[Show abstract][Hide abstract] ABSTRACT: Autosomal recessive osteopetrosis is a human bone disease mainly caused by TCIRG1 gene mutations that prevent osteoclasts resorbing activity, recapitulated by the oc/oc mouse model. Bone marrow transplantation is the only available treatment, limited by the need for a matched donor. The use of induced pluripotent stem cells (iPSCs) as an unlimited source of autologous cells to generate gene corrected osteoclasts might represent a powerful alternative. We generated iPSCs from oc/oc mice, corrected the mutation using a BAC carrying the entire Tcirg1 gene locus as a template for homologous recombination, and induced hematopoietic differentiation. Similarly to physiologic fetal hematopoiesis, iPSC-derived CD41(+) cells gradually gave rise to CD45(+) cells, which comprised both mature myeloid cells and high proliferative potential colony-forming cells. Finally, we differentiated the gene corrected iPSC-derived myeloid cells into osteoclasts with rescued bone resorbing activity. These results are promising for a future translation into the human clinical setting.
[Show abstract][Hide abstract] ABSTRACT: Helicobacter pylori is 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 that H. pylori could 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 onto H. pylori selective medium and by screening PCR for Helicobacteraceae, followed by specific PCRs for H. pylori, Wolinella spp., and “Candidatus Helicobacter bovis.” Three out of 163 bulk milk samples tested positive for Helicobacteraceae, but not for the subsequent PCRs. H. pylori was not isolated in any case. However, given similar growth conditions, Arcobacter butzleri, A. cryaerophilus, and A. skirrowii were recovered. In conclusion, the prevalence of Helicobacteraceae in raw milk was negligible (1.8%), and H. pylori was 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.
[Show abstract][Hide abstract] ABSTRACT: Leptospirosis is an important zoonotic disease diffused worldwide, and wildlife species are commonly considered to be important epidemiological carriers. Four-hundred and forty-one serological and 198 renal samples from red deer, roe deer and chamois collected in the Province of Sondrio were analysed using the microscopic agglutination test and histopathologic examination. Positive serological findings were found only in 15 red deer and 19 positive serologic reactions were recorded. The most frequent serovars were Bratislava and Grippotyphosa, followed by Pomona, Hardjo and Copenhagheni. Twenty-two per cent of renal samples from seropositive red deer were affected by mild to moderate multifocal chronic lymphoplasmacytic and fibrosing tubulo-interstitial nephritis, mainly involving the cortical parenchyma. In this study, antibodies to Leptospira spp. were infrequent in wild ruminants, and only red deer seemed to be sensitive to the infection. Given the low presence and the fact that there was no record of Leptospira spp. infections in cattle, sheep, goats and also hunters in area during the study period, wild ruminants in Alpine environments cannot be considered as reservoirs or important sources of Leptospira spp. infection for humans or domestic animals.
Veterinaria italiana 12/2014; 50(4):285-91. DOI:10.12834/VetIt.1309.06 · 0.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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 a rHSA matrix to yield rHSA-NPs. The resulting NPs were analysed by transmission electron microscopy, photon correlation spectroscopy, and atomic absorption. The sizes of the manufactured NP were 170±10nm, and the zeta-potential was -50±3mV. The NPs remained stable when stored after lyophilisation 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 favourable properties containing iron oxide for detection by MRI.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: p53 influences genomic stability, apoptosis, autophagy, response to stress, and DNA damage. New p53-target genes could elucidate mechanisms through which p53 controls cell integrity and response to damage.
DRAGO (drug-activated gene overexpressed, KIAA0247) was characterized by bioinformatics methods as well as by real-time polymerase chain reaction, chromatin immunoprecipitation and luciferase assays, time-lapse microscopy, and cell viability assays. Transgenic mice (94 p53(-/-) and 107 p53(+/-) mice on a C57BL/6J background) were used to assess DRAGO activity in vivo. Survival analyses were performed using Kaplan-Meier curves and the Mantel-Haenszel test. All statistical tests were two-sided.
We identified DRAGO as a new p53-responsive gene induced upon treatment with DNA-damaging agents. DRAGO is highly conserved, and its ectopic overexpression resulted in growth suppression and cell death. DRAGO(-/-) mice are viable without macroscopic alterations. However, in p53(-/-) or p53(+/-) mice, the deletion of both DRAGO alleles statistically significantly accelerated tumor development and shortened lifespan compared with p53(-/-) or p53(+/-) mice bearing wild-type DRAGO alleles (p53(-/-), DRAGO(-/-) mice: hazard ratio [HR] = 3.25, 95% confidence interval [CI] = 1.7 to 6.1, P < .001; p53(+/-), DRAGO(-/-) mice: HR = 2.35, 95% CI = 1.3 to 4.0, P < .001; both groups compared with DRAGO(+/+) counterparts). DRAGO mRNA levels were statistically significantly reduced in advanced-stage, compared with early-stage, ovarian tumors, but no mutations were found in several human tumors. We show that DRAGO expression is regulated both at transcriptional-through p53 (and p73) and methylation-dependent control-and post-transcriptional levels by miRNAs.
DRAGO represents a new p53-dependent gene highly regulated in human cells and whose expression cooperates with p53 in tumor suppressor functions.
Journal of the National Cancer Institute 03/2014; 106(8). DOI:10.1093/jnci/dju053 · 12.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Context:Tyrosine kinase inhibitors (TKI) represent a new treatment option for patients with advanced medullary thyroid cancer (MTC). However, cures have not been achieved with current available agents used in monotherapy.Objective:Since RET has been shown to negatively regulate CD95 death receptor activation in preclinical models of RET-dependent MTC, we investigated the potential of the combination approach with the RET targeting TKI sunitinib and cisplatin to enhance apoptosis activation through the extrinsic pathway.Design:The effects of sunitinib and cisplatin were examined in human MTC cell lines harboring oncogenic RET mutations. Experiments were designed to determine drug effects on RET signaling, cell growth, apoptosis, autophagy, tumor growth in mice, and to investigate the mechanisms of the drug interaction.Results:Sunitinib and cisplatin synergistically inhibited the growth of MZ-CRC-1 cells harboring the RET M918T activating mutation. The combination enhanced apoptosis activation through CD95-mediated, caspase-8 dependent, pathway. Moreover, sunitinib induced a severe perturbation of the autophagic flux characterized by autophagosome accumulation and a remarkable lysosomal dysfunction which was further enhanced, with lysosomal leakage induction, by cisplatin. Administration of the drug combination to mice xenografted with MZ-CRC-1 cells improved the antitumor efficacy, as compared to single agent treatments, inducing complete responses in 30% of treated mice, a significant increase in caspase-3 activation (P < 0.01 vs cisplatin, P < 0.0005 vs sunitinib) and apoptosis in tumor cells.Conclusions:Addition of cisplatin to sunitinib potentiates apoptotic cell death and has promising preclinical activity in MTCs harboring the RET M918T oncogene.
The Journal of Clinical Endocrinology and Metabolism 11/2013; 99(2). DOI:10.1210/jc.2013-2574 · 6.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Treatment of ovarian carcinoma often fails to be curative due to 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 pro-apoptotic agents of this class with improved pharmacological profile are needed. To identify novel treatment options for ovarian carcinoma by interfering with anti-apoptotic 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 due to 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. Since the tumor microenviroment 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 in immuno-deficient mice. A significant tumor volume inhibition was observed together with activation of caspase 3 and of apoptotic cell death. A biochemical analysis of Tumor Necrosis Factor (TNF) and TRAIL content in specimens from xenografted mice indicated that SM83 down-modulated the levels of human TNF in plasma samples and tended to up-modulate human TRAIL levels in tumors. Thus, TRAIL appears to contribute to the antitumor activity of the novel SMACm SM83 in subcutaneously grown ovarian carcinoma. Overall, our results indicate SM83 as an attractive candidate for further development.
[Show abstract][Hide abstract] ABSTRACT: Effects of post-resuscitation treatment with argon on neurological recovery were investigated in a porcine model of cardiac arrest (CA) with an underlying acute myocardial infarction.
The left anterior descending coronary artery (LAD) was occluded in 12 pigs and CA was induced. After 8 min of untreated CA, cardiopulmonary resuscitation was performed for 5 min prior to defibrillation. Following resuscitation, animals were subjected to 4 hr ventilation with 70% argon/30% oxygen or 70% nitrogen/30% oxygen. Myocardial function was echocardiographically assessed and serum neuron specific enolase (NSE) was measured. Animals were observed up to 72 hr for assessment of survival and neurological recovery.
All the animals were resuscitated and survived for 72 hr, except for a control pig. Ventilation with argon did not have any detrimental effects on hemodynamics and respiratory gas exchange. All the 6 argon treated animals had a fast and complete 72 hr neurological recovery, in contrast to only 2 of the 6 controls (p < 0.05). Seventy-two hr neurological alertness score and neurological deficit score were respectively 100 and 0 in the argon group and 79 and 29 in the control one (p < 0.01 and p < 0.05). Significantly lower increases in serum NSE (12% vs. 234%) and minimal histological brain injury (neuronal degeneration: 0 vs. 1) were also observed in argon treated animals, in comparison to controls.
In this model, post-resuscitation treatment with argon allowed for a faster and complete neurological recovery, without detrimental effects on hemodynamics and respiratory gas exchanges.
[Show abstract][Hide abstract] ABSTRACT: The development of resistance and progressive disease after treatment with angiogenesis inhibitors is becoming a controversial issue. We investigated the experimental conditions that cause multikinase receptor inhibitors (RTKI) to augment metastasis and whether opportune combinations with chemotherapy could counteract this effect. The renal Renca-luc tumor was transplanted orthotopically in the kidney of Balb/c mice, which were then or not nephrectomized. The Lewis Lung carcinoma (LLC) was transplanted in the tibial muscle of C57/Bl6 mice. Treatment with the RTKI sunitinib started at different stages of tumor progression, mimicking neo-adjuvant or adjuvant settings. Combination studies with paclitaxel, doxorubicin, cisplatin, gemcitabine and topotecan were done on the LLC model, using opportune regimens. In a neo-adjuvant setting sunitinib inhibited Renca-luc tumor growth, prolonging survival despite an increase in lung metastasis; treatment after primary tumor surgery (adjuvant setting) or on established metastasis prolonged survival and decreased metastasis. Sunitinib increased lung metastasis from mice bearing early-stage LLC, but did not affect established metastases (no acceleration) from advanced tumors. Combinations with doxorubicin, topotecan, gemcitabine, but not cisplatin and paclitaxel, counteracted the increase in metastasis from LLC, partly reflecting their antitumor activity. Histology analysis after sunitinib confirmed tumor vascular changes and increased hypoxia. Topotecan at suboptimal daily doses reduced sunitinib-related metastasis, reducing tumor hypoxia. Tyrosin kinase inhibitors, as sunitinib, can have adverse malignant effects mainly in the neo-adjuvant setting. The addition of chemotherapy might influence metastasis, depending on each drug mechanism of action and its regimen of administration.
Molecular Cancer Therapeutics 08/2013; 12(10). DOI:10.1158/1535-7163.MCT-13-0244 · 5.68 Impact Factor