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Background: Alopecia X in Pomeranians is caused by a hair cycle deregulation, associated with downregulation of key regulatory genes of the Wnt and Shh pathways, and stem‐cell markers. However, the pathogenesis remains unclear. p63 is an important transcription factor correlated with the aforementioned hair cycle modulating genes. Hypothesis/Objectives: The aim of this study was to highlight possible changes of p63 immunohistochemical expression within the hair follicles in canine alopecia X compared with normal skin. Animals: Skin biopsies from 19 alopecia X‐affected and six control Pomeranians were analysed. Materials and Methods: Serial histological sections of skin biopsies harbouring anagen, telogen and kenogen hair follicles were immunohistochemically evaluated for differences in p63 expression in the affected and control samples. Results: Dogs with alopecia X had a significantly decreased immunoexpression of p63 in telogen and kenogen hair follicles. Conclusions and Clinical Relevance: The decrease of p63 immunoexpression observed in canine alopecia X suggests an involvement of p63 in hair cycle. [ABSTRACT FROM AUTHOR]

Simple Summary: Melanoma represents only 1% of human skin cancers, but in several cases can lead to the death of the patient. Nowadays, there are different systemic therapies used for the treatment of human melanoma. Although these substantially improve patients' lifespan, they are still associated with resistance. Extracellular vesicles (EVs), tiny vesicles released by tumor cells involved in intercellular communication, play an important role in melanoma pathogenesis and progression. They are crucially involved in several mechanisms of cancer drug resistance in several types of cancer, and there is a strong indication that EVs released by melanoma cells might play a role in the development of resistance, modulating the response towards anti-cancer drugs. Understanding their role will help improve the outcome of melanoma treatment. Extracellular vesicles (EVs) are involved in the pathogenesis of neoplastic diseases. Their role in mediating drug resistance has been widely described in several types of cancers, including melanoma. EVs can mediate drug resistance through several different mechanisms, such as drug-sequestration, transfer of pro-survival proteins and RNA, induction of cancer stem cell-like features and interaction with cells of the tumor microenvironment and immune-system. Melanoma is a highly immunogenic tumor originating from the malignant transformation of melanocytes. Several therapeutic strategies currently used in the treatment of melanoma and the combination of BRAF and MEK-inhibitors, as well as immune check-point inhibitors (ICI), have consistently improved the overall survival time of melanoma patients. However, the development of resistance is one of the biggest problems leading to a poor clinical outcome, and EVs can contribute to this. EVs isolated from melanoma cells can contain "sequestered" chemotherapeutic drugs in order to eliminate them, or bioactive molecules (such as miRNA or proteins) that have been proven to play a crucial role in the transmission of resistance to sensitive neoplastic cells. This leads to the hypothesis that EVs could be considered as resistance-mediators in sensitive melanoma cells. These findings are a pivotal starting point for further investigations to better understand EVs' role in drug resistance mechanisms and how to target them. The purpose of this review is to summarize knowledge about EVs in order to develop a deeper understanding of their underlying mechanisms. This could lead to the development of new therapeutic strategies able to bypass EV-mediated drug-resistance in melanoma, such as by the use of combination therapy, including EV release inhibitors. [ABSTRACT FROM AUTHOR]

One of the primary objectives of the Oncology Pathology Working Group (OPWG) is for oncologists and pathologists to collaboratively generate consensus documents to standardize aspects of and provide guidelines for veterinary oncologic pathology. Consensus is established through review of relevant peer‐reviewed literature relative to a subgroup's particular focus. In this article, the authors provide a critical review of the current literature for the diagnosis of, and histopathologic prognostication for, canine cutaneous and oral/lip melanocytic neoplasms, suggest guidelines for reporting, provide recommendations for clinical interpretation, and discuss future directions. This document represents the opinions of the working group and the authors and does not constitute a formal endorsement by the American College of Veterinary Pathologists, American College of Veterinary Internal Medicine or the Veterinary Cancer Society. [ABSTRACT FROM AUTHOR]

Circulating nucleic acids and extracellular vesicles (EV) represent novel biomarkers to diagnose cancer. The non‐invasive nature of these so‐called liquid biopsies provides an attractive alternative to tissue biopsy‐based cancer diagnostics. This study aimed to investigate if circulating cell cycle‐related E2F target transcripts can be used to diagnose tumours in canine tumour patients with different types of tumours. Furthermore, we assessed if these mRNAs are localised within circulating EV. We isolated total RNA from the plasma of 20 canine tumour patients and 20 healthy controls. Four E2F target genes (CDC6, DHFR, H2AFZ and ATAD2) were selected based on the analysis of published data of tumour samples available in public databases. We performed reverse transcription and quantitative real‐time PCR to analyse the plasma levels of selected E2F target transcripts. All four E2F target transcripts were detectable in the plasma of canine tumour patients. CDC6 mRNA levels were significantly higher in the plasma of canine tumour patients compared to healthy controls. A subset of canine tumour patient and healthy control plasma samples (n = 7) were subjected to size exclusion chromatography in order to validate association of the E2F target transcripts to circulating EV. For CDC6, EV analysis enhanced their detectability compared to total plasma analysis. In conclusion, our study reveals circulating CDC6 as a promising non‐invasive biomarker to diagnose canine tumours. [ABSTRACT FROM AUTHOR]

CASE DESCRIPTION A 5-year-old male Dwarf rabbit and 4-year-old female Mini-Rex rabbit were evaluated because of anorexia and urine scalding of the perineum. CLINICAL FINDINGS Abdominal radiography revealed a diffuse increase in the opacity of the urinary bladder attributable to urinary sludge. In I rabbit, abdominal ultrasonography revealed several mass-like lesions protruding from the mucosal surface into the lumen of the urinary bladder. Rabbits were anesthetized, and cystoscopy was performed with a rigid 2.7-mm, 30° endoscope. Histologic analysis of tissue samples obtained through the cystoscope operating channel revealed findings consistent with polypoid cystitis. TREATMENT AND OUTCOME To remove the urinary sludge from each rabbit, the urinary bladder was filled with sterile saline (0.9% NaCI) solution and emptied with a gentle massage several times until the ejected fluid was transparent. Rabbits were treated with NSAIDs, antimicrobials (chosen following microbial culture of urine and antimicrobial susceptibility testing), bathing of the perineum, and a low-calcium diet. The male rabbit died of unrelated causes 18 months later; postmortem examination findings confirmed the polypoid cystitis. The female rabbit remained disease free through to last follow-up (12 months after initial evaluation). CLINICAL RELEVANCE This was the first report of polypoid cystitis in pet rabbits. Although ultrasonographic findings supported this diagnosis, a definitive diagnosis was achieved through cystoscopy and lesion biopsy. Treatments administered were intended to reduce the potential sources of irritation. Research is needed to investigate the effectiveness of the applied interventions and the association between excessive urinary calcium excretion and polyploid cystitis in rabbits. [ABSTRACT FROM AUTHOR]

Background Recent investigations have highlighted the controversial role of Wnt/β-catenin pathway activation in human cutaneous melanoma. Survivin has been proposed as a valid prognostic marker for invasive and metastatic melanomas and lymph node melanoma metastasis in human cutaneous melanoma and is a promising therapeutic target. Hypothesis/Objectives Our aim was to investigate the immunohistochemical expression of survivin and β-catenin in canine cutaneous melanocytic tumours, in order to understand their prognostic significance. Methods Twenty-one melanocytic tumours (10 melanocytomas and 11 melanomas) were investigated by immunohistochemistry using specific anti-survivin and anti-β-catenin antibodies. A semi-quantitative method was used to analyse the results; β-catenin immunolabelling in neoplastic cells was evaluated as cytoplasmic, membranous or nuclear. The number of survivin-positive cells was counted within ~1000 neoplastic cells. Results were related to histopathological features, evaluated in haematoxylin- and eosin-stained slides, and to the clinical data obtained through a telephone survey with referring veterinarians. Results Despite a low level of expression in the majority of cases, β-catenin was found to be correlated strongly with malignant behaviour ( P < 0.01). An overexpression of nuclear survivin was statistically related to histological features of malignancy, presence of metastasis and death related to melanoma spread ( P < 0.01). Conclusions and clinical importance The low nuclear β-catenin expression, mainly found in metastatic cases, would indicate that β-catenin activation may have only limited importance in the development or progression of canine cutaneous melanoma. The correlation of nuclear survivin expression with malignancy would indicate that survivin is possibly a useful prognostic marker and therapeutic target in canine melanoma patients. [ABSTRACT FROM AUTHOR]

Background Survivin has been identified as one of the most cancer-specific molecules, with a dual function of apoptosis inhibitor and orchestrator of cell division. Hypothesis/Objectives Based on our recent results obtained during the study of the role of survivin in epithelial-to-mesenchymal transition, we investigate its potential role in maintenance of stemness in both the normal canine hair follicle and related tumours. Methods We performed a simultaneous evaluation, by immunofluorescence, of the expression of survivin and CK15. CK15 was selected as a marker for epidermal and hair follicle stem cells, based on its ability to identify hair follicle stem cells in the normal hair follicle and in canine follicular tumours. In this study, six cases were selected from the cases of hair follicle tumours evaluated in previous studies, based on the highest immunoreactivity for survivin and CK15. Three samples of healthy canine skin were also included as a normal control. Results A partial co-localization of the molecules was observed in normal hair follicles, as well as in trichoepitheliomas and trichoblastomas. In particular, a different co-expression was observed in relationship to the hair follicle cycle stage. Conclusions and clinical importance These findings suggest that survivin could play an important role in the maintenance of the hair follicle cycle as well as in tumour initiation and maintenance of cancer stem cells. [ABSTRACT FROM AUTHOR]

Background - Sebaceous glands are specialized cutaneous adnexal glands, which work under constant hormonal control to produce sebum. They can give rise to several proliferative lesions, such as hamartoma, hyperplasia and neoplasms (adenoma, epithelioma and carcinoma). Their nomenclature is currently confusing, both in veterinary and in human medicine, owing to the difficulty of differentiating between some of these lesions. Methods - The present study used immunohistochemistry to determine the expression levels and patterns of survivin and Ki67 in five samples of normal canine skin and 44 cases of canine cutaneous lesions with sebaceous differentiation (10 hamartomas, nine hyperplasia, eight adenomas, eight epitheliomas and nine carcinomas). Results - In normal glands, survivin, as well as Ki67, was expressed in scattered reserve cells. In hamartomas, survivin was more highly expressed than in normal skin, indicating a possible role of this molecule in the pathogenesis of these congenital lesions. In tumours, a moderate or high level of survivin and Ki67 expression (more than two and four and more than two positive cells, respectively) were significantly correlated with a malignant histotype, infiltrative growth and a moderate or high number of mitoses (more than two). Conclusions and clinical importance - The level of survivin expression increased with increasing malignancy, designating survivin as a new diagnostic marker in the assessment of malignancy of sebaceous tumours. [ABSTRACT FROM AUTHOR]

Background: Osteosarcoma (OSA) represents the most common canine primary bone tumour. Despite several pathways have been investigated so far, few molecules have been identified as prognostic tools or potential therapeutic targets, and there is still the need to find out molecular pathways with specific influence over OSA progression to facilitate earlier prognosis and treatment. Aims of the present study were to evaluate the immunohistochemical pattern and levels of expression of a panel of molecules (survivin, β-catenin, caspase 3 -inactive and active forms- and p53) involved in cell cycle and apoptosis regulation in canine OSA samples, known to be of interest in the study also of human OSA, and to detect specific relations among them and with histological tumour grade, disease free interval (DFI) and overall survival (OS). Results: Nuclear β-catenin immunostaining was detected in normal osteoblasts adjacent to the tumour, and in 47% of the cases. Cytoplasmic and/or membranous immunostaining were also observed. Nuclear survivin and p53 positive cells were found in all cases. Moderate/high cytoplasmic β-catenin expression (≥10% positive cells) was significantly associated with the development of metastasis (P = 0.014); moderate/high nuclear p53 expression (≥10% positive cells) was significantly associated with moderate/high histological grade (P = 0.017) and shorter OS (P = 0.049). Moderate/high nuclear survivin expression (≥15% positive cells) showed a tendency toward a longer OS (P = 0,088). Conclusions: The present results confirmed p53 as negative prognostic marker, while suggested survivin as a potential positive prognostic indicator, rather than indicative of a poor prognosis. The detection of nuclear β-catenin immunostaining in normal osteoblasts and the absent/low expression in most of the OSAs, suggested that this pathway could not play a major role in oncogenic transformation of canine osteoblasts. Further studies are needed to confirm these hypotheses. [ABSTRACT FROM AUTHOR]

Zinc deficiency causes skin diseases both in humans and in animals. The underlying pathogenic mechanisms remain unclear, but a growing body of evidence indicates a role for zinc in skin protection against free radical-induced oxidative damage. The immunohistochemical expression of heat shock proteins (HSPs; Hsp27, Hsp72, Hsp73 and Hsp90), Cu/Zn superoxide dismutase (SOD), metallothionein (MT), Ki-67 antigen and active caspase-3 were evaluated in normal canine skin and in samples from eight dogs with zinc-responsive dermatosis. All investigated HSPs showed intense cytoplasmic immunostaining in the affected epidermis. Focal nuclear positivity of Hsp72 was also detected in keratinocytes. Although Cu/Zn SOD expression was similar to that observed in normal skin, MT immunoreactivity occurred in both the cytoplasm and the nucleus of basal cells in normal skin but was absent from the affected epidermis. Caspase-3 activation was also absent in the involved epidermis, which revealed a high Ki-67 index (a 3.5- to 9-fold increase compared with normal skin). These results support the hypothesis that cellular response to stress, particularly oxidative stress, is involved in the pathogenesis of skin lesions in canine zinc-responsive dermatosis. The lack of MT immunoreactivity in the affected epidermis may be indicative of low zinc levels, thus resulting in vulnerability to oxidative damage. In contrast, high expression levels of HSPs in skin during zinc deficiency may confer protection against a variety of dangerous stimuli, contributing to inhibition of apoptosis and to cell cycle regulation of proliferating keratinocytes. [ABSTRACT FROM AUTHOR]

Survivin, a member of the inhibitor of apoptosis protein (IAP) family, is ubiquitously expressed during tissue development, undetectable in most normal tissues, but re-expressed in most cancers, including skin malignancies. Expression of survivin was evaluated retrospectively in 19 canine cutaneous squamous cell carcinomas (SCCs; one in situ; 16 well differentiated; one invasive, one lymph node metastasis) and 19 well differentiated SCCs from human beings. Seven specimens of normal canine skin were included. Immunohistochemical expression of full-length survivin was determined using a commercially available antibody. In addition, apoptotic rate [Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labelling index (TUNEL) index] and mitotic index (MI), counting mitoses in 10 high power fields (HPF), were determined. Scattered survivin positive nuclei were identified in the epidermal basal cell layer of normal canine skin. Nuclear survivin expression was identified in 18 of 19 human and in all canine SCCs, mainly along the base of the tumour cell population. Cytoplasmic survivin expression was rarely observed in human SCCs and in 84.2% of canine SCCs. The TUNEL index ranged from 0.1 to 2.6 in human beings and from 7.5 to 69.4 in dogs, while MIs ranged from 0 to 4 in human beings and dogs. No correlation was found between survivin expression and apoptotic or mitotic rates. Canine and human tumours showed similar nuclear survivin expression, indicating similar functions of the molecule. We demonstrated survivin expression in normal adult canine epidermis. Increased nuclear survivin expression in pre-neoplastic and neoplastic lesions demonstrates a possible association of survivin with development of SCCs in human beings and dogs. [ABSTRACT FROM AUTHOR]

Cell stress and death are linked in the neoplastic process, and heat shock proteins appear to play an important role by inhibiting apoptotic pathways. The apoptotic rates in 9 canine infundibular keratinizing acanthomas (IKAs) and 17 canine squamous cell carcinomas (SCCs) were correlated with the immunohistochemical expression of caspase-3 and the antiapoptotic heat shock proteins Hsp27, 72 and 73. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) method. The absence of a correlation between the TUNEL index and active-caspase-3 expression, a paucity of active-caspase-3-positive cells and Hsp72 over-expression were considered to be indicative of inhibition of apoptosis, and suggestive that inhibition of cell death plays a key role in oncogenesis and tumour growth of some canine skin neoplasms. [ABSTRACT FROM AUTHOR]

A 4-year-old, male cat was presented with a fixed, subcutaneous mass in the lumbosacral region. A histopathological examination revealed a well-defined but nonencapsulated neoplasm characterized by a proliferation of predominantly spindle cells, with high mitotic activity. Interspersed between these cells were single cellular elements with chondroid differentiation. Large areas of cartilaginous tissue with foci of endochondral ossification, necrosis and myxoid tissue were also observed within the neoplastic parenchyma. A diagnosis of extraskeletal mesenchymal chondrosarcoma was made based on the histological pattern– characterized by the coexistence of cartilaginous islands and undifferentiated mesenchymal cells, results of Alcian blue staining at various pH, immunohistochemical reactivity against vimentin and S-100, and the absence of skeletal involvement or other primary tumour sites. Clinical history of the cat excluded traumas, vaccinations or other types of subcutaneous inoculation. Six months on from surgical treatment, neither recurrence nor metastases have been detected. [ABSTRACT FROM AUTHOR]

Heat shock proteins (HSPs) are strongly implicated in the control of cell growth, differentiation and biological behaviour of many human cutaneous neoplasms. To our knowledge, no data have been published in the veterinary literature concerning either normal or neoplastic skin. In this study, the immunohistochemical expression of Hsp27, Hsp72 and Hsp73 was evaluated in normal canine skin, 14 intracutaneous cornifying epitheliomas (ICE), 10 well-differentiated and 5 moderately differentiated squamous cell carcinomas (SCC). Expression was correlated with the histological degree of keratinocyte differentiation and proliferation, and investigated as to its usefulness in the differential diagnosis of these canine tumours. In normal epidermis, Hsp27 exhibited cytoplasmic labelling in the spinous and granular layers, whereas in neoplastic tissues it was detected particularly in those areas showing squamous differentiation. Hsp72 immunoreactivity was more intense in ICE and well-differentiated SCC than in normal skin; however, reduced immunolabelling was observed in moderately differentiated SCC. Unlike Hsp72, Hsp73 showed less intense labelling in ICE and well-differentiated SCC than in normal epithelium and an increased positivity in moderately differentiated SCC. These results indicate that HSP immunoreactivity differs between normal and neoplastic canine skin. Hsp27 expression seems to correlate directly with cellular differentiation; by contrast, the involvement of Hsp72/73 in proliferation and differentiation of tumour cells remains controversial. The pattern and intensity of immunolabelling of each investigated HSP did not show, however, significant differences between ICE and SCC; therefore, they do not seem to be useful in the differential diagnosis of these two canine tumours. [ABSTRACT FROM AUTHOR]

The tumour suppressor PTEN is a negative regulator of the PI3K/AKT signalling pathway. Liver‐specific deletion of Pten in mice results in the hyper‐activation PI3K/AKT signalling accompanied by enhanced genome duplication (polyploidization), marked lipid accumulation (steatosis) and formation of hepatocellular carcinomas. However, it is unknown whether polyploidization in this model has an impact on the development of steatosis and the progression towards liver cancer. Here, we used a liver‐specific conditional knockout approach to delete Pten in combination with deletion of E2f7/8, known key inducers of polyploidization. As expected, Pten deletion caused severe steatosis and liver tumours accompanied by enhanced polyploidization. Additional deletion of E2f7/8 inhibited polyploidization, alleviated Pten‐induced steatosis without affecting lipid species composition and accelerated liver tumour progression. Global transcriptomic analysis showed that inhibition of polyploidization in Pten‐deficient livers resulted in reduced expression of genes involved in energy metabolism, including PPAR‐gamma signalling. However, we find no evidence that deregulated genes in Pten‐deficient livers are direct transcriptional targets of E2F7/8, supporting that reduction in steatosis and progression towards liver cancer are likely consequences of inhibiting polyploidization. Lastly, flow cytometry and image analysis on isolated primary wildtype mouse hepatocytes provided further support that polyploid cells can accumulate more lipid droplets than diploid hepatocytes. Collectively, we show that polyploidization promotes steatosis and function as an important barrier against liver tumour progression in Pten‐deficient livers. [ABSTRACT FROM AUTHOR]

Background - Epithelial-to-mesenchymal transition (EMT) is a multistep process, important in tumour invasion and metastasis, characterized by loss of epithelial markers, redistribution of β-catenin and gain of mesenchymal markers. Hyposthesis/Objectives - Our aim was to investigate the immunohistochemical aberrant expression of cytokeratin, vimentin, survivin and heat shock protein 72 (Hsp72) in canine cutaneous epithelial tumours, to understand the association of expression of these molecules with features of malignancy and their role in the EMT phenotype. Methods - Ten canine squamous cell carcinomas (SCCs; one with lymph node metastasis), 30 canine hair follicle tumours (six pilomatricomas, eight infundibular keratinizing acanthomas, six trichoepitheliomas and 10 trichoblastomas) and five normal skin samples were investigated by immunohistochemistry using specific anti-vimentin, -cytokeratin, -survivin and -Hsp72 antibodies. A semi-quantitative method was used to analyse the results, as follows: 0 to <5%; ≥5 to <10%; ≥10 to <25%; and ≥25% of positive cells. Immunofluorescence was performed to investigate survivin-vimentin and survivin-Hsp72 colocalization in selected SCCs. Results - In malignant hair follicle tumours and SCCs, a reduced intensity of cytokeratin and increased survivin and Hsp72 expression were observed. In SCCs, loss of cytokeratin expression and vimentin immunolabelling, suggestive of the EMT phenotype, were evident in <5% of neoplastic cells in the front of tumour invasion. In the same areas, strong nuclear survivin and cytoplasmic Hsp72 staining was evident, often colocalizing. Only a few neoplastic cells in the front of tumour invasion showed vimentin-survivin colocalization. Conclusions and clinical importance - A possible simultaneous involvement of survivin and Hsp72 in tumour invasion and the multistep process of EMT of cutaneous epithelial tumours of dogs is suggested. [ABSTRACT FROM AUTHOR]

Hyperglycaemia in pregnancy (HIP) is a pregnancy complication characterized by mild to moderate hyperglycaemia that negatively impacts short‐ and long‐term health of mother and child. However, relationships between severity and timing of pregnancy hyperglycaemia and postpartum outcomes have not been systemically investigated. We investigated the impact of hyperglycaemia developing during pregnancy (gestational diabetes mellitus, GDM) or already present pre‐mating (pre‐gestational diabetes mellitus, PDM) on maternal health and pregnancy outcomes. GDM and PDM were induced in C57BL/6NTac mice by combined 60% high fat diet (HF) and low dose streptozotocin (STZ). Animals were screened for PDM prior to mating, and all underwent an oral glucose tolerance test on gestational day (GD)15. Tissues were collected at GD18 or at postnatal day (PN)15. Among HFSTZ‐treated dams, 34% developed PDM and 66% developed GDM, characterized by impaired glucose‐induced insulin release and inadequate suppression of endogenous glucose production. No increased adiposity or overt insulin resistance was observed. Furthermore, markers of non‐alcoholic fatty liver disease (NAFLD) were significantly increased in PDM at GD18 and were positively correlated with basal glucose levels at GD18 in GDM dams. By PN15, NAFLD markers were also increased in GDM dams. Only PDM affected pregnancy outcomes such as litter size. Our findings indicate that GDM and PDM, resulting in disturbances of maternal glucose homeostasis, increase the risk of postpartum NAFLD development, related to the onset and severity of pregnancy hyperglycaemia. These findings signal a need for earlier monitoring of maternal glycaemia and more rigorous follow‐up of maternal health after GDM and PDM pregnancy in humans. Key points: We studied the impact of high‐fat diet/streptozotocin induced hyperglycaemia in pregnancy in mice and found that this impaired glucose tolerance and insulin release.Litter size and embryo survival were compromised by pre‐gestational, but not by gestational, diabetes.Despite postpartum recovery from hyperglycaemia in a majority of dams, liver disease markers were further elevated by postnatal day 15.Maternal liver disease markers were associated with the severity of hyperglycaemia at gestational day 18.The association between hyperglycaemic exposure and non‐alcoholic fatty liver disease signals a need for more rigorous monitoring and follow‐up of maternal glycaemia and health in diabetic pregnancy in humans. [ABSTRACT FROM AUTHOR]

Simple Summary: Neuroblastoma mostly affects young children and despite intensive treatment, many children die of progressive disease. It remains challenging to identify those patients at risk. Analyzing blood, as liquid biopsies, is not invasive and can help to identify these patients. We studied whether RNA molecules can be detected in these liquid biopsies. In blood plasma, RNA can be free-floating or packed in small particles, 'extracellular vesicles'. We present a workflow to analyze this cell-free RNA from small volumes of blood plasma of children with neuroblastoma. We have used neuroblastoma-specific markers and markers involved in cell proliferation. These latter genes can be upregulated in many different tumor types. We demonstrate that both types of markers have a higher expression in patients with metastatic disease, compared to healthy controls and patients with localized disease. These findings are essential for future studies on cell-free RNA, hopefully leading to improved survival for these patients. Neuroblastoma affects mostly young children, bearing a high morbidity and mortality. Liquid biopsies, e.g., molecular analysis of circulating tumor-derived nucleic acids in blood, offer a minimally invasive diagnostic modality. Cell-free RNA (cfRNA) is released by all cells, especially cancer. It circulates in blood packed in extracellular vesicles (EV) or attached to proteins. We studied the feasibility of analyzing cfRNA and EV, isolated by size exclusion chromatography (SEC), from platelet-poor plasma from healthy controls (n = 40) and neuroblastoma patients with localized (n = 10) and metastatic disease (n = 30). The mRNA content was determined using several multiplex droplet digital PCR (ddPCR) assays for a neuroblastoma-specific gene panel (PHOX2B, TH, CHRNA3) and a cell cycle regulation panel (E2F1, CDC6, ATAD2, H2AFZ, MCM2, DHFR). We applied corrections for the presence of platelets. We demonstrated that neuroblastoma-specific markers were present in plasma from 14/30 patients with metastatic disease and not in healthy controls and patients with localized disease. Most cell cycle markers had a higher expression in patients. The mRNA markers were mostly present in the EV-enriched SEC fractions. In conclusion, cfRNA can be isolated from plasma and EV and analyzed using multiplex ddPCR. cfRNA is an interesting novel liquid biopsy-based target to explore further. [ABSTRACT FROM AUTHOR]

Survivin is a member of the inhibitor of apoptosis (IAP) protein family acting at the intersection between proliferation and cell survival. This protein exhibits low or undetectable expression in most adult tissues but is increased in the majority of cancers. Suggested to be one of the most cancer-specific proteins identified to date, survivin acts as a signalling node in tumour maintenance and, after first promising results, is now attracting increasing attention as a target in anti-cancer therapy. In the skin, survivin has been implicated in a number of pathological conditions such as psoriasis and tumours of melanocytic and epithelial origin. Its expression can correlate with tumour severity, metastasis and decreased patient survival and has been inversely correlated with the sensitivity to cytotoxic agents used in anti-cancer therapy. Survivin may also be of importance for normal epidermal homeostasis possibly supporting self-renewal of epidermal stem cells. In this review, the authors summarize and discuss current data of survivin in skin biology and provide a comprehensive compilation of survivin expression in skin pathologies with focus on future therapeutical use. [ABSTRACT FROM AUTHOR]

Simple Summary: Feline calicivirus (FCV) is a common viral pathogen affecting domestic cats, which is responsible for diverse clinical presentations, commonly including upper respiratory tract signs, oral ulcerations, and a fever. In addition, FCV infection can be associated with severe pneumonia, lameness, and virulent systemic disease. In this study, we clinically and pathologically describe a rare case of FCV-associated paw and mouth disease in a febrile household cat. The FCV strains detected in the animal and in an overtly healthy cohabiting cat were analyzed to assess the phenotype and antigenic properties. Feline calicivirus (FCV) infection in cats can led to several diverse clinical presentations, ranging from mild upper respiratory signs to virulent systemic disease. Herein, we report a paw and mouth disease case in a 7-year-old household cat due to an FCV infection. An asymptomatic cat living in the same household was also infected with FCV. Clinical and pathological investigations were combined with the molecular and phenotypical characterization of the FCV strains. The RNA of the FCV was detected using qualitative and quantitative reverse transcription (RT)-PCR assays, and FCV antigen was confirmed by immunohistochemistry. After the whole genome analysis, the strains detected in the two cats appeared to be genetically diverse from FCVs previously detected in association with paw and mouth disease and with virulent systemic disease. Interestingly, the isolates obtained in this study were resistant to low pH conditions and slightly susceptible to bile salts, but they were susceptible to a trypsin treatment, revealing a phenotype pattern that is different from that which has been observed for respiratory FCVs. [ABSTRACT FROM AUTHOR]

The elongated soft palate is an abnormality that characterizes most brachycephalic dogs and contributes to the brachycephalic obstructive airway syndrome (BOAS). Palatoplasty is routinely performed in brachycephalic dogs; several surgical techniques exist. The use of surgical instruments such as monopolar electrocoagulation, CO 2 or diode laser, bipolar vessel sealing device and harmonic shears has become routine to reduce the operating time, the intraoperative risk of bleeding and the postoperative oedema. This prospective study aimed to compare the histomorphological effect of a CO 2 laser and LigaSure device in palates of dogs undergoing palatoplasty. Twenty owned brachycephalic dogs were included, 10 palatoplasties were performed using CO 2 laser and 10 using LigaSure™ device. The dogs were positioned in sternal recumbency. A transoral approach was performed: the elongated soft palate was grasped with Allis forceps and brought rostrally, the palatoplasty was performed using the tonsillar crypts as anatomical landmarks. Surgical specimens were routinely fixed in 10 % formalin. Two sections perpendicular to the surgical margins were trimmed from each sample, paraffin-embedded and stained with hematoxylin and eosin (H&E). Tissue damage induced by the two types of surgical devices was graded (1–4, from minimal to severe) and the depth of thermal injury measured in μm on captured images (using an image analysis program - ImageJ). Mean values and standard deviations (SD) were calculated based on six measurements for each sample. The tissue damage was graded 3.7±0.48 in group LigaSure™ and 2.8±1 in group Laser. The mean depth of thermal injury was 874.94±184.92 μm in the LigaSure™ group and 451,76±137,86 μm in the Laser group. The comparison between the two groups showed significant lower grade and extension of thermal injury in the palate samples obtained with CO 2 laser (p<0.05). Additionally, there is a lack of literature that correlates the histological changes with the clinical outcomes of the different palatoplasty methods in brachycephalic dogs. By comparing histological changes and clinical outcomes, we aim to provide valuable insights for optimizing the surgical approach for palatoplasty in brachycephalic dogs, ultimately improving postoperative outcomes for these patients. • In all palate samples, indications of thermal injury were noted along the surgical incision margins induced by both devices. • Soft palate ablation by a CO 2 laser induced less thermal tissue damage than an excision using a LigaSure™ device. • Both devices had comparable clinical results and intra- and post-operative complication rates. [ABSTRACT FROM AUTHOR]

Insufficient vascularization is a recurring cause of impaired pedicled skin flap healing. The administration of adipose tissue-derived stromal cells' (ASCs') secretome is a novel approach to augment vascularization. Yet, the secretome comprised of soluble factors that require a sustained-release vehicle to increase residence time. We hypothesized that administration of a hydrogel derived from decellularized extracellular matrix (ECM) of porcine skin with bound trophic factors from ASCs enhances skin flap viability and wound repair in a rat model. Porcine skin was decellularized and pepsin-digested to form a hydrogel at 37°C. Conditioned medium (CMe) of human ASC was collected, concentrated 20-fold, and mixed with the hydrogel. Sixty Wistar rats were included. A dorsal skin flap (caudal based) of 3 × 10 cm was elevated for topical application of DMEM (group I), a prehydrogel with or without ASC CMe (groups II and III), or ASC CMe (group IV). After 7, 14, and 28 days, perfusion was measured, and skin flaps were harvested for wound healing assessment and immunohistochemical analysis. Decellularized skin ECM hydrogel contained negligible amounts of DNA (11.6 ± 0.6 ng/mg), was noncytotoxic and well tolerated by rats. Irrespective of ASC secretome, ECM hydrogel application resulted macroscopically and microscopically in similar dermal wound healing in terms of proliferation, immune response, and matrix remodeling as the control group. However, ASC CMe alone increased vessel density after 7 days. Porcine skin-derived ECM hydrogels loaded with ASC secretome are noncytotoxic but demand optimization to significantly augment wound healing of skin flaps. [ABSTRACT FROM AUTHOR]

Simple Summary: Extracellular vesicles (EVs) are small vesicles released by human and animal cells, parasites, microorganisms, and plants. They travel within bodily fluids, transferring the content of their cell of origin to other cells, being both intra- and inter-organism messengers. This EV-mediated method of communication governs many normal functions as well as disease processes. Because of this important role, EVs have been largely studied since 1984, mainly in humans, but more recently also in animals, parasites, and bacteria. In this review, we explore the literature on EVs in animals between 1984 and 2021 and summarize the most important results of approximately 220 scientific papers. Results are presented based on the main topic of research, such as EVs in physiology and pathophysiology, use of EVs as markers to diagnose diseases, or as possible natural transporters of therapies or vaccines. Since working with EVs is challenging, we also address the critical technical points found in the veterinary literature. Finally, we included a brief summary on EVs shed within animal milk, an area of large interest for the multiple applications for human health. Extracellular vesicles (EVs) are cell-derived membrane-bound vesicles involved in many physiological and pathological processes not only in humans but also in all the organisms of the eukaryotic and prokaryotic kingdoms. EV shedding constitutes a fundamental universal mechanism of intra-kingdom and inter-kingdom intercellular communication. A tremendous increase of interest in EVs has therefore grown in the last decades, mainly in humans, but progressively also in animals, parasites, and bacteria. With the present review, we aim to summarize the current status of the EV research on domestic and wild animals, analyzing the content of scientific literature, including approximately 220 papers published between 1984 and 2021. Critical aspects evidenced through the veterinarian EV literature are discussed. Then, specific subsections describe details regarding EVs in physiology and pathophysiology, as biomarkers, and in therapy and vaccines. Further, the wide area of research related to animal milk-derived EVs is also presented in brief. The numerous studies on EVs related to parasites and parasitic diseases are excluded, deserving further specific attention. The literature shows that EVs are becoming increasingly addressed in veterinary studies and standardization in protocols and procedures is mandatory, as in human research, to maximize the knowledge and the possibility to exploit these naturally produced nanoparticles. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: γ9δ2T cells, which express Vγ9 and Vδ2 chains of the T cell receptor (TCR), mediate cancer immune surveillance by sensing early metabolic changes in malignant leukemic blast and not their healthy hematopoietic stem counterparts via the γ9δ2TCR targeting joined conformational and spatial changes of CD277 at the cell membrane (CD277J). This concept led to the development of next generation CAR-T cells, so-called TEGs: αβT cells Engineered to express a defined γδTCR. The high affinity γ9δ2TCR clone 5 has recently been selected within the TEG format as a clinical candidate (TEG001). However, exploring safety and efficacy against a target, which reflects an early metabolic change in tumor cells, remains challenging given the lack of appropriate tools. Therefore, we tested whether TEG001 is able to eliminate established leukemia in a primary disease model, without harming other parts of the healthy hematopoiesis in vivo. Methods: Separate sets of NSG mice were respectively injected with primary human acute myeloid leukemia (AML) blasts and cord blood-derived human progenitor cells from healthy donors. These mice were then treated with TEG001 and mock cells. Tumor burden and human cells engraftment were measured in peripheral blood and followed up over time by quantifying for absolute cell number by flow cytometry. Statistical analysis was performed using non-parametric 2-tailed Mann-Whitney t-test. Results: We successfully engrafted primary AML blasts and healthy hematopoietic cells after 6-8 weeks. Here we report that metabolic cancer targeting through TEG001 eradicated established primary leukemic blasts in vivo, while healthy hematopoietic compartments derived from human cord-blood remained unharmed in spite of TEGs persistence up to 50 days after infusion. No additional signs of off-target toxicity were observed in any other tissues. Conclusion: Within the limitations of humanized PD-X models, targeting CD277J by TEG001 is safe and efficient. Therefore, we have initiated clinical testing of TEG001 in a phase I first-in-human clinical trial (NTR6541; date of registration 25 July 2017). [ABSTRACT FROM AUTHOR]

OBJECTIVE To explore sources of serum aldosterone concentration variability in a population of healthy and diseased ferrets, determine a preliminary I-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. [ABSTRACT FROM AUTHOR]

The presence of polyploid cells in the endocrine and exocrine pancreas has been reported for four decades. In rodents, pancreatic polyploidization is initiated after weaning and the number of polyploid cells increases with age. Surprisingly the molecular regulators and biological functions of polyploidization in the pancreas are still unknown. We discovered that atypical E2f activity is essential for polyploidization in the pancreas, using an inducible Cre/LoxP approach in new-born mice to delete ubiquitously the atypical E2f transcription factors, E2f7 and E2f8. In contrast to its critical role in embryonic survival, conditional deletion of both of both atypical E2fs in newborn mice had no impact on postnatal survival and mice lived until old age. However, deficiency of E2f7 or E2f8 alone was sufficient to suppress polyploidization in the pancreas and associated with only a minor decrease in blood serum levels of glucose, insulin, amylase and lipase under 4 hours starvation condition compared to wildtype littermates. In mice with fewer pancreatic polyploid cells that were fed ad libitum, no major impact on hormones or enzymes levels was observed. In summary, we identified atypical E2fs to be essential for polyploidization in the pancreas and discovered that postnatal induced loss of both atypical E2fs in many organs is compatible with life until old age. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to the article "Frustrations, Requirements and Expectations of Skin Biopsy for Diagnosing Skin Disease," by David Pinson in a previous issue.

Aelurostrongylus abstrusus is a metastrongyloid nematode infesting the respiratory system of domestic cats worldwide. Troglostrongylus brevior and Troglostrongylus subcrenatus, two lungworms thought to infest wild felids, have been found recently in domestic cats from Spain and Italy. These unexpected findings have raised doubts about the assumed past and present occurrence of Troglostrongylus spp., especially T. brevior, in domestic hosts and suggest that there may have been missed detection or misdiagnosis. The present retrospective study evaluated the presence of lungworms in cats from Italy with a diagnosis of respiratory parasitism or with compatible lung lesions from 2002 to 2013. Sixty-eight samples of DNA and larvae from cats with a diagnosis of aelurostrongylosis, and 53 formalin-fixed paraffin-embedded lung samples from cats confirmed as lungworm infested or with compatible lesions, were investigated using two DNA-based assays specific for A. abstrusus or T. brevior. All DNA and larval samples were positive for A. abstrusus and one was additionally positive for T. brevior. Most paraffin-embedded lung tissues were positive only for A. abstrusus, but two samples tested positive for both lungworms and one for T. brevior only. This study supports the major role of A. abstrusus in causing feline respiratory parasitism in endemic areas of Italy. [ABSTRACT FROM AUTHOR]

Background A novel bivalent vaccine to protect against myxomatosis and rabbit haemorrhagic disease is commercially available for pet rabbits. Hypothesis/Objectives To describe the appearance of cutaneous lesions arising in pet rabbits positive for myxoma virus (MV) by RT-PCR evaluation shortly after vaccination. Animals Four pet rabbits presenting with papular, crusting skin lesions ~10 days after vaccination. Methods Histological evaluation of formalin-fixed skin biopsies obtained from lesional skin (case 1). Real-time polymerase chain reaction (RT-PCR) evaluation of paraffin-embedded tissue from skin biopsies (case 1) and crusts obtained from the lesion surface (cases 2-4) for myxoma virus are reported as cycle threshold ( Ct) values. Results Lesions affecting the ear pinna, dorsal aspect of the nose, vulva and/or conjunctiva are reported. Histopathological findings included severe ulcerative, necrotizing dermatitis and intralesional cytoplasmic inclusion bodies in myxoma cells. DNA was amplified from all the paraffin-embedded skin biopsies ( Ct = 34-35) and crusts ( Ct = 20-24). Conclusions and clinical importance Although a wild virus challenge cannot be definitively excluded, veterinarians and pet-owners should be aware that cutaneous lesions have been observed after vaccination with this novel vaccine in low numbers of rabbits. [ABSTRACT FROM AUTHOR]

CPV1 (also called COPV) is a papillomavirus responsible for oral papillomatosis in young dogs. The involvement of this viral type in oral oncogenesis has been hypothesized in oral squamous cell carcinomas (SCCs), but has never been investigated in other neoplastic and hyperplastic oral lesions of dogs. Aim of this study was to investigate the presence of CPV1 in different neoplastic and hyperplastic lesions in order to assess its role in canine oral oncogenesis; according to the results obtained, a second aim of the study was to define if the dog can be considered a valid animal model for oral high risk HPV-induced tumors. Eighty-eight formalin-fixed, paraffin-embedded (FFPE) canine oral lesions including 78 oral tumors (papillomas, SCCs, melanomas, ameloblastomas, oral adenocarcinomas) and 10 hyperplastic lesions (gingival hyperplasia) were investigated with immunohistochemistry for the presence of papillomavirus L1 protein and with Real-Time PCR for CPV1 DNA. RT-PCR for RNA was performed on selected samples. All viral papillomas tested were positive for immunohistochemistry and Real-time PCR. In 3/33 (10%) SCCs, viral DNA was demonstrated but no viral RNA could be found. No positivity was observed both with immunohistochemistry and Real-Time PCR in the other hyperplastic and neoplastic lesions of the oral cavity of dogs. Even though the finding of CPV1 DNA in few SCCs in face of a negative immunohistochemistry could support the hypothesis of an abortive infection in the development of these lesions, the absence of viral RNA points out that CPV1 more likely represents an innocent bystander in SCC oncogenesis. The study demonstrates a strong association between CPV1 and oral viral papillomas whereas viral contribution to the pathogenesis of other oral lesions seems unlikely. Moreover, it suggests that a canine model of CPV1 infection for HPV-induced oncogenesis could be inappropriate. [ABSTRACT FROM AUTHOR]

Background - Although cutaneous stem cells have been implicated in skin tumourigenesis in humans, no studies have been conducted to elucidate the presence and the possible role of stem cells in hair follicle tumours in the dog. Hypothesis - Stem cell markers are expressed in canine epidermal and follicular tumours and can be used to better understand the biology and origin of these tumours. Animals and Methods - In the present study, normal skin sections and 44 follicular tumours were retrospectively investigated for the immunohistochemical expression of keratin 15 (K15) and nestin. In addition, 30 squamous cell carcinomas were evaluated for K15 expression. Results - In normal skin, K15 and nestin were expressed in the outer root sheath cells of the isthmic portion of the hair follicle (bulge region), and K15 expression was also scattered in the basal cell layer of the epidermis. Infundibular keratinizing acanthomas, pilomatricomas and squamous cell carcinomas were mostly negative for K15, trichoblastomas were moderately to strongly positive, tricholemmomas were either negative or strongly positive, and trichoepitheliomas had heterogeneous staining. Nestin expression was generally faint in all follicular tumours. Conclusions and clinical importance - Our results show that K15 can be a reliable marker for investigating the role of stem cells in hair follicle tumours of the dog, while nestin was judged to be a nonoptimal marker. Furthermore, our study suggests that hair follicle stem cells are present in the bulge region of hair follicles and could possibly play a role in tumourigenesis of canine tumours originating from this portion of the follicle, namely trichoblastomas, tricholemmomas and trichoepitheliomas. The loss of K15 expression in squamous cell carcinomas compared with normal skin suggests that this event could be important in the malignant transformation. [ABSTRACT FROM AUTHOR]

Platelet derived growth factor receptor (PDGFR)α and PDGFRβ are tyrosine kinase receptors that are over-expressed in 70-80% of human osteosarcomas (OSAs) and may be suitable therapeutic targets for specific kinase inhibitors (TKIs). Canine OSA shows histopathological and clinical features similar to human OSA, and is considered an excellent model in comparative oncology. This study investigated PDGF-A, PDGF-B, PDGFRα and PDGFRβ expression in 33 canine OSA samples by immunohistochemistry and in seven primary canine OSA cell lines by Western blot and quantitative PCR analysis. Immunohistochemical data showed that PDGF-A and PDGF-B are expressed in 42% and 60% of the OSAs analysed, respectively, while PDGFRα and PDGFRβ were expressed in 78% and 81% of cases, respectively. Quantitative PCR data showed that all canine OSA cell lines overexpressed PDGFRα, while 6/7 overexpressed PDGFRβ and PDGF-A relative to a normal osteoblastic cell line. Moreover, in vitro treatment with a specific PDGFR inhibitor, AG1296, caused a dose- and time-dependent decrease in AKT phosphorylation. Collectively, these data show that PDGFRs/PDGFs are co-expressed in canine osteosarcomas, which suggests that an autocrine and/or paracrine loop is involved and that they play an important role in the aetiology of OSA. PDGFRs may be suitable targets for the treatment of canine OSA with a specific TKI. [ABSTRACT FROM AUTHOR]

Simple Summary: In 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. [ABSTRACT FROM AUTHOR]

CD62L (L-Selectin) dependent lymphocyte infiltration is known to induce inflammatory bowel disease (IBD), while its function in the liver, especially in non-alcoholic steatohepatitis (NASH), remains unclear. We here investigated the functional role of CD62L in NASH in humans as well as in two mouse models of steatohepatitis. Hepatic expression of a soluble form of CD62L (sCD62L) was measured in patients with steatosis and NASH. Furthermore, CD62L−/− mice were fed with a methionine and choline deficient (MCD) diet for 4 weeks or with a high fat diet (HFD) for 24 weeks. Patients with NASH displayed increased serum levels of sCD62L. Hepatic CD62L expression was higher in patients with steatosis and increased dramatically in NASH patients. Interestingly, compared to wild type (WT) mice, MCD and HFD-treated CD62L−/− mice were protected from diet-induced steatohepatitis. This was reflected by less fat accumulation in hepatocytes and a dampened manifestation of the metabolic syndrome with an improved insulin resistance and decreased cholesterol and triglyceride levels. Consistent with ameliorated disease, CD62L−/− animals exhibited an enhanced hepatic infiltration of Treg cells and a strong activation of an anti-oxidative stress response. Those changes finally resulted in less fibrosis in CD62L−/− mice. Additionally, this effect could be reproduced in a therapeutic setting by administrating an anti-CD62L blocking antibody. CD62L expression in humans and mice correlates with disease activity of steatohepatitis. CD62L knockout and anti-CD62L-treated mice are protected from diet-induced steatohepatitis suggesting that CD62L is a promising target for therapeutic interventions in NASH. [ABSTRACT FROM AUTHOR]

E2F transcription factors control the cytokinesis machinery and thereby ploidy in hepatocytes. If or how these proteins limit proliferation of polyploid cells with extra centrosomes remains unknown. Here, we show that the PIDDosome, a signaling platform essential for caspase-2-activation, limits hepatocyte ploidy and is instructed by the E2F network to control p53 in the developing as well as regenerating liver. Casp2 and Pidd1 act as direct transcriptional targets of E2F1 and its antagonists, E2F7 and E2F8, that together co-regulate PIDDosome expression during juvenile liver growth and regeneration. Of note, whereas hepatocyte aneuploidy correlates with the basal ploidy state, the degree of aneuploidy itself is not limited by PIDDosome-dependent p53 activation. Finally, we provide evidence that the same signaling network is engaged to control ploidy in the human liver after resection. Our study defines the PIDDosome as a primary target to manipulate hepatocyte ploidy and proliferation rates in the regenerating liver. • The PIDDosome controls hepatocyte ploidy during postnatal development & regeneration • The PIDDosome defines the speed of liver regeneration posthepatectomy • Aneuploidy in the liver correlates with basal ploidy state but is not limited by CASP2 • E2F family members regulate expression of CASP2 and PIDD1 for liver ploidy control Sladky et al. report a key role for the PIDDosome in regulating p53 activation to limit hepatocyte polyploidy during juvenile liver growth and regeneration. Expression of essential PIDDosome components is controlled by a E2F-family regulated circuitry. The study defines the PIDDosome as a putative target to enhance liver regeneration. [ABSTRACT FROM AUTHOR]

During fasting, mitochondrial fatty-acid β-oxidation (mFAO) is essential for the generation of glucose by the liver. Children with a loss-of-function deficiency in the mFAO enzyme medium-chain acyl-Coenzyme A dehydrogenase (MCAD) are at serious risk of life-threatening low blood glucose levels during fasting in combination with intercurrent disease. However, a subset of these children remains asymptomatic throughout life. In MCAD-deficient (MCAD-KO) mice, glucose levels are similar to those of wild-type (WT) mice, even during fasting. We investigated if metabolic adaptations in the liver may underlie the robustness of this KO mouse. WT and KO mice were given a high- or low-fat diet and subsequently fasted. We analyzed histology, mitochondrial function, targeted mitochondrial proteomics, and transcriptome in liver tissue. Loss of MCAD led to a decreased capacity to oxidize octanoyl-CoA. This was not compensated for by altered protein levels of the short- and long-chain isoenzymes SCAD and LCAD. In the transcriptome, we identified subtle adaptations in the expression of genes encoding enzymes catalyzing CoA- and NAD(P)(H)-involving reactions and of genes involved in detoxification mechanisms. We discuss how these processes may contribute to robustness in MCAD-KO mice and potentially also in asymptomatic human subjects with a complete loss of MCAD activity. [ABSTRACT FROM AUTHOR]