Protein engineering of cytochrome c by semisynthesis: substitutions at Glutamic acid 66

We have used protein semisynthesis to prepare four analogues of horse cytochrome c, in which the glutamic acid residue at position 66 has been removed and replaced by norvaline, glutamine, lysine and, as a methodological control, glutamic acid. This residue is quite strongly conserved in mitochondrial cytochrome c, and forms part of a cluster of acidic residues that occurs in all cytochromes c but whose function is obscure. Comparative studies of the physical and biochemical properties of the analogues have now disclosed two specific roles for Glu66 in the protein. It contributes significantly to the stabilization of the active conformation of the protein, probably by salt bridge formation, and it appears to participate in the redox-state-dependent ATP-binding site of cytochrome c. Our results also support two general views of the role of surface charged residues in cytochrome c, namely that their disposition influences both redox potential, through the electrostatic field felt at the redox centre, and the kinetics of electron transfer, through the dipole moment they generat

Oral Immunization of Mice with Lactic Acid Bacteria Producing Helicobacter pylori Urease B Subunit Partially Protects against Challenge with Helicobacter felis

BackgroundThe development of an efficacious vaccine against infection with Helicobacter pylori the causative agent of chronic gastritis, peptic ulcer disease, and gastric adenocarcinoma, remains a challenge. Since the use of mucosal adjuvants is limited in human application, we have evaluated the potential of recombinant Lactobacillus strains producing H. pylori urease B (UreB) subunit to deliver this antigen to the gastrointestinal tract MethodsMice were injected orally 3 times with a triple dose of recombinant Lactobacillus plantarum NCIMB8826, the recombinant isogenic cell-wall mutant (alr− MD007 strain) expressing UreB, or a mixture of recombinant UreB and cholera toxin (rUreB/CT) as a control. Urease-specific seric immunoglobulin (Ig) G and IgA were measured by use of an enzyme-linked immunosorbent assay. After challenge with Helicobacter felis stomach infection was examined by use of the rapid urease test and by polymerase chain reaction detection of Helicobacter genomic DNA ResultsIntragastric immunization with both recombinant Lactobacillus strains and rUreB/CT elicited UreB-specific antibodies. After challenge, reduction of H. felis load in the stomachs of mice was observed only after immunization with the recombinant mutant strain MD007 or with rUreB/CT ConclusionsThis is the first report of successful induction of partial protection against H. felis with a mucosal prime-boost regimen in which recombinant Lactobacillus strains were used as antigen-delivery vehicle

Rotavirus specific plasma secretory immunoglobulin in children with acute gastroenteritis and children vaccinated with an attenuated human rotavirus vaccine

Q2Q1Artículo de investigación2409-2417Rotavirus (RV)–specific secretory immunoglobulin (RV-SIg) has been previously detected in serum of naturally RV infected children and shown to reflect the intestinal Ig immune response. Total plasma SIgA and plasma RV-SIg were evaluated by ELISA in children with gastroenteritis due or not due to RV infection and in 50 children vaccinated with the attenuated RIX4414 human RV vaccine and 62 placebo recipients. RV-SIg was only detected in children with evidence of previous RV infection or with acute RV gastroenteritis. Vaccinees had higher RV-SIg titers than placebo recipients and RV-SIg titers increased after the second vaccine dose. RV-SIg measured after the second dose correlated with protection when vaccinees and placebo recipients were analyzed jointly. RV-SIg may serve as a valuable correlate of protection for RV vaccines

Secretory immunoglobulin A: well beyond immune exclusion at mucosal surfaces.

At mucosal surfaces, secretory IgA (SIgA) antibodies serve as the first line of defense against microorganisms through a mechanism called immune exclusion that prevents interaction of neutralized antigens with the epithelium. In addition, SIgA plays a role in the immune balance of the epithelial barrier through selective adhesion to M cells in intestinal Peyer's patches. This mediates the transepithelial retro-transport of the antibody and associated antigens from the intestinal lumen to underlying gut-associated organized lymphoid tissue. In Peyer's patches, SIgA-based immune complexes are internalized by underlying antigen-presenting cells, leaving the antigen with masked epitopes, a form that limits the risk of overwhelming the local immune protection system with danger signals. This translates into the onset of mucosal and systemic responses associated with production of anti-inflammatory cytokines and limited activation of antigen-presenting cells. In the gastrointestinal tract, SIgA exhibits thus properties of a neutralizing agent (immune exclusion) and of an immunopotentiator inducing effector immune responses in a noninflammatory context favorable to preserve local homeostasis

Molecular definition of the role of secretory component in secretory IgA-mediated protection at mucosal surfaces

International audienceNo abstrac

Novel functions of the polymeric Ig receptor: well beyond transport of immunoglobulins

International audienceThe polymeric Ig receptor (pIgR) ensures efficient secretion of polymeric IgA (pIgA) at mucosal surfaces. On basal to apical transport across epithelial cells, the pIgR extracellular domain is cleaved, releasing secretory component (SC) in association with pIgA. This finds its raison d'être in the recent observation that SC is directly involved in the protective function of secretory IgA. In addition, free SC exhibits scavenger properties with respect to enteric pathogens. However, although pIgR dedicates its life to mucosal protection, it also seems to permit pathogen entrance through the epithelial barrier. The multiple mechanisms that they are involved in make pIgR and SC instrumental to mucosal immunity