Indocyanine-enhanced mouse model of bleomycin-induced lung fibrosis with hallmarks of progressive emphysema.

The development of new drugs for idiopathic pulmonary fibrosis strongly relies on preclinical experimentation, which requires the continuous improvement of animal models and integration with in vivo imaging data. Here, we investigated the lung distribution of bleomycin (BLM) associated with the indocyanine green (ICG) dye by fluorescence imaging. A long-lasting lung retention (up to 21 days) was observed upon oropharyngeal aspiration (OA) of either ICG or BLM + ICG, with significantly more severe pulmonary fibrosis, accompanied by the progressive appearance of emphysema-like features, uniquely associated with the latter combination. More severe and persistent lung fibrosis, together with a progressive air space enlargement uniquely associated with the BLM + ICG group, was confirmed by longitudinal micro-computed tomography (CT) and histological analyses. Multiple inflammation and fibrosis biomarkers were found to be increased in the bronchoalveolar lavage fluid of BLM- and BLM + ICG-treated animals, but with a clear trend toward a much stronger increase in the latter group. Similarly, in vitro assays performed on macrophage and epithelial cell lines revealed a significantly more marked cytotoxicity in the case of BLM + ICG-treated mice. Also unique to this group was the synergistic upregulation of apoptotic markers both in lung sections and cell lines. Although the exact mechanism underlying the more intense lung fibrosis phenotype with emphysema-like features induced by BLM + ICG remains to be elucidated, we believe that this combination treatment, whose overall effects more closely resemble the human disease, represents a valuable alternative model for studying fibrosis development and for the identification of new antifibrotic compounds.

Chemico-Physical Properties of Some 1,1'-Bis-alkyl-2,2'-hexane-1,6-diyl-bispyridinium Chlorides Hydrogenated and Partially Fluorinated for Gene Delivery.

The development of very efficient and safe non-viral vectors, constituted mainly by cationic lipids bearing multiple charges, is a landmark for in vivo gene-based medicine. To understand the effect of the hydrophobic chain's length, we here report the synthesis, and the chemico-physical and biological characterization, of a new term of the homologous series of hydrogenated gemini bispyridinium surfactants, the 1,1'-bis-dodecyl-2,2'-hexane-1,6-diyl-bispyridinium chloride (GP12_6). Moreover, we have collected and compared the thermodynamic micellization parameters (cmc, changes in enthalpy, free energy, and entropy of micellization) obtained by isothermal titration calorimetry (ITC) experiments for hydrogenated surfactants GP12_6 and GP16_6, and for the partially fluorinated ones, FGPn (where n is the spacer length). The data obtained for GP12_6 by EMSA, MTT, transient transfection assays, and AFM imaging show that in this class of compounds, the gene delivery ability strictly depends on the spacer length but barely on the hydrophobic tail length. CD spectra have been shown to be a useful tool to verify the formation of lipoplexes due to the presence of a "tail" in the 288-320 nm region attributed to a chiroptical feature named ψ-phase. Ellipsometric measurements suggest that FGP6 and FGP8 (showing a very interesting gene delivery activity, when formulated with DOPE) act in a very similar way, and dissimilar from FGP4, exactly as in the case of transfection, and confirm the hypothesis suggested by previously obtained thermodynamic data about the requirement of a proper length of the spacer to allow the molecule to form a sort of molecular tong able to intercalate DNA.

Gene-Delivery Ability of New Hydrogenated and Partially Fluorinated Gemini bispyridinium Surfactants with Six Methylene Spacers.

The pandemic emergency determined by the spreading worldwide of the SARS-CoV-2 virus has focused the scientific and economic efforts of the pharmaceutical industry and governments on the possibility to fight the virus by genetic immunization. The genetic material must be delivered inside the cells by means of vectors. Due to the risk of adverse or immunogenic reaction or replication connected with the more efficient viral vectors, non-viral vectors are in many cases considered as a preferred strategy for gene delivery into eukaryotic cells. This paper is devoted to the evaluation of the gene delivery ability of new synthesized gemini bis-pyridinium surfactants with six methylene spacers, both hydrogenated and fluorinated, in comparison with compounds with spacers of different lengths, previously studied. Results from MTT proliferation assay, electrophoresis mobility shift assay (EMSA), transient transfection assay tests and atomic force microscopy (AFM) imaging confirm that pyridinium gemini surfactants could be a valuable tool for gene delivery purposes, but their performance is highly dependent on the spacer length and strictly related to their structure in solution. All the fluorinated compounds are unable to transfect RD-4 cells, if used alone, but they are all able to deliver a plasmid carrying an enhanced green fluorescent protein (EGFP) expression cassette, when co-formulated with 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE) in a 1:2 ratio. The fluorinated compounds with spacers formed by six (FGP6) and eight carbon atoms (FGP8) give rise to a very interesting gene delivery activity, greater to that of the commercial reagent, when formulated with DOPE. The hydrogenated compound GP16_6 is unable to sufficiently compact the DNA, as shown by AFM images.

Oncolytic Virotherapy and Microenvironment in Multiple Myeloma.

Multiple myeloma (MM) is a hematologic malignancy characterized by the accumulation of bone marrow (BM) clonal plasma cells, which are strictly dependent on the microenvironment. Despite the improvement of MM survival with the use of new drugs, MM patients still relapse and become always refractory to the treatment. The development of new therapeutic strategies targeting both tumor and microenvironment cells are necessary. Oncolytic virotherapy represent a promising approach in cancer treatment due to tumor-specific oncolysis and activation of the immune system. Different types of human viruses were checked in preclinical MM models, and the use of several viruses are currently investigated in clinical trials in MM patients. More recently, the use of alternative non-human viruses has been also highlighted in preclinical studies. This strategy could avoid the antiviral immune response of the patients against human viruses due to vaccination or natural infections, which could invalid the efficiency of virotherapy approach. In this review, we explored the effects of the main oncolytic viruses, which act through both direct and indirect mechanisms targeting myeloma and microenvironment cells inducing an anti-MM response. The efficacy of the oncolytic virus-therapy in combination with other anti-MM drugs targeting the microenvironment has been also discussed.

Fighting breast cancer stem cells through the immune-targeting of the xCT cystine-glutamate antiporter.

Tumor relapse and metastatic spreading act as major hindrances to achieve complete cure of breast cancer. Evidence suggests that cancer stem cells (CSC) would function as a reservoir for the local and distant recurrence of the disease, due to their resistance to radio- and chemotherapy and their ability to regenerate the tumor. Therefore, the identification of appropriate molecular targets expressed by CSC may be critical in the development of more effective therapies. Our studies focused on the identification of mammary CSC antigens and on the development of CSC-targeting vaccines. We compared the transcriptional profile of CSC-enriched tumorspheres from an Her2+ breast cancer cell line with that of the more differentiated parental cells. Among the molecules strongly upregulated in tumorspheres we selected the transmembrane amino-acid antiporter xCT. In this review, we summarize the results we obtained with different xCT-targeting vaccines. We show that, despite xCT being a self-antigen, vaccination was able to induce a humoral immune response that delayed primary tumor growth and strongly impaired pulmonary metastasis formation in mice challenged with tumorsphere-derived cells. Moreover, immunotargeting of xCT was able to increase CSC chemosensitivity to doxorubicin, suggesting that it may act as an adjuvant to chemotherapy. In conclusion, our approach based on the comparison of the transcriptome of tumorspheres and parental cells allowed us to identify a novel CSC-related target and to develop preclinical therapeutic approaches able to impact on CSC biology, and therefore, hampering tumor growth and dissemination.

Dependence on glutamine uptake and glutamine addiction characterize myeloma cells: a new attractive target.

The importance of glutamine (Gln) metabolism in multiple myeloma (MM) cells and its potential role as a therapeutic target are still unknown, although it has been reported that human myeloma cell lines (HMCLs) are highly sensitive to Gln depletion. In this study, we found that both HMCLs and primary bone marrow (BM) CD138(+) cells produced large amounts of ammonium in the presence of Gln. MM patients have lower BM plasma Gln with higher ammonium and glutamate than patients with indolent monoclonal gammopathies. Interestingly, HMCLs expressed glutaminase (GLS1) and were sensitive to its inhibition, whereas they exhibited negligible expression of glutamine synthetase (GS). High GLS1 and low GS expression were also observed in primary CD138(+) cells. Gln-free incubation or treatment with the glutaminolytic enzyme l-asparaginase depleted the cell contents of Gln, glutamate, and the anaplerotic substrate 2-oxoglutarate, inhibiting MM cell growth. Consistent with the dependence of MM cells on extracellular Gln, a gene expression profile analysis, on both proprietary and published datasets, showed an increased expression of the Gln transporters SNAT1, ASCT2, and LAT1 by CD138(+) cells across the progression of monoclonal gammopathies. Among these transporters, only ASCT2 inhibition in HMCLs caused a marked decrease in Gln uptake and a significant fall in cell growth. Consistently, stable ASCT2 downregulation by a lentiviral approach inhibited HMCL growth in vitro and in a murine model. In conclusion, MM cells strictly depend on extracellular Gln and show features of Gln addiction. Therefore, the inhibition of Gln uptake is a new attractive therapeutic strategy for MM.

IL21R expressing CD14+CD16+ monocytes expand in multiple myeloma patients leading to increased osteoclasts.

Bone marrow monocytes are primarily committed to osteoclast formation. It is, however, unknown whether potential primary alterations are specifically present in bone marrow monocytes from patients with multiple myeloma, smoldering myeloma or monoclonal gammopathy of undetermined significance. We analyzed the immunophenotypic and transcriptional profiles of bone marrow CD14+ monocytes in a cohort of patients with different types of monoclonal gammopathies to identify alterations involved in myeloma-enhanced osteoclastogenesis. The number of bone marrow CD14+CD16+ cells was higher in patients with active myeloma than in those with smoldering myeloma or monoclonal gammopathy of undetermined significance. Interestingly, sorted bone marrow CD14+CD16+ cells from myeloma patients were more pro-osteoclastogenic than CD14+CD16-cells in cultures ex vivo Moreover, transcriptional analysis demonstrated that bone marrow CD14+ cells from patients with multiple myeloma (but neither monoclonal gammopathy of undetermined significance nor smoldering myeloma) significantly upregulated genes involved in osteoclast formation, including IL21RIL21R mRNA over-expression by bone marrow CD14+ cells was independent of the presence of interleukin-21. Consistently, interleukin-21 production by T cells as well as levels of interleukin-21 in the bone marrow were not significantly different among monoclonal gammopathies. Thereafter, we showed that IL21R over-expression in CD14+ cells increased osteoclast formation. Consistently, interleukin-21 receptor signaling inhibition by Janex 1 suppressed osteoclast differentiation from bone marrow CD14+ cells of myeloma patients. Our results indicate that bone marrow monocytes from multiple myeloma patients show distinct features compared to those from patients with indolent monoclonal gammopathies, supporting the role of IL21R over-expression by bone marrow CD14+ cells in enhanced osteoclast formation.

Virus-Mediated Metalloproteinase 1 Induction Revealed by Transcriptome Profiling of Bovine Herpesvirus 4-Infected Bovine Endometrial Stromal Cells.

Viral infections can cause genital tract disorders (including abortion) in cows, and bovine herpesvirus 4 (BoHV-4) is often present in endometritis-affected animals. A major problem with cattle uterine viral infections in general, and BoHV-4 in particular, is our limited understanding of the pathogenic role(s) that these infections play in the endometrium. A similar lack of knowledge holds for the molecular mechanisms utilized, and the host cell pathways affected, by BoHV-4. To begin to fill these gaps, we set up optimized conditions for BoHV-4 infection of a pure population of bovine endometrial stromal cells (BESCs) to be used as source material for RNA sequencing-based transcriptome profiling. Many genes were found to be upregulated (417) or downregulated (181) after BoHV-4 infection. As revealed by enrichment functional analysis on differentially expressed genes, BoHV-4 infection affects various pathways related to cell proliferation and cell surface integrity, at least three of which were centered on upregulation of matrix metalloproteinase 1 (MMP1) and interleukin 8 (IL8). This was confirmed by reverse transcription PCR, real-time PCR, Western-immunoblot analysis, and a luciferase assay with a bovine MMP1-specific promoter reporter construct. Further, it was found that MMP1 transcription was upregulated by the BoHV-4 transactivator IE2/RTA, leading to abnormally high metalloproteinase tissue levels, potentially leading to defective endometrium healing and unresolved inflammation. Based on these findings, we propose a new model for BoHV-4 action centered on IE2-mediated MMP1 upregulation and novel therapeutic interventions based on IFN gamma-mediated MMP1 downregulation.

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.

In vivo monitoring of lung inflammation in CFTR-deficient mice.

BACKGROUND: Experimentally, lung inflammation in laboratory animals is usually detected by the presence of inflammatory markers, such as immune cells and cytokines, in the bronchoalveolar lavage fluid (BALF) of sacrificed animals. This method, although extensively used, is time, money and animal life consuming, especially when applied to genetically modified animals. Thus a new and more convenient approach, based on in vivo imaging analysis, has been set up to evaluate the inflammatory response in the lung of CFTR-deficient (CF) mice, a murine model of cystic fibrosis. METHODS: Wild type (WT) and CF mice were stimulated with P. aeruginosa LPS, TNF-alpha and culture supernatant derived from P. aeruginosa (strain VR1). Lung inflammation was detected by measuring bioluminescence in vivo in mice transiently transgenized with a luciferase reporter gene under the control of a bovine IL-8 gene promoter. RESULTS: Differences in bioluminescence (BLI) signal were revealed by comparing the two types of mice after intratracheal challenge with pro-inflammatory stimuli. BLI increased at 4 h after stimulation with TNF-alpha and at 24 h after administration of LPS and VR1 supernatant in CF mice with respect to untreated animals. The BLI signal was significantly more intense and lasted for longer times in CF animals when compared to WT mice. Analysis of BALF markers: leukocytes, cytokines and histology revealed no significant differences between CF and WT mice. CONCLUSIONS: In vivo gene delivery technology and non-invasive bioluminescent imaging has been successfully adapted to CFTR-deficient mice. Activation of bIL-8 transgene promoter can be monitored by non-invasive BLI imaging in the lung of the same animal and compared longitudinally in both CF or WT mice, after challenge with pro-inflammatory stimuli. The combination of these technologies and the use of CF mice offer the unique opportunity of evaluating the impact of therapies aimed to control inflammation in a CF background.

In vivo imaging of the lung inflammatory response to Pseudomonas aeruginosa and its modulation by azithromycin.

BACKGROUND: Chronic inflammation of the airways is a central component in lung diseases and is frequently associated with bacterial infections. Monitoring the pro-inflammatory capability of bacterial virulence factors in vivo is challenging and usually requires invasive methods. METHODS: Lung inflammation was induced using the culture supernatants from two Pseudomonas aeruginosa clinical strains, VR1 and VR2, isolated from patients affected by cystic fibrosis and showing different phenotypes in terms of motility, colony characteristics and biofilm production as well as pyoverdine and pyocyanine release. More interesting, the strains differ also for the presence in supernatants of metalloproteases, a family of virulence factors with known pro-inflammatory activity. We have evaluated the benefit of using a mouse model, transiently expressing the luciferase reporter gene under the control of an heterologous IL-8 bovine promoter, to detect and monitoring lung inflammation. RESULTS: In vivo imaging indicated that VR1 strain, releasing in its culture supernatant metalloproteases and other virulence factors, induced lung inflammation while the VR2 strain presented with a severely reduced pro-inflammatory activity. The bioluminescence signal was detectable from 4 to 48 h after supernatant instillation. The animal model was also used to test the anti-inflammatory activity of azithromycin (AZM), an antibiotic with demonstrated inhibitory effect on the synthesis of bacterial exoproducts. The inflammation signal in mice was in fact significantly reduced when bacteria grew in the presence of a sub-lethal dose of AZM causing inhibition of the synthesis of metalloproteases and other bacterial elements. The in vivo data were further supported by quantification of immune cells and cytokine expression in mouse broncho-alveolar lavage samples. CONCLUSIONS: This experimental animal model is based on the transient transduction of the bovine IL-8 promoter, a gene representing a major player during inflammation, essential for leukocytes recruitment to the inflamed tissue. It appears to be an appropriate molecular read-out for monitoring the activation of inflammatory pathways caused by bacterial virulence factors. The data presented indicate that the model is suitable to functionally monitor in real time the lung inflammatory response facilitating the identification of bacterial factors with pro-inflammatory activity and the evaluation of the anti-inflammatory activity of old and new molecules for therapeutic use.

Aurora and IKK kinases cooperatively interact to protect multiple myeloma cells from Apo2L/TRAIL.

Constitutive activation of the canonical and noncanonical nuclear factor-κB (NF-κB) pathways is frequent in multiple myeloma (MM) and can compromise sensitivity to TRAIL. In this study, we demonstrate that Aurora kinases physically and functionally interact with the key regulators of canonical and noncanonical NF-κB pathways IκB kinase ß (IKKß) and IKKα to activate NF-κB in MM, and the pharmacological blockade of Aurora kinase activity induces TRAIL sensitization in MM because it abrogates TRAIL-induced activation of NF-κB. We specifically found that TRAIL induces prosurvival signaling by increasing the phosphorylation state of both Aurora and IKK kinases and their physical interactions, and the blockade of Aurora kinase activity by pan-Aurora kinase inhibitors (pan-AKIs) disrupts TRAIL-induced survival signaling by effectively reducing Aurora-IKK kinase interactions and NF-κB activation. Pan-AKIs consistently blocked TRAIL induction of the antiapoptotic NF-κB target genes A1/Bfl-1 and/or Mcl-1, both important targets for TRAIL sensitization in MM cells. In summary, these results identify a novel interaction between Aurora and IKK kinases and show that these pathways can cooperate to promote TRAIL resistance. Finally, combining pan-AKIs with TRAIL in vivo showed dramatic efficacy in a multidrug-resistant human myeloma xenograft model. These findings suggest that combining Aurora kinase inhibitors with TRAIL may have therapeutic benefit in MM.

Cyclodextrin- and calixarene-based polycationic amphiphiles as gene delivery systems: a structure-activity relationship study.

Multi-head/multi-tail facial amphiphiles built on cyclodextrin (CD) and calixarene (CA) scaffolds are paradigmatic examples of monodisperse gene delivery systems. The possibility to precisely control the architectural features at the molecular level offers unprecedented opportunities for conducting structure-activity relationship studies. A major requirement for those channels is the design of a sufficiently diverse ensemble of compounds for parallel evaluation of their capabilities to condense DNA into transfection nanoparticles where the gene material is protected from the environment. Here we have undertaken the preparation of an oriented library of ß-cyclodextrin (ßCD) and calix[4]arene (CA4) vectors with facial amphiphilic character designed to ascertain the effect of the cationic head nature (aminothiourea-, arginine- or guanidine-type groups) and the macrocyclic platform on the abilities to complex plasmid DNA (pDNA) and in the efficiency of the resulting nanocomplexes to transfect cells in vitro. The hydrophobic domain, formed by hexanoyl or hexyl chains, remains constant in each series, matching the overall structure found to be optimal in previous studies. DLS, TEM and AFM data support that all the compounds self-assemble in the presence of pDNA through a process that involves initially electrostatic interactions followed by formation of ßCD or CA4 bilayers between the oligonucleotide filaments. Spherical transfectious nanoparticles that are monomolecular in DNA are thus obtained. Evaluation in epithelial COS-7 and human rhabdomyosarcoma RD-4 cells evidenced the importance of having primary amino groups in the vector to warrant high levels of transfection, probably because of their buffering capacity. The results indicate that the optimal cationic head depends on the macrocyclic core, aminothiourea groups being preferred in the ßCD series and arginine groups in the CA4 series. Whereas the transfection efficiency relationships remain essentially unchanged within each series, irrespective of the cell type, the optimal platform (ßD or CA4) strongly depends on the cell type. The results illustrate the potential of monodisperse vector prototypes and diversity-oriented strategies on identifying the optimal candidates for gene therapy applications.

BoHV-4 immediate early 1 gene is a dispensable gene and its product is not a bone marrow stromal cell antigen 2 counteracting factor.

BACKGROUND: Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus whose genome was cloned as Bacterial Artificial Chromosome (BAC) and exploited as a gene delivery vector for vaccine purposes. Although BoHV-4 genome has been completely sequenced and its open reading frames (ORFs) structurally defined in silico, most of them are not functionally characterized. In BoHV-4 genome two major immediate early genes (IE) are present, IE1 and IE2. IE2 is an essential gene because its removal from the viral genome renders the virus unable to replicate, whereas for IE1 no many functional information are available. RESULTS: In this work, IE1 contribution in initiating and maintaining BoHV-4 lytic replication was assessed generating a recombinant BoHV-4 genome lacking of IE1 gene, BoHV-4ΔIE1. In contrast to BoHV-4IE2 deleted mutant, BoHV-4ΔIE1 infectious replicating viral particles (IRVPs) could be reconstituted following viral DNA electroporation in permissive cells. However the titer of BoHV-4ΔIE1 IRVPs produced into the cell supernatant and BoHV-4ΔIE1 plaques size were reduced respect to BoHV-4 undeleted control. Further the impaired BoHV-4ΔIE1 IRVPs produced into the cell supernatant could be rescued by expressing IE1 gene product in trans, confirming the implication of IE1 in BoHV-4 lytic replication. Next, the possible role of BoHV-4IE1 as bone marrow stromal cell antigen 2 (BST-2) counteracting factor, as hypothesized by IE1 amino-terminal gene product homology with Kaposi Sarcoma Associated Herpesvirus (KSHV) K5, was excluded too. CONCLUSIONS: Although the real function of BoHV-4IE1 is still elusive, a new BoHV-4 genome gene locus as a target site for the insertion of foreign DNA and resulting in the attenuation of the virus has been revealed. These data can be considered of relevance to improve BoHV-4 gene delivery properties.

Interferon gamma-mediated BoHV-4 replication restriction in bovine endometrial stromal cells is host IDO1 gene expression independent and BoHV-4 IE2 gene expression dependent.

In the present work the interaction between bovine herpesvirus 4 (BoHV-4)-infected bovine endometrial stromal cells (BESCs) and interferon gamma (IFNG) was investigated. Starting from the particular tropism of BoHV-4 toward BESCs, a pure population of these cells, free of CD45-positive cells, was prepared and proven to have a bona fide mesenchymal derivation as shown by vimentin-positive and cytokeratin-negative staining. BESCs expressed functional IFNG receptors (IFNGR) 1 and 2 but not IFNG ligand. BESCs transfected with a new reporter construct made by cloning the bovine indoleamine 2, 3-dioxygenase 1 (IDO1) promoter in front of the luciferase reporter gene responded to exogenous IFNG treatment. Further, IFNG-treated or constitutively secreting IFNG BESCs strongly restricted BoHV-4 replication and consequent cytopathic effect. IDO1 expression in BESCs was tightly induced by IFNG and IDO1 was previously shown to be the mediator for some of the IFNG pathogenostatic effects. However, IDO1 inhibitors and IDO1 constitutive expression could not respectively abrogate or recapitulate IFNG effect on BoHV-4-infected BESCs, whereas BoHV-4 immediate early (IE2) gene expression was transcriptionally depressed by IFNG axis activation independently from IDO1 expression; this was further confirmed by revealing a BoHV-4 IE2 gene promoter area containing potential responsive elements interacting with inhibitory transcription factors induced by IFNG in BESCs. The data achieved in this work highlight at least two issues: first, the role of BESCs as target/effector cells for the IFNG; second, the importance of uterine IFNG integrity to control BoHV-4 infection recrudescence from a persistent/latent state to a chronic disease, endometritis.

Enlightened Mannhemia haemolytica lung inflammation in bovinized mice.

Polymorphonuclear cells diapedesis has an important contribution to the induced Mannhemia haemolytica (M. haemolytica) infection lung inflammation and IL-8 is the primary polymorphonuclear chemoattractant. Using a bovine IL-8/luciferase transiently transgenized mouse model, the orchestration among M. haemolytica, IL-8 promoter activation and neutrophilia was followed in real time by in vivo image analysis.

A novel magnetic ligand-based assay for the electrochemical determination of BRD4.

The first magnetic ligand-based electrochemical assay aimed at the determination of BRD4 was developed and validated. BRD4 is an epigenetic regulator of great interest in oncology in relation to its overexpression observed in the pathogenesis of several cancer diseases. BRD4 also represents a major target for the development of innovative treatments aimed at protein inhibition or degradation. Despite the relevance of BRD4 both for diagnostics and therapeutic purposes, current methodologies for its determination are limited to commercial ELISA kits. We present a novel magnetic ligand-based assay for the electrochemical determination of BRD4. The developed assay is based on the use of a small synthetic fragment of the natural protein ligand for BRD4 as receptor, thus exploiting the intrinsic biological protein-protein recognition mechanism. In addition, the assay features the use of magnetic beads as immobilization platforms and peroxidase-conjugated monoclonal anti-BRD4 antibody for the generation of the electrochemical signal. The ligand-based assay shows outstanding performance in terms of rapidity, with results achievable in less than 20 min, no matrix effect when applied to human plasma or cell lysate samples, and excellent specificity. The proposed method exhibits a limit of detection of 2.66 nM and a response range tunable as a function of the amount of immobilized receptor. The developed ligand-based assay was successfully applied to the accurate determination of BRD4 in untreated cell lysates, as proven by the ELISA reference method. The good performance of the proposed bioassay for determination of BRD4 showed potential application of this strategy in convenient point-of-care testing.

Ovine Herpesvirus 2 Glycoprotein B Complementation Restores Infectivity to a Bovine Herpesvirus 4 gB-Null Mutant.

Ovine herpesvirus 2 (OvHV-2) and bovine herpesvirus 4 (BoHV-4) are gamma herpesviruses that belong to the genera Macavirus and Rhadinovirus, respectively. As with all herpesviruses, both OvHV-2 and BoHV-4 express glycoprotein B (gB), which plays an essential role in the infection of host cells. In that context, it has been demonstrated that a BoHV-4 gB-null mutant is unable to infect host cells. In this study, we used homologous recombination to insert OvHV-2 ORF 8, encoding gB, into the BoHV-4 gB-null mutant genome, creating a chimeric BoHV-4 virus carrying and expressing OvHV-2 gB (BoHV-4∆gB/OvHV-2-gB) that was infectious and able to replicate in vitro. We then evaluated BoHV-4∆gB/OvHV-2-gB as a potential vaccine candidate for sheep-associated malignant catarrhal fever (SA-MCF), a fatal disease of ungulates caused by OvHV-2. Using rabbits as a laboratory model for MCF, we assessed the safety, immunogenicity, and efficacy of BoHV-4∆gB/OvHV-2-gB in an immunization/challenge trial. The results showed that while BoHV-4∆gB/OvHV-2-gB was safe and induced OvHV-2 gB-specific humoral immune responses, immunization conferred only 28.5% protection upon challenge with OvHV-2. Therefore, future studies should focus on alternative strategies to express OvHV-2 proteins to develop an effective vaccine against SA-MCF.

Evaluation of the immunogenicity of an mRNA vectored Nipah virus vaccine candidate in pigs.

Nipah virus (NiV) poses a significant threat to human and livestock populations across South and Southeast Asia. Vaccines are required to reduce the risk and impact of spillover infection events. Pigs can act as an intermediate amplifying host for NiV and, separately, provide a preclinical model for evaluating human vaccine candidate immunogenicity. The aim of this study was therefore to evaluate the immunogenicity of an mRNA vectored NiV vaccine candidate in pigs. Pigs were immunized twice with 100 µg nucleoside-modified mRNA vaccine encoding soluble G glycoprotein from the Malaysia strain of NiV, formulated in lipid nanoparticles. Potent antigen-binding and virus neutralizing antibodies were detected in serum following the booster immunization. Antibody responses effectively neutralized both the Malaysia and Bangladesh strains of NiV but showed limited neutralization of the related (about 80% amino acid sequence identity for G) Hendra virus. Antibodies were also capable of neutralizing NiV glycoprotein mediated cell-cell fusion. NiV G-specific T cell cytokine responses were also measurable following the booster immunization with evidence for induction of both CD4 and CD8 T cell responses. These data support the further evaluation of mRNA vectored NiV G as a vaccine for both pigs and humans.