New insight into the biological activity of Salmo salar NK-lysin antimicrobial peptides.

NK-lysin is a potent antimicrobial peptide (AMP) with antimicrobial activity against bacteria, fungi, viruses, and parasites. NK-lysin is a type of granulysin, a member of the saposin-like proteins family first isolated from a pig's small intestine. In previous work, for the first time, we identified four variants of nk-lysin from Atlantic salmon (Salmo salar) using EST sequences. In the present study, we reported and characterized two additional transcripts of NK-lysin from S. salar. Besides, we evaluated the tissue distribution of three NK-lysins from S. salar and assessed the antimicrobial, hemolytic, and immunomodulatory activities and signaling pathways of three NK-lysin-derived peptides. The synthetic peptides displayed antimicrobial activity against Piscirickettsia salmonis (LF-89) and Flavobacterium psychrophilum. These peptides induced the expression of immune genes related to innate and adaptive immune responses in vitro and in vivo. The immunomodulatory activity of the peptides involves the mitogen-activated protein kinases-mediated signaling pathway, including p38, extracellular signal-regulated kinase 1/2, and/or c-Jun N-terminal kinases. Besides, the peptides modulated the immune response induced by pathogen-associated molecular patterns (PAMPs). Our findings show that NK-lysin could be a highly effective immunostimulant or vaccine adjuvant for use in fish aquaculture.

Design and functional characterization of Salmo salar TLR5 agonist peptides derived from high mobility group B1 acidic tail.

Toll-like receptor 5 (TLR5) responds to the monomeric form of flagellin and induces the MyD88-depending signaling pathway, activating proinflammatory transcription factors such as NF-κB and the consequent induction of cytokines. On the other hand, HMGB1 is a highly conserved non-histone chromosomal protein shown to interact with and activate TLR5. The present work aimed to design and characterize TLR5 agonist peptides derived from the acidic tail of Salmo salar HMGB1 based on the structural knowledge of the TLR5 surface using global molecular docking platforms. Peptide binding poses complexed on TLR5 ectodomain model from each algorithm were filtrated based on docking scoring functions and predicted theoretical binding affinity of the complex. Circular dichroism spectra were recorded for each peptide selected for synthesis. Only intrinsically disordered peptides (6W, 11W, and SsOri) were selected for experimental functional assay. The functional characterization of the peptides was performed by NF-κB activation assays, RT-qPCR gene expression assays, and Piscirickettsia salmonis challenge in SHK-1 cells. The 6W and 11W peptides increased the nuclear translation of p65 and phosphorylation. In addition, the peptides induced the expression of genes related to the TLR5 pathway activation, pro- and anti-inflammatory response, and differentiation and activation of T lymphocytes towards phenotypes such as TH1, TH17, and TH2. Finally, it was shown that the 11W peptide protects immune cells against infection with P. salmonis bacteria. Overall, the results indicate the usefulness of novel peptides as potential immunostimulants in salmonids.

Isolation, characterization, and immunomodulatory activity evaluation of probiotic strains from colostrum and canine milk.

Background: This study aimed to characterize potential probiotic strains for use in dogs to prevent infectious enteropathies. Lactic acid bacteria (LAB) isolated from canine milk and colostrum were characterized according to their functional properties, including their resistance to gastrointestinal conditions, inhibitory effect against pathogens, and intestinal adhesion. Methods: The immunomodulatory effects of the strains were also analyzed in in vitro and in vivo studies. Among the strains evaluated, two LAB strains (TUCO-16 and TUCO-17) showed remarkable resistance to pH 3.0, bile salts, and pancreatin, as well as inhibitory effects against pathogenic Escherichia coli, Salmonella sp., and Clostridium perfringens. Results: The TUCO-16 and TUCO-17 strains induced a significant increase in the expression of TNF-α, IL-8, and TLR2 in canine macrophages. The oral administration of TUCO-16 and TUCO-17 strains to mice significantly augmented their resistance to pathogenic E. coli or Salmonella intestinal infections. Both canine strains reduced intestinal damage and pathogen counts in the liver and spleen and avoided their dissemination into the bloodstream. These protective effects were related to the ability of TUCO-16 and TUCO-17 strains to differentially modulate the production of IFN-γ, IFN-ß, TNF-α, IL-6, KC, MCP-1, and IL-10 in the intestinal mucosa. Conclusion: Both strains, TUCO-16 and TUCO-17, are potential probiotic candidates for improving intestinal health in dogs, particularly for their ability to inhibit the growth of Gram-negative pathogens common in gastrointestinal infections and modulate the animal's immune response. Further studies are required to effectively demonstrate the beneficial effects of TUCO-16 and TUCO-17 strains in dogs.

Immunomodulatory role of vasoactive intestinal peptide and ghrelin in Oncorhynchus mykiss.

Neuropeptides are a group of peptides derived from precursor proteins synthesized in neuronal and nonneuronal cells. The classical functions of neuropeptides have been extensively studied in mammals, including neuromodulation in the central nervous system, molecular signaling in the peripheral nervous system, and immunomodulation associated mainly with anti-inflammatory activity. In contrast, in teleosts, studies of the immunomodulatory function of these neuropeptides are limited. In Oncorhynchus mykiss, vasoactive intestinal peptide (VIP) mRNA sequences have not been cloned, and the role of VIP in modulating the immune system has not been studied. Furthermore, in relation to other neuropeptides with possible immunomodulatory function, such as ghrelin, there are also few studies. Therefore, in this work, we performed molecular cloning, identification, and phylogenetic analysis of three VIP precursor sequences (prepro-VIP1, VIP2 and VIP3) in rainbow trout. In addition, the immunomodulatory function of both neuropeptides was evaluated in an in vitro model using the VIP1 sequence identified in this work and a ghrelin sequence already studied in O. mykiss. The results suggest that the prepro-VIP2 sequence has the lowest percentage of identity with respect to the other homologous sequences and is more closely related to mammalian orthologous sequences. VIP1 induces significant expression of both pro-inflammatory (IFN-γ, IL-1ß) and anti-inflammatory (IL-10 and TGF-ß) cytokines, whereas ghrelin only induces significant expression of proinflammatory cytokines such as IL-6 and TNF-α.

First report of bovine viral diarrhea virus subgenotypes 1d and 1e in southern Chile.

Bovine viral diarrhea virus (BVDV) affects cattle worldwide causing severe productive and economic loss. In this study, we investigated the subgenotypes of BVDV circulating in cattle samples from the Aysén region, an active cattle breeding area located in southern Chile. Partial amplification of the 5' untranslated region (UTR) was performed by polymerase chain reaction (PCR), and twelve samples were analyzed by Sanger sequencing and phylogenetic analysis. Eight samples were identified as belonging to Pestivirus bovis subgenotype 1e, three to 1-b, and one to 1-d. The phylogenetic analyses performed revealed a marked distance between these now-identified strains and those previously reported in the country. These findings support the need to continually expand the analysis of the variability of the viral phylogeny for the currently circulating BVDV strains and to update the vaccines recommended for this livestock area and surrounding areas.

Design and Construction of a Synthetic Nanobody Library: Testing Its Potential with a Single Selection Round Strategy.

Nanobodies (Nbs) are single domain antibody fragments derived from heavy-chain antibodies found in members of the Camelidae family. They have become a relevant class of biomolecules for many different applications because of several important advantages such as their small size, high solubility and stability, and low production costs. On the other hand, synthetic Nb libraries are emerging as an attractive alternative to animal immunization for the selection of antigen-specific Nbs. Here, we present the design and construction of a new synthetic nanobody library using the phage display technology, following a structure-based approach in which the three hypervariable loops were subjected to position-specific randomization schemes. The constructed library has a clonal diversity of 108 and an amino acid variability that matches the codon distribution set by design at each randomized position. We have explored the capabilities of the new library by selecting nanobodies specific for three antigens: vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF) and the glycoprotein complex (GnGc) of Andes virus. To test the potential of the library to yield a variety of antigen-specific Nbs, we introduced a biopanning strategy consisting of a single selection round using stringent conditions. Using this approach, we obtained several binders for each of the target antigens. The constructed library represents a promising nanobody source for different applications.

Effects of recombinant FSH (bscrFSH) and pituitary FSH (FSH-p) on embryo production in superovulated dairy heifers inseminated with unsorted and sex-sorted semen.

Superovulation is a drug-based method used in cattle to stimulate the ovarian folliculogenesis and the number of oocytes and transferable embryos. The present study aimed to test the effects of recombinant FSH (bscrFSH) and pituitary FSH (FSH-p) on ovarian response and in vivo embryo production in superovulated dairy heifers inseminated with unsorted and sex-sorted semen. Forty healthy Holstein heifers subjected to a superovulation (SOV) protocol by using FSH-p or bscrFSH were divided randomly into four groups: a) FSH-p inseminated with unsorted semen (USP; n = 10), b) FSH-p inseminated with sex-sorted semen (SSP; n = 10), c) bscrFSH inseminated with unsorted semen (USR; n = 10), and d) bscrFSH inseminated with sex-sorted semen (SSR; n = 10). Ultrasonography was carried out on Day 8 (estrus) and Day 15 (embryo collection) to evaluate the ovarian structures [follicles (FL), corpora lutea (CL), and non-ovulated follicles (NOFL)]. Embryonic-derived parameters were scored on Day 15 [total structures collected (TS), unfertilised oocytes (UFOs), total embryos (TEs), transferable embryos (TFEs), freezable embryos (FEs), and degenerated embryos (DEs)]. No differences were observed regarding ovarian structures (FL and NOFL) irrespective of SOV protocol or group assessed (P > 0.05). CL increased in bscrFSH-derived SOV protocol (P < 0.05). On Day 15, the embryonic-derived parameters TEs, TFEs, and FEs decreased in SSP/SSR compared to USP/USR (P < 0.05). Differences were observed regarding UFOs, with a greater number in SSP and SSR (P = 0.01). In conclusion, the bscrFSH-derived SOV protocol showed improved results compared to FSH-p-derived SOV protocol regarding ovarian (CL) and embryo-derived (TFE) parameters irrespective of the type of semen used.

Forms and Methods for Interferon's Encapsulation.

Interferons (IFNs) are cytokines involved in the immune response that act on innate and adaptive immunity. These proteins are natural cell-signaling glycoproteins expressed in response to viral infections, tumors, and biological inducers and constitute the first line of defense of vertebrates against infectious agents. They have been marketed for more than 30 years with considerable impact on the global therapeutic protein market thanks to their diversity in terms of biological activities. They have been used as single agents or with combination treatment regimens, demonstrating promising clinical results, resulting in 22 different formulations approved by regulatory agencies. The 163 clinical trials with currently active IFNs reinforce their importance as therapeutics for human health. However, their application has presented difficulties due to the molecules' size, sensitivity to degradation, and rapid elimination from the bloodstream. For some years now, work has been underway to obtain new drug delivery systems to provide adequate therapeutic concentrations for these cytokines, decrease their toxicity and prolong their half-life in the circulation. Although different research groups have presented various formulations that encapsulate IFNs, to date, there is no formulation approved for use in humans. The current review exhibits an updated summary of all encapsulation forms presented in the scientific literature for IFN-α, IFN-ß, and IFN-γ, from the year 1996 to the year 2021, considering parameters such as: encapsulating matrix, route of administration, target, advantages, and disadvantages of each formulation.

Expression and characterization of a novel single-chain anti-vascular endothelial growth factor antibody in the goat milk.

Vascular endothelial growth factor (VEGF) has essential functions in angiogenesis, endothelial cell proliferation, migration, and tumor invasion. Different approaches have been developed to suppress tumor angiogenesis, which is considered a hallmark of cancer. Anti-VEGF monoclonal antibodies constitute an important strategy for cancer immunotherapy, which has been produced on several platforms. In this study, a novel single-chain anti-VEGF monoclonal antibody (scVEGFmAb) was produced in the goat mammary gland by adenoviral transduction. scVEGFmAb was purified by affinity chromatography. N-glycans were analyzed by exoglycosidase digestion and hydrophilic interaction ultra-performance liquid chromatography coupled to electrospray ionization mass spectrometry. The biological activity of scVEGFmAb was assessed by scratch and mouse aortic ring assays. scVEGFmAb was produced at 0.61 g/L in the goat milk, and its purification rendered 95 % purity. N-glycans attached to scVEGFmAb backbone were mainly neutral biantennary core fucosylated with Galß1,4GlcNAc motif, and charged structures were capped with Neu5Ac and Neu5Gc. The chimeric molecule significantly prevented cell migration and suppressed microvessel sprouting. These results demonstrated for the first time the feasibility of producing an anti-VEGF therapeutic antibody in the milk of non-transgenic goats with the potential to counteract tumor angiogenesis.

Design and characterization of a novel dimeric blood-brain barrier penetrating TNFα inhibitor.

Tumor necrosis factor-alpha (TNFα) inhibitors could prevent neurological disorders systemically, but their design generally relies on molecules unable to cross the blood-brain barrier (BBB). This research was aimed to design and characterize a novel TNFα inhibitor based on the angiopeptide-2 as a BBB shuttle molecule fused to the extracellular domain of human TNFα receptor 2 and a mutated vascular endothelial growth factor (VEGF) dimerization domain. This new chimeric protein (MTV) would be able to trigger receptor-mediated transcytosis across the BBB via low-density lipoprotein receptor-related protein-1 (LRP-1) and inhibit the cytotoxic effect of TNFα more efficiently because of its dimeric structure. Stably transformed CHO cells successfully expressed MTV, and its purification by Immobilized-Metal Affinity Chromatography (IMAC) rendered high purity degree. Mutated VEGF domain included in MTV did not show cell proliferation or angiogenic activities measured by scratch and aortic ring assays, which corroborate that the function of this domain is restricted to dimerization. The pairs MTV-TNFα (Kd 279 ± 40.9 nM) and MTV-LRP1 (Kd 399 ± 50.5 nM) showed high affinity by microscale thermophoresis, and a significant increase in cell survival was observed after blocking TNFα with MTV in a cell cytotoxicity assay. Also, the antibody staining in CHOK1 and bEnd3 cells demonstrated the adhesion of MTV to the LRP1 receptor located in the cell membrane. These results provide compelling evidence for the proper functioning of the three main domains of MTV individually, which encourage us to continue the research with this new molecule as a potential candidate for the systemic treatment of neurological disorders.

In vivo modification of the goat mammary gland glycosylation pathway.

Complex recombinant glycoproteins produced as potential biopharmaceuticals in goat's milk have an aberrant pattern of N-glycosylation due to the lack of multi-antennary structures. Overexpression of glycosyltransferases may increase oligosaccharide branching of the desired glycoproteins. Here, human erythropoietin fused to human IgG Fc (EPO-Fc) was co-expressed with N-acetyl-glucosaminyltransferase-IVa (GnT-IVa) by adenoviral transduction in goat mammary gland to evaluate the in vivo modification of N-glycosylation pattern in this tissue. Adenoviral vectors, containing the EPO-Fc and GnT-IVa sequences were assembled for in vitro and in vivo expression in mammalian cell culture or in goat mammary gland. Protein detection was assessed by gel electrophoresis and western blot, and N-glycans were identified by HPLC and mass spectrometry. GnT-IVa overexpression and its colocalization with EPO-Fc in the Golgi apparatus of SiHa cells were demonstrated. N-glycan analysis of in vitro and in vivo expression of EPO-Fc modified by GnT-IVa (EPO-Fc/GnT-IVa) showed an increase in high molecular weight structures, which corresponded to tri- and tetra-antennary N-glycans in SiHa cells and mostly tri-antennary N-glycans in goat's milk from transformed mammary tissue. The results confirmed that successful modification of the goat mammary gland secretion pathway could be achieved by co-expressing glycoenzymes together with the glycoprotein of interest. This is the first report of modification of the N-glycosylation pattern in the goat mammary gland in vivo, and constitutes a step forward for improving the use of the mammary gland as a bioreactor for the production of complex recombinant proteins.

Fluorescence-assisted sequential insertion of transgenes (FASIT): an approach for increasing specific productivity in mammalian cells.

Currently, the generation of cell lines for the production of recombinant proteins has the limitation of unstable gene expression due to the repeat-induced gene silencing or the loss of transgene copies resulting from recombination events. In this work, we developed a new strategy based on the sequential insertion of transgenes for generating stable clones producing high levels of a chimeric human follicle-stimulating hormone (hscFSH). Gene insertion was done by transducing HEK-293 cells with a lentiviral vector containing a bicistronic transcriptional unit for expressing hscFSH and GFP genes. Clone selection was performed by flow cytometry coupled to cell sorting, and the GFP gene was further removed by CRE-mediated site-specific recombination. High-producing clones of hscFSH were obtained after three rounds of lentiviral transduction. Expression levels increased in a step-wise manner from 7 to 23 pg/cell/day, with a relatively constant rate of 7 pg/cell/day in each round of transduction. The GFP gene was successfully removed from the cell genome without disturbing the hscFSH gene expression. Clones generated using this approach showed stable expression levels for more than two years. This is the first report describing the sequential insertion of transgenes as an alternative for increasing the expression levels of transformed cell lines. The methodology described here could notably impact on biotechnological industry by improving the capacity of mammalian cells to produce biopharmaceuticals.

Lectin-like oxidized LDL receptor-1 is an enhancer of tumor angiogenesis in human prostate cancer cells.

Altered expression and function of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) has been associated with several diseases such as endothelial dysfunction, atherosclerosis and obesity. In these pathologies, oxLDL/LOX-1 activates signaling pathways that promote cell proliferation, cell motility and angiogenesis. Recent studies have indicated that olr1 mRNA is over-expressed in stage III and IV of human prostatic adenocarcinomas. However, the function of LOX-1 in prostate cancer angiogenesis remains to be determined. Our aim was to analyze the contribution of oxLDL and LOX-1 to tumor angiogenesis using C4-2 prostate cancer cells. We analyzed the expression of pro-angiogenic molecules and angiogenesis on prostate cancer tumor xenografts, using prostate cancer cell models with overexpression or knockdown of LOX-1 receptor. Our results demonstrate that the activation of LOX-1 using oxLDL increases cell proliferation, and the expression of the pro-angiogenic molecules VEGF, MMP-2, and MMP-9 in a dose-dependent manner. Noticeably, these effects were prevented in the C4-2 prostate cancer model when LOX-1 expression was knocked down. The angiogenic effect of LOX-1 activated with oxLDL was further demonstrated using the aortic ring assay and the xenograft model of tumor growth on chorioallantoic membrane of chicken embryos. Consequently, we propose that LOX-1 activation by oxLDL is an important event that enhances tumor angiogenesis in human prostate cancer cells.

First report of bovine herpesvirus 5 in bull semen.

BoHV-5 was detected in one of several extended semen samples from a healthy donor bull during routine virus screening. This was achieved by polymerase chain reaction assay (PCR) and virus isolation, with primary identification by the fluorescent antibody test. The isolated virus, B4180, was characterized by sequencing a cloned fragment of the gC gene and by restriction enzyme analysis (REA). The nucleotide sequence shared 99 % similarity with published sequences of BoHV-5, and the REA showed that the isolate was of the BoHV-5a subtype. This study provides the first evidence of intermittent BoHV-5 shedding in bull semen as well as information about its geographic distribution.

Avian CD154 enhances humoral and cellular immune responses induced by an adenovirus vector-based vaccine in chickens.

Recombinant adenoviral vectors have emerged as an attractive system for veterinary vaccines development. However, for poultry vaccination a very important criterion for an ideal vaccine is its low cost. The objective of this study was to test the ability of chicken CD154 to enhance the immunogenicity of an adenoviral vector-based vaccine against avian influenza virus in order to reduce the amount of antigen required to induce an effective immune response in avian. Chickens were vaccinated with three different doses of adenoviral vectors encoding either HA (AdHA), or HA fused to extracellular domain chicken's CD154 (AdHACD). Hemagglutination inhibition (HI) assay and relative quantification of IFN-γ showed that the adenoviral vector encoding for the chimeric antigen is able to elicit an improved humoral and cellular immune response, which demonstrated that CD154 can be used as a molecular adjuvant allowing to reduce in about 50-fold the amount of adenoviral vector vaccine required to induce an effective immune response.

Human αIFN co-formulated with milk derived E2-CSFV protein induce early full protection in vaccinated pigs.

Subunit vaccines are a suitable alternative for the control of classical swine fever. However, such vaccines have as the main drawback the relatively long period of time required to induce a protective response, which hampers their use under outbreak conditions. In this work, type I interferon is used as an immunostimulating molecule in order to increase the immunogenicity of a vaccine candidate based on the E2-CSFV antigen produced in goat milk. Pigs vaccinated with E2-CSFV antigen co-formulated with recombinant human alpha interferon were protected against clinical signs and viremia as early as 7 days post-vaccination. It was also demonstrated that interferon stimulates a response of specific anti-CSFV neutralizing antibodies. The present work constitutes the first report of a subunit vaccine able to confer complete protection by the end of the first week after vaccination. These results suggest that the E2-CSFV antigen combined with type I interferons could be potentially used under outbreak conditions to stop CSFV spread and for eradication programs in CSF enzootic areas.