Long-term efficacy of encapsulated xenogeneic islet transplantation: Impact of encapsulation techniques and donor genetic traits.

AIMS/INTRODUCTION: To investigate the long-term efficacy of various encapsulated xenogeneic islet transplantation, and to explore the impact of different donor porcine genetic traits on islet transplantation outcomes. MATERIALS AND METHODS: Donor porcine islets were obtained from wild-type, α1,3-galactosyltransferase knockout (GTKO) and GTKO with overexpression of membrane cofactor protein genotype. Naked, alginate, alginate-chitosan (AC), alginate-perfluorodecalin (A-PFD) and AC-perfluorodecalin (AC-PFD) encapsulated porcine islets were transplanted into diabetic mice. RESULTS: In vitro assessments showed no differences in the viability and function of islets across encapsulation types and donor porcine islet genotypes. Xenogeneic encapsulated islet transplantation with AC-PFD capsules showed the most favorable long-term outcomes, maintaining normal blood glucose levels for 180 days. A-PFD capsules showed comparable results to AC-PFD capsules, followed by AC capsules and alginate capsules. Conversely, blood glucose levels in naked islet transplantation increased to >300 mg/dL within a week after transplantation. Naked islet transplantation outcomes showed no improvement based on donor islet genotype. However, alginate or AC capsules showed delayed increases in blood glucose levels for GTKO and GTKO with overexpression of membrane cofactor protein porcine islets compared with wild-type porcine islets. CONCLUSION: The AC-PFD capsule, designed to ameliorate both hypoxia and inflammation, showed the highest long-term efficacy in xenogeneic islet transplantation. Genetic modifications of porcine islets with GTKO or GTKO with overexpression of membrane cofactor protein did not influence naked islet transplantation outcomes, but did delay graft failure when encapsulated.

Development of a Chimeric Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)-2 Vaccine Candidate Expressing Hypo-Glycosylated Glycoprotein-5 Ectodomain of Korean Lineage-1 Strain.

Vaccination is a practical method to provide protection against porcine reproductive and respiratory syndrome virus (PRRSV), but current PRRSV vaccines show limited efficacy against divergent field strains. Lineage 1 PRRSV includes virulent strains such as NADC30 and MN184 and now has become one of the most prevalent viruses in Korea. Accordingly, there is an urgent need to develop a new vaccine for Korean lineage-1 strains. In this study, a vaccine candidate against Korean lineage-1 PRRSV, vCSL1-GP5-N33D, was developed by reverse genetics technology. vCSL1-GP5-N33D was designed as a hypo-glycosylated chimeric virus containing the glycoprotein 5 ectodomain region of the Korean lineage-1 wild-type strain. An inactivated vaccine of vCSL1-GP5-N33D was applied to a PRRS-endemic farm and elicited high serum virus neutralization (SVN) antibody titers. The vaccinated group induced SVN antibody titers of 4.40 (log2) ± 2.46, which were approximately 2-fold higher than those of the negative control at 8-weeks post-vaccination. Moreover, 60% of pigs in the vaccinated group displayed SVN antibody titers of ≥5 (log2), while none of the pigs in the negative control exhibited SVN antibody titers of ≥5 (log2). The overall results of the animal experiment suggest that the vCSL1-GP5-N33D inactivated vaccine is a promising vaccine candidate.

A chimeric porcine reproductive and respiratory syndrome virus (PRRSV)-2 vaccine is safe under international guidelines and effective both in experimental and field conditions.

Vaccination is currently the most effective strategy to control porcine reproductive and respiratory syndrome (PRRS). New-generation PRRS vaccines are required to be safe and broadly cross-protective. We have recently created the chimeric PRRS virus K418DM which proved to be a good vaccine candidate under field conditions. In the present study, we designed safety and efficacy tests under experimental and field conditions for further evaluation of K418DM1.1, a plaque-purified K418DM. In the homologous challenge study, K418DM1.1 induced high serum virus neutralization (SVN) antibody titers (i.e., 4.2 log2 ± 1.7) at 21 days post-challenge (dpc) and provided protection as demonstrated by the significantly lower levels of viremia at 3 and 7 dpc and significantly lower microscopic lung lesion scores compared to the unvaccinated group. K418DM1.1 was also protective in the heterologous challenge study, with vaccinated pigs showing significantly lower levels of viremia at 14 dpc compared to the unvaccinated pigs. A field study was performed to evaluate the efficacy of K418DM1.1 against heterologous exposure and vaccinated pigs presented significantly lower viremia than unvaccinated pigs. According to the safety test for the examination of virulence reversion, no infectivity was observed in tissue homogenate filtrate both in the vaccinated and comingled groups. Thus, the risk of virulence, as well as transmission, appeared negligible. These overall results indicate that K418DM1.1 is a good vaccine candidate based on its safety and protective efficacy.

Construction of a Dual-Fluorescence Reporter System to Monitor the Dynamic Progression of Pluripotent Cell Differentiation.

Oct4 is a crucial germ line-specific transcription factor expressed in different pluripotent cells and downregulated in the process of differentiation. There are two conserved enhancers, called the distal enhancer (DE) and proximal enhancer (PE), in the 5' upstream regulatory sequences (URSs) of the mouse Oct4 gene, which were demonstrated to control Oct4 expression independently in embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs). We analyzed the URSs of the pig Oct4 and identified two similar enhancers that were highly consistent with the mouse DE and PE. A dual-fluorescence reporter was later constructed by combining a DE-free-Oct4-promoter-driven EGFP reporter cassette with a PE-free-Oct4-promoter-driven mCherry reporter cassette. Then, it was tested in a mouse ESC-like cell line (F9) and a mouse EpiSC-like cell line (P19) before it is formally used for pig. As a result, a higher red fluorescence was observed in F9 cells, while green fluorescence was primarily detected in P19 cells. This fluorescence expression pattern in the two cell lines was consistent with that in the early naïve pluripotent state and late primed pluripotent state during differentiation of mouse ESCs. Hence, this reporter system will be a convenient tool for screening out ESC-like naïve pluripotent stem cells from other metastable state cells in a heterogenous population.

Strict vegetarian diet improves the risk factors associated with metabolic diseases by modulating gut microbiota and reducing intestinal inflammation.

Low-grade inflammation of the intestine results in metabolic dysfunction, in which dysbiosis of the gut microbiota is intimately involved. Dietary fibre induces prebiotic effects that may restore imbalances in the gut microbiota; however, no clinical trials have been reported in patients with metabolic diseases. Here, six obese subjects with type 2 diabetes and/or hypertension were assigned to a strict vegetarian diet (SVD) for 1 month, and blood biomarkers of glucose and lipid metabolisms, faecal microbiota using 454-pyrosequencing of 16S ribosomal RNA genes, faecal lipocalin-2 and short-chain fatty acids were monitored. An SVD reduced body weight and the concentrations of triglycerides, total cholesterol, low-density lipoprotein cholesterol and haemoglobin A1c, and improved fasting glucose and postprandial glucose levels. An SVD reduced the Firmicutes-to-Bacteroidetes ratio in the gut microbiota, but did not alter enterotypes. An SVD led to a decrease in the pathobionts such as the Enterobacteriaceae and an increase in commensal microbes such as Bacteroides fragilis and Clostridium species belonging to clusters XIVa and IV, resulting in reduced intestinal lipocalin-2 and short-chain fatty acids levels. This study underscores the benefits of dietary fibre for improving the risk factors of metabolic diseases and shows that increased fibre intake reduces gut inflammation by changing the gut microbiota.

Nucleofection-mediated α1,3-galactosyltransferase gene inactivation and membrane cofactor protein expression for pig-to-primate xenotransplantation.

Xenotransplantation of pig organs into primates leads to hyperacute rejection (HAR). Functional ablation of the pig α 1,3-galactosyltransferase (GalT) gene, which abrogates expression of the Gal α 1-3Gal ß 1-4GlcNAc-R (Gal) antigen, which inhibits HAR. However, antigens other than Gal may induce immunological rejection by their cognate antibody responses. Ultimately, overexpression of complement regulatory proteins reduces acute humoral rejection by non-Gal antibodies when GalT is ablated. In this study, we developed a vector-based strategy for ablation of GalT function and concurrent expression of membrane cofactor protein (MCP, CD46). We constructed an MCP expression cassette (designated as MCP-IRESneo) and inserted between the left and the right homologous arms to target exon 9 of the GalT gene. Nucleofection of porcine ear skin fibroblasts using the U-023 and V-013 programs resulted in high transfection efficiency and cell survival. We identified 28 clones in which the MCP-IRESneo vector had been successfully targeted to exon 9 of the GalT gene. Two of those clones, with apparent morphologically mitotic fibroblast features were selected through long-term culture. GalT gene expression was downregulated in these 2 clones. Importantly, MCP was shown to be efficiently expressed at the cell surface and to efficiently protect cell lysis against normal human complement serum attack in vitro.

Fringe field switching of a twisted nematic liquid crystal device for a single-cell-gap transflective display.

We propose an optical configuration of a twisted nematic liquid crystal device driven by a fringe field for a single-cell-gap transflective display. The dark state of the reflective part is realized by a nematic liquid crystal layer with a twist angle of 63.6 degrees and retardation of 194 nm, while a quarter-wave plate is inserted for the dark state of the transmissive part. Wavelength dispersion of the liquid crystal layer is suppressed by introducing a half-wave plate. Different directions of electric fields rotate liquid crystals to 15 degrees for the bright state of the reflective part, but to -30 degrees for that of the transmissive part. With the proposed configuration, we can realize a high-brightness single-gamma transflective display in a single-cell-gap structure without any in-cell retardation layers.

Pregnancy-associated plasma protein-a production in rat granulosa cells: stimulation by follicle-stimulating hormone and inhibition by the oocyte-derived bone morphogenetic protein-15.

Pregnancy-associated plasma protein-A (PAPP-A) is the major IGF binding protein-4 (IGFBP-4) protease in follicular fluid, consistent with its proposed role in folliculogenesis. Despite growing interest, almost nothing is known about how PAPP-A expression is regulated in any tissue. Here we show that FSH and oocytes regulate PAPP-A expression in granulosa cells (GCs). By in situ hybridization, ovary PAPP-A mRNA was markedly increased by pregnant mare serum gonadotropin treatment, and the message was localized to the membrana GCs but not cumulus GCs (CGCs) of dominant follicles. To explore the mechanism, we used primary cultures of rat GCs. Control (untreated) cells produced little or no PAPP-A spontaneously. Conversely, FSH markedly stimulated PAPP-A mRNA and protein in a dose- and time-dependent fashion. Interestingly, PAPP-A expression in isolated CGCs was also strongly induced by FSH, and the induction was inhibited by added oocytes. To investigate the nature of the inhibition, we tested the effect of oocyte-derived bone morphogenetic protein-15 (BMP-15). BMP-15 alone had no effect on basal levels of PAPP-A expression by cultures of membrana GCs or CGCs. However, BMP-15 markedly inhibited the FSH stimulation of PAPP-A production in a dose-dependent manner. The cleavage of IGFBP-4 by conditioned media from FSH-treated GCs was completely inhibited by anti-PAPP-A antibody, indicating the IGFBP-4 protease secreted by GCs is PAPP-A. These results demonstrate stimulatory and inhibitory roles for FSH and BMP-15, respectively, in regulating PAPP-A production by GCs. We propose that FSH and oocyte-derived BMP-15 form a controlling network that ensures the spatiotemporal pattern of GC PAPP-A expression in the dominant follicle.

Recombinant FSH and pregnancy-associated plasma protein.

OBJECTIVES: To evaluate the effect of rec-FSH on the ovarian follicular environment in controlled ovarian hyperstimulation (COH) cycle, we compared the expression of the markers that have been reported to reflect the follicle health. STUDY DESIGN: A total of 31 women (<35 years) with normal ovarian function were allocated to three different COH protocols (urinary FSH (u-FSH)+hMG, rec-FSH only, or u-FSH only). E2, P4, leptin and IGF-I levels in serum or follicular fluid (FF) were measured with radioimmunoassay; cyclin D2, EGF receptor (EGF-R), and inhibin-alpha mRNA levels in luteinized granulosa cells were measured with RT-PCR; and IGFBP-4 and PAPP-A levels in FF were measured with Western blot analysis. RESULTS: There were no significant differences in serum E2, P4 and leptin levels and follicular leptin and IGF-I levels. Cyclin D2, EGF-R, and inhibin-alpha mRNA expression in luteinized granulosa cells were not different among three groups. However, follicular IGFBP-4 and PAPP-A levels in rec-FSH group were significantly higher than other groups (P<0.05). CONCLUSIONS: The use of rec-FSH increases the expression of follicular PAPP-A reflecting IGFBP-4 proteolytic activity and it may influence positively the follicular environment.