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Spontaneous infections of the preputial glands represent overlooked health problems in mice that could raise welfare concerns and potentially confound scientific experiments. Agents involved in preputial gland infections have rarely been investigated, with opportunistic pathogens of laboratory animals usually detected in inflamed preputial glands. The aim of this study was to investigate the prevalence of bacterial infection in the preputial glands and the relationship between haematological and pathological changes and infection status. We analysed 40 preputial glands from 20 one-year-old C57BL/6NCrl male mice by using bacteriology, haematology and pathology. Bacteria were isolated from 16/20 (80%) mice, for a total of 32/40 (80%) examined preputial glands. Enterobacter cloacae, Pasteurella spp., Klebsiella spp. and Staphylococcus aureus were identified in 35%, 17.5%, 15% and 12.5% of the examined glands, respectively. Preputial gland inflammation was identified in 29/40 (72.5%) glands and was classified as chronic interstitial adenitis in 27 cases and suppurative adenitis in the remaining two glands. No haematological changes were found in mice with infected glands. Histologically, the presence of intralesional bacteria, intraluminal necrotic material, intraluminal keratin accumulation, interstitial inflammatory cell infiltrate and granulocytes (intraluminal and/or interstitial), along with total inflammatory score and total histopathological score, were significantly increased in infected glands and correlated with the bacterial load. Most severe inflammatory changes were identified after S. aureus infection, while ductal hyperkeratosis was significantly increased in glands infected with Klebsiella spp. In conclusion, preputial gland infection was a common event in one-year-old C57BL/6NCrl mice, and bacterial load correlated with pathological findings, while systemic effects were not highlighted by haematology.

Background: HDL (high-density lipoprotein) and its major protein component, apoA-I (apolipoprotein A-I), play a unique role in cholesterol homeostasis and immunity. ApoA-I deficiency in hyperlipidemic, atheroprone mice was shown to drive cholesterol accumulation and inflammatory cell activation/proliferation. The present study was aimed at investigating the impact of apoA-I deficiency on lipid deposition and local/systemic inflammation in normolipidemic conditions. Methods: ApoE deficient mice, apoE/apoA-I double deficient (DKO) mice, DKO mice overexpressing human apoA-I, and C57Bl/6J control mice were fed normal laboratory diet until 30 weeks of age. Plasma lipids were quantified, atherosclerosis development at the aortic sinus and coronary arteries was measured, skin ultrastructure was evaluated by electron microscopy. Blood and lymphoid organs were characterized through histological, immunocytofluorimetric, and whole transcriptome analyses. Results: DKO were characterized by almost complete HDL deficiency and by plasma total cholesterol levels comparable to control mice. Only DKO showed xanthoma formation and severe inflammation in the skin-draining lymph nodes, whose transcriptome analysis revealed a dramatic impairment in energy metabolism and fatty acid oxidation pathways. An increased presence of CD4 + T effector memory cells was detected in blood, spleen, and skin-draining lymph nodes of DKO. A worsening of atherosclerosis at the aortic sinus and coronary arteries was also observed in DKO versus apoE deficient. Human apoA-I overexpression in the DKO background was able to rescue the skin phenotype and halt atherosclerosis development. Conclusions: HDL deficiency, in the absence of hyperlipidemia, is associated with severe alterations of skin morphology, aortic and coronary atherosclerosis, local and systemic inflammation.

Alterations in extracellular matrix (ECM) components that modulate inflammatory cell behavior have been shown to serve as early starters for multifactorial diseases such as fibrosis and cancer. Here, we demonstrated that loss of the ECM glycoprotein EMILIN-1 alters the inflammatory context in skin during IMQ-induced psoriasis, a disease characterized by a prominent inflammatory infiltrate and alteration of vessels that appear dilated and tortuous. Abrogation of EMILIN-1 expression or expression of the EMILIN-1 mutant E933A impairs macrophage polarization and leads to imbalanced tissue homeostasis. We found that EMILIN-1 deficiency is associated with dilated lymphatic vessels, increased macrophage recruitment and psoriasis severity. Importantly, the null or mutant EMILIN-1 background was characterized by the induction of a myofibroblast phenotype, which in turn drove macrophages towards the M1 phenotype. By using the transgenic mouse model carrying the E933A mutation in the gC1q domain of EMILIN-1, which abolishes the interaction with α4- and α9-integrins, we demonstrated that the observed changes in TGFβ signaling were due to both the EMI and gC1q domains of EMILIN-1. gC1q may exert multiple functions in psoriasis, in the context of a final, more consistent inflammatory condition by controlling skin homeostasis via interaction with both keratinocytes and fibroblasts, influencing non-canonical TGFβ signaling, and likely acting on lymphatic vessel structure and function. The analyses of human psoriatic lesions, in which lower levels of EMILIN-1 were present with a very rare association with lymphatic vessels, support the multifaceted role of this ECM component in the skin inflammatory scenario.

Background and purpose Fenretinide, a synthetic retinoid derivative first investigated for cancer prevention and treatment, has been shown to ameliorate glucose tolerance, improve plasma lipid profile and reduce body fat mass. These effects, together with its ability to inhibit ceramide synthesis, suggest that fenretinide may have an anti-atherosclerotic action. Experimental approach To this aim, nine-week-old apoE-knockout (EKO) female mice were fed for twelve weeks a Western diet, without (control) or with (0.1% w/w) fenretinide. As a reference, wild-type (WT) mice were treated similarly. Growth and metabolic parameters were monitored throughout the study. Atherosclerosis development was evaluated in the aorta and at the aortic sinus. Blood and lymphoid organs were further characterized with thorough cytological/histological and immunocytofluorimetric analyses. Key results Fenretinide treatment significantly lowered body weight, glucose levels and plasma levels of total cholesterol, triglycerides, and phospholipids. In the liver, fenretinide remarkably reduced hepatic glycogenosis and steatosis driven by the Western diet. Treated spleens were abnormally enlarged, with severe follicular atrophy and massive extramedullary haematopoiesis. Severe renal hemosiderin deposition was observed in treated EKO mice. Treatment resulted in a threefold increase of total leukocytes (WT and EKO) and raised the activated/resting monocyte ratio in EKO mice. Finally, atherosclerosis development was markedly increased at the aortic arch, thoracic and abdominal aorta of fenretinide-treated mice. Conclusions and implications We provide the first evidence that, despite beneficial metabolic effects, fenretinide treatment may enhance the development of atherosclerosis.

The recent development of mouse cages equipped with monitoring wireless technology raised questions on the potential effects on animals induced by electromagnetic fields (EMFs) generated by electronic boards positioned underneath the cages. The aims of this study were to characterize the EMF produced by digitally ventilated cages (DVC) and perform a clinicopathological study on mice maintained in DVC for up to 1 year. The EMFs were measured in empty individually ventilated cages (IVC) and DVC. Male (n = 160) and female (n = 160) C57BL/6NCrl mice were randomly housed in IVC and DVC in a single rack, 4 mice per cage. Body weight and food and water consumption were recorded at 14-day intervals. At sacrifice (days 60, 120, 180, and 365), body and testes weight was measured, and necropsy, hematology, bone marrow cytology, histology, and immunohistochemistry for cleaved-caspase 3 on the testes were performed. Digitally ventilated cages produced extremely low-intensity electric fields ranging from 5 Hz to 3 GHz. No exposure-related clinical signs and mortality occurred. Occasional statistical differences in body weight, food and water consumption, hematology, bone marrow, and histopathology were recorded, but considered without biological or clinical relevance. In conclusion, long-term maintenance in DVC had no definite effects on C57BL/6NCrl mice.

Background Nonsteroidal anti-inflammatory drugs (NSAIDs) have been proposed as chemopreventive agents for many tumours; however, the mechanism responsible for their anti-neoplastic activity remains elusive and the side effects due to cyclooxygenase (COX) inhibition prevent this clinical application. Methods Molecular biology, in silico, cellular and in vivo tools, including innovative in vivo imaging and classical biochemical assays, were applied to identify and characterise the COX-independent anti-cancer mechanism of NSAIDs. Results Here, we show that tumour-protective functions of NSAIDs and exisulind (a sulindac metabolite lacking anti-inflammatory activity) occur through a COX-independent mechanism. We demonstrate these NSAIDs counteract carcinogen-induced proliferation by inhibiting the sirtuin 1 (SIRT1) deacetylase activity, augmenting acetylation and activity of the tumour suppressor p53 and increasing the expression of the antiproliferative gene p21. These properties are shared by all NSAIDs except for ketoprofen lacking anti-cancer properties. The clinical interest of the mechanism identified is underlined by our finding that p53 is activated in mastectomy patients undergoing intraoperative ketorolac, a treatment associated with decreased relapse risk and increased survival. Conclusion Our study, for the first-time, links NSAID chemopreventive activity with direct SIRT1 inhibition and activation of the p53/p21 anti-oncogenic pathway, suggesting a novel strategy for the design of tumour-protective drugs.

Primary vasopressor efficacy of epinephrine during cardiopulmonary resuscitation (CPR) is due to its α-adrenergic effects. However, epinephrine plays β1-adrenergic actions, which increasing myocardial oxygen consumption may lead to refractory ventricular fibrillation (VF) and poor outcome. Effects of a single dose of esmolol in addition to epinephrine during CPR were investigated in a porcine model of VF with an underlying acute myocardial infarction. VF was ischemically induced in 16 pigs and left untreated for 12 min. During CPR, animals were randomized to receive epinephrine (30 µg/kg) with either esmolol (0.5 mg/kg) or saline (control). Pigs were then observed up to 96 h. Coronary perfusion pressure increased during CPR in the esmolol group compared to control (47 ± 21 vs. 24 ± 10 mmHg at min 5, p p p p

Background Ventilation with the noble gas argon (Ar) has shown neuroprotective and cardioprotective properties in different in vitro and in vivo models. Hence, the neuroprotective effects of Ar were investigated in a severe, preclinically relevant porcine model of cardiac arrest. Methods and Results Cardiac arrest was ischemically induced in 36 pigs and left untreated for 12 minutes before starting cardiopulmonary resuscitation. Animals were randomized to 4‐hour post‐resuscitation ventilation with: 70% nitrogen–30% oxygen (control); 50% Ar–20% nitrogen–30% oxygen (Ar 50%); and 70% Ar–30% oxygen (Ar 70%). Hemodynamic parameters and myocardial function were monitored and serial blood samples taken. Pigs were observed up to 96 hours for survival and neurological recovery. Heart and brain were harvested for histopathology. Ten animals in each group were successfully resuscitated. Ninety‐six‐hour survival was 60%, 70%, and 90%, for the control, Ar 50%, and Ar 70% groups, respectively. In the Ar 50% and Ar 70% groups, 60% and 80%, respectively, achieved good neurological recovery, in contrast to only 30% in the control group ( P P P =0.007), after Ar compared with control treatment. A lower increase in circulating biomarkers of brain injury, together with less kynurenine pathway activation ( P P Conclusions Post‐resuscitation ventilation with Ar significantly improves neurologic recovery and ameliorates brain injury after cardiac arrest with long no‐flow duration. Benefits are greater after Ar 70% than Ar 50%.

Forty‑one tumors were detected in a population of 1,649,003 cattle slaughtered in 4 abattoirs in Lombardy over a 5‑year period, for an overall prevalence of 2.5 tumors per 100,000 cattle. Tumors were classified according to the WHO histological classification of tumors of domestic animals. Alimentary and hemopoietic systems were commonly affected with 9 cases each. Other affected sites were the respiratory (n = 3), urinary (n = 2), endocrine (n = 2), musculoskeletal (n = 2), nervous (n = 1), and cardiovascular (n = 1) systems. The peritoneum was affected by 6 cases, while the primary location of 3 tumors of the connective tissues and 3 metastatic carcinomas was unidentified. Liver tumors and mesotheliomas, for which environmental risk factors are well‑known in humans, were common, as well as tumors typically encountered in pediatric human patients (tumors of mesenchymal tissues, pulmonary blastomas and nephroblastomas). These findings suggest the useful role of bovines as sentinel and model for human carcinogenesis. Our study indicates that the establishment of a bovine cancer registry in Lombardy is feasible considering its potential contribution to understanding the role of environmental risk factors in the genesis of tumors in animals and humans.

Synovial sarcoma (SS) is an aggressive tumor with propensity for lung metastases which significantly impact patients' prognosis. New therapeutic approaches are needed to improve treatment outcome. Targeting the heparanase/heparan sulfate proteoglycan system by heparin derivatives which act as heparanase inhibitors/heparan sulfate mimetics is emerging as a therapeutic approach that can sensitize the tumor response to chemotherapy. We investigated the therapeutic potential of a supersulfated low molecular weight heparin (ssLMWH) in preclinical models of SS. ssLMWH showed a potent anti-heparanase activity, dose-dependently inhibited SS colony growth and cell invasion, and downregulated the activation of receptor tyrosine kinases including IGF1R and IR. The combination of ssLMWH and the IGF1R/IR inhibitor BMS754807 synergistically inhibited proliferation of cells exhibiting IGF1R hyperactivation, also abrogating cell motility and promoting apoptosis in association with PI3K/AKT pathway inhibition. The drug combination strongly enhanced the antitumor effect against the CME-1 model, as compared to single agent treatment, abrogating orthotopic tumor growth and significantly repressing spontaneous lung metastatic dissemination in treated mice. These findings provide a strong preclinical rationale for developing drug regimens combining heparanase inhibitors/HS mimetics with IGF1R antagonists for treatment of metastatic SS.

INTRODUCTION The study investigated the effect of untreated cardiac arrest (CA), that is, "no-flow" time, on postresuscitation myocardial and neurological injury, and survival in a pig model to identify an optimal duration that adequately reflects the most frequent clinical scenario. METHODS An established model of myocardial infarction followed by CA and cardiopulmonary resuscitation was used. Twenty-two pigs were subjected to three no-flow durations: short (8-10 min), intermediate (12-13 min), and long (14-15 min). Left ventricular ejection fraction (LVEF) was assessed together with thermodilution cardiac output (CO) and high sensitivity cardiac troponin T (hs-cTnT). Neurological impairment was evaluated by neurological scores, serum neuron specific enolase (NSE), and histopathology. RESULTS More than 60% of animals survived when the duration of CA was ≤13 min, compared to only 20% for a duration ≥14 min. Neuronal degeneration and neurological scores showed a trend toward a worse recovery for longer no-flow durations. No animals achieved a good neurological recovery for a no-flow ≥14 min, in comparison to a 56% for a duration ≤13 min (P = 0.043). Serum NSE levels significantly correlated with the no-flow duration (r = 0.892). Longer durations of CA were characterized by lower LVEF and CO compared to shorter durations (P

The XPO1/CRM1 inhibitor selinexor (KPT-330), is currently being evaluated in multiple clinical trials as an anticancer agent. XPO1 participates in the nuclear export of FoxO-1, which we previously found to be decreased in platinum-resistant ovarian carcinoma. The aim of this study was to determine whether enriching FoxO-1 nuclear localization using selinexor would increase ovarian cancer cell sensitivity to cisplatin. Selinexor, as a single agent, displayed a striking antiproliferative effect in different ovarian carcinoma cell lines. A schedule-dependent synergistic effect of selinexor in combination with cisplatin was found in cisplatin-sensitive IGROV-1, the combination efficacy being more evident in sensitive than in the resistant cells. In IGROV-1 cells, the combination was more effective when selinexor followed cisplatin exposure. A modulation of proteins involved in apoptosis (p53, Bax) and in cell cycle progression (p21WAF1) was found by Western blotting. Selinexor-treated cells exhibited enriched FoxO-1 nuclear staining. Knock-down experiments with RNA interference indicated that FOXO1-silenced cells displayed a reduced sensitivity to selinexor. FOXO1 silencing also tended to reduce the efficacy of the drug combination at selected cisplatin concentrations. Selinexor significantly inhibited tumor growth, induced FoxO-1 nuclear localization and improved the efficacy of cisplatin in IGROV-1 xenografts. Taken together, our results support FoxO-1 as one of the key factors promoting sensitivity towards selinexor and the synergistic interaction between cisplatin and selinexor in ovarian carcinoma cells with selected molecular backgrounds, highlighting the need for treatment regimens tailored to the molecular tumor features.

Assessment of the host immune response pattern is of increasing importance as highly prognostic and diagnostic, in immune-related diseases and in some types of cancer. Chronic inflammation is a major hallmark in colon cancer formation, but, despite the extent of local inflammatory infiltrate has been demonstrated to be extremely informative, its evaluation is not routinely assessed due to the complexity and limitations of classical immunohistochemistry (IHC). In the last years, technological advance helped in bypassing technical limits, setting up multiplex IHC (mIHC) based on tyramide signal amplification (TSA) method and designing software suited to aid pathologists in cell scoring analysis. Several studies verified the efficacy of this method, but they were restricted to the analysis of human samples. In the era of translational medicine the use of animal models to depict human pathologies, in a more complete and complex approach, is really crucial. Nevertheless, the optimization and validation of this method to species other than human is still poor. We took advantage of Multispectral Imaging System to identify the immunoprofile of Dextran Sulphate Sodium (DSS)-treated mouse colon. We optimized a protocol to sequentially stain formalin fixed paraffin embedded murine colon samples for CD3, CD8a, CD4, and CD4R5B0 antigens. With this approach we obtained a detailed lymphocyte profile, while preserving the morphological tissue context, generally lost with techniques like gene expression profiling or flow cytometry. This study, comparing the results obtained by mIHC with immunophenotyping performed with cytofluorimetric and standard IHC methods validates the potentiality and the applicability of this innovative approach.

The enzyme nicotinamide phosphoribosyltransferase is both a key intracellular enzyme for NAD biosynthesis (iNAMPT) and an extracellular cytokine (eNAMPT). The relationship between this latter role and the catalytic activity of the enzyme is at present unknown. With the intent of discovering inhibitors specifically able to target eNAMPT, we increased the polarity of MV78 (EC50 = 5.8 nM; IC50 = 3.1 nM), a NAMPT inhibitor previously discovered by us. The replacement of a phenyl ring with a 1,2,3-triazole bearing a protonable N,N-dialkyl methanamine group gave a series of molecules which maintained the inhibition of the enzymatic activity but were unable to cross the plasma membrane and affect cell viability in vitro. Compounds 30b and 30f can therefore be considered as the first experimental/pharmacological tools for scientists that wish to understand the role of the catalytic activity of eNAMPT. Serendipitously, we also discovered a compound (25) which, notwithstanding its high polarity, was able to cross the plasma membrane being cytotoxic, a potent NAMPT inhibitor and effective in reducing growth of triple negative mammary carcinoma in mice. In our hands, 25 lacked retinal and cardiac toxicity, although we observed a lesser toxicity of NAMPT inhibitors in general compared to other reports.

Colon cancer is one of the first tumor types where a functional link between inflammation and tumor onset has been described; however, the microenvironmental cues affecting colon cancer progression are poorly understood. Here we demonstrate that the expression of the ECM molecule EMILIN-1 halts the development of AOM-DSS induced tumors. In fact, upon AOM-DSS treatment the Emilin1-/- (E1-/-) mice were characterized by a higher tumor incidence, bigger adenomas and less survival. Similar results were obtained with the E933A EMILIN-1 (E1-E933A) transgenic mouse model, expressing a mutant EMILIN-1 unable to interact with α4/α9β1 integrins. Interestingly, upon chronic treatment with DSS, E1-/- and E1-E933A mice were characterized by the presence of increased inflammatory infiltrates, higher colitis scores and more severe mucosal injury respect to the wild type (E1+/+) mice. Since alterations of the intestinal lymphatic network are a well-established feature of human inflammatory bowel disease and EMILIN-1 is a key structural element in the maintenance of the integrity of lymphatic vessels, we assessed the lymphatic vasculature in this context. The analyses revealed that both E1-/- and E1-E933A mice displayed a higher density of LYVE-1 positive vessels; however, their functionality was severely compromised after colitis induction. Taken together, these results suggest that the loss of EMILIN-1 expression may cause the reduction of the inflammatory resolution during colon cancer progression due to a decreased lymph flow and impaired inflammatory cell drainage.

In spite of the progress in gene editing achieved in recent years, a subset of genetic diseases involving structural chromosome abnormalities, including aneuploidies, large deletions and complex rearrangements, cannot be treated with conventional gene therapy approaches. We have previously devised a strategy, dubbed chromosome transplantation (CT), to replace an endogenous mutated chromosome with an exogenous normal one. To establish a proof of principle for our approach, we chose as disease model the chronic granulomatous disease (CGD), an X-linked severe immunodeficiency due to abnormalities in CYBB (GP91) gene, including large genomic deletions. We corrected the gene defect by CT in induced pluripotent stem cells (iPSCs) from a CGD male mouse model. The Hprt gene of the endogenous X chromosome was inactivated by CRISPR/Cas9 technology thus allowing the exploitation of the hypoxanthine–aminopterin–thymidine selection system to introduce a normal donor X chromosome by microcell-mediated chromosome transfer. X-transplanted clones were obtained, and diploid XY clones which spontaneously lost the endogenous X chromosome were isolated. These cells were differentiated toward the myeloid lineage, and functional granulocytes producing GP91 protein were obtained. We propose the CT approach to correct iPSCs from patients affected by other X-linked diseases with large deletions, whose treatment is still unsatisfactory. Stem Cells 2019;37:876–887

Objective To investigate the biological effects of a novel endovenous scaffold in a porcine model. Methods Petalo is a compliant venous scaffold implanted into the internal jugular veins of 12 healthy pigs. The pigs were sacrificed at one, two, three, and six months, respectively. Microscopic investigations were performed at two blinded laboratories. Results Neo-intima formation progressively covering up the stent metallic bars was observed. The inflammatory response of the venous wall showed a peak after three months by the implant, followed by marked reduction after six months. The device induced a significant ( p Conclusions The implant of Petalo compliant venous scaffold in the venous wall of this porcine model is characterized by neointima formation and by an inflammatory reaction which tends to decrease after six months. Our data point against the induction of smooth muscle cells proliferation and migration as confirmed by electronic transmission microscopy analyses.

Topiramate is an anticonvulsant drug also prescribed for migraine prophylaxis that acts through several mechanisms of action. Several studies indicate that topiramate induces weight loss and a moderate reduction of plasma lipids and glucose. Based on these favourable metabolic effects, aim of this study was to evaluate if topiramate could modulate atherosclerosis development and protect target organs of dysmetabolic conditions. Thirty apoE-deficient mice were divided into three groups and fed for 12 weeks a high fat diet (Control) or the same diet containing topiramate at 0.125% and 0.250%. Body weight, water and food intake were monitored throughout the study. Plasma lipids and glucose levels were measured and a glucose tolerance test was performed. Atherosclerosis development was evaluated in the whole aorta and at the aortic sinus. Histological analysis of liver, kidney and adipose tissue was performed. Topiramate did not affect weight gain and food intake. Glucose tolerance and plasma lipids were not changed and, in turn, atherosclerosis development was not different among groups. Topiramate did not modify liver and adipose tissue histology. Conversely, in the kidneys, the treatment reduced the occurrence of glomerular lipidosis by decreasing foam cells accumulation and reducing the expression of inflammatory markers. Blood urea nitrogen levels were also reduced by treatment. Our results indicate that topiramate does not affect atherosclerosis development, but preserves kidney structure and function. The study suggests that topiramate could be investigated in drug repurposing studies for the treatment of glomerular lipidosis.

Aim of the study: To evaluate in an established porcine post cardiac arrest model the effect of a mild hypercapnic ventilatory strategy on outcome. Methods: The left anterior descending coronary artery was occluded in 14 pigs and ventricular fibrillation induced and left untreated for 12 min. Cardiopulmonary resuscitation was performed for 5 min prior to defibrillation. After resuscitation, pigs were assigned to either normocapnic (end-tidal carbon dioxide (EtCO2) target: 35-40 mmHg) or hypercapnic ventilation (EtCO2 45-50 mmHg). Hemodynamics was invasively measured and EtCO2 was monitored with an infrared capnometer. Blood gas analysis, serum neuron-specific enolase (NSE) and high sensitive cardiac troponin T (hs-cTnT) were assessed. Survival and functional recovery were evaluated up to 96 h. Results: Twelve pigs were successfully resuscitated and eight survived up to 96 h, with animals in the hypercapnic group showing trend towards a longer survival. EtCO2 and arterial partial pressure of CO2 were higher in the hypercapnic group compared to the normocapnic one (p

Background. Stenting has become the first-line treatment of obstructive venous disease because of poor results of balloon angioplasty. This preclinical study aimed to investigate the safety and efficacy profile of a novel compliant venous scaffold (CVS) denominated Petalo CVS, specifically designed for venous diseases. Materials and Methods. Twelve healthy pigs weighing 90 kg were used to test Petalo CVS. The devices were implanted into the internal jugular veins (IJVs) using a femoral vein percutaneous approach. The safety profile including the success rate of device releasing, anchoring, and positioning was evaluated immediately. Fracture, migration, primary patency, and endothelial response were assessed at 1, 2, 3, and 6 months after the study procedure. Results. A total of 32 devices were successfully released in both IJVs. No procedure- or device-related complications were reported, and all pigs successfully completed the different scheduled follow-up periods. The primary patency rate was 100%, and no fracture or migration of the device into the brachiocephalic trunk was reported. Histological examination revealed only minimal lesions with minimal or absent inflammatory reaction surrounding the incorporated metallic rods. Conclusions. This porcine model study showed a promising safety and efficacy profile of Petalo CVS, a novel endovenous device based on specific concepts.

The 129 mouse strain is commonly used for the generation of genetically engineered mice. Genetic drift or accidental contamination during outcrossing has resulted in several 129 substrains. Comprehensive data on spontaneous age-related pathology exist for the 129S4/SvJae substrain, whereas only limited information is available for other 129 substrains. This longitudinal aging study describes the life span and spontaneous lesions of 44 male and 18 female mice of the 129S6/SvEvTac substrain. Median survival time was 778 and 770 days for males and females, respectively. Tumors of lung and Harderian gland were the most common neoplasms in both sexes. Hepatocellular tumors occurred mainly in males. Hematopoietic tumors were observed at low frequency. Suppurative and ulcerative blepharoconjunctivitis was the most common nonneoplastic condition in both sexes. Corynebacteria (primarily Corynebacterium urealyticum and C. pseudodiphtheriticum) were isolated from animals with blepharoconjunctivitis and in some cases from unaffected mice, although a clear causal association between corynebacterial infections and blepharoconjunctivitis could not be inferred. Polyarteritis occurred only in males and was identified as the most common nonneoplastic contributory cause of death. Eosinophilic crystalline pneumonia occurred in both sexes and was a relevant cause of death or comorbidity. Epithelial hyalinosis at extrapulmonary sites was noted at higher frequency in females. This study contributes important data on the spontaneous age-related pathology of the 129S6/SvEvTac mouse substrain and is a valuable reference for evaluation of the phenotype in genetically engineered mice obtained with this 129 substrain.

Highly aggressive cancer types such as pancreatic cancer possess a mortality rate of up to 80% within the first 6months after diagnosis. To reduce this high mortality rate, more sensitive diagnostic tools allowing an early stage medical imaging of even very small tumours are needed. For this purpose, magnetic, biodegradable nanoparticles prepared using recombinant human serum albumin (rHSA) and incorporated iron oxide (maghemite, γ-Fe2O3) nanoparticles were developed. Galectin-1 has been chosen as target receptor as this protein is upregulated in pancreatic cancer and its precursor lesions but not in healthy pancreatic tissue nor in pancreatitis. Tissue plasminogen activator derived peptides (t-PA-ligands), that have a high affinity to galectin-1 have been chosen as target moieties and were covalently attached onto the nanoparticle surface. Improved targeting and imaging properties were shown in mice using single photon emission computed tomography-computer tomography (SPECT-CT), a handheld gamma camera, and magnetic resonance imaging (MRI).

Non-Small Cell Lung Cancer (NSCLC) remains an aggressive and fatal disease with low responsiveness to chemotherapy, frequent drug resistance development and metastatic behavior. Platinum-based therapy is the standard of care for NSCLC with limited benefits. Since epigenetic alterations have been implicated in the aggressive behavior of lung cancer, the purpose of the present study was to examine the capability of the pan-histone deacetylase inhibitor SAHA and of ST3595, a novel hydroxamate-based compound, to interfere with the proliferative and invasive potential of NSCLC cells. We used two NSCLC cell lines (H460 and A549) and the cisplatin-resistant variants (H460/Pt and A549/Pt), to mimic a frequent clinical condition. The resistant models exhibited increased invasive properties as compared to parental cells, features associated with a wide modulation of the level of angiogenesis- and invasion-related factors in the cell conditioned media. The levels of urokinase-type plasminogen activator, IL-8, and macrophage migration inhibitory factor were increased in the conditioned media from both H460/Pt and A549/Pt cells. SAHA and ST3595 induced a strong inhibition of cell invasive properties, which was more marked after ST3595 exposure. Both HDAC inhibitors up-regulated the metastasis suppressor KiSS1 at the mRNA level. Forced expression of KiSS1 significantly decreased the invasive capability of drug-resistant cells. ST3595 displayed an anti-metastatic effect in tumors associated with decreased of phosphorylation of Src. Our data indicate that HDAC inhibitors are effective in NSCLC cell systems. The ability of ST3595 to counteract the invasive potential of resistant cells through mechanisms involving KiSS1 is an interesting novel finding.

Background Canine cancer registry data can be put to good use in epidemiological studies. Quantitative comparison of tumour types may reveal unusual cancer frequencies, providing directions for research and generation of hypotheses of cancer causation in a specific area, and suggest leads for identifying risk factors. Here we report canine cancer incidence rates calculated from a population-based registry in an area without any known specific environmental hazard. Results In its 90 months of operation from 2001 to 2008 (the observation period in this study), the population-based Piedmont Canine Cancer Registry collected data on 1175 tumours confirmed by histopathological diagnosis. The incidence rate was 804 per 100,000 dog-years for malignant tumours and 897 per 100,000 dog-years for benign tumours. Higher rates for all cancers were observed in purebred dogs, particularly in Yorkshire terrier and Boxer. The most prevalent malignant neoplasms were cutaneous mastocytoma and hemangiopericytoma, and mammary gland complex carcinoma and simplex carcinoma. Conclusions The Piedmont canine cancer registry is one of few of its kind whose operations have been consistently supported by long-term public funding. The registry-based cancer incidence rates were estimated with particular attention to the validity of data collection, thus minimizing the potential for bias. The findings on cancer incidence rates may provide a reliable reference for comparison studies. Researches conducted on dogs, used as sentinels for community exposure to environmental carcinogens, can be useful to detect excess risks in the incidence of malignant tumours in the human population.

It has been shown that individual acute myeloid leukemia (AML) patients are characterized by one of few initiating DNA mutations and 5-10 cooperating mutations not yet defined among hundreds identified by massive sequencing of AML genomes. We report an in vivo insertional-mutagenesis screen for genes cooperating with one AML initiating mutations (PML-RARA, oncogene of acute promyelocytic leukemia, APL), which allowed identification of hundreds of genetic cooperators. The cooperators are mutated at low frequency in APL or AML patients but are always abnormally expressed in a cohort of 182 APLs and AMLs analyzed. These deregulations appear non-randomly distributed and present in all samples, regardless of their associated genomic mutations. Reverse-engineering approaches showed that these cooperators belong to a single transcriptional gene network, enriched in genes mutated in AMLs, where perturbation of single genes modifies expression of others. Their gene-ontology analysis showed enrichment of genes directly involved in cell proliferation control. Therefore, the pool of PML-RARA cooperating mutations appears large and heterogeneous, but functionally equivalent and deregulated in the majority of APLs and AMLs. Our data suggest that the high heterogeneity of DNA mutations in APLs and AMLs can be reduced to patterns of gene expression deregulation of a single 'mutated' gene network.Leukemia advance online publication, 20 January 2017; doi:10.1038/leu.2016.386.

Ringtail is a pathologic condition of laboratory rodents characterized by annular constrictions of the tail. Traditionally, it is classified as an environmental disorder caused by low relative humidity, but other factors (temperature, dietary deficiencies, genetic susceptibility, and caging type) have also been proposed. Twenty litters of mice with ringtail lesions occurred from September 2010 to August 2013 in a facility located in the northern Italy. Mice were maintained under controlled environmental conditions and fed a standard diet. Retrospective analysis of environmental data (relative humidity, temperature) was carried out. Gross, histopathologic, scanning, and transmission electron microscopy examination of tails and limbs was performed. The incidence of ringtail was 0.075% (20/26 800) of all weaned litters over the 3-year period of examination. Temperature and relative humidity remained within accepted limits in all cases except one. We observed annular constrictions in tail, digits of pes, crus, and antebrachium in 116 (100.0%), 47 (40.5%), 11 (9.5%), and 2 (1.7%) of 116 affected mice, respectively. Histologic and ultrastructural examination revealed abnormal keratin desquamation and presence of a keratin ring encircling the tail, causing progressive strangulation of the growing tail with subsequent compression and ulceration of underlying soft tissues, resulting in circulatory changes (edema, hyperemia, thrombosis, hemorrhages), ischemic necrosis, and eventually auto-amputation distal to the constriction. On the basis of our findings, we suggest a disorder of cornification as the primary lesion of ringtail in mice. The cause of these cases, however, remained undetermined, even though traditional etiologic factors (relative humidity, temperature, diet, caging type) were reasonably excluded.

"Tissue-selective estrogen complex" or TSEC is a novel concept of estrogen replacement therapy for the postmenopause based on the combined use of estrogens and selective estrogen receptor modulators (SERMs). The aim of this study was to exploit the potential of a novel transgenic mouse where luciferase expression is associated with cell proliferation (the MITO-luc mouse) to investigate cell proliferation in reproductive and nonreproductive tissues in mice exposed to repetitive treatments with TSEC.Ovariectomized MITO-Luc mice were subjected to a daily oral treatment with bazedoxifene, conjugated estrogen (CE), TSEC, or raloxifene for 21 days. During the treatment, the proliferative effects of treatments were monitored by bioluminescence-based in vivo imaging. At the end of the treatment, mice were euthanized and cell proliferation assessed in selected tissues by quantitative analysis of luciferase activity and by immunohistochemistry (IHC).In uterus treatment with CE, but not TSEC, induced a large increase in luciferase activity underlying the proliferative effect of the hormone. No accumulation of luciferase was observed in other organs and tissues target of estrogen action. We observed an increase of Ki67 immunoreactivity only in the uterus of mice treated with CE.Pairing of an SERM with estrogens results in a complete blockage of CE proliferative effects in uterus and the absence of any undesired proliferative effects in other organs; moreover, the MITO-Luc mouse is an efficacious tool for the global, rapid, and reliable analysis of drug-induced proliferation.

Continuous or primary epithelial cell lines have been extensively used to study the mammary gland immune response, but they are constituted by a single cell population. Our aim was to test whether an explant of heifer gland, where the tissue structure is maintained, might be a valid model to investigate the innate immune response to infection. The study was carried out on 2 mm 3 -sections of heifer udders, in 2 consecutive trials, using LPS or LTA in the first trial and two different concentrations of Staphylococcus aureus ( Staph . aureus ) in the second. Treated and untreated sections were collected after 1 h, 3 h and 6 h incubation; in the first trial, a final time-point at 18 h was considered. The mRNA expression of TNFα, IL-1β, IL-6, IL-8 and LAP was analyzed by quantitative real-time PCR. Histological examination showed well-preserved morphology of the tissue, and apoptosis only showed a slight, not significant increase throughout the experiment. IL-1β and IL-6 were significantly up-regulated, in response to LPS or Staph . aureus , while TNF-α and IL-8 significantly increased only under LPS treatment. LAP expression showed a significant late increase when stimulated by LPS. The immunochemical staining of the sections demonstrated a higher number of T lymphocytes within the alveolar epithelium, in comparison with interstitial localization. Since the explants belonged to pubertal non-pregnant heifers, T cells may be regarded as resident cells, suggesting their participation in the regulation of mammary homeostasis. Therefore, applying our model would give new insights in the investigation of udder pathophysiology.

Iron oxide-containing magnetic nanoparticles (MNPs) have certain advantages over currently used contrast agents for tumor imaging by magnetic resonance imaging (MRI) as they offer the possibility of functionalization with ligands and tracers. Functionalized MNPs also may be used for targeted tumor therapy. In the current study nanoparticles (NPs) consisting of recombinant human serum albumin (rHSA) with incorporated hydrophilic (NH4)2Ce(IV)(NO3)6-γ-Fe2O3 particles (CAN maghemite particles) for medical imaging were produced and characterized. For this purpose CAN maghemite particles were incorporated into an rHSA matrix to yield rHSA-NPs. The resulting NPs were analyzed by transmission electron microscopy, photon correlation spectroscopy, and atomic absorption. The sizes of the manufactured NP were 170 ± 10 nm, and the zeta-potential was − 50 ± 3 mV. The NPs remained stable when stored after lyophilization with sucrose 3% [w/v] as a cryoprotector. They showed pro-inflammatory properties without cell and animal toxicity in vivo and were highly contrasting in MRI. In conclusion, this report introduces novel rHSA NP with favorable properties containing iron oxide for detection by MRI.

The development of tools for the early diagnosis of pancreatic adenocarcinoma is an urgent need in order to increase treatment success rate and reduce patient mortality. Here, we present a modular nanosystem platform integrating soft nanoparticles with a targeting peptide and an active imaging agent for diagnostics. Biocompatible single-chain polymer nanoparticles (SCPNs) based on poly(methacrylic acid) were prepared and functionalized with the somatostatin analogue PTR86 as the targeting moiety, since somatostatin receptors are overexpressed in pancreatic cancer. The gamma emitter 67Ga was incorporated by chelation and allowed in vivo investigation of the pharmacokinetic properties of the nanoparticles using single photon emission computerized tomography (SPECT). The resulting engineered nanosystem was tested in a xenograph mouse model of human pancreatic adenocarcinoma. Imaging results demonstrate that accumulation of targeted SCPNs in the tumor is higher than that observed for nontargeted nanoparticles due to improved retention in this tissue. ¸ 2016 American Chemical Society.

Large bone defects still represent a major burden in orthopedics, requiring bone-graft implantation to promote the bone repair. Along with autografts that currently represent the gold standard for complicated fracture repair, the bone tissue engineering offers a promising alternative strategy combining bone-graft substitutes with osteoprogenitor cells able to support the bone tissue ingrowth within the implant. Hence, the optimization of cell loading and distribution within osteoconductive scaffolds is mandatory to support a successful bone formation within the scaffold pores. With this purpose, we engineered constructs by seeding and culturing autologous, osteodifferentiated bone marrow mesenchymal stem cells within hydroxyapatite (HA)-based grafts by means of a perfusion bioreactor to enhance the in vivo implant-bone osseointegration in an ovine model. Specifically, we compared the engineered constructs in two different anatomical bone sites, tibia, and femur, compared with cell-free or static cell-loaded scaffolds. After 2 and 4 months, the bone formation and the scaffold osseointegration were assessed by micro-CT and histological analyses. The results demonstrated the capability of the acellular HA-based grafts to determine an implant-bone osseointegration similar to that of statically or dynamically cultured grafts. Our study demonstrated that the tibia is characterized by a lower bone repair capability compared to femur, in which the contribution of transplanted cells is not crucial to enhance the bone-implant osseointegration. Indeed, only in tibia, the dynamic cell-loaded implants performed slightly better than the cell-free or static cell-loaded grafts, indicating that this is a valid approach to sustain the bone deposition and osseointegration in disadvantaged anatomical sites.

Malignant Mesothelioma is a highly aggressive cancer, which is difficult to diagnose and treat. Here we describe the molecular, cellular and morphological characterization of a syngeneic system consisting of murine AB1, AB12 and AB22 mesothelioma cells injected in immunocompetent BALB/c mice, which allows the study of the interplay of tumor cells with the immune system. Murine mesothelioma cells, like human ones, respond to exogenous High Mobility Group Box 1 protein, a Damage-Associated Molecular Pattern that acts as a chemoattractant for leukocytes and as a proinflammatory mediator. The tumors derived from AB cells are morphologically and histologically similar to human MM tumors, and respond to treatments used for MM patients. Our system largely recapitulates human mesothelioma, and we advocate its use for the study of MM development and treatment. Malignant Mesothelioma is a highly aggressive cancer, which is difficult to diagnose and treat. Here we describe the molecular, cellular and morphological characterization of a syngeneic system consisting of murine AB1, AB12 and AB22 mesothelioma cells injected in immunocompetent BALB/c mice, which allows the study of the interplay of tumor cells with the immune system. Murine mesothelioma cells, like human ones, respond to exogenous High Mobility Group Box 1 protein, a Damage-Associated Molecular Pattern that acts as a chemoattractant for leukocytes and as a proinflammatory mediator. The tumors derived from AB cells are morphologically and histologically similar to human MM tumors, and respond to treatments used for MM patients. Our system largely recapitulates human mesothelioma, and we advocate its use for the study of MM development and treatment

An 18-month-old male basset hound was presented with vomiting, diarrhoea and depression. Abdominal ultrasonography revealed a mass in the left kidney. An ultrasound-guided core-biopsy indicated aggregates of spindle cells, but did not allow a definitive diagnosis. Nephrectomy was performed after a period of six months, when ultrasound examination revealed a slight increase in mass dimensions. Histologically the mass was composed of neoplastic spindle cells forming interlacing fascicles, bundles and whorls, within a loose myxoid to dense collagenous stroma. Immunohistochemically neoplastic cells were positive for vimentin and smooth muscle actin. Based on these findings the tumour was diagnosed as a congenital mesoblastic nephroma, classical variant. After a two-and-a-half-year follow-up the dog was clinically healthy, indicating a benign behaviour. To the authors' knowledge, this report describes the first case of canine congenital mesoblastic nephroma successfully treated surgically, with a reasonable postsurgical follow-up.

Enterohepatic Helicobacter spp. have been described colonizing the large intestine and liver of healthy and symptomatic subjects and are thought to have a role in the development of inflammatory bowel disease (IBD). The prevalence of enterohepatic Helicobacter spp. infection in dogs is largely unknown and to our knowledge there are no data about their potential pathogenic role. In light of these considerations, the aims of this study were (i) to assess the prevalence of enterohepatic Helicobacter spp. in colonic biopsies of symptomatic pet dogs and (ii) to evaluate a possible association between Helicobacter spp. colonization status (heavily colonized, poorly colonized and uncolonized biopsies) and histological lesions. Colonic biopsies from 27 pet dogs of different ages were evaluated by family Helicobacteraceae and enterohepatic Helicobacter spp. PCR, histology, and immunohistochemistry for the in situ detection of Helicobacter spp. organisms. 85% and 52% of colonic biopsies were positive by Helicobacteraceae and enterohepatic Helicobacter spp. PCR, respectively. Immunohistochemistry revealed Helicobacter spp. were localized both in the superficial mucus (55%) and within intestinal crypts (33%). Dogs with heavy enterohepatic Helicobacter spp. colonization were significantly younger and had a higher level of mucosal fibrosis/atrophy than dogs with uncolonized or poorly colonized biopsies (p < 0.05). These findings contribute to widen current knowledge regarding canine enterohepatic Helicobacter spp., suggesting the infection is rather common in dogs and acquired at an early age. Furthermore, heavy colonization of colonic crypts is associated with chronic inflammatory lesions (fibrosis/atrophy), supporting the role of enterohepatic Helicobacter spp. in the development of canine IBD.

Objective—To determine the Helicobacter spp present in the oral cavity of dogs and the relationship of those organisms with gastric Helicobacter spp to better define the potential for dog-human and dog-dog transmission. Sample—Saliva and dental plaque from 28 dogs and gastric biopsy specimens from a subset of 8 dogs. Procedures—PCR-based screening for Helicobacter spp was conducted on samples obtained from the oral cavity of 28 dogs. Comparative analysis was conducted on Helicobacteraceae 16S rDNA clone libraries from the oral cavity and stomach of a subset of 8 dogs (5 vomiting and 3 healthy) that had positive PCR results for Helicobacter spp. Results—Helicobacteraceae DNA was identified in the oral cavity of 24 of 28 dogs. Analysis of cloned 16S rDNA amplicons from 8 dogs revealed that Wolinella spp was the most common (8/8 dogs) and abundant (52/57 [91%] clones) member of the Helicobacteraceae family in the oral cavity. Only 2 of 8 dogs harbored Helicobacter spp in the oral cavity, and 1 of those was coinfected with Helicobacter heilmannii and Helicobacter felis in samples obtained from the stomach and saliva. Evaluation of oral cavity DNA with Wolinella-specific PCR primers yielded positive results for 16 of 20 other dogs (24/28 samples were positive for Wolinella spp). Conclusions and Clinical Relevance—Wolinella spp rather than Helicobacter spp were the predominant Helicobacteraceae in the oral cavity of dogs. The oral cavity of dogs was apparently not a zoonotically important reservoir of Helicobacter spp that were non–Helicobacter pylori organisms.

SummaryTP53 missense mutations dramatically influence tumor progression, however, their mechanism of action is still poorly understood. Here we demonstrate the fundamental role of the prolyl isomerase Pin1 in mutant p53 oncogenic functions. Pin1 enhances tumorigenesis in a Li-Fraumeni mouse model and cooperates with mutant p53 in Ras-dependent transformation. In breast cancer cells, Pin1 promotes mutant p53 dependent inhibition of the antimetastatic factor p63 and induction of a mutant p53 transcriptional program to increase aggressiveness. Furthermore, we identified a transcriptional signature associated with poor prognosis in breast cancer and, in a cohort of patients, Pin1 overexpression influenced the prognostic value of p53 mutation. These results define a Pin1/mutant p53 axis that conveys oncogenic signals to promote aggressiveness in human cancers.

Human parthenogenetic embryos have recently been proposed as an alternative, less controversial source of embryonic stem cell (ESC) lines; however many aspects related to the biology of parthenogenetic embryos and parthenogenetic derived cell lines still need to be elucidated. We present here results on human cell lines (HP1 and HP3) derived from blastocysts obtained by oocyte parthenogenetic activation. Cell lines showed typical ESC morphology, expressed Oct-4, Nanog, Sox-2, Rex-1, alkaline phosphatase, SSEA-4, TRA 1-81 and had high telomerase activity. Expression of genes specific for different embryonic germ layers was detected from HP cells differentiated upon embryoid body (EBs) formation. Furthermore, when cultured in appropriate conditions, HP cell lines were able to differentiate into mature cell types of the neural and hematopoietic lineages. However, the injection of undifferentiated HP cells in immunodeficient mice resulted either in poor differentiation or in tumour formation with the morphological characteristics of myofibrosarcomas. Further analysis of HP cells indicated aberrant levels of molecules related to spindle formation as well as the presence of an abnormal number of centrioles and autophagic activity. Our results confirm and extend the notion that human parthenogenetic stem cells can be derived and can differentiate in mature cell types, but also highlight the possibility that, alteration of the proliferation mechanisms may occur in these cells, suggesting great caution if a therapeutic use of this kind of stem cells is considered.

Background: In dogs, the gastric Helicobacter spp. have been well studied, but there is little information regarding the other parts of the alimentary system. We sought to determine the spatial distribution of Helicobacter spp. in the gastrointestinal tract and the hepatobiliary system of dogs using culture-independent methods. Materials and methods: Samples of stomach, duodenum, ileum, cecum, colon, pancreas, liver, and bile from six dogs were evaluated for Helicobacter spp. by genus, gastric, and enterohepatic Helicobacter spp. Polymerase chain reaction, 16S rRNA gene sequence analysis, immunohistochemistry, and fluorescence in situ hybridization (FISH). Results: In the stomach, Helicobacter spp. DNA was detected in all six dogs, with H. bizzozeronii and H. felis identified by specific polymerase chain reaction. Helicobacter organisms were localized within the surface mucus, the lumen of gastric glands, and inside parietal cells. The small intestine harbored gastric and enterohepatic Helicobacter spp. DNA/antigen in low amounts. In the cecum and colon, Helicobacter spp. DNA, with highest similarity to H. bilis/flexispira taxon 8, H. cinaedi, and H. canis, was detected in all six dogs. Helicobacter organisms were localized at the mucosal surface and within the crypts. Gastric Helicobacter spp. DNA was detected occasionally in the large intestine, but no gastric Helicobacter spp. were present in clone libraries or detected by FISH. Conclusions: This study demonstrates that in addition to the stomach, the large intestine of dogs is also abundantly colonized by Helicobacter spp. Additional studies are necessary to investigate the association between enterohepatic Helicobacter spp. and presence of intestinal inflammatory or proliferative disorders in dogs.

Prolactin-secreting pituitary adenomas are common spontaneous lesions in aging FVB females. Prolactin-secreting pituitary proliferations play a significant role in mouse mammary tumorigenesis generally producing adenosquamous carcinomas. Since genetically engineered FVB mice are frequently used to study mammary tumor biology, we have examined a cohort of 64 aging wild-type FVB/N females to establish the prevalence and the nature of spontaneous mammary and pituitary tumors. Tissues from mammary and pituitary glands were studied by histopathology and immunohistochemistry. Of the 64 examined mice, 20 had pituitary tumors and 20 had mammary tumors. Mammary and pituitary tumors were associated in 17 mice. All pituitary tumors were prolactin-positive by immunohistochemistry and classified as prolactinomas. Fourteen mammary tumors, including 12 cases with and 2 without concurrent prolactinomas, were adenocarcinomas with different combinations of epithelial growth patterns. Five mice with prolactinomas had mammary tumors characterized by the epithelial-mesenchymal transition (EMT) phenotype. Estrogen receptor alpha (ERα)-positivity was observed for 14 of the 18 mammary tumors tested, including both adenocarcinomas with nuclear immunoreactivity and EMT-phenotype tumors with both nuclear and cytoplasmic immunoreactivity. No immunoreactivity for the progesterone receptor was observed. This study confirms that spontaneous prolactinomas and mammary tumors are both common and significantly associated lesions in FVB mice. Parity and age represented risk factors for the development of these tumors. Compared with previous reports, prolactinoma-associated mammary tumors displayed a broader morphologic spectrum, including cases with the EMT phenotype. The elevated number of prolactinoma-associated and ERα-positive mammary tumors opens intriguing possibilities concerning the role of ERα cytoplasmic localization during EMT tumorigenesis.

The mouse aldehyde oxidase AOH2 (aldehyde oxidase homolog 2) is a molybdoflavoenzyme. Harderian glands are the richest source of AOH2, although the protein is detectable also in sebaceous glands, epidermis, and other keratinized epithelia. The levels of AOH2 in the Harderian gland and skin are controlled by genetic background, being maximal in CD1 and C57BL/6 and minimal in DBA/2, CBA, and 129/Sv strains. Testosterone is a negative regulator of AOH2 in Harderian glands. Purified AOH2 oxidizes retinaldehyde into retinoic acid, while it is devoid of pyridoxal-oxidizing activity. Aoh2(-/-) mice, the first aldehyde oxidase knockout animals ever generated, are viable and fertile. The data obtained for this knockout model indicate a significant role of AOH2 in the local synthesis and biodisposition of endogenous retinoids in the Harderian gland and skin. The Harderian gland's transcriptome of knockout mice demonstrates overall downregulation of direct retinoid-dependent genes as well as perturbations in pathways controlling lipid homeostasis and cellular secretion, particularly in sexually immature animals. The skin of knockout mice is characterized by thickening of the epidermis in basal conditions and after UV light exposure. This has correlates in the corresponding transcriptome, which shows enrichment and overall upregulation of genes involved in hypertrophic responses.

The acetyl-transferase Tip60 might influence tumorigenesis in multiple ways. First, Tip60 is a co-regulator of transcription factors that either promote or suppress tumorigenesis, such as Myc and p53. Second, Tip60 modulates DNA-damage response (DDR) signalling, and a DDR triggered by oncogenes can counteract tumour progression. Using E(mu)-myc transgenic mice that are heterozygous for a Tip60 gene (Htatip) knockout allele (hereafter denoted as Tip60+/- mice), we show that Tip60 counteracts Myc-induced lymphomagenesis in a haplo-insufficient manner and in a time window that is restricted to a pre- or early-tumoral stage. Tip60 heterozygosity severely impaired the Myc-induced DDR but caused no general DDR defect in B cells. Myc- and p53-dependent transcription were not affected, and neither were Myc-induced proliferation, activation of the ARF-p53 tumour suppressor pathway or the resulting apoptotic response. We found that the human TIP60 gene (HTATIP) is a frequent target for mono-allelic loss in human lymphomas and head-and-neck and mammary carcinomas, with concomitant reduction in mRNA levels. Immunohistochemical analysis also demonstrated loss of nuclear TIP60 staining in mammary carcinomas. These events correlated with disease grade and frequently concurred with mutation of p53. Thus, in both mouse and human, Tip60 has a haplo-insufficient tumour suppressor activity that is independent from-but not contradictory with-its role within the ARF-p53 pathway. We suggest that this is because critical levels of Tip60 are required for mounting an oncogene-induced DDR in incipient tumour cells, the failure of which might synergize with p53 mutation towards tumour progression.

Large and circumferential tracheal defects remain at this time an unsolved problem for reconstructive surgery. Many types of prosthetic and tissue grafts have been used but with limited comfortable results. Major complications are anastomotic dehiscence, graft ischemia and stenosis due to the poor vascularization of the prosthetic complex. We studied the in vivo behaviour of a prefabricated flap composed of a partially bioresorbable tracheal prosthesis and an arterio-venous vascular carrier. The prosthesis was made of a tubular skeleton of knitted Dacron (20 microm porosity) embedded within a bioresorbable poly-lactic-co-glycolic acid polymer (PLA(75)GA(25)) covering both sides. Fifteen New Zealand White rabbits were divided in three groups, depending on the time of examination (30, 90 and 180 days post-implantation). The prosthesis was implanted in the visceral space of the neck using the common carotid trunk and the internal jugular vein as vascular pedicle. The histological, immunohistochemical, and ESEM analyses of collected samples, showed a time-dependent process of tissue neoformation and neovascularization on the prosthetic material with a significant increase from 30 to 90 days post-implantation. In contrast, there was no statistically significant difference in the fibrovascular connective deposition from 90 to 180 days. This finding indicated the three months time as the best period for the tissue deposition and consequent hypothetical orthotopic transplantation of the prosthesis. Further in vivo studies are intended to confirm the results.

Helicobacter pyloriis responsible for gastritis and gastric adenocarcinoma in humans, but the routes of transmission of this bacterium have not been clearly defined. Few studies led to supposing thatH. pyloricould be transmitted through raw milk, and no one investigated the presence of other Helicobacteraceae in milk. In the current work, the presence of Helicobacteraceae was investigated in the bulk tank milk of dairy cattle herds located in northern Italy both by direct plating ontoH. pyloriselective medium and by screening PCR for Helicobacteraceae, followed by specific PCRs forH. pylori,Wolinellaspp., and “Candidatus Helicobacter bovis.” Three out of 163 bulk milk samples tested positive for Helicobacteraceae, but not for the subsequent PCRs.H. pyloriwas not isolated in any case. However, given similar growth conditions,Arcobacter butzleri,A. cryaerophilus, andA. skirrowiiwere recovered. In conclusion, the prevalence of Helicobacteraceae in raw milk was negligible (1.8%), andH. pyloriwas not identified in any of the positive samples, suggesting that, at least in the farming conditions of the investigated area, bovine milk does not represent a potential source of infection.

// Marianna Paulis 1,2 , Alessandra Castelli 1,2 , Lucia Susani 1,2 , Michela Lizier 2,3 , Irina Lagutina 4 , Maria Luisa Focarelli 1,2 , Camilla Recordati 5 , Paolo Uva 6 , Francesca Faggioli 1,2 , Tui Neri 1,2 , Eugenio Scanziani 5,7 , Cesare Galli 4,8 , Franco Lucchini 3 , Anna Villa 1,2,* and Paolo Vezzoni 1,2,* 1 Milan Unit, Istituto di Ricerca Genetica e Biomedica, CNR, Milan, Italy 2 Humanitas Clinical and Research Center, Rozzano, Milan, Italy 3 Centro Ricerche Biotecnologiche, Universita Cattolica del Sacro Cuore, Cremona, Italy 4 Avantea, Cremona, Italy 5 Mouse and Animal Pathology Laboratory, Fondazione Filarete, Milan, Italy 6 CRS4 Bioinformatics Laboratory, Parco Scientifico e Tecnologico POLARIS, Pula, Cagliari, Italy 7 Department of Veterinary Sciences and Public Health, University of Milan, Milan, Italy 8 Department of Veterinary Medical Sciences, University of Bologna, Ozzano Emilia, Bologna, Italy * These authors jointly supervised this work Correspondence to: Paolo Vezzoni, email: // Marianna Paulis, email: // Keywords : embryonic stem cells, chromosome transplantation, microcell fusion, genomic disorders, cell therapy Received : September 28, 2015 Accepted : October 01, 2015 Published : October 17, 2015 Abstract Genomic disorders resulting from large rearrangements of the genome remain an important unsolved issue in gene therapy. Chromosome transplantation, defined as the perfect replacement of an endogenous chromosome with a homologous one, has the potential of curing this kind of disorders. Here we report the first successful case of chromosome transplantation by replacement of an endogenous X chromosome carrying a mutation in the Hprt genewith a normal one in mouse embryonic stem cells (ESCs), correcting the genetic defect. The defect was also corrected by replacing the Y chromosome with an X chromosome. Chromosome transplanted clones maintained in vitro and in vivo features of stemness and contributed to chimera formation. Genome integrity was confirmed by cytogenetic and molecular genome analysis. The approach here proposed, with some modifications, might be used to cure various disorders due to other X chromosome aberrations in induced pluripotent stem (iPS) cells derived from affected patients.

Summary Nephroblastoma, a relatively common renal neoplasm of young swine, represents the animal counterpart of Wilms' tumour of children. Five porcine nephroblastomas were examined histologically, and immunohistochemically with antibodies against vimentin (VIM), cytokeratins (CKs), smooth-muscle actin, Factor VIII, and laminin. Histologically all showed the three components typical of this tumour: mesenchymal blastema, epithelium (tubuli, and glomeruloid bodies) and stroma. The only antibody recognizing mesenchymal cells was VIM. One-third of tubular structures were positive for VIM. All of the tubules were positive for CK19, two-thirds expressed CK AE1/AE3, and only one-third expressed CKs 8–18. Small round tubuli, located in the stromal septa, were positive for CK7 (ureteric branches). Stromal cells expressed both VIM and actin, demonstrating myofibroblastic differentiation. The kidney originates from mesenchymal blastema, which changes to epithelium, losing VIM and acquiring CK expression. In the adult mammalian kidney, CK 19 is expressed only by the parietal epithelium of Bowman's capsule and the distal tubules. Nevertheless, CK19 is also considered a “transient” CK, expressed by different kinds of epithelia during differentiation. CK 19 was also detected in several undifferentiated neoplasms. This finding, together with the co-expression of VIM detected in some tubules, demonstrates the embryonic origin of nephroblastoma.

The p73 gene has a complex regulation, which leads to the expression of different isoforms, often with opposite biological effects. We have generated in the human colocarcinoma cell line HCT116, expressing a wild-type p53, an inducible DNp73alpha expressing system. Two clones (HCT116/DN3 and HCT116/DN14), upon doxycycline addition, show a strong expression of DNp73alpha. In vitro the two DNp73alpha overexpressing clones grow at similar rate of the control transfected clone (HCT116/8a) and similarly respond to DNA damage. When injected in mice, HCT116/DN3, HCT116/DN14, and HCT116/8a cells grew similarly in the absence or presence of tetracycline. In HCT116/DN3 and HCT116/DN14 tumors, tetracycline induced a strong expression of DNp73alpha both as mRNA and protein. These results indicate that in this system the overexpression of the DNp73alpha does not induce a more aggressive phenotype and does not seem to be associated with a reduced response of the cells to treatment with anticancer agents.

Epithelioid trophoblastic tumor (ETT) is an unusual type of trophoblastic tumor, with features resembling carcinoma. In this study, we describe a 4-year-old cynomolgus monkey ( Macaca fascicularis) showing, at necropsy, a lobulated mass replacing the left ovary and several nodular lesions within the lungs. Histologically, the mass in the ovary and lung metastases were characterized by nests of epithelioid cells, with intermingled, occasional, multinucleate tumor cells consistent with syncytiotrophoblasts and moderate amount of eosinophilic, hyaline-like material. Immunohistochemically, the tumor cells were diffusely positive for cytokeratins (AE1/AE3) and inhibin-α, but only focal immunoreactivity was observed for human chorionic gonadotropin, whereas placental alkaline phosphatase was always negative. On the basis of morphology and immunohistochemical reactivity, tumor cells were identified as intermediate trophoblast.

Rituximab is an anti-CD20 chimeric mAb effective for the treatment of B-NHL. It can lyse lymphoma cells in vitro through both C- and Ab-dependent cellular cytotoxicity. The mechanism of action of rituximab in vivo is however still unclear. We have set up a new in vivo model in nonimmunodeficient mice by stable transduction of the human CD20 cDNA in the murine lymphoma line EL4. Animals injected i.v. with the EL4-CD20+ lymphoma cells died within 30 days with evident liver, spleen, and bone marrow involvement, confirmed by immunohistochemistry and PCR analysis. A single injection of rituximab or the murine anti-CD20 Ab 1F5, given i.p. 1 day after the tumor, cured 100% of the animals. Indeed, at week 4 after tumor cell inoculation, CD20+ cells were undetectable in all organs analyzed in rituximab-treated animals, as determined by immunohistochemistry and PCR. Rituximab had no direct effect on tumor growth in vitro. Depletion of either NK cells or neutrophils or both in tumor-injected animals did not affect the therapeutic activity of the drug. Similarly, rituximab was able to eradicate tumor cells in athymic nude mice, suggesting that its activity is T cell independent. In contrast, the protective activity of rituximab or the 1F5 Ab was completely abolished in syngeneic knockout animals lacking C1q, the first component of the classical pathway of C (C1qa−/−). These data demonstrate that C activation is fundamental for rituximab therapeutic activity in vivo.

The early consequences ofHelicobacter pyloriinfection and the role of bacterial virulence determinants in disease outcome remain to be established. The present study sought to measure the development of host inflammatory and immune responses and their relationship to the putative bacterial virulence factorscagpathogenicity island (cagPAI),vacAallele, andoipAin combination with bacterial colonization density in a feline model of the early stages ofH. pyloriinfection. Gastric tissues obtained from infected and uninfected cats were evaluated forH. pylori ureB,cagPAI,vacAallele, andoipAand colonization density (urease, histology, and real-time PCR). Inflammation was assessed by measuring mRNA upregulation of gamma interferon (IFN-γ), interleukin (IL)-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, and IL-12 p40 and histopathology. The mucosal immune response was characterized by morphometric analysis of lymphoid follicles and by differentiating lymphocyte populations with antibodies against surface markers. InfectingH. pyloristrains were positive forvacAs1but lackedcagPAI and an activeoipAgene. Colonization density was uniform throughout the stomach. Upregulation of IFN-γ, IL-1α, IL-1β, and IL-8 and increased severity of inflammatory infiltrates and fibrosis were observed in infected cats. The median number and total area of lymphoid aggregates were 5 and 10 times greater, respectively, in the stomachs of infected than uninfected cats. Secondary lymphoid follicles in uninfected cats were rare and positive for BLA.36 and B220 but negative for CD3 and CD79α, whereas in infected cats they were frequent and positive for BLA.36, CD79α, and CD3 but negative for B220. Upregulation of IFN-γ, IL-1α, IL-1β, and IL-8 and marked hyperplasia of secondary lymphoid follicles are early consequences ofH. pyloriinfection in cats. The response appears to be similar to that of infected people, particularly children, can develop independently of the pathogenicity factorscagPAI andoipA, and is not correlated with the degree of colonization density or urease activity.

Breast tumor growth and metastasization are both hormone-sensitive and angiogenesis-dependent. Recent work carried out in our laboratory on a transgenic model of breast cancer displaying many similarities to its human counterpart, has shown that liposome-mediated angiostatin cDNA delivery partially inhibits both local and metastatic growth. However, it is now recognized that anti-angiogenesis strategy alone cannot completely arrest tumor growth and spread, and this led to the suggestion that approaches based on different molecular mechanisms could usefully be combined. In the present work, we investigated whether tamoxifen, a classical antiestrogen agent widely used in human therapy, could improve the results obtained with angiostatin alone. Further reduction of local growth was achieved with the combined regimen with respect to angiostatin or tamoxifen alone, while, as expected, no metastatic growth was detected in either group. We therefore conclude that a combination of angiogenesis inhibitors with antiestrogen drugs might be useful in humans and that other associations between conventional and gene transfer-mediated therapy are worth investigating and will soon become important components of anticancer therapy.