Efeitos da BMP-7 na cicatrização de feridas em murganhos diabéticos - um estudo piloto

Dissertação de Mestrado em Biologia Celular e Molecular apresentada à Faculdade de Ciências e TecnologiaResumo: A proteína óssea morfogenética 7 (BMP-7) é uma proteína da superfamília fator de transformação de crescimento (TGF-β). Esta proteína foi descoberta pela sua capacidade de induzir células mesenquimais a se diferenciarem em osteoblastos, por promover a proliferação celular e a síntese de proteoglicanos e colágenio tipo II da matriz extracelular. No entanto, os papéis principais da BMP-7 na pele não são bem conhecidos e nunca foram estudados, particularmente na cicatrização de feridas diabéticas. Objetivo: Este estudo pretende avaliar o papel fundamental da BMP-7 na cicatrização de feridas diabéticas na pele. Materiais e métodos: Foram utilizados murganhos adultos C57Bl/6 e a indução de diabetes foi feita com o uso de estreptozotocina (50mg/kg, i.p., por 5 dias consecutivos). Subsequentemente, foram induzidas duas feridas de 6 mm em murganhos diabéticos e as feridas foram tratadas topicamente com proteína óssea morfogénica 7 humana recombinante (rhBMP-7), aplicando 2 ou 10 µg por ferida todos os dias ou solução salina como o tratamento controlo, até 10 dias após a indução de feridas. As células inflamatórias, macrófagos e linfócitos, foram quantificados por imunohistoquímica. A presença de macrófagos foi avaliada com CD68, os macrófagos de fenótipo M1 foram avaliados com dupla marcação de CD68 e TNFα, enquanto os macrófagos de fenótipo M2 foram avaliados com dupla marcação de CD68 e CD206. Além disso, a presença de linfócitos foi avaliada usando CD3, para quantificação de vasos sanguíneos/angiogénese utilizamos CD31. O factor de crescimento VEGF foi analisado uma vez que esta relacionado com a angiogénese. IL-6 e TNFα, marcadores de inflamação, também foram analisados. Ainda, o KI-67 foi usado para avaliar a proliferação celular. Hematoxilina e Eosina, bem como coloração de Herovici foram usados para avaliar a histologia da pele e a organização da matriz extracelular (ECM), bem como a deposição de colágenio. Além disso, também avaliamos por qRT-PCR a expressão génica de IL-6, KC, TNF-α, IL-β1, MCP-1, MMP-9, VEGF, VEGFR2, TGF-β1, SMAD-4 e BMP- 7. Resultados: O tratamento com BMP-7 foi induziu significativamente o fecho da ferida em ambos os grupos, 2 µg (**p <0.01, n = 6 feridas) e 10 µg (*p <0.05, n = 8 feridas) quando comparados com a solução salina (n = 6 feridas). O tratamento com BMP-7 reduziu a expressão de moléculas inflamatórias, nos grupos de 2 µg e 10µg de BMP-7, tais como IL-6, TNF-α, IL-β1, MCP-1, MMP-9, KC ( n = 3 em 2 e n = 4 em 10 de BMP-7, n = 3 em solução salina). A redução do fenótipo de macrófagos pró-inflamatórios M1 foi significativamente reduzida pelo tratamento com BMP-7 nos grupos de 2 µg e 10 µg de BMP-7 (***p <0.001, n = 3 em 2 µg e n = 4 em 10 µg de BMP-7 , n = 3 em solução salina) quando comparado ao grupo tratado com solução salina. Por outro lado, 2 e 10 µg de BMP-7 induziram um aumento da angiogénese, uma vez que o número de vasos aumentou (**p <0.01 e ***p <0.001). A expressão do VEGF aumentou tanto em 2 µg como em 10 µg de BMP-7 (**p<0.01 and *p<0.05). Além disso, KI-67 aumentou nos tratamentos com BMP-7 (***p <0.001) quando comparado ao grupo tratado com solução salina, indicando que a BMP-7 estimula a fase proliferativa na cicatrização de feridas. A deposição de colágenio também foi estimulada pela BMP-7. Não foram observadas diferenças estatisticamente significantes no número de macrófagos anti inflamatórios M2, células T, assim como nas expressões dos genes TGF-β1, SMAD-4 e BMP-7, nessas condições. Conclusões: Em conclusão, o tratamento tópico da ferida com BMP-7 foi capaz de melhorar o fecho da ferida devido a uma diminuição significativa na infiltração de células inflamatórias, incluindo macrófagos e particularmente macrófagos M1. Um aumento significativo na neovascularização, juntamente com a estimulação da fase proliferativa, também foi verificado no tratamento com BMP7. BMP-7 apresenta várias características benéficas para cicatrização de feridas. No entanto, devido à capacidade da BMP-7 promover a diferenciação de uma matriz óssea na pele, após tratamento tópico, mais estudos são necessários para avaliar se a BMP-7 em concentrações diferentes das que usamos será um bom candidato para tratar feridas na pele diabética.Abstract: Bone morphogenetic protein 7 (BMP-7) is a protein from the transforming growth factor (TGF-β) superfamily. It was discovered for its ability to induce mesenchymal cells to differentiate into osteoblasts, for the promotion of cell proliferation and extracellular matrix proteoglycan and collagen type II synthesis. However, the key roles of BMP-7 in the skin are not well known, and have never been studied, particularly in diabetic wound healing. Objective: This study evaluates keys role of BMP-7 in diabetic wound healing at the skin level. Materials and methods: We used C57Bl/6 adult mice and diabetes was induced using streptozotocin (50mg/kg, ip, for 5 consecutive days). Subsequently two 6 mm wounds were induced in diabetic mice and wounds were then treated topically with recombinant human bone morphogenetic protein-7 (rhBMP-7), using either 2 or 10µg per wound every day, or saline as the control treatment up to 10 days post wounding. Inflammatory cells, macrophages and lymphocytes, were evaluated by immunohistochemistry. The presence of macrophages was evaluated with CD68 staining, the M1 macrophage phenotype was evaluated with CD68 and TNFα costaining, while the M2 phenotype was evaluated with CD68 and CD206 co-staining. Moreover, the presence of lymphocytes was evaluated using CD3, for quantification of blood vessels/angiogenesis we used CD31. The growth factor VEGF was evaluated since its important for angiogenesis. IL-6 and TNFα, markers of inflammation, were also analysed. In addition, KI-67 was measured to evaluate the cell proliferation. Hematoxilyn & Eosin, as well as Herovici staining were used to evaluate skin histology and the organization of the extracellular matrix (ECM), as well as collagen deposition. In addition, we also evaluated by qRT-PCR the gene expression of IL-6, KC, TNF-α, IL-β1, MCP-1, MMP-9, VEGF, VEGFR2, TGF-β1, SMAD-4 and BMP-7. Results: BMP-7 treatment was able to induce significant reduction in wound closure in both 2µg (**p<0.01, n=6 wounds) and 10µg (*p<0.05, n=8 wounds) BMP-7 groups when compared to the saline treated group (n=6 wounds). In addition, BMP-7 treatment tended to reduce the expression of inflammatory molecules, in both 2µg and 10µg BMP-7 groups, such as IL-6, TNF-α, IL-β1, MCP-1, MMP-9, KC (n=3 in 2µg and n=4 in 10µg of BMP-7, n=3 in saline). The reduction of the M1 pro-inflammatory macrophages phenotype was significantly reduced by BMP-7 treatment in both 2µg and 10µg BMP-7 groups (***p<0.001, n=3 in 2µg and n=4 in 10µg of BMP-7, n=3 in saline) when compared to the saline treated group. On the other hand, 2µg and 10µg of BMP-7 induced an increase in angiogenesis since the number of blood vessels was increased (**p<0.01 and ***p<0.001). VEGF expression increased in both 2µg and 10µg of BMP-7 (**p<0.01 and *p<0.05). In addition, KI-67 was also increased by 2µg and 10µg of BMP-7 (***p<0.001) when compared to the saline treated group, indicating that BMP-7 stimulates the proliferative phase in wound healing. Collagen deposition was also stimulated by BMP-7. There were no statistically significant differences observed in the number of M2 antiinflammatory macrophages, T- cells, as well as for TGF-β1, SMAD-4 and BMP-7 gene expression, under these conditions. Conclusions: In conclusion, the BMP-7 topical wound treatment was able to improve wound closure due to a significant decrease in inflammatory cell infiltration including, macrophages and particularly M1 macrophage phenotype. A significant increase in neovascularization, along with stimulation of the proliferative phase, was also stimulated by BMP-7. BMP-7 has several characteristics that are known to be beneficial in wound healing. However, due to the ability of BMP-7 to promote the differentiation of a bone matrix in the skin, post tropical treatment, more studies are needed to evaluate whether BMP-7 at concentrations different from the ones we used will be a good candidate to treat diabetic skin wounds.Outro - European Foundation for the Study of Diabetes, Sociedade Portuguesa Diabetologia, Fundação para a Ciência e a Tecnologia and Programa Operacional Factores de Competitividade – COMPETE. We thank Dr. YuHua Tseng (Joslin, Boston) for providing the recombinant human BMP-7 (rhBMP-7)

Effective Management of Nasal Vestibule Squamous Cell Carcinoma with Cemiplimab: A Case Report

Nasal vestibule squamous cell carcinoma (SCC) is a rare malignancy with limited treatment options. This case report presents an 83-year-old female with SCC of the nasal vestibule who was ineligible for surgery or radiotherapy due to various factors. The patient was successfully treated with cemiplimab, a systemic anti-PD-1 antibody, resulting in a remarkable tumor reduction without any observed side effects. This is the first reported case of nasal vestibule SCC treated with cemiplimab, highlighting its potential as a promising therapeutic option

A Retrospective Study of Cemiplimab Effectiveness in Elderly Patients with Squamous Cell Carcinoma of the Skin: Insights from a Real-Life Scenario

Abstract Introduction This retrospective study investigates the efficacy of cemiplimab, a monoclonal antibody targeting the PD-1 receptor, in treating squamous cell carcinoma (SCC) of the skin. Methods The study analyzes data from 50 patients with SCC, focusing on various clinical parameters, including patient demographics, tumor characteristics, treatment history, disease status at the beginning of therapy, and survival outcomes. Results Of the patients who received at least one cycle of cemiplimab, 42% showed a clinical response. Adverse reactions were generally low, with the safety profile deemed excellent. During a median follow-up of 9.6 months, 17 patients experienced progression or death. Among these, 15 patients had died at the time of the analysis. The median progression-free survival (PFS) for the entire cohort was approximately 20.8 months, while median overall survival (OS) was not reached. Univariate Cox regression analysis for PFS showed that tumors in the arms and legs were associated with higher progression risk, while age above 65 years was not statistically significant. Distant metastasis exhibited a trend towards improved PFS. In terms of OS, distant metastasis was a significant predictor of reduced survival, while age above 65 years was not statistically significant. In a multivariate model, only the absence of distant metastasis remained significant, with an adjusted odds ratio (OR) of 12.3 (95% confidence interval 1.3–112.1). Conclusion These findings provide valuable insights into the real-world effectiveness of cemiplimab in SCC management