Exploring the sheep (Ovis aries) immunoglobulin repertoire by next generation sequencing.

Next-generation sequencing (NGS) has revolutionized the way we determine the antibody repertoires encoded by B cells in the blood or lymphoid organs and transformed our understanding of adaptive immune responses in many species. Sheep (Ovis aries) have been widely used as a host for therapeutic antibody production since the early 1980s, however, little is known about their immune repertoires or immunological processes affecting the antibody generation. The objective of this study was to employ NGS for a comprehensive analysis of immunoglobulin heavy and light chain repertoires in four healthy sheep. We obtained > 90 % complete antibody sequences and nearly 130,000, 48,000 and 218,000 unique CDR3 reads for the heavy chain (IGH), kappa chain (IGK), and lambda chain (IGL) loci, respectively. Consistent with other species, we observed biased usage of germline variable (V), diversity (D) and joining (J) genes in the heavy and kappa loci, but not in the lambda loci. Moreover, the enormous diversity of CDR3 sequences was observed through sequence clustering and convergent recombination. These data will build a foundation for future studies investigating immune repertoires in health and disease as well as contribute to further refinement of ovine-derived therapeutic antibody drugs.

Comparative proteomic analysis of growth hormone secretagogue A233 treatment of murine macrophage cells J774A.2 indicates it has a role in antiviral innate response.

BACKGROUND: Growth hormone secretagogues (GHS), among other factors, regulate the release of GH. The biological activity of the secretagogue peptide A233 as a promoter of growth and innate immunity in teleost fish has previously been demonstrated, but its role in the immune system of mammals is not well understood. METHODS: The effect of the peptide was investigated in J774A.2 macrophage cells using a comparative proteomics approach after 6 and 12 h of peptide stimulation. RESULTS: The functional analysis of differentially modulated proteins showed that A233 peptide treatment appears to promote activation and ROS-dependent cytotoxic functions in macrophages and enhanced expression of antiviral protein complexes such as MAVS. In accordance with this hypothesis, we found that A233 treatment enhanced superoxide anion production and the IFN-γ level in J774A.2 cells and mouse splenocytes, respectively, and reduced viral load in a dengue virus mouse model of infection. CONCLUSIONS: The growth hormone secretagogue A233 peptide promotes activation of ROS-dependent cytotoxic functions and exerts immunomodulatory effects that enable an antiviral state in a dengue virus mouse model. GENERAL SIGNIFICANCE: The increase of IFN-γ level and the differential modulation of antiviral proteins by the A233 peptide suggest that the molecule could activate an innate immune response with a possible further impact in the treatment of acute and chronic diseases.

Bioinformatics tools for the functional interpretation of quantitative proteomics results.

Proteins are the principal mediators of the functions in the cell; therefore, any abnormal variations on their abundance levels may reflect the presence of pathological processes. In this sense, many researchers rely on the functional interpretation of protein lists generated by quantitative proteomics experiments to analyze, for instance, these variations in the context of diseases' molecular basis and drug discovery. Since no analytical strategy or bioinformatics tool by itself is capable of extract all the information covered by a single experiment; herein we seek to provide the biologists with four groups of different but complementary bioinformatics tools for the functional interpretation of quantitative proteomics results. To this end we will review the basic concepts of a set of different bioinformatics approaches and we will give examples of freely available tools for each one of these approaches.