Characterization of the Molecular Mechanism of the Tissue-Specific and Developmentally Regulated Alternative Splicing of <i>SCN9A</i> Exon 5

The voltage-gated sodium channel Nav 1.7, encoded by SCN9A gene, is expressed in dorsal root ganglia (DRG) and in sympathetic ganglia and it is considered to be one of the major player in the regulation of pain perception. The linkage to several human pain disorders confirms its pivotal role in pain pathway. The gene contains an exon 5 pair, 5N and 5A, that are mutually exclusive spliced. These exons encode part of the voltage sensor of the channel; although the alternative splice variants differ by only two amino acids, they differentially affect the amplitude of the currents generated and the excitability of nociceptive neurons. In this study I mapped some of the splicing regulatory elements that determine SCN9A exon 5 developmentally regulated mutually exclusive splicing. In the first stages of development, characterized by a major inclusion of the neonatal form, ETR3 plays an essential role acting on both sides, favouring 5N and inhibiting 5A inclusion. For the switching towards a major inclusion of exon 5A some elements have been identified. Fox1 helps the recognition of the weak 5A 5' splice site, interacting with four cis-acting elements downstream of exon 5A. Furthermore an exonic splicing enhancer (ESE), bound by SRSF1 and SRSF6, improves the usage of 5A weak 3' splice site. At the same time 5N inclusion is inhibited by the presence of two silencers, one exonic recognized by PTB and one intronic close to 5N 5' splice site. The duplication of this exon is well conserved in six out of nine voltage-gated sodium channel (VGSC) genes and some elements mapped for SCN9A gene display a great homology in other family members. The investigation of possible common factors reveals some interesting results: for example the ESE in the adult form is present in at least other three VGSCs

TRAIL receptors are expressed in both malignant and stromal cells in pancreatic ductal adenocarcinoma

: This study assesses the expression of all TNF-related apoptosis-inducing ligand (TRAIL) receptors in pancreatic ductal adenocarcinoma (PDAC) tumor tissue. We aimed to include TRAIL receptor expression as an inclusion parameter in a future clinical study using a TRAIL-based therapy approach for PDAC patients. Considering the emerging influence of PDAC desmoplastic stroma on the efficacy of anti-PDAC therapies, this analysis was extended to tumor stromal cells. Additionally, we performed PDAC stroma characterization. Our retrospective cohort study (N=50) included patients with histologically confirmed PDAC who underwent surgery. The expression of TRAIL receptors (DR4, DR5, DcR1, DcR2, and OPG) in tumor and stromal cells was evaluated by immunohistochemistry (IHC). The amount of tumor stroma was assessed by anti-vimentin IHC and Mallory's trichrome staining. The prognostic impact was determined by the univariate Cox proportional hazards regression model. An extensive expression of functional receptors DR4 and DR5 and a variable expression of decoy receptors were detected in PDAC tumor and stromal cells. Functional receptors were detected also in metastatic tumor and stromal cells. A poor prognosis was associated with low or absent expression of decoy receptors in tumor cells of primary PDAC. After assessing that almost 80% of tumor mass was composed of stroma, we correlated a cellular-dense stroma in primary PDAC with reduced relapse-free survival. We demonstrated that TRAIL functional receptors are widely expressed in PDAC, representing a promising target for TRAIL-based therapies. Further, we demonstrated that a low expression of DcR1 and the absence of OPG in tumor cells, as well as a cellular-dense tumor stroma, could negatively impact the prognosis of PDAC patients

Human Adipose Mesenchymal Stromal/Stem Cells Improve Fat Transplantation Performance

The resorption rate of autologous fat transfer (AFT) is 40-60% of the implanted tissue, requiring new surgical strategies for tissue reconstruction. We previously demonstrated in a rabbit model that AFT may be empowered by adipose-derived mesenchymal stromal/stem cells (AD-MSCs), which improve graft persistence by exerting proangiogenic/anti-inflammatory effects. However, their fate after implantation requires more investigation. We report a xenograft model of adipose tissue engineering in which NOD/SCID mice underwent AFT with/without human autologous AD-MSCs and were monitored for 180 days (d). The effect of AD-MSCs on AFT grafting was also monitored by evaluating the expression of CD31 and F4/80 markers. Green fluorescent protein-positive AD-MSCs (AD-MSC-GFP) were detected in fibroblastoid cells 7 days after transplantation and in mature adipocytes at 60 days, indicating both persistence and differentiation of the implanted cells. This evidence also correlated with the persistence of a higher graft weight in AFT-AD-MSC compared to AFT alone treated mice. An observation up to 180 d revealed a lower resorption rate and reduced lipidic cyst formation in the AFT-AD-MSC group, suggesting a long-term action of AD-MSCs in support of AFT performance and an anti-inflammatory/proangiogenic activity. Together, these data indicate the protective role of adipose progenitors in autologous AFT tissue resorption

Autologous anti-GD2 CAR T cells efficiently target primary human glioblastoma

Glioblastoma (GBM) remains a deadly tumor. Treatment with chemo-radiotherapy and corticosteroids is known to impair the functionality of lymphocytes, potentially compromising the development of autologous CAR T cell therapies. We here generated pre-clinical investigations of autologous anti-GD2 CAR T cells tested against 2D and 3D models of GBM primary cells. We detected a robust antitumor effect, highlighting the feasibility of developing an autologous anti-GD2 CAR T cell-based therapy for GBM patients

The adhesion molecule L1 regulates transendothelial migration and trafficking of dendritic cells

The adhesion molecule L1, which is extensively characterized in the nervous system, is also expressed in dendritic cells (DCs), but its function there has remained elusive. To address this issue, we ablated L1 expression in DCs of conditional knockout mice. L1-deficient DCs were impaired in adhesion to and transmigration through monolayers of either lymphatic or blood vessel endothelial cells, implicating L1 in transendothelial migration of DCs. In agreement with these findings, L1 was expressed in cutaneous DCs that migrated to draining lymph nodes, and its ablation reduced DC trafficking in vivo. Within the skin, L1 was found in Langerhans cells but not in dermal DCs, and L1 deficiency impaired Langerhans cell migration. Under inflammatory conditions, L1 also became expressed in vascular endothelium and enhanced transmigration of DCs, likely through L1 homophilic interactions. Our results implicate L1 in the regulation of DC trafficking and shed light on novel mechanisms underlying transendothelial migration of DCs. These observations might offer novel therapeutic perspectives for the treatment of certain immunological disorders

Stem Cells and Ocular Regeneration.

The surface of the eye is a peculiar and extraordinary area, since the functions of all regions of the ocular surface system (OSS) are closely integrated forming a unique functional unit composed of different tissues and organs. Anatomically the ocular surface is composed principally by cornea and conjunctiva, but it also includes the lacrimal gland and the lacrimal drainage system for the tear film homeostasis, and the eyelids. Additional components as Meibomian glands, eyelashes and eyebrows complete the picture, contributing to the outermost lipid layer and to particle clearance (Fig. 1). Cornea and conjunc- tiva are generally covered by a complex liquid known as the tear film, providing a continuous moist environment connecting all areas at the front of the eye ball and protecting them from pathogens and small solid particles. This interconnected system provides the necessary conditions for the maintenance of functions needed for the vision process, as the final goal of eye function. The dysfunction of this apparatus could lead to several pathologies, such as corneal opacity or dry eye diseases (DED). Each component plays an important role for ocular surface maintenance, therefore some recent findings in stem cell biology aimed at the regeneration of different components of the ocular surface by tissue engineering, are examined in the following paragraphs

Dissecting Immunotherapy Strategies for Small Cell Lung Cancer: Antibodies, Ionizing Radiation and CAR-T

Small cell lung cancer (SCLC) is a highly aggressive malignancy that accounts for about 14% of all lung cancers. Platinum-based chemotherapy has been the only available treatment for a long time, until the introduction of immune checkpoint inhibitors (ICIs) recently changed first-line standard of care and shed light on the pivotal role of the immune system. Despite improved survival in a subset of patients, a lot of them still do not benefit from first-line chemo-immunotherapy, and several studies are investigating whether different combination strategies (with both systemic and local treatments, such as radiotherapy) may improve patient outcomes. Moreover, research of biomarkers that may be used to predict patients&rsquo; outcomes is ongoing. In addition to ICIs, immunotherapy offers other different strategies, including naked monoclonal antibodies targeting tumor associated antigens, conjugated antibody, bispecific antibodies and cellular therapies. In this review, we summarize the main evidence available about the use of immunotherapy in SCLC, the rationale behind combination strategies and the studies that are currently ongoing in this setting, in order to give the reader a clear and complete view of this rapidly expanding topic

Delayed Effect of Dendritic Cells Vaccination on Survival in Glioblastoma: A Systematic Review and Meta-Analysis

Background: Dendritic cell vaccination (DCV) strategies, thanks to a complex immune response, may flare tumor regression and improve patients’ long-term survival. This meta-analysis aims to assess the efficacy of DCV for newly diagnosed glioblastoma patients in clinical trials. Methods: The study databases, including PubMed, Web of Knowledge, Google Scholar, Scopus, and Cochrane, were searched by two blinded investigators considering eligible studies based on the following keywords: “glioblastoma multiforme”, “dendritic cell”, “vaccination”, “immunotherapy”, “immune system”, “immune response”, “chemotherapy”, “recurrence”, and “temozolomide”. Among the 157 screened, only 15 articles were eligible for the final analysis. Results: Regimens including DCV showed no effect on 6-month progression-free survival (PFS, HR = 1.385, 95% CI: 0.822–2.335, p = 0.673) or on 6-month overall survival (OS, HR = 1.408, 95% CI: 0.882–2.248, p = 0.754). In contrast, DCV led to significantly longer 1-year OS (HR = 1.936, 95% CI: 1.396–2.85, p = 0.001) and longer 2-year OS (HR = 3.670, 95% CI: 2.291–5.879, p = 0.001) versus control groups. Hence, introducing DCV could lead to increased 1 and 2-year survival of patients by 1.9 and 3.6 times, respectively. Conclusion: Antitumor regimens including DCV can effectively improve mid-term survival in patients suffering glioblastoma multiforme (GBM), but its impact emerges only after one year from vaccination. These data indicate the need for more time to achieve an anti-GBM immune response and suggest additional therapeutics, such as checkpoint inhibitors, to empower an earlier DCV action in patients affected by a very poor prognosis

GD2 CAR T cells against human glioblastoma

Glioblastoma is the most malignant primary brain tumor and is still in need of effective medical treatment. We isolated patient-derived glioblastoma cells showing high GD2 antigen expression representing a potential target for CAR T strategy. Data highlighted a robust GD2 CAR antitumor potential in 2D and 3D glioblastoma models associated with a significant and CAR T-restricted increase of selected cytokines. Interestingly, immunosuppressant TGF β1, expressed in all co-cultures, did not influence antitumor activity. The orthotopic NOD/SCID models using primary glioblastoma cells reproduced human histopathological features. Considering still-conflicting data on the delivery route for targeting brain tumors, we compared intracerebral versus intravenous CAR T injections. We report that the intracerebral route significantly increased the length of survival time in a dose-dependent manner, without any side effects. Collectively, the proposed anti-GD2 CAR can counteract human glioblastoma potentially opening a new therapeutic option for a still incurable cancer