microRNAs as New Biomolecular Markers to Estimate Time since Death: A Systematic Review.

Estimating the post-mortem interval is still one of the most complex challenges in forensics. In fact, the main tools currently used are burdened by numerous limitations, which sometimes allow the time of death to be placed only within too large time intervals. In recent years, researchers have tried to identify new tools to try to narrow down the interval within which to place the time of death; among these, the analysis of microRNAs seems to be promising. An evidence-based systematic review of the literature has been conducted to evaluate the state of the art of knowledge, focusing on the potential correlation between miRNA degradation and PMI estimation. The research has been performed using the electronic databases PubMed, Scopus, and WOS. The results allowed us to highlight the usefulness of miRNAs both as markers for PMI estimation and for normalization, especially due to their stability. In fact, some miRNAs remain particularly stable for long periods and in different tissues, while others degrade faster. Furthermore, there are numerous factors capable of influencing the behavior of these molecules, among which the type of tissue, the cause of death, and the circadian rhythm appear to be the most relevant. Despite the promising results of the few articles present in the literature, because of the numerous limitations they are burdened by, further research is still necessary to achieve more solid and shareable results.

Immune checkpoint inhibitors and neurotoxicity: a focus on diagnosis and management for a multidisciplinary approach.

INTRODUCTION: Although immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment, the consequential over activation of the immune system is often complicated by adverse events that can affect several organs and systems, including the nervous system. The precise pathophysiology underlying neurological irAEs (n-irAEs) is not completely known. Around 3.8% of patients receiving anti-CTLA-4 agents, 6.1% of patients receiving anti-PD-1/PD-L1, and 12% of patients receiving combination therapies have n-irAEs. Most n-irAEs are low-grade, while severe toxicities have rarely been reported. in this article, we performed an updated literature search on immuno-related neurotoxicity on main medical research database, from February 2017 to December 2023. AREAS COVERED: We have also compared the latest national and international guidelines on n-irAEs management with each other in order to better define patient management. EXPERT OPINION: A multidisciplinary approach appears necessary in the management of oncological patients during immunotherapy. Therefore, in order to better manage these toxicities, we believe that it is essential to collaborate with neurologists specialized in the diagnosis and treatment of n-irAEs, and that a global neurological assessment, both central and peripheral, is necessary before starting immunotherapy, with regular reassessment during treatment.

Single Injection AAV2-FGF18 Gene Therapy Reduces Cartilage Loss and Subchondral Bone Damage in a Mechanically Induced Model of Osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is a highly debilitating, degenerative pathology of cartilaginous joints affecting over 500 million people worldwide. The global economic burden of OA is estimated at $260-519 billion and growing, driven by aging global population and increasing rates of obesity. To date, only the multi-injection chondroanabolic treatment regimen of Fibroblast Growth Factor 18 (FGF18) has demonstrated clinically meaningful disease-modifying efficacy in placebo-controlled human trials. Our work focuses on the development of a novel single injection disease-modifying gene therapy, based on FGF18's chondroanabolic activity. METHODS: OA was induced in Sprague-Dawley rats using destabilization of the medial meniscus (DMM) (3 weeks), followed by intra-articular treatment with 3 dose levels of AAV2-FGF18, rh- FGF18 protein, and PBS. Durability, redosability, and biodistribution were measured by quantifying nLuc reporter bioluminescence. Transcriptomic analysis was performed by RNA-seq on cultured human chondrocytes and rat knee joints. Morphological analysis was performed on knee joints stained with Safranin O/Fast Green and anti-PRG antibody. RESULTS: Dose-dependent reductions in cartilage defect size were observed in the AAV2-FGF18- treated joints relative to the vehicle control. Total defect width was reduced by up to 76% and cartilage thickness in the thinnest zone was increased by up to 106%. Morphologically, the vehicle- treated joints exhibited pronounced degeneration, ranging from severe cartilage erosion and bone void formation, to subchondral bone remodeling and near-complete subchondral bone collapse. In contrast, AAV2-FGF18-treated joints appeared more anatomically normal, with only regional glycosaminoglycan loss and marginal cartilage erosion. While effective at reducing cartilage lesions, treatment with rhFGF18 injections resulted in significant joint swelling (19% increase in diameter), as well as a decrease in PRG4 staining uniformity and intensity. In contrast to early-timepoint in vitro RNA-seq analysis, which showed a high degree of concordance between protein- and gene therapy-treated chondrocytes, in vivo transcriptomic analysis, revealed few gene expression changes following protein treatment. On the other hand, the gene therapy treatment exhibited a high degree of durability and localization over the study period, upregulating several chondroanabolic genes while downregulating OA- and fibrocartilage-associated markers. CONCLUSION: FGF18 gene therapy treatment of OA joints can provide benefits to both cartilage and subchondral bone, with a high degree of localization and durability.

Development and characterization of a first-in-class adjustable-dose gene therapy system.

INTRODUCTION: Despite significant potential, gene therapy has been relegated to the treatment of rare diseases, due in part to an inability to adjust dosage following initial administration. Other significant constraints include cost, specificity, antigenicity, and systemic toxicity of current generation technologies. To overcome these challenges, we developed a first-in-class adjustable-dose gene therapy system, with optimized biocompatibility, localization, durability, and cost. METHODS: A lipid nanoparticle (LNP) delivery system was developed and characterized by dynamic light scattering for size, zeta potential, and polydispersity. Cytocompatibility and transfection efficiency were optimized in vitro using primary human adipocytes and preadipocytes. Durability, immunogenicity, and adjustment of expression were evaluated in C57BL/6 and B6 albino mice using in vivo bioluminescence imaging. Biodistribution was assessed by qPCR and immunohistochemistry; therapeutic protein expression was quantified by ELISA. RESULTS: Following LNP optimization, in vitro transfection efficiency of primary human adipocytes reached 81.3 % ± 8.3 % without compromising cytocompatibility. Critical physico-chemical properties of the system (size, zeta potential, polydispersity) remained stable over a broad range of genetic cassette sizes (1,871-6,203 bp). Durable expression was observed in vivo over 6 months, localizing to subcutaneous adipose tissues at the injection site with no detectable transgene in the liver, heart, spleen, or kidney. Gene expression was adjustable using several physical and pharmacological approaches, including cryolipolysis, focused ultrasound, and pharmacologically inducible apoptosis. The ability of transfected adipocytes to express therapeutic transgenes ranging from peptides to antibodies, at potentially clinically relevant levels, was confirmed in vitro and in vivo. CONCLUSION: We report the development of a novel, low-cost therapeutic platform, designed to enable the replacement of subcutaneously administered protein treatments with a single-injection, adjustable-dose gene therapy.

Adeno-Associated Virus-Delivered Fibroblast Growth Factor 18 Gene Therapy Promotes Cartilage Anabolism.

OBJECTIVE: To determine the characterization of chondrogenic properties of adeno-associated virus type 2 (AAV2)-delivered hFGF18, via analysis of effects on primary human chondrocyte proliferation, gene expression, and in vivo cartilage thickness changes in the tibia and meniscus. DESIGN: Chondrogenic properties of AAV2-FGF18 were compared with recombinant human FGF18 (rhFGF18) in vitro relative to phosphate-buffered saline (PBS) and AAV2-GFP negative controls. Transcriptome analysis was performed using RNA-seq on primary human chondrocytes treated with rhFGF18 and AAV2-FGF18, relative to PBS. Durability of gene expression was assessed using AAV2-nLuc and in vivo imaging. Chondrogenesis was evaluated by measuring weight-normalized thickness in the tibial plateau and the white zone of the anterior horn of the medial meniscus in Sprague-Dawley rats. RESULTS: AAV2-FGF18 elicits chondrogenesis by promoting proliferation and upregulation of hyaline cartilage-associated genes, including COL2A1 and HAS2, while downregulating fibrocartilage-associated COL1A1. This activity translates to statistically significant, dose-dependent increases in cartilage thickness in vivo within the area of the tibial plateau, following a single intra-articular injection of the AAV2-FGF18 or a regimen of 6 twice-weekly injections of rhFGF18 protein relative to AAV2-GFP. In addition, we observed AAV2-FGF18-induced and rhFGF18-induced increases in cartilage thickness of the anterior horn of the medial meniscus. Finally, the single-injection AAV2-delivered hFGF18 offers a potential safety advantage over the multi-injection protein treatment as evidenced by reduced joint swelling over the study period. CONCLUSION: AAV2-delivered hFGF18 represents a promising strategy for the restoration of hyaline cartilage by promoting extracellular matrix production, chondrocyte proliferation, and increasing articular and meniscal cartilage thickness in vivo after a single intra-articular injection.

Comparative evaluation of rhFGF18 and rhGDF11 treatment in a transient ischemia stroke model.

Background: Pharmacological treatments for ischemic stroke remain limited to thrombolysis, which is associated with increased risk of potentially fatal hemorrhage. Treatments with Recombinant Human Fibroblast Growth Factor 18 (rhFGF18) and Growth and Differentiation Factor 11 (rhGDF11) appear promising based on different preclinical models. The goal of this study was to compare the effects of rhFGF18 and rhGDF11 directly on survival, behavioral deficits, and histological fingerprint of cerebral ischemia in the Wistar rat middle cerebral artery occlusion (MCAO) model of stroke. Methods: Ischemia-reperfusion injury was induced using a 2-hour transient MCAO. Animals were administered rhFGF18 (infusion), rhGDF11 (multi-injection), or Phosphate Buffered Saline (PBS) vehicle control and followed for 42 days. Motor-Cognitive deficits were evaluated using the Morris Water Maze at Days 0 (pre-MCAO), 7, 21, and 42. Histopathological assessments were performed on Days 21 and 42. Results: Day 7 post-ischemia water maze performance times increased 38.3%, 2.1%, and 23.1% for PBS, rhFGF18, and rhGDF11-treated groups, respectively. Fraction of neurons with abnormal morphology (chromatolysis, pyknotic nuclei, somal degeneration) decreased in all groups toward Day 42 and was lowest for rhFGF18. AChE-positive fiber density and activity increased over time in the rhFGF18 group, remained unchanged in the rhGDF11 treatment arm, and declined in the PBS control. Metabolic increases were greatest in rhGDF11 treated animals, with both rhFGF18 and rhGDF11 achieving improvements over PBS, as evidenced by increased succinate dehydrogenase and lactate dehydrogenase activity. Finally, rhFGF18 treatment exhibited a trend for reduced mortality relative to PBS (5.6%, 95% CI [27.3%, 0.1% ] vs. 22.2%, 95% CI [47.6%, 6.4% ]). Conclusions: rhFGF18 treatment appears promising in improving survival and promoting motor-cognitive recovery following cerebral ischemia-reperfusion injury.