Identification of the Porcine Vascular Endothelial Cell-Specific Promoter ESAM1.0 Using Transcriptome Analysis.

The vascular endothelium of xenografted pig organs represents the initial site of rejection after exposure to recipient immune cells. In this study, we aimed to develop a promoter specific to porcine vascular endothelial cells as a step toward overcoming xenograft rejection. Transcriptome analysis was performed on porcine aortic endothelial cells (PAECs), ear skin fibroblasts isolated from GGTA knockout (GTKO) pigs, and the porcine renal epithelial cell line pk-15. RNA sequencing confirmed 243 differentially expressed genes with expression changes of more than 10-fold among the three cell types. Employing the Human Protein Atlas database as a reference, we identified 34 genes exclusive to GTKO PAECs. The endothelial cell-specific adhesion molecule (ESAM) was selected via qPCR validation and showed high endothelial cell specificity and stable expression across tissues. We selected 1.0 kb upstream sequences of the translation start site of the gene as the promoter ESAM1.0. A luciferase assay revealed that ESAM1.0 promoter transcriptional activity was significant in PAECs, leading to a 2.8-fold higher level of expression than that of the porcine intercellular adhesion molecule 2 (ICAM2) promoter, which is frequently used to target endothelial cells in transgenic pigs. Consequently, ESAM1.0 will enable the generation of genetically modified pigs with endothelium-specific target genes to reduce xenograft rejection.

Comparative transcriptome analysis between long- and short-term survival after pig-to-monkey cardiac xenotransplantation reveals differential heart failure development.

Cardiac xenotransplantation is the potential treatment for end-stage heart failure, but the allogenic organ supply needs to catch up to clinical demand. Therefore, genetically-modified porcine heart xenotransplantation could be a potential alternative. So far, pig-to-monkey heart xenografts have been studied using multi-transgenic pigs, indicating various survival periods. However, functional mechanisms based on survival period-related gene expression are unclear. This study aimed to identify the differential mechanisms between pig-to-monkey post-xenotransplantation long- and short-term survivals. Heterotopic abdominal transplantation was performed using a donor CD46-expressing GTKO pig and a recipient cynomolgus monkey. RNA-seq was performed using samples from POD60 XH from monkey and NH from age-matched pigs, D35 and D95. Gene-annotated DEGs for POD60 XH were compared with those for POD9 XH (Park et al. 2021). DEGs were identified by comparing gene expression levels in POD60 XH versus either D35 or D95 NH. 1,804 and 1,655 DEGs were identified in POD60 XH versus D35 NH and POD60 XH versus D95 NH, respectively. Overlapped 1,148 DEGs were annotated and compared with 1,348 DEGs for POD9 XH. Transcriptomic features for heart failure and inhibition of T cell activation were observed in both long (POD60)- and short (POD9)-term survived monkeys. Only short-term survived monkey showed heart remodeling and regeneration features, while long-term survived monkey indicated multi-organ failure by neural and hormonal signaling as well as suppression of B cell activation. Our results reveal differential heart failure development and survival at the transcriptome level and suggest candidate genes for specific signals to control adverse cardiac xenotransplantation effects.

Mortality, growth, and egg production do not differ between nontransgenic and transgenic female chickens with ubiquitous expression of the 3D8 single chain variable fragment gene.

To date, many transgenic (TG) chicken lines have been developed, but few studies have performed a comparative analysis of their mortality, growth, and egg productivity. Previously, we reported the production of 3D8 scFv TG chickens showing antiviral activity. Here, we performed a biometric characterization of TG offspring female chickens. We selected 40 TG and 40 non-TG offspring female chicks among newly hatched chicks produced via artificial insemination of semen from heterotypic 3D8 scFv males into wild-type female chickens. Serum was collected at 14 wk of age, and serum concentrations of biochemical parameters, cytokines, and sex hormones were analyzed. Mortality and growth were monitored daily from 1 to 34 wk, egg productivity was monitored daily from 20 to 34 wk, and the weekly average values were used for analyses. Some serum parameters and cytokines were significantly different between non-TG and TG offspring female chickens. The levels of phosphorus (PHOS), total protein (TP), albumin (ALB), globulin (GLOB), and alanine aminotransferase (ALT) were significantly higher in non-TG chickens (P < 0.05). The levels of alkaline phosphatase (ALP) and gamma-glutamyltransferase (GGT) were significantly higher in TG chickens (P < 0.05). The levels of insulin growth factor-1 (IGF-1), interferon-gamma (INF-γ), interleukin-4 (IL-4), and IL-8 were significantly lower in TG chickens (P < 0.05). Despite these differences, the mortality rates, body weight, egg production rates, and egg weight were not significantly different in the experimental groups of non-TG and TG offspring female chickens (P > 0.05). In conclusion, ubiquitous expression of the 3D8 scFv gene in TG offspring female chickens does not affect some biometric characteristics, including mortality, growth, and egg productivity.

Comparison of Graft Survival Between Full-Thickness and Lamellar Pig-to-Monkey Corneal Xenotransplantation from the Same Genetically Engineered Pig Model with Minimal Immunosuppression.

BACKGROUND: The graft survival rate of full-thickness corneal xenotransplantation (XTP) with minimal immunosuppression in genetically engineered pigs is unknown, whereas lamellar corneal XTP shows satisfactory results. We compared graft survival between full-thickness and lamellar transplantations in the same genetically engineered pig. METHODS: Six pig-to-monkey corneal transplantations were performed on 3 transgenic pigs. Two corneas harvested from 1 pig were transplanted into 2 monkeys using full-thickness and lamellar corneal xenotransplantation. The transgenic donor pigs used were α1,3-galactosyltransferase gene-knockout + membrane cofactor protein (GTKO+CD46) in one recipient and GTKO+CD46+ thrombomodulin (TBM) in the other. RESULTS: The graft survival time for GTKO+CD46 XTP was 28 days. With the addition of TBM, the survival differences between lamellar and full-thickness XTP were 98 days versus 14 days and >463 days (ongoing) versus 21 days, respectively. An excessive number of inflammatory cells was observed in failed grafts, but none were in the recipient's stromal bed. CONCLUSIONS: Unlike full-thickness corneal XTP, lamellar xenocorneal transplantation does not exhibit surgical complications, such as retrocorneal membrane or anterior synechia. The graft survival of lamellar XTP in this study was not as good as in our previous experiments, although the survival period was superior to that of full-thickness XTP. The difference in graft survival based on transgenic type is not definitive. Further studies using transgenic pigs and minimal immunosuppression need to focus on improving graft survival of lamellar XTP and using a larger sample size to determine the potential of full-thickness corneal XTP.

Current Status of Genetically Engineered Pig to Monkey Kidney Xenotransplantation in Korea.

BACKGROUND: In South Korea, pig-to-nonhuman primate trials of solid organs have only been performed recently, and the results are not sufficiently satisfactory to initiate clinical trials. Since November 2011, we have performed 30 kidney pig-to-nonhuman primate xenotransplantations at Konkuk University Hospital. METHODS: Donor αGal-knockout-based transgenic pigs were obtained from 3 institutes. The knock-in genes were CD39, CD46, CD55, CD73, and thrombomodulin, and 2-4 transgenic modifications with GTKO were done. The recipient animal was the cynomolgus monkey. We used the immunosuppressants anti-CD154, rituximab, anti-thymocyte globulin, tacrolimus, mycophenolate mofetil, and steroids. RESULTS: The mean survival duration of the recipients was 39 days. Except for a few cases for which survival durations were <2 days because of technical failure, 24 grafts survived for >7 days, with an average survival duration of 50 days. Long-term survival was observed 115 days after the removal of the contralateral kidney, which is currently the longest-recorded graft survival in Korea. We confirmed functioning grafts for the surviving transplanted kidneys after the second-look operation, and no signs of hyperacute rejection were observed. CONCLUSIONS: Although our survival results are relatively poor, they are the best-recorded results in South Korea, and the ongoing results are improving. With the support of government funds and the volunteering activities of clinical experts, we aim to further improve our experiments and contribute to the commencement of clinical trials of kidney xenotransplantation in Korea.

cd26 Knockdown Negatively Affects Porcine Parthenogenetic Preimplantation Embryo Development.

cd26 is ubiquitously distributed in the body, particularly in the endothelial and epithelial cells, with the highest expression in the kidney, liver, and small intestine. In humans, cd26 serves as a marker for the embryo implantation phase. However, little is known about the role of cd26 in porcine pre-implantation embryo development. Here, we aimed to examine siRNA-induced cd26 downregulation in the cytoplasm of MII oocytes, to determine whether cd26 is involved in the regulation of porcine pre-implantation embryonic development. The cd26 siRNA was micro-injected into the cytoplasm of MII oocytes, which were then parthenogenetically activated electrically in a medium containing 0.3M Mannitol. Inhibition of the cd26 expression did not affect cleavage but stopped development in the blastocyst stage. Additionally, the cd26 siRNA-treated blastocysts had significantly more apoptotic cells than the untreated blastocysts. Among the 579 transcripts evaluated with transcriptome resequencing, 38 genes were differentially expressed between the treatment and control blastocysts (p < 0.05). Twenty-four genes were upregulated in cd26 siRNA-injected blastocysts, whereas 14 were downregulated. These genes are involved in apoptosis, accumulation of reactive oxygen species, and aberrant expression of ribosomal protein genes. Our results indicate that cd26 is required for proper porcine parthenogenetic activation during embryonic development.

Effect of Factor H on Complement Alternative Pathway Activation in Human Serum Remains on Porcine Cells Lacking N-Glycolylneuraminic Acid.

Background: Triple knockout (TKO) donor pigs lacking alpha-1,3-galactose (Gal), N-glycolylneuraminic acid (Neu5Gc), and Sd(a) expressions were developed to improve the clinical success of xenotransplantation. Neu5Gc, a sialic acid expressed on cell surfaces, recruits factor H to protect cells from attack by the complement system. Lack of Neu5Gc expression may cause unwanted complement activation, abrogating the potential benefit of gene-modified donor pigs. To investigate whether TKO porcine cells display increased susceptibility to complement activation in human serum, pathway-specific complement activation, apoptosis, and human platelet aggregation by porcine cells were compared between alpha-1,3-galactosyltransferase gene-knockout (GTKO) and TKO porcine cells. Methods: Primary porcine peripheral blood mononuclear cells (pPBMCs) and endothelial cells (pECs) from GTKO and TKO pigs were used. Cells were incubated in human serum diluted in gelatin veronal buffer (GVB++) or Mg++-EGTA GVB, and C3 deposition and apoptotic changes in these cells were measured by flow cytometry. C3 deposition levels were also measured after incubating these cells in 10% human serum supplemented with human factor H. Platelet aggregation in human platelet-rich plasma containing GTKO or TKO pECs was analyzed. Results: The C3 deposition level in GTKO pPBMCs or pECs in GVB++ was significantly higher than that of TKO pPBMCs or pECs, respectively, but C3 deposition levels in Mg++-EGTA-GVB were comparable between them. The addition of factor H into the porcine cell suspension in 10% serum in Mg++ -EGTA-GVB inhibited C3 deposition in a dose-dependent manner, and the extent of inhibition by factor H was similar between GTKO and TKO porcine cells. The percentage of late apoptotic cells in porcine cell suspension in GVB++ increased with the addition of human serum, of which the net increase was significantly less in TKO pPBMCs than in GTKO pPBMCs. Finally, the lag time of platelet aggregation in recalcified human plasma was significantly prolonged in the presence of TKO pECs compared to that in the presence of GTKO pECs. Conclusion: TKO genetic modification protects porcine cells from serum-induced complement activation and apoptotic changes, and delays recalcification-induced human platelet aggregation. It does not hamper factor H recruitment on cell surfaces, allowing the suppression of alternative complement pathway activation.

Research Note: Embryonic viability by weight difference between donor and surrogate eggs in a surrogate eggshell incubation system.

A surrogate eggshell incubation system is a well-defined method to apply to avian genetic modification. In this study, we tried to investigate whether the egg weight differences between donor and surrogate eggs have an effect on donor viability. The groups were divided by egg weight differences between the donor and surrogate eggs into 4 in each system. The viability at d 4 was evaluated at the end of System II, the embryos alive were transferred into the second surrogate eggshells, and the viability at d 5, 6 was evaluated at early phase of System III. Then, the viability of System III was evaluated at different incubation period: d 6-12, d 13-18, d 19-21, and hatching rate was evaluated at d 22. Although the effect of egg weight differences between the donor and surrogate eggs was not observed, a specific group in System III showed higher survival and hatching rate than other group (P > 0.05).

Early Interferon-Gamma Response in Nonhuman Primate Recipients of Solid-Organ Xenotransplantation.

BACKGROUND: To understand changes in biological responses in nonhuman primate (NHP) recipients of xenotransplantation (XTP), we retrospectively investigated chronological changes in cytokine profiles of NHP recipients after solid-organ XTP. METHODS: Plasma samples were collected from 7 NHP recipients of pig heart or kidney XTP with α-1,3-galactosyltransferase gene knockout (GTKO) under anti-CD154-based immune suppression at the following time points: immediately before; 2 hours, 3 days, and 7 days after XTP; and weekly thereafter until the graft failed. The plasma levels of the following cytokines were measured: interleukin (IL)-1α, IL-1ß, IL-6, IL-12p70, IL-8, IL-10, IL-15, tumor necrosis factor, interferon gamma (IFN-γ), D-dimer, C3a, and histone-complexed DNA fragments. For in vitro experiments, human natural killer (NK) cells were cocultured with wild-type porcine endothelial cells (PECs), GTKO-PECs, and human umbilical vein endothelial cells, with or without anti-CD154 antibody. IFN-γ levels in the culture supernatants were compared. RESULTS: IFN-γ levels peaked on day 7 or 10 of XTP and then decreased to basal levels, whereas proinflammatory cytokine levels increased along with the elevation of histone-complexed DNA fragments and were sustained until xenograft failure. In vitro, human NK cells produced more IFN-γ when in contact with wild-type PECs than with human umbilical vein endothelial cells, which was not reduced by the use of GTKO-PECs or addition of anti-CD154 antibody to the mixture. CONCLUSIONS: In NHP recipients of XTP, the early peak of IFN-γ priming subsequent inflammatory responses may be attributed to NK cell activation in response to xenografts.

Select Porcine Elongation Factor 1α Sequences Mediate Stable High-Level and Upregulated Expression of Heterologous Genes in Porcine Cells in Response to Primate Serum.

Genetically engineered (GE) pigs with various combinations of genetic profiles have been developed using heterologous promoters. This study aimed to identify autologous promoters for high and ubiquitous expression of xenotransplantation relevant genes in GE pigs. A 1.4 kb upstream regulatory sequence of porcine elongation factor 1α (pEF1α) gene was selected and isolated for use as a promoter. Activity of the pEF1α promoter was subsequently compared with that of the cytomegalovirus (CMV) promoter, CMV enhancer/chicken ß-actin (CAG) promoter, and human EF1α (hEF1α) promoter in different types of pig-derived cells. Comparative analysis of luciferase and mutant human leukocyte antigen class E-F2A-ß-2 microglobulin (HLA-E) expression driven by pEF1α, CMV, CAG, and hEF1α promoters revealed the pEF1α promoter mediated comparable expression levels with those of the CAG promoter in porcine ear skin fibroblasts (PEFs) and porcine kidney-15 (PK-15) cells, but lower than those of the CAG promoter in porcine aortic endothelial cells (PAECs). The pEF1α promoter provided long-term stable HLA-E expression in PEFs, but the CAG promoter failed to sustain those levels of expression. For xenogeneic serum-induced cytotoxicity assays, the cells were cultured for several hours in growth medium supplemented with primate serum. Notably, the pEF1α promoter induced significant increases in luciferase and HLA-E expression in response to primate serum in PAECs compared with those driven by the CAG promoter, suggesting the pEF1α promoter could regulate temporal expression of heterologous genes under xenogeneic-cytotoxic conditions. These results suggest the pEF1α promoter may be valuable for development of GE pigs spatiotemporally and stably expressing immunomodulatory genes for xenotransplantation.

Development and in vitro evaluation of recombinant chicken promoters to efficiently drive transgene expression in chicken oviduct cells.

Virus injection into EGK-X embryos is a well-defined approach in avian transgenesis. This system uses a chicken ovalbumin gene promoter to induce transgene expression in the chicken oviduct. Although a reconstructed chicken ovalbumin promoter that links an ovalbumin promoter and estrogen-responsive enhancer element (ERE) is useful, a large viral vector containing the ovalbumin promoter and a target gene restricts viral packaging capacity and produces low-titer virus particles. We newly developed recombinant chicken promoters by linking regulatory regions of ovalbumin and other oviduct-specific genes. Putative enhancer fragments of the genes, such as ovotransferrin (TF), ovomucin alpha subunit (OVOA), and ovalbumin-related protein X (OVALX), were placed at the 5`-flanking region of the 2.8-kb ovalbumin promoter. Basal promoter fragments of the genes, namely, pTF, lysozyme (pLYZ), and ovomucoid (pOVM), were placed at the 3`-flanking region of the 1.6-kb ovalbumin ERE. The recombinant promoters cloned into each reporter vector were evaluated using a dual luciferase assay in human and chicken somatic cells, and LMH/2A cells treated with 0-1,000 nM estrogen, and cultured primary chicken oviduct cells. The recombinant promoters with linking ovalbumin and TF, OVOA, pOVM, and pLYZ regulatory regions had 2.1- to 19.5-fold (P < 0.05) higher luciferase activity than the reconstructed ovalbumin promoter in chicken oviduct cells. Therefore, recombinant promoters may be used to efficiently drive transgene expression in transgenic chickens.

Immunosuppression-enhancing effect of the administration of allogeneic canine adipose-derived mesenchymal stem cells (cA-MSCs) compared with autologous cA-MSCs in vitro.

BACKGROUND: Recently, mesenchymal stem cells therapy has been performed in dogs, although the outcome is not always favorable. OBJECTIVES: To investigate the therapeutic efficacy of mesenchymal stem cells (MSCs) using dog leukocyte antigen (DLA) matching between the donor and recipient in vitro. METHODS: Canine adipose-derived MSCs (cA-MSCs) isolated from the subcutaneous tissue of Dog 1 underwent characterization. For major DLA genotyping (DQA1, DQB1, and DRB1), peripheral blood mononuclear cells (PBMCs) from two dogs (Dogs 1 and 2) were analyzed by direct sequencing of polymerase chain reaction (PCR) products. The cA-MSCs were co-cultured at a 1:10 ratio with activated PBMCs (DLA matching or mismatching) for 3 days and analyzed for immunosuppressive (IDO, PTGS2, and PTGES), inflammatory (IL6 and IL10), and apoptotic genes (CASP8, BAX, TP53, and BCL2) by quantitative real-time reverse transcriptase-PCR. RESULTS: cA-MSCs were expressed cell surface markers such as CD90+/44+/29+/45- and differentiated into osteocytes, chondrocytes, and adipocytes in vitro. According to the Immuno Polymorphism Database, DLA genotyping comparisons of Dogs 1 and 2 revealed complete differences in genes DQA1, DQB1, and DRB1. In the co-culturing of cA-MSCs and PBMCs, DLA mismatch between the two cell types induced a significant increase in the expression of immunosuppressive (IDO/PTGS2) and apoptotic (CASP8/BAX) genes. CONCLUSIONS: The administration of cA-MSCs matching the recipient DLA type can alleviate the need to regulate excessive immunosuppressive responses associated with genes, such as IDO and PTGES. Furthermore, easy and reliable DLA genotyping technology is required because of the high degree of genetic polymorphisms of DQA1, DQB1, and DRB1 and the low readability of DLA 88.

N-acetyl-L-cysteine Improves the Developmental Competence of Bovine Oocytes and Embryos Cultured In Vitro by Attenuating Oxidative Damage and Apoptosis.

Oxidative stress has been suggested to negatively affect oocyte and embryo quality and developmental competence, resulting in failure to reach full term. In this study, we investigated the effect of N-acetyl-L-cysteine (NAC), a cell-permeating antioxidant, on developmental competence and the quality of oocytes and embryos upon supplementation (0.1-10 mM) in maturation and culture medium in vitro using slaughterhouse-derived oocytes and embryos. The results show that treating oocytes with 1.0 mM NAC for 8 h during in vitro maturation attenuated the intracellular reactive oxygen species (ROS) (p < 0.05) and upregulated intracellular glutathione levels (p < 0.01) in oocytes. Interestingly, we found that NAC affects early embryonic development, not only in a dose-dependent, but also in a stage-specific, manner. Significantly (p < 0.05) decreased cleavage rates (90.25% vs. 81.46%) were observed during the early stage (days 0-2), while significantly (p < 0.05) increased developmental rates (38.20% vs. 44.46%) were observed during the later stage (from day 3) of embryonic development. In particular, NAC supplementation decreased the proportion of apoptotic blastomeres significantly (p < 0.05), resulting in enhanced hatching capability and developmental rates during the in vitro culture of embryos. Taken together, our results suggest that NAC supplementation has beneficial effects on bovine oocytes and embryos through the prevention of apoptosis and the elimination of oxygen free radicals during maturation and culture in vitro.

Effect of sex-specific differences on function of induced hepatocyte-like cells generated from male and female mouse embryonic fibroblasts.

BACKGROUND: The liver is one of the vital organs involved in detoxification and metabolism. The sex-based differences between the functionality of male and female liver have been previously reported, i.e., male's liver are good in alcohol clearance and lipid metabolism, while female's liver are better in cholesterol metabolism. To date, studies on novel drug toxicity have not considered the sex-specific dimorphic nature of the liver. However, the use of hepatocyte-like cells to treat liver diseases has increased recently. METHODS: Mouse embryos were isolated from a pregnant female C57BL/6J mouse where mouse embryonic fibroblasts (MEFs) were isolated from back skin tissue of each embryo. MEFs were transduced with human transcription factors hHnf1α, hHnf4α, and hFoxa3 using the lentiviral system. The transduced MEFs were further treated with hepatocyte-conditioned media followed by its analysis through RT-qPCR, immunofluorescence, functional assays, and finally whole-transcriptome RNA sequencing analysis. For in vivo investigation, the mouse hepatocyte-like cells (miHep) were transplanted into CCl4-induced acute liver mouse model. RESULTS: In this study, we evaluated the sex-specific effect of miHep induced from male- and female-specific mouse embryonic fibroblasts (MEFs). We observed miHeps with a polygonal cytoplasm and bipolar nucleus and found that male miHeps showed higher mHnf4a, albumin secretion, and polyploidization than female miHeps. Transcriptomes from miHeps were similar to those from the liver, especially for Hnf4a of male miHeps. Male Cyps were normalized to those from females, which revealed Cyp expression differences between liver and miHeps. In both liver and miHeps, Cyp 4a12a and Cyp 4b13a/2b9 predominated in males and females, respectively. After grafting of miHeps, AST/ALT decreased, regardless of mouse sex. CONCLUSION: In conclusion, activation of endogenic Hnf4a is important for generation of successful sex-specific miHeps; furthermore, the male-derived miHep exhibits comparatively enhanced hepatic features than those of female miHep.

Comprehensive Analysis of Cardiac Xeno-Graft Unveils Rejection Mechanisms.

Porcine heart xenotransplantation is a potential treatment for patients with end-stage heart failure. To understand molecular mechanisms of graft rejection after heart transplantation, we transplanted a 31-day-old alpha-1,3-galactosyltransferase knockout (GTKO) porcine heart to a five-year-old cynomolgus monkey. Histological and transcriptome analyses were conducted on xenografted cardiac tissue at rejection (nine days after transplantation). The recipient monkey's blood parameters were analyzed on days -7, -3, 1, 4, and 7. Validation was conducted by quantitative real-time PCR (qPCR) with selected genes. A non-transplanted GTKO porcine heart from an age-matched litter was used as a control. The recipient monkey showed systemic inflammatory responses, and the rejected cardiac graft indicated myocardial infarction and cardiac fibrosis. The transplanted heart exhibited a total of 3748 differentially expressed genes compared to the non-transplanted heart transcriptome, with 2443 upregulated and 1305 downregulated genes. Key biological pathways involved at the terminal stage of graft rejection were cardiomyopathies, extracellular interactions, and ion channel activities. The results of qPCR evaluation were in agreement with the transcriptome data. Transcriptome analysis of porcine cardiac tissue at graft rejection reveals dysregulation of the key molecules and signaling pathways, which play relevant roles on structural and functional integrities of the heart.

Inclusion of Lactobacillus salivarius strain revealed a positive effect on improving growth performance, fecal microbiota and immunological responses in chicken.

Probiotics are defined as live microorganisms that when administered in an appropriate amount, provide health benefits to the host. This study aimed to evaluate the effect of the oral administration of Lactobacillus salivarius (L. salivarius) on growth performance, immunological responses, fecal microbial flora and intestinal mucosal morphology in chickens. Chickens were fed with 109 colony-forming units (CFUs) of wild-type (WT) L. salivarius or phosphate-buffered saline (PBS) for 5 weeks. Chickens body weight was significantly increased by administration of L. salivarius groups compared than control group. The microbial taxonomy in the small intestine and cecum was identified via the chicken feces sample. A total of 286,331 bacterial species were obtained from the chicken fecal samples in overall experimental group. From these, 145,012 bacterial species were obtained from oral administration of L. salivarius treatment group, while 141,319 bacterial species were obtained from control group. Almost 98% of all 16S rRNA sequences from the chicken fecal sample of the two groups were classified into known phyla. Firmicutes, Cyanobacteria, Proteobacteria, Bacteroidetes and Actinobacteria were highly abundant in both groups. Compared with the control birds, the chickens orally administered L. salivarius showed no significant differences in villus length and crypt length. Serum concentrations of the cytokines IL-8, TNF-α, IFN-γ, and IL-4 were markedly reduced in the L. salivarius group. In summary, our findings reveal that L. salivarius can act as a potential probiotic to improve performance and overall gut health in of chickens.

The 3D8 single chain variable fragment protein suppresses Newcastle disease virus transmission in transgenic chickens.

BACKGROUND: The 3D8 single chain variable fragment (scFv) is a mini-antibody sequence that exhibits independent nuclease activity against all types of nucleic acids. In this research, crossing a 3D8 scFv G1 transgenic rooster with wild-type hens produced 3D8 scFv G2 transgenic chickens to evaluate suppression of viral transmission. RESULT: The transgenic chickens were identified using genomic PCR and immunohistochemistry. To evaluate Newcastle disease virus (NDV) protection conferred by 3D8 scFv expression, transgenic, non-transgenic, and specific pathogen-free (SPF) chickens were challenged with virulent NDV by direct injection or aerosol exposure. The three groups of chickens showed no significant differences (p < 0.05) in mean death time after being directly challenged with NDV; however, in contrast to chickens in the non-transgenic and SPF groups, chickens in the transgenic group survived after aerosol exposure. Although the transgenic chickens did not survive after direct challenge, we found that the chickens expressing the 3D8 scFv survived aerosol exposure to NDV. CONCLUSIONS: Our finding suggest that the 3D8 scFv could be a useful tool to prevent chickens from spreading NDV and control virus transmission.

Administration of L. salivarius expressing 3D8 scFv as a feed additive improved the growth performance, immune homeostasis, and gut microbiota of chickens.

Probiotics have been defined as live microorganisms that are administered in an appropriate amount to provide health benefits to the host animal. In this study, we investigated the effect of L. salivarius DJ-sa-01 secreting the 3D8 single-chain variable fragment (3D8 scFv) on the growth performance, cytokine secretion, and intestinal microbial flora of chickens. The experiment was divided into the control group and L. salivarius expressing 3D8 scFv experimental group. Chicken was fed 109 colony-forming units (CFUs) of wild-type (WT) L. salivarius or 3D8 scFv-secreting L. salivarius daily for 35 days. The administration of L. salivarius expressing 3D8 scFv significantly improved the body weight of chickens compared with the administration of WT L. salivarius. A 16S ribosomal RNA metagenomic analysis showed that Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes were the dominant phyla in both experimental groups. At the genus level, Lactobacillus was more abundant (22.82%) in the L. salivarius/3D8 group compared with the WT L. salivarius group. The serum levels of cytokines, such as IL-8, TNF-α, IL-1ß, IFN-γ, IL-4, and IGF1, were significantly reduced in the L. salivarius/3D8-treated chickens. In summary, our results suggest that L. salivarius expressing 3D8 scFv could be considered a feed additive for improving the growth performance, immune function, and disease resistance of poultry.

Evaluation of Intestinal Epithelial Barrier Function in Inflammatory Bowel Diseases Using Murine Intestinal Organoids.

BACKGROUND: Intestinal organoids have evolved as potential molecular tools that could be used to study host-microbiome interactions, nutrient uptake, and drug screening. Gut epithelial barrier functions play a crucial role in health and diseases, especially in autoimmune diseases, such as inflammatory bowel diseases (IBDs), because they disrupt the epithelial mucosa and impair barrier function. METHODS: In this study, we generated an in vitro IBD model based on dextran sodium sulfate (DSS) and intestinal organoids that could potentially be used to assess barrier integrity. Intestinal organoids were long-term cultivated and characterized with several specific markers, and the key functionality of paracellular permeability was determined using FITC-dextran 4 kDa. Intestinal organoids that had been treated with 2 µM DSS for 3 h were developed and the intestinal epithelial barrier function was sequentially evaluated. RESULTS: The results indicated that the paracellular permeability represented epithelial characteristics and their barrier function had declined when they were exposed to FITC-dextran 4 kDa after DSS treatment. In addition, we analyzed the endogenous mRNA expression of pro-inflammatory cytokines and their downstream effector genes. The results demonstrated that the inflammatory cytokines genes significantly increased in inflamed organoids compared to the control, leading to epithelial barrier damage and dysfunction. CONCLUSION: The collective results showed that in vitro 3D organoids mimic in vivo tissue topology and functionality with minor limitations, and hence are helpful for testing disease models.

Morphometric analysis of alpha-1, 3-galactosyltransferase knockout pig kidney and heart.

We report a morphometric evaluation of α1,3-galactosyltransferase knockout (GTKO) pig heart and kidney (n = 9) at the end of one, three and seven months. Organs parameters gradually increased with the age (p < 0.05) and body weight (p < 0.05) of the pigs. Organs morphometries were significantly correlated to the age and body weight of the animal. We were able to conclude the average size of GTKO pig heart and kidney based on age and body weight, which could be helpful in estimating the size of these organs non-invasively for transplantation.