Discovery and characterization of a new class of NAD+-independent SIRT1 activators.

The activity of sirtuin 1 (SIRT1, a member of the NAD+-dependent deacetylases family) decreases during aging as NAD+ levels naturally decline, thus increasing the risk of several age-associated diseases. Several sirtuin-activating compounds (STACs) have been developed to counteract the age-associated reduction in SIRT1 activity, and some of them are currently under development in clinical trials. STACs induce SIRT1 activation, either through allosteric activation of the enzyme in the presence of NAD+, or by increasing NAD+ levels by inhibiting its degradation or by supplying a key precursor in biosynthesis. In this study, we have identified (E)-2'-des-methyl sulindac analogues as a novel class of STACs that act also in the absence of NAD+, a peculiar behavior demonstrated through enzymatic and mass spectrometry experiments, both in vitro and in cell lines. The activation of the SIRT1 pathway was confirmed in vivo through gene expression and metabolomics analysis. Our data suggest that these compounds could serve as candidate leads for a novel therapeutic strategy aimed at addressing a key metabolic deficiency that may contribute to metabolic and age-associated diseases.

The extracellular matrix protein EMILIN-1 impacts on the microenvironment by hampering gastric cancer development and progression.

BACKGROUND: The contribution of the tumor microenvironment and extracellular matrix to the aggressive biology of Gastric Cancer (GC) has been recently characterized; however, the role of EMILIN-1 in this context is unknown. EMILIN-1 is an essential structural element for the maintenance of lymphatic vessel (LV) integrity and displays anti-proliferative properties as demonstrated in skin and colon cancer. Given the key role of LVs in GC progression, the aim of this study was to investigate the role of EMILIN-1 in GC mouse models. METHODS: We used the syngeneic YTN16 cells which were injected subcutaneously and intraperitoneally in genetically modified EMILIN-1 mice. In alternative, carcinogenesis was induced using N-Methyl-N-nitrosourea (MNU). Mouse-derived samples and human biopsies were analyzed by IHC and IF to the possible correlation between EMILIN-1 expression and LV pattern. RESULTS: Transgenic mice developed tumors earlier compared to WT animals. 20 days post-injection tumors developed in EMILIN-1 mutant mice were larger and displayed a significant increase of lymphangiogenesis. Treatment of transgenic mice with MNU associated with an increased number of tumors, exacerbated aggressive lesions and higher levels of LV abnormalities. A significant correlation between the levels of EMILIN-1 and podoplanin was detected also in human samples, confirming the results obtained with the pre-clinical models. CONCLUSIONS: This study demonstrates for the first time that loss of EMILIN-1 in GC leads to lymphatic dysfunction and proliferative advantages that sustain tumorigenesis, and assess the use of our animal model as a valuable tool to verify the fate of GC upon loss of EMILIN-1.

Pneumocystis murina lesions in lungs of experimentally infected Cd40l-/- mice.

The Cd40l-/- mouse is a well-established model of X-linked hyper-immunoglobulin M (IgM) syndrome, an immunodeficiency disorder of human beings characterized by the lack of expression of the CD40 ligand (CD40L) on activated T-cells, predisposing to infections with opportunistic pathogens like Pneumocystis jirovecii. The aim of our study was to describe the pulmonary lesions in Cd40l-/- mice experimentally infected with Pneumocystis murina, in comparison with naturally infected severe combined immunodeficient (SCID) mice. Formalin-fixed paraffin-embedded lungs from 26 Cd40l-/-, 11 SCID, and 5 uninfected Cd40l-/- mice were examined by histology and immunohistochemistry for the presence of the pathogen and for leukocyte populations (CD3, CD4, CD45R/B220, CD8a, Iba-1, Ly-6G, CD206, MHC II, and NKp46/NCR1). Infection was confirmed by immunohistochemistry in 18/26 (69%) Cd40l-/- mice and in 11/11 (100%) SCID mice. Fourteen out of 26 (54%) Cd40l-/- mice had interstitial pneumonia. Twenty-three out of 26 (88%) Cd40l-/- mice had peribronchiolar/perivascular lymphoplasmacytic infiltrates, rich in B-cells and Mott cells. Acidophilic macrophage pneumonia was additionally found in 20/26 (77%) Cd40l-/- mice. Only 4/11 (36%) SCID mice had interstitial pneumonia, but no peribronchiolar/perivascular infiltrates or acidophilic macrophage pneumonia were observed in this strain. This study represents the first description of pulmonary histopathological lesions in Cd40l-/- mice infected with P. murina. We speculate that the singular characteristics of the inflammatory infiltrates observed in Cd40l-/- mice could be explained by the specific immune phenotype of the model.

Chronic cholesterol administration to the brain supports complete and long-lasting cognitive and motor amelioration in Huntington's disease.

Evidence that Huntington's disease (HD) is characterized by impaired cholesterol biosynthesis in the brain has led to strategies to increase its level in the brain of the rapidly progressing R6/2 mouse model, with a positive therapeutic outcome. Here we tested the long-term efficacy of chronic administration of cholesterol to the brain of the slowly progressing zQ175DN knock-in HD mice in preventing ("early treatment") or reversing ("late treatment") HD symptoms. To do this we used the most advanced formulation of cholesterol loaded brain-permeable nanoparticles (NPs), termed hybrid-g7-NPs-chol, which were injected intraperitoneally. We show that one cycle of treatment with hybrid-g7-NPs-chol, administered in the presymptomatic ("early treatment") or symptomatic ("late treatment") stages is sufficient to normalize cognitive defects up to 5 months, as well as to improve other behavioral and neuropathological parameters. A multiple cycle treatment combining both early and late treatments ("2 cycle treatment") lasting 6 months generates therapeutic effects for more than 11 months, without severe adverse reactions. Sustained cholesterol delivery to the brain of zQ175DN mice also reduces mutant Huntingtin aggregates in both the striatum and cortex and completely normalizes synaptic communication in the striatal medium spiny neurons compared to saline-treated HD mice. Furthermore, through a meta-analysis of published and current data, we demonstrated the power of hybrid-g7-NPs-chol and other strategies able to increase brain cholesterol biosynthesis, to reverse cognitive decline and counteract the formation of mutant Huntingtin aggregates. These results demonstrate that cholesterol delivery via brain-permeable NPs is a therapeutic option to sustainably reverse HD-related behavioral decline and neuropathological signs over time, highlighting the therapeutic potential of cholesterol-based strategies in HD patients. DATA AVAILABILITY: This study does not include data deposited in public repositories. Data are available on request to the corresponding authors.

Lack of ApoA-I in ApoEKO Mice Causes Skin Xanthomas, Worsening of Inflammation, and Increased Coronary Atherosclerosis in the Absence of Hyperlipidemia.

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.

Repeated oral administration of low doses of silver in mice: tissue distribution and effects on central nervous system.

BACKGROUND: Widespread use of silver in its different forms raises concerns about potential adverse effects after ingestion, the main exposure route for humans. The aim of this study was to investigate in CD-1 (ICR) male mice the tissue distribution and in vivo effects of 4-week oral exposure to 0.25 and 1 mg Ag/kg bw 10 nm citrate coated silver nanoparticles (AgNPs) and 1 mg Ag/kg bw silver acetate (AgAc) at the end of treatment (EoT) and after 4 weeks of recovery. RESULTS: There were no treatment-related clinical signs and mortality, and no significant effects on body and organ weights at the EoT and after recovery. Treatment-related changes in hematology and clinical chemistry were found after recovery, the most relevant being a dose-dependent lymphopenia and increased triglycerides in AgNP-treated mice, and increased levels of urea in all treated groups, associated with decreased albumin only in AgAc-treated mice. At the EoT the highest silver concentration determined by Triple Quadrupole ICP-MS analysis was found in the brain, followed by testis, liver, and spleen; much lower concentrations were present in the small intestine and kidney. Tissue silver concentrations were slightly higher after exposure to AgAc than AgNPs and dose dependent for AgNPs. After recovery silver was still present in the brain and testis, highlighting slow elimination. No histopathological changes and absence of silver staining by autometallography were observed in the organs of treated mice. At the EoT GFAP (astrocytes) immunoreactivity was significantly increased in the hippocampus of AgNP-treated mice in a dose-dependent manner and Iba1 (microglial cells) immunoreactivity was significantly increased in the cortex of 1 mg/kg bw AgNP-treated mice. After recovery, a significant reduction of Iba1 was observed in the cortex of all treated groups. TEM analysis of the hippocampus revealed splitting of basement membrane of the capillaries and swelling of astrocytic perivascular end-feet in 1 mg/kg bw AgNP- and AgAc-treated mice at the EoT. CONCLUSIONS: Our study revealed accumulation and slow clearance of silver in the brain after oral administration of 10 nm AgNPs and AgAc at low doses in mice, associated with effects on glial cells and ultrastructural alterations of the Blood-Brain Barrier.

Total laryngectomy in a cat with a laryngeal peripheral nerve sheath tumor.

OBJECTIVE: To report the surgical technique and outcome of total laryngectomy in a single clinical case. STUDY DESIGN: Case report. ANIMAL: A 5-year-old female spayed domestic shorthair cat. METHODS: A cat presented for acute, severe respiratory distress caused by an invasive laryngeal mass. Incisional biopsy was indicative of sarcoma. Computed tomography of head, neck, and thorax was performed revealing no evidence of metastasis. A total laryngectomy and permanent tracheostomy were performed, and the cat could breathe without difficulties immediately postoperatively. Histopathology confirmed a laryngeal low-grade peripheral nerve sheath tumor (PNST). RESULTS: Surgical margins were free of tumor cells. Surgical revision of the tracheostomy stoma due to obstructive granulation tissue was necessary 24 days after the initial surgery. Nine days after revision surgery, the cat was discharged from the hospital. No evidence of local recurrence or metastasis was detected on repeat computed tomography of the head, neck, and thorax at 6 months, nor on chest radiographs at 12 months postoperatively. At the time of writing (13 months postoperatively), the cat is still alive with a good quality of life. CONCLUSION: Total laryngectomy with permanent tracheostomy allowed the complete removal of an obstructive laryngeal PNST and provided a good quality of life in a cat. CLINICAL SIGNIFICANCE: To the authors' knowledge, this case report represents the first detailed description of the surgical procedure and clinical outcome for a total laryngectomy in a cat.

Chromosome Transplantation: Correction of the Chronic Granulomatous Disease Defect in Mouse Induced Pluripotent Stem Cells.

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.

Inhibition of SIRT1 deacetylase and p53 activation uncouples the anti-inflammatory and chemopreventive actions of NSAIDs.

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.

The atypical receptor CCRL2 is required for CXCR2-dependent neutrophil recruitment and tissue damage.

CCRL2 is a 7-transmembrane domain receptor that shares structural and functional similarities with the family of atypical chemokine receptors (ACKRs). CCRL2 is upregulated by inflammatory signals and, unlike other ACKRs, it is not a chemoattractant-scavenging receptor, does not activate ß-arrestins, and is widely expressed by many leukocyte subsets. Therefore, the biological role of CCRL2 in immunity is still unclear. We report that CCRL2-deficient mice have a defect in neutrophil recruitment and are protected in 2 models of inflammatory arthritis. In vitro, CCRL2 was found to constitutively form homodimers and heterodimers with CXCR2, a main neutrophil chemotactic receptor. By heterodimerization, CCRL2 could regulate membrane expression and promote CXCR2 functions, including the activation of ß2-integrins. Therefore, upregulation of CCRL2 observed under inflammatory conditions is functional to finely tune CXCR2-mediated neutrophil recruitment at sites of inflammation.

Topiramate protects apoE-deficient mice from kidney damage without affecting plasma lipids.

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.

Long-Term Study on the Effects of Housing C57BL/6NCrl Mice in Cages Equipped With Wireless Technology Generating Extremely Low-Intensity Electromagnetic Fields.

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.

Tumor progression and metastatic dissemination in ovarian cancer after dose-dense or conventional paclitaxel and cisplatin plus bevacizumab.

The efficacy of therapeutic regimens incorporating weekly or every-3-weeks paclitaxel (PTX) for ovarian cancer is debated. We investigated the addition of bevacizumab in regimens of chemotherapy with different PTX doses and schedules in preclinical models. Treatments were cisplatin (DDP) with weekly PTX (conventional), or dose-dense-equi (every other day to the conventional cumulative dose), or dose-dense-high (total dose 1.5 times higher), with or without bevacizumab. Treatment efficacy was evaluated analyzing tumor growth in different time-windows in two patient-derived ovarian cancer xenografts with different sensitivity to cisplatin. Tumor progression, metastasis and survival were studied in ovarian cancer models growing orthotopically and disseminating in the mouse peritoneal cavity. Short-term effects on cell cycle, tumor cell proliferation/apoptosis and vasculature were evaluated by flow cytometry and immunohistochemistry. PTX dose-dense (with/without DDP) was superior to the conventional scheme in a dose-dependent manner; the high efficacy was confirmed by the lower ratio of tumor to normal cells. All schemes benefited from bevacizumab, which reduced tumor vessels. However, DDP/PTX dose-dense-high (only chemotherapy) was at least as active as DDP/PTX conventional plus bevacizumab. DDP/PTX dose-dense-high plus bevacizumab was the most effective in delaying tumor progression, though it did not prolong mouse survival and the continuous treatment with bevacizumab was associated with a malignant disease. These findings indicate that the effect of bevacizumab in combination with chemotherapy may depend on the schedule-dose of the treatment and help to explain the unclear benefits after bevacizumab.

Estimating canine cancer incidence: findings from a population-based tumour registry in northwestern Italy.

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.

Functional Single-Chain Polymer Nanoparticles: Targeting and Imaging Pancreatic Tumors in Vivo.

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.

Tissue distribution and acute toxicity of silver after single intravenous administration in mice: nano-specific and size-dependent effects.

BACKGROUND: Silver nanoparticles (AgNPs) are an important class of nanomaterials used as antimicrobial agents for a wide range of medical and industrial applications. However toxicity of AgNPs and impact of their physicochemical characteristics in in vivo models still need to be comprehensively characterized. The aim of this study was to investigate the effect of size and coating on tissue distribution and toxicity of AgNPs after intravenous administration in mice, and compare the results with those obtained after silver acetate administration. METHODS: Male CD-1(ICR) mice were intravenously injected with AgNPs of different sizes (10 nm, 40 nm, 100 nm), citrate-or polyvinylpyrrolidone-coated, at a single dose of 10 mg/kg bw. An equivalent dose of silver ions was administered as silver acetate. Mice were euthanized 24 h after the treatment, and silver quantification by ICP-MS and histopathology were performed on spleen, liver, lungs, kidneys, brain, and blood. RESULTS: For all particle sizes, regardless of their coating, the highest silver concentrations were found in the spleen and liver, followed by lung, kidney, and brain. Silver concentrations were significantly higher in the spleen, lung, kidney, brain, and blood of mice treated with 10 nm AgNPs than those treated with larger particles. Relevant toxic effects (midzonal hepatocellular necrosis, gall bladder hemorrhage) were found in mice treated with 10 nm AgNPs, while in mice treated with 40 nm and 100 nm AgNPs lesions were milder or negligible, respectively. In mice treated with silver acetate, silver concentrations were significantly lower in the spleen and lung, and higher in the kidney than in mice treated with 10 nm AgNPs, and a different target organ of toxicity was identified (kidney). CONCLUSIONS: Administration of the smallest (10 nm) nanoparticles resulted in enhanced silver tissue distribution and overt hepatobiliary toxicity compared to larger ones (40 and 100 nm), while coating had no relevant impact. Distinct patterns of tissue distribution and toxicity were observed after silver acetate administration. It is concluded that if AgNPs become systemically available, they behave differently from ionic silver, exerting distinct and size-dependent effects, strictly related to the nanoparticulate form.

Neuroanatomy-based matrix-guided trimming protocol for the rat brain.

Brain trimming through defined neuroanatomical landmarks is recommended to obtain consistent sections in rat toxicity studies. In this article, we describe a matrix-guided trimming protocol that uses channels to reproduce coronal levels of anatomical landmarks. Both setup phase and validation study were performed on Han Wistar male rats (Crl:WI(Han)), 10-week-old, with bodyweight of 298 ± 29 (SD) g, using a matrix (ASI-Instruments(®), Houston, TX) fitted for brains of rats with 200 to 400 g bodyweight. In the setup phase, we identified eight channels, that is, 6, 8, 10, 12, 14, 16, 19, and 21, matching the recommended landmarks midway to the optic chiasm, frontal pole, optic chiasm, infundibulum, mamillary bodies, midbrain, middle cerebellum, and posterior cerebellum, respectively. In the validation study, we trimmed the immersion-fixed brains of 60 rats using the selected channels to determine how consistently the channels reproduced anatomical landmarks. Percentage of success (i.e., presence of expected targets for each level) ranged from 89 to 100%. Where 100% success was not achieved, it was noted that the shift in brain trimming was toward the caudal pole. In conclusion, we developed and validated a trimming protocol for the rat brain that allow comparable extensiveness, homology, and relevance of coronal sections as the landmark-guided trimming with the advantage of being quickly learned by technicians.

Magnetic resonance imaging visualization of vulnerable atherosclerotic plaques at the brachiocephalic artery of apolipoprotein E knockout mice by the blood-pool contrast agent B22956/1.

The aim of this study was to identify, by magnetic resonance imaging (MRI), the ability of the blood-pool contrast agent B22956/1 to detect atherosclerotic plaques developing at the brachiocephalic artery of apolipoprotein E knockout (apoE-KO) mice and to possibly identify vulnerable atherosclerotic lesions. After high-fat feeding for 8 or 12 weeks, MRIs of brachiocephalic arteries were acquired before and after B22956/1 administration; then vessels were removed and analyzed by histology. B22956/1 injection caused a rapid increase in plaque signal enhancement and plaque to muscle contrast values, which remained stable up to 70 minutes. A linear correlation between signal enhancement and macrophage content was found 10 minutes after B22956/1 injection (p < .01). Signal enhancement and plaque to muscle contrast values correlated with macrophage content 40 minutes after contrast agent administration (p < .01). Finally, 70 minutes after B22956/1 infusion, plaque to muscle contrast significantly correlated with the percentage of stenosis (p < .005). B22956/1 administration to high fat-fed apoE-KO mice resulted in a rapid enhancement of atherosclerotic plaques and in a great ability to rapidly visualize vulnerable plaques, characterized by a high macrophage content. These results suggest that B22956/1 could represent an interesting tool for the identification of atherosclerotic plaques potentially leading to acute cardiovascular events.

Antitumor activity of a novel homodimeric SMAC mimetic in ovarian carcinoma.

Treatment of ovarian carcinoma often fails to be curative because of drug resistance, and many efforts are directed to overcome tumor cell resistance by increasing apoptosis induction. The potential of second mitochondria-derived activator of caspases (SMAC) mimetics (SMACm) has appeared in preclinical studies, but novel proapoptotic agents of this class with improved pharmacological profile are needed. To identify novel treatment options for ovarian carcinoma by interfering with antiapoptotic factors, in the present study a novel homodimeric SMACm (SM83) was employed in preclinical models both in vitro and in vivo. An investigation of the structural features of dimeric SM83 as compared to a closely related reference compound indicated slight differences, likely because of the interaction between one of the terminal phenyl groups and triazole rings of SM83 with the BIR2 domain. Although SM83 per se did not inhibit cell proliferation, it displayed a synergistic effect in combination with TNF-related apoptosis inducing ligand (TRAIL) in cell sensitivity assays. Because the tumor microenvironment is a reservoir of cytokines that may act in conjunction with SMACm to affect tumor growth, the activity of the novel compound was tested in vivo in ovarian carcinoma cells subcutaneously xenografted into immunodeficient mice. A significant tumor volume inhibition was observed together with activation of caspase 3 and apoptotic cell death. A biochemical analysis of tumor necrosis factor (TNF) and TRAIL content in specimens from xenografted mice indicated that SM83 downmodulated the levels of human TNF in plasma samples and tended to upmodulate human TRAIL levels in tumors. Thus, TRAIL appears to contribute to the antitumor activity of novel SMACm SM83 in subcutaneously grown ovarian carcinoma. Overall, our results indicate that SM83 is an attractive candidate for further development.