Bovine papillomaviruses (BPVs) types 1 and 2 are the only known papillomaviruses able to jump the species. In fact, BPVs 1/2 induce neoplasia in their natural bovine host but infection is also associated to neoplastic skin lesions in equids termed sarcoids. The equine sarcoid is considered to be the most common equine cutaneous tumour worldwide for which no effective therapy is available. Very little is known about the molecular mechanisms underlying tumourigenesis, although genes contributing to sarcoid development have been identified. Several studies associate the development of cancer to the loss of function of a number of oncosuppressor genes. In this study the putative role of O6-methylguanine-DNA methyltrasferase (MGMT) was investigated for sarcoids. The expression of the oncosuppressor protein was assessed in normal and sarcoid cells and tissues. In addition, the DNA methylation profile was analysed to assess the role of epigenetic mechanism in regulation of MGMT expression.

BPA (bisphenol A or 2,2-bis(4-hydroxy-phenol)propane) is present in the manufacture of polycarbonate plastic and epoxy resins, which can be used in impact-resistant safety equipment and baby bottles, as protective coatings inside metal food containers, and as composites and sealants in dentistry. Recently, attention has focused on the estrogen-like and carcinogenic adverse effects of BPA. Thus, it is necessary to investigate the cytotoxicity and apoptosis-inducing activity of this compound.

The Ovalbumin(257-264) CTL epitope on the major coat protein of the filamentous bacteriophage in different antigen formulations was displayed and the immune response in C57BL6/J mice studied. The display of single cytotoxic epitope on the surface of the virion is sufficient to induce priming and sustain long-term major histocompatibility complex class I restricted cytotoxic T lymphocytes response in vivo. The filamentous bacteriophage is a versatile carrier able to display simultaneously either single or multiple epitopes and can elicit a cellular response carrying very little peptide (<1.5 microg).

Kidney transplanted recipients (KTR) are at high risk of severe SARS-CoV-2 infection due to immunosuppressive therapy. Although several studies reported antibody production in KTR after vaccination, data related to immunity to the Omicron (B.1.1.529) variant are sparse. Herein, we analyzed anti-SARS-CoV-2 immune response in seven KTR and eight healthy controls after the second and third dose of the mRNA vaccine (BNT162b2). A significant increase in neutralizing antibody (nAb) titers were detected against pseudoviruses expressing the Wuhan-Hu-1 spike (S) protein after the third dose in both groups, although nAbs in KTR were lower than controls. nAbs against pseudoviruses expressing the Omicron S protein were low in both groups, with no increase after the 3rd dose in KTR. Reactivity of CD4+ T cells after boosting was observed when cells were challenged with Wuhan-Hu-1 S peptides, while Omicron S peptides were less effective in both groups. IFN-γ production was detected in KTR in response to ancestral S peptides, confirming antigen-specific T cell activation. Our study demonstrates that the 3rd mRNA dose induces T cell response against Wuhan-Hu-1 spike peptides in KTR, and an increment in the humoral immunity. Instead, humoral and cellular immunity to Omicron variant immunogenic peptides were low in both KTR and healthy vaccinated subjects.

Bi-allelic hypomorphic mutations in DNMT3B disrupt DNA methyltransferase activity and lead to immunodeficiency, centromeric instability, facial anomalies syndrome, type 1 (ICF1). Although several ICF1 phenotypes have been linked to abnormally hypomethylated repetitive regions, the unique genomic regions responsible for the remaining disease phenotypes remain largely uncharacterized. Here we explored two ICF1 patient-derived induced pluripotent stem cells (iPSCs) and their CRISPR-Cas9-corrected clones to determine whether DNMT3B correction can globally overcome DNA methylation defects and related changes in the epigenome. Hypomethylated regions throughout the genome are highly comparable between ICF1 iPSCs carrying different DNMT3B variants, and significantly overlap with those in ICF1 patient peripheral blood and lymphoblastoid cell lines. These regions include large CpG island domains, as well as promoters and enhancers of several lineage-specific genes, in particular immune-related, suggesting that they are premarked during early development. CRISPR-corrected ICF1 iPSCs reveal that the majority of phenotype-related hypomethylated regions reacquire normal DNA methylation levels following editing. However, at the most severely hypomethylated regions in ICF1 iPSCs, which also display the highest increases in H3K4me3 levels and/or abnormal CTCF binding, the epigenetic memory persists, and hypomethylation remains uncorrected. Overall, we demonstrate that restoring the catalytic activity of DNMT3B can reverse the majority of the aberrant ICF1 epigenome. However, a small fraction of the genome is resilient to this rescue, highlighting the challenge of reverting disease states that are due to genome-wide epigenetic perturbations. Uncovering the basis for the persistent epigenetic memory will promote the development of strategies to overcome this obstacle.

The control of intracellular vesicle trafficking is an ideal target to weigh the role of alternative splicing in shaping genomes to make cells. Alternative splicing has been reported for several Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptors of the vesicle (v-SNAREs) or of the target membrane (t-SNARES), which are crucial to intracellular membrane fusion and protein and lipid traffic in Eukaryotes. However, splicing has not yet been investigated in Longins, i.e. the most widespread v-SNAREs. Longins are essential in Eukaryotes and prototyped by VAMP7, Sec22b and Ykt6, sharing a conserved N-terminal Longin domain which regulates membrane fusion and subcellular targeting. Human VAMP7/TI-VAMP, encoded by gene SYBL1, is involved in multiple cell pathways, including control of neurite outgrowth.

Active infection by bovine papillomavirus type 2 (BPV-2) was documented for fifteen urinary bladder tumors in cattle. Two were diagnosed as papillary urothelial neoplasm of low malignant potential (PUNLMP), nine as papillary and four as invasive urothelial cancers.

Several efforts have been invested in the identification of CTL and Th epitopes, as well as in the characterization of their immunodominance and MHC restriction, for the generation of a peptide-based HCMV vaccine. Small synthetic peptides are, however, poor antigens and carrier proteins are important for improving the efficacy of synthetic peptide vaccines. Recombinant bacteriophages appear as promising tools in the design of subunit vaccines. To investigate the antigenicity of peptides carried by recombinant bacteriophages we displayed different HCMV MHCII restricted peptides on the capsid of filamentous bacteriophage (fd) and found that hybrid bacteriophages are processed by human APC and activate HCMV-specific CD4 T-cells. Furthermore we constructed a reporter T-cell hybridoma expressing a chimeric TCR comprising murine alphabeta constant regions and human variable regions specific for the HLA-A2 restricted immunodominant NLV peptide of HCMV. Using the filamentous bacteriophage as an epitope carrier, we detected a more robust and long lasting response of the reporter T-cell hybridoma compared to peptide stimulation. Our results show a general enhancement of T-cell responses when antigenic peptides are carried by phages.

Multiple papillomatous nodules were observed scattered over the amniotic membrane in six water buffaloes that had recently aborted. Grossly, some of the nodules had multiple villous projections while others appeared as single prominent conical or cylindrical horns. Histology revealed folded hyperplastic and hyperkeratotic epithelium supported by a narrow fibro-vascular stalk. Using PCR, sequences of the bovine Deltapapillomavirus type 2 (BPV-2) E5 gene were amplified from the amniotic papillomas. Furthermore, expression of the E5 gene was detected using reverse transcription (RT)-PCR. Western blotting revealed BPV-2 E5 oncoprotein as well as L1 protein, suggesting both abortive and productive infection. Additionally, a functional complex composed of BPV-2 E5 oncoprotein and the phosphorylated PDGFβR was detected, which is consistent with the activation of PDGFβR by the interaction with BPV-2 E5 oncoprotein. These results demonstrate that BPV-2 can infect the amnion of water buffaloes and suggest that this infection may cause proliferation of the epithelial cells of the amnion. While the precise pathogenesis in uncertain, it is possible that BPV-2 infection of stratified squamous epithelial cells within squamous metaplasia foci and/or amniotic plaques could lead to papilloma formation. Papillomavirus-associated amniotic papillomas have not previously been reported in any species, including humans.

ERas is a new gene recently found in mouse embryonic stem (ES) cells and localized on the X chromosome. It plays a role in mouse ES cell survival and is constitutively active without any mutations. It was also found to be responsible for the maintenance of quiescence of the hepatic stellate cells (HSCs), liver-resident mesenchymal stem cells, the activation of which results in liver fibrosis. This gene was not present in human ES cells. ERas was found to be activated in a significant population of human gastric cancer, where ERAS may play a crucial role in gastric cancer cell survival and metastases to liver via down-regulation of E-cadherin. ERas gene has been found to be expressed both in ES cells and adult tissues of cynomolgus monkey. Cynomolgus ERAS did not promote cell proliferation or induce tumor formation. ERAS was also detected in normal and neoplastic urothelium of the urinary bladder in cattle, where bovine ERAS formed a constitutive complex with platelet derived growth factor β receptor (PDGFβR) resulting in the activation of AKT signaling. Here, molecular and morphological findings of ERAS in the full term placenta of pregnant cows have been investigated for the first time. ERAS was studied by reverse transcriptase PCR (RT-PCR). Alignment of the sequence detects a 100% identity with all transcript variant bovine ERas mRNAs, present in the GenBank database (http://www.ncbi.nlm.nih.gov). Furthermore, ERAS was detected by Western blot and investigated by real time PCR that revealed an amount of ERAS more than ERAS found in normal bovine urothelium but less than ERAS present in the liver. Immunohistochemical examination revealed the presence of ERAS protein both at the level of plasma membrane and in cytoplasm of epithelial cells lining caruncular crypts and in trophoblasts of villi. An evident ERAS immunoreactivity was also seen throughout the chorionic and uterine gland epithelium. Although this is not a functional study and further investigations will be warranted, it is conceivable that ERAS may have pleiotropic effects in the placenta, some of which, like normal urothelial cells, might lead to activation of AKT pathway. We speculate that ERAS may play a key role in cellular processes such as cell differentiation and movement. Accordingly, we believe it may be an important factor involved in trophoblast invasiveness via AKT signaling pathway. Therefore, ERas gene is a functional gene which contributes to homeostasis of bovine placenta.

Congenital fibropapillomatosis of the gingiva and oral mucosa and epidermal hyperplasia of the lip are described, for the first time, in two newborn lambs. Expression of the E5 oncoprotein of bovine deltapapillomavirus types 2 (BPV-2) and -13 (BPV-13) was detected in both fibropapillomas and the hyperplastic epidermal cells suggesting the BPV infection was the cause of the proliferative lesions. No DNA sequences of BPV-1 and BPV-14 were detected. Both BPV-2 and BPV-13 DNA were also amplified from peripheral blood mononuclear cells (PBMCs) of the newborn lambs' dams. The concordance between BPV genotypes detected in the blood of dam and the oral and skin pathological samples of their offspring suggests that a vertical hematogeneous transmission was most likely source of BPV infection. Immunoblotting revealed the presence of E5 dimers allowing the viral protein to be biologically active. E5 dimers bind and activate the platelet derived growth factor β receptor (PDGFβR), a major molecular mechanism contributing to disease. The detection of E5 protein within the proliferating cells therefore adds further evidence that the BPV infection was the cause of the proliferative lesions seen in these lambs. This is the first evidence of vertical transmission of BPVs in sheep resulting in a clinical disease.

Many solid tumours including melanoma, glioblastoma, and breast carcinomas express MHC class II molecules (MHC II). The surface expression of these molecules confers to non-hematopoietic tumour cells the role of non-professional antigen presenting cells and the ability to potentially stimulate tumour-specific CD4+ T cell response. We studied EBP1, an ErbB3 binding protein, and the effects of p48 and p42 isoforms on the MHC II expression in U87 glioblastoma, M14 melanoma and MCF7 mammary carcinoma cell lines. We found that overexpression of p48 increases MHC II transcription in U87 and M14, through upregulation of CIITA transactivator and STAT1 phosphorylation. In addition, p48 protein influences MHC II expression by increasing mRNA stability. In melanoma and glioblastoma cell lines, p48 isoform functions as oncogene promoting tumour growth, while p42 isoform, that does not affect MHC II expression, acts as a tumour suppressor by blocking cell growth and inducing apoptosis. In contrast, p48 seems to act as tumour suppressor in breast carcinoma inhibiting proliferation, favouring apoptosis, and inducing a slight increase of MHC II expression similar to p42. Our data highlight the tissue specificity function of EBP1 isoforms and demonstrate that only the oncogene p48 activates MHC II expression in human solid tumours, via STAT1 phosphorylation, in order to affect tumour progression by triggering specific immune response.

In human cancer cells, BAG3 protein is known to sustain cell survival. Here, for the first time, we demonstrate the expression of BAG3 protein both in equine sarcoids in vivo and in EqS04b cells, a sarcoid-derived fully transformed cell line harbouring bovine papilloma virus (BPV)-1 genome. Evidence of a possible involvement of BAG3 in equine sarcoid carcinogenesis was obtained by immunohistochemistry analysis of tumour samples. We found that most tumour samples stained positive for BAG3, even though to a different grade, while normal dermal fibroblasts from healthy horses displayed very weak staining pattern for BAG3 expression. By siRNA technology, we demonstrate in EqS04b the role of BAG3 in counteracting basal as well as chemical-triggered pro-death signals. BAG3 down-modulation was indeed shown to promote cell death and cell cycle arrest in G0/G1. In addition, we found that BAG3 silencing sensitized EqS04b cells to phenethylisothiocyanate (PEITC), a promising cancer chemopreventive/chemotherapeutic agent present in edible cruciferous vegetables. Notably, such a pro-survival role of BAG3 was less marked in E. Derm cells, an equine BPV-negative fibroblast cell line taken as a normal counterpart. Altogether our findings might suggest a mutual cooperation between BAG3 and viral oncoproteins to sustain cell survival.

The transcriptome profiles were compared for buffalo embryos with normal growth and embryos with retarded growth on Day 25 after mating. Embryos with retarded growth on Day 25 after mating have a reduced likelihood of undergoing attachment to the uterine endometrium and establishing a pregnancy. Italian Mediterranean buffaloes were mated by AI and on Day 25 underwent trans-rectal ultrasonography to ascertain embryo development. Embryos with an embryonic width (EW)>2.7 mm were classed as normal embryos and embryos with an EW<2.7 mm were classed as retarded embryos. Three buffaloes with embryos of the largest EW (3.7, 3.7 and 3.9 mm) and three buffaloes with embryos of the smallest EW (1.5, 1.6 and 1.9 mm) were slaughtered on Day 27 to recover embryos for transcriptome analysis using a bovine custom designed oligo array. A total of 1,047 transcripts were differentially expressed between embryos with normal growth and embryos with retarded growth. Retarded embryos showed 773/1,047 (74%) transcripts that were down-regulated and 274/1,047 (26%) transcripts that were up-regulated relative to normal embryos; in silico analyses focused on 680/1,047 (65%) of the differentially expressed transcripts. The most altered transcripts observed in retarded embryos were associated with membrane structure and function and with metabolic and homeostasis maintenance functions. Other notable functions altered in retarded embryos were developmental processes and in particular nervous system differentiation and function. Specific biochemical pathways such as the complement cascade and coagulation were also altered in retarded embryos. It was concluded from the findings that buffalo embryos with retarded growth on Day 25 after mating show altered gene expression compared with normal embryos, and some de-regulated functions are associated with attachment to the uterine endometrium.

We report in this work that a cellular and humoral autoreactive response can be induced against liver-specific self-determinants by repeated immunization with a chimeric tissue-specific self-antigen carrying a heterologous T(h) epitope. Epitope spreading rendering the autoimmune reaction independent of the presence of the cognate heterologous help is also demonstrated. Although neutrophil infiltrates can be demonstrated in the livers of treated mice, no clinical sign of organ damage is observed. These findings suggest that breakage of tolerance by this means leads the process only up to the next checkpoint in the progression of autoimmune disease and that further events are required to precipitate functional organ impairment.

The DR5-DQ7/DR7-DQ2 genotype is very frequent among patients affected by celiac disease (CD), in Europe. This genotype, associated to high risk of CD, carries the HLA-DQA1*05 and HLA-DQB1*02 predisposing alleles, in trans configuration. The alleles encode the DQ2.5 heterodimer responsible of gluten peptide presentation on the surface of antigen-presenting cells (APCs), and consequent pathogenic CD4+ T cell activation. We demonstrated that DR5/DR7 APCs induce an anti-gluten CD4+ T cell response, of comparable intensity to that observed with APCs carrying DR1/DR3 genotype, which risk alleles are in cis configuration. In addition, we showed that DR5/DR7 APCs from celiac patients stimulated an effector CD4+ T cell response higher with respect to that induced by DR5/DR7 APCs from healthy subjects. To explain these findings, we assessed the DQ2.5 RNA and protein quantity. We showed that the expression of DQA1*05 and DQB1*02 risk alleles is much higher than the expression of non-CD-associated alleles, in agreement with the previous results obtained with DR1/DR3 genotype. The differential expression of transcripts influences the quantity of DQα1*05 and DQβ1*02 chains and, as consequence, the cell surface density of DQ2.5 heterodimers. Moreover, both RNA and proteins, are more abundant in APCs from celiac patients than controls. Finally, to unravel the mechanism regulating the expression of predisposing DQA1*05 and DQB1*02 alleles, we quantified the new synthetized RNA and found that the differential expression is explained by their transcription rate. Our results confirmed that the strength of antigen-specific CD4+ T cell response is mainly determined by the amount of gluten in the diet and provided a new possible approach for a personalized diagnosis and for risk stratification.

In humans, muscle-invasive bladder cancer (MIBC) is highly aggressive and associated with a poor prognosis. With a high mutation load and large number of altered genes, strategies to delineate key driver events are necessary. Dogs and cats develop urothelial carcinoma (UC) with histological and clinical similarities to human MIBC. Cattle that graze on bracken fern also develop UC, associated with exposure to the carcinogen ptaquiloside. These species may represent relevant animal models of spontaneous and carcinogen-induced UC that can provide insight into human MIBC.

Hepcidin (HEP) and ferroportin (FPN) play a central role in systemic iron homeostasis. The HEP/FPN axis controls both extracellular iron concentration and total body iron levels. HEP is synthesized mainly by hepatocytes and controls the absorption of dietary iron and the distribution of iron to the various cell types; its synthesis is regulated by both iron and innate immunity. FPN is a membrane protein and the major exporter of iron from mammalian cells, including iron recycling macrophages, iron absorbing duodenal enterocytes, and iron storing hepatocytes. HEP limits the pool of extracellular iron by binding FPN and mediating its degradation, thus preventing its release from intracellular sources. Here we investigated, for the first time, the molecular and morphological expression of HEP and FPN in placenta of pregnant cows at term. Their expression has been evaluated investigating their mRNAs by reverse transcriptase PCR (RT-PCR). Sequencing of related amplicons revealed a 100% identity with HEP and FPN sequences from Bos taurus as reported in the GeneBank (mRNASequence ID: NM_001114508.2 and ID: NM_001077970.1, respectively). HEP and FPN proteins have also been revealed by Western blot analysis and immunohistochemistry. The strongest immunoreactivity for both proteins was observed in the cytoplasm of the trophoblastic cells of the villi and the caruncular crypts of the placentome. Hep mRNA was more representative in caruncular rather cotyledonar areas; on the contrary, Fpn mRNA was more expressed in cotyledonar rather than in caruncular areas. Transcripts of ferritin, transferrin and its receptor have been also documented by real time RT-PCR. HEP and FPN placental proteins may play a dual role. HEP/FPN axis seems to have a central role in infections, with microorganisms within macrophages or that survive in the bloodstream or other cellular spaces. In addition, HEP may be responsible for iron flux regulation as a molecular bridge for iron trafficking and response to infection. FPN may also have a significant role for embryonic development, growth and organogenesis.

Calpain 3 (Capn3), also named p94, is a skeletal muscle tissue-specific protein known to be responsible for limb-girdle muscular dystrophy type 2A (LGMD2A). Recent experimental studies have hypothesized a pro-apoptotic role of Capn3 in some melanoma cell lines. So far the link between calpain3 and tumors comes from in vitro studies. The objective of this study was to describe Capn3 activation in naturally occurring urothelial tumors of the urinary bladder in cattle.

Many pseudogenes possess biological activities and play important roles in the pathogenesis of various types of cancer including bladder cancer (BlCa), which still lacks suitable molecular biomarkers. Recently, pseudogenes were found to be significantly enriched in a pan-cancer classification based on the Cancer Genome Atlas gene expression data. Among them, the top-ranking pseudogene was the proliferation-associated 2G4 pseudogene 4 (PA2G4P4).