Réal Lemieux

Inhibition of B cell-mediated antigen presentation by intravenous immunoglobulins (IVIg).

Previous work from our laboratory revealed that IVIg interacted with intracellular proteins involved in antigen presentation in B cells, suggesting that IVIg might interfere with the process of antigen presentation in these cells. In the present work, we used an in vitro assay with ovalbumin as model antigen and showed that IVIg inhibited both BCR-dependent and BCR-independent antigen presentation. The inhibition could not be explained by a modulation of expression of MHC II molecules expressed on B cells and was shown to occur in an FcgammaRIIb-independent manner, suggesting that the events responsible for the inhibitory effect occur at the intracellular level. This was supported by the observation of a direct correlation between the level of spontaneous internalization of two different proteins (IVIg and HSA) and their inhibitory potential. The inhibition of B cell-mediated antigen presentation reported here may help explain some of the anti-inflammatory effects of IVIg observed in treated patients, such as a decrease in autoantibody production.

Urea substitutes toxic formamide as destabilizing agent in nucleic acid hybridizations with RNA probes

Since their introduction some three decades ago, methods for hybridization analysis of nucleic acids immobilized on solid supports have evolved to improve the sensitivity, speed, and convenience of their application. However, in many cases these methods still require the use of solutions containing formamide, a recognized hazardous solvent with potential toxicity. Here, we have compared the efficiency of urea to that of formamide as denaturing agent in nucleic acid hybridization with RNA probes. We show that urea at concentrations of 2–4 molar in solution performs as good as 50% formamide to reduce heterologous background hybridization in Northern blotting experiments realized at 68°C. Presence of urea at higher concentrations resulted in reduced hybridization sensitivity, possibly due to increased viscosity. When tested in Southern blot analysis of genomic DNA, our results revealed that the use of urea in hybridization solution is also suitable to carry out single‐copy gene detection. Together, these findings show that urea can efficiently and safely replace formamide in solutions.

Activation of cryptic IgG reactive with BAFF, amyloid beta peptide and GM-CSF during the industrial fractionation of human plasma into therapeutic intravenous immunoglobulins.

The mechanisms of therapeutic action of IVIg are still unclear in most autoimmune and inflammatory diseases. IVIg have been shown to bind to a variety of human proteins including BAFF, amyloid beta peptide and GM-CSF. It has been suggested that this autoreactivity could contribute to the therapeutic immunomodulatory effects of IVIg. In this work, we showed that native IgG purified from plasma under non-denaturing conditions were much less autoreactive than IVIg. However the native IgG autoreactivity with BAFF, amyloid beta peptide and GM-CSF was significantly increased by short incubation under the slightly denaturing conditions used during industrial plasma fractionation. We conclude that the relatively mild conditions used in industrial plasma fractionation are sufficiently denaturing to activate a significant amount of cryptic autoreactive plasma IgG which could be involved not only in the therapeutic immunomodulatory effects of IVIg but also in the adverse allergic reactions often observed in IVIg-infused patients.

Prevention of T cell activation by interference of internalized intravenous immunoglobulin (IVIg) with MHC II-dependent native antigen presentation.

Activation of self-reactive CD4(+) T cells plays a central role in the initiation and maintenance of autoimmune diseases. We recently reported that intravenous immunoglobulin (IVIg) inhibits the MHC II-restricted CD4(+) T cell activation induced by the presentation of immune complexes. Because native antigens can also play a role in the induction of several autoimmune diseases, we determined whether IVIg could also affect CD4(+) T cell activation following presentation of native antigens by APCs. Here we report that IVIg significantly reduces the activation of CD4(+) T cells by native ovalbumin. The inhibitory effect is FcγR-independent and occurs following internalization of IVIg inside APCs, where it interferes with the intracellular events leading to MHC II-dependent antigen presentation. The effect of IVIg on native antigen presentation could therefore contribute to dampen the autoimmune reaction by reducing CD4(+) T cell activation and the subsequent inflammatory response induced by these cells.

TELOMERE-INDEPENDENT REDUCTION OF HUMAN B LYMPHOCYTE: PROLIFERATION DURING LONG-TERM CULTURE

Telomeres and telomerase, the telomere lengthening enzyme, have been shown to play a central role in the long-term ability of cells to proliferate and maintain viability. In opposition to transformed cells, normal somatic cells express a low level of telomerase, which results in the gradual shortening of their telomeres after each division and in cell senescence once a critical telomere length is reached. We have tested the hypothesis that shortening of telomeres could limit the expansion of normal human B lymphocytes maintained in long-term culture using a CD40/CD154 system. Measurement of temolerase activity in cell lysates showed a rapid up-regulation of telomerase following the initiation of the culture that was dependent on the CD40 signaling. The high level of telomerase activity and the corresponding long telomere structures remained constant for the 35 day culture period in which a gradual reduction of the cell expansion rate is observed. We conclude that the gradual in vitro senescence of cultured B cells does not correlate with a corresponding loss of telomerase activity and of telomere length. Rather the phenomenon may be related to an intrinsic property of the proliferating B cells to differentiate into Ig-secreting cells.

Immunomodulatory effects of therapeutic preparations of human albumin

Background and Objectivesu2002 Albumin is the most abundant protein in plasma and is considered to be immunologically inert. However, we recently observed that therapeutic human albumin preparations, used as protein control in studies involving high doses of IVIg, modulated the MHC II‐restricted activation of antigen‐specific T cells. In the present work, we characterized this effect in more details.