Biomedical Research: Engineered C57BL/6 as Mouse Models for the Study of Treatments for Hutchinson-Gilford Progeria Syndrome (HGPS), and HER2-positive cancers.

This thesis focuses on two engineered C57BL/6 mice models used to study treatments for Progera Syndrome and HER2-positive cancers. The first study, conducted in collaboration with the National Research Council, Institute of Molecular Genetics - Unit of Bologna, is on LmnaG609G transgenic mice homologous for the genetic cause of Progeria Syndrome. The objective was to describe the model, giving information on the housing, breeding, welfare and progression of the disease. Similarities and differences between the model and human patients were highlighted and the acquired data will be essential in programming future studies using this animal model. Following this, a preliminary study was conducted on the mice, using a treatment of all-trans retinoic acid (0.4 mg/kg) combined with low doses of rapamycin (1 mg/kg) administered twice weekly intraperitoneally, in order to evaluate whether the mice's lifespan was improved. Results comparing treated and untreated groups were not significant. However, the animal groups were small in number and only males were considered, so future research will be needed. The second study reports the use of HER2 tolerant transgenic mice to investigate the efficacy of immunotherapy with oncolytic viruses. This study was performed in collaboration with the Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna. The mouse demonstrated to be a reliable model since it enabled the development of implanted HER2 cancer and allowed the virus replication within the tumour. Animals did not show any side effects and the safe profile of the virus was confirmed. To date we tested the efficacy of two viruses: R-LM113 and R-LM113-mIL-12. The latter gave better results, underlining the importance of the immune system in oncology and immune competent models in oncology research. In conclusion, we can state that both animal models considered in this thesis have demonstrated their appropriateness for testing specific therapies.Questa tesi si focalizza su due modelli murini C57BL/6 geneticamente modificati per lo studio di terapie per la Sindrome Progerica e tumori HER2-positivi. La prima ricerca, in collaborazione con il Consiglio Nazionale delle Ricerche - Istituto di Genetica Molecolare di Bologna, studia il topo transgenico LmnaG609G portatore della modificazione genetica che causa la Sindrome Progerica. L'obiettivo è stato di descrivere il modello, dando informazioni sull'allevamento, sul benessere e sulla progressione della malattia. Similitudini e differenze tra il modello animale e il paziente umano sono messe in evidenza e tutte le informazioni acquisite saranno utili per programmare studi futuri. Sul medesimo modello, è stato condotto uno studio preliminare per valutare l'efficacia del trattamento con una combinazione di acido retinoico (0.4 mg/kg) e rapamicina (1 mg/kg) somministrato intraperineo due volte a settimana nel prolungare l'aspettativa di vita. I risultati non sono stati significativi, ma data la preliminarietà dello studio saranno necessari ulteriori approfondimenti. La seconda ricerca riporta l'utilizzo di un topo transgenico reso tollerante al recettore HER2 (sovraespresso in alcuni tumori) per investigare l'efficacia di virus oncolitici in immunoterapia. Questo studio è stato condotto in collaborazione con il Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale dell'Università di Bologna. Il topo si è mostrato un modello affidabile in quanto è stato in grado di sviluppare masse tumorali dopo iniezione di cellule murine trasdotte con HER2 e ha permesso al virus di replicare. Gli animali non hanno mostrato effetti indesiderati e l'alto profilo di sicurezza virale è stato confermato. Ad oggi è stata valutata l'attività di due virus: R-LM113 and R-LM113-mIL-12. Il secondo ha dato migliori risultati, sottolineando l'importanza del sistema immunitario in oncologia e l'utilizzo di modelli animali immunocompetenti. In conclusione, si può affermare che entrambi i modelli animali si sono rivelati appropriati per testare l'efficacia di terapie mirate

High-dose therapy and autologous stem cell transplantation in patients with POEMS syndrome: a retrospective study of the Plasma Cell Disorder sub-committee of the Chronic Malignancy Working Party of the European Society for Blood & Marrow Transplantation

POEMS syndrome is a rare para-neoplastic syndrome secondary to a plasma cell dyscrasia. Effective treatment can control the diseaserelated symptom complex. We describe the clinical outcome of autologous stem cell transplantation for patients with POEMS syndrome, determining the impact of patient- and disease-specific factors on prognosis. One hundred and twenty-seven patients underwent an autologous stem cell transplantation at 1997-2010 with a median age of 50 years (range 26-69 years). Median time from diagnosis to autologous stem cell transplantation was 7.5 months with 32% of patients receiving an autologous stem cell transplantation more than 12 months from diagnosis. Engraftment was seen in 97% patients and engraftment syndrome was documented in 23% of autologous stem cell transplantation recipients. Hematologic response was characterized as complete response in 48.5%, partial response in 20.8%, less than partial response in 30.7%. With a median follow up of 48 months (95%CI: 38.3, 58.6), 90% of patients are alive and 16.5% of patients have progressed. The 1-year non-relapse mortality was 3.3%. The 3-year probabilities of progression-free survival and overall survival are 84% and 94%, respectively, with 5-year probabilities of progression-free survival and overall survival of 74% and 89%. In a cohort of graft recipients, detailed organ-specific symptom response demonstrated clear symptom benefit after autologous stem cell transplantation especially in relation to neurological symptom control. The data analysed in this study demonstrate the clinical utility of autologous stem cell transplantation for patients with POEMS syndrome

Tolerability and pharmacokinetics of two antimony products after subcutaneous administration in dogs

Introduction: Pentavalent antimony is the first choice drug for leishmaniasis in dog. Leishmaniasis has a complex pathogenesis and it manifests various clinical signs, some of which are often similar to those associated with the toxicity induced by antimonial treatment. Among the reasons for this toxicity, also a general problem of drug\u2019s quality has been reported. Methodology: The general and local tolerability of two commercially available meglumine antimoniate based veterinary products was evaluated in 12 healthy dogs, 6 receiving Antimania (Fatro, Italy) and 6 receiving Glucantime (Merial, Spain), following repeated subcutaneous administrations of therapeutic doses for 14 days. Results: Individual and mean values of haematological and biochemical parameters in both groups remained in the physiological range, with no considerable differences within the two groups. The general tolerability of the drugs was also supported by clinical observations and physical examination of the dogs throughout the whole study period. Only slight local reactions at the injection sites, that spontaneously disappeared, were observed for both products starting from 12-84 hours after the administration. The pharmacokinetic parameters indicated no antimony accumulation. Conclusions: These results suggest that meglumine antimoniate administered at the recommended dosage regimen is well tolerated by healthy dogs, and that there is no significant difference between the two tested products

A quick and simple method for the determination of ivermectin in dog plasma by LC–MS/MS

Ivermectin is an endectocide belonging to the macrocyclic lactone class, commonly used in dogs as a heartworm preventative and for the treatment of several external and internal parasite infections. Among the analytical methods for ivermectin determination in plasma available in literature, many require a laborious clean-up step on SPE cartridge, and use fluorescence detection instead of the more reliable mass spectrometry. In the context of a project aimed at its pharmacokinetic evaluation in this species, a liquid chromatography-tandem mass spectrometry method for the determination of ivermectin in dog plasma was developed and validated, using blank plasma provided by a local canine blood transfusion service. Samples underwent a quick liquid-liquid extraction before analysis in the LC–MS/MS system, operating in selected reaction monitoring (SRM) mode. The method provided satisfactory linearity (R2 >0.99), accuracy (bias <3%) and precision (CV <10%) over the 0.5–20.0 ng/mL range. • This is to our knowledge the first validated LC–MS/MS method for ivermectin determination in dog plasma. • Sample preparation is simple, rapid and inexpensive, without compromising sensitivity. • The modest amount of plasma required makes the proposed technique suitable for pharmacokinetic studies also in small dogs. Method name: Ivermectin in dog plasma by LC–MS/MS, Keywords: Ivermectin, Plasma, Dog, LC–MS/M

Survey on antimicrobial prescribing patterns in small animal veterinary practice in Emilia Romagna, Italy

This investigation provides for the first time a general view of the prescribing patterns of antimicrobials in small animal practice in Emilia Romagna, Italy. In the context of a project on antimicrobial resistance managed by the Regional Veterinary Service, veterinary clinicians were invited to voluntarily complete an online questionnaire. This was designed to gather information on antimicrobial prescribing practices and biosecurity measures and to understand the perception of the issue specific to this region of Italy. In total, 266 questionnaires correctly completed were collected. Although clinicians seemed to follow different approaches when using antimicrobials, the data analysis revealed a general awareness on resistance. Penicillins were the most commonly prescribed class, followed by (fluoro)quinolones and cephalosporins. Among those who use laboratory testing more or less frequently (microbiological analysis and susceptibility testing) to support their prescribing habits, only 7 per cent make a habit of always waiting for the results before starting the treatment. Seventy-eight per cent of the respondents declared the use of antimicrobials licensed for human beings. Biosecurity measures were carefully taken into account by the majority of the veterinarians. The results identified the antimicrobial classes that are commonly prescribed and highlighted that perioperative hygiene measures and the use of laboratory diagnosis are critical aspects that need to be emphasised in drawing up guidelines on the prudent use of these drugs in pets