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 bleomycintreated mice. A reporter gene construct, containing the luciferase open reading frame under the matrix metalloproteinase-1 promoter control region, was tested on doublebleomycin-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.Fil: Stellari, Fabio Franco. Chiese Farmaceutici; ItaliaFil: Ruscitti, Francesca. Chiese Farmaceutici; ItaliaFil: Pompilio, Daniela. Chiese Farmaceutici; ItaliaFil: Ravanetti, Francesca. Università di Parma. Dipartimento di Scienze Medico Veterinarie; ItaliaFil: Tebaldi, Giulia. Università di Parma. Dipartimento di Scienze Medico Veterinarie; ItaliaFil: Macchi, Francesca. Università di Parma. Dipartimento di Scienze Medico Veterinarie; ItaliaFil: Verna, Andrea Elizabeth. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Chiese Farmaceutici; ItaliaFil: Villetti, Gino. Chiese Farmaceutici; ItaliaFil: Donofrio, Gaetano. Università di Parma. Dipartimento di Scienze Medico Veterinarie ; Itali

Hypertension, uncontrolled hypertension and resistant hypertension: prevalence, comorbidities and prescribed medications in 228,406 adults resident in urban areas. A population-based observational study

Although hypertension is the leading cause of cardiovascular disease and premature death worldwide, it remains difficult to control. The prevalence of uncontrolled and resistant hypertension (RH) may be underestimated and can reach up to 50% of all hypertensive patients. The aim of this observational study was to analyze the prevalence of hypertension, uncontrolled hypertension and RH, and their associations with risk factors or diseases in a large cohort of patients referred to primary care physician. In a population of 228406 adults, we only collected data from people with a diagnosis of arterial hypertension for a total of 43,526 patients. For this purpose, we used the MySQL database, run by Azalea.NET, built on the medical records of 150 General Practitioners (GPs). Patient data included sex, age, blood pressure (BP) values, number of antihypertensive drugs and presence of major cardiovascular comorbidities. We classified patients with RH as those treated with 3 different antihypertensive agents, with recorded BP & GE; 140/90 mmHg, or patients taking & GE; 4 medications. The prevalence of hypertension was 19.06%, that of resistant hypertension was 2.46% of the whole population and 20.85% of the hypertensive group. Thirteen thousand hundred, forty-six patients (30.20% of the hypertensive group) had uncontrolled BP (& GE; 140/90 mmHg), whereas 16,577 patients did not have BP measurements done in the last 2 years (38.09% of the hypertensive group). Patients with uncontrolled BP were mainly female, used less drugs and showed a lower prevalence of all major cardiovascular comorbidities, except for diabetes. Instead, patients with RH had a significantly higher prevalence of all considered comorbidities compared to those without RH. Our results evidence that a broad number of patients with hypertension, especially those without comorbidities or with a low number of antihypertensive drugs, do not achieve adequate BP control. To improve the clinical management of these patients it is very important to increase the collaboration between GPs and clinical specialists of hypertension

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-syEBOVgD106TK, was generated. Results: EBOV GP was abundantly expressed by BoHV-4-syEBOVgD106TK transduced cells without decreasing viral replication. BoHV-4-syEBOVgD106TK 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-syEBOVgD106TK 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

Dissecting Molecular Heterogeneity of Circulating Tumor Cells (CTCs) from Metastatic Breast Cancer Patients through Copy Number Aberration (CNA) and Single Nucleotide Variant (SNV) Single Cell Analysis

Circulating tumor cells' (CTCs) heterogeneity contributes to counteract their introduction in clinical practice. Through single-cell sequencing we aim at exploring CTC heterogeneity in metastatic breast cancer (MBC) patients. Single CTCs were isolated using DEPArray NxT. After whole genome amplification, libraries were prepared for copy number aberration (CNA) and single nucleotide variant (SNV) analysis and sequenced using Ion GeneStudio S5 and Illumina MiSeq, respectively. CTCs demonstrate distinctive mutational signatures but retain molecular traces of their common origin. CNA profiling identifies frequent aberrations involving critical genes in pathogenesis: gains of 1q (CCND1) and 11q (WNT3A), loss of 22q (CHEK2). The longitudinal single-CTC analysis allows tracking of clonal selection and the emergence of resistance-associated aberrations, such as gain of a region in 12q (CDK4). A group composed of CTCs from different patients sharing common traits emerges. Further analyses identify losses of 15q and enrichment of terms associated with pseudopodium formation as frequent and exclusive events. CTCs from MBC patients are heterogeneous, especially concerning their mutational status. The single-cell analysis allows the identification of aberrations associated with resistance, and is a candidate tool to better address treatment strategy. The translational significance of the group populated by similar CTCs should be elucidated

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/relA, 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

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/relA, 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

Profilo trascrizionale di cellule stromali endometriali bovine infettate da Bovine Herpesvirus 4

ABSTRACT Bovine postpartum uterine diseases affect half of all dairy cattle after parturition, causing infertility by disrupting uterine and ovarian function. Female genital tract infections are a primary economic problem for efficient performance of dairy herds, as they cause reduced pregnancies as well as reduced milk production. Although the etiology of bovine uterine diseases is mainly ascribed to bacterial pathogens, such as Escherichia coli and Arcanobacterium pyogenes, more extensive diagnostics have in many cases also detected the presence of Bovine Herpesvirus 4 (BoHV-4) in affected cattle, implying that co-infection may be involved in a possible vicious circle. Following recognition of bacterial lipopolysaccharide (LPS) by the Toll-like receptor 4 (TLR4) expressed at the bovine endometrial epithelium level, expression of immune factors such as prostaglandin E (PGE) and Tumor necrosis factor alpha (TNF-α) are activated. This cytokine cascade triggers BoHV-4 replication by binding specific sites within the immediate early IE2 gene promoter. Conversely, Interferon gamma (IFN-γ) produced by the cells of the immune system, inhibits viral replication, always acting at the level of the IE2 gene promoter. Although BoHV-4 is often isolated from animals affected by these uterine pathologies, and for this reason, consistently associated with postpartum metritis and chronic infertility in cattle, it is difficult to directly correlate the presence of the virus to the pathology in progress, partly because, the cellular pathways and the molecular mechanisms involved are not well defined, as well as which extracellular stimuli related to the intrauterine environment could influence the BoHV-4 replicative cycle. In order to shed light on the true pathogenic role and to better investigate the molecular mechanisms of the physio-pathological response underlying the pathogen-host interaction, an experimental BoHV-4 infection was carried out with the aim to optimize infection conditions and a transcriptomic analysis of a pure bovine endometrial stromal cells population (BESCs) infected with BoHV-4 was performed. The RNA-seq technology used for this purpose allowed to discover a set of differentially expressed genes (DE genes), among which 417 were up-regulated while 118 were down-regulated during BoHV-4 infection. DE genes have been functionally classified through enrichment functional analysis on the basis of their involvement in various cellular pathways. Many pathways, related to cell proliferation and cell surface integrity, were found to be affected by BoHV-4 infection. Some DE genes that were found to be up-regulated also play a key role in pathogenesis as in the case of IL-8 and MMP-1. MMP-1 up-regulation was not unexpected, as it belongs to the zinc-dependent endo-peptidases family involved in numerous and important cellular networks. Thanks to the ECM remodeling and immuno-modulation processes, MMPs assume a relevant connotation in the particular context of metritis, where under suitable conditions favor pathogen eradication, inflammatory state resolution as well as tissue anatomical and functional restoration. Alternatively, pathogenic dysregulation and MMPs hyper-activation leads to host morbidity and mortality thereby favoring pathogen persistence and dissemination, preventing tissue healing and finally defining an immuno-pathological status. By virtue of the fact that these enzymes play a pivotal role in maintaining the delicate balance between normal and hyper-activated immune response and for the putative involvement of MMP-1 in the bovine uterine diseases development, it was selected as a candidate gene for further studies as a key host factor involved in BoHV-4 pathogenesis. The in silico observations were further corroborated by reverse transcription PCR, real time PCR, Western immunoblotting and finally a luciferase assay where a bovine MMP-1 specific promoter reporter construct was exploited. BESC cells transfected with the reporter construct and subsequently infected with BoHV-4 showed an increasing luciferase expression with increasing viral load. BoHV-4 replication is promoted by IE2 gene expression, while the IE2 protein product RTA/50 is able to transactivate both viral and host cell genes, such as those coding for the chemokine IL-8. IL-8 expression in infected BESCs was shown to increase in a time- and dose-dependent manner. This suggested a potential relationship between MMP-1 up-regulation, IE2 gene expression and viral replication. Following BESCs transfection with an IE2 over-expressing construct, a direct correlation between RTA/50 over-expression and MMP-1 up-regulation was observed. This justified the abnormally high metalloproteinase levels in tissues, and suggested a possible connection to the defective endometrium healing and unresolved inflammation state. These data, combined with those obtained by transcriptomic profile analysis, represent significant steps toward understanding the vicious cycle mentioned above, more precisely the existing interactions between virus, endometrial layer and host immune response, which sustain viral replication thereby eliciting tissue damage. Elucidation of the molecular mechanisms and host cell pathways involved in this pathogenesis is central to the discovery of new targets for novel therapeutic treatments based on MMP-1 down-regulation.RIASSUNTO Le malattie bovine uterine colpiscono la metà delle bovine da latte dopo il parto, provocando infertilità attraverso la distruzione della funzione ovarica e uterina. Le infezioni del tratto genitale femminile rappresentano un problema economico primario inficiando sulle efficienti prestazioni delle mandrie da latte poiché causano riduzione delle gravidanze e della produzione di latte. Sebbene l'eziologia delle malattie uterine dei bovini sia principalmente attribuita a batteri patogeni, come per esempio Escherichia coli ed Arcanobacterium pyogenes, studi diagnostici più approfonditi, hanno rilevato anche la presenza del Bovine Herpesvirus 4 (BoHV-4) in molti dei bovini colpiti, implicando quindi che l'infezione virale possa essere coinvolta in un possibile circolo vizioso. A seguito del riconoscimento del Lipopolisaccaride batterico (LPS) da parte del recettore Toll-like receptor 4 (TLR4) espresso a livello dell'epitelio endometriale bovino, viene attivata l'espressione di fattori immunitari quali la Prostaglandina E (PGE) ed il fattore di Necrosi Tumorale α (TNF-α). Questa cascata di citochine innesca a sua volta, la replicazione di BoHV-4, interagendo con specifici siti all'interno del promotore dell’Immediate Early gene 2 (IE2). Viceversa, l'Interferon gamma (IFN-γ) prodotto dalle cellule del sistema immunitario, inibisce la replicazione virale, agendo sempre a livello del promotore del gene IE2. Per chiarirne il vero ruolo patogenetico e studiare meglio i meccanismi molecolari della risposta fisiopatologica alla base dell'interazione ospite-patogeno, è stata condotta un'infezione sperimentale con BoHV-4 al fine di ottimizzare le condizioni di infezione ed in seguito è stata eseguita un'analisi trascrittomica di una popolazione di cellule stromali endometriali bovine pure (BESC) infettate con BoHV-4. Per l’analisi trascrittomica, è stata applicata la tecnologia dell’RNA-seq che ha permesso di scoprire un insieme di geni differenzialmente espressi (geni DE), tra questi più precisamente, 417 sono risultati essere up-regolati mentre 118 down-regolati durante l'infezione da BoHV-4. I geni DE sono stati successivamente funzionalmente classificati attraverso l'analisi funzionale dell'arricchimento in base al loro coinvolgimento nei vari pathways cellulari. Molti pathways, come per esempio quelli correlati alla proliferazione cellulare ed all'integrità della superficie cellulare, sono risultati essere influenzati dall'infezione con BoHV-4. Inoltre, è stato interessante notare come alcuni geni DE, che si è osservato essere up-regolati, svolgano anche un ruolo chiave nella patogenesi, come nel caso del gene dell’Interleuchina 8 (IL-8) e della metalloproteasi di matrice 1 (MMP-1). L'up-regolazione di MMP-1 non è un dato inaspettato, in quanto MMP-1 appartiene alla famiglia delle endo-peptidasi zinco-dipendenti che sono coinvolte in numerosi e importanti networks cellulari. Grazie ai processi di rimodellamento della Matrice Extracellulare (ECM) ed immuno-modulazione, le metalloproteasi (MMPs) assumono una connotazione rilevante nel particolare contesto della metrite, dove in condizioni favorevoli facilitano l'eradicazione degli agenti patogeni, la risoluzione dello stato infiammatorio ripristinando funzionalmente e anatomicamente i tessuti coinvolti. In alternativa, la disregolazione patogenica e l'iper-attivazione delle MMPs portano ad un incremento della morbidità e mortalità dell’ospite, favorendo così la persistenza e la disseminazione dei patogeni, prevenendo la guarigione dei tessuti e definendo infine uno stato immuno-patologico. In virtù del fatto che questi enzimi svolgono un ruolo fondamentale nel mantenimento del delicato equilibrio tra risposta immunitaria normale ed iper-attivata e per il presunto coinvolgimento di MMP-1 nello sviluppo delle patologie dell'utero bovino, tale gene è stato selezionato come candidato per ulteriori studi come fattore chiave nell’ospite coinvolto nella patogenesi di BoHV-4. Le osservazioni in silico sono state ulteriormente confermate mediante reverse transcription PCR, Real time PCR, Western immunoblotting ed infine un saggio di Luciferasi in cui è stato sfruttato un costrutto reporter caratterizzato dalla presenza dello specifico promotore bovino per MMP-1. Le cellule BESC transfettate con il costrutto reporter e successivamente infettate con BoHV-4 hanno mostrato una crescente espressione della luciferasi all’aumentare della carica virale. È noto che la replicazione di BoHV-4 è promossa dall'espressione del gene IE2, mentre il prodotto proteico di IE2, la proteina RTA/50 è in grado di transattivare sia geni virali che quelli delle cellule ospiti, come quelli per esempio che codificano per la chemochina IL-8. È stato infatti dimostrato come l'espressione dell’IL-8 nelle BESCs infettate aumenta in maniera tempo e dose dipendente, suggerendo quindi una potenziale relazione tra l’up-regolazione di MMP-1, l’espressione del gene IE2 e la replicazione virale. In seguito alla transfezione delle BESCs con un costrutto over esprimente IE2, è stata osservata una correlazione diretta tra l’over espressione della proteina RTA/50 e l’up-regolazione di MMP-1. Questo dato ha giustificato i livelli anormalmente alti di metalloproteinasi nei tessuti suggerendo inoltre una possibile connessione tra il deficit della guarigione endometriale e la permanenza dello stato infiammatorio. Questi dati, in associazione a quelli ottenuti dall'analisi del profilo trascrittomico, rappresentano un significativo passo avanti verso la comprensione del circolo vizioso sopra menzionato; più precisamente sulle relazioni esistenti tra virus, strato endometriale e risposta immunitaria dell'ospite che sostengono la replicazione virale, incrementando così il danno tissutale. La delucidazione quindi dei meccanismi molecolari e dei pathways delle cellule ospiti coinvolti in questa patogenesi è di fondamentale importanza per la scoperta di nuovi target terapeutici basati sulla down-regolazione di MMP-1

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