Genetic engineering of porcine endothelial cell lines for evaluation of human-to-pig xenoreactive immune responses.
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ABSTRACT:
SUBMITTER: Li P
PROVIDER: S-EPMC8222275 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Improving animal robustness and resistance to pathogens by adding health criteria in selection schemes is one of the challenging objectives of the next decade. In order to better understand the genetic control of immunity in French Large White pigs, we have launched a program combining genetic and genomic studies not focussing on any particular pathogen. Animals recorded for production traits were scored for a wide range of immunity parameters three weeks after vaccination against Mycoplasma hyopneumoniae: i) total white blood cells and lymphocyte counts and proportions of various leucocyte subsets including cells harbouring IgM, ??TCR, CD4/CD8, CD16/CD2 and CD16/CD172a/MHCII, ii) innate immune response parameters (phagocytosis and in vitro production of IL1B, IL6, IL8, TNF, IL12 and IFN?after blood stimulation), iii) adaptive immune response parameters (lymphocyte proliferation, in vitro production of IL2, IL4, IL10 and IFN? after blood stimulation, total IgG, IgA, IgM and specific IgG levels) and iv) two acute phase proteins (C-reactive protein and haploglobin). Across traits, heritability estimates reached 0.4 on average (se=0.1) and 42 of the 54 measured parameters showed moderate to high heritabilities (?0.2), confirming that many parameters are under genetic control and could be included in selection protocols. Functional analyses revealed that the blood transcriptome is informative for part of the immunity traits and should provide relevant phenotypic information to better characterize some immunity traits.
| S-EPMC3108228 | biostudies-literature
Project description:Vaccine development and pathogenesis studies for human enterovirus 71 are limited by a lack of suitable animal models. Here, we report the development of a novel neonatal gnotobiotic pig model using the non-pig-adapted neurovirulent human enterovirus 71 strain BJ110, which has a C4 genotype. Porcine small intestinal epithelial cells, peripheral blood mononuclear cells and neural cells were infected in vitro. Oral and combined oral-nasal infection of 5-day-old neonatal gnotobiotic pigs with 5×10(8) fluorescence forming units (FFU) resulted in shedding up to 18 days post-infection, with viral titers in rectal swab samples peaking at 2.22×10(8) viral RNA copies/mL. Viral capsid proteins were detected in enterocytes within the small intestines on post-infection days (PIDs) 7 and 14. Additionally, viral RNA was detected in intestinal and extra-intestinal tissues, including the central nervous system, the lung and cardiac muscle. The infected neonatal gnotobiotic pigs developed fever, forelimb weakness, rapid breathing and some hand, foot and mouth disease symptoms. Flow cytometry analysis revealed increased frequencies of both CD4(+) and CD8(+) IFN-γ-producing T cells in the brain and the blood on PID 14, but reduced frequencies were observed in the lung. Furthermore, high titers of serum virus-neutralizing antibodies were generated in both orally and combined oral-nasally infected pigs on PIDs 7, 14, 21 and 28. Together, these results demonstrate that neonatal gnotobiotic pigs represent a novel animal model for evaluating vaccines for human enterovirus 71 and for understanding the pathogenesis of this virus and the associated immune responses.
| S-EPMC4051366 | biostudies-literature
Project description:BackgroundIn recent years, animal welfare and health has become more and more important in pig breeding. So far, numerous parameters have been considered as important biomarkers, especially in the immune reaction and inflammation. Previous studies have shown moderate to high heritabilities in most of these traits. However, the genetic background of health and robustness of pigs needs to be extensively clarified. The objective of this study was to identify genomic regions with a biological relevance for the immunocompetence of piglets. Genome-wide Association Studies (GWAS) in 535 Landrace (LR) and 461 Large White (LW) piglets were performed, investigating 20 immune relevant traits. Besides the health indicators of the complete and differential blood count, eight different cytokines and haptoglobin were recorded in all piglets and their biological dams to capture mediating processes and acute phase reactions. Additionally, all animals were genotyped using the Illumina PorcineSNP60v2 BeadChip.ResultsIn summary, GWAS detected 25 genome-wide and 452 chromosome-wide significant SNPs associated with 17 immune relevant traits in the two maternal pig lines LR and LW. Only small differences were observed considering the maternal immune records as covariate within the statistical model. Furthermore, the study identified across- and within-breed differences as well as relevant candidate genes. In LR more significant associations and related candidate genes were detected, compared with LW. The results detected in LR and LW are partly in accordance with previously identified quantitative trait loci (QTL) regions. In addition, promising novel genomic regions were identified which might be of interest for further detailed analysis. Especially putative pleiotropic regions on SSC5, SSC12, SSC15, SSC16 and SSC17 are of major interest with regard to the interacting structure of the immune system. The comparison with already identified QTL gives indications on interactions with traits affecting piglet survival and also production traits.ConclusionIn conclusion, results suggest a polygenic and breed-specific background of immune relevant traits. The current study provides knowledge about regions with biological relevance for health and immune traits. Identified markers and putative pleiotropic regions provide first indications in the context of balancing a breeding-based modification of the porcine immune system.
| S-EPMC8491387 | biostudies-literature
Project description:BACKGROUND:Currently, an in vitro immunogenicity screening system for the immunological assessment of potential porcine reproductive and respiratory syndrome virus (PRRSV) vaccine candidates is highly desired. Thus, in the present study, two genetically divergent PRRSVs were characterized in vitro and in vivo to identify an in vitro system and immunological markers that predict the host immune response. Porcine alveolar macrophages (PAMs) and peripheral blood mononuclear cells (PBMCs) collected from PRRSV-negative pigs were used for in vitro immunological evaluation, and the response of these cells to VR2332c or JA142c were compared with those elicited in pigs challenged with the same viruses. RESULTS:Compared with VR2332c or mock infection, JA142c induced increased levels of type I interferons and pro-inflammatory cytokines (TNF-?, IL-1?/?, IL-6, IL-8, and IL-12) in PAMs, and these elevated levels were comparable to the cytokine induction observed in PRRSV-challenged pigs. Furthermore, significantly greater numbers of activated CD4+ T cells, type I helper T cells, cytotoxic T cells and total IFN-?+ cells were observed in JA142c-challenged pigs than in VR2332c- or mock-challenged pigs. CONCLUSIONS:Based on these results, the innate immune response patterns (particularly IFN-?, TNF-? and IL-12) to specific PRRSV strains in PAMs might reflect those elicited by the same viruses in pigs.
| S-EPMC6278023 | biostudies-literature
Project description:BackgroundGenetically engineered porcine donors are a potential solution for the shortage of human organs for transplantation. Incompatibilities between humans and porcine donors are largely due to carbohydrate xenoantigens on the surface of porcine cells, provoking an immune response which leads to xenograft rejection.Materials and methodsMultiplex genetic knockout of GGTA1, β4GalNT2, and CMAH is predicted to increase the rate of xenograft survival, as described previously for GGTA1. In this study, the clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 system was used to target genes relevant to xenotransplantation, and a method for highly efficient editing of multiple genes in primary porcine fibroblasts was described.ResultsEditing efficiencies greater than 85% were achieved for knockout of GGTA1, β4GalNT2, and CMAH.ConclusionThe high-efficiency protocol presented here reduces scale and cost while accelerating the production of genetically engineered primary porcine fibroblast cells for in vitro studies and the production of animal models.
| S-EPMC8074785 | biostudies-literature
Project description:BackgroundBackfat thickness is an important carcass composition trait for pork production and is commonly included in swine breeding programmes. In this paper, we report the results of a large genome-wide association study for backfat thickness using data from eight lines of diverse genetic backgrounds.MethodsData comprised 275,590 pigs from eight lines with diverse genetic backgrounds (breeds included Large White, Landrace, Pietrain, Hampshire, Duroc, and synthetic lines) genotyped and imputed for 71,324 single-nucleotide polymorphisms (SNPs). For each line, we estimated SNP associations using a univariate linear mixed model that accounted for genomic relationships. SNPs with significant associations were identified using a threshold of p < 10-6 and used to define genomic regions of interest. The proportion of genetic variance explained by a genomic region was estimated using a ridge regression model.ResultsWe found significant associations with backfat thickness for 264 SNPs across 27 genomic regions. Six genomic regions were detected in three or more lines. The average estimate of the SNP-based heritability was 0.48, with estimates by line ranging from 0.30 to 0.58. The genomic regions jointly explained from 3.2 to 19.5% of the additive genetic variance of backfat thickness within a line. Individual genomic regions explained up to 8.0% of the additive genetic variance of backfat thickness within a line. Some of these 27 genomic regions also explained up to 1.6% of the additive genetic variance in lines for which the genomic region was not statistically significant. We identified 64 candidate genes with annotated functions that can be related to fat metabolism, including well-studied genes such as MC4R, IGF2, and LEPR, and more novel candidate genes such as DHCR7, FGF23, MEDAG, DGKI, and PTN.ConclusionsOur results confirm the polygenic architecture of backfat thickness and the role of genes involved in energy homeostasis, adipogenesis, fatty acid metabolism, and insulin signalling pathways for fat deposition in pigs. The results also suggest that several less well-understood metabolic pathways contribute to backfat development, such as those of phosphate, calcium, and vitamin D homeostasis.
| S-EPMC8459476 | biostudies-literature
Project description:The host-associated defence system responsible for the clearance of porcine reproductive and respiratory syndrome virus (PRRSV) from infected pigs is currently poorly understood. To better understand the dynamics of host-pathogen interactions, seventy-five of 100 pigs infected with PRRSV-JA142 and 25 control pigs were euthanized at 3, 10, 21, 28 and 35 days post-challenge (dpc). Blood, lung, bronchoalveolar lavage (BAL) and bronchial lymph node (BLN) samples were collected to evaluate the cellular immune responses. The humoral responses were evaluated by measuring the levels of anti-PRRSV IgG and serum virus-neutralizing (SVN) antibodies. Consequently, the highest viral loads in the sera and lungs of the infected pigs were detected between 3 and 10 dpc, and these resulted in moderate to mild interstitial pneumonia, which resolved accompanied by the clearance of most of the virus by 28 dpc. At peak viremia, the frequencies of alveolar macrophages in infected pigs were significantly decreased, whereas the monocyte-derived DC/macrophage and conventional DC frequencies were increased, and these effects coincided with the early induction of local T-cell responses and the presence of proinflammatory cytokines/chemokines in the lungs, BAL, and BLN as early as 10 dpc. Conversely, the systemic T-cell responses measured in the peripheral blood mononuclear cells were delayed and significantly induced only after the peak viremic stage between 3 and 10 dpc. Taken together, our results suggest that activation of immune responses in the lung could be the key elements for restraining PRRSV through the early induction of T-cell responses at the sites of virus replication.
| S-EPMC7222343 | biostudies-literature
Project description:Endothelial progenitor cells (EPCs) play a significant role in multiple biological processes such as vascular homeostasis, regeneration, and tumor angiogenesis. This makes them a promising cell of choice for studying a variety of biological processes, toxicity assays, biomaterial-cell interaction studies, as well as in tissue-engineering applications. In this study, we report the generation of two clones of SV40-immortalized EPCs from umbilical cord blood. These cells retained most of the functional features of mature endothelial cells and showed no indication of senescence after repeated culture for more than 240 days. Extensive functional characterization of the immortalized cells by western blot, flow cytometry, and immunofluorescence studies substantiated that these cells retained their ability to synthesize nitric oxide, von Willebrand factor, P-Selectin etc. These cells achieved unlimited proliferation potential subsequent to inactivation of the cyclin-dependent kinase inhibitor p21, but failed to form colonies on soft agar. We also show their enhanced growth and survival on vascular biomaterials compared to parental cultures in late population doubling. These immortalized EPCs can be used as a cellular model system for studying the biology of these cells, gene manipulation experiments, cell-biomaterial interactions, as well as a variety of tissue-engineering applications.
| S-EPMC3483052 | biostudies-literature
Project description:Recent advances toward using pig tissues in human transplantation have made it necessary to determine the risk of transmitting zoonotic viruses from pigs to humans or vice versa. We investigated the suitability of the porcine encephalomyocarditis virus (EMCV) model for such studies by determining its ability to persist in pigs, escape detection by routine serological methods, and infect human cells. Intraperitoneal inoculation of 5-week-old pigs with EMCV-30, a strain isolated from commercial pigs, resulted in acute cellular degeneration, infiltration of lymphocytes, and apoptosis in myocardium in 13 of 15 (86.7%) pigs during the acute phase of disease (3 to 21 days postinfection), followed by less-severe lymphocytic infiltration and apoptosis in 5 of 10 (50%) pigs during the chronic phase of the disease (day 45 to 90 postinfection). In the brain, lymphocytic infiltration, neuronal degeneration, and gliosis were observed in 26 to 33% of pigs in the acute phase of disease whereas perivascular cuffing was the predominant feature during chronic disease. EMCV antigens and RNA were demonstrated in the myocardium and brain during the chronic phase of disease. Analysis of 100 commercial pigs that were negative for EMCV antibodies identified two pig hearts positive for EMCV RNA. Porcine EMCV productively infected primary human cardiomyocytes as demonstrated by immunostaining using a monoclonal antibody specific for EMCV RNA polymerase, which is expressed only in productively infected cells, and by a one-step growth curve that showed production of 100 to 1,000 PFU of virus per cell within 6 h. The findings that porcine EMCV can persist in pig myocardium and can infect human myocardial cells make it an important infectious agent to screen for in pig-to-human cardiac transplants and a good model for xenozoonosis.
| S-EPMC114749 | biostudies-literature
Project description:BACKGROUND: Photocopiers emit nanoparticles with complex chemical composition. Short-term exposures to modest nanoparticle concentrations triggered upper airway inflammation and oxidative stress in healthy human volunteers in a recent study. To further understand the toxicological properties of copier-emitted nanoparticles, we studied in-vitro their ability to induce cytotoxicity, pro-inflammatory cytokine release, DNA damage, and apoptosis in relevant human cell lines. METHODS: Three cell types were used: THP-1, primary human nasal- and small airway epithelial cells. Following collection in a large volume photocopy center, nanoparticles were extracted, dispersed and characterized in the cell culture medium. Cells were doped at 30, 100 and 300 ?g/mL administered doses for up to 24 hrs. Estimated dose delivered to cells, was ~10% and 22% of the administered dose at 6 and 24 hrs, respectively. Gene expression analysis of key biomarkers was performed using real time quantitative PCR (RT-qPCR) in THP-1 cells at 5 ?g nanoparticles/mL for 6-hr exposure for confirmation purposes. RESULTS: Multiple cytokines, GM-CSF, IL-1?, IL-6, IL-8, IFN?, MCP-1, TNF-? and VEGF, were significantly elevated in THP-1 cells in a dose-dependent manner. Gene expression analysis confirmed up-regulation of the TNF-? gene in THP-1 cells, consistent with cytokine findings. In both primary epithelial cells, cytokines IL-8, VEGF, EGF, IL-1?, TNF-?, IL-6 and GM-CSF were significantly elevated. Apoptosis was induced in all cell lines in a dose-dependent manner, consistent with the significant up-regulation of key apoptosis-regulating genes P53 and Casp8 in THP-1 cells. No significant DNA damage was found at any concentration with the comet assay. Up-regulation of key DNA damage and repair genes, Ku70 and Rad51, were also observed in THP-1 cells, albeit not statistically significant. Significant up-regulation of the key gene HO1 for oxidative stress, implicates oxidative stress induced by nanoparticles. CONCLUSIONS: Copier-emitted nanoparticles induced the release of pro-inflammatory cytokines, apoptosis and modest cytotoxicity but no DNA damage in all three-human cell lines. Taken together with gene expression data in THP-1 cells, we conclude that these nanoparticles are directly responsible for inflammation observed in human volunteers. Further toxicological evaluations of these nanoparticles, including across different toner formulations, are warranted.
| S-EPMC3766213 | biostudies-literature