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Background and Aims Patients with glycogen storage disease type 1a (GSD-1a) primarily present with life-threatening hypoglycemia and display severe liver disease characterized by hepatomegaly. Despite strict dietary management, long-term complications still occur, such as liver tumor development. Variations in residual glucose-6-phosphatase (G6PC1) activity likely contribute to phenotypic heterogeneity in biochemical symptoms and complications between patients. However, lack of insight into the relationship between G6PC1 activity and symptoms/complications and poor understanding of the underlying disease mechanisms pose major challenges to provide optimal health care and quality of life for GSD-1a patients. Currently available GSD-1a animal models are not suitable to systematically investigate the relationship between hepatic G6PC activity and phenotypic heterogeneity or the contribution of gene-gene interactions (GGIs) in the liver. Approach and Results To meet these needs, we generated and characterized a hepatocyte-specific GSD-1a mouse model using somatic CRISPR/CRISPR-associated protein 9 (Cas9)-mediated gene editing. Hepatic G6pc editing reduced hepatic G6PC activity up to 98% and resulted in failure to thrive, fasting hypoglycemia, hypertriglyceridemia, hepatomegaly, hepatic steatosis (HS), and increased liver tumor incidence. This approach was furthermore successful in simultaneously modulating hepatic G6PC and carbohydrate response element-binding protein, a transcription factor that is activated in GSD-1a and protects against HS under these conditions. Importantly, it also allowed for the modeling of a spectrum of GSD-1a phenotypes in terms of hepatic G6PC activity, fasting hypoglycemia, hypertriglyceridemia, hepatomegaly and HS. Conclusions In conclusion, we show that somatic CRISPR/Cas9-mediated gene editing allows for the modeling of a spectrum of hepatocyte-borne GSD-1a disease symptoms in mice and to efficiently study GGIs in the liver. This approach opens perspectives for translational research and will likely contribute to personalized treatments for GSD-1a and other genetic liver diseases.

γδT cell receptors (γδTCRs) recognize a broad range of malignantly transformed cells in mainly a major histocompatibility complex (MHC)-independent manner, making them valuable additions to the engineered immune effector cell therapy that currently focuses primarily on αβTCRs and chimeric antigen receptors (CARs). As an exception to the rule, we have previously identified a γδTCR, which exerts antitumor reactivity against HLA-A*24:02-expressing malignant cells, however without the need for defined HLA-restricted peptides, and without exhibiting any sign of off-target toxicity in humanized HLA-A*24:02 transgenic NSG (NSG-A24:02) mouse models. This particular tumor-HLA-A*24:02-specific Vγ5Vδ1TCR required CD8αα co-receptor for its tumor reactive capacity when introduced into αβT cells engineered to express a defined γδTCR (TEG), referred to as TEG011; thus, it was only active in CD8+ TEG011. We subsequently explored the concept of additional redirection of CD4+ T cells through co-expression of the human CD8α gene into CD4+ and CD8+ TEG011 cells, later referred as TEG011_CD8α. Adoptive transfer of TEG011_CD8α cells in humanized HLA-A*24:02 transgenic NSG (NSG-A24:02) mice injected with tumor HLA-A*24:02+ cells showed superior tumor control in comparison to TEG011, and to mock control groups. The total percentage of mice with persisting TEG011_CD8α cells, as well as the total number of TEG011_CD8α cells per mice, was significantly improved over time, mainly due to a dominance of CD4+CD8+ double-positive TEG011_CD8α, which resulted in higher total counts of functional T cells in spleen and bone marrow. We observed that tumor clearance in the bone marrow of TEG011_CD8α-treated mice associated with better human T cell infiltration, which was not observed in the TEG011-treated group. Overall, introduction of transgenic human CD8α receptor on TEG011 improves antitumor reactivity against HLA-A*24:02+ tumor cells and further enhances in vivo tumor control.

OBJECTIVE To explore sources of serum aldosterone concentration variability in a population of healthy and diseased ferrets, determine a preliminary 1 -sided reference interval for serum aldosterone concentration in healthy ferrets, and identify a decision limit to differentiate healthy from diseased ferrets on the basis of serum aldosterone concentration. DESIGN Prospective threshold definition and diagnostic accuracy study. ANIMALS 78 healthy (n = 56) and diseased (22) ferrets. PROCEDURES Serum aldosterone concentrations were measured on consecutively admitted ferrets, and an upper reference limit for aldosterone concentrations was established. Sensitivity and specificity of aldosterone concentration cutoffs to differentiate healthy from diseased ferrets were estimated with receiver operating characteristic curve analysis. RESULTS Measurements of serum aldosterone concentrations in the ferrets showed wide variability, with a median concentration of 4.75 pg/mL (interquartile range, 0.55 to 17.9 pg/mL; range, 0.02 to 283.9 pg/mL) and 76% (59/78) of samples having concentrations < 18 pg/mL. Ferrets that were healthy, older, or sexually inactive had significantly lower aldosterone concentrations. The upper limit of the reference interval for healthy ferrets was 13.3 pg/mL (90% confidence interval, 9.9 to 16.9 pg/mL). Analysis of receiver operating characteristic curves indicated that an aldosterone concentration cutoff value of 7.6 pg/mL differentiated healthy ferrets from diseased ferrets with a sensitivity of 72.7% and specificity of 73.2% (area under the curve, 0.79; 95% confidence interval, 0.67 to 0.91). CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that high aldosterone concentrations should not be considered diagnostic of primary hyperaldosteronism in ferrets. A need exists to develop better tests to identify primary hyperaldosteronism.

Uncontrolled proliferation is a key feature of tumor progression and malignancy. This suggests that cell-cycle related factors could be exploited as cancer biomarkers and that pathways specifically involved in the cell cycle, such as the Rb-E2F pathway, could be targeted as an effective anti-tumor therapy. We investigated 34 formalin-fixed paraffin-embedded (FFPE) tissue samples of canine cutaneous melanocytoma, cutaneous melanoma, and oral melanoma. Corresponding clinical follow-up data were used to determine the prognostic value of the mRNA expression levels of several cell cycle regulated E2F target genes (E2F1, DHFR, CDC6, ATAD2, MCM2, H2AFZ, GINS2, and survivin/BIRC5). Moreover, using four canine melanoma cell lines, we explored the possibility of blocking the Rb-E2F pathway by using a CDK4/6 inhibitor (Palbociclib) as a potential anti-cancer therapy. We investigated the expression levels of the same E2F target gene transcripts before and after treatment to determine the potential utility of these molecules as predictive markers. The E2F target gene H2AFZ was expressed in 91.43% of the primary tumors and H2AFZ expression was significantly higher in cases with unfavorable clinical outcome. Among the other tested genes, survivin/BIRC5 showed as well-promising results as a prognostic marker in canine melanoma. Three of the four tested melanoma cell lines were sensitive to the CDK4/6 inhibitor. The resistant cell line displayed higher expression levels of H2AFZ before treatment compared to the CDK4/6 inhibitor-sensitive cell lines. The present results suggest that CDK4/6 inhibitors could potentially be used as a new anti-cancer treatment for canine melanoma and that H2AFZ could serve as a prognostic and predictive marker for patient selection.

With a size range from 30 to 1000 nm, extracellular vesicles (EVs) are one of the smallest cell components able to transport biologically active molecules. They mediate intercellular communications and play a fundamental role in the maintenance of tissue homeostasis and pathogenesis in several types of diseases. In particular, EVs actively contribute to cancer initiation and progression, and there is emerging understanding of their role in creation of the metastatic niche. This fact underlies the recent exponential growth in EV research, which has improved our understanding of their specific roles in disease and their potential applications in diagnosis and therapy. EVs and their biomolecular cargo reflect the state of the diseased donor cells, and can be detected in body fluids and exploited as biomarkers in cancer and other diseases. Relatively few studies have been published on EVs in the veterinary field. This review provides an overview of the features and biology of EVs as well as recent developments in EV research including techniques for isolation and analysis, and will address the way in which the EVs released by diseased tissues can be studied and exploited in the field of veterinary pathology. Uniquely, this review emphasizes the important contribution that pathologists can make to the field of EV research: pathologists can help EV scientists in studying and confirming the role of EVs and their molecular cargo in diseased tissues and as biomarkers in liquid biopsies.

Simple SummaryIn virtually all human malignancies, the CDK-RB-E2F pathway is dysregulated resulting in the activation of the E2F transcriptional network. Rb and atypical E2Fs are the most important negative regulators of E2F-dependent transcription during tumorigenesis. However, it is unknown whether they cooporate or act independently in tumor development. Here we show that combined loss of RB and atypical E2Fs in mice enhances tumorigenesis in the liver, while in the pituitary gland, we observe inhibition of tumorigenesis. These findings suggest that the interaction between RB and atypical E2Fs in controlling tumorigenesis occurs in a tissue cell-type specific manner.E2F-transcription factors activate many genes involved in cell cycle progression, DNA repair, and apoptosis. Hence, E2F-dependent transcription must be tightly regulated to prevent tumorigenesis, and therefore metazoan cells possess multiple E2F regulation mechanisms. The best-known is the Retinoblastoma protein (RB), which is mutated in many cancers. Atypical E2Fs (E2F7 and -8) can repress E2F-target gene expression independently of RB and are rarely mutated in cancer. Therefore, they may act as emergency brakes in RB-mutated cells to suppress tumor growth. Currently, it is unknown if and how RB and atypical E2Fs functionally interact in vivo. Here, we demonstrate that mice with liver-specific combinatorial deletion of Rb and E2f7/8 have reduced life-spans compared to E2f7/8 or Rb deletion alone. This was associated with increased proliferation and enhanced malignant progression of liver tumors. Hence, atypical repressor E2Fs and RB cooperatively act as tumor suppressors in hepatocytes. In contrast, loss of either E2f7 or E2f8 largely prevented the formation of pituitary tumors in Rb+/- mice. To test whether atypical E2Fs can also function as oncogenes independent of RB loss, we induced long-term overexpression of E2f7 or E2f8 in mice. E2F7 and -8 overexpression increased the incidence of tumors in the lungs, but not in other tissues. Collectively, these data show that atypical E2Fs can promote but also inhibit tumorigenesis depending on tissue type and RB status. We propose that the complex interactions between atypical E2Fs and RB on maintenance of genetic stability underlie this context-dependency.

Background and Aims Up-regulation of the E2F-dependent transcriptional network has been identified in nearly every human malignancy and is an important driver of tumorigenesis. Two members of the E2F family, E2F7 and E2F8, are potent repressors of E2F-dependent transcription. They are atypical in that they do not bind to dimerization partner proteins and are not controlled by retinoblastoma protein. The physiological relevance of E2F7 and E2F8 remains incompletely understood, largely because tools to manipulate their activity in vivo have been lacking.Approach and Results Here, we generated transgenic mice with doxycycline-controlled transcriptional activation of E2f7 and E2f8 and induced their expression during postnatal development, in adulthood, and in the context of cancer. Systemic induction of E2f7 and, to lesser extent, E2f8 transgenes in juvenile mice impaired cell proliferation, caused replication stress, DNA damage, and apoptosis, and inhibited animal growth. In adult mice, however, E2F7 and E2F8 induction was well tolerated, yet profoundly interfered with DNA replication, DNA integrity, and cell proliferation in diethylnitrosamine-induced liver tumors.Conclusion Collectively, our findings demonstrate that atypical E2Fs can override cell-cycle entry and progression governed by other E2F family members and suggest that this property can be exploited to inhibit proliferation of neoplastic hepatocytes when growth and development have subsided during adulthood.

E-cadherin, a glycoprotein involved in cell-cell adhesion, has a pivotal role in epithelial-mesenchymal transition, a process through which neoplastic epithelial cells develop an invasive phenotype. In human cutaneous melanomas, decreased E-cadherin expression is associated with shorter survival and increased Breslow thickness, whereas in the dog its role is poorly understood. Tumor thickness and modified Clark level were recently proposed as useful features to assess canine melanocytic tumors, but no studies investigated their association with E-cadherin expression. We performed immunohistochemistry on 77 formalin-fixed, paraffin-embedded primary canine melanocytic tumors. A 3-tier and a 2-tier classification system for assessing E-cadherin expression were tested, with the latter being more informative for the assessment of canine melanocytic tumors. E-cadherin expression was lower in cutaneous melanomas than melanocytomas, as well as in amelanotic tumors compared to pigmented tumors. In amelanotic melanomas, absent E-cadherin expression was associated with an unfavorable outcome, suggesting a potential use of this marker in defining the prognosis of amelanotic melanomas. E-cadherin expression was lower in tumors with greater tumor thickness and modified Clark level ≥IV, suggesting its possible utility in identifying the most invasive tumors. The expression of E-cadherin in oral melanomas was heterogeneous, but was associated with pigmentation and clinical outcome; thus, E-cadherin evaluation could be advantageous to detect the most aggressive neoplasms. However, cutaneous melanomas without E-cadherin expression frequently had a favorable clinical outcome. Hence, its importance as prognostic factor should be carefully considered depending on the tumor origin.

Evidence is accumulating that tumour development is driven by cancer stem cells (CSCs). In order to understand the presence and potential contribution of stem cells (SCs) as tumour-initiating cells in canine cutaneous tumours, we selected three putative SC markers (Lgr5, Lgr6 and Sox9) and investigated their expression pattern, level of protein and mRNA expression, in 43 canine hair follicle (HF) and 18 canine cutaneous epidermal tumours by immunohistochemistry and qRT-PCR, using normal skin samples as controls. Lgr5 protein expression was not detected in epidermal and HF tumours, however, Lgr5 mRNA overexpression was evident in some HF tumours. Sox9 was expressed in several tumour cases, both at the protein and mRNA level. The Lgr6 antibody tested was not suitable for formalin-fixed paraffin-embedded tissue samples, but Lgr6 gene showed higher expression in several samples of both HF and epidermal tumours compared with normal skin. Significantly higher mRNA expression levels of the three SC markers were found in trichoblastomas (TB) compared with basal cell carcinomas (BCC). The present results indicated that canine HF and epidermal tumours might have common tumour-initiating cells. The mRNA expression of the three selected SC markers, especially Lgr5, could be potentially useful in the distinction between canine TB and BCC.

Combined_supplemental_materials-Silvestri_et_al for E-Cadherin Expression in Canine Melanocytic Tumors: Histological, Immunohistochemical, and Survival Analysis by Serenella Silvestri, Ilaria Porcellato, Luca Mechelli, Laura Menchetti, Selina Iussich, Raffaella De Maria, Monica Sforna, Laura Bongiovanni and Chiara Brachelente in Veterinary Pathology

Many studies have focused on Intellectual Capital (IC) applied to the Third Sector in the past few years. Despite the growing interest in intellectual capital in the field, the concept remains unclear. Few scholars and practitioners deal with the subject, however, as far as we know there are no studies that show the relationship between social impact generated by non-profit organizations and IC. This is the first study to be focused on this topic. This paper aims to fill the gap in the literature and demonstrate the relation between social impact and IC in the Social Work Integration Cooperatives (SWICs). This paper contributes to the literature by theoretically arguing that the measurement of social value improves SWICs’ economic value as a consequence of improvements of relationships and trust with external stakeholders (intangible assets). To ground our theoretical hypothesis, we measure the social impact value achieved by Italian SWICs through an aggregate analysis. That is the starting point and the findings can generate further research from both non-profit practitioners and scholars through the measurement of hypotheses over time.

The study of the immune response in several types of tumours has been rapidly increasing in recent years with the dual aim of understanding the interactions between neoplastic and immune cells and their importance in cancer pathogenesis and progression, as well as identifying targets for cancer immunotherapy. Despite being considered one of the most immunogenic tumour types, melanoma can progress in the presence of abundant lymphocytic infiltration, therefore suggesting that the immune response is not able to efficiently control tumour growth. The purpose of this study was to investigate whether the density, distribution and grade of tumour-infiltrating lymphocytes (TILs) in 97 canine melanocytic tumours is associated with histologic indicators of malignancy and can be considered a prognostic factor in the dog. As a further step in the characterization of the immune response in melanocytic tumours, an immunohistochemical investigation was performed to evaluate the two main populations of TILs, T-lymphocytes (CD3+ ) and B-lymphocytes (CD20+ ). The results of our study show that TILs are present in a large proportion of canine melanocytic tumours, especially in oral melanomas, and that the infiltrate is usually mild. The quantity of CD20+ TILs was significantly associated with some histologic prognostic factors, such as the mitotic count, the cellular pleomorphism and the percentage of pigmented cells. Remarkably, a high infiltration of CD20+ TILs was associated with tumour-related death, presence of metastasis/recurrence, shorter overall and disease-free survival, increased hazard of death and of developing recurrence/metastasis, hence representing a potential new negative prognostic factor in canine melanocytic tumours.