Clinical and laboratory characterization of patients with localized scleroderma and response to UVA-1 phototherapy: In vivo and in vitro skin models

Background/Purpose Localized scleroderma (LS) is a rare disease leading to progressive hardening and induration of the skin and subcutaneous tissues. LS is responsive to UVA-1 phototherapy, though its exact mechanism of action dermal fibrosis is yet to be fully elucidated. We aimed to investigate the molecular changes induced by UVA-1 rays in human primary fibroblasts cultures. Methods A total of 16 LS patients were treated with medium-dose UVA-1 phototherapy. At baseline, during and after therapy, Localized Scleroderma Assessment Tool, Dermatology Life Quality Index and lesions' staging and mapping were performed along with high-frequency ultrasound (HFUS) examination for dermal thickness assessment. Gene expression analysis for 23 mRNA transcripts, in vitro UVA-1 irradiation and viability tests were realized on lesional fibroblasts' primary cultures, before and 3 months after therapy. Results The dermal thickness, the LoSCAT and the DLQI progressively decreased starting from the last phototherapy session up to the 6 and 9 month follow-ups (-57% and -60%, respectively). Molecular gene analysis (rt-PCR) revealed that UVA-1 phototherapy exerts multiple effects: the activation of specific anti-fibrotic pathways (e.g., overexpression of CTHRC1 and metalloproteases 1, 2, 7, 8, 9, 12, suppression of TIMP-1), the downregulation of peculiar pro-fibrotic pathways (e.g., downregulation of TGF-ss, TGF-ssrII, Grb2, SMAD 2/3, TNRSF12A, CTGF) through a significant overexpression of IL-1ss; the stabilization of collagen synthesis acting on genes COL1A1, COL3A1, COL8A1, COL10A1, COL12A1. Conclusion UVA-1 phototherapy adds significant benefits in local tissue remodeling, rebalancing the alteration between pro-fibrotic and anti-fibrotic pathways; these changes can be well monitored by HFUS. © 2022 The Authors

Monitoring Anti-PEG Antibodies Level upon Repeated Lipid Nanoparticle-Based COVID-19 Vaccine Administration

PEGylated lipids are one of the four constituents of lipid nanoparticle mRNA COVID-19 vaccines. Therefore, various concerns have been raised on the generation of anti-PEG antibodies and their potential role in inducing hypersensitivity reactions following vaccination or in reducing vaccine efficacy due to anti-carrier immunity. Here, we assess the prevalence of anti-PEG antibodies, in a cohort of vaccinated individuals, and give an overview of their time evolution after repeated vaccine administrations. Results indicate that, in our cohort, the presence of PEG in the formulation did not influence the level of anti-Spike antibodies generated upon vaccination and was not related to any reported, serious adverse effects. The time-course analysis of anti-PEG IgG showed no significant booster effect after each dose, whereas for IgM a significant increase in antibody levels was detected after the first and third dose. Data suggest that the presence of PEG in the formulation does not affect safety or efficacy of lipid-nanoparticle-based COVID-19 vaccines

TiO2@BSA nano-composites investigated through orthogonal multi-techniques characterization platform.

Abstract Biocompatible coating based on bovine serum albumin (BSA) was applied on two different TiO2 nanoparticles (aeroxide P25 and food grade E171) to investigate properties and stability of resulting TiO2@BSA composites, under the final perspective to create a "Safe-by-Design" coating, able to uniform, level off and mitigate surface chemistry related phenomena, as naturally occurring when nano-phases come in touch with proteins enriched biological fluids. The first step towards validating the proposed approach is a detailed characterization of surface chemistry with the quantification of amount and stability of BSA coating deposited on nanoparticles' surfaces. At this purpose, we implemented an orthogonal multi-techniques characterization platform, providing important information on colloidal behavior, particle size distribution and BSA-coating structure of investigated TiO2 systems. Specifically, the proposed orthogonal approach enabled the quantitative determination of bound and free (not adsorbed) BSA, a key aspect for the design of intentionally BSA coated nano-structures, in nanomedicine and, overall, for the control of nano-surface reactivity. In fact, the BSA-coating strategy developed and the orthogonal characterisation performed can be extended to different designed nanomaterials in order to further investigate the protein-corona formation and promote the implementation of BSA engineered coating as a strategy to harmonize the surface reactivity and minimize the biological impact

Human neutralizing antibodies to cold linear epitopes and subdomain 1 of the SARS-CoV-2 spike glycoprotein

Emergence of SARS-CoV-2 variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike glycoprotein that are both functionally relevant and averse to change, we identified human neutralizing antibodies to highly conserved viral epitopes. Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four genera, including the nine human coronaviruses, through recognition of a conserved motif that includes the S2´ site of proteolytic cleavage. Antibody hr2.016 targets the stem helix and neutralizes SARS-CoV-2 variants. Antibody sd1.040 binds to subdomain 1, synergizes with antibody rbd.042 for neutralization and, like fp.006 and hr2.016, protects mice expressing human ACE2 against infection when present as bispecific antibody. Thus, coldspot-guided antibody discovery reveals donor-derived neutralizing antibodies that are cross-reactive with Orthocoronavirinae, including SARS-CoV-2 variants

Hedgehog signalling pathway and Carbonic Anhydrases in Breast cancer cell physiology

Breast cancer (BC) is one of the most diffused types of cancer worldwide. It affects predominantly women and is a highly curable disease if diagnosed at early stage. Several classifications of BC do exist according to different histopathological and molecular features. The worst subtype of BC are triple negative breast cancers (TNBCs) that do not express Estrogen (ER), Progesterone (PgR) and epidermal growth factor 2 (HER2) receptors consequently, these tumors are not sensitive to hormonal therapy. Nowadays, there is no specific clinical guideline approved, therefore, TNBCs treatment consists of cytotoxic agents and radiotherapy. The Hedgehog (Hh) pathway plays a pivotal role during the development of the organism and its expression is tightly controlled and normally kept silent in adult tissue. The Hh pathway indeed is reactivated for homeostasis maintenance after tissue injury or for the physiological tissue renewal. Deregulation of the Hh pathway is associated with development disturbs, underlining the relevance of its precise controlled expression. More and more Hh signalling is found to be involved in cancer development and progression. A growing body of literature underlines the correlation between the Hh pathway and bad prognosis for BCs. Indeed, the Hh pathway is normally silenced in normal breast epithelium and aberrantly activated in TNBCs, where it is associated with a more aggressive phenotype, enhancing proliferation, migration and invasion, furthermore, its activation is related to chemoresistant phenotypes of TNBCs. Carbonic anhydrases (CAs) are evolutionary conserved enzymes, mostly known for their role in pH regulation. CAs catalyze the reversible hydration of H2CO3 into CO2 and H2O. Several other roles have been described and attributed to CAs, from cell survival and migration, to the priming of the stem cell niche. Several CAs classes have been characterized and are conserved among the species. The classification is done according to the subcellular localization of CAs. Membrane bound CAs namely CAIX and CAXII, are overexpressed and active in cancer. While the role of CAIX is associated to bad prognosis, the role of CAXII has a contradictory outcome and still need to be elucidated. In BC, downregulation of CAXII reduced MDAMB231 cell migration through a reduction in p38 phosphorylation, while some researchers confer to the presence of this protein, a good prognostic value. We demonstrate for the first time a correlation between the Hh pathway and CAXII in controlling BCs pathology, emphasising how the activation of the Hh pathway is crucial in the control of CAXII expression, with consistent effects on cell proliferation, migration and invasion. Understanding the reciprocal regulation could be of main interest and should be taken into account in the design of new molecules to improve BC patient’s life expectancy

Inhibition of smoothened in breast cancer cells reduces CAXII expression and cell migration

Breast cancer (BC) relapse and metastasis are the leading cause of death and, together with drug resistance, keep mortality still high. The Hedgehog (Hh) pathway is expressed during embryogenesis, organogenesis and in adult tissue homeostasis and its aberrant activation is often associated with cancer. Carbonic anhydrase (CA) enzymes are important during development; they play a key role in controlling several cellular mechanisms, such as pH regulation, survival, and migration, and they are aberrantly expressed in cancer. The goal of this study was to investigate the interplay between the Hh pathway and CAXII in terms of BC cell migration. We here demonstrated that smoothened (SMO) silencing resulted in a reduction of CAXII expression at mRNA and protein level. This led to a decrease in cell migration, which was restored when cells were treated with an SMO agonist, Sag dihydrochloride (SAG), but not when cells were cotreated with SAG and the CAs inhibitor Acetazolamide. This suggested that the ability of SAG to promote cell migration was impaired when CAXII was inhibited. The reduction was also confirmed within hypoxic and inflammatory microenvironment, typical of BC, indicating a key role of the Hh pathway in controlling CAXII expression. Our results may contribute to further understand the physiology of BC cells and indicate that the Hh pathway controls BC cell migration and cell invasion also through CAXII, with important implications in identifying novel therapeutic targets

Analytical Ultracentrifugation to Assess the Quality of LNP-mRNA Therapeutics

The approval of safe and effective LNP-mRNA vaccines during the SARS-CoV-2 pandemic is catalyzing the development of the next generation of mRNA therapeutics. Proper characterization methods are crucial for assessing the quality and efficacy of these complex formulations. Here, we show that analytical ultracentrifugation (AUC) can measure, simultaneously and without any sample preparation step, the sedimentation coefficients of both the LNP-mRNA formulation and the mRNA molecules. This allows measuring several quality attributes, such as particle size distribution, encapsulation efficiency and density of the formulation. The technique can also be applied to study the stability of the formulation under stress conditions and different buffers

Sizing up the nanomedicines: a thorough examination of techniques and data interpretation

Size is a universal and first in mind property relevant for nanomedicine/nanomaterial characterization, representing the most suitable measurand. However, due to the acknowledged lack of standardized protocols that are able to provide reliable results, especially in the biorelevant context, researchers often engage with the procedures that are successful in acquiring data, but not necessarily the correct ones. ..

The Shc protein RAI promotes an adaptive cell survival program in hypoxic neuroblastoma cells

Neuroblastoma (NB) is a highly malignant pediatric solid tumor where a hypoxic signature correlates with unfavorable patient outcome. The hypoxia-inducible factor (HIF)-1\u3b1 plays an important role in NB progression, contributing to cell proliferation and invasiveness. RAI belongs to the Shc family proteins, it is mainly neuron specific and protects against cerebral ischemia. RAI is also expressed in several NB cell lines, where it promotes cell survival. In this work, hypoxia differently affected cell survival and pro-apoptotic program in two NB cell lines, either expressing RAI (SKNBE) or not (SKNMC). RAI expression appeared to promote NB cell survival and to reduce some pro-apoptotic markers under hypoxia. Accordingly, the RAI silencing in SKNBE cells resulted in a reduction of cell survival and HIF-1\u3b1 expression. Furthermore, using SKNMC cells stably expressing RAI, we defined a role of RAI in NB cell responses to hypoxia. Of interest, in hypoxic SKNMC cells expressing RAI HIF-1\u3b1 protein levels were higher than in control cells. This was associated with a) an increased cell survival; b) an increased expression of anti-apoptotic markers; c) a pro-autophagic and not pro-apoptotic phenotype; and d) an increased metabolic activity. We may conclude that RAI plays an important role in hypoxic signaling in NB cells and the interplay between RAI and HIF-1\u3b1 may be relevant in the protection of NB cells against hypoxia. Our results may contribute to a further understanding the physiology of NB cells and the molecular mechanisms involved in their survival, with important implications in NB progression