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The present study investigated the biocompatibility of chitosan films and scaffolds modified with d-(+)raffinose and their capability to support the growth and maintenance of the differentiation of articular chondrocytes in vitro. Primary equine articular chondrocytes were cultured on films and scaffolds of modified d-(+) raffinose chitosan. Their behavior was compared to that of chondrocytes grown in conventional bi- and three-dimensional culture systems, such as micromasses and alginate beads. Chitosan films maintained the phenotype of differentiated chondrocytes (typical round morphology) and sustained the synthesis of cartilaginous extracellular matrix (ECM), even at 4weeks of culture. Indeed, starting from 2weeks of culture, chondrocytes seeded on chitosan scaffolds were able to penetrate the surface pores and to colonize the internal matrix. Moreover they produced ECM expressing the genes of typical chondrocytes differentiation markers such as collagen II and aggrecan. In conclusion, chitosan modified with d-raffinose represents an ideal support for chondrocyte adhesion, proliferation and for the maintenance of cellular phenotypic and genotypic differentiation. This novel biomaterial could potentially be a reliable support for the re-differentiation of dedifferentiated chondrocytes., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

An engineered killer peptide (KP) based on a recombinant anti-idiotypic antibody representing the functional image of a yeast killer toxin (KT) was demonstrated to mediate antimicrobial effects against fungi and viruses. KP binds to murine dendritic cells and macrophages and up-regulate co-receptor expression, thus sustaining CD4+ lymphocyte activation. No immunological data are available in domestic animals thus KP-induced immunomodulation was evaluated in porcine monocyte and lymphocyte subsets. PBMC from healthy adult pigs were stimulated with KP or a scramble peptide (SP), or kept unstimulated for 24, 48 and 72h, and subsequently analyzed by flow cytometry. In monocytes, KP induced a strong dose-dependent shift from a major fraction of CD172α+CD14+ low cells to a predominant fraction of CD172α+CD14+ high cells, known to sustain leukocyte activation/differentiation and inflammatory responses. The CD16+ cell percentages, specifically the CD3+CD16+ natural killer T (NKT) cell fraction and CD16 expression showed an intense and stable dose-dependent increase while the CD3-CD16+ NK cell fraction decreased. CD4+ and CD8+ T cells increased and CD8α and CD8β expression were up-regulated. CD8β+ cytotoxic T cells and CD16+ cells comparably increased. A marked stimulation of activated CD16+CD25+ and CD8β+CD25+ cells was observed at 24h. The increase of CD8α+ cells and CD8α expression were due to increased CD4+CD8α+ (memory T helper) cells, also showing a CD8α+ high phenotype. Concomitantly, the CD4+CD8α- T helper lymphocyte fraction significantly decreased. Overall, KP induced a wide modulation of innate immune and T cells that can exert regulatory and cytotoxic functions, which are fundamental for an efficient Th1 response., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The study aims at evaluating gene expression of pro-inflammatory (IL-1β, IL-8, TNF-α), pro-immune (IFN-γ), anti-inflammatory (IL-10) cytokines and of the immunoregulatory signal FoxP3 in association with PRRSV-specific IFN-γ secreting cell (SC) responsiveness upon PRRSV natural infection. Forty PRRSV-negative pigs were assigned to two groups: 20 pigs were vaccinated at 3 weeks of age (weaning) against PRRSV (V-PRRSV) with a modified live virus vaccine (MLV) and 20 pigs were kept non-vaccinated (NV) as controls. Blood samples were collected at 3 (vaccination), 6, 8, 10, 12, 14, and 16 weeks of age. Natural infection occurred from 8 weeks of age onward in both groups and viremia lasted 8 weeks. In the early phase of infection, pro-inflammatory cytokines (IL-1β, IL-8, TNF-α) showed a delayed increase concomitant with the peak of viremia in both groups. In both groups, IL-10 peaked at 12 weeks in association with the increase of pro-inflammatory cytokines. Conversely, in vaccinated pigs (V-PRRSV), IFN-γ showed higher gene expression during the early phase of infection and a more intense secreting cell (SC) response in the late phase. Differently, gene expression of the transcription factor FoxP3, expressed by T regulatory lymphocytes (Tregs), increased significantly in controls only and was associated with the rise of the viral load. Moreover, FoxP3 levels remained significantly higher during the late phase of infection and paralleled with lower levels of IFN-γ SC detected by ELISPOT. The expression/production of immunoregulatory signals involved in Treg activation could be a promising marker to study the immunobiology of PRRSV infection.

Porcine epidemic diarrhoea (PED) is a contagious enteric disease of pigs caused by a coronavirus. A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) based on the use of monoclonal antibodies was developed for the detection of porcine epidemic diarrhoea virus (PEDV). The DAS-ELISA was compared with RT-PCR in the examination of 506 specimens collected during 2006-2007 from pigs originating from different farms located in the Po valley. Both faecal samples obtained directly from the rectum of live animals showing clinical signs and intestinal samples collected from the caecum of deceased pigs were included in the study. The correlation between the two methods was higher when testing faecal samples (K=0.97, 95% CI: 0.94-1.00) than testing intestinal samples (K=0.62, 95% CI: 0.35-0.89). The use of ELISA technology provided an efficient and effective mean of evaluating the presence of coronavirus PED antigen in field samples and indicates that this procedure is a very useful tool in epidemiological studies., (Copyright 2009 Elsevier Ltd. All rights reserved.)