Biomedical subjects

N Bewarder

Publications and source records attributed to N Bewarder.

5 recordsLinked to original sources

PubMed1996

Epitope mapping of new monoclonal antibodies recognizing distinct human FcRII (CD32) isoforms.

The class II Fc gamma receptors are widely distributed on cells of the immune system. Nevertheless, the exact cell type distribution of the FcRII isoforms is still unclear because of the lack of appropriate antibodies that discriminate between the various isoforms. In this study we describe the generation and characterization of three monoclonal antibodies (MAbs) raised against recombinant human FcRIIb2 as well as a synthetic peptide (amino acids 30-39) of this receptor. Analyses of the isoform specificity of these antibodies using ELISA and Western blots revealed that the MAbs II1A5 (mIgG1) and ID2.7 (mIgM) are pan FcRII antibodies recognizing all known FcRII isoforms. In contrast, the MAb II8D2 (mIgG1) specifically reacts with FcRIIb but not with FcRIIa. The observed antibody reactivities could be confirmed by examination of the exact epitopes using overlapping 15-mer peptides spanning the entire FcRIIb2. So far these antibodies are the only ones described that detect FcRII in Western blots. Moreover, they can be used to analyze the cellular FcRII isoform distribution at the protein level, which was otherwise not possible.

Amino Acid Sequence↗

PubMed1996

In vivo and in vitro specificity of protein tyrosine kinases for immunoglobulin G receptor (FcgammaRII) phosphorylation.

Human B cells express four immunoglobulin G receptors, FcgammaRIIa, FcgammaRIIb1, FcgammaRIIb2, and FcgammaRIIc. Coligation of either FcgammaRII isoform with the B-cell antigen receptor (BCR) results in the abrogation of B-cell activation, but only the FcgammaRIIa/c and FcgammaIIb1 isoforms become phosphorylated. To identify the FcgammaRII-phosphorylating protein tyrosine kinase (PTK), we used the combination of an in vitro and an in vivo approach. In an in vitro assay using recombinant cytoplasmic tails of the different FcgammaRII isoforms as well as tyrosine exchange mutants, we show that each of the BCR-associated PTKs (Lyn, Blk, Fyn, and Syk) shows different phosphorylation patterns with regard to the different FcgammaR isoforms and point mutants. While each PTK phosphorylated FcgammaRIIa/c, FcgammaRIIb1 was phosphorylated by Lyn and Blk whereas FcgammaRIIb2 became phosphorylated only by Blk. Mutants lacking both tyrosine residues of the immune receptor tyrosine-based activation motif (ITAM) of FcgammaRIIa/c were not phosphorylated by Blk and Fyn, while Lyn-mediated phosphorylation was dependent on the presence of the C-terminal tyrosine of the ITAM. Results obtained in assays using an FcgammaR- B-cell line transfected with wild-type or mutated FcgammaRIIa demonstrated that exchange of the C-terminal tyrosine of the ITAM of FcgammaRIIa/c was sufficient to abolish FcgammaRIIa/c phosphorylation in B cells. Additionally, we could show that Lyn and Fyn bind to FcgammaRIIa/c, with the ITAM being necessary for association. Comparison of the phosphorylation pattern of each PTK observed in vitro with the phosphorylation pattern observed in vivo suggests that Lyn is the most likely candidate for FcgammaRIIa/c and FcgammaRIIb1 phosphorylation in vivo.

Amino Acid Sequence↗

PubMed1994

Tyrosine-containing sequence motifs of the human immunoglobulin G receptors FcRIIb1 and FcRIIb2 essential for endocytosis and regulation of calcium flux in B cells.

Human B cells express two closely related immunoglobulin G receptors, FcRIIb1 and FcRIIb2, which differ by a 19 amino acid insertion in the cytoplasmic tail of FcRIIb1. The cytoplasmic tails of both isoforms contain a conserved sequence motif (AENTITYSLL) essential for mediating endocytosis via FcRIIb2. Truncation of this motif abolished endocytosis, while replacement of tyrosine (Tyr273) in FcRIIb2 by phenylalanine had no effect on the amount and kinetics of ligand uptake. Co-cross-linking of FcRIIb1 or FcRIIb2 with the antigen receptor on B cells led to an abortive calcium signal. Neither isoform interfered with the early intracellular calcium mobilization, but both prevented the opening of a plasma membrane calcium channel essential for a sustained elevated intracellular calcium level. Modulation of calcium channel activity is mediated by the same sequence motif essential for endocytosis but requires the presence of Tyr292 in FcRIIb1 and Tyr273 in FcRIIb2. Co-cross-linking of FcRIIb1 with surface IgG is associated with tyrosine phosphorylation of Tyr292, whereas Tyr272 in FcRIIb2 was not phosphorylated. Thus, FcRIIb phosphorylation is probably not directly involved in the modulation of the calcium signal but may be essential for further diversification of signals transduced via the coexpressed isoforms FcRIIb1 and FcRIIb2.

Animals↗

PubMed1994

Biological functions of human Fc gamma RIIa/Fc gamma RIIc in B cells.

The human immunoglobulin receptor IIa (FcRIIa) was transfected into the FcR- mouse B cell line IIA1.6 to study its role in mediating endocytosis of human IgG complexes as well as its possible function in inhibiting the attenuated increased calcium level in B cells after antigen receptor cross-linking. Using FcRIIa mutants with truncated cytoplasmic domains we show that the region within the cytoplasmic region necessary for endocytosis differs from that necessary for the abrogation of the elevated calcium level in B cells induced after antigen receptor cross-linking. Deletion of 14 amino acids at the carboxy terminus led to a slow internalization of FcRIIa bound human IgG, whereas the mutant lacking 30 amino acids completely failed to mediate IgG uptake. The mutant lacking 14 amino acids at the carboxy terminus is not tyrosine phosphorylated in the course of receptor-mediated IgG uptake. This suggests that phosphorylation of FcRIIa is not necessary to mediate this function. FcRIIa cross-linking leads to a rapid transient rise in the intracellular calcium concentration due to calcium release from intracellular stores. Using the FcRIIa mutants we could further show that the signal delivered by FcRIIa is strongly dependent on the last 14 amino acids of the cytoplasmic tail. Analyses of the tyrosine phosphorylation of FcRIIa revealed that the calcium release mediated by FcRIIa cross-linking is dependent on tyrosine phosphorylation. In contrast, inhibition of the antigen receptor (sIgG) induced rise in intracellular calcium concentration by FcRIIa-sIgG co-cross-linking was not impaired when 30 amino acids were deleted at the carboxy terminus. FcRIIa wild type was rapidly phosphorylated when co-cross-linked with sIgG, but phosphorylation is not a prerequisite to inhibit calcium influx induced by sIgG cross-linking. These results show that distinct regions within the cytoplasmic tail of FcRIIa are necessary for the various signals transmitted to the cell.

Amino Acid Sequence↗

PubMedDate not supplied

External quality control program for inter-laboratory quality control.

BACKGROUND: The accuracy and precision of tumor marker testing is of high clinical importance requiring the implementation of effective quality control procedures in the diagnostic laboratory. An external quality control program including an inter-laboratory comparison could be of help in improving the quality of test results. MATERIALS AND METHODS: Laboratories using BIOREF reference material sent their routine internal quality control results of each monthly control period to the evaluation center for statistical analysis and inter-laboratory comparison. RESULTS: The inter-laboratory comparison showed that the mean values of test results obtained by different laboratories can vary considerably especially when different test kits are used. A comparison of test results of laboratories using the same test kit showed much better correlation but discrepancies of coefficients of variation between these laboratories were still observed. CONCLUSION: An external quality control program revealed problems in the accuracy and precision of tumor marker test results in the individual laboratory, which could serve as a basis for the improvement of test performance.

Biomarkers, Tumor↗