Genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) has been based on ORF5/GP5 and ORF7/N protein variations. Complete viral genome studies are limited and focused on a single or a few set of strains. Moreover, there is a general tendency to extrapolate results obtained from a single isolate to the overall PRRSV population. In the present study, six genotype-I isolates of PRRSV were sequenced from ORF1a to ORF7. Phylogenetic comparisons and the variability degree of known linear B-epitopes were done considering other available full-length genotype-I sequences. Cytokine induction of all strains was also evaluated in different cellular systems. Non structural protein 2 (nsp2) was the most variable part of the virus with 2 out of 6 strains harboring a 74 aa deletion. Deletions were also found in ORF3 and ORF4. Phylogenetic analyses showed that isolates could be grouped differently depending on the ORF examined and the highest similarity with the full genome cluster was found for the nsp9. Interestingly, most of predicted linear B-epitopes in the literature, particularly in nsp2 and GP4 regions, were found deleted or varied in some of our isolates. Moreover, 4 strains, those with deletions in nsp2, induced TNF-α and 3 induced IL-10. These results underline the high genetic diversity of PRRSV mainly in nsp1, nsp2 and ORFs 3 and 4. This variability also affects most of the known linear B-epitopes of the virus. Accordingly, different PRRSV strains might have substantially different immunobiological properties. These data can contribute to the understanding of PRRSV complexity.

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Laminitis, a highly debilitating disease of the foot in ungulates, is characterized by pathological changes of the complex lamellar structures that maintain the appendicular skeleton within the hoof. Laminitis is a multifactorial disease that involves perturbation of the vascular, hematological, and inflammatory homeostasis of the foot. Interestingly, the pathogenesis of the disease resembles what is observed in metabolic syndromes and sepsis-induced organ failure in humans and animals. We hypothesized that local administration of mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) might contribute to establishing an anti-inflammatory and pro-angiogenic environment, and could stimulate the injured tissue in order to restore its functional integrity. According to this assumption, an experimental protocol based on the local intravenous administration of adipose tissue-derived MSCs (aMSCs) in combination with PRP was developed for the treatment of horses affected by chronic laminitis. Nine horses with severely compromised venograms (showing grade III and IV laminitis) that had been unsuccessfully treated with conventional therapies were enrolled. aMSCs and PRP (15 × 106 cells resuspended in 15 mL of PRP) were injected into the lateral or medial digital vein three times, at one-month intervals. The first administration was performed with allogeneic aMSCs, while for the following administrations, autologous aMSCs were used. There was no adverse short-term reaction to the intravenous injection of aMSCs. In the long term, venograms outlined, in all subjects, a progressive amelioration of the vascularization of the foot. An improvement in the structure and function of the hoof was also observed. No adverse events were reported during the follow-up, and the horses returned to a comfortable quality of life. Although the number of animals enrolled in the study is limited, both clinical observations and venography demonstrated an enhancement in the condition of all horses, suggesting that the regenerative therapies in chronic laminitis could be useful, and are worthy of further investigation.

Regenerative medicine aims to restore the normal function of diseased or damaged cells, tissues, and organs using a set of different approaches, including cell-based therapies. In the veterinary field, regenerative medicine is strongly related to the use of mesenchymal stromal cells (MSCs), which belong to the body repair system and are defined as multipotent progenitor cells, able to self-replicate and to differentiate into different cell types. This review aims to take stock of what is known about the MSCs and their use in the veterinary medicine focusing on clinical reports on dogs and horses in musculoskeletal diseases, a research field extensively reported in the literature data. Finally, a perspective regarding the use of the secretome and/or extracellular vesicles (EVs) in the veterinary field to replace parental MSCs is provided. The pharmaceuticalization of EVs is wished due to the realization of a Good Manufacturing Practice (GMP product suitable for clinical trials.

Drug-induced hepatotoxicity is a leading cause of clinical trial withdrawal. Therefore, in vitro modeling the hepatic behavior and functionalities is not only crucial to better understand physiological and pathological processes but also to support drug development with reliable high-throughput platforms. Different physiological and pathological models are currently under development and are commonly implemented both within platforms for standard 2D cultures and within tailor-made chambers. This paper introduces Hep3Gel: a hybrid alginate−extracellular matrix (ECM) hydrogel to produce 3D in vitro models of the liver, aiming to reproduce the hepatic chemomechanical niche, with the possibility of adapting its shape to different manufacturing techniques. The ECM, extracted and powdered from porcine livers by a specifically set-up procedure, preserved its crucial biological macromolecules and was embedded within alginate hydrogels prior to crosslinking. The viscoelastic behavior of Hep3Gel was tuned, reproducing the properties of a physiological organ, according to the available knowledge about hepatic biomechanics. By finely tuning the crosslinking kinetics of Hep3Gel, its dualistic nature can be exploited either by self-spreading or adapting its shape to different culture supports or retaining the imposed fiber shape during an extrusion-based 3D-bioprinting process, thus being a shape-shifter hydrogel. The self-spreading ability of Hep3Gel was characterized by combining empirical and numerical procedures, while its use as a bioink was experimentally characterized through rheological a priori printability evaluations and 3D printing tests. The effect of the addition of the ECM was evident after 4 days, doubling the survival rate of cells embedded within control hydrogels. This study represents a proof of concept of the applicability of Hep3Gel as a tool to develop 3D in vitro models of the liver.

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The advent of stem cells and stem cell-based therapies for specific diseases requires particular knowledge of laboratory procedures, which not only guarantee the continuous production of cells, but also provide them an identity and integrity as close as possible to their origin. Their cryopreservation at temperatures below -80°C and typically below -140°C is of paramount importance. This target can be achieved by incorporating high molar concentrations of cryoprotectant mixtures that preserve cells from deleterious ice crystal formation. Usually, dimethyl sulfoxide (DMSO) and animal proteins are used as protectant reagents, but unexpected changes in stem cell fate and downstream toxicity effects have been reported, limiting their wide use in clinical settings. In scientific reviews, there are not much data regarding viability of mesenchymal stromal cells (MSCs) after the freezing/thawing process. During our routine analysis, a poor resistance to cryopreservation of these cells was observed, as well as their weak ability to replicate. This is an important point in the study of MSCs; moreover, it represents a limit for preservation and long-term storage. For this reason, MSCs isolated from equine, ovine, and rodent bone marrow and equine adipose tissue were compared using different cryopreservation solutions for this study of vitality. Our findings showed the best results regarding cell viability using a solution of fetal bovine serum with addition of 10% DMSO. In particular, we noted an increase in survival of equine bone marrow MSCs. This parameter has been evaluated by Trypan blue staining at fixed times (0, 24, and 48 hours post-thaw). This result highlights the fact that equine bone marrow MSCs are the frailest we analyzed. Therefore, it could be useful to delve further into this topic in order to improve the storage possibility for these cells and their potential use in cell-based therapies.

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