Erythropoietin (Epo) is a 166 amino acids protein containing three N-glycosylation sites (Asn-24, Asn-38, and Asn-83) and 1 O- glycosylation site (Ser-126) and involved in the regulation of the level ...of red blood cells. Today, only one recombinant human Epo (rHuEpo), produced in CHO cell line, is extensively used in therapy to cure severe anemia. The structure of the glycan chains of this rHuEpo slightly differ of those of the urinary human Epo (uHuEpo), considered as the natural Epo molecule. In an attempt to produce a rHuEpo as close as possible to the uHuEpo, Epo gene was expressed in a human lymphoblastoid cell line, named RPMI 1788. In order to fully characterize the Epo-RPMI, structural characterizations of the protein skeleton as well as glycan chains were undergone. As expected, the amino acid sequence of the Epo-RPMI conformed to that of uHuEpo. Surprisingly, the structure of some N-glycan chains, as mainly determined by ESI-MS, revealed some unusual characteristics. Thus, 80% of N-glycans possess a bisecting GlcNAc residue, 25% bear a second fucose residue which is present, in a large part, in a sialyl Le(x)motif, and 13% contain more than three LacNAc repeats (up to five per molecule). Despite these unusual structural characteristics, the data concerning the in vitro and in vivo biological activities were not impaired when compared to Epo-CHO and uHuEpo.
The substitution of plasmatic anti-RhD polyclonal antibodies by a monoclonal antibody (mAb) for preventing the hemolytic disease of the newborn (HDN) is an important issue due to supply and safety ...concerns. Since it has been suggested that FcγR are involved in the prevention of HDN, the in vitro functional properties of two anti-RhD mAbs differing through their glycosylation profiles were compared using FcγR-based assays to select a candidate mAb. T125(YB2/0), a low fucosylated antibody, bound strongly to both activating FcγRIII and inhibitory FcγRII, as opposed to its highly fucosylated counterpart. It also exerted a strong ADCC against RhD
+ RBCs and a potent FcγRIIB-mediated inhibition of cytokine release. Moreover, an in vivo RhD
+ red blood cells (RBCs) clearance assay showed that this antibody exhibits a RhD
+ RBCs clearance as potent as polyclonal anti-RhD antibodies in NOD-SCID mice. Thus, T125(YB2/O) has been selected to be tested for the prevention of anti-RhD allo-immunization.
We have identified the liver-regulating protein (LRP), a cell surface protein involved in the maintenance of hepatocyte differentiation when cocultured with rat liver epithelial cells (RLEC). LRP was ...defined by immunoreactivity to a monoclonal antibody (mAb L8) prepared from RLEC. mAb L8 specifically detected two polypeptides of 85 and 73 kD in immunoprecipitation of both hepatocyte- and RLEC-iodinated plasma membranes. The involvement of these polypeptides, which are integral membrane proteins, in cell interaction-mediated regulation of hepatocytes was assessed by evaluating the perturbing effects of the antibody on cocultures with RLEC. Several parameters characteristic of differentiated hepatocytes were studied, such as liver-specific and house-keeping gene expression, cytoskeletal organization and deposition of extracellular matrix (ECM). An early cytoskeletal disturbance was evidenced and a marked alteration of hepatocyte functional capacity was observed in the presence of the antibody, together with a loss of ECM deposition. By contrast, cell-cell aggregation or cell adhesion to various extracellular matrix components were not affected. These findings suggest that LRP is distinct from an extracellular matrix receptor. The fact that early addition of mAb L8 during cell contact establishment was necessary to be effective may indicate that LRP is a novel plasma membrane protein that plays an early pivotal role in the coordinated metabolic changes which lead to the differentiated phenotype of mature hepatocytes.
NK cells can kill antibody-coated target cells following engagement of Fc gamma RIIIA, the major activating Fc gamma R expressed by these cells. The presence of Fc gamma RIIC (CD32C) has also been ...reported, but its contribution to the Fc gamma R-dependent effector functions of NK cells remains debated. We demonstrate here that inhibitory Fc gamma RIIB is also expressed by a small subset of CD56 super(+)/NKp46 super(+) NK cells and can efficiently down-modulate their Fc gamma R-dependent effector function. Immunofluorescence analyses of NK cells from 52 healthy donors showed the presence of CD56 super(bright)/Fc gamma RII super(-) (5.2% plus or minus 3.4), CD56 super(dim)/Fc gamma RII super(lo/-) (94.1% plus or minus 3.4), and CD56 super(dim)/Fc gamma RII super(bright) (0.64% plus or minus 0.72) cells. QRT-PCR and protein analyses performed on isolated Fc gamma RII super(bright) NK cells indicated that Fc gamma RIIB is strongly expressed by these cells but not by Fc gamma RII super(lo/-) cells. In addition, Fc gamma RII super(bright) cells showed a weaker antibody-dependent degranulation when incubated with IgG-coated target cells compared with Fc gamma RII super(lo/-) NK cells, although a strong Fc gamma RIIIA expression was detected in both cells. Furthermore, the addition of anti-Fc gamma RII Fab paralleled a higher degranulation of Fc gamma RII super(bright) NK cells, indicating a direct role for Fc gamma RIIB in this down-modulating effect. Thus, it is proposed that Fc gamma RIIB super(bright) NK cells represent a new NK cell compartment able to down-modulate NK cell functions triggered by the engagement of activating Fc gamma R.
Intravenous immunoglobulins (IVIg) are therapeutic preparations of normal human polyclonal Ig G (IgG) that exert immunomodulatory effects in patients with autoimmune or systemic inflammatory ...diseases. Two different IgG subfractions were evaluated for their respective immunomodulatory effects in the treatment of experimental autoimmune diseases: a fraction enriched in antibodies that recognize the F(ab‘)2 portion of IVIg and a fraction of natural polyreactive autoantibodies purified on a dinitrophenyl (DNP)-Affiprep immunoadsorbent. A very small fraction of IgG interacting with DNP but not with F(ab‘)2 fragments expressed an increased ability to bind to self-antigens. The anti-DNP fraction, but not the anti-idiotype fraction, protected against inflammation observed in collagen-induced arthritis and experimental autoimmune encephalomyelitis in rats. Furthermore, it was able to reduce the occurrence of spontaneous diabetes mellitus in nonobese diabetic mice at lower concentrations than unfractionated IVIg. The therapeutic benefit of the anti-DNP fraction was associated with the inhibition of secretion of proinflammatory cytokines and stimulation of secretion of IL-1 receptor antagonist. Our results provide evidence that polyreactive autoantibodies play a role in the protective effect of IVIg in experimental models of autoimmune diseases in which inflammatory reactions are part of the disease process.
Perspectives Cortey, A.; Brossard, Y.; Beliard, R. ...
Journal de gynécologie, obstétrique et biologie de la reproduction,
2/2006, Volume:
35
Journal Article
Perspectives Cortey, A.; Brossard, Y.; Beliard, R. ...
Journal de gynécologie, obstétrique et biologie de la reproduction,
2006, Volume:
35
Journal Article
Actuellement, l’immunoprophylaxie Rh anténatale concerne toutes les femmes RH :-1 bien que 30 à 40 % d’entre elles portent un enfant RH :-1. La connaissance du génotype RhD fœtal permettrait de ...modifier une pratique de prophylaxie médicalement peu rationnelle (car exposant inutilement beaucoup de patientes à un médicament dérivé du sang), sans en réduire l’efficacité.
Le génotypage RhD fœtal par PCR déterminé sur le liquide amniotique a une sensibilité excellente. L’existence de gènes D silencieux perturbe sa spécificité qui reste pourtant acceptable. Les patientes doivent néanmoins être systématiquement informées de la possibilité de ces faux positifs.
Le génotypage RhD fœtal sur sang maternel est plus complexe. Sa sensibilité est bonne à partir de 10 SA et excellente à partir de 15 SA. Toutefois, en cas de premier résultat négatif, il est recommandé de contrôler le RhD fœtal à partir d’un second prélèvement effectué quelques semaines après.
Une autre perspective d’avenir pour l’immunoprophylaxie Rhésus est représentée par les tentatives de substitution des Ig polyclonales anti-D par des anticorps monoclonaux humains anti-D. L’élaboration d’anticorps monoclonaux ayant des capacités de neutralisation des globules rouges RhD positif comparables aux IgRh polyclonales est délicate. Une nouvelle génération d’anticorps monoclonaux humains anti-D (anti-D recombinants produits dans des lignées cellulaires assurant une glycosylation post-traductionnelle efficace) a été mise au point et les résultats cliniques obtenus permettent d’espérer qu’ils prendront bientôt place à côté des Ig polyclonales dans l’immunoprophylaxie Rh de la femme enceinte. On ne pourra cependant juger de cette place qu’après une évaluation à grande échelle de ces nouveaux produits (efficacité comparée, résistances éventuelles…) sur le long terme (plusieurs grossesses).
At present, rhesus prophylaxis concerns RhD negative pregnant women, even though 30 to 40% of them are bearing a RhD negative child. Knowing the RhD fetal genotype could change this quite irrational practice of prophylaxis (exposing many more women than needed to blood derived products) without reducing its efficacy.
RhD fetal genotype determined on amniotic fluid has an excellent sensitivity. Presence of silent D genes slightly impairs its specificity which remains acceptable. However women have to be informed of possible false positives.
Fetal RhD genotyping on maternal blood is more complex. Sensitivity is good from 10 GW and excellent after 15 GW. In case of a first negative result, it is recommended to control fetal RhD on a second sample drawn a few weeks later.
Another new perspective for rhesus prophylaxis is the attempt to substitute polyclonal IgG anti-D into human monoclonal IgG anti-D. The main difficulty is to elaborate monoclonal antibodies with a capacity to neutralize RhD positive red blood cells equivalent to those of polyclonal anti-D. A new generation of antibodies is in process and preliminary clinical results are suggesting a possible use of these monoclonal antibodies for future rhesus prophylaxis but long-term follow-up is required to draw further conclusions.
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