predominantly of λ type but rarely in a monoclonal form. METHODS A gammopathy case with HSP provided us with a unique molecularly defined nephritogenic IgA1λ. Immunological analysis, biological activities, glycosylation analysis and finally IgA sequence were determined. RESULTS Compared to IgA1 from healthy subjects or IgAN patients, IgA1 CAT showed hyposialylation but no hypogalactosylation, in agreement with underexpression of sialyltransferase genes by the plasma cell clone. IgA variable domains had low pIs with negatively charged complementarity-determining regions. Weak reactivity appeared against the cationic autoantigen lactoferrin, which was, however, absent from kidney deposits. Deposition also occurred in mice upon injection of only the polymeric form of IgA1 CAT, despite whether or not co-injected with lactoferrin. CONCLUSIONS This monoclonal model of IgA nephritogenicity strongly suggests that beside hinge region glycosylation, V domains play a role in IgA stability and pathogenicity and supports the hypothesis that responses against cationic epitopes from pathogens or autoantigens may select negatively charged complementarity-determining regions prone either to bind charged structures of the mesangium or to promote by themselves IgA aggregation and deposition.

Clinico-biological characteristics of flow cytometry applied to hypersensitivity to NSAIDs

Non steroidal anti-infl ammatory drug (NSAIDs) induced reactions are polymorphic, mainly angio-oedema and shock [1], urticaria [2] and respiratory manifestations (asthma, rhinitis, F Widal triad) [3]. In contrast, IgE dependent reactions are relatively rare. However, the underlying mechanisms are still controversial. NSAIDs are potent Cox-1 inhibitors and this activity could induce an over-production of leukotrienes, which may be also associated with an overexpression of enzymes of the leukotriene cascade e. g. LTC4 synthase [4]. NSAIDs could be also immunogenic by inducing specifi c antibody (IgE?) synthesis leading to a specifi c antibody-dependent cellular activation [5]. Based on previous results showing that basophil activation leads to up-regulation of membrane markers e. g. CD63 [6] and down-regulation of membrane IgE (decrease of the mean fl uorescence intensity, (MFI-IgE) of the basophil population labelled with anti-IgE FITC) [7], we tested NSAIDs-induced basophil activation in patients suffering from NSAIDs-induced urticaria, angio-oedema and asthma.