Human Immunoglobulin Heavy Gamma Chain Polymorphisms: Molecular Confirmation Of Proteomic Assessment.

Immunoglobulin G (IgG) proteins are known for the huge diversity of the variable domains of their heavy and light chains, aimed at protecting each individual against foreign antigens. The IgG also harbor specific polymorphism concentrated in the CH2 and CH3-CHS constant regions located on the Fc fragment of their heavy chains. But this individual particularity relies only on a few amino acids among which some could make accurate sequence determination a challenge for mass spectrometry-based techniques.The purpose of the study was to bring a molecular validation of proteomic results by the sequencing of encoding DNA fragments. It was performed using ten individual samples (DNA and sera) selected on the basis of their Gm (gamma marker) allotype polymorphism in order to cover the main immunoglobulin heavy gamma (IGHG) gene diversity. Gm allotypes, reflecting part of this diversity, were determined by a serological method. On its side, the IGH locus comprises four functional IGHG genes totalizing 34 alleles and encoding the four IgG subclasses. The genomic study focused on the nucleotide polymorphism of the CH2 and CH3-CHS exons and of the intron. Despite strong sequence identity, four pairs of specific gene amplification primers could be designed. Additional primers were identified to perform the subsequent sequencing. The nucleotide sequences obtained were first assigned to a specific IGHG gene, and then IGHG alleles were deduced using a home-made decision tree reading of the nucleotide sequences. IGHG amino acid (AA) alleles were determined by mass spectrometry. Identical results were found at 95% between alleles identified by proteomics and those deduced from genomics. These results validate the proteomic approach which could be used for diagnostic purposes, namely for a mother-and-child differential IGHG detection in a context of suspicion of congenital infection.

Evidence of airborne excretion of Pneumocystis carinii during infection in immunocompetent rats. Lung involvement and antibody response.

To better understand the role of immunocompetent hosts in the diffusion of Pneumocystis in the environment, airborne shedding of Pneumocystis carinii in the surrounding air of experimentally infected Sprague Dawley rats was quantified by means of a real-time PCR assay, in parallel with the kinetics of P. carinii loads in lungs and specific serum antibody titres. Pneumocystis-free Sprague Dawley rats were intratracheally inoculated at day 0 (d0) and then followed for 60 days. P. carinii DNA was detected in lungs until d29 in two separate experiments and thereafter remained undetectable. A transient air excretion of Pneumocystis DNA was observed between d14 and d22 in the first experiment and between d9 and d19 in the second experiment; it was related to the peak of infection in lungs. IgM and IgG anti-P. carinii antibody increase preceded clearance of P. carinii in the lungs and cessation of airborne excretion. In rats receiving a second challenge 3 months after the first inoculation, Pneumocystis was only detected at a low level in the lungs of 2 of 3 rats at d2 post challenge and was never detected in air samples. Anti-Pneumocystis antibody determinations showed a typical secondary IgG antibody response. This study provides the first direct evidence that immunocompetent hosts can excrete Pneumocystis following a primary acquired infection. Lung infection was apparently controlled by the immune response since fungal burdens decreased to become undetectable as specific antibodies reached high titres in serum. This immune response was apparently protective against reinfection 3 months later.