Endocanalicular transendothelial crossing (ETC): A novel intravasation mode used by HEK-EBNA293-VEGF-D cells during the metastatic process in a xenograft model.

In cancer metastasis, intravasation of the invasive tumor cell (TCi) represents one of the most relevant events. During the last years, models regarding cancer cell intravasation have been proposed, such as the "endocanalicular transendothelial crossing" (ETC) theory. This theory describes the interplay between two adjacent endothelial cells and the TCi or a leukocyte during intravasation. Two endothelial cells create a channel with their cell membranes, in which the cell fits in without involving endothelial cell intercellular junctions, reaching the lumen through a transendothelial passage. In the present study, ten SCID mice were subcutaneously xenotransplanted with the HEK-EBNA293-VEGF-D cell line and euthanized after 35 days. Post-mortem examinations were performed and proper specimens from tumors were collected. Routine histology and immunohistochemistry for Ki-67, pAKT, pERK, ZEB-1, TWIST-1, F-actin, E-cadherin and LYVE-1 were performed followed by ultrastructural serial sections analysis. A novel experimental approach involving Computed Tomography (CT) combined with 3D digital model reconstruction was employed. The analysis of activated transcription factors supports that tumor cells at the periphery potentially underwent an epithelial-to-mesenchymal transition (EMT)-like process. Topographical analysis of LYVE-1 immunolabeled lymphatics revealed a peritumoral localisation. TEM investigations of the lymphatic vessels combined with 3D digital modelling enhanced the understanding of the endotheliocytes behavior during TCi intravasation, clarifying the ETC theory. Serial ultrastructural analysis performed within tumor periphery revealed numerous cells during the ETC process. Furthermore, this study demonstrates that ETC is an intravasation mode more frequently used by the TCi than by leukocytes during intravasation in the HEK-EBNA293-VEGF-D xenograft model and lays down the potential basis for promising future studies regarding intravasation blocking therapy.

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.

Transformation and Tumorigenicity Testing of Simian Cell Lines and Evaluation of Poliovirus Replication.

The key role of cell cultures in different scientific fields is worldwide recognized, both as in vitro research models alternative to laboratory animals and substrates for biological production. However, many safety concerns rise from the use of animal/human cell lines that may be tumorigenic, leading to potential adverse contaminations in cell-derived biologicals. In order to evaluate the suitability of 13 different cell lines for Poliovirus vaccine production, safety and quality, in vitro/in vivo tumorigenicity and Poliovirus propagation properties were evaluated. Our results revealed that non-human primate cell lines CYNOM-K1, FRhK-4, 4MBr-5 and 4647 are free of tumorigenic features and represent highly susceptible substrates for attenuated Sabin Poliovirus strains. In particular, FRhK-4 and 4647 cell lines are characterized by a higher in vitro replication, resulting indicated for the use in large-scale production field.

Protein mutations following adaptation of avian influenza viruses in different biological systems.

Traditionally, embryonated chicken eggs (ECE) are considered the gold standard for Influenza virus isolation and vaccine production. Nowadays, different biological systems have been improved and performed, in order to evaluate a feasible alternative to ECE. In fact, in a previous study, mammalian and avian cell cultures were successfully used for avian influenza viruses primary isolation from target tissues and virus propagation. This research is focused on the investigation of adaptive mutations that occur after influenza A virus amplification in ECE and cell cultures. The results of the study shows that avian influenza viruses after multiple passages in different biological systems undergo mutations, in particular, the largest number of amino acid substitutions occurred in all biological substrates in the hemagglutinin.

Expression of a single siRNA against a conserved region of NP gene strongly inhibits in vitro replication of different Influenza A virus strains of avian and swine origin.

Influenza A virus is the principal agent responsible of the respiratory tract's infections in humans. Every year, highly pathogenic and infectious strains with new antigenic assets appear, making ineffective vaccines so far developed. The discovery of RNA interference (RNAi) opened the way to the progress of new promising drugs against Influenza A virus and also to the introduction of disease resistance traits in genetically modified animals. In this paper, we show that Madin-Darby Canine Kidney (MDCK) cell line expressing short hairpin RNAs (shRNAs) cassette, designed on a specific conserved region of the nucleoprotein (NP) viral genome, can strongly inhibit the viral replication of four viral strains sharing the target sequence, reducing the viral mRNA respectively to 2.5×10(-4), 7.5×10(-5), 1.7×10(-3), 1.9×10(-4) compared to the control, as assessed by real-time PCR. Moreover, we demonstrate that during the challenge with a viral strain bearing a single mismatch on the target sequence, although a weaker inhibition is observed, viral mRNA is still lowered down to 1.2×10(-3) folds in the shRNA-expressing clone compared to the control, indicating a broad potential use of this approach. In addition, we developed a highly predictive and fast screening test of siRNA sequences based on dual-luciferase assay, useful for the in vitro prediction of the potential effect of viral inhibition. In conclusion, these findings reveal new siRNA sequences able to inhibit Influenza A virus replication and provide a basis for the development of siRNAs as prophylaxis and therapy for influenza infection both in humans and animals.

Veterinary biobank facility: development and management for diagnostic and research purposes.

Biobanking is an essential tool for ensuring easy availability of high-quality biomaterial collections that combine essential samples and epidemiological, clinical, and research data for the scientific community. Specimen collection is an integral part of clinical research. Indeed, every year throughout the world, millions of biological samples are stored for diagnostics and research, but in many fields the lack of biological material and models is a major hindrance for ongoing research. A biobank facility provides suitable samples for large-scale screening studies and database repositories. Software dedicated to biological banks simplify sample registration and identification, the cataloging of sample properties (type of sample/specimen, associated diseases and/or therapeutic protocols, environmental information, etc.), sample tracking, quality assurance, and specimen availability characterized by well-defined features. Biobank facilities must adopt good laboratory practices (GLPs) and a stringent quality control system and also comply with ethical issues, when required. The creation of a veterinary network can be useful under different aspects: the first one is related to the importance of animal sciences itself to improve research and strategies in the different branches of the veterinary area, and the second aspect is related to the possibility of data management harmonization to improve scientific cooperation.

Ex vivo expanded mesenchymal stromal cell minimal quality requirements for clinical application.

Mesenchymal stromal cells (MSCs), as advanced therapy products, must satisfy all the requirements for human use of medicinal products, aiming to maintain the quality and safety of the cells. The MSC manufacturing process for clinical use should comply with the principles of Good Manufacturing Practice (GMP). This ensures that cell preparations are produced and controlled, from the collection and manipulation of raw materials, through the processing of intermediate products, to the quality controls, storage, labeling and packaging, and release. The objective of this document is to provide the minimal quality requirements for the MSC production and its delivery for clinical use, so that the safety of the final cell therapy product will not be compromised. For this purpose, the document evaluates the most important steps of GMP-compliant MSC production: the isolation and expansion process; the validation phase of the process, including all quality controls for the characterization, functionality, potency, and safety of MSCs; and the quality control at the batch release to guarantee the safety of patient infusion.

Mesenchymal stromal cells primed with Paclitaxel attract and kill leukaemia cells, inhibit angiogenesis and improve survival of leukaemia-bearing mice.

Current leukaemia therapy focuses on increasing chemotherapy efficacy. Mesenchymal stromal cells (MSCs) have been proposed for carrying and delivery drugs to improve killing of cancer cells. We have shown that MSCs loaded with Paclitaxel (PTX) acquire a potent anti-tumour activity. We investigated the effect of human MSCs (hMSCs) and mouse SR4987 loaded with PTX (hMSCsPTX and SR4987PTX) on MOLT-4 and L1210, two leukaemia cell (LCs) lines of human and mouse origin, respectively. SR4987PTX and hMSCsPTX showed strong anti-LC activity. hMSCsPTX, co-injected with MOLT-4 cells or intra-tumour injected into established subcutaneous MOLT-4 nodules, strongly inhibited growth and angiogenesis. In BDF1-mice-bearing L1210, the intraperitoneal administration of SR4987PTX doubled mouse survival time. In vitro, both hMSCs and hMSCsPTX released chemotactic factors, bound and formed rosettes with LCs. In ultrastructural analysis of rosettes, hMSCsPTX showed no morphological alterations while the attached LCs were apoptotic and necrotic. hMSCs and hMSCsPTX released molecules that reduced LC adhesion to microvascular endothelium (hMECs) and down-modulated ICAM1 and VCAM1 on hMECs. Priming hMSCs with PTX is a simple procedure that does not require any genetic cell manipulation. Once the effectiveness of hMSCsPTX on established cancers in mice is proven, this procedure could be proposed for leukaemia therapy in humans.

Human skin-derived fibroblasts acquire in vitro anti-tumor potential after priming with Paclitaxel.

The main goal in cancer chemotherapy is to drive the drug into the tumor microenvironment to kill as many cancer cells as possible while producing the lowest collateral toxicity. Previously, we have shown that human bone marrow derived mesenchymal stromal cells (hBM-MSCs) exposed to Paclitaxel (PTX) were able to uptake and subsequently release the drug in the culture medium. PTX primed hBM-MSCs (hBM-MSCsPTX) located in the vicinity of cancer cells produced a strong inhibition of tumor cell growth both in vitro and in vivo. To expand these observations, in the present study we exposed human skin derived fibroblasts (hSDFs) to 2,000 ng/ml of PTX and then tested both cells and their conditioned medium (CM) in vitro for their capacity to inhibit the proliferation of human tumor cell lines (MOLT-4, DU-145, U87-MG, SH-SY5Y(+) and LAN-5). We found that hSDFs primed with PTX (hSDFsPTX) were able to uptake and subsequently release PTX in a time dependent manner. hSDFsPTX-derived CM(hSDFsPTX-CM) from 1:4 to 1:10 dilutions produced a significant (p < 0.05) in vitro tumor growth inhibition. hSDFsPTX co-cultured with leukemia cells at 1:1 to 1:10 ratio, completely inhibited cells growth whereas no inhibition was induced by normal hSDFs cells. Our results demonstrate for the first time that hSDFs can be loaded in vitro with PTX and thus can acquire a potent anti-tumor activity. Since hSDFs can be easily isolated from skin biopsies without any particular pain and discomfort to donor patients, we conclude that hSDFs may represent a valid cell type option for carrying and delivering anti-cancer drugs.

Evaluation of Different DNA Vaccines against Porcine Reproductive and Respiratory Syndrome (PRRS) in Pigs.

In veterinary medicine, there have been different experiences with the plasmid DNA vaccination. In this area and with the hypothesis to demonstrate the effectiveness of different plasmids encoding porcine respiratory and reproductive syndrome (PRRS), five DNA vaccines against PRRS were evaluated for their innocuity and efficacy in pigs. Eighteen animals were divided into five groups which were injected with five (A, B, C, D, E) different DNA vaccines. Albeit, none of the proposed vaccines were able to protect the animals against PRRS virus. Only vaccines A and B were able to reduce the clinical signs of the infection. ELISA IgM were detected 30 days after the first vaccination in the pigs injected by Vaccine A or B. ELISA IgG were detected 90 days after the first vaccination in the pigs injected by Vaccine B or C. Neutralizing antibody were detected Post Challenge Days 61 (PCD) in all groups. In the pigs inoculated with Vaccine C, IFN-g were detected 90 days after first vaccination, and after challenge exposure they increased. In the other groups, the IFN-g were detected after challenge infection. Pigs injected with each of the vaccines A, B, C, D and E showed a significantly higher level of CD4(-)CD8⁺ lymphocytes (p < 0.001) after infection in comparison with their controls.

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.

Transforming growth factor-beta1 induces microvascular abnormalities through a down-modulation of neural cell adhesion molecule in human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a very angiogenic and malignant cancer. Conventional chemotherapy is poorly effective because of the abnormal structural organization of HCC-infiltrating vessels. In previous work, we demonstrated that HCC angiogenesis is driven by transforming growth factor beta-1(TGF-ß1)/CD105 axis, stimulating liver-derived microvascular endothelial cells (Ld-MECs) migration. As TGF-ß1 also affects mural cells (MCs) recruitment and maturation, we asked whether it may contribute to HCC-induced vascular abnormalities. HCC and adjacent non-neoplastic liver (nNL) biopsies obtained from 12 patients were analyzed by immunohistochemistry for angiogenic markers CD105, TGF-ß1, CD44 and vascular endothelial growth factor-a (VEGFa) and for MC markers NG2, α-smooth muscle actin (αSMA) and neural cell adhesion molecule (NCAM). The same markers were also investigated by immunocytochemistry on cultured HCC-derived stromal cells (HCC-StCs) and nNL-derived StCs (nNL-StCs) isolated from the same liver biopsies. Angiogenic factors released by StCs were analyzed by ELISA and the interaction between StCs and Ld-MECs by adhesion assay. Compared with nNL, HCC biopsies showed increased angiogenic markers and αSMA that was localized in vessels. By contrast, NG2 and NCAM were substantially localized in tumor cells but absent in vessels and stroma. Cultured HCC-StCs showed less expression of NG2, αSMA and NCAM. They also demonstrated a lower capacity to release angiogenic factors and adhered on Ld-MECs. HCC-StCs and nNL-StCs treated with TGF-ß1 or with of HepG2 (a human hepatoma cell line) derived conditioned medium (CM), down-modulated NCAM expression, whereas anti-NCAM antibodies significantly reduced the adhesion of StCs to Ld-MECs. By further blocking TGF-ß1 with anti-TGF-ß1 antibodies or with Ly-364947 (a specific inhibitor TGF-ß1-receptor) adhesion to Ld-MECs and NCAM expression respectively was partially restored. TGF-ß1 contributes to HCC-induced vascular alterations by affecting the interaction between HCC-StCs and Ld-MECs through a down-modulation of NCAM expression.

Evaluation of a robotic system for the recovery of peripheral blood mononuclear cells.

Investigations into immune responses are often based upon recovery of peripheral blood mononuclear cells (PBMC). To this purpose, the recovery of PBMC by gradient centrifugation is labour-intensive and requires a reasonable level of skill by the laboratory technician. Thus, we set out to determine whether laboratory automation equipment could be used for the recovery of PBMC from blood samples of horses, pigs and cattle, based on the Ficoll-Paque gradient centrifugation technique. Mixing of blood samples with PBS, layering of diluted blood onto Ficoll-Paque gradients, recovery of separated PBMC in RPMI 1640 medium were performed using an automated robotic system, the SBF200 (AM Robotic Systems, Warrington, UK) under laminar air flow conditions. Tubes were tagged with bar codes and manually placed after gradient centrifugation into a tube reader to measure the volume and position of the PBMC layer. The results of the automated procedure compared very well to those of the manual one in terms of percent cell recovery, sterility and cell viability. Also, a high throughput of samples could be implemented: with the integration of cell counting it should be possible for 96 blood samples to be processed, including the production of aliquots, by one person in a day.

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.

Mesenchymal stromal cells primed with paclitaxel provide a new approach for cancer therapy.

BACKGROUND: Mesenchymal stromal cells may represent an ideal candidate to deliver anti-cancer drugs. In a previous study, we demonstrated that exposure of mouse bone marrow derived stromal cells to Doxorubicin led them to acquire anti-proliferative potential towards co-cultured haematopoietic stem cells (HSCs). We thus hypothesized whether freshly isolated human bone marrow Mesenchymal stem cells (hMSCs) and mature murine stromal cells (SR4987 line) primed in vitro with anti-cancer drugs and then localized near cancer cells, could inhibit proliferation. METHODS AND PRINCIPAL FINDINGS: Paclitaxel (PTX) was used to prime culture of hMSCs and SR4987. Incorporation of PTX into hMSCs was studied by using FICT-labelled-PTX and analyzed by FACS and confocal microscopy. Release of PTX in culture medium by PTX primed hMSCs (hMSCsPTX) was investigated by HPLC. Culture of Endothelial cells (ECs) and aorta ring assay were used to test the anti-angiogenic activity of hMSCsPTX and PTX primed SR4987(SR4987PTX), while anti-tumor activity was tested in vitro on the proliferation of different tumor cell lines and in vivo by co-transplanting hMSCsPTX and SR4987PTX with cancer cells in mice. Nevertheless, despite a loss of cells due to chemo-induced apoptosis, both hMSCs and SR4987 were able to rapidly incorporate PTX and could slowly release PTX in the culture medium in a time dependent manner. PTX primed cells acquired a potent anti-tumor and anti-angiogenic activity in vitro that was dose dependent, and demonstrable by using their conditioned medium or by co-culture assay. Finally, hMSCsPTX and SR4987PTX co-injected with human cancer cells (DU145 and U87MG) and mouse melanoma cells (B16) in immunodeficient and in syngenic mice significantly delayed tumor takes and reduced tumor growth. CONCLUSIONS: These data demonstrate, for the first time, that without any genetic manipulation, mesenchymal stromal cells can uptake and subsequently slowly release PTX. This may lead to potential new tools to increase efficacy of cancer therapy.

Evaluation of a transcutaneous carbon dioxide monitor in patients with acute respiratory failure.

BACKGROUND: Non-invasive measurement of oxygenation is a routine procedure in clinical practice, but transcutaneous monitoring of PCO(2)(PtCO(2)) is used much less than expected. METHODS: The aim of our study was to analyze the value of a commercially available combined SpO(2)/PtCO(2) monitor (TOSCA-Linde Medical System, Basel, Switzerland) in adult non-invasive ventilated patients with acute respiratory failure. Eighty critically ill adult patients, requiring arterial blood sample gas analyses, underwent SpO(2) and PtCO(2) measurements (10 min after the probe was attached to an earlobe) simultaneously with arterial blood sampling. The level of agreement between PaCO(2) - PtCO(2) and SaO(2) - SpO(2)was assessed by Bland-Altman analyses. RESULTS: Both, SaO(2) from blood gas analysis and SpO(2) from the transcutaneous monitor, and PaCO(2) and PtCO(2) were equally useful. No measurements were outside of the acceptable clinical range of agreement of ± 7.5 mmHg. CONCLUSIONS: The accuracy of estimation of the TOSCA transcutaneous electrode (compared with the "gold standard" blood sample gas analysis) was generally good. Moreover, TOSCA presents the advantage of the possibility of continuous non-invasive measurement. The level of agreement of the two methods of measurement allows us to state that the TOSCA sensor is useful in routine monitoring of adults admitted to an intermediate respiratory unit and undergoing non-invasive ventilation.