Feed pellets containing chitosan nanoparticles as plasmid DNA oral delivery system for fish: In vivo assessment in gilthead sea bream (Sparus aurata) juveniles.

The aim of this study was the assessment of preloaded feed pellets as a delivery system for plasmid DNA (pDNA), with the purpose of evaluating the potential administration of DNA vaccines orally in aquacultured fish. Pellets were made up by usual feed ingredients, which were mixed with chitosan nanoparticles entrapping a model plasmid (pCMVß) expressible in eukaryotic cells before being elaborated. The plasmid is characterized by the insertion of the reporter gene lacZ, encoding for the bacterial enzyme ß-galactosidase (ß-gal). The possible in vivo expression of the exogenous gene was measured in different fish tissues of gilthead sea bream (Sparus aurata) juveniles by two different procedures. On the one hand, the activity of the enzyme ß-gal was detected and quantified in muscle, liver and intestine; on the other, specific IgM against ß-gal antigen was titrated in blood samples. Intramuscular (i.m.) injection of equal amounts of plasmid was also carried out for the purpose of comparison with oral administration. The expression of the reporter gene was detected in fish tissues following both oral and i. m. administration of pDNA up to 60 days. However, organ distribution of the gene expression was more evident after oral (ß-gal activity measured in gut, liver and muscle) than after parenteral administration (restricted to adjacent muscle tissues). In agreement, specific IgM titration indicated that humoral immune response was more intense and sustained throughout the experimental period after oral than after i. m. delivery of equal amounts of pDNA. These results suggest that feed pellets containing chitosan nanoparticles might enable efficient oral delivery of pDNA, a fact that might imply valuable applications in terms of on-farm mass immunization purposes, especially with regard to DNA-based vaccines and small size fish, in which i. m. administration remains unfeasible.

In vivo assessment of specific cytotoxic T lymphocyte killing.

The direct killing of target cells by cytotoxic T lymphocytes (CTLs) plays a fundamental role in protective immunity to viral, bacterial, protozoan and fungi infections, as well as to tumor cells. In vivo cytotoxic assays take into account the interaction of target and effector cells in the context of the proper microenvironment making the analysis biologically more relevant than in vitro cytotoxic assays. Thus, the development, improvement and validation of in vivo methods are necessary in view of the importance of the results they may provide. We describe and discuss in this manuscript a method to evaluate in vivo specific cytotoxic T lymphocyte killing. We used as model system mice immunized with human recombinant replication-deficient adenovirus 5 (HAd5) containing different transgenes as the trigger of a CTL-mediated immune response. To these mice, we adoptively transferred syngeneic cells labeled with different vital fluorescent dyes. Donor cells were pulsed (target) or not (control non-target) with distinct CD8 T-cell epitopes, mixed in a 1:1 ratio and injected i.v. into immunized or non-immunized recipient mice. After 18-24h, spleen cells are collected and analysed by flow cytometry. A deviation from the 1:1 ratio of control and target cell populations indicates antigen specific lysis of target cells.

Assessment of DNA vaccine potential for gilthead sea bream (Sparus aurata) by intramuscular injection of a reporter gene.

Naked circular plasmid DNA containing the cytomegalovirus (CMV)-promoter-driven lacZ reporter gene (pCMV-LacZ) was injected in the epaxial muscle of gilthead sea bream (Sparus aurata). A mosaic pattern of expression of beta-galactosidase (beta-gal) in the myofibres at the site of injection was visualised by in situ histochemical staining using 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. As measured by o-nitrophenyl-beta-D-galactopyranoside assay, beta-gal enzymatic activity was found to steadily increase for at least 50 days post injection (p.i.) in pCMV-LacZ-injected muscle. In parallel, foreign DNA was detected by polymerase chain reaction in injected muscles (but not in other tissues) up to 60 days p.i., persisting most probably in an extrachromosomal, non-replicative, circular form. Neither beta-gal activity nor pCMV-LacZ-related amplification products were found 90 days p.i. Antibodies against beta-gal were demonstrated in pCMV-LacZ-injected fish sampled 45 days p.i. The results suggest that intramuscular delivery of foreign genes represents a realistic approach for DNA vaccine technology for the prevention of infectious diseases in gilthead sea bream.

Comprehensive assessment of determinant specificity, frequency, and cytokine signature of the primed CD8 cell repertoire induced by a minor transplantation antigen.

T cell immunity is often focused on one peptide segment of a complex protein Ag, with other epitopes inducing weaker, low frequency responses or no responses at all. Such determinant hierarchy has been well characterized for MHC class II-restricted CD4 cell immunity, but is less well understood for class I-restricted CD8 cell responses. We studied class I determinant recognition in a skin transplant model with beta-galactosidase (beta-gal) as a minor transplantation Ag. CD8 T cells from C57BL/6 mice that rejected congenic C57BL/6 beta-gal transgenic skin were tested in enzyme-linked immunospot assays for recall responses to single-step, overlapping, 9-mer peptides that spanned a 94-aa region of the beta-gal sequence. This approach provided every possible class I-restricted peptide for CD8 cell recognition, allowing us to define the in vivo frequency of CD8 cells specific for each of the 86 individual peptides. While four peptides were predicted to bind to the Kb or Db molecules, only one (beta-gal96-103) actually induced an immune response. No peptides outside of the motifs were recognized. Tolerization to beta-gal96-103 significantly prolonged beta-gal transgenic skin graft survival, confirming its immune dominance. Therefore, single-determinant dominance characterized this CD8 cell response. The data demonstrate the feasibility of large-scale, comprehensive, class I determinant mapping, an approach that should be indispensable in measuring CD8 cell immunity in humans.

A cross-sectional survey of sensitization to Aspergillus oryzae-derived lactase in pharmaceutical workers.

BACKGROUND: The presence of IgE-mediated occupational respiratory sensitization to microbial enzymes has been well documented in a variety of industries. Aspergillus oryzae -derived lactase is used as a dietary aid for patients with lactose intolerance. OBJECTIVE: In 1993, a cross-sectional survey of 94 pharmaceutical workers exposed to lactase for a mean duration of 23 months and 24 nonexposed recently hired employees was initiated to identify lactase-sensitized workers and potential risk factors that could be used in making recommendations for preventing future cases of lactase sensitization. METHODS: The survey included a physician-administered questionnaire, skin prick testing to lactase enzyme and a panel of common aeroallergens, and spirometry. RESULTS: Twenty-seven of 94 lactase-exposed workers (29%) had positive skin test responses to lactase. These workers were 9 times more likely to have upper or lower respiratory symptoms compared with workers with negative skin test responses. Atopic workers were 4 times more likely to have lactase skin sensitivity than nonatopic workers. However, atopy was not a risk factor for the development of upper and/or lower respiratory symptoms. Lactase skin reactivity was not observed in the 24 nonexposed employees. CONCLUSION: This cross-sectional survey revealed that atopic workers were more likely to have lactase sensitization and that lactase-sensitized workers were more likely to have upper and/or lower respiratory symptoms, but atopy was not a risk factor for upper or lower respiratory symptoms. In spite of these findings, the company allowed only nonatopic, nonlactase-sensitized workers to continue working in high lactase-exposure areas with careful symptom monitoring and use of protective clothing. Although this strategy was successful in total prevention of new cases of occupational respiratory disease after 5 years, the results of this cross-sectional survey do not support exclusion of atopic workers from working with industrial enzymes.