Daciana Silvia Marta

NLRP1 is an inflammasome sensor protein expressed in barrier tissues of humans. Its activation in response to microbes or cellular stress triggers a cascade of molecular events, leading up to IL1β-driven inflammation and pyroptosis. Rare germline mutations of NLRP1 cause its persistent activation, resulting in autoinflammatory syndromes. Multiple self-healing palmoplantar carcinoma (MSPC) is one such syndrome, characterized by the appearance of recurrent keratoacanthomas (KAs) on the palms and soles. Here, we aimed to compare the subcellular localization of mutant NLRP1 in lesions from an MSPC patient to wild-type NLRP1 in non-MSPC-KAs and in skin from healthy donors. Using mass spectrometry, immunohistochemistry and immunoelectron tomography, we found that NLRP1 localized to mast cell granules in all MSPC lesions but also in healthy skin, a novel finding which implicates these cells in NLRP1-associated responses in human skin. Moreover, we found that mast cells expressing the A66V pathogenic variant of NLRP1 overpopulated MSPC-KAs, infiltrated the epidermis and degranulated, a behavior not seen in other lesions from this study. The released granules had the highest NLRP1 protein content and also contained NLRP3 and IL1β, suggesting the coexistence of inflammasome pathways within mast cells. Taken together, our findings propose cutaneous mast cells as a previously unrecognized NLRP1 reservoir in health and disease.

Coated magnetic nanoparticles (MNPs) comprising Fe₃O₄ are a powerful drug delivery system for cancer treatment. They can be preferentially internalized into the tumour cells and release the antitumoral agents on-site. In the present work, we have functionalized the MNPs with glutamic acid which, even if non-essential, is strongly involved in the protein synthesis as a nitrogen donor, being used as a conjugate for specific antitumoral drugs due to its capability to internalize into tumour versus healthy cells faster. The functionalized MNPs were stabilized by polyethylene glycol (PEG) coating. This system improves drug efficacy by concentrating therapeutic agents at the tumour site, and can also induce ferroptosis in cancer cells, an iron-dependent cell death process, which has a beneficial therapeutic effect. Additionally, PEG molecules on the surface of MNPs facilitate drug conjugation for targeted delivery to cancer cells. These MNPs were designed as a delivery system for antitumoral drugs like irinotecan, carboplatin and cisplatin for personalized therapies. Based on the obtained results, it was found that the functionalization with glutamic acid and stabilization with PEG can improve the internalization efficiency of the magnetic carriers into the tumour cells. Owing to their stability, the MNPs can reach and penetrate mitochondria and organize around lipid vesicles, with the most effective results observed for the cisplatin-loaded system from a concentration of 0.1 mg/mL.

The antitumoral activity, and in general, the biological activity is strongly altered by the low uptake of the active agents within the targeted cells. Therefore, lots of efforts have been made to ensure better cellular uptake by using specific carriers. In the present research we have obtained magnetic nanoparticles stabilized by polyethylene glycol (PEG) coating, and functionalized with aspartic acid, which is an important amino acid for protein synthesis and energy production in the body. Such decorated nanoparticles can be internalized by the tumoral cell due to their higher metabolic rate. The nanoparticles were used as a delivery system for antitumoral drugs as cisplatin, carboplatin or irinotecan in a Trojan Horse strategy. Based on the obtained results, it was found that aspartic acid can improve the internalization efficiency of the magnetic carriers after being loaded with antitumoral agents. The nanoparticles are quite stable, can reach and enter the mitochondria and organize around lipid vesicles in quite a high concentration, best results being obtained for the system loaded with cisplatin starting from 0.1 mg/mL.

Vaccines against COVID-19 based on the mRNA technology have broken many records, from the speed of development and production, to the number of doses administered and have overall proven safe, with only very rare reported adverse events. The accelerated rollout and the permissive regulatory framework had the major caveat that manufacturers did not provide biodistribution and pharmacokinetics data for their products in humans, despite this being essential for interpreting both the dynamics of the immune response and any potential toxic effects. Thankfully, in the past two years, the scientific community has attempted to fill the gaps, which will undoubtedly help in fine-tuning the next generation of mRNA vaccines. Here we review existing data on the biodistribution and pharmacokinetics of the commercially available mRNA vaccine platforms, focusing on human studies, where available. We structure this review by tissue type and we discuss potential correlations between vaccine mRNA uptake and pathogenic effects, if applicable. We find that many studies have focused on the heart, due to the medical and social impact of myocarditis, especially in adolescents. We conclude by observing critical data is still missing for many organs and we suggest potential avenues for future research.

Mistletoe (Viscum album L.), a semi-parasitic medicinal plant, continues to be of interest, due to its phytochemical composition. The leaves of mistletoe contain phenols, which have a variety of biological effects. The main goal was to characterize the mistletoe that parasitized Robinia pseudoacacia L., (called VAR) in terms of phenolic compounds and to assess the wound healing potential in vitro using the scratch method. Furthermore, the antioxidant capacity was evaluated both by spectrophotometric techniques (DPPH, FRAP, TEAC), as well as by the ability of the mistletoe extract to synthesize green selenium nanoparticles. Among the phenolic acids, dihydroxybenzoic acid is in high level (2.86±0.03 mg/g dw), whereas isorhamnetin-glucuronides dominate the flavonol class (0.593±0.03 mg/g dw). The presence of phenolic compounds in the VAR leaves provides antioxidant capacity. The reducing capacity of VAR extract was demonstrated for the first time by the biosynthesis of nanoselenium particles (NSePs) with a regular, spherical shape and a diameter of around 130 nm. The VAR concentrations of 25-200 µg/mL showed no toxic effect on normal human dermal fibroblasts (NHDF), and the concentration of 100 µg/mL exhibited the best percentage of wound surface closure in vitro (94.08%). The results show that mistletoe is a promising plant because of its phytochemical composition and antioxidant capacity, which can modulate the wound repair process in vitro.

Mutant calreticulin (CALR) proteins resulting from a −1/+2 frameshifting mutation of the CALR exon 9 carry a novel C-terminal amino acid sequence and drive the development of myeloproliferative neoplasms (MPNs). Mutant CALRs were shown to interact with and activate the thrombopoietin receptor (TpoR/MPL) in the same cell. We report that mutant CALR proteins are secreted and can be found in patient plasma at levels up to 160 ng/mL, with a mean of 25.64 ng/mL. Plasma mutant CALR is found in complex with soluble transferrin receptor 1 (sTFR1) that acts as a carrier protein and increases mutant CALR half-life. Recombinant mutant CALR proteins bound and activated the TpoR in cell lines and primary megakaryocytic progenitors from patients with mutated CALR in which they drive thrombopoietin-independent colony formation. Importantly, the CALR-sTFR1 complex remains functional for TpoR activation. By bioluminescence resonance energy transfer assay, we show that mutant CALR proteins produced in 1 cell can specifically interact in trans with the TpoR on a target cell. In comparison with cells that only carry TpoR, cells that carry both TpoR and mutant CALR are hypersensitive to exogenous mutant CALR proteins and respond to levels of mutant CALR proteins similar to those in patient plasma. This is consistent with CALR-mutated cells that expose TpoR carrying immature N-linked sugars at the cell surface. Thus, secreted mutant CALR proteins will act more specifically on the MPN clone. In conclusion, a chaperone, CALR, can turn into a rogue cytokine through somatic mutation of its encoding gene.

COVID-19 mRNA vaccines effectively reduce incidence of severe disease, hospitalisation and death. The biodistribution and pharmacokinetics of the mRNA-containing lipid nanoparticles (LNPs) in these vaccines are unknown in humans. In this study, we used qPCR to track circulating mRNA in blood at different time-points after BNT162b2 vaccination in a small cohort of healthy individuals. We found that vaccine-associated synthetic mRNA persists in systemic circulation for at least 2 weeks. Furthermore, we used transmission electron microscopy (TEM) to investigate SARS-CoV-2 spike protein expression in human leukemic cells and in primary mononuclear blood cells treated in vitro with the BNT162b2 vaccine. TEM revealed morphological changes suggestive of LNP uptake, but only a small fraction of K562 leukemic cells presented spike-like structures at the cell surface, suggesting reduced levels of expression for these specific phenotypes.

Objective. Respiratory bacterial infections are associated with important coagulation disturbances that amplify the pulmonary lesions and determine a more severe course of the disease. The aim of our study was to investigate the correlation between the evolution of the general clinical parameters and the occurrence of thrombotic events on one side, and plasma levels of selected proteins involved in inflammation and coagulation on the other side, with the intent to establish and to validate a laboratory test panel for the assessment of the vascular risk in patients with bacterial respiratory infections.