Mucosal tolerance induction generally requires multiple or large Ag doses. Because microfold (M) cells have been implicated as being important for mucosal tolerance induction and because reovirus attachment protein σ1 (pσ1) is capable of binding M cells, we postulated that targeting a model Ag to M cells via pσ1 could induce a state of unresponsiveness. Accordingly, a genetic fusion between OVA and the M cell ligand, reovirus pσ1, termed OVA-pσ1, was developed to enhance tolerogen uptake. When applied nasally, not parenterally, as little as a single dose of OVA-pσ1 failed to induce OVA-specific Abs even in the presence of adjuvant. Moreover, the mice remained unresponsive to peripheral OVA challenge, unlike mice given multiple nasal OVA doses that rendered them responsive to OVA. The observed unresponsiveness to OVA-pσ1 could be adoptively transferred using cervical lymph node CD4+ T cells, which failed to undergo proliferative or delayed-type hypersensitivity responses in recipients. To discern the cytokines responsible as a mechanism for this unresponsiveness, restimulation assays revealed increased production of regulatory cytokines, IL-4, IL-10, and TGF-β1, with greatly reduced IL-17 and IFN-γ. The induced IL-10 was derived predominantly from FoxP3+CD25+CD4+ T cells. No FoxP3+CD25+CD4+ T cells were induced in OVA-pσ1-dosed IL-10-deficient (IL-10−/−) mice, and despite showing increased TGF-β1 synthesis, these mice were responsive to OVA. These data demonstrate the feasibility of using pσ1 as a mucosal delivery platform specifically for low-dose tolerance induction.
Absence of suitable mucosal adjuvants for humans prompted us to consider alternative vaccine designs for mucosal immunization. Because adenovirus is adept in binding to the respiratory epithelium, we tested the adenovirus 2 fiber protein (Ad2F) as a potential vaccine-targeting molecule to mediate vaccine uptake. The vaccine component (the host cell-binding domain to botulinum toxin (BoNT) serotype A) was genetically fused to Ad2F to enable epithelial binding. The binding domain for BoNT was selected because it lies within the immunodominant H chain as a β-trefoil (Hcβtre) structure; we hypothesize that induced neutralizing Abs should be protective. Mice were nasally immunized with the Hcβtre or Hcβtre-Ad2F, with or without cholera toxin (CT). Without CT, mice immunized with Hcβtre produced weak secretory IgA (sIgA) and plasma IgG Ab response. Hcβtre-Ad2F-immunized mice produced a sIgA response equivalent to mice coimmunized with CT. With CT, Hcβtre-Ad2F-immunized mice showed a more rapid onset of sIgA and plasma IgG Ab responses that were supported by a mixed Th1/Th2 cells, as opposed to mostly Th2 cells by Hcβtre-dosed mice. Mice immunized with adjuvanted Hcβtre-Ad2F or Hcβtre were protected against lethal BoNT serotype A challenge. Using a mouse neutralization assay, fecal Abs from Hcβtre-Ad2F or Hcβtre plus CT-dosed mice could confer protection. Parenteral immunization showed that the inclusion of Ad2F enhances anti-Hcβtre Ab titers even in the absence of adjuvant. This study shows that the Hcβtre structure can confer protective immunity and that use of Hcβtre-Ad2F gives more rapid and sustained mucosal and plasma Ab responses.
Recent investigations have suggested that novel food should be evaluated not only from the nutritional but also from the physiological point of view. Opioid peptides derived from milk belong to compounds whose physiological importance has not yet been fully recognized. The commercial Humana formula for newborns sold on the Polish market was assayed in the study. Four opioid peptides with agonistic and antagonistic activity were found in a peptide extract from the Humana formula and its pepsin-trypsin hydrolysate: β-casomorphin-5, casoxin C and 6 and lactoferroxin A. The opioid activity of the peptide extracts was determined by examining their influence on the motor activity of isolated rabbit intestine. In conclusion, it was suggested that the opioid activity of the formulas could be an additional indicator of their diversity.
Nagy A., Marciniak-Darmochwał K., Krawczuk S., Mierzejewska D., Kostyra H., Gelencsér É.(2009): Influence of glycation and pepsin hydrolysis on immunoreactivity of albumin/globulin fraction of herbicide resistant wheat line. Czech J. Food Sci., 27: 320-329.The aim of this study was to investigate the influence of non-enzymatic glycosylation on the immunogenic properties of soluble wheat proteins. Albumin/globulin fractions of herbicide resistant wheat line were non-enzymatically glycosylated using glucose for seven days at 37°C. The changes in their structures and immunoreactivity were then determined. The protein fractions were also hydrolysed with pepsin to determine the resistance to digestion. Albumin/globulin fractions before and after non-enzymatic glycosylation were analysed using o-phthaldialdehyde method and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The immunoreactivity of the protein fractions was determined using enzyme-linked immunosorbent assay methods, as well as affinity chromatography. The soluble wheat proteins showed smaller amounts of available α-amino groups after non-enzymatic glycosylation, and were stronger immunogens after glycation, but their antigenicity was not been affected significantly. However, pepsin hydrolysis of wheat proteins decreased their immunoreactivity.
Oral Salmonella vaccine expressing enterotoxigenic E. coli colonization factor antigen 1 (CFA/I) fimbriae induced production of Treg cells. Adoptive transfer of CD25+ Treg cells obtained from oral Salmonella-CFA/I-vaccinated mice conferred superior protection to proteolipid protein (PLP)139-151-dependent EAE to those from Salmonella vector-immunized mice; this was attributed to their differential cytokine profiles. Salmonella-CFA/I-vaccinated mice produced more TGF-β than Salmonella vector-immunized mice. To test the hypothesis Salmonella-CFA/I-induced Treg cells, independent of PLP-specificity, confers protection via TGF-β, SJL/J mice were immunized with either Salmonella-CFA/I or Salmonella vector. Two wks later, CD25+ and CD25- CD4+ T cells were sorted, adoptively transferred to naïve SJL/J mice, concomitantly treated with anti-TGF-β mAb or isotype IgG, and then induced with EAE. Mice adoptively transferred with Treg cells neutralized of their TGF-β showed earlier disease onset and greater disease severity than mice adoptively transferred with Treg cells treated with IgG control. Moreover, greater proinflammatory cytokine production was observed. This work shows Treg cells can be induced to high potency by simply vaccinating against irrelevant Ags using a live, bacterial vaccine, thus, offering a novel approach to treat autoimmune diseases independent of the auto-Ag. Supported by NIH AI-41123.
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