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Background: Presence of micrometastases in the sentinel lymph node (SLN) is currently used to assess prognosis of melanoma patients. The immunoactivity within the SLN is known to be influenced by the primary tumor (PT), which may in turn impact the SLNs' metastatic state., Aim: We characterize the temporal dependence and underlying mechanisms of the immunological effects of the PT on the SLN., Methods: The prognostic value of SLN state as a function of PT removal time was evaluated. To put the results into a functional context, selected PTs and corresponding SLNs were analyzed for gene and protein expression patterns., Results: In a cohort of 202 patients with known distant metastasis and similar PT prognostic characteristics, SLNs removed before or within one week after the PT (IM-SLN) had a higher incidence of micrometastases than those removed at least one week after the PT (DEL-SLN). The immunoactivity in IM-SLN was found to be lower than in DEL-SLN. Specifically, in IM-SLNs, T helper 17 / regulatory T-cells were predominant, whereas in DEL-SLNs, cytotoxic γδT-cells were more frequent. The higher immune activity in DEL-SLNs was probably facilitated by CD209 + antigen-presenting cells. Indeed, in PT with high TGFβ expression CD209 + cells appear to be trapped and no increased immunoactivity was observed in DEL-SLN., Conclusions: Presence of micrometastases in DEL-SLNs have a higher negative prognostic value as in IM-SLNs since they indicate not only a melanoma's propensity to metastasize, but possibly also its capacity to escape immune responses., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. VKD received a research grant, honoraria for talks and travel support from SUN Pharma. CR is employed by Merck Serono. TG reports receiving speakers and/or advisory board honoraria from BMS, Sanofi-Genzyme, MSD, Novartis Pharma, Roche, Abbvie, Almirall, Janssen, Lilly, Pfizer, Pierre Fabre, outside the submitted work. SH has acted as a consultant and / or received travel assistance from BMS, Kyowa Kirin. RG received honoraria as speaker from BMS, MSD, Novartis, Amgen, Merck Serono, Almirall Hermal, SUN, Sanofi, Pierre-Fabre, as advisory board member from BMS, Novartis, Almirall Hermal, MSD, Amgen, SUN, Sanofi, Pierre-Fabre, 4SC, MerckSerono, Pfizer, Immunocore, Delcath, for meeting support from SUN, Pierre-Fabre, Boehringer Ingelheim and for research projects (to institution) from Novartis, Pfizer, Johnson & Johnson, Amgen, Merck-Serono, SUN Pharma, Sanofi, Kyowa-Kirin, Admiral-Hermal. SU declares research support from Bristol Myers Squibb and Merck Serono; speakers and advisory board honoraria from Bristol Myers Squibb, Merck Sharp & Dohme, Merck Serono, Novartis and Roche, and travel support from Bristol Myers Squibb, Merck Sharp & Dohme, and Pierre Fabre. JCB is receiving speaker’s bureau honoraria from Amgen, Pfizer, Sanofi, and Sun Pharma, is a paid consultant/advisory board member/DSMB member for Almirall, Boehringer Ingelheim, ICON, MerckSerono, Pfizer, and Sanofi. His group receives research grants from Merck Serono, IQVIA, and Alcedis. The other authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Mycosis fungoides (MF) is the most common subtype of cutaneous T-cell lymphoma (CTCL). Comprehensive analysis of MF cells in situ and ex vivo is complicated by the fact that is challenging to distinguish malignant from reactive T cells with certainty., Methods: To overcome this limitation, we performed combined single-cell RNA (scRNAseq) and T-cell receptor TCR sequencing (scTCRseq) of skin lesions of cutaneous MF lesions from 12 patients. A sufficient quantity of living T cells was obtained from 9 patients, but 2 had to be excluded due to unclear diagnoses (coexisting CLL or revision to a fixed toxic drug eruption)., Results: From the remaining patients we established single-cell mRNA expression profiles and the corresponding TCR repertoire of 18,630 T cells. TCR clonality unequivocally identified 13,592 malignant T cells. Reactive T cells of all patients clustered together, while malignant cells of each patient formed a unique cluster expressing genes typical of naive/memory, such as CD27, CCR7 and IL7R , or cytotoxic T cells, e.g., GZMA, NKG7 and GNLY . Genes encoding classic CTCL markers were not detected in all clusters, consistent with the fact that mRNA expression does not correlate linearly with protein expression. Nevertheless, we successfully pinpointed distinctive gene signatures differentiating reactive malignant from malignant T cells: keratins ( KRT81 , KRT86 ), galectins ( LGALS1 , LGALS3 ) and S100 genes ( S100A4 , S100A6 ) being overexpressed in malignant cells., Conclusions: Combined scRNAseq and scTCRseq not only allows unambiguous identification of MF cells, but also revealed marked heterogeneity between and within patients with unexpected functional phenotypes. While the correlation between mRNA and protein abundance was limited with respect to established MF markers, we were able to identify a single-cell gene expression signature that distinguishes malignant from reactive T cells., Competing Interests: SU declares research support from Bristol Myers Squibb and Merck Serono; speakers and advisory board honoraria from Bristol Myers Squibb, Merck Sharp and Novartis, and travel support from Bristol Myers Squibb, Merck Sharp & Dohme and Pierre Fabre. EL reports personal fees, honoraria, and support for attending meetings or travel grants from Bristol Myers Squibb, Medac, Novartis, Pierre Fabre, and Sun Pharma, and honoraria from MSD, Recordati, and Sanofi. EL participated on a drug safety monitoring or advisory board for Bristol Myers Squibb, Novartis, Sanofi, and Sun Pharma. TG has received speakers and/or advisory board honoraria from BMS, Sano-fi-Genzyme, MSD, Novartis Pharma, Roche, Abbvie, Almirall, Janssen, Lilly, Pfizer, Pierre Fabre, Merck-Serono, outside the submitted work. JB is receiving speaker’s bureau honoraria from Amgen, Pfizer, Recordati and Sanofi, and is a paid consultant/advisory board member/DSMB member for Almirall, Boehringer Ingelheim, InProTher, ICON, Merck Serono, Pfizer, 4SC and Sanofi/Regeneron. His group receives research grants from Bristol Myers Squibb, Merck Serono, HTG, IQVIA and Alcedis GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Srinivas, Peiffer, Horny, Lei, Buus, Kubat, Luo, Yin, Spassova, Sucker, Farahpour, Kehrmann, Ugurel, Livingstone, Gambichler, Ødum and Becker.)

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer associated with integration of Merkel cell polyomavirus (MCPyV). MCPyV-encoded T-antigens (TAs) are pivotal for sustaining MCC's oncogenic phenotype, i.e., repression of TAs results in reactivation of the RB pathway and subsequent cell cycle arrest. However, the MCC cell line LoKe, characterized by a homozygous loss of the RB1 gene, exhibits uninterrupted cell cycle progression after shRNA-mediated TA repression. This unique feature allows an in-depth analysis of the effects of TAs beyond inhibition of the RB pathway, revealing the decrease in expression of stem cell-related genes upon panTA-knockdown. Analysis of gene regulatory networks identified members of the E2F family (E2F1, E2F8, TFDP1) as key transcriptional regulators that maintain stem cell properties in TA-expressing MCC cells. Furthermore, minichromosome maintenance (MCM) genes, which encodes DNA-binding licensing proteins essential for stem cell maintenance, were suppressed upon panTA-knockdown. The decline in stemness occurred simultaneously with neural differentiation, marked by the increased expression of neurogenesis-related genes such as neurexins, BTG2, and MYT1L. This upregulation can be attributed to heightened activity of PBX1 and BPTF, crucial regulators of neurogenesis pathways. The observations in LoKe were confirmed in an additional MCPyV-positive MCC cell line in which RB1 was silenced before panTA-knockdown. Moreover, spatially resolved transcriptomics demonstrated reduced TA expression in situ in a part of a MCC tumor characterized by neural differentiation. In summary, TAs are critical for maintaining stemness of MCC cells and suppressing neural differentiation, irrespective of their impact on the RB-signaling pathway., (© 2024 The Author(s). Journal of Medical Virology published by Wiley Periodicals LLC.)

Mesoporous silica nanoparticles (MSNs) are inorganic nanocarriers presenting versatile properties and the possibility to deliver drug molecules via different routes of application. Their modification with lipids could diminish the burst release profile for water-soluble molecules. In the case of oleic acid (OA) as a lipid component, an improvement in skin penetration can be expected. Therefore, in the present study, aminopropyl-functionalized MSNs were modified with oleic acid through carbodiimide chemistry and were subsequently incorporated into a semisolid hydrogel for dermal delivery. Doxorubicin served as a model drug. The FT-IR and XRD analysis as well as the ninhydrin reaction showed the successful preparation of the proposed nanocarrier with a uniform particle size (352-449 nm) and negative zeta potential. Transmission electron microscopy was applied to evaluate any possible changes in morphology. High encapsulation efficiency (97.6 ± 1.8%) was achieved together with a sustained release profile over 48 h. The composite hydrogels containing the OA-modified nanoparticles were characterized by excellent physiochemical properties (pH of 6.9; occlusion factor of 53.9; spreadability of factor 2.87 and viscosity of 1486 Pa·s) for dermal application. The in vitro permeation study showed 2.35 fold improvement compared with the hydrogel containing free drug. In vitro cell studies showed that loading in OA-modified nanoparticles significantly improved doxorubicin's cytotoxic effects toward epidermoid carcinoma cells (A431). All of the results suggest that the prepared composite hydrogel has potential for dermal delivery of doxorubicin in the treatment of skin cancer.

Resveratrol could be applied in wound healing therapies because of its antioxidant, anti-inflammatory and antibacterial effects. However, the main limitation of resveratrol is its low aqueous solubility. In this study, resveratrol was included in hydroxypropyl-β-cyclodextrin complexes and further formulated in Pluronic F-127 hydrogels for wound treatment therapy. IR-spectroscopy and XRD analysis confirmed the successful incorporation of resveratrol into complexes. The wound-healing ability of these complexes was estimated by a scratch assay on fibroblasts, which showed a tendency for improvement of the effect of resveratrol after complexation. The antimicrobial activity of resveratrol in aqueous dispersion and in the complexes was evaluated on methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli , and Candida albicans strains. The results revealed a twofold decrease in the MIC and stronger inhibition of the metabolic activity of MRSA after treatment with resveratrol in the complexes compared to the suspended drug. Furthermore, the complexes were included in Pluronic hydrogel, which provided efficient drug release and appropriate viscoelastic properties. The formulated hydrogel showed excellent biocompatibility which was confirmed via skin irritation test on rabbits. In conclusion, Pluronic hydrogel containing resveratrol included in hydroxypropyl-β-cyclodextrin complexes is a promising topical formulation for further studies directed at wound therapy.

Background: Recent evidence suggests additional immunomodulatory properties of RANKL inhibition possibly boosting the clinical efficacy of immune checkpoint inhibitors (ICI)., Methods: We conducted a prospective, multicentre clinical trial in unresectable stage IV melanoma patients with bone metastases who received denosumab in parallel with dual ICI (BONEMET) and performed comprehensive immune monitoring at baseline and 4, 12, and 24 weeks after initiation of therapy. Secondary endpoints included tolerability and efficacy. For comparison, biospecimens from melanoma patients treated with dual ICI without denosumab were analyzed accordingly and served as retrospective reference cohort., Results: In both the BONEMET (n = 16) and the reference cohort (n = 18) serum levels of 17 cytokines, including IFNγ were significantly increased after 4 weeks of treatment. Patients who received ICI and denosumab showed a significantly higher increase in serum CXCL-13 and a significant decrease in VEGFc compared with the reference cohort. While no changes in T cell composition were observed at 4 weeks, patients in the BONEMET cohort showed a significant decrease in the peripheral naïve T-cell population and an increase in CD8 + effector cells after 12 weeks. Treatment-related adverse events occurred with comparable frequency (93.8% in the BONEMET cohort versus 83.3% in the reference cohort). 7/16 patients in the BONEMET cohort and 8/18 patients in the reference cohort achieved disease control., Conclusion: Denosumab in combination with dual ICI modulates cytokine expression and T-cell composition in peripheral blood. The upregulation of CXCL-13, a key factor for initiating tertiary lymphoid structures, strengthens the hypothesis that denosumab indeed boost immunological effects., Competing Interests: Declaration of Competing Interest The other authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Agar, a naturally occurring polysaccharide, has been modified by grafting it with acrylic (AcA) and methacrylic (McA) acid monomers, resulting in acrylic or methacrylic acid grafted polymer (AA-g-AcA or AA-g-McA) with pH-sensitive swelling behavior. Different ratios between agar, monomers, and initiator were applied. The synthesized grades of both new polymer series were characterized using FTIR spectroscopy, NMR, TGA, DSC, and XRD to ascertain the intended grafting. The percentage of grafting (% G), grafting efficiency (% GE), and % conversion (% C) were calculated, and models with optimal characteristics were further characterized. The swelling behavior of the newly synthesized polymers was studied over time and in solutions with different pH. These polymers were subsequently crosslinked with varying amounts of glutaraldehyde to obtain 5-fluorouracil-loaded nanogels. The optimal ratios of polymer, drug, and crosslinker resulted in nearly 80% loading efficiency. The performed physicochemical characterization (TEM and DLS) showed spherical nanogels with nanometer sizes (105.7-250 nm), negative zeta potentials, and narrow size distributions. According to FTIR analysis, 5-fluorouracil was physically incorporated. The swelling and release behavior of the prepared nanogels was pH-sensitive, favoring the delivery of the chemotherapeutic to tumor cells. The biocompatibility of the proposed nanocarrier was proven using an in vitro hemolysis assay.

The key technological implementation of sodium-ion batteries is converting biomass-derived hard carbons into effective anode materials. This becomes feasible if appropriate knowledge of the relations between the structure of carbonized biomass products, the mineral ash content in them, and Na storage properties is gained. In this study, we examine the simultaneous impact of the ash phase composition and carbon structure on the Na storage properties of hard carbons derived from spent coffee grounds (SCGs). The carbon structure is modified using the pre-carbonization of SCGs at 750 °C, followed by annealing at 1100 °C in an Ar atmosphere. Two variants of the pre-carbonization procedure are adopted: the pre-carbonization of SCGs in a fixed bed and CO 2 flow. For the sake of comparison, the pre-carbonized products are chemically treated to remove the ash content. The Na storage performance of SCG-derived carbons is examined in model two and three Na-ion cells. It was found that ash-containing carbons outperformed the ash-free analogs with respect to cycling stability, Coulombic efficiency, and rate capability. The enhanced performance is explained in terms of the modification of the carbon surface by ash phases (mainly albite) and its interaction with the electrolyte, which is monitored by ex situ XPS.

In this study, doxorubicin was loaded in a chitosan-albumin nanogel with the aim of improving its stability and exploring the potential of the system in the treatment of skin cancer. Infrared spectroscopy and X-ray diffraction confirmed the encapsulation of the drug. Transmission electron microscopy revealed the spherical shape of the nanogel particles. The drug-loaded nanogel was characterized with a small diameter of 29 nm, narrow polydispersity (0.223) and positive zeta potential (+34 mV). The exposure of encapsulated doxorubicin to light (including UV irradiation and daylight) did not provoke any degradation, whereas the nonencapsulated drug was significantly degraded. In vitro studies on keratinocytes (HaCaT) and epidermoid squamous skin carcinoma cells (A-431) disclosed that the encapsulated doxorubicin was more cytotoxic on both cell lines than the pure drug was. More importantly, the cytotoxic concentration of encapsulated doxorubicin in carcinoma cells was approximately two times lower than that in keratinocytes, indicating that it would not affect them. Thus, the loading of doxorubicin into the developed chitosan-albumin nanogel definitely stabilized the drug against photodegradation and increased its antineoplastic effect on the skin cancer cell line.

Budesonide is a mineral corticoid applied in the local therapy of pediatric atopic dermatitis. Unfortunately, its dermal administration is hindered by the concomitant adverse effects and its physicochemical properties. The characteristic pH change in the atopic lesions can be utilized for the preparation of a pH-sensitive nanocarrier. In this view, the formulation of Eudragit L 100 nanoparticles as a budesonide delivery platform could provide more efficient release to the desired site, improve its penetration, and subsequently lower the undesired effects. In this study, budesonide-loaded Eudragit L100 nanoparticles were prepared via the nanoprecipitation method (mean diameter 57 nm, -31.2 mV, and approx. 90% encapsulation efficiency). Their safety was proven by cytotoxicity assays on the HaCaT keratinocyte cell line. Further, the drug-loaded nanoparticles were incorporated into two types of hydrogels based on methylcellulose or Pluronic F127. The formulated hydrogels were characterized with respect to their pH, occlusion, rheology, penetration, spreadability, and drug release. In conclusion, the developed hydrogels containing budesonide-loaded nanoparticles showed promising potential for the pediatric treatment of atopic dermatitis.