INTRACELLULAR DNA DELIVERY USING POLYMERIC NANOPARTICLES FOR LYSOSOMAL STORAGE DISORDERS GENE THERAPY

INTRODUCTION: Gene therapy can be currently performed by viral or non-viral vectors. Although viral ones have already come to a clinical translation, concerns remain about both safety and the industrial applicability of this system. We therefore started the study of a transposon-based gene transfer system. We used nanoparticles (NPs) as vector to carry the transposon within the blood stream, then in cells. These had to be able to hold large plasmids, such as transposon. The NPs must then be able to reach the tissue most affected by the genetic disorder. Coating the NPs with specific peptides, they acquire the ability to accumulate in the target tissue, in our case the Central Nervous System (CNS) penetrating the blood-brain barrier (BBB). We started to develop a non-viral gene therapy approach in order to treat a neurological disease, globoid cell leukodystrophy in (GLD), an inherited, rapidly fatal disorder affecting myelin. METHODS: We tested the ability of a transposon-based system called Sleeping Beauty (SB), to convey the luciferase marker gene in the control cells. We then verified the ability of NPS of chitosan to contain large plasmids. We then verified the quality (size, shape) of the NPS obtained and their ability to integrate the gene of interest (GALC) within the cellular genome. Lastly, we verified the ability of a heptameric synthetic glycopeptide, of opioid origin, g7, to convey inside the CNS NPs of Poly Lactic-Co-Glycolic Acid (PLGA). We injected PLGA NPs coated with g7 in tail vein\u2019s Balb/c mice and followed through fluorescence spectroscopy their distribution. RESULTS: We verified the high SB transduction efficiency on control cells by their expression of luciferase marker gene. We then assessed the copies number of the integrated gene. We demonstrated the high transduction efficiency of the transgene within the genome of fibroblasts grown from biopsies of patients with GLD. We assessed the quality and features of chitosan-SB NPs. We could observe that g7 coated PLGA NPs accumulate in the CNS 10 minutes after infusion, with accumulation even and up to 2 hours. DISCUSSION: SB demonstrated high efficiency in stable transduction of control cells. So we thought to encapsulate SB in chitosan NPs. These have proven to be highly efficient in stably transducing deficient fibroblasts. We can confirm that g7 is today the best way to address the NPs to the CNS. CONCLUSIONS: Future studies will test the ability of g7 to coat the NPs of chitosan, verify its efficiency in conveying the chitosan NPs to the CNS as well as they did for PLGA NPs. Finally, by cloning into SB the gene whose mutation causes GLD, can be assessed the therapeutic efficacy of the system in the mouse model of the disease

Impact of standardization in tissue processing: the performance of different fixatives

Most tissues in clinical practice are formalin-fixed and paraffin-embedded for histological as well as molecular analyses. The reproducibility and uniformity of molecular analyses is strictly dependent on the quality of the biomolecules, which is highly influenced by pre-analytical processes. In this study, the effect of different fixatives was compared, including formalin, Bouin's solution, RCL2® and TAG-1™ fixatives, by stringent application of ISO standards in mouse liver tissue processing, including formalin-free transport of tissues and tissue grossing in a refrigerated environment. The effect of fixatives was studied in terms of nucleic acid quality at the time of tissue processing and after one year of tissue storage at room temperature in the dark. Furthermore, a microcomputed tomography (CT) scan analysis was applied to investigate the paraffin embedding. The results show that the application of ISO standards in tissue processing allows analysis of 400 bases amplicons from RNA and 1000 bases from DNA, even in extracts from formalin-fixed and paraffin-embedded tissues. However, after one year storage at room temperature in the dark, a degradation of the nucleic acids was observed. Nevertheless, extracts can still be analyzed, but for metachronous tests it is highly recommended to repeat the quantitation of housekeeping genes in order to standardize the extent of nucleic acid degradation

Self-efficacy for coping. Utility of the Cancer behavior inventory (Italian) for use in palliative care

Background: Newer models of palliative and supportive cancer care view the person as an active agent in managing physical and psychosocial challenges. Therefore, personal efficacy is an integral part of this model. Due to the lack of instruments in Italian to assess coping self-efficacy, the present study included the translation and validation of the Italian version of the Cancer Behavior Inventory-Brief (CBI-B/I) and an initial analysis of the utility of self-efficacy for coping in an Italian sample of palliative care patients. Methods: 216 advanced cancer patients who attended palliative care clinics were enrolled. The CBI-B/I was administered along with the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 (EORTC QLQ-C30), the Mini Mental Adjustment to Cancer Scale (Mini-MAC), the Cancer Concerns Checklist (CCL), and the Hospital Anxiety and Depression Scale (HADS). The Eastern Cooperative Oncology Group Performance Status (ECOG-PS) ratings of functional capacity were completed by physicians. Results: Factor analysis confirmed that the structure of the CBI-B/I was consistent with the English version. Internal consistency reliability and significant correlations with the EORTC QLQ-C30, Mini-MAC, and HADS supported the concurrent validity of the CBI-B/I. Differences in CBI-B/I scores for high versus low levels of the CCL and ECOG-PS supported the clinical utility of the CBI-B/I. Conclusions: The CBI-B/I has strong psychometric properties and represents an important addition to newer model of palliative and supportive care. In order to improve clinical practice, the CBI-B/I could be useful in identifying specific self-efficacy goals for coping in structured psychosocial intervention

A "SURFin' Leukemia": Acute Lymphoblastic Leukemia Masquerading as a Syndrome of Undifferentiated Recurrent Fever

Periodic fever is not uncommon in childhood and is often ascribed to autoinflammatory conditions; however, it may be present also in children with cancer. We here describe the case of a 3-year-old boy with acute lymphoblastic leukemia who initially presented with a 4-month history of recurrent, stereotyped episodes of fever and localized joint pain, separated by completely symptom-free intervals. These symptoms were initially interpreted as a possible syndrome of undifferentiated recurrent fever until more signs of leukemia became apparent. Our report confirms that acute lymphoblastic leukemia can rarely present with periodic fever, thus possibly leading to diagnostic errors unless a high index of suspicion is maintained

An update on microbiological causes of canine otitis externa in Campania Region – Italy.

Objective: To update the recent knowledge of the microbiological causes of canine otitis externa in Campania Region (Italy) and the antibiotic susceptibility patterns of the isolated strains. Methods: A total of 122 dogs were examined by otoscopy, and auricular swab samples were collected from both ears in 74 dogs presenting clinical bilateral otitis and from single ears in 48 dogs displaying clinical unilateral otitis. Cytological examination, bacteriological analysis and antimicrobial susceptibility tests were performed. Results: Thirty-one out of 122 dogs were positive for yeast species (25.4%, 95% confidence interval (CI): 18.2%–34.2%) with a higher prevalence of Malassezia pachydermatis (21/31 isolates, 67.7%, CI: 48.5%–82.7%), and a total of 91 out of 122 dogs were positive for bacterial species (74.6%; CI: 65.8%–81.8%) with a higher prevalence of Staphylococcus pseudintermedius (45/143 isolates, 31.5%, CI: 24.1%–39.8%). These results are the first description of Streptococcus agalactiae-associated otitis. The yeasts isolated showed high levels of susceptibility to all antifungal agents tested; on the contrary all the isolated bacterial strains were highly resistant to at least four out of ten antimicrobial classes. Both Gram-positive and Gram-negative bacteria showed high resistance to amoxicillin/clavulanate and kanamycin hence they are not recommended as initial empirical therapy for the otitis treatment. Conclusions: This update illustrates an increase in antibiotic resistances providing an insight into the current knowledge of the therapeutic procedures followed on canine otitis externa in Italy. It also emphasizes the importance of considering the results of the microbiological and sensitivity tests to decide on an appropriate antibiotic therapy

Genetic profiling of autoinflammatory disorders in patients with periodic fever: a prospective study

Periodic fever syndromes (PFS) are an emerging group of autoinflammatory disorders. Clinical overlap exists and multiple genetic analyses may be needed to assist diagnosis. We evaluated the diagnostic value of a 5-gene sequencing panel (5GP) in patients with undiagnosed PFS

Evidence and antibiotic resistance profiles of clinical Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) and non-ACB complex members in companion animals: A 2020-2022 retrospective study

To evaluate the frequency of Acinetobacter spp., belonging to both Acinetobacter calcoaceticus-baumannii (ACB) and non-ACB complex, and their antibiotic resistance profiles in veterinary medicine, a three-year (2020–2022) retrospective study was carried out on sick companion animals. Epidemiological data from different clinical canine, feline, and equine samples, were acquired. For each strain, MALDI-TOF MS identification and susceptibility to a panel of 11 antibiotics, by Kirby-Bauer and E-test methods, were performed. Out of 628 bacteriological examinations, 2.5% resulted positive for strains belonging to Acinetobacter genus. Frequencies of 2.3%, 1.9%, and 3% were obtained from both in-visiting and hospitalized dogs, cats, and horses, respectively. Members of ACB-complex accounted for 50% of isolates. Since all strains resulted susceptible to aminoglycosides and polymyxins, no pandrug-resistant (PDR) species were recorded. While 12.5% A. baumannii resulted extensively-drug resistant (XDR), a higher percentage of multidrug-resistant strains was recorded among non-ACB strains (35.5%) than ACB strains (25%). Susceptibility was observed in the same percentage in both groups (62.5%). All ACB strains confirmed their intrinsic resistances. Non-ACB species showed lower resistances against antipseudomonal penicillins plus beta-lactamase inhibitors (P=0.1306), III generation cephalosporins (P=0.0547), and tetracyclines (P=0.0209) than ACB species. Carbapenem-resistance was observed for XDR A. baumannii (12.5%) and, in particular for MDR non-ACB complex members (25%). To our knowledge, A. lactucae represents the first description in two sick dogs in Italy. Furthermore, our results emphasize the role of non-ACB-complex species as important zoonotic pathogens, which could be reservoirs of clinically relevant resistance profiles

Contribution to the ecology of the Italian hare (Lepus corsicanus)

the italian hare (Lepus corsicanus) is endemic to Central-Southern Italy and Sicily, classified as vulnerable due to habitat alterations, low density and fragmented populations and ecological competition with the sympatric european hare (Lepus europaeus). Despite this status, only few and local studies have explored its ecological features. We provided some key traits of the ecological niche of the italian hare as well as its potential distribution in the italian peninsula. All data derived from genetically validated presences. We generated a habitat suitability model using maximum entropy distribution model for the italian hare and its main competitor, the european hare. the dietary habits were obtained for the italian hare with DnA metabarcoding and High-throughput Sequencing on faecal pellets. The most relevant environmental variables affecting the potential distribution of the italian hare are shared with the european hare, suggesting a potential competition. the variation in the observed altitudinal distribution is statistically significant between the two species.The diet of the Italian hare all year around includes 344 plant taxa accounted by 62 families. The Fagaceae, Fabaceae, Poaceae, Rosaceae and Solanaceae (counts > 20,000) represented the 90.22% of the total diet. Fabaceae (60.70%) and Fagaceae (67.47%) were the most abundant plant items occurring in the Spring/Summer and Autumn/Winter diets, respectively. the Spring/Summer diet showed richness (N = 266) and diversity index values (Shannon: 2.329, Evenness: 0.03858, Equitability: 0.4169) higher than the Autumn/Winter diet (N = 199, Shannon: 1.818, Evenness: 0.03096, Equitability: 0.3435). Our contribution adds important information to broaden the knowledge on the environmental (spatial and trophic) requirements of the Italian hare, representing effective support for fitting management actions in conservation planning