Identification of a porcine liver eomes-high-T-bet-low NK cell subset that resembles human liver resident NK cells

Natural killer (NK) cells are cells of the innate immunity and play an important role in the defense against viral infections and cancer, but also contribute to shaping adaptive immune responses. Long-lived tissue-resident NK cells have been described in man and mouse, particularly in the liver, contributing to the idea that the functional palette of NK cells may be broader than originally thought, and may include memory-like responses and maintaining tissue homeostasis. Remarkably, liver resident (lr)NK cells in man and mouse show substantial species-specific differences, in particular reverse expression patterns of the T-box transcription factors Eomesodermin (Eomes) and T-bet (Eomes(high)T-bet(low) in man and vice versa in mouse). In pig, compared to blood NK cells which are CD3-CD8 alpha(high) cells, the porcine liver contains an abundant additional CD3-CD8 alpha(dim) NK cell subpopulation. In the current study, we show that this porcine CD3-CD8 alpha(dim) liver NK population is highly similar to its human lrNK counterpart and therefore different from mouse lrNK cells. Like human lrNK cells, this porcine NK cell population shows an Eomes(high)T-bet(low) expression pattern. In addition, like its human counterpart, the porcine liver NK population is CD49e(-) and CXCR6(+). Furthermore, the porcine Eomes(high)T-bet(low) liver NK cell population is able to produce IFN-gamma upon IL-2/12/18 stimulation but lacks the ability to kill K562 or pseudorabies virus-infected target cells, although limited degranulation could be observed upon incubation with K562 cells or upon CD16 crosslinking. All together, these results show that porcine Eomes(high)T-bet(low) NK cells in the liver strongly resemble human lrNK cells, and therefore indicate that the pig may represent a unique model to study the function of these lrNK cells in health and disease

Performance of the Roche Total Mycophenolic Acid® assay on the Cobas Integra 400®, Cobas 6000® and comparison to LC-MS/MS in liver transplant patients

Background: Mycophenolic acid (MPA) is an immunosuppressant for which therapeutic drug monitoring (TDM) is performed for optimal prophylaxis and avoidance of toxicity in transplant patients. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is ideally suited for TDM of MPA. There have been several method comparisons of the Roche Total MPA assay, but none have been performed with respect to liver transplant patients. Methods: We validated the Roche Total MPA assay on the Cobas Integra 400 and Cobas 6000 and compared it to a validated LC-MS/MS (API 2000 (TM)) method. Fifty-five EDTA plasma samples from liver transplant patients were measured with the Roche assay on these platforms and compared to the LC-MS/MS results. Results: Validation of the LC-MS/MS, Cobas Integra 400 and 6000 was performed with good results. The LC-MS/MS/Integra 400/Cobas 6000 were linear up to 30, 15 and 17 mg/L, respectively. Imprecision was <10% for LC-MS/MS and <7% for the Roche assay on both platforms. The samples showed good agreement with LC-MS/MS. Passing-Bablok regression analysis showed Cobas Integra (mg/L) = 1.02 x LC-MS/MS (mg/L)-0.50 and Cobas 6000 (mg/L) = 0.98 x LC-MS/MS-0.47. Conclusions: The Roche Total Mycophenolic Acid-assay is suitable for measuring total MPA in plasma from liver transplant patients and is a good alternative for LC-MS/MS

Development and use of a polarized equine upper respiratory tract mucosal explant system to study the early phase of pathogenesis of a European strain of equine arteritis virus

The upper respiratory tract mucosa represents the first line of defense, which has to be overcome by pathogens before invading the host. Considering the economic and ethical aspects involved in using experimental animals for pathogenesis studies, respiratory mucosal explants, in which the tissue's three-dimensional architecture is preserved, may be ideal alternatives. Different respiratory mucosal explant cultures have been developed. However, none of them could be inoculated with pathogens solely at the epithelium side. In the present study, equine nasal and nasopharyngeal explants were embedded in agarose (3%), leaving the epithelium side exposed to allow apical inoculation. Morphometric analysis did not show degenerative changes during 72 h of cultivation. The number of apoptotic cells in the mucosa slightly increased over time. After validation, the system was used for apical infection with a European strain (08P178) of equine arteritis virus (EAV) (107.6TCID50/mL per explant). Impermeability of agarose to virus particles was demonstrated by the absence of labeled microspheres (40nm) and a lack of EAV-antigens in RK13 cells seeded underneath the agarose layer in which inoculated explants were embedded. At 72 hpi, 27% of the EAV-positive cells were CD172a+ and 19% were CD3+ in nasal explants and 45% of the EAV-positive cells were CD172a+ and 15% were CD3+ in nasopharyngeal explants. Only a small percentage of EAV-positive cells were IgM+. This study validates the usefulness of a polarized mucosal explant system and shows that CD172a+ myeloid cells and CD3+ T lymphocytes represent important EAV-target cells in the respiratory mucosa

Interferon alpha suppresses alphaherpesvirus immediate early protein levels in sensory neurons, leading to the establishment of a latent infection

Alphaherpesviruses are a subfamily of the herpesviruses containing closely related human and animal pathogens, including human herpes simplex virus (HSV-1) and porcine pseudorabies virus (PRV)

The in ovo CAM-assay as a xenograft model for sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions. This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft-(viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products

Single step spray-dried polyelectrolyte microparticles enhance the antigen cross-presentation capacity of porcine dendritic cells

C

An unbiased approach to mapping the signaling network of the pseudorabies virus US3 protein

The US3 serine/threonine protein kinase is conserved among the alphaherpesvirus family and represents an important virulence factor. US3 plays a role in viral nuclear egress, induces dramatic alterations of the cytoskeleton, represses apoptosis, enhances gene expression and modulates the immune response. Although several substrates of US3 have been identified, an unbiased screen to identify US3 phosphorylation targets has not yet been described. Here, we perform a shotgun and phosphoproteomics analysis of cells expressing the US3 protein of pseudorabies virus (PRV) to identify US3 phosphorylation targets in an unbiased way. We identified several cellular proteins that are differentially phosphorylated upon US3 expression and validated the phosphorylation of lamin A/C at serine 404, both in US3-transfected and PRV-infected cells. These results provide new insights into the signaling network of the US3 protein kinase and may serve as a basis for future research into the role of the US3 protein in the viral replication cycle