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Biomedical subjects

Réal Lemieux

Publications and source records attributed to Réal Lemieux.

At least 19 recordsLinked to original sources
PubMed2006

Reversal of immune thrombocytopenia in mice by cross-linking human immunoglobulin G with a high-affinity monoclonal antibody.

Intravenous immunoglobulins (IVIgs) are used to treat an increasing number of autoimmune diseases, but their exact mechanism of action remains unknown. This study showed that cross-linking of human IgG present in IVIg preparations using a mouse monoclonal anti-human IgG generated complexes that prevented or reversed thrombocytopenia in mice more efficiently than IVIg. Furthermore, biologically active complexes were obtained simply by adding the monoclonal antibody to human serum. These results suggest the possible development of an IVIg-free substitute through the ex vivo, and possibly in vivo, formation of immune complexes containing autologous IgG of immune thrombocytopenic purpura patients.

Animals↗
PubMed2006

High-throughput molecular profiling of blood donors for minor red blood cell and platelet antigens.

BACKGROUND: ABO and D phenotyping of both blood donors and patients receiving transfusions is routinely performed by blood banks to ensure compatibility. These analyses are performed by antibody-based agglutination assays. Blood is not tested for minor blood group antigens on a regular basis, however, because of cost and time constraints. This can result in alloimmunization of the patient against one to several minor antigens and may complicate future transfusions. STUDY DESIGN AND METHODS: To address this problem, an assay has been generated on the GenomeLab SNPstream genotyping system to test simultaneously polymorphisms linked to 22 different blood antigens with donor's DNA isolated from minute amounts of white blood cells. RESULTS: The results showed that both the error rate of the assay, as measured by the strand concordance rate, and the no-call rate were very low (0.1%). The concordance rate with the actual red blood cell (RBC) and platelet (PLT) serology data varied from 97 to 100 percent. Experimental or database errors as well as rare polymorphisms contributing to antigen conformation could explain the observed differences. These rates, however, are well above requirements because phenotyping and cross-matching will always be performed before transfusion. CONCLUSION: Molecular profiling of blood donors for minor RBC and PLT antigens will give blood banks instant access to many different matched donors through the setup of a centralized data storage system.

ABO Blood-Group System↗
PubMed2006

Characterization of blood components prepared from whole-blood donations after a 24-hour hold with the platelet-rich plasma method.

BACKGROUND: The preparation of platelet (PLT) concentrates (PCs) from PLT-rich plasma (PRP) requires that whole blood (WB) be processed within 8 hours of collection. Increasing WB storage time to 24 hours would be logistically attractive. This study compares the in vitro quality of blood components prepared from WB stored for 8 and 24 hours at room temperature before processing with the PRP method. STUDY DESIGN AND METHODS: WB units were collected from ABO-matched blood donors. To reduce individual variations, paired donations were drawn in parallel, pooled, and split back in the collection bag. One unit was held for 6 to 8 hours and the other for 22 to 24 hours at 20 to 24 degrees C. Prestorage leukoreduced components were prepared with the PRP as intermediate product and analyzed during storage. RESULTS: RBC units prepared after an 8- or 24-hour hold were comparable in terms of hemolysis, sodium, pH, and ATP levels. RBC 2,3- diphosphoglycerate (2,3-DPG) was significantly lower in RBCs prepared from 24-hour hold donations immediately after processing but not after 20 days of storage. Residual white blood cells were approximately fivefold higher (p < 0.05) in 24-hour RBC units. For PCs, measurements for glucose, ATP, lactate, pH, extent of shape change, hypotonic shock response, and CD62p activation were similar. No differences were observed in the von Willebrand factor, factor (F)V, FVIII, and fibrinogen content of fresh-frozen plasma. CONCLUSIONS: The decrease in FVIII and RBC 2,3-DPG can be acceptable as a compromise to improve blood component logistics, but leukoreduction efficiency must be improved before considering the adoption of an overnight storage of WB before PRP processing.

ABO Blood-Group System↗
PubMed2006

Autoantibody-induced formation of immune complexes in normal human serum.

Intravenous immunoglobulins (IVIg) are concentrated preparations of purified human plasma-derived IgG routinely used in the treatment of many autoimmune diseases. Their precise mechanisms of therapeutic action have remained unclear in most diseases and are attracting much interest due to the rapidly increasing use of this precious plasma-derived product. The presence in IVIg of IgG reactive with various human structures has been known for many years. In this review, we first briefly discuss the formation and role of natural autoreactive human IgG in healthy individuals. A role for IgG autoantibodies in the in vivo immunomodulatory effects of IVIg has been proposed several years ago but the underlying mechanism has remained unclear. Recent work has shown that the large IVIg doses infused in many patients could oversaturate the normal anti-idiotype-dependent inhibition of autoreactive IgG present in the plasma of all healthy individuals since the formation of autoimmune complexes could be observed in normal serum in presence of therapeutic amounts of IVIg. These autoimmune complexes could have potent in vivo immunomodulatory effects by interacting with various IgG and complement receptors. Furthermore the autoreactive IgG can be easily purified from IVIg by affinity chromatography, raising the interesting possibility of further fractionating IVIg into different sub-products for use in the treatment of different diseases. These results indicate that natural autoantibodies have important in vivo roles not only for the protection of the body against infectious agents but also for the efficiency of passive immunotherapy procedures used in the treatment of many diseases.

Animals↗
PubMed2005

Iron specific growth inhibition of Burkitt's lymphoma cells in vitro, associated with a decrease in translocated c-myc expression.

The cellular proto-oncogene c-myc is an important transcription factor that plays a role in several cellular activities such as proliferation, differentiation, and apoptosis. It follows that regulation of c-myc expression is crucial for maintaining cell integrity. Amplification or translocation can convert this proto-oncogene into an activated oncogene, thereby deregulating c-myc expression. Changes in the expression of c-myc leading to malignant cell growth and tumor progression can also be triggered by extrinsic factors. It has been reported that iron can increase cell proliferation, mainly by stimulating DNA synthesis as well as by enhancing c-myc expression. Here, we studied the effect of iron on cells in which c-myc expression is deregulated by either chromosomal translocation or gene amplification. When added to Burkitt's lymphoma cell lines, iron markedly inhibits cell proliferation. This effect is mediated by a cell cycle arrest in G2/M, followed by an important decrease in c-myc expression, whereas no effect could be observed in a cell line harboring amplified c-myc. Moreover, the down-regulation of c-myc expression, which is independent from cell cycle blockade, leads to cell death by apoptosis. Collectively, our results suggest the existence of a novel iron-dependent cell cycle regulatory mechanism involving modulation of translocated c-myc gene expression.

Apoptosis↗
PubMed2005

The survival of IL-6-dependent myeloma cells critically relies on their capability to transit the G1 to S phase interval of the cell cycle.

Interleukin-6 (IL-6) has an essential role in the initial progression of myeloma cell tumours. IL-6 triggers proliferation of these cells via the Ras-mitogen-activated protein kinase (MAPK) cascade and is thought to promote their survival via signal transducer and activator of transcription (STAT) pathway-dependent regulation of Bcl-2 family antiapoptotic members. Using IL-6-dependent murine B9 hybridoma/plasmacytoma cells, we here report that exiting the cell cycle G1 phase is a crucial step contributing to maintain viability. We show that (1) drug-mediated reversible G1 arrest triggered apoptosis despite the presence of IL-6; (2) a short IL-6 pulse to G1-arrested cells was sufficient to induce S phase entry and prevent apoptosis; and (3) phorbol ester and related derivatives promoted S phase entry and survival of IL-6-starved cells without up-regulating bcl-XL expression. Furthermore, that the MAPK kinase (MEK) 1/2 inhibitor, U0126, blocked proliferation and induced death of B9 cells indicate that IL-6 may not exert its survival effect primarily through bcl-XL and emphasizes the key role of Ras-MAPK cascade elements in the regulation of myeloma growth/viability.

Animals↗
PubMed2005

Efficient in vitro megakaryocyte maturation using cytokine cocktails optimized by statistical experimental design.

OBJECTIVE: A multi-step statistical strategy was applied to quantify individual and interactive effects of cytokines on megakaryopoiesis and to determine the concentration of the selected cytokines that optimize ex vivo megakaryocyte (MK) expansion, maturation, and platelet production in stromal- and serum-free conditions. MATERIALS AND METHODS: Immature MK were first generated from human CD34(+)-enriched cord blood cells cultured for 7 days in conditions favoring MK commitment. Then, the effect of different combinations of cytokines at various concentrations on MK differentiation and platelet production was tested on the day-7 MK. RESULTS: A large-scale screening of 13 cytokines in the presence of thrombopoietin (TPO) using Placket-Burman designs (PBD) was initially performed to identify stimulators of MK maturation. Afterwards, a statistical analysis of the two-level factorial designs revealed that in the presence of TPO, MK maturation was significantly stimulated by stem cell factor (SCF), interleukin (IL)-6, and IL-9, whereas Flt-3 ligand (FL) had a positive effect only on the expansion of MK progenitors. In contrast, erythropoietin (EPO) and IL-8 were inhibitors of MK maturation. A response surface methodology was then used to optimize the concentrations of the selected cytokines (TPO, SCF, IL-6, and IL-9) and defined a new cytokine cocktail that maximized MK expansion and maturation. Importantly, the increased MK output was accompanied by a very high MK purity ( approximately 90%). Another optimum was also found at a higher SCF concentration, which further improved MK expansion and maturation, but reduced MK purity. CONCLUSION: These statistical methods provide an efficient tool to analyze complex systems of cytokines and to develop promising ex vivo MK culture systems for clinical applications.

Blood Platelets↗
PubMed2005

Therapeutic intravenous immunoglobulins.

Intravenous immunoglobulins (IVIg) are concentrated formulations of human IgG prepared by industrial fractionation of large pools of individual plasma donations. IVIg were developed 20 years ago for the prophylaxis support of immunodeficient patients. However, IVIg have been increasingly used since 10 years, in the treatment of many autoimmune and inflammatory diseases raising the possibility of product shortages and ever increasing costs in the near future. Surprisingly, the immunomodulatory mechanisms of action of IVIg are unclear because of the diversity and often contradictory Fc, F(ab')(2), and non-IgG-related mechanisms that have been proposed from clinical observations and from results obtained in various in vitro and in vivo experimental models. These concepts are reviewed here and we discuss in more details three areas of active research, namely the mechanisms of IVIg action in Idiopathic Thrombocytopenic Purpura (ITP), the effects of IVIg on activated B lymphocytes and the possible involvement of autoantibodies of IgG isotype (auto-IgG) in the immunomodulatory effects of IVIg. The elucidation of the mechanisms of action of IVIg is crucial for a more rationalized clinical use of IVIg and for developing substitutes for some of the immunomodulatory indications in order to ensure long-term availability of plasma-derived IVIg for immunodeficient patients.

Autoantibodies↗
PubMed2004

Increased megakaryopoiesis in cultures of CD34-enriched cord blood cells maintained at 39 degrees C.

Based on previous evidence suggesting positive effects of fever on in vivo hematopoiesis, we tested the effect of hyperthermia on megakaryopoiesis (MK) in ex vivo cultures of CD34-enriched cord blood (CB) cells. The cells were cultured at 37 degrees C or 39 degrees C for 14 days in cytokine conditions optimized for megakaryocyte development and analyzed periodically. Compared to 37 degrees C, the cultures maintained at 39 degrees C produced significantly more (up to 10-fold) total cells, myeloid and MK progenitors, and total MKs, and showed accelerated and enhanced MK maturation with increased yields of proplatelets and platelets. This observation could facilitate clinical applications requiring ex vivo expansion of hematopoietic cells.

Antigens, CD34↗
PubMed2004

Autoantibodies purified from therapeutic preparations of intravenous immunoglobulins (IVIg) induce the formation of autoimmune complexes in normal human serum: a role in the in vivo mechanisms of action of IVIg?

Although intravenous immunoglobulins (IVIg) are widely used in the treatment of many autoimmune and inflammatory diseases, the mechanisms of action are still unclear in most cases. We have recently reported the presence of soluble autoimmune complexes (auto-IC) in human serum after the addition of a dose of IVIg similar to the one used in therapy. Here, we report the isolation and characterization of the responsible auto-IgG present in IVIg. The auto-IgG were purified by affinity chromatography on serum proteins immobilized on Sepharose. The purified auto-IgG constituted approximately 3% of the IgG present in IVIg and recognized a wide variety of structures in ELISA as well as many serum proteins on western blots. Auto-IC were formed in human serum following the addition of an amount of purified auto-IgG sufficient to over-saturate the auto-IgG inhibitory mechanisms known to be present in normal serum. These results indicate that most of the IgG present in IVIg are not involved in the formation of the soluble auto-IC, raising the possibility of preparing from IVIg a novel product which could be used for the treatment of the autoimmune diseases in which IC are thought to play an important role.

Autoantibodies↗
PubMed2004

Sensitive detection of human IgG in ELISA using a monoclonal anti-IgG-peroxidase conjugate.

Enzyme-antibody (Ab) conjugates specific for IgG are widely used in indirect immunological assays and have been until recently routinely prepared with polyclonal IgG-specific animal Abs. The use of monoclonal Abs (MAbs) could permit a better standardization of the IgG-specific conjugate reagents but is expected to result in lower reactivity due to the recognition of a single epitope by the MAbs. In this work, we have characterized a monoclonal anti-human IgG-peroxidase (HRP) reagent and compared its reactivity with commercial reagents. The murine C5-1 anti-human IgG MAb was selected for conjugation because of its high affinity (K(a) = 1.9 x 10(10)M), pan-IgG reactivity and absence of cross-reactivity with various structures including animal IgGs. The specific activity and binding kinetics of the C5-1:HRP conjugate were similar to the ones of two polyclonal anti-IgG:HRP conjugates when tested with immobilized human IgG. The C5-1:HRP conjugate could detect low amounts of human IgG much more effectively than two commercial monoclonal conjugates although it was slightly less effective than a polyclonal conjugate. However, the C5-1 conjugate yielded reduced background reactivity compared to the polyclonal conjugate, resulting in similar signal-to-noise ratios. These results indicate that the C5-1:HRP conjugate could be a suitable substitute for anti-human IgG conjugates prepared from animal antisera.

Animals↗
PubMed2004

Tetramolecular immune complexes are more efficient than IVIg to prevent antibody-dependent in vitro and in vivo phagocytosis of blood cells.

Intravenous immunoglobulins (IVIg) have immunomodulatory effects in vivo and are widely used in the treatment of autoimmune diseases, such as idiopathic thrombocytopenic purpura (ITP). The mechanisms by which IVIg can prevent platelet clearance in ITP patients are not fully understood but are known to require the participation of low affinity Fcgamma receptors (FcgammaRs), which interact poorly with monomeric immunoglobulin G (IgG). Given the importance of low affinity FcgammaRs in the treatment of ITP, we hypothesized that immune complexes (IC) produced in vitro could reproduce the effects of IVIg. Small-size tetramolecular IC were prepared using mouse monoclonal anti-human IgG and human Fc fragments. The effects of tetramolecular IC and IVIg on the in vitro and in vivo inhibition of phagocytosis of opsonized blood cells were compared. The results obtained showed that tetramolecular IC were at least six times more efficient than IVIg to prevent phagocytosis of opsonized red blood cells in vitro, and clearance of platelets in the thrombocytopenic mouse model.

Animals↗
PubMed2003

Monoclonal C5-1 antibody produced in transgenic alfalfa plants exhibits a N-glycosylation that is homogenous and suitable for glyco-engineering into human-compatible structures.

Structural analysis of the N-glycosylation of alfalfa proteins was investigated in order to evaluate the capacity of this plant to perform this biologically important post-translational modification. We show that, in alfalfa, N-linked glycans are processed into a large variety of mature oligosaccharides having core-xylose and core alpha(1,3)-fucose, as well as terminal Lewis(a) epitopes. In contrast, expression of the C5-1 monoclonal antibody in alfalfa plants results in the production of plant-derived IgG1 which is N-glycosylated by a predominant glycan having a alpha(1,3)-fucose and a beta(1,2)-xylose attached to a GlcNAc2Man3GlcNAc2 core. Since this core is common to plant and mammal N-linked glycans, it therefore appears that alfalfa plants have the ability to produce recombinant IgG1 having a N-glycosylation that is suitable for in vitro or in vivo glycan remodelling into a human-compatible plantibody. For instance, as proof of concept, in vitro galactosylation of the alfalfa-derived C5-1 mAb resulted in a homogenous plantibody harbouring terminal beta(1,4)-galactose residues as observed in the mammalian IgG.

Journal Article↗
PubMed2003

Modulations of anti-D affinity following promiscuous binding of the heavy chain with naïve light chains.

BACKGROUND: It is generally accepted that the antibody heavy (H) chain is more important than the light (L) chain for determining antigen specificity. In the case of anti-D, the predominant role of H chains in antigen binding is well recognized, but much less is known about the function of L chains. In this work, the contribution of L chains from non-D-immunized donors to the specificity and reactivity of anti-Ds was studied with L-chain shuffling. STUDY DESIGN AND METHODS: A kappa L chain library was recombined with the H chain of the 43F10 anti-D in a phagemid vector system (pComb3H, Scripps Institute). D-specific F(ab) phages were selected by panning on RBCs. Soluble F(ab)s were prepared, and their reactivity was assessed by RBC agglutination. The nucleotide and amino acid sequences of the variable region of the L chains were analyzed. RESULTS: The L chains of the six D-specific 43F10 F(ab) clones studied used five different germline genes from three Vkappa families and three different Jkappa segments. The L chains were all cationic with isoelectric points ranging from 8.1 to 10.2. CONCLUSION: The 43F10 anti-D H chain could bind promiscuously to a diversity of L chains from non-D-immunized donors without losing the D-antigen specificity. Relationships between the anti-D affinity and the cationic charge of the L chain as well as with the presence of an arginine residue in the L-chain complementarity-determining region 1 were observed.

Antibody Affinity↗
PubMed2003

Extended blood grouping of blood donors with automatable PCR-ELISA genotyping.

BACKGROUND: In the past 10 years, PCR-based methods have been described to allow the detection of gene polymorphisms responsible for many blood group antigens. These methods are routinely used to test samples of fetal origin and to resolve serologic discrepancies. Another interesting application of blood group genotyping could be the extended typing of blood donors for minor antigens to facilitate the procurement of compatible blood for alloimmunized patients. STUDY DESIGN AND METHODS: PCR-based tests have been modified to allow multiplex amplification of specific fragments of blood group genes and the convenient detection of hybridized amplicons by ELISA in a microplate format. RESULTS: The results obtained show that fragments of the Rh (D, c, C, e, E), Kell (K, k), Duffy (Fya, Fyb), and Kidd (Jka, Jkb) genes could be amplified along with controls in multiplex PCR reactions. Labeling of amplicons with digoxigenin allowed their solid-phase detection in microplate wells previously coated with individual blood group-specific oligonucleotides. A comparative study performed with 100 individuals showed a 99.7 percent concordance between genotypes and phenotypes for the 11 antigens assayed, with only three discrepant Fyb genotypes. CONCLUSION: Extended genotyping could be performed once on regular donors and confirmed when needed by standard serologic RBC assays. The format of these tests will allow easy automation of the procedure including the interpretation and downloading of the results with existing ELISA software.

Automation↗
PubMed2003

Preferential ex vivo expansion of megakaryocytes from human cord blood CD34+-enriched cells in the presence of thrombopoietin and limiting amounts of stem cell factor and Flt-3 ligand.

The high proliferative potential of cord blood (CB) stem cells and the identification of the key factor of megakaryopoiesis, thrombopoietin (TPO), permit the ex vivo expansion of megakaryocytes (MKs) for possible use in early post-transplant support of patients and the production of functional platelets for transfusion. However, culture conditions for the generation of adequate MKs for this purpose are not yet optimized. Therefore, we sought to define the mixture of early-acting cytokines and TPO that would promote the expansion of MK progenitors over other lineages and result in overall better MK expansion and platelet yields. CB CD34(+)-enriched cells were cultured in serum-free medium for 17 days in presence of TPO alone or in various combinations with early-acting cytokines used at different concentrations and addition times. MK expansion and polyploidy and platelet production were monitored by flow cytometry analysis using specific surface markers (CD41 and CD42b) and propidium iodide labeling. Our results showed that the use of high concentrations of stem cell factor (SCF) and Flt-3 ligand (FL) in early CB TPO-supplemented cultures was more favorable to monocytic and granulocytic cell expansion. However, we observed that their presence in limiting amounts was required for the preferential expansion of MK progenitors. The addition of SCF, FL, TPO, and interleukin-6 (IL-6) at high concentrations in secondary cultures of these expanded MKs resulted in optimal MK proportion (approximately 25% of MKs) and expansion (>300 MK per seeded cell), highest proportions of polyploid MKs (22% of mature MKs > or = 8N), and best platelet yields. Our results indicate that TPO-induced MK progenitors are more sensitive to early-acting cytokines than non-MK cells. We propose that MKs generated in the optimized conditions, in combination with immature stem/progenitor cells, could prove useful for the short-term platelet recovery following CB transplantation.

Antigens, CD34↗
PubMed2002

Intravenous immunoglobulins induce the in vitro differentiation of human B lymphocytes and the secretion of IgG.

The therapeutic effects of intravenous immunoglobulins (IVIGs) in several autoimmune diseases are characterized by a decrease in pathologic autoantibody levels. Although little direct evidence has been reported in humans, the large repertoire of natural immunoglobulin G (IgG) antibodies in IVIGs is expected to be involved in the regulation of autoreactive B lymphocytes. In normal adult mice, IVIGs have been reported to modulate immature B cells as well as peripheral B lymphocytes through V-region connections. Studies with human serum also indicated that anti-idiotypic antibodies, present in IVIG preparations, could recognize both natural and pathologic autoantibodies. We have used an in vitro culture system to characterize the direct effect of IVIGs on human B lymphocytes. This in vitro culture system involves CD40 activation of B lymphocytes by its ligand CD154 in the presence of cytokines. In this system, addition of IVIGs decreased by 50% to 80% the expansion of B lymphocytes. This reduced expansion was due to a decrease in the proliferation rate. In addition, a portion of B lymphocytes was differentiated into IgG-secreting cells in the presence of IVIGs and the secreted IgGs were reactive with antigens such as nucleoprotamine, dsDNA, tetanus toxin, and human IgG F(ab')(2) fragments. These observations indicate that IVIGs can have direct effects on B lymphocytes and suggest that such IVIG regulation of B lymphocytes could be involved in the therapeutic effects of IVIGs in autoimmune diseases.

Antibody Formation↗