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

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

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 < 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.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

Preliminary and Accelerated Stability Study of Melissa Officinalis Syrup Cultivated in the South of Tocantins

Melissa officinalis L. (Lamiaceae) is known as a true lemon balm and is popularly used in traditional medicine as a tranquilizer. The present research aimed to propose a source of syrup based on the tincture of M. officinalis and perform preliminary and accelerated stability tests, and microbiological evaluation. The leaves of M. officinalis were collected and after botanical identification, the raw material processing step was followed to obtain the dye. The tincture obtained was used to prepare the syrup based on M. Officinalis. The syrup was used as steps of physical-chemical and microbiological quality control. It was found that the M. Officinalis syrup, in the analysis of pH, organoleptic characteristics, density, volume, and microbiological assay, is by the consulted literature. The observed results indicate that the syrup of M. Officinalis is within the defined parameters, which guarantees the patient the efficacy and safety of the herbal medicine

Comparing Johnson's Sb and Weibull functions to model the diameter distribution of forest plantations through ALS data

The analysis of the diameter distribution is important for forest management since the knowledge of tree density and growing stock by diameter classes is essential to define management plans and to support operational decisions. The modeling of diameter distributions from airborne laser scanning (ALS) data has been performed through the two-parameterWeibull probability density function (PDF), but the more flexible PDF Johnson’s SB has never been tested for this purpose until now. This study evaluated the performance of the Johnson’s SB to predict the diameter distributions based on ALS data from two of the most common forest plantations in the northwest of the Iberian Peninsula (Eucalyptus globulus Labill. and Pinus radiata D. Don). The Weibull PDF was taken as a benchmark for the diameter distributions prediction and both PDFs were fitted with ALS data. The results show that the SB presented a comparable performance to the Weibull for both forest types. The SB presented a slightly better performance for the E. globulus, while theWeibull PDF had a small advantage when applied to the P. radiata data. The Johnson’s SB PDF is more flexible but also more sensitive to possible errors arising from the higher number of stand variables needed for the estimation of the PDF parametersinfo:eu-repo/semantics/publishedVersio

Generalized linear mixed models for the genetic evaluation of binary reproductive traits: a simulation study

Funda??o de Amparo ? Pesquisa do Estado de Minas Gerais (FAPEMIG)Empresa de Pesquisa Agropecu?ria de Minas Gerais (EPAMIG)Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq)Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES)The objective of this study was to evaluate the use of probit and logit link functions for the genetic evaluation of early pregnancy using simulated data. The following simulation/analysis structures were constructed: logit/logit, logit/probit, probit/logit, and probit/probit. The percentages of precocious females were 5, 10, 15, 20, 25 and 30% and were adjusted based on a change in the mean of the latent variable. The parametric heritability (h?) was 0.40. Simulation and genetic evaluation were implemented in the R software. Heritability estimates (h^2) were compared with h? using the mean squared error. Pearson correlations between predicted and true breeding values and the percentage of coincidence between true and predicted ranking, considering the 10% of bulls with the highest breeding values (TOP10) were calculated. The mean h^? values were under- and overestimated for all percentages of precocious females when logit/probit and probit/logit models used. In addition, the mean squared errors of these models were high when compared with those obtained with the probit/probit and logit/logit models. Considering (h^2), probit/probit and logit/logit were also superior to logit/probit and probit/logit, providing values close to the parametric heritability. Logit/probit and probit/logit presented low Pearson correlations, whereas the correlations obtained with probit/probit and logit/logit ranged from moderate to high. With respect to the TOP10 bulls, logit/probit and probit/logit presented much lower percentages than probit/probit and logit/logit. The genetic parameter estimates and predictions of breeding values of the animals obtained with the logit/logit and probit/probit models were similar. In contrast, the results obtained with probit/logit and logit/probit were not satisfactory. There is need to compare the estimation and prediction ability of logit and probit link functions

Ser ou não ser praxado? A teoria da ação planeada como modelo para compreender a participação dos caloiros na praxe académica

The main goal of this study was to investigate the predictors of the freshmen’s intentions to participate in academic hazing. With this goal, we used the theory of planned behavior (Azjen, 1991) which allows an analysis of the relative weight of attitudes, subjective norm, and perceived behavioral control in the intentions to participate in academic hazing. The freshmen who participated in this study (N = 353) filled in a questionnaire. The results supported the theory of planned behavior predictive power, with the attitude and perceived behavioral control as significant predictors of the freshmen’s intentions to participate in academic hazing. The subjective norm was not a significant predictor. The theoretical and social implications of these results are discussed.Com este estudo pretendemos investigar quais são os preditores da intenção de participação dos caloiros na praxe académica. Para tal, recorremos à teoria da ação planeada (Ajzen, 1991) que permite analisar o peso relativo das atitudes, da norma subjetiva, e do controlo comportamental percebido nas intenções de ser praxado. Os participantes, estudantes universitários (N = 353) a frequentar o 1º ano de uma Licenciatura, preencheram um questionário. Os resultados confirmaram o valor preditivo da teoria, tendo sido a atitude e o controlo comportamental percebido preditores significativos da intenção dos caloiros em serem praxados. A norma subjetiva não se mostrou um preditor significativo. As implicações teóricas e sociais destes resultados são discutidas