Regucalcin regulation by extracellular calcium in prostate cells

Prostate cancer is one of the most diagnosed diseases in men at the present time. It is well known that changes in calcium (Ca2+) homeostasis are derived from modifications in Ca2+ regulating elements. Regucalcin (RGN) is a Ca2+-binding protein which plays an important role in maintenance of intracellular Ca2+ homeostasis and regulation of apoptosis and proliferation. RGN is underexpressed in prostate cancer cells, suggesting that a loss of RGN expression may be associated with tumor development. In vivo studies have also shown that Ca2+ administration acts as a regulator of RGN expression in liver and kidney tissues. However, no studies on the characterization of RGN regulation by extracelular Ca2+ in prostate cells have been conducted. To attain this goal, prostate cells were stimulated with different doses of CaCl2 during several periods of time. To assess RGN mRNA and protein expression, Real Time PCR and Western Blot were carried out, respectively. Moreover, the cell viability in response to treatments was evaluated through MTS assays. Our results show that nonneoplastic PNT1A cells present higher levels of RGN when compared to neoplastic LNCaP or PC3 cells. We also verified that RGN expression in PNT1A cells is up-regulated by extracellular Ca2+ at 1,5h after stimuli, but its expression decreases after 3h of stimulation. We also showed that high doses of extracellular Ca2+ induce different effects on cell proliferation between PNT1A and LNCaP cells. This study led us to conclude that RGN appears to be regulated by extracellular Ca2+ levels in prostate cells and that an elevation of extracellular Ca2+ promotes high rates of cell proliferation in LNCaP cells, possibly due to the down-regulation in RGN expression in cancer cells.O cancro da próstata é de umas das doenças mais diagnosticadas em homens de países Ocidentais. É sabido que as alterações na homeostase do cálcio (Ca2+) derivam de modificações ao nível dos elementos reguladores do Ca2+ . A regucalcina (RGN) é uma proteína de ligação ao Ca2+ que desempenha um papel importante ao nível da manutenção da homeostase do Ca2+ intracelular, tal como na regulação da apoptose e da proliferação. A RGN é sub-expressa em células do cancro da próstata, sugerindo que a perda de expressão da RGN poderá estar associada com o desenvolvimento tumoral. Estudos in vivo mostram que a administração de Ca2+ actua como um regulador da expressão de RGN em fígado e de rim de rato. No entanto, ainda nenhum estudo acerca da caracterização da regulação da RGN pelo Ca2+ extracelular foi conduzido em células de próstata. Para atingir este objectivo, células da próstata foram estimuladas com diferentes doses de CaCl2 durante vários períodos de tempo. Para avaliar a expressão do mRNA e da proteína, estudos de Real Time PCR e de Western Blot foram efectuados, respectivamente. A viabilidade celular em resposta aos diferentes tratamentos foi avaliada através de ensaios de MTS. Os nossos resultados mostram que células não neoplásicas PNT1A apresentam níveis elevados de RGN quando comparados com células neoplásicas LNCaP e PC3. Também verificamos que a expressão de RGN em células PNT1A é regulada positivamente pelo Ca2+ extracelular às 1,5h de estímulo, sendo que a sua expressão começa a diminuir após as 3h. Mostramos ainda que elevadas doses de Ca2+ extracelular induzem diferentes efeitos ao nível da proliferação entre as células PNT1A e LNCaP. Este estudo levou-nos a concluir que a RGN aparenta ser regulada através do Ca2+ extracelular em células da próstata e que uma elevação nos níveis de Ca2+ extracelular poderá promover elevadas taxas de proliferação em células LNCaP, possivelmente devido a sub-expressão de RGN verificada em células tumorais

Bioengineering strategies for cancer therapy and modelling

Tese de doutoramento em Engenharia de Tecidos, Medicina Regenerativa e Células EstaminaisCancer is a global pandemic with a high incidence among the world population and effective treatments are for the most part elusive. The tumor microenvironment is a highly complex and heterotypic mixture of cells that interact to regulate central control mechanisms, driving immunosuppression and promoting both survival and invasion of cancer cells into surrounding tissues. It has been this complexity that has made finding effective therapeutics such a demanding task and therefore cancer still remains a burden worldwide in health as well as in economic terms. While the progression in the field of cancer research has been clear over the years, there are still several challenges that need to be addressed. Herein, two different sides to this disease are explored: treatment and in vitro models. Adoptive T cell therapy has shown impressive results, however not without its limitations. The use of the T cell mitogen IL-2 within culture systems is known to lead to early exhaustion of T cell subsets while high density of co-stimulating molecules has been linked to undesired immune responses. As an alternative, a nanoparticle system based on the natural polymer gellan-gum was proposed, with tailorable surface functionalization with co-stimulatory molecules. High levels of T cell expansion were observed over the studied period, with secreted IL-2 levels overcoming those of commercial alternatives. With this system, increased expression of cytotoxic molecules Granzyme B and Perforin were also detected in vitro. On the other spectrum, 3D cancer models have sustained a great number of developments observed by an increase in similarity towards native tissues; however, a requirement for even more complex architectures capable of better mimicking cellular interactions is still present. Therefore, an assembloid-based approach was proposed to develop a 3D in vitro melanoma model to further study cellular interactions. These heterotypic tumor assembloids presented a complex architecture capable of sustaining endothelial cell function as well as a high expression of stemness-related markers. These models were subjected to functionality assays where they showed a capacity for “cooperative invasion” which was coincident with an observed increased production of MMP-2. To further unravel the role of stromal cells in the invasive potential of cancer cells a 3D chemotaxis chamber was developed to study cellular interactions observed in the tumor microenvironment, where stem cells and fibroblasts showed to have a crucial role. Ultimately, this thesis allowed to explore biomedical engineering approaches to further contribute to the knowledge in the field opening new doors to be explored in the future.O cancro é uma pandemia global com uma elevada incidência e cujo desenvolvimento de tratamentos eficazes continua a ser difícil. O microambiente tumoral é uma mistura altamente complexa e heterotípica de células que interagem para regular mecanismos centrais que provocam imunossupressão promovendo a sobrevivência e invasão de células tumorais para os tecidos circundantes. É esta complexidade que tem tornado desafiante encontrar terapias eficazes, tornando esta doença um fardo a nível global em termos de saúde e economia. Enquanto a progressão na área da investigação oncológica tem sido clara ao longo dos anos, existem ainda vários desafios que precisam de serem encarados para permitir futuros desenvolvimentos. Aqui, foram exploradas duas vertentes diferentes desta doença: o tratamento e os modelos in vitro. A terapia celular adotiva tem demonstrado resultados impressionantes, no entanto não sem as suas limitações. O uso do mitogénio IL-2 nestes sistemas de cultura é conhecido por levar rapidamente à exaustão das células T, enquanto o uso de moléculas co-estimulatórias em elevadas densidades está associado a respostas imunes não desejadas. Como alternativa, foi proposto um sistema de nanopartículas baseado no polímero natural goma gelana e funcionalizado com moléculas co estimulatórias. Foram observados elevados níveis de expansão de células T e quantidade de IL-2 secretada superior à de alternativas comerciais. Foi ainda verificado in vitro um aumento de expressão das moléculas citotóxicas Grazima B e Perforina. No outro espectro, têm sido desenvolvidos modelos tumorais 3D com uma cada vez maior similaridade para tecidos nativos; no entanto, a necessidade de arquiteturas ainda mais complexas capazes de melhor representar interações celulares persiste. Assim, foi proposta uma abordagem baseada em “assemblóides” para obter modelos 3D in vitro de melanoma para estudar interações celulares. Estes “assemblóides” tumorais heterotípicos apresentaram uma arquitetura complexa capaz de suportar a função de células endoteliais, bem como a elevada expressão de marcadores de pluripotência. Estes modelos foram sujeitos a ensaios de funcionalidade onde mostraram a capacidade de “invasão cooperativa” que foi coincidente com uma produção aumentada de MMP-2. Para tornar mais claro o papel das células estaminais no potencial invasivo de células tumorais, uma câmara 3D de quimiotaxia foi desenvolvida para estudar as interações celulares observadas no microambiente tumoral onde as células estaminais e fibroblastos mostraram ter um papel determinante. Em última análise, esta tese permitiu explorar abordagens da engenharia biomédica de forma a contribuir para o conhecimento da área e abrir novas portas a serem exploradas no futuro

Unravelling the path to create a cell sheet-based model of skin scar-like tissue

Regardless of the advances in understanding the mechanisms and the pathophysiology behind skin deformities, scaring continues to be an unsolved clinical problem. The underlying wound healing process involves a series of key cells which play different key roles. Fibroblasts are known to suffer the influence of local biochemical (e.g TGF-B1) and biomechanical signaling upon a wound scenario leading to a phenotypical change into myofibroblasts. The latter enhance immature extracellular matrix (ECM) synthesis and generate tensional forces that leads to ECM reorganization. Certain skin pathologies (e.g hypertrophic scars) rise from a dysfunction of this underlying regulatory mechanism which in turn drives myofibroblast persistence in the wound. When trying to study the mechanisms behind scarring human ex vivo samples are many times scarce and most of the current in vitro systems rely on standard 2D cultures of keloid/hypertrophic scar fibroblasts. Taking all of this into consideration we propose the use of cell sheet technology to create an in vitro 3D scar model. Herein we report the effect of TGF-B1 in human dermal fibroblast cell sheets as the first step to attain cell sheets with a myofibroblast-like phenotype in which cells are embedded in a scar-like ECM. To further strengthen our concept we performed the stacking of pre-formed cell sheets generating a cohesive 3D scar-like tissue. Human dermal fibroblast (hDFbs) cell sheets were produced as previously described1, and stimulated with TGF-B1 (10ng/ml) over 7, 14 and 21 days. Following phenotype and ECM characterization, cell sheets were stacked in order to obtain a 3D structure composed of 2 or 3 cell-sheets. The analysis of key genes (q-PCR) and proteins (Western blot and immunocytochemistry) showed that hDFbs cell sheets, when stimulated with TGF-B1 present an increased expression of a-SMA, fibronectin (FN) ED- A and FN ED-B, characteristic of a myofibroblast-like phenotype. When looking into the expression of scar ECM-associated proteins, hDFbs cell sheets obtained in the presence of TGF-B1 produced higher amounts of fibronectin and collagen I. Stable 3D constructs with a noticeable level of integration after a total of 21 days of culture, were further created upon stacking of the cell sheets obtained after 7days of culture in the presence of TGF-B1. In conclusion, this work suggested that it is possible to promote the secretion of scar-like ECM in hDFbs cell sheets due to phenotypic changes into myofibroblast-like cells when stimulated with TGF-B1. Cohesive 3D scar-like tissue structures were obtained which opens the possibility to develop a highly accurate in vitro 3D scar model to study underlying cellular mechanisms involved in the wound healing deregulation. Grant IF/00945/2014 funded by FCT/MCTES, Project “NORTE-08-5369-FSE-000044”, funded by Programa Operacional Norte 2020 Fundo Social Europeu, and GENE2SKIN Twinning Project, Horizon 2020, funded by the European Commissioninfo:eu-repo/semantics/publishedVersio

In vitro 3D cell sheet-based model for unraveling scar pathophysiology

Fibroblasts are key players in the scarring process. In hypertrophic scars, fibroblasts suffer phenotypical changes into myofibroblasts persisting in the wound under the influence of local biochemical (TGFb1) and biomechanical signaling leading to enhanced immature extracellular matrix (ECM) synthesis. Benchtop models of hypertrophic scars rely on scarce human ex vivo samples or standard 2D cultures of hypertrophic scar fibroblasts. We therefore propose the use of human dermal fibroblast cell sheets (hDFbsCS) as the first step to attain cell sheets with a myofibroblast-like phenotype to generate cohesive in vitro 3D scar-like tissues. hDFbsCS were produced as previously described (Cerqueira, 2014), and stimulated with TGFb1 up to 21 days. Following phenotype and ECM characterization, 3 hDFbsCS were stacked to obtain a 3D structure. Gene and protein analysis showed that upon TGFb1 stimulation, hDFbsCS present a higher expression of aSMA, fibronectin EDA and EDB, characteristic of amyofibroblast-like phenotype. Regarding the expression of scar ECM-associated proteins, TGFb1 stimulated hDFbsCS produced increased fibronectin and collagen I. Upon stacking of hDFbsCS obtained after 7 days of culture in the presence of TGFb1, stable and integrated 3D constructs were obtained. This work suggests that it is possible to create cohesive 3D scar-like tissue structures from hDFbsCS opening the possibility to develop in vitro 3D scar models to study wound healing deregulation pathophysiology. Acknowledgments: Grant IF.00945.2014 and SFRH.BD. 119756.2016 (FCT MCTES), NORTE.08.5369.FSE.000044 (funded by Programa Operacional Norte 2020 Fundo Social Europeu), GENE2SKIN Twinning Project, Horizon 2020 (European Commission).Grant IF.00945.2014 and SFRH.BD.119756.2016 (FCT_MCTES), NORTE.08.5369.FSE.000044 (funded by Programa_Operacional_Norte_2020 Fundo Social Europeu), GENE2SKIN Twinning Project, Horizon_2020 (European Commission).info:eu-repo/semantics/publishedVersio

Exploring Past, Present and Future

UID/HIS/04209/2019 POCI-01-0145-FEDER-007460.publishersversionpublishe

Stem cell-containing hyaluronic acid-based spongy hydrogels for integrated diabetic wound healing

The detailed pathophysiology of diabetic foot ulcers is yet to be established and improved treatments are still required. We propose a strategy that directs inflammation, neovascularization, and neoinnervation of diabetic wounds. Aiming to potentiate a relevant secretome for nerve regeneration, stem cells were precultured in hyaluronic acid-based spongy hydrogels under neurogenic/standard media before transplantation into diabetic mice full-thickness wounds. Acellular spongy hydrogels and empty wounds were used as controls. Reepithelialization was attained 4 weeks after transplantation independently of the test groups, whereas a thicker and more differentiated epidermis was observed for the cellular spongy hydrogels. A switch from the inflammatory to the proliferative phase of wound healing was revealed for all the experimental groups 2 weeks after injury, but a significantly higher M2(CD163 þ )/M1(CD86 þ ) subtype ratio was observed in the neurogenic preconditioned group that also failed to promote neoinnervation. A higher number of intraepidermal nerve fibers were observed for the unconditioned group probably due to a more controlled transition from the inflammatory to the proliferative phase. Overall, stem cell-containing spongy hydrogels represent a promising approach to enhance diabetic wound healing by positively impacting re-epithelialization and by modulating the inflammatory response to promote a successful neoinnervation.The authors would like to acknowledge Gene2Skin Project (H2020-TWINN2015-692221) and Fundac¸a˜o para a Cieˆncia e Tecnologia for SFRH/BD/ 78025/2011 (LPdS), SFRH/BPD/96611/2013 (MTC), SFRH/BPD/101886/2014 (RPP), SFRH/BPD/101952/2014 (TCS) grants. Moreover, the authors would also like to acknowledge Teresa Oliveira for histology support, Andreia Carvalho for hASCs supply, Luca Gasperini for cell profiler analysis, and Manuela E. L. Lago and Carla M. Abreu for intraepidermal nerve fiber quantification.info:eu-repo/semantics/publishedVersio

Integrin-specific hydrogels for growth factor-free vasculogenesis

Integrin-binding biomaterials have been extensively evaluated for their capacity to enable de novo formation of capillary-like structures/vessels, ultimately supporting neovascularization in vivo. Yet, the role of integrins as vascular initiators in engineered materials is still not well understood. Here, we show that αvβ3 integrin-specific 3D matrices were able to retain PECAM1+ cells from the stromal vascular fraction (SVF) of adipose tissue, triggering vasculogenesis in vitro in the absence of extrinsic growth factors. Our results suggest that αvβ3-RGD-driven signaling in the formation of capillary-like structures prevents the activation of the caspase 8 pathway and activates the FAK/paxillin pathway, both responsible for endothelial cells (ECs) survival and migration. We also show that prevascularized αvβ3 integrin-specific constructs inosculate with the host vascular system fostering in vivo neovascularization. Overall, this work demonstrates the ability of the biomaterial to trigger vasculogenesis in an integrin-specific manner, by activating essential pathways for EC survival and migration within a self-regulatory growth factor microenvironment. This strategy represents an improvement to current vascularization routes for Tissue Engineering constructs, potentially enhancing their clinical applicability.The authors would like to acknowledge the financial support from the Consolidator Grant “ECM_INK” (ERC-2016-COG-726061) and the Starting Grant “CapBed” (ERC2018-STG-805411), to the FSE/POCH (Fundo Social Europeu através do Programa Operacional do Capital Humano) under the scope of the PD/169/2013, NORTE-08- 5369-FSE-000037 (H.R.M.), and to FCT/MCTES (Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia, e Ensino Superior) through the grants SFRH/BD/119756/2016 (D.B.R.), Ph.D. grant PD/BD/135252/2017 (S.F.R.) and IF/00347/ 2015 (R.P.P.)

Spongy-like hydrogels prevascularization with the adipose tissue vascular fraction delays cutaneous wound healing by sustaining inflammatory cell influx

In vitro prevascularization is one of the most explored approaches to foster engineered tissue vascularization. We previously demonstrated a benefit in tissue neovascularization by using integrin-specific biomaterials prevascularized by stromal vascular fraction (SVF) cells, which triggered vasculogenesis in the absence of extrinsic growth factors. SVF cells are also associated to biological processes important in cutaneous wound healing. Thus, we aimed to investigate whether in vitro construct prevascularization with SVF accelerates the healing cascade by fostering early vascularization vis-à-vis SVF seeding prior to implantation. Prevascularized constructs delayed re-epithelization of full-thickness mice wounds compared to both non-prevascularized and control (no SVF) groups. Our results suggest this delay is due to a persistent inflammation as indicated by a significantly lower M2(CD163+)/M1(CD86+) macrophage subtype ratio. Moreover, a slower transition from the inflammatory to the proliferative phase of the healing was confirmed by reduced extracellular matrix deposition and increased presence of thick collagen fibers from early time-points, suggesting the prevalence of fiber crosslinking in relation to neodeposition. Overall, while prevascularization potentiates inflammatory cell influx, which negatively impacts the cutaneous wound healing cascade, an effective wound healing was guaranteed in non-prevascularized SVF cell-containing spongy-like hydrogels confirming that the SVF can have enhanced efficacy.Authors would like to acknowledge the financial support from the Consolidator Grant “ECM_INK” (ERC-2016-COG-726061) and the Starting Grant “CapBed” (ERC-2018-STG-805411), to the FSE/POCH (Fundo Social Europeu através do Programa Operacional do Capital Humano) under the scope of the PD/169/2013, NORTE-08-5369-FSE-000037 (H.R.M.), and to FCT/MCTES (Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia, e Ensino Superior) through the grants SFRH/BD/119756/2016 (D.B.R.), PhD grant PD/BD/135252/2017 (S.F.R.) and IF/00347/2015 (R.P.P.). Authors would also like to acknowledge BioRender.com as a platform for image creation

Is the reason to switch relevant?

57763]. AS is supported by a doctoral grant from “Fundação para a Ciência e Tecnologia” (SFRH/BD/108246/2015).Background: To investigate whether the reason to discontinue the first TNF inhibitor (TNFi) affects the response to the second TNFi in axial spondyloarthritis (axSpA). Methods: Patients with axSpA from the Rheumatic Diseases Portuguese Register (ReumaPt), who discontinued their first TNFi and started the second TNFi between June 2008 and May 2018, were included. Response was assessed by the Ankylosing Spondylitis Disease Activity Score (ASDAS) clinically important improvement (ASDAS-CII), major important improvement (ASDAS-MI), low disease activity (ASDAS-LDA), and inactive disease (ASDAS-ID). The reason for discontinuation of the first TNFi was defined, according to ASDAS-CII as primary failure (no response ≤ 6 months), secondary failure (response ≤ 6 months but lost thereafter), adverse events, and others. The association between the reason for discontinuation of the first TNFi and response to the second TNFi over time was assessed in multivariable generalized equation (GEE) models. Results: In total, 193 patients were included. The reason for discontinuation of the first TNFi did not influence the response to the second TNFi, according to the ASDAS-CII. However, a difference was found with more stringent outcomes, e.g., there was a higher likelihood to achieve ASDAS-ID with the second TNFi for patients discontinuing the first TNFi due to secondary failure (OR 7.3 [95%CI 1.9; 27.7]), adverse events (OR 9.1 [2.5; 33.3]), or other reasons (OR 7.7 [1.6; 37.9]) compared to primary failure. Conclusion: Patients with axSpA with secondary failure to their first TNFi, compared to those with primary failure, have a better response to the second TNFi according to stringent outcomes.publishersversionpublishe

The impact of a cash transfer programme on tuberculosis treatment success rate: a quasi-experimental study in Brazil.

BACKGROUND: Evidence suggests that social protection policies such as Brazil's Bolsa Família Programme (BFP), a governmental conditional cash transfer, may play a role in tuberculosis (TB) elimination. However, study limitations hamper conclusions. This paper uses a quasi-experimental approach to more rigorously evaluate the effect of BFP on TB treatment success rate. METHODS: Propensity scores were estimated from a complete-case logistic regression using covariates from a linked data set, including the Brazil's TB notification system (SINAN), linked to the national registry of those in poverty (CadUnico) and the BFP payroll. RESULTS: The average effect of treatment on the treated was estimated as the difference in TB treatment success rate between matched groups (ie, the control and exposed patients, n=2167). Patients with TB receiving BFP showed a treatment success rate of 10.58 percentage points higher (95% CI 4.39 to 16.77) than patients with TB not receiving BFP. This association was robust to sensitivity analyses. CONCLUSIONS: This study further confirms a positive relationship between the provision of conditional cash transfers and TB treatment success rate. Further research is needed to understand how to enhance access to social protection so to optimise public health impact