Role of the neurotrophic factor receptor RET in haematopoiesis

Tese de doutoramento, Ciências Biomédicas (Imunologia), Universidade de Lisboa, Faculdade de Medicina, 2013Haematopoiesis is a developmental process that ensures the generation of all blood cell lineages throughout life. As a consequence, this is a highly complex and dynamic developmental cascade subject to tight regulatory mechanisms. Thus, the study of novel molecular signals is critical to further understand how haematopoiesis operates to ensure the balance between cell lineage commitment, cell homeostasis and efficient haematopoietic responses to insults and disturbances. The tyrosine kinase RET is the receptor for the GDNF (glial cell line-derived neurotrophic factor) neurotrophic factor family (GDNF family ligands – GFLs). Productive RET signalling controls the development and maintenance of the enteric nervous system, kidneys and spermatogenesis. Interestingly, Ret expression was detected in haematopoietic cells and lymphoid organs and RET signalling was shown to regulate enteric lymphoid organogenesis. However the role of RET in haematopoiesis remains completely unexplored. Haematopoietic stem cells (HSCs) are at the onset of the developmental cascade that generate all blood cells, thus we initially investigated the role of RET in HSC function and how modulation of RET signalling can be used to control HSC responses. Finally, we investigated the role of RET in late stages of haematopoietic cell precursor differentiation into the T cell lineage. We found that the tyrosine kinase RET is critical to HSC survival and function. HSCs express RET signalling molecules and HSCs microenvironment provides RET ligands. Moreover Ret ablation leads to reduced HSC numbers and recruitment of quiescent cells into proliferation. Although RET null progenitors have normal differentiation potential, they exhibit impaired in vivo stress response and reconstitution potential. Remarkably RET downstream signalling results in p38/MAP kinase phosphorylation and CREB transcription factor activation, providing HSCs with critical surviving cues. In agreement, recue of Ret null progenitors was efficiently achieved in vivo by forcing the expression of RET downstream targets, Bcl2 or Bcl2l1. Thus, RET activation improves HSC survival and in vivo transplantation efficiency, unveiling exciting new possibilities in transplantation and HSC ex vivo expansion. In addition, our work demonstrates that HSC use neurotrophic factors to regulate and maintain their fitness. RET signalling molecules are also expressed in thymocytes, more specifically, we found their expression in CD4/CD8 double negative thymocytes (DN). Nevertheless, ablation of Ret or it co-receptors Gfra1 and Gfra2 had a minor impact in foetal thymopoiesis. In agreement, Ret conditional knockout mice had similar thymocyte development and fitness when compared to their WT counterparts. Thus, while RET signalling is critical to HSC function, it is dispensable for T cell development in vivo. Altogether, our work show that molecular mechanisms usually assigned to specific tissues, can be more widely used by unrelated cell types, such as haematopoietic stem cells. Our findings also illustrate how a same signalling pathway can be regulated to originate different cell responses. Unlike several neuronal populations that use GFLs depending on the specific expressed co-receptor, HSCs express multiple RET coreceptors and respond to GFLs in a redundant fashion. There is increasing evidence that nervous signals can control haematopoiesis, namely by regulating osteoblast and mesenchymal stem cell function in HSC niches. Herein, we show that neurons and HSC employ common regulatory mechanisms. Thus, our work paves the way to further studies employing neurotrophic factors in HSC expansion and transplantation protocols.O sistema hematopoiético, que inclui as células do sistema imunitário, é altamente complexo e dinâmico, e como tal, está sujeito a uma regulação apertada. Assim, o estudo dos mecanismos moleculares responsáveis pelo desenvolvimento de células hematopoiéticas é essencial para compreender a forma como este sistema funciona de modo a manter o equilíbrio entre o número de células necessárias em homeostasia e uma rápida resposta em situações de desequilíbrio. A tirosina cinase RET é o receptor para os factores neurotróficos da família do GDNF (glial cell line-derived neurotrophic factor) (GDNF family ligands – GFLs) e tem uma função crucial no desenvolvimento e manutenção do sistema nervoso, no desenvolvimento embrionário do rim e na espermatogénese. Curiosamente, a expressão de Ret já foi detectada em várias populações de células hematopoiéticas e em órgãos linfoides primários como o fígado fetal, a medula óssea e o timo. Para além disso, foi também demonstrado um papel crucial da sinalização por RET em células hematopoiéticas envolvidas na organogénese das placas de Peyer no intestino durante a vida embrionária. No entanto, apesar de a expressão de RET ter sido identificada em várias populações celulares, uma possível função de RET no desenvolvimento ou função de células hematopoiéticas não é ainda conhecida. As células estaminais hematopoiéticas, pelas suas capacidades de autorrenovação e proliferação, são a base do programa de desenvolvimento e diferenciação que origina todas as células do sangue. Por este motivo, é crucial compreender os complexos mecanismos que regulam as células estaminais hematopoiéticas. Assim, no laboratório estamos particularmente interessados no papel de RET na função das células estaminais hematopoiéticas e no modo como a sinalização por RET pode ser usada de forma a modular a sua resposta em situações de desequilíbrio como a transplantação. Uma vez que foi proposto um efeito do ligando de RET GDNF na sobrevivência de timócitos in vitro, estamos também interessados em compreender a função de RET no desenvolvimento de células T in vivo. O timo é responsável pela produção de todas as populações de células T, que são essenciais para respostas imunitárias eficientes. Como tal é indispensável perceber de que modo o microambiente tímico fornece uma complexa rede de sinais que levam ao desenvolvimento de células T a partir de precursores da medula óssea. No laboratório descobrimos que a tirosina cinase RET tem uma função crítica na sobrevivência e função das células estaminais hematopoiéticas. Estas expressam a maquinaria de sinalização por RET, que inclui o receptor RET e os seus co-receptores, enquanto o microambiente onde as células estaminais hematopoéticas se encontram providencia os ligandos de RET necessários. Para além disso, a ablação de Ret leva à redução do número de células estaminais hematopoiéticas e ao recrutamento de células quiescentes para o estado proliferativo. Apesar de os progenitores hematopoiéticos deficientes em RET terem um potencial de diferenciação normal, apresentam uma fraca resposta ao stresse in vivo e um potencial de reconstituição reduzido. Importante, a sinalização de RET fornece factores de sobrevivência às células estaminais hematopoiéticas, por a jusante resultar na fosforilação da cinase p38/MAPK e na activação do factor de transcrição CREB. Em concordância, a sobreexpressão dos genes alvo a jusante de RET, Bcl2 ou Bcl2l1, resgata in vivo a função hematopoiética de progenitores deficientes em Ret, aumentado o seu potencial te transplantação. Na verdade, a activação de RET aumenta a sobrevivência de células estaminais hematopoiéticas e a sua eficiência de transplantação in vivo, revelando novas possibilidades de intervenção em terapias de transplantação e expansão ex vivo de células estaminais hematopoiéticas. Assim, o nosso trabalho mostra que factores neurotróficos presentes no nicho das células estaminais hematopoiéticas regulam a sua função através do receptor RET. Embora tenhamos confirmado que as moléculas envolvidas na sinalização de RET são expressas no timo, especialmente na população de timócitos negativa para os coreceptores CD4 e CD8 (DN), a remoção de Ret ou dos seus co-receptores Gfra1 ou Gfra2 não afecta a timopoiese fetal. Concordantemente, animais adultos com eliminação condicional de Ret em timócitos revelam que a capacidade de desenvolvimento de timócitos deficientes em Ret é semelhante à dos controlos selvagens. Do mesmo modo mutações que conferem um ganho de função no receptor RET não influenciam o desenvolvimento tímico. Assim, apesar de a sinalização por RET poder fornecer sinais de sobrevivência a timócitos, esta é dispensável para o desenvolvimento de células T in vivo, mesmo em condições de competição entre progenitores deficientes ou competentes em Ret. No seu conjunto, o nosso trabalho demonstra que mecanismos moleculares geralmente atribuídos a tecidos específicos, podem ser mais amplamente utilizado por tipos de células não relacionadas, tais como células estaminais hematopoiéticas. Os nossos resultados também ilustram como uma mesma via de sinalização pode ser 3 regulada de forma a originar diferentes respostas celulares. Contrariamente a diversas populações de neurónios, que usam GFLs específicos dependendo do coreceptor que expressam, as HSCs expressam múltiplos coreceptores de RET respondem aos GFLs de forma redundante. Existem cada vez mais evidências de que os sinais provenientes do sistema nervoso podem controlar hematopoiese, nomeadamente através da regulação da função dos osteoblastos e células estaminais mesenquimais presentes nos nichos das HSCs. Surpreendentemente, nós mostramos que os neurônios e as HSCs utilizam mecanismos comuns de regulação.. Deste modo, o nosso trabalho abre caminho a novos estudos na utilização factores neurotróficos em protocolos de expansão e transplantação de HSCs

Redução dos níveis de proteína bruta nas dietas pré-iniciais e iniciais de frangos de corte

O presente trabalho, teve por objetivo estudar o efeito da redução do nível de proteína bruta das rações pré-iniciais e iniciais de frangos de corte, mantendo-se constante os níveis de aminoácidos essencias, sem comprometer o desempenho zootécnico, com reflexos sobre o custo final das rações. O estudo foi conduzido na Granja Experimental de Corte, Fazenda do Glória, Universidade Federal de Uberlândia. O experimento foi projetado num delineamento inteiramente casualisado, composto de 3 tratamentos e 6 repetições, sendo envolvidas 540 aves mistas. As rações pré-iniciais (300 g/ave) e iniciais (900 g/ave), foram produzidas a base de sorgo grão moído, farelo de soja, óleo degomado de soja, fosfato bicálcico, calcário calcítico, cloreto de sódio, aminoácidos sintéticos (DL-metionina, L-lisina e L-treonina), vitamínico e minerais. Os tratamentos foram assim distribuídos: Tratamento A: Ração A, Tratamento B: Ração A com menos 0,5 de proteína bruta e Tratamento C: Ração A com menos 1,0 de proteína bruta. As variáveis analisadas aos 7 e 21 dias foram: consumo médio de ração, peso vivo médio, conversão alimentar e viabilidade. Aos 7 dias de idade para as variáveis consumo médio de ração, peso vivo e viabilidade, não observou-se diferenças estatísticas entre os respectivos tratamentos. Já em relação a conversão alimentar, o tratamento C demonstrou melhor resultado que o tratamento A, não diferindo estatisticamente do tratamento B. Na análise efetuada aos 21 dias de idade observou-se que não houve diferenças estatísticas entre os tratamentos. Pode-se concluir que é possível reduzir os níveis de proteína bruta da dieta de frangos de corte, mantendo-se constante os níveis de aminoácidos, sem comprometer o rendimento das aves até os 21 dias de idade, podendo-se utilizar de rações com menor custo

A Rare Cause of Abdominal Pain: IgG4-Related Sclerosing Mesenteritis

A 67-year-old man with previous cardiovascular disease was referred to our consultation due to a 5-month history of recurrent epigastric pain. Esophagogastroduodenoscopy and full blood workup presented no alterations. CT scan showed an irregularly shaped mass at the root of the mesentery, measuring 40x25x47mm, with spiculated contours and retractile behaviour (a). Simultaneous densification of the adjacent fat and infracentimetric ganglionic formations scattered throughout the mesentery were shown. Surgical biopsy revealed extensive storiform fibrosclerosis, with the presence of interstitial lymphoplasmocytic infiltrate and obliterative phlebitis (b); the plasma cells had mostly IgG expression, with IgG4:IgG ratio >40% (c), accounting for more than 30- 40 IgG4 plasma cells per field. The serum IgG4 level was 137mg/dL. A diagnosis of IgG4-related sclerosing mesenteritis was made, without other organ involvement. Prednisolone (0.6mg/kg/d) improved partially the abdominal pain, so steroid sparing strategy with off-label rituximab was associated. Due to its low prevalence, the understanding of this entity is scarce, and its diagnosis is challenging. Unlike other manifestations of IgG4-related disease, the intra-abdominal disease is identified in later stages, due to unspecific symptoms. This case aims to raise awareness about this condition as a differential diagnosis of abdominal pain.info:eu-repo/semantics/publishedVersio

IL-9 expression by invariant NKT cells is not imprinted during thymic development

Copyright © 2015 by The American Association of Immunologists, Inc. All rights reservedInvariant NKT (iNKT) cell thymic development can lead to distinct committed effector lineages, namely NKT1, NKT2, and NKT17. However, following identification of IL-9-producing iNKT cells involved in mucosal inflammation, their development remains unaddressed. In this study, we report that although thymic iNKT cells from naive mice do not express IL-9, iNKT cell activation in the presence of TGF-β and IL-4 induces IL-9 secretion in murine and human iNKT cells. Acquisition of IL-9 production was observed in different iNKT subsets defined by CD4, NK1.1, and neuropilin-1, indicating that distinct functional subpopulations are receptive to IL-9 polarization. Transcription factor expression kinetics suggest that regulatory mechanisms of IL-9 expression are shared by iNKT and CD4 T cells, with Irf4 and Batf deficiency deeply affecting IL-9 production. Importantly, adoptive transfer of an enriched IL-9(+) iNKT cell population leads to exacerbated allergic inflammation in the airways upon intranasal immunization with house dust mite, confirming the ability of IL-9-producing iNKT cells to mediate proinflammatory effects in vivo, as previously reported. Taken together, our data show that peripheral iNKT cells retain the capacity of shaping their function in response to environmental cues, namely TGF-β and IL-4, adopting an IL-9-producing NKT cell phenotype able to mediate proinflammatory effects in vivo, namely granulocyte and mast cell recruitment to the lungs.This work was supported by Fundação Para a Ciência e Tecnologia, Portugal Grants PTDC/SAU-TOX/114424/2009 and HMSP-ICT/0034/2013 and the Novo Nordisk/European Foundation for the Study of Diabetes (to L.G.), as well as by grants from the Fundação Para a Ciência e Tecnologia, Portugal, the European Molecular Biology Organization, the European Research Council, and the U.S. National Blood Foundation (to H.V.-F.). A.A.-D., C.F.A., D.F.-P., and M.M. were supported by fellowships from the Fundação Para a Ciência e Tecnologia, Portugal.info:eu-repo/semantics/publishedVersio

Extensively drug-resistant Pseudomonas aeruginosa: clinical features and treatment with Ceftazidime-Avibactam and Ceftolozane-Tazobactam in a tertiary care university hospital center in Portugal: a cross-sectional and retrospective observational study

Copyright © 2024 Mendes Pedro, Paulo, Santos, Fonseca, Melo Cristino, Pereira and Caneiras. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Introduction: Extensively drug-resistant Pseudomonas aeruginosa (XDR-PA) is a growing concern due to its increasing incidence, limited therapeutic options, limited data on the optimal treatment, and high mortality rates. The study aimed to characterize the population, the outcome and the microbiological characteristics of XDR-PA identified in a Portuguese university hospital center. Methods: All XDR-PA isolates between January 2019 and December 2021 were identified. XDR-PA was defined as resistance to piperacillin-tazobactam, third and fourth generation cephalosporins, carbapenems, aminoglycosides and fluoroquinolones. A retrospective analysis of the medical records was performed. Results: One hundred seventy-eight individual episodes among 130 patients with XDR-PA detection were identified. The most common sources of infection were respiratory (32%) and urinary tracts (30%), although skin and soft tissue infections (18%) and primary bacteremia (14%) were also prevalent. Colonization was admitted in 64 cases. Several patients had risk factors for complicated infections, most notably immunosuppression, structural lung abnormalities, major surgery, hemodialysis or foreign intravascular or urinary devices. XDR-PA identification was more frequent in male patients with an average age of 64.3 ± 17.5 years. One non-susceptibility to colistin was reported. Only 12.4% were susceptible to aztreonam. Ceftazidime-avibactam (CZA) was susceptible in 71.5% of the tested isolates. Ceftolozane-tazobactam (C/T) was susceptible in 77.5% of the tested isolates. Antibiotic regimens with XDR-PA coverage were reserved for patients with declared infection, except to cystic fibrosis. The most frequently administered antibiotics were colistin (41 cases), CZA (39 cases), and C/T (16 cases). When combination therapy was used, CZA plus colistin was preferred. The global mortality rate among infected patients was 35.1%, significantly higher in those with hematologic malignancy (50.0%, p < 0.05), followed by the ones with bacteremia (44.4%, p < 0.05) and those medicated with colistin (39.0%, p < 0.05), especially the ones with respiratory infections (60.0%). Among patients treated with CZA or C/T, the mortality rate seemed to be lower. Discussion: XDR-PA infections can be severe and difficult to treat, with a high mortality rate. Even though colistin seems to be a viable option, it is likely less safe and efficient than CZA and C/T. To the best of the authors' knowledge, this is the first description of the clinical infection characteristics and treatment of XDR-PA in Portugal.info:eu-repo/semantics/publishedVersio

VIDEOAULAS NO PROCESSO DE ENSINO-APRENDIZAGEM DE QUÍMICA NO ENSINO MÉDIO

O presente artigo tem como objetivo o uso de vídeos em sala de aula, suas aplicações e como o mesmo pode ser de fácil utilidade nas aulas de química. Bem como identificar o impacto causado pelo uso da tecnologia em sala de aula. A abordagem metodológica adotada foi de natureza bibliográfica, exploratória e qualitativa, usando como estratégia um estudo de caso assim como a elaboração de um vídeo com o assunto de Propriedades Coligativas realizado pelos discentes do segundo ano da Escola Estadual João Manoel Pessoa, na cidade de Itajá/RN. A elaboração e a aplicação dos vídeos em sala, possibilitou aos discentes experimentarem diferentes vertentes nos diversos contextos dos assuntos de química, tornando assim, a aprendizagem mais significativa e diferenciada

Extensively drug-resistant Pseudomonas aeruginosa: clinical features and treatment with ceftazidime/avibactam and ceftolozane/tazobactam in a tertiary care university hospital center in Portugal – A cross-sectional and retrospective observational study

IntroductionExtensively drug-resistant Pseudomonas aeruginosa (XDR-PA) is a growing concern due to its increasing incidence, limited therapeutic options, limited data on the optimal treatment, and high mortality rates. The study aimed to characterize the population, the outcome and the microbiological characteristics of XDR-PA identified in a Portuguese university hospital center.MethodsAll XDR-PA isolates between January 2019 and December 2021 were identified. XDR-PA was defined as resistance to piperacillin-tazobactam, third and fourth generation cephalosporins, carbapenems, aminoglycosides and fluoroquinolones. A retrospective analysis of the medical records was performed.ResultsOne hundred seventy-eight individual episodes among 130 patients with XDR-PA detection were identified. The most common sources of infection were respiratory (32%) and urinary tracts (30%), although skin and soft tissue infections (18%) and primary bacteremia (14%) were also prevalent. Colonization was admitted in 64 cases. Several patients had risk factors for complicated infections, most notably immunosuppression, structural lung abnormalities, major surgery, hemodialysis or foreign intravascular or urinary devices. XDR-PA identification was more frequent in male patients with an average age of 64.3 ± 17.5 years. One non-susceptibility to colistin was reported. Only 12.4% were susceptible to aztreonam. Ceftazidime-avibactam (CZA) was susceptible in 71.5% of the tested isolates. Ceftolozane-tazobactam (C/T) was susceptible in 77.5% of the tested isolates. Antibiotic regimens with XDR-PA coverage were reserved for patients with declared infection, except to cystic fibrosis. The most frequently administered antibiotics were colistin (41 cases), CZA (39 cases), and C/T (16 cases). When combination therapy was used, CZA plus colistin was preferred. The global mortality rate among infected patients was 35.1%, significantly higher in those with hematologic malignancy (50.0%, p &lt; 0.05), followed by the ones with bacteremia (44.4%, p &lt; 0.05) and those medicated with colistin (39.0%, p &lt; 0.05), especially the ones with respiratory infections (60.0%). Among patients treated with CZA or C/T, the mortality rate seemed to be lower.DiscussionXDR-PA infections can be severe and difficult to treat, with a high mortality rate. Even though colistin seems to be a viable option, it is likely less safe and efficient than CZA and C/T. To the best of the authors’ knowledge, this is the first description of the clinical infection characteristics and treatment of XDR-PA in Portugal

Hospital-acquired intestinal toxemia botulism in a newly diagnosed adult colon cancer patient

This manuscript reports a case of intestinal toxemia botulism in an adult with recently diagnosed metastatic colon cancer in whom botulism symptoms began 23 days after hospital admission. Representing the rarest form of botulism presentation in clinical practice, this infectious disease may have developed due to a cluster of predisposing factors that favored Clostridium botulinum colonization and the endogenous production of neurotoxins, among which are previous use of broad-spectrum antibiotics and colon changes related to the development of the neoplasia. This case highlights the importance of considering intestinal toxemia botulism in the differential diagnosis of a patient presenting with symmetrical descending flaccid paralysis, since immediate treatment with botulinum antitoxin may improve clinical outcomes