siRNAs pools generated in Escherichia coli exhibit strong RNA-interference activity against influenza virus genomic sequences.

Due to the recurrent pandemic outbreaks that occurred during the last century, Influenza A viruses are considered a serious potential danger to human health. Among the innate immune pathways in eukaryotes, RNA interference plays a significant role in the interaction between viruses and host cells. RNA interference is addressed by small dsRNA molecules produced by the host itself (miRNAs, i.e. "micro-RNAs") but can be triggered also by the administration of exogenous short RNAs (siRNAs, "short interfering RNAs"). In this work, artificial siRNA pools targeting NP and PB genomic regions of the Influenza virus were produced in engineered Escherichia coli, adapting a published protocol. In a MDCK cell in vitro model, these preparations were challenged against reporter vectors bearing viral genomic sequences. A strong and specific RNA interference activity was observed, and the details of this action were indagated.

The Contribution of Adipose Tissue-Derived Mesenchymal Stem Cells and Platelet-Rich Plasma to the Treatment of Chronic Equine Laminitis: A Proof of Concept.

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.

Susceptibility of different cell lines to Avian and Swine Influenza viruses.

Influenza outbreaks are widespread in swine and avian populations. Disease control is jeopardized by the extreme antigenic variability of virus strains. Primary isolation of Influenza virus is performed using embryonated chicken eggs (ECE), but alternatives to ECE are badly needed. Although various cultured cells have been used for propagating Influenza A viruses, few types of cells can efficiently support virus replication. One of the most commonly cell lines used in order to isolate Influenza A virus, is represented by the Madin Darby Canine Kidney (MDCK) cell line, but cells derived from primary swine organs (kidney, testicle, lung and trachea) can also be employed. The aim of this study was the evaluation of NSK, MDCK, UMNSAH/DF1 cell lines suitability, compared to ECE for isolation and propagation of Avian and Swine virus subtypes. The results indicated both NSK and MDCK could provide an appropriate substrate for cultivating either Avian (AIV) or Swine (SIV) Influenza virus strains, especially for high pathogenicity Avian Influenza ones. Furthermore, NSK appeared more susceptible than MDCK cells for primary isolation of AIV. In contrast, UMNSAH/DF1 cell line seemed to be less permissive to support Avian virus growth. Furthermore, no SIV replication was detected except for one subtype. Additionally, the results of this study indicated that not all virus strains seemed to adapt with the same efficiency to the different cell lines. On the contrary, chicken embryos were shown to be the most suitable biological system for AIV isolation.

Mesenchymal stromal cell cryopreservation.

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.