Transplantation of a bioengineered tissue patch promotes uterine repair in the sheep.

Innovative bioengineering strategies utilizing extracellular matrix (ECM) based scaffolds derived from decellularized tissue offer new prospects for restoring damaged uterine tissue. Despite successful fertility restoration in small animal models, the translation to larger and more clinically relevant models have not yet been assessed. Thus, our study investigated the feasibility to use a 6 cm2 graft constructed from decellularized sheep uterine tissue, mimicking a future application to repair a uterine defect in women. Some grafts were also recellularized with fetal sheep bone marrow-derived mesenchymal stem cells (SF-MSCs). The animals were followed for six weeks post-surgery during which blood samples were collected to assess the systemic immune cell activation by fluorescence-activated cell sorting (FACS) analysis. Tissue regeneration was assessed by histology, immunohistochemistry, and gene expression analyses. There was a large intra-group variance which prompted us to implement a novel scoring system to comprehensively evaluate the regenerative outcomes. Based on the regenerative score each graft received, we focused our analysis to map potential differences that may have played a role in the success or failure of tissue repair following the transplantation therapy. Notably, three out of 15 grafts exhibited major regeneration that resembled native uterine tissue, and an additional three grafts showed substantial regenerative outcomes. For the better regenerated grafts, it was observed that the systemic T-cell subgroups were significantly different compared with the failing grafts. Hence, our data suggest that the T-cell response play an important role for determining the uterus tissue regeneration outcomes. The remarkable regeneration seen in the best-performing grafts after just six weeks following transplantation provides compelling evidence that decellularized tissue for uterine bioengineering holds great promise for clinically relevant applications.

Mesenchymal stem cells establish a pro-regenerative immune milieu after decellularized rat uterus tissue transplantation.

Decellularized tissue is generally considered immune privileged after transplantation and is an attractive scaffold type for tissue regeneration, including applications for infertility treatment. However, the immune response following transplantation of decellularized tissue is insufficiently studied, in particular after they have been recellularized with mesenchymal stem cells (MSCs). Therefore, we replaced a large uterus segment with a bioengineered graft developed from decellularized uterus tissue and analyzed the immune response during the first 4 months in acellular or MSCs-recellularized scaffolds in the rat. Immunohistochemistry-stained infiltrating immune cells and plasma levels for 16 cytokines and chemokines were quantified. Results revealed that MSCs created an advantageous microenvironment by increasing anti-inflammatory interleukin 10 levels, and increasing the population of FOXP3+ TRegs and CD163+ M2 macrophages, and by reducing the CD8+ cytotoxic T cell population. Hence, MSCs should be considered an immunotherapeutic cell source with the ability to dictate regeneration success after decellularized tissue transplantation.

Patterns of cellular gene expression in swine macrophages infected with highly virulent classical swine fever virus strain Brescia.

Experimental exposure of swine to highly virulent classical swine fever virus (CSFV) strain Brescia causes an invariably fatal disease of all infected animals by 8-14 days post-infection. Host mechanisms involved in this severe outcome of infection have not been clearly established. To understand these mechanisms, we analyzed the response of primary cultured swine macrophages, a CSFV primary target cell, to infection with Brescia strain. Steady state levels of mRNA accumulation were assessed for 58 genes involved in modulation of the host immune response, at 24 and 48 h post-infection (hpi), by means of quantitative reverse transcription real-time PCR analysis (qrt-PCR). Eighteen genes showed altered expression upon infection with CSFV strain Brescia including: cytokines (IL-1alpha, IL-1beta, IL-6, and IL-12p35); cytokine receptors (IL-2Ralpha, IL-12Rbeta, and TGF-betaIIIR); chemokines (IL-8, AMCF-1, AMCF-2, MCP-2, and RANTES); interferons (INFalpha and INFbeta); and toll-like receptors (TLR3, TLR5, TLR9, and TLR10). Although these genes are associated with mechanisms of innate immune response and antiviral activity, their altered expression does not curtail CSFV Brescia growth kinetics and virus yield in swine macrophages. Data gathered here suggests that the observed gene expression profile might explain immunological and pathological changes associated with virulent CSFV infections.

Infection of porcine alveolar macrophages with recombinant chimeric porcine reproductive and respiratory syndrome virus: effects on cellular gene transcription and virus growth.

Porcine reproductive and respiratory syndrome virus (PRRSV) genetic determinants affecting the response of the host primary target cell, the macrophage, to infection are yet to be defined. Here we have used recombinant viruses encompassing ORF 1A to identify PRRSV determinants associated with growth and modulation of pro- and anti-inflammatory cytokine expression in primary pulmonary alveolar macrophages (PAMs) cultures. Three genomic chimeras encompassing ORF 1A of PRRSV live attenuated vaccine Prime Pac (LAV SP) in the genetic background of pathogenic strain NVSL 97-7895 (FL12v) were characterized in vitro. Unlike parental viruses, two of the recombinant viruses encompassing the area of the genome encoding for NSP2 to NSP8 showed reduced growth in PAM cultures. The effect of virus infections on gene activation was studied for 25 immunomodulatory cellular genes in PAMs at 24 and 48h post-infection (hpi). Steady state mRNA levels in PAMs infected with recombinant and LAV SP viruses were compared to levels observed in cells infected with parental virus FL12v. Recombinant viruses induced patterns of transcriptional activation differing from patterns induced by parental FL12v, suggesting a regulatory role of PRRSV ORF1A on PAM gene expression.