Commentary : mesenchymal stem cells : a new piece in the puzzle of COVID-19 treatment

A Commentary on Mesenchymal Stem Cells: A New Piece in the Puzzle of COVID-19 Treatment By Saldanha-Araujo F, Melgaço Garcez E, Silva-Carvalho AE and Carvalho JL (2020). Front. Immunol. 11:1563. doi: 10.3389/fimmu.2020.01563

The inflammatory status of soluble microenvironment influences the capacity of melanoma cells to control t-cell responses

The development of immunotherapeutic approaches for the treatment of melanoma requires a better understanding of immunoescape mechanisms of tumor cells and how they interact with other tumor-resident cell types. Here, we evaluated how the conditioned media of resting (rCM) and immune-activated PBMCs (iCM) influence the ability of a metastatic melanoma cell line (MeWo) to control T-cells function. MeWo cells were expanded in RPMI, rCM, or iCM and the secretome generated after cell expansion was identified as MeSec (RPMI), niSec (non-inflammatory), or iSec (inflammatory secretome), respectively. Then, the immunomodulatory potential of such secretomes was tested in PHA-activated PBMCs. iCM induced higher levels of IFN-γ and IL-10 in treated melanoma cells compared to rCM, as well as higher IDO and PD-L1 expression. The iSec was able to inhibit T-cell activation and proliferation. Interestingly, PBMCs treated with iSec presented a reduced expression of the regulators of Th1 and Th2 responses T-BET and GATA-3, as well as low expression of IFN-γ, and co-stimulatory molecules TIM-3 and LAG-3. Importantly, our findings show that melanoma may benefit from an inflammatory microenvironment to enhance its ability to control the T-cell response. Interestingly, such an immunomodulatory effect involves the inhibition of the checkpoint molecules LAG-3 and TIM-3, which are currently investigated as important therapeutic targets for melanoma treatment. Further studies are needed to better understand how checkpoint molecules are modulated by paracrine and cell contact-dependent interaction between melanoma and immune cells. Such advances are fundamental for the development of new therapeutic approaches focused on melanoma immunotherapy

Effects of in vitro short- and long-term treatment with telomerase inhibitor in U-251 glioma cells

BACKGROUND: The inhibition of the enzyme telomerase (TERT) has been widely investigated as a new pharmacological approach for cancer treatment, but its real potential and the biochemical consequences are not totally understood. OBJECTIVE: Here, we investigated the effects of the telomerase inhibitor MST-312 on a human glioma cell line after both short- and long-term (290 days) treatments. METHODS: Effects on cell growth, viability, cell cycle, morphology, cell death and genes expression were assessed. RESULTS: We found that short-term treatment promoted cell cycle arrest followed by apoptosis. Importantly, cells with telomerase knock-down revealed that the toxic effects of MST-312 are partially TERT dependent. In contrast, although the long-term treatment decreased cell proliferation at first, it also caused adaptations potentially related to treatment resistance and tumor aggressiveness after long time of exposition. CONCLUSIONS: Despite the short-term effects of telomerase inhibition not being due to telomere erosion, they are at least partially related to the enzyme inhibition, which may represent an important strategy to pave the way for tumor growth control, especially through modulation of the non-canonical functions of telomerase. On the other hand, long-term exposure to the inhibitor had the potential to induce cell adaptations with possible negative clinical implications

The Peptide Salamandrin-I Modulates Components Involved in Pyroptosis and Induces Cell Death in Human Leukemia Cell Line HL-60

Amphibian secretions have been extensively investigated for the production of bioactive molecules. Salamandrin-I is an antioxidant peptide, isolated from the skin secretion of the fire salamander, that has induced no toxicity in microglia or erythrocytes. Importantly, the administration of antioxidants may constitute an adequate therapeutic approach to cancer treatment. Here, with the purpose of better characterizing the therapeutic potential of salamandrin-I, we investigated whether this antioxidant peptide also exerts anticancer activity, using the human leukemia cell line HL-60 as a cancer model. Salamandrin-I treatment induced a significant reduction in HL-60 proliferation, which was accompanied by cell cycle arrest. Furthermore, the peptide-induced cell death showed a significant increase in the LDH release in HL-60 cells. The cellular toxicity exerted by salamandrin-I is possibly related to pyroptosis, since the HL-60 cells showed loss of mitochondrial membrane potential and hyperexpression of inflammasome components following the peptide treatment. This is the first demonstration of the anticancer potential of the salamandrin-I peptide. Such results are important, as they offer relevant insights into the field of cancer therapy and allow the design of future bioactive molecules using salamandrin-I as a template

GVHD-derived plasma as a priming strategy of mesenchymal stem cells

Background: Mesenchymal stem cell (MSC) therapy is an important alternative for GVHD treatment, but a third of patients fail to respond to such therapy. Therefore, strategies to enhance the immunosuppressive potential of MSCs constitute an active area of investigation. Here, we proposed an innovative priming strategy based on the plasma obtained from GVHD patients and tested whether this approach could enhance the immunosuppressive capacity of MSCs. Methods: We obtained the plasma from healthy as well as acute (aGVHD) and chronic (cGVHD) GVHD donors. Plasma samples were characterized according to the TNF-α, IFN-γ, IL-10, IL-1β, IL-12p40, and IL-15 cytokine levels. The MSCs primed with such plasmas were investigated according to surface markers, morphology, proliferation, mRNA expression, and the capacity to control T cell proliferation and Treg generation. Results: Interestingly, 57% of aGVHD and 33% of cGVHD plasmas significantly enhanced the immunosuppressive potential of MSCs. The most suppressive MSCs presented altered morphology, and those primed with cGHVD displayed a pronounced overexpression of ICAM-1 on their surface. Furthermore, we observed that the ratio of IFN-γ to IL-10 cytokine levels in the plasma used for MSC priming was significantly correlated with higher suppressive potential and Treg generation induction by primed MSCs, regardless of the clinical status of the donor. Conclusions: This work constitutes an important proof of concept which demonstrates that it is possible to prime MSCs with biological material and also that the cytokine levels in the plasma may affect the MSC immunosuppressive potential, serving as the basis for the development of new therapeutic approaches for the treatment of immune diseases

Neuroprotective effects on microglia and insights into the structure–activity relationship of an antioxidant peptide isolated from Pelophylax perezi

© 2022 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly citedTryptophyllins constitute a heterogeneous group of peptides that are one of the first classes of peptides identified from amphibian's skin secretions. Here, we report the structural characterization and antioxidant properties of a novel tryptophyllin-like peptide, named PpT-2, isolated from the Iberian green frog Pelophylax perezi. The skin secretion of P. perezi was obtained by electrical stimulation and fractionated using RP-HPLC. De novo peptide sequencing was conducted using MALDI MS/MS. The primary structure of PpT-2 (FPWLLS-NH2 ) was confirmed by Edman degradation and subsequently investigated using in silico tools. PpT-2 shared physicochemical properties with other well-known antioxidants. To test PpT-2 for antioxidant activity in vitro, the peptide was synthesized by solid phase and assessed in the chemical-based ABTS and DPPH scavenging assays. Then, a flow cytometry experiment was conducted to assess PpT-2 antioxidant activity in oxidatively challenged murine microglial cells. As predicted by the in silico analyses, PpT-2 scavenged free radicals in vitro and suppressed the generation of reactive species in PMA-stimulated BV-2 microglia cells. We further explored possible bioactivities of PpT-2 against prostate cancer cells and bacteria, against which the peptide exerted a moderate antiproliferative effect and negligible antimicrobial activity. The biocompatibility of PpT-2 was evaluated in cytotoxicity assays and in vivo toxicity with Galleria mellonella. No toxicity was detected in cells treated with up to 512 µg/ml and in G. mellonella treated with up to 40 mg/kg PpT-2. This novel peptide, PpT-2, stands as a promising peptide with potential therapeutic and biotechnological applications, mainly for the treatment/prevention of neurodegenerative disorders.This work was financed by FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalization (POCI), and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia in the framework of the project POCI-01-0145-FEDER-031158 – PTDC/BII-BIO/31158/2017. The authors would like to thank the participation and scientific support of the Unit projects UIDB/50006/2020 | UIDP/50006/2020, and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Universal Faixa ‘B’ (grant number 32103/2018-0). A.P. is a recipient of a post-doctoral grant from the project PTDC/BII-BIO/31158/2017. The authors would like to thank the researcher Roberto Resendes (CiBio, University of the Azores, Ponta Delgada, São Miguel, Azores, Portugal) for the logistical support in the collection of samples. C.P.A acknowledges FCT-MCTES fellowship PD/BD/136860/2018. A.B.-N. and F.C.D.A.L. acknowledge CNPq (grants 420449/2018-3 and 428211/2018-6) for financial support.info:eu-repo/semantics/publishedVersio

Promising self-emulsifying drug delivery system loaded with lycopene from red guava (Psidium guajava L.): in vivo toxicity, biodistribution and cytotoxicity on DU-145 prostate cancer cells

Background Self-emulsifying drug delivery systems (SEDDSs) have attracted attention because of their effects on solubility and bioavailability of lipophilic compounds. Herein, a SEDDS loaded with lycopene purified from red guava (nanoLPG) was produced. The nanoemulsion was characterized using dynamic light scattering (DLS), zeta potential measurement, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), lycopene content quantification, radical scavenging activity and colloidal stability in cell culture medium. Then, in vivo toxicity and tissue distribution in orally treated mice and cytotoxicity on human prostate carcinoma cells (DU-145) and human peripheral blood mononuclear cells (PBMC) were evaluated. Results NanoLPG exhibited physicochemical properties with a size around 200 nm, negative zeta-potential, and spherical morphology. The size, polydispersity index, and zeta potential parameters suffered insignificant alterations during the 12 month storage at 5 °C, which were associated with lycopene stability at 5 °C for 10 months. The nanoemulsion showed partial aggregation in cell culture medium at 37 °C after 24 h. NanoLPG at 0.10 mg/mL exhibited radical scavenging activity equivalent to 0.043 ± 0.002 mg Trolox/mL. The in vivo studies did not reveal any significant changes in clinical, behavioral, hematological, biochemical, and histopathological parameters in mice orally treated with nanoLPG at 10 mg/kg for 28 days. In addition, nanoLPG successfully delivered lycopene to the liver, kidney and prostate in mice, improved its cytotoxicity against DU-145 prostate cancer cells—probably by pathway independent on classical necrosis and apoptosis—and did not affect PBMC viability. Conclusions Thus, nanoLPG stands as a promising and biosafe lycopene delivery system for further development of nanotechnology-based health products

Influence of plasma from patients with graft versus host disease over the immunosuppressive potential of mesenchymal stem cells

Dissertação (mestrado) — Universidade de Brasília, Faculdade de Medicina, Programa de Pós-Graduação em Patologia Molecular, 2019.As Células-tronco mesenquimais (CTMs) têm recebido destaque como uma importante ferramenta para terapia celular devido às suas inúmeras características biológicas associadas à facilidade de obtenção e de expansão in vitro. Dentre as suas propriedades, a que mais chama atenção é a sua capacidade de interagir e modular o sistema imune. Os resultados mais expressivos desse potencial imunorregulador das CTMs têm sido observados no manejo e tratamento da Doença do Enxerto contra o hospedeiro (DECH). Essa doença é caracterizada por um processo inflamatório sistêmico mediado por células T do doador que reagem contra antígenos do receptor, resultando em danos em uma série de órgãos e tecidos. Entretanto, nesse cenário, nota-se que é necessário um elevado número de CTMs para se obter o efeito terapêutico desejado e nem todos os pacientes com DECH se beneficiam com essa terapia. Diante disso, há uma busca constante por estratégias que possam realçar as propriedades dessas células. No presente estudo estabelecemos uma estratégia inovadora de licenciamento submetendo as CTMs ao plasma de pacientes com DECH aguda e crônica, e investigamos os impactos dessa estratégia sobre as propriedades biológicas das CTMs. Apesar de apresentarem uma constituição heterogenia, de modo geral, os plasmas de pacientes com DECH apresentaram maior concentração das citocinas TNF-α, INF-γ, IL-1β e IL-15 quando comparados com o plasma de doadores saudáveis. Além disso, os plasmas de DECHc apresentaram maior concentração de IL-10 em relação aos plasmas de DECHa. Mais importante, alguns plasmas de DECH foram capazes de realçar o potencial imunossupressivo das CTMs, alterando também sua morfologia. Observamos ainda um aumento significativo na expressão de ICAM-1 e VCAM-1 em CTMs expostas ao plasma de pacientes com DECH. Pontualmente, alguns plasmas aumentaram a expressão de transcritos imunorreguladores, como TGF-β1, HGF e GALECTINA-1. Em suma, nossos achados podem servir de base para o desenvolvimento de novas estratégias terapêuticas para tratamento da DECH, onde se poderia explorar a própria inflamação promovida pela doença como forma de realçar a capacidade imunossupressiva das células que serão infundidas no paciente.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).Mesenchymal stem-cells (MSCs) have been gaining prominence as an important tool in cell therapy due to its innumerable biological characteristics, associated to ease of obtaining and "in vitro" expansion. Among their properties, the most attractive one is the ability to interact and modulate the immune system. The most expressive results of MSCs immunoregulatory potential have been observed in the management and treatment of Graft versus Host Disease (GvHD). This disease is characterized by a systemic inflammatory process mediated by donor T cells that react against receptor antigens, resulting in damage to several organs and tissues. However, in this scenario, a high number of MSCs is necessary to achieve the therapeutic effect and not all patients are benefited from such therapy. Moreover, there is a constant search for strategies that can enhance the properties of these cells. In the present study we established an innovative licensing strategy by subjecting the MSCs to the plasma of patients with acute and chronic GvHD and investigating the impacts of this strategy on MSCs biological properties. In general, the plasmas of GvHD patients showed a higher concentration of the TNF-α, INF-γ, IL- 1β and IL-15 cytokines when compared to plasma from healthy donors. In addition, the cGvHD plasmas presented a higher concentration of IL-10 in relation to the aGvHD plasmas. Some plasmas of GvHD were able to highlight the suppressive potential of MSCs, also altering their morphology. We also observed a significant increase in the expression of ICAM-1 and VCAM- 1 in MSCs exposed to plasma from patients with GvHD. Singly, some plasmas increased the expression of immunoregulatory transcripts, such as TGF-β1, HGF and GALECTIN-1. Taken together, our findings may serve as a basis for the development of new therapeutic strategies for the treatment of GvHD, where inflammation promoted by the disease is explored as a tool to enhance the immunosuppressive capacity of the cells that will be infused in the patient

Regulatory T-Cell enhancement, expression of adhesion molecules, and production of anti-inflammatory factors are differentially modulated by spheroid-cultured mesenchymal stem cells

The culture of mesenchymal stem cells (MSCs) as spheroids promotes a more physiological cellular behavior, as it more accurately reflects the biological microenvironment. Nevertheless, mixed results have been found regarding the immunosuppressive properties of spheroid-cultured MSCs (3D-MSCs), the mechanisms of immunoregulation of 3D-MSCs being scarcely described at this point. In the present study, we constructed spheroids from MSCs and compared their immunosuppressive potential with that of MSCs cultured in monolayer (2D-MSCs). First, we evaluated the ability of 2D-MSCs and 3D-MSCs to control the activation and proliferation of T-cells. Next, we evaluated the percentage of regulatory T-cells (Tregs) after the co-culturing of peripheral blood mononuclear cells (PBMCs) with 2D-MSCs and 3D-MSCs. Finally, we investigated the expression of adhesion molecules, as well as the expressions of several anti-inflammatory transcripts in 2D-MSCs and 3DMSCs maintained in both inflammatory and non-inflammatory conditions. Interestingly, our data show that several anti-inflammatory genes are up-regulated in 3D-MSCs, and that these cells can control T-cell proliferation. Nevertheless, 2D-MSCs are more efficient in suppressing the immune cell proliferation. Importantly, contrary to what was observed in 3D-MSCs, the expressions of ICAM-1 and VCAM-1 are significantly upregulated in 2D-MSCs exposed to an inflammatory environment. Furthermore, only 2D-MSCs are able to promote the enhancement of Tregs. Taken together, our data clearly show that the immunosuppressive potential of MSCs is significantly impacted by their shape, and highlights the important role of cell–cell adhesion molecules for optimal MSC immunomodulatory function.Instituto de Ciências Biológicas (IB)Faculdade de Ciências da Saúde (FS)Departamento de Farmácia (FS FAR