Endogenous Extracellular Matrix Regulates the Response of Osteosarcoma 3D Spheroids to Doxorubicin.

The extracellular matrix (ECM) modulates cell behavior, shape, and viability as well as mechanical properties. In recent years, ECM disregulation and aberrant remodeling has gained considerable attention in cancer targeting and prevention since it may stimulate tumorigenesis and metastasis. Here, we developed an in vitro model that aims at mimicking the in vivo tumor microenvironment by recapitulating the interactions between osteosarcoma (OS) cells and ECM with respect to cancer progression. We long-term cultured 3D OS spheroids made of metastatic or non-metastatic OS cells mixed with mesenchymal stromal cells (MSCs); confirmed the deposition of ECM proteins such as Type I collagen, Type III collagen, and fibronectin by the stromal component at the interface between tumor cells and MSCs; and found that ECM secretion is inhibited by a neutralizing anti-IL-6 antibody, suggesting a new role of this cytokine in OS ECM deposition. Most importantly, we showed that the cytotoxic effect of doxorubicin is reduced by the presence of Type I collagen. We thus conclude that ECM protein deposition is crucial for modelling and studying drug response. Our results also suggest that targeting ECM proteins might improve the outcome of a subset of chemoresistant tumors.

Immunization With Bovine Herpesvirus-4-Based Vector Delivering PPRV-H Protein Protects Sheep From PPRV Challenge.

The Morbillivirus peste des petits ruminants virus (PPRV) is the causal agent of a highly contagious disease that mostly affects sheep and goats and produces considerable losses in developing countries. Current PPRV control strategies rely on live-attenuated vaccines, which are not ideal, as they cannot differentiate infected from vaccinated animals (DIVA). Recombinant vector-based vaccines expressing viral subunits can provide an alternative to conventional vaccines, as they can be easily paired with DIVA diagnostic tools. In the present work, we used the bovine herpesvirus-4-based vector (BoHV-4-A) to deliver PPRV hemagglutinin H antigen (BoHV-4-A-PPRV-H-ΔTK). Vaccination with BoHV-4-A-PPRV-H-ΔTK protected sheep from virulent PPRV challenge and prevented virus shedding. Protection correlated with anti-PPRV IgGs, neutralizing antibodies and IFN-γ-producing cells induced by the vaccine. Detection of antibodies exclusively against H-PPRV in animal sera and not against other PPRV viral proteins such as F or N could serve as a DIVA diagnostic test when using BoHV-4-A-PPRV-H-ΔTK as vaccine. Our data indicate that BoHV-4-A-PPRV-H-ΔTK could be a promising new approach for PPRV eradication programs.

Persistency of Mesenchymal Stromal/Stem Cells in Lungs.

Mesenchymal stromal/stem cells (MSCs) are a fibroblast-like cell population with high regenerative potential that can be isolated from many different tissues. Several data suggest MSCs as a therapeutic tool capable of migrating to a site of injury and guide tissue regeneration mainly through their secretome. Pulmonary first-pass effect occurs during intravenous administration of MSCs, where 50 to 80% of the cells tend to localize in the lungs. This phenomenon has been exploited to study MSC potential therapeutic effects in several preclinical models of lung diseases. Data demonstrated that, regardless of the lung disease severity and the delivery route, MSCs were not able to survive longer than 24 h in the respiratory tract but still surprisingly determined a therapeutic effect. In this work, two different mouse bone marrow-derived mesenchymal stromal/stem cell (mBM-MSC) lines, stably transduced with a third-generation lentiviral vector expressing luciferase and green fluorescent protein reporter genes tracking MSCs in vivo biodistribution and persistency, have been generated. Cells within the engrafted lung were in vivo traced using the high-throughput bioluminescence imaging (BLI) technique, with no invasiveness on animal, minimizing biological variations and costs. In vivo BLI analysis allowed the detection and monitoring of the mBM-MSC clones up to 28 days after implantation independently from the delivery route. This longer persistency than previously observed (24 h) could have a strong impact in terms of pharmacokinetics and pharmacodynamics of MSCs as a therapeutic tool.

A Simplified SARS-CoV-2 Pseudovirus Neutralization Assay.

COVID-19 is an ongoing pandemic caused by the highly infectious coronavirus SARS-CoV-2 that is engaging worldwide scientific research to find a timely and effective eradication strategy. Great efforts have been put into anti-COVID-19 vaccine generation in an effort to protect the world population and block SARS-CoV-2 spread. To validate the protective efficacy of the vaccination campaign and effectively control the pandemic, it is necessary to quantify the induction of neutralizing antibodies by vaccination, as they have been established to be a correlate of protection. In this work, a SARS-CoV-2 pseudovirus neutralization assay, based on a replication-incompetent lentivirus expressing an adapted form of CoV-2 S protein and an ACE2/TMPRSS2 stably expressing cell line, has been minimized in terms of protocol steps without loss of accuracy. The goal of the present simplified neutralization system is to improve SARS-CoV-2 vaccination campaign by means of an easy and accessible approach to be performed in any medical laboratory, maintaining the sensitivity and quantitative reliability of classical serum neutralization assays. Further, this assay can be easily adapted to different coronavirus variants by simply modifying the pseudotyping vector.

Conformable hierarchically engineered polymeric micromeshes enabling combinatorial therapies in brain tumours.

The poor transport of molecular and nanoscale agents through the blood-brain barrier together with tumour heterogeneity contribute to the dismal prognosis in patients with glioblastoma multiforme. Here, a biodegradable implant (µMESH) is engineered in the form of a micrometre-sized poly(lactic-co-glycolic acid) mesh laid over a water-soluble poly(vinyl alcohol) layer. Upon poly(vinyl alcohol) dissolution, the flexible poly(lactic-co-glycolic acid) mesh conforms to the resected tumour cavity as docetaxel-loaded nanomedicines and diclofenac molecules are continuously and directly released into the adjacent tumour bed. In orthotopic brain cancer models, generated with a conventional, reference cell line and patient-derived cells, a single µMESH application, carrying 0.75 mg kg-1 of docetaxel and diclofenac, abrogates disease recurrence up to eight months after tumour resection, with no appreciable adverse effects. Without tumour resection, the µMESH increases the median overall survival (∼30 d) as compared with the one-time intracranial deposition of docetaxel-loaded nanomedicines (15 d) or 10 cycles of systemically administered temozolomide (12 d). The µMESH modular structure, for the independent coloading of different molecules and nanomedicines, together with its mechanical flexibility, can be exploited to treat a variety of cancers, realizing patient-specific dosing and interventions.

Pathogenic LRRK2 requires secondary factors to induce cellular toxicity.

Pathogenic mutations in the leucine-rich repeat kinase 2 (LRRK2) gene belong to the most common genetic causes of inherited Parkinson's disease (PD) and variations in its locus increase the risk to develop sporadic PD. Extensive research efforts aimed at understanding how changes in the LRRK2 function result in molecular alterations that ultimately lead to PD. Cellular LRRK2-based models revealed several potential pathophysiological mechanisms including apoptotic cell death, LRRK2 protein accumulation and deficits in neurite outgrowth. However, highly variable outcomes between different cellular models have been reported. Here, we have investigated the effect of different experimental conditions, such as the use of different tags and gene transfer methods, in various cellular LRRK2 models. Readouts included cell death, sensitivity to oxidative stress, LRRK2 relocalization, α-synuclein aggregation and neurite outgrowth in cell culture, as well as neurite maintenance in vivo. We show that overexpression levels and/or the tag fused to LRRK2 affect the relocalization of LRRK2 to filamentous and skein-like structures. We found that overexpression of LRRK2 per se is not sufficient to induce cellular toxicity or to affect α-synuclein-induced toxicity and aggregate formation. Finally, neurite outgrowth/retraction experiments in cell lines and in vivo revealed that secondary, yet unknown, factors are required for the pathogenic LRRK2 effects on neurite length. Our findings stress the importance of technical and biological factors in LRRK2-induced cellular phenotypes and hence imply that conclusions based on these types of LRRK2-based assays should be interpreted with caution.

Immunotargeting of the xCT Cystine/Glutamate Antiporter Potentiates the Efficacy of HER2-Targeted Immunotherapies in Breast Cancer.

Despite HER2-targeted therapies improving the outcome of HER2+ breast cancer, many patients experience resistance and metastatic progression. Cancer stem cells (CSC) play a role in this resistance and progression, thus combining HER2 targeting with CSC inhibition could improve the management of HER2+ breast cancer. The cystine-glutamate antiporter, xCT, is overexpressed in mammary CSCs and is crucial for their redox balance, self-renewal, and resistance to therapies, representing a potential target for breast cancer immunotherapy. We developed a combined immunotherapy targeting HER2 and xCT using the Bovine Herpes virus-4 vector, a safe vaccine that can confer immunogenicity to tumor antigens. Mammary cancer-prone BALB-neuT mice, transgenic for rat Her2, were immunized with the single or combined vaccines. Anti-HER2 vaccination slowed primary tumor growth, whereas anti-xCT vaccination primarily prevented metastasis formation. The combination of the two vaccines exerted a complementary effect by mediating the induction of cytotoxic T cells and of HER2 and xCT antibodies that induce antibody-dependent cell-mediated cytotoxicity and hinder cancer cell proliferation. Antibodies targeting xCT, but not those targeting HER2, directly affected CSC viability, self-renewal, and migration, inducing the antimetastatic effect of xCT vaccination. Our findings present a new therapy for HER2+ breast cancer, demonstrating that CSC immunotargeting via anti-xCT vaccination synergizes with HER2-directed immunotherapy.

Bovine Herpesvirus-4-Vectored Delivery of Nipah Virus Glycoproteins Enhances T Cell Immunogenicity in Pigs.

Nipah virus (NiV) is an emergent pathogen capable of causing acute respiratory illness and fatal encephalitis in pigs and humans. A high fatality rate and broad host tropism makes NiV a serious public and animal health concern. There is therefore an urgent need for a NiV vaccines to protect animals and humans. In this study we investigated the immunogenicity of bovine herpesvirus (BoHV-4) vectors expressing either NiV attachment (G) or fusion (F) glycoproteins, BoHV-4-A-CMV-NiV-GΔTK or BoHV-4-A-CMV-NiV-FΔTK, respectively in pigs. The vaccines were benchmarked against a canarypox (ALVAC) vector expressing NiV G, previously demonstrated to induce protective immunity in pigs. Both BoHV-4 vectors induced robust antigen-specific antibody responses. BoHV-4-A-CMV-NiV-GΔTK stimulated NiV-neutralizing antibody titers comparable to ALVAC NiV G and greater than those induced by BoHV-4-A-CMV-NiV-FΔTK. In contrast, only BoHV-4-A-CMV-NiV-FΔTK immunized pigs had antibodies capable of significantly neutralizing NiV G and F-mediated cell fusion. All three vectored vaccines evoked antigen-specific CD4 and CD8 T cell responses, which were particularly strong in BoHV-4-A-CMV-NiV-GΔTK immunized pigs and to a lesser extent BoHV-4-A-CMV-NiV-FΔTK. These findings emphasize the potential of BoHV-4 vectors for inducing antibody and cell-mediated immunity in pigs and provide a solid basis for the further evaluation of these vectored NiV vaccine candidates.

Uniparental disomy and pretreatment IGF-1 may predict elevated IGF-1 levels in Prader-Willi patients on GH treatment.

Pediatric patients with Prader-Willi syndrome (PWS) can be treated with recombinant human GH (rhGH). These patients are highly sensitive to rhGH and the standard doses suggested by the international guidelines often result in IGF-1 above the normal range. We aimed to evaluate 1 the proper rhGH dose to optimize auxological outcomes and to avoid potential overtreatment, and 2 which patients are more sensitive to rhGH. In this multicenter real-life study, we recruited 215 patients with PWS older than 1 year, on rhGH at least for 6 months, from Italian Centers for PWS care. We collected auxological parameters, rhGH dose, IGF-1 at recruitment and (when available) at start of treatment. The rhGH dose was 4.3 (0.7/8.4) mg/m2/week. At recruitment, IGF-1 was normal in 72.1% and elevated in 27.9% of the patients. In the group of 115 patients with IGF-1 available at start of rhGH, normal pretreatment IGF-1 and uniparental disomy were associated with elevated IGF-1 during the therapy. No difference in height and growth velocity was found between patients treated with the highest and the lowest range dose. The rhGH dose prescribed in Italy seems lower than the recommended one. Normal pretreatment IGF-1 and uniparental disomy are risk factors for elevated IGF-1. The latter seems to be associated with higher sensitivity to GH. In case of these risk factors, we recommend a more accurate titration of the dose to avoid overtreatment and its potential side effects.

Capacity to Elicit Cytotoxic CD8 T Cell Activity Against Mycobacterium avium subsp. paratuberculosis Is Retained in a Vaccine Candidate 35 kDa Peptide Modified for Expression in Mammalian Cells.

Studies focused on development of an attenuated vaccine against Mycobacterium avium subsp. paratuberculosis (Map), the causative agent of paratuberculosis (Ptb) in cattle and other species, revealed that deletion of relA, a global gene regulator, abrogates the ability of Map to establish a persistent infection. In the absence of relA, cattle develop CD8 cytotoxic T cells (CTL) with the ability to kill intracellular bacteria. Analysis of the recall response to a relA mutant, Map/ΔrelA, with cells from a vaccinated steer demonstrated that a 35-kDa membrane peptide (MMP) is one of the targets of the response. This observation suggested that it might be possible to develop a peptide-based vaccine. As reported here, the gene encoding the hypothetical MMP ORF, MAP2121c, was modified for expression in mammalian cells as a first step in developing an expression cassette for incorporation into a mammalian expression vector. The modified sequence of MMP, tPA-MMP, was mutated to generate two additional sequences for the study, one with substitutions to replace five potential residues that could be glycosylated, tPA-MMP-5mut, and one with substitutions to replace the first two potential residues that could be glycosylated, tPA-MMP-2mut. The sequences were placed in an expression cassette to produce peptides for analysis. An ex vivo platform was used with flow cytometry and a bacterium viability assay to determine if modifications in the gene encoding MMP for expression in mammalian cells altered its capacity to elicit development of CD8 CTL, essential for its use in a peptide-based vaccine. Monocyte-depleted PBMC (mdPBMC) were stimulated with antigen-presenting cells (APC) pulsed with different MMP constructs. CD4 and CD8 T cells proliferated in response to stimulation with MMP (control) expressed in Escherichia coli (eMMP), tPA-MMP, and tPA-MMP-2mut. CD8 T cells retained the capacity to kill intracellular bacteria. The tPA-MMP-5mut failed to elicit a proliferative response and was not included in further studies. The data show that the expression cassettes containing MMP and MMP-2mut can be used to screen and select a mammalian expression vector for the development of an efficacious peptide-based vaccine against Ptb.

Bovine herpesvirus 4-based vector delivering the full length xCT DNA efficiently protects mice from mammary cancer metastases by targeting cancer stem cells.

Despite marked advancements in its treatment, breast cancer is still the second leading cause of cancer death in women, due to relapses and distal metastases. Breast cancer stem cells (CSCs), are a cellular reservoir for recurrence, metastatic evolution and disease progression, making the development of novel therapeutics that target CSCs, and thereby inhibit metastases, an urgent need. We have previously demonstrated that the cystine-glutamate antiporter xCT (SLC7A11), a protein that was shown to be overexpressed in mammary CSCs and that plays a key role in the maintenance of their redox balance, self-renewal and resistance to chemotherapy, is a potential target for mammary cancer immunotherapy. This paper reports on the development of an anti-xCT viral vaccine that is based on the bovine herpesvirus 4 (BoHV-4) vector, which we have previously showed to be a safe vaccine that can transduce cells in vivo and confer immunogenicity to tumor antigens. We show that the vaccination of BALB/c mice with BoHV-4 expressing xCT (BoHV-4-mxCT), impaired lung metastases induced by syngeneic mammary CSCs both in preventive and therapeutic settings. Vaccination induced T lymphocyte activation and the production of anti-xCT antibodies that can mediate antibody-dependent cell cytotoxicity (ADCC), and directly impair CSC phenotype, self-renewal and redox balance. Our findings pave the way for the potential future use of BoHV-4-based vector targeting xCT in metastatic breast cancer treatment.

Bovine Herpesvirus-4-Based Vector Delivering Peste des Petits Ruminants Virus Hemagglutinin ORF Induces both Neutralizing Antibodies and Cytotoxic T Cell Responses.

Peste des Petits Ruminants Virus (PPRV) is an extremely infective morbillivirus that primarily affects goats and sheep. In underdeveloped countries where livestock are the main economical resource, PPRV causes considerable economic losses. Protective live attenuated vaccines are currently available but they induce antibody responses similar to those produced in PPRV naturally infected animals. Effective vaccines able to distinguish between vaccinated and naturally infected animals are required to PPRV control and eradication programs. Hemagglutinin (H) is a highly immunogenic PPRV envelope glycoprotein displaying both hemagglutinin and neuraminidase activities, playing a crucial role in virus attachment and penetration. In this study, a recombinant Bovine Herpesvirus-4 (BoHV-4)-based vector delivering an optimized PPRV-Hemagglutinin expression cassette, BoHV-4-A-PPRV-H-ΔTK, was assessed in immunocompetent C57BL/6 mice. BoHV-4-A-PPRV-H-ΔTK-immunization elicited both cellular and humoral immune responses with specific T cell, cytotoxic T lymphocyte, and sero-neutralizing antibody against PPRV. These data suggest recombinant BoHV-4-A-PPRV-H-ΔTK as an effective vaccine candidate to protect against PPRV herd infection and potentially applicable for eradication programs.

Pharmacokinetic and Pharmacodynamic Characteristics of Dasiglucagon, a Novel Soluble and Stable Glucagon Analog.

OBJECTIVE: Treatment of severe hypoglycemia outside of the hospital setting is limited to glucagon formulations requiring reconstitution before use, which may lead to erroneous or delayed glucagon administration. We compared the pharmacokinetic (PK) and pharmacodynamic (PD) characteristics and safety and tolerability of different doses of dasiglucagon, a novel soluble glucagon analog, with approved pediatric and full doses of GlucaGen in insulin-induced hypoglycemia in patients with type 1 diabetes. RESEARCH DESIGN AND METHODS: In this single-center, randomized, double-blind trial, 58 patients with type 1 diabetes received single subcutaneous injections of 0.1, 0.3, 0.6, or 1.0 mg dasiglucagon or 0.5 or 1.0 mg GlucaGen in a state of hypoglycemia (blood glucose target 55 mg/dL) induced by an intravenous insulin infusion. RESULTS: Dasiglucagon demonstrated a dose-dependent and rapid increase in plasma concentrations, reaching a maximum at ∼35 min with a half-life of ∼0.5 h. Dasiglucagon rapidly increased plasma glucose (PG) by ≥20 mg/dL (9-14 min) to PG ≥70 mg/dL (within 6-10 min), similar to GlucaGen, but with a longer-lasting and greater effect on PG. All patients on both treatments reached these end points within 30 min (predefined success criteria). Both treatments were well tolerated. Nausea was the most frequent adverse event, occurring at a similar rate (44-56%). CONCLUSIONS: Dasiglucagon was well tolerated and showed an early PD response similar to that of GlucaGen at corresponding doses, suggesting comparable clinical effects of the two glucagon formulations. Dasiglucagon has the potential to become an effective and reliable rescue treatment for severe hypoglycemia in a ready-to-use pen.

Induction of Antihuman C-C Chemokine Receptor Type 5 Antibodies by a Bovine Herpesvirus Type-4 Based Vector.

Bovine herpesvirus 4 (BoHV-4) is a promising vector for the delivery and intracellular expression of recombinant antigens and can thus be considered as a new prototype vaccine formulation system. An interesting, and actively pursued, antigen in the context of human immunodeficiency virus (HIV) infection prophylaxis (and therapy) is the C-C chemokine receptor type 5 (CCR5) co-receptor, whose blockage by specific antibodies has been shown to inhibit both viral entry and cell-to-cell transmission of the virus. Building on our previous work on the BoHV-4 vector system, we have engineered and tested a replication-competent derivative of BoHV-4 (BoHV-4-CMV-hCCR5ΔTK) bearing a human CCR5 (hCCR5) expression cassette. We show here that CCR5 is indeed expressed at high levels in multiple types of BoHV-4-CMV-hCCR5ΔTK-infected cells. More importantly, two intravenous inoculations of CCR5-expressing BoHV-4 virions into rabbits led to the production of anti-CCR5 antibodies capable of reacting with the CCR5 receptor exposed on the surface of HEK293T cells through specific recognition of the amino-terminal region (aa 14-34) of the protein. Given the growing interest for anti-CCR5 immunization as an HIV control strategy and the many advantages of virus-based immunogen formulations (especially for poorly immunogenic or self-antigens), the results reported in this study provide preliminary validation of BoHV-4 as a safe viral vector suitable for CCR5 vaccination.

Assessment and optimization of Theileria parva sporozoite full-length p67 antigen expression in mammalian cells.

Delivery of various forms of recombinant Theileria parva sporozoite antigen (p67) has been shown to elicit antibody responses in cattle capable of providing protection against East Coast fever, the clinical disease caused by T. parva. Previous formulations of full-length and shorter recombinant versions of p67 derived from bacteria, insect, and mammalian cell systems are expressed in non-native and highly unstable forms. The stable expression of full-length recombinant p67 in mammalian cells has never been described and has remained especially elusive. In this study, p67 was expressed in human-derived cells as a full-length, membrane-linked protein and as a secreted form by omission of the putative transmembrane domain. The recombinant protein expressed in this system yielded primarily two products based on Western immunoblot analysis, including one at the expected size of 67 kDa, and one with a higher than expected molecular weight. Through treatment with PNGase F, our data indicate that the larger product of this mammalian cell-expressed recombinant p67 cannot be attributed to glycosylation. By increasing the denaturing conditions, we determined that the larger sized mammalian cell-expressed recombinant p67 product is likely a dimeric aggregate of the protein. Both forms of this recombinant p67 reacted with a monoclonal antibody to the p67 molecule, which reacts with the native sporozoite. Additionally, through this work we developed multiple mammalian cell lines, including both human and bovine-derived cell lines, transduced by a lentiviral vector, that are constitutively able to express a stable, secreted form of p67 for use in immunization, diagnostics, or in vitro assays. The recombinant p67 developed in this system is immunogenic in goats and cattle based on ELISA and flow cytometric analysis. The development of a mammalian cell system that expresses full-length p67 in a stable form as described here is expected to optimize p67-based immunization.

Heterologous Matrix Metalloproteinase Gene Promoter Activity Allows In Vivo Real-time Imaging of Bleomycin-Induced Lung Fibrosis in Transiently Transgenized Mice.

Idiopathic pulmonary fibrosis is a very common interstitial lung disease derived from chronic inflammatory insults, characterized by massive scar tissue deposition that causes the progressive loss of lung function and subsequent death for respiratory failure. Bleomycin is used as the standard agent to induce experimental pulmonary fibrosis in animal models for the study of its pathogenesis. However, to visualize the establishment of lung fibrosis after treatment, the animal sacrifice is necessary. Thus, the aim of this study was to avoid this limitation by using an innovative approach based on a double bleomycin treatment protocol, along with the in vivo images analysis of bleomycin-treated mice. A reporter gene construct, containing the luciferase open reading frame under the matrix metalloproteinase-1 promoter control region, was tested on double bleomycin-treated mice to investigate, in real time, the correlation between bleomycin treatment, inflammation, tissue remodeling and fibrosis. Bioluminescence emitted by the lungs of bleomycin-treated mice, corroborated by fluorescent molecular tomography, successfully allowed real time monitoring of fibrosis establishment. The reporter gene technology experienced in this work could represent an advanced functional approach for real time non-invasive assessment of disease evolution during therapy, in a reliable and translational living animal model.

Immunophenotypical characterization of canine mesenchymal stem cells from perivisceral and subcutaneous adipose tissue by a species-specific panel of antibodies.

Immunophenotypical characterization of mesenchymal stem cells is fundamental for the design and execution of sound experimental and clinical studies. The scarce availability of species-specific antibodies for canine antigens has hampered the immunophenotypical characterization of canine mesenchymal stem cells (MSC). The aim of this study was to select a panel of species-specific direct antibodies readily useful for canine mesenchymal stem cells characterization. They were isolated from perivisceral and subcutaneous adipose tissue samples collected during regular surgeries from 8 dogs. Single color flow cytometric analysis of mesenchymal stem cells (P3) deriving from subcutaneous and perivisceral adipose tissue with a panel of 7 direct anti-canine antibodies revealed two largely homogenous cell populations with a similar pattern: CD29+, CD44+, CD73+, CD90+, CD34-, CD45- and MHC-II- with no statistically significant differences among them. Antibody reactivity was demonstrated on canine peripheral blood mononuclear cells. The similarities are reinforced by their in vitro cell morphology, trilineage differentiation ability and RT-PCR analysis (CD90+, CD73+, CD105+, CD44+, CD13+, CD29+, Oct-4+ gene and CD31- and CD45- expression). Our results report for the first time a comparison between the immunophenotypic profile of canine MSC deriving from perivisceral and subcutaneous adipose tissue. The substantial equivalence between the two populations has practical implication on clinical applications, giving the opportunity to choose the source depending on the patient needs. The results contribute to routine characterization of MSC populations grown in vitro, a mandatory process for the definition of solid and reproducible laboratory and therapeutic procedures.

BoHV-4-based vector delivering Ebola virus surface glycoprotein.

BACKGROUND: Ebola virus (EBOV) is a Category A pathogen that is a member of Filoviridae family that causes hemorrhagic fever in humans and non-human primates. Unpredictable and devastating outbreaks of disease have recently occurred in Africa and current immunoprophylaxis and therapies are limited. The main limitation of working with pathogens like EBOV is the need for costly containment. To potentiate further and wider opportunity for EBOV prophylactics and therapies development, innovative approaches are necessary. METHODS: In the present study, an antigen delivery platform based on a recombinant bovine herpesvirus 4 (BoHV-4), delivering a synthetic EBOV glycoprotein (GP) gene sequence, BoHV-4-syEBOVgD106ΔTK, was generated. RESULTS: EBOV GP was abundantly expressed by BoHV-4-syEBOVgD106ΔTK transduced cells without decreasing viral replication. BoHV-4-syEBOVgD106ΔTK immunized goats produced high titers of anti-EBOV GP antibodies and conferred a long lasting (up to 6 months), detectable antibody response. Furthermore, no evidence of BoHV-4-syEBOVgD106ΔTK viremia and secondary localization was detected in any of the immunized animals. CONCLUSIONS: The BoHV-4-based vector approach described here, represents: an alternative antigen delivery system for vaccination and a proof of principle study for anti-EBOV antibodies generation in goats for potential immunotherapy applications.

High-content analysis of α-synuclein aggregation and cell death in a cellular model of Parkinson's disease.

BACKGROUND: Alpha-synuclein (α-SYN) aggregates represent a key feature of Parkinson's disease, but the exact relationship between α-SYN aggregation and neurodegeneration remains incompletely understood. Therefore, the availability of a cellular assay that allows medium-throughput analysis of α-SYN-linked pathology will be of great value for studying the aggregation process and for advancing α-SYN-based therapies. NEW METHOD: Here we describe a high-content neuronal cell assay that simultaneously measures oxidative stress-induced α-SYN aggregation and apoptosis. RESULTS: We optimized an automated and reproducible assay to quantify both α-SYN aggregation and cell death in human SH-SY5Y neuroblastoma cells. COMPARISON WITH EXISTING METHODS: Quantification of α-SYN aggregates in cells has typically relied on manual imaging and counting or cell-free assays, which are time consuming and do not allow a concurrent analysis of cell viability. Our high-content analysis method for quantification of α-SYN aggregation allows simultaneous measurements of multiple cell parameters at a single-cell level in a fast, objective and automated manner. CONCLUSIONS: The presented analysis approach offers a rapid, objective and multiparametric approach for the screening of compounds and genes that might alter α-SYN aggregation and/or toxicity.

FK506 reduces neuroinflammation and dopaminergic neurodegeneration in an α-synuclein-based rat model for Parkinson's disease.

Alpha-synuclein (α-synuclein) is considered a key player in Parkinson's disease (PD), but the exact relationship between α-synuclein aggregation and dopaminergic neurodegeneration remains unresolved. There is increasing evidence that neuroinflammatory processes are closely linked to dopaminergic cell death, but whether the inflammatory process is causally involved in PD or rather reflects secondary consequences of nigrostriatal pathway injury is still under debate. We evaluated the therapeutic effect of the immunophilin ligand FK506 in a rAAV2/7 α-synuclein overexpression rat model. Treatment with FK506 significantly increased the survival of dopaminergic neurons in a dose-dependent manner. No reduction in α-synuclein aggregation was apparent in this time window, but FK506 significantly lowered the infiltration of both T helper and cytotoxic T cells and the number and subtype of microglia and macrophages. These data suggest that the anti-inflammatory properties of FK506 decrease neurodegeneration in this α-synuclein-based PD model, pointing to a causal role of neuroinflammation in the pathogenesis of PD.