Senotherapeutic Peptide 14 Suppresses Th1 and M1 Human T Cell and Monocyte Subsets In Vitro.

Inflammation contributes to the onset and exacerbation of numerous age-related diseases, often manifesting as a chronic condition during aging. Given that cellular senescence fosters local and systemic inflammation, senotherapeutic interventions could potentially aid in managing or even reducing inflammation. Here, we investigated the immunomodulatory effects of the senotherapeutic Peptide 14 (Pep 14) in human peripheral blood mononuclear cells (PBMCs), monocytes, and macrophages. We found that, despite failing to significantly influence T cell activation and proliferation, the peptide promoted a Th2/Treg gene expression and cytokine signature in PBMCs, characterized by increased expression of the transcription factors GATA3 and FOXP3, as well as the cytokines IL-4 and IL-10. These observations were partially confirmed through ELISA, in which we observed increased IL-10 release by resting and PHA-stimulated PBMCs. In monocytes from the U-937 cell line, Pep 14 induced apoptosis in lipopolysaccharide (LPS)-stimulated cells and upregulated IL-10 expression. Furthermore, Pep 14 prevented LPS-induced activation and promoted an M2-like polarization in U-937-derived macrophages, evidenced by decreased expression of M1 markers and increased expression of M2 markers. We also showed that the conditioned media from Pep 14-treated macrophages enhanced fibroblast migration, indicative of a functional M2 phenotype. Taken together, our findings suggest that Pep 14 modulates immune cell function towards an anti-inflammatory and regenerative phenotype, highlighting its potential as a therapeutic intervention to alleviate immunosenescence-associated dysregulation.

The Combination of Synoeca-MP Antimicrobial Peptide with IDR-1018 Stimulates Proliferation, Migration, and the Expression of Pro-Regenerative Genes in Both Human Skin Cell Cultures and 3D Skin Equivalents.

In skin lesions, the development of microbial infection affects the healing process, increasing morbidity and mortality rates in patients with severe burns, diabetic foot, and other types of skin injuries. Synoeca-MP is an antimicrobial peptide (AMP) that exhibits activity against several bacteria of clinical importance, but its cytotoxicity can represent a problem for its positioning as an effective antimicrobial compound. In contrast, the immunomodulatory peptide IDR-1018 presents low toxicity and a wide regenerative potential due to its ability to reduce apoptotic mRNA expression and promote skin cell proliferation. In the present study, we used human skin cells and a 3D skin equivalent models to analyze the potential of the IDR-1018 peptide to attenuate the cytotoxicity of synoeca-MP, as well as the influence of synoeca-MP/IDR-1018 combination on cell proliferation, regenerative processes, and wound repair. We found that the addition of IDR-1018 significantly improved the biological properties of synoeca-MP on skin cells without modifying its antibacterial activity against S. aureus. Likewise, in both melanocytes and keratinocytes, the treatment with synoeca-MP/IDR-1018 combination induces cell proliferation and migration, while in a 3D human skin equivalent model, it can accelerate wound reepithelization. Furthermore, treatment with this peptide combination generates an up-regulation in the expression of pro-regenerative genes in both monolayer cell cultures and in 3D skin equivalents. This data suggests that the synoeca-MP/IDR-1018 combination possesses a good profile of antimicrobial and pro-regenerative activity, opening the door to the development of new strategies for the treatment of skin lesions.

In vitro and in vivo toxicity assessment of the senotherapeutic Peptide 14.

Senotherapeutic molecules decrease cellular senescence burden, constituting promising approaches to combat the accumulation of senescent cells observed in chronological aging and age-related diseases. Numerous molecules have displayed senotherapeutic potential, but toxicity has been frequently observed. Recently, a new senotherapeutic compound, Peptide 14, was developed to modulate cellular senescence in the skin. In order to assess the potential toxic and genotoxic effects of the peptide, we observed the viability of human primary dermal fibroblasts and epidermal keratinocytes with Peptide 14 treatment, and show that it is mostly non-toxic in concentrations up to 100 µM. Cancer lines were also used to investigate its potential of modulating proliferation. Different concentrations of the peptide promoted a discrete reduction in the proliferation of cancerous cells of the MeWo and HeLa lineages. In full-thickness human skin equivalents, topically formulated Peptide 14 also failed to exert any significant irritation, nor cellular toxicity when added to the culture media. Genotoxic assays including the Ames, micronucleus, and karyotyping tests also indicate the safety of the peptide. Finally, the irritative potential of the peptide was assessed in human subjects in a repeated insult patch test executed using 1 mM peptide. No visible skin reactions were observed in any of the 54 participants. Taken together, the present data support that Peptide 14 is a senotherapeutic molecule with a positive safety profile as tested with cruelty-free models, justifying further studies involving the peptide.

Pouteria macrophylla Fruit Extract Microemulsion for Cutaneous Depigmentation: Evaluation Using a 3D Pigmented Skin Model.

Here, we verify the depigmenting action of Pouteria macrophylla fruit extract (EXT), incorporate it into a safe topical microemulsion and assess its effectiveness in a 3D pigmented skin model. Melanocytes-B16F10- were used to assess the EXT effects on cell viability, melanin synthesis, and melanin synthesis-related gene transcription factor expression, which demonstrated a 32% and 50% reduction of intra and extracellular melanin content, respectively. The developed microemulsion was composed of Cremophor EL®/Span 80 4:1 (w/w), ethyl oleate, and pH 4.5 HEPES buffer and had an average droplet size of 40 nm (PdI 0.40 ± 0.07). Skin irritation test with reconstituted epidermis (Skin Ethic RHETM) showed that the formulation is non-irritating. Tyrosinase inhibition was maintained after skin permeation in vitro, in which microemulsion showed twice the inhibition of the conventional emulsion (20.7 ± 2.2% and 10.7 ± 2.4%, respectively). The depigmenting effect of the microemulsion was finally confirmed in a 3D culture model of pigmented skin, in which histological analysis showed a more pronounced effect than a commercial depigmenting formulation. In conclusion, the developed microemulsion is a promising safe formulation for the administration of cutite fruit extract, which showed remarkable depigmenting potential compared to a commercial formulation.

Nanostructured lipid carriers loaded with an association of minoxidil and latanoprost for targeted topical therapy of alopecia.

Alopecia is a condition associated with different etiologies, ranging from hormonal changes to chemotherapy, that affects over 80 million people in the USA. Nevertheless, there are currently few FDA-approved drugs for topical treatment, and existing formulations still present skin irritation issues, compromising treatment adherence. This work aimed to develop a safe formulation based on nanostructured lipid carriers (NLC) that entrap an association of minoxidil and latanoprost and target drug delivery to the hair follicles. To do so, thermal techniques combined with FTIR were used to assess the chemical compatibility of the proposed drug association. Then, NLC with 393.5 ± 36.0 nm (PdI < 0.4) and +22.5 ± 0.2 mV zeta potential were produced and shown to entrap 86.9% of minoxidil and 99.9% of latanoprost efficiently. In vitro, the free drug combination was indicated to exert positive effects over human primary epidermal keratinocytes, supporting cell proliferation, migration and inducing the mRNA expression of MKI67 proliferation marker and VEGF - a possible effector for minoxidil-mediated hair growth. Interestingly, such a favorable drug combination profile was optimized when delivered using our NLC. Furthermore, according to the HET-CAM and reconstructed human epidermis assays, the nanoformulation was well tolerated. Finally, drug penetration was evaluated in vitro using porcine skin. Such experiments indicated that the NLC could be deposited preferentially into the hair follicles, causing a considerable increase in the penetration of the two drugs in such structures, compared to the control (composed of the free compounds) and generating a target-effect of approximately 50% for both drugs. In summary, present results suggest that hair follicle-targeted delivery of the minoxidil and latanoprost combination is a promising alternative to treat alopecia.

Skin Regenerative Potential of Cupuaçu Seed Extract (Theobroma grandiflorum), a Native Fruit from the Amazon: Development of a Topical Formulation Based on Chitosan-Coated Nanocapsules.

Scarless skin regeneration is a challenge in regenerative medicine. Herein, we explore the regenerative potential of a Cupuaçu seed extract (Theobroma grandiflorum) to develop an innovative skin regeneration formulation based on chitosan-coated nanocapsules. Cupuaçu seed extract significantly stimulated cell proliferation and migration. A reparative gene expression profile could be verified following extract treatment, which included high levels of MKI67, a cellular proliferation marker, and extracellular matrix genes, such as ELN and HAS2, which code for elastin and hyaluronic acid synthase 2. Formulations with Cupuaçu seed extract successfully entrapped into nanocapsules (EE% > 94%) were developed. Uncoated or coated nanocapsules with low-molecular-weight chitosan presented unimodal size distribution with hydrodynamic diameters of 278.3 ± 5.0 nm (PDI = 0.18 ± 0.02) and 337.2 ± 2.1 nm (PDI = 0.27 ± 0.01), respectively. Both nanosystems were physically stable for at least 120 days and showed to be non-irritating to reconstructed human epidermis. Chitosan coating promoted active penetration into undamaged skin areas, which were still covered by the stratum corneum. In conclusion, the present study demonstrated for the first time the biotechnological potential of the frequently discarded Cupuaçu seed as a valuable pharmaceutical ingredient to be used in regenerative skin products.

Dissecting the relationship between antimicrobial peptides and mesenchymal stem cells.

Among the various biological properties presented by Mesenchymal Stem Cells (MSCs), their ability to control the immune response and fight pathogen infection through the production of antimicrobial peptides (AMPs) have been the subject of intense research in recent years. AMPs secreted by MSCs exhibit activity against a wide range of microorganisms, including bacteria, fungi, yeasts, and viruses. The main AMPs produced by these cells are hepcidin, cathelicidin LL-37, and ß-defensin-2. In addition to acting against pathogens, those AMPs have also been shown to interact with MSCs to modulate MSC proliferation, migration, and regeneration, indicating that such peptides exert a more diverse biological effect than initially thought. In the present review, we discuss the production of AMPs by MSCs, revise the multiple functions of these peptides, including their influence over MSCs, and present an overview of clinical situations in which the antimicrobial properties of MSCs may be explored for therapy. Finally, we discuss possibilities of combining MSCs and AMPs to generate improved therapeutic strategies.

Advanced Therapies and Regulatory Framework in Different Areas of the Globe: Past, Present, and Future.

PURPOSE: The field of human medicine is in a constant state of evolution, developing and incorporating technological advances from diverse scientific fields. In recent years, cellular and gene therapies have come of age, challenging regulatory agencies to define the path for commercial registration. Approval necessarily demands robust evidence for safety and efficacy, but these exigencies must not be such that they render unviable the development and testing of the therapeutic agent. Furthermore, reimbursement strategies are required to guarantee commercial viability of these products, to avoid the risk that they will be removed from the market or become unavailable to most patients through lack of financial resources. To address such challenges, several countries have created strategies to manage advanced therapy products. METHODS: Based on official documents published by regulatory agencies worldwide, this review summarizes the current scenario in the United States, Europe, Brazil, Japan, South Korea, and China in this regard, discussing the harmonized and dissonant aspects of the regulatory framework in different regions of the world and exploring perspectives for the future. FINDINGS: The technical aspects of advanced therapies are increasingly complex, bringing challenges for high mass commercialization and demanding specific regulation. The regulatory framework of the analyzed regions is mainly recent and discordant, but many harmonizing initiatives were observed. IMPLICATIONS: The comparative analysis of regulatory frameworks in different parts of the world is informative, as scientists must be aware of the rationale of regulators to assertively develop new technology and products that will be commercialized. The comparative analysis also provides insight into the main dissonances that must be addressed, fostering the harmonization of local regulatory frameworks. Many unanswered questions still lie ahead for the field of advanced therapies, and empirical evidence will be the most effective way to separate hype from hope and to establish the most sustainable mechanisms to regulate and finance such products in each part of the world.

Hallmarks of aging and immunosenescence: Connecting the dots.

Aging is a natural physiological process that features various and variable challenges, associated with loss of homeostasis within the organism, often leading to negative consequences for health. Cellular senescence occurs when cells exhaust the capacity to renew themselves and their tissue environment as the cell cycle comes to a halt. This process is influenced by genetics, metabolism and extrinsic factors. Immunosenescence, the aging of the immune system, is a result of the aging process, but can also in turn act as a secondary inducer of senescence within other tissues. This review aims to summarize the current state of knowledge regarding hallmarks of aging in relation to immunosenescence, with a focus on aging-related imbalances in the medullary environment, as well as the components of the innate and adaptive immune responses. Aging within the immune system alters its functionality, and has consequences for the person's ability to fight infections, as well as for susceptibility to chronic diseases such as cancer and cardiovascular disease. The senescence-associated secretory phenotype is described, as well as the involvement of this phenomenon in the paracrine induction of senescence in otherwise healthy cells. Inflammaging is discussed in detail, along with the comorbidities associated with this process. A knowledge of these processes is required in order to consider possible targets for the application of senotherapeutic agents - interventions with the potential to modulate the senescence process, thus prolonging the healthy lifespan of the immune system and minimizing the secondary effects of immunosenescence.

IDR-1018 induces cell proliferation, migration, and reparative gene expression in 2D culture and 3D human skin equivalents.

Skin lesions are associated with functional/cosmetic problems for those afflicted. Scarless regeneration is a challenge, not limited to the skin, and focus of active investigation. Recently, the host defense peptide innate defense regulatory peptide 1018 (IDR-1018) has shown exciting regenerative properties. Nevertheless, literature regarding IDR-1018 regenerative potential is scarce and limited to animal models. Here, we evaluated the regenerative potential of IDR-1018 using human 2D and 3D human skin equivalents. First, we investigated IDR-1018 using human cells found in skin-primary fibroblasts, primary keratinocytes, and the MeWo melanocytes cell line. IDR-1018 promoted cell proliferation and expression of marker of proliferation Ki-67, matrix metalloproteinase 1, and hyaluronan synthase 2 by fibroblasts. In keratinocytes, a drastic increase in expression was observed for Ki-67, matrix metalloproteinase 1, C-X-C motif chemokine receptor type 4, C-X-C motif chemokine receptor type 7, fibroblast growth factor 2, hyaluronan synthase 2, vascular endothelial growth factor, and elastin, reflecting an intense stimulation of these cells. In melanocytes, increased migration and proliferation were observed following IDR-1018 treatment. The capacity of IDR-1018 to promote dermal contraction was verified using a dermal model. Finally, using a 3D human skin equivalent lesion model, we revealed that the regenerative potential of IDR1018, previously tested in mice and pigs, is valid for human skin tissue. Lesions closed faster in IDR-1018-treated samples, and the gene expression signature observed in 2D was reproduced in the 3D human skin equivalents. Overall, the present data show the regenerative potential of IDR-1018 in an experimental system comprising human cells, underscoring the potential application for clinical investigation.

Mesenchymal stem cells immunomodulation: The road to IFN-γ licensing and the path ahead.

Mesenchymal Stem Cells (MSCs) have gained prominence as an important tool in cell therapy, especially considering their capacity to control the immune system. Due to this property, the application of MSCs has been investigated for the treatment of several immune disorders, such as diabetes, rheumatoid arthritis, Crohn's disease, systemic lupus erythematosus, and graft-versus-host-disease (GvHD). The application of MSCs to treat inflammatory diseases has led to impressive results. However, individual response to treatment is still heterogeneous, and the number of cells required to treat humans is very high. The possibility of increasing the immunosuppressive potential of MSCs is seen at this point as a promising alternative to overcome such limitations. One of the most exploited strategies for this purpose has been the licensing of MSCs prior to clinical application. In this review, we will discuss the mechanisms by which MSCs modulate the immune response and the main advances in the licensing of these cells, with a special focus on the use of interferon gamma (IFN-γ). Also, we will address the main challenges ahead before licensed MSCs are finally used successfully in clinical practice.

Breaking the frontiers of cosmetology with antimicrobial peptides.

Antimicrobial peptides (AMPs) are mostly endogenous, cationic, amphipathic polypeptides, produced by many natural sources. Recently, many biological functions beyond antimicrobial activity have been attributed to AMPs, and some of these have attracted the attention of the cosmetics industry. AMPs have revealed antioxidant, self-renewal and pro-collagen effects, which are desirable in anti-aging cosmetics. Additionally, AMPs may also be customized to act on specific cellular targets. Here, we review the recent literature that highlights the many possibilities presented by AMPs, focusing on the relevance and impact that this potentially novel class of active cosmetic ingredients might have in the near future, creating new market outlooks for the cosmetic industry with these molecules as a viable alternative to conventional cosmetics.