Functional and metabolic characterization of antitumour macrophages

Tese de mestrado, Oncobiologia, Universidade de Lisboa, Faculdade de Medicina, 2021O microambiente tumoral é um ecossistema muito heterogéneo, constituído não apenas por células tumorais mas também por matriz extracelular, fibroblastos e ainda células do sistema imunitário, tanto da linhagem mieloide como da linfoide. Estas células do nosso sistema de defesa possuem a capacidade natural de vigiar e eliminar tumores em desenvolvimento, principalmente por meio das propriedades citotóxicas das células T. No entanto, as células imunológicas muitas vezes não conseguem controlar o crescimento desregulado dos tumores devido a mecanismos de evasão – por eles mesmos induzidos – que surgem e se desenvolvem ao longo da carcinogénese, prejudicando a imunidade antitumoral e comprometendo a sobrevivência do hospedeiro. Com efeito, à medida que o tumor se desenvolve, as próprias células malignas educam o estroma que as rodeia para superar os obstáculos da vigilância imunológica, tanto por meio de fatores solúveis imunossupressores, como por comunicações célula a célula inibidoras do sistema imunitário ou ainda através de competição metabólica por nutrientes. Estes factores, todos em conjunto, impedem a erradicação do cancro por meio do sistema imunitário. Entre os elementos do microambiente tumoral que são directa ou indirectamente modulados pelas células malignas, destacam-se as células mieloides por geralmente constituírem a população imunitária mais representativa dos infiltrados imunes e, mais ainda, pela plasticidade funcional que demonstram, podendo exercer funções tanto antitumorais como protumorais, de acordo com os estímulos que recebem nos tecidos em que se encontram. Com isto em mente, verificámos que tem sido dada uma atenção desproporcional entre as células mieloides que promovem o crescimento de tumores (protumorais) em relação às células que os tentam combater (antitumorais). Por isso, estamos convencidos de que mais esforços devem ser feitos para perceber como é que podemos aumentar a atividade protetora das células mieloides no contexto do cancro. O conhecimento dos mecanismos utilizados pelo nosso sistema de defesas sempre foi fundamental ao longo da história para o desenho de terapias eficientes, em qualquer contexto clínico. Assim, o conhecimento biológico e consequente manipulação terapêutica das células mieloides podem colaborar com as terapias existentes contra o cancro, por exemplo a quimio- e a imunoterapia, para melhorar os cuidados de saúde e a sobrevivência dos pacientes oncológicos. No sentido de estudar o potencial antitumoral das células mieloides, neste trabalho utilizámos um modelo ortotópico de células tumorais mamárias de ratinho (E0771) que nos permite estudar as actividades antitumorais das células mieloides in vivo. Tirando partido da capacidade inerente que estas células possuem de responder a agentes de maturação, como ligandos dos receptores Toll-like receptor (TLR) e moléculas co-estimuladoras, pudemos demonstrar nesta tese que a injecção intratumoral de um ligando do TLR3 (Poly I:C) em combinação com um agonista da molécula co-estimuladora CD40 (tratamento MCT) foi capaz de induzir remissão total dos tumores implantados, na esmagadora maioria dos ratinhos tratados. Demonstrando posteriormente que este tratamento combinatório não é prejudicial, em si mesmo, para as células tumorais, concluímos que o efeito alcançado dever-se-ia ao impacto do tratamento no microambiente tumoral. Com o objectivo de dissecar os mecanismos subjacentes ao efeito marcante que alcançámos com este tratamento e tendo em consideração a capacidade inerente que as células mieloides têm para responder ao mesmo, eliminámos os macrófagos selectivamente e reparámos que o tratamento perdeu a sua eficácia. Concluímos, assim, que o efeito terapêutico era dependente da ação dos macrófagos. Analisando de seguida os infiltrados imunitários após o tratamento, observámos que os macrófagos associados ao tumor (TAMs) sobre expressaram duas moléculas pro-inflamatórias com propriedades antitumorais conhecidas, a saber TNF e iNOS, por comparação com os ratinhos não tratados. Para estudar o papel que estas moléculas poderiam ter na resposta antitumoral mediada pelos macrófagos, utilizámos um sistema in vitro de macrófagos derivados da medula óssea de ratinhos. Após a diferenciação, tratámo-los com diferentes estímulos para podermos comparar o comportamento dos macrófagos estimulados com o MCT com macrófagos antitumorais (M1) e protumorais (M2a e M2c). Verificámos, em primeiro lugar, que os macrófagos MCT sobre expressaram igualmente TNF e iNOS, dando credibilidade à nossa metodologia. De seguida, observámos que o sobrenadante (SN) dos macrófagos MCT era capaz de induzir morte das células E0771, por semelhança com o SN dos M1 e por diferença com o SN dos M2a e M2c. Para perceber se este efeito se devia à produção de TNF, utilizámos o mesmo sistema em cima descrito com o suplemento de anticorpos monoclonais que bloqueavam a ação do TNF. No entanto, a presença do anticorpo não foi capaz de aumentar viabilidade celular das células E0771. Para estudar a importância desta molécula in vivo, injectámos os mesmos anticorpos bloqueadores do TNF concomitantemente com o tratamento MCT. Em sintonia com os resultados obtidos in vitro, não registámos qualquer diferença na eficácia do tratamento. Como abordagem alternativa, utilizámos clones das células E0771 resistentes à ação do TNF. Após a transplantação ortotópica destas células e da injecção do MCT, reparámos que o tratamento foi igualmente eficaz. Estes resultados, no seu conjunto, levaram-nos a concluir que o TNF não tem um papel importante enquanto mediador da resposta antitumoral dos macrófagos. Procurámos, então, perceber se a expressão de iNOS poderia ser responsável pelas propriedades antitumorais dos macrófagos estimulados com o MCT. Aproveitando ratinhos geneticamente modificados para não expressar iNOS (ratinhos iNOS-ko), diferenciámos macrófagos in vitro e tratámo-los como em cima está descrito. Interessantemente, observámos que na ausência da expressão de iNOS as propriedades antitumorais dos SN dos macrófagos MCT estavam significativamente reduzidas, sugerindo que o MCT é capaz de induzir a expressão de iNOS em macrófagos para mediar a sua resposta antitumoral. Impulsionados pela caracterização funcional dos macrófagos antitumorais MCT, e sabendo que o metabolismo celular pode ditar o resultado funcional das células imunes, investigámos os requisitos metabólicos dos macrófagos MCT. Para nossa surpresa, descobrimos que eles diferiam dos macrófagos M1, que eram marcadamente glicolíticos, e que, ao invés disso, exibiam uma dependência mitocondrial semelhante à dos macrófagos M2, perfil metabólico este que se pensa ser ajustado às características nutricionais do microambiente tumoral. Essa semelhança refletiu-se depois num conteúdo celular de ATP semelhante entre os macrófagos MCT e M2. Dadas estas semelhanças, perguntámo-nos quais seriam os substratos metabólicos que suportavam o metabolismo mitocondrial dos macrófagos MCT. Curiosamente, observámos que os macrófagos MCT eram capazes de captar glicose de forma semelhante aos M1 e que possuíam um conteúdo lipídico distinto dos M2. Juntando a evidência de que os macrófagos M1 tinham a maior capacidade de acidificar o meio de entre todos os macrófagos, concluímos que os macrófagos MCT não usam os lípidos como fonte de energia, como fazem os macrófagos M2, mas antes desviam o piruvato derivado da glicose para alimentar o TCA e o metabolismo mitocondrial. Para nossa surpresa, observámos que os macrófagos MCT apresentaram a menor massa mitocondrial e o menor potencial de membrana mitocondrial quando comparado com os outros fenótipos de macrófagos (M0, M1 e M2a). Futuros estudos mais detalhados permitir-nos-ão, por um lado, compreender a biologia das mitocôndrias dos macrófagos e, por outro lado, desvendar se e como é que as mitocôndrias orquestram as funções antitumorais de macrófagos MCT. Em conclusão, o trabalho aqui desenvolvido demonstra que o tratamento MCT induz macrófagos antitumorais que expressam iNOS e que apresentam um metabolismo marcadamente dependente da mitocôndria. Acreditamos que estes estudos promissores estabelecem as bases para o desenvolvimento de estratégias terapêuticas que abranjam a reprogramação funcional e metabólica dos TAMs, com vista à sua combinação com outras terapias contra o cancro.The tumour microenvironment (TME) is a heterogeneous ecosystem populated by myeloid and lymphoid immune cells that have the natural capacity to patrol and eliminate nascent tumours, mainly through the cytotoxic properties of T cells. However, immune cells often fail to control tumour growth due to escape mechanisms induced by tumour cells themselves that halt antitumour immunity and ultimately prevent tumour eradication. These include immunosuppressive soluble factors, deleterious cell to cell interactions and metabolic competition for nutrients. Among the cellular participants of the TME modulated by tumour cells, the myeloid compartment has a prominent role as they can exert antitumoural or protumoural functions according to the surrounding environmental cues. With this is mind, we believe there has been a disproportionate attention on protumour over antitumour myeloid cell functions, and that more efforts should be put on understanding how to enhance the protective activity of myeloid cells in the context of cancer. In this thesis, we took advantage of myeloid cell inherent capacity to respond to maturing agents, such as TLR ligands and co-stimulatory agonists, to study their antitumour potential. Using the E0771 TNBC orthotopic mouse model, we observed that the injection of TLR3 ligand plus an anti-CD40 agonistic mAb (Myeloid-Cell Treatment; MCT hereafter) was able to induce tumour regression in the vast majority of treated mice. The prevention of this regression by clodronate-encapsulated liposomes suggested a key antitumour role for macrophages. Furthermore, MCT-activated tumour-associated macrophages (TAMs) overexpressed TNF and iNOS, which are known tumoricidal molecules, when compared to the respective controls. Taking advantage of in vitro bone marrow-derived macrophages (BMDMs) to dissect the contributions of each molecule, we first observed that the supernatant derived from MCT-stimulated BMDMs increased E0771 cell death, demonstrating their antitumour properties. BMDMs also presented increased expression of TNF and iNOS. However, in vivo and in vitro studies with anti-TNF blocking antibodies suggested a neglectable role of TNF in MCT-induced antitumour immunity. Together with in vivo experiments with an TNF-resistant E0771 clone, we excluded TNF as the soluble mediator of antitumour MCT-TAMs in this model. Finally, iNOS expression in MCT-BMDMs was found critical for the decreased tumour cell viability in vitro, thus suggesting MCT programmes TAMs with iNOS-dependent antitumour properties. Given that cellular metabolism can dictate the functional outcome of immune cells, we then investigated the metabolic requirements of MCT BMDMs. To our surprise, we found that MCT BMDMs differed from pro-inflammatory/antitumoural in vitro-differentiated M1 macrophages, which were markedly glycolytic, as they instead displayed a mitochondrial dependency that was similar to anti inflammatory/protumoural M2 macrophages, which reflected on comparable ATP contents in MCT and M2 BMDMs. These data contradicted previous reports that linked glucose oxidation with antitumour functions, and led us to investigate the metabolic intermediates that supported MCT-BMDMs mitochondrial metabolism. We observed MCT-BMDMs had similar glucose uptake compared to M1, and distinct lipid content when compared to M2 BMDMs. Collectively with the evidence that M1 BMDMs had the highest capacity to acidify the medium, we concluded that MCT BMDMs presumably divert glucose-derived pyruvate to feed into the TCA and support mitochondrial metabolism. To our surprise, MCT BMDMs displayed the lower mitochondrial mass and potential when compared to all other BMDMs phenotypes (M0, M1 and M2a). Further studies will be needed, on one hand, to understand BMDMs mitochondrial biology and, on the other hand, to unravel if and how mitochondria orchestrate antitumour functions of MCT-stimulated macrophages. In conclusion, our data reveal that MCT effectively induces iNOS-expressing antitumour macrophages that feature a mitochondrial metabolism, presumably fitted to the nutrient availability of the TME. We believe this thesis lays the groundwork for the design of therapeutic strategies that encompass functional and metabolic reprogramming of TAMs. Harnessing the myeloid compartment therapeutically could synergize with standard and emerging anticancer therapies to improve oncologic patient healthcare and survival

Bringing macrophages to the frontline against cancer: current Immunotherapies targeting macrophages

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).Macrophages are found in all tissues and display outstanding functional diversity. From embryo to birth and throughout adult life, they play critical roles in development, homeostasis, tissue repair, immunity, and, importantly, in the control of cancer growth. In this review, we will briefly detail the multi-functional, protumoral, and antitumoral roles of macrophages in the tumor microenvironment. Our objective is to focus on the ever-growing therapeutic opportunities, with promising preclinical and clinical results developed in recent years, to modulate the contribution of macrophages in oncologic diseases. While the majority of cancer immunotherapies target T cells, we believe that macrophages have a promising therapeutic potential as tumoricidal effectors and in mobilizing their surroundings towards antitumor immunity to efficiently limit cancer progression.This work was supported by an iMM-Laço research grant and the Fundacão para a Ciência e Tecnologia through a research grant (PTDC/MED-IMU/30948/2017) and a personal fellowship (CEECIND/00697/2018) received by K.S. and a PhD fellowship (SFRH/BD/144792/2019) to C.J. This work was also kindly backed by the COST Action BM1404 Mye-EUNITER (http://www.mye-euniter.eu accessed on 26 November 2018). COST is supported by the EU Framework Program Horizon 2020.info:eu-repo/semantics/publishedVersio

AVALIAÇÃO TECNOLÓGICA DA EXTRAÇÃO ALCOÓLICA NO PROCESSAMENTO DE LICOR DE BANANA

Neste trabalho objetivou-se estabelecer a proporção de banana, a composição da solução extratora, bem como o tempo de extração para produzir licor com qualidade. Efetuou-se experimento fatorial com três quantidades de polpa de banana (500, 800 e 1100 g de banana para produção de 4 L de licor) e dois teores alcoólicos da solução extratora (70 e 95°GL). O processo de extração foi estudado durante 21 dias em que se analisaram as coordenadas de cor, pH, teor de sólidos solúveis e densidade a 20°C do extrato. O licor jovem foi caracterizado e submetido à análise sensorial após 45 dias de envelhecimento. O tempo de 16 dias mostrou-se suficiente para finalizar a etapa de extração, evidenciando que o tempo de infusão deve ser de 15 a 16 dias. A solução extratora com teor alcoólico de 95°GL apresentou melhores resultados, principalmente no que se refere à cor final do produto e facilidade na filtração. Como não houve diferença significativa entre as quantidades de polpa de banana optouse pela menor proporção em razão de fatores econômicos. Portanto, a utilização de 500 g de polpa de banana é suficiente para produzir 4 L de licor

ATLANTIC EPIPHYTES: a data set of vascular and non-vascular epiphyte plants and lichens from the Atlantic Forest

Epiphytes are hyper-diverse and one of the frequently undervalued life forms in plant surveys and biodiversity inventories. Epiphytes of the Atlantic Forest, one of the most endangered ecosystems in the world, have high endemism and radiated recently in the Pliocene. We aimed to (1) compile an extensive Atlantic Forest data set on vascular, non-vascular plants (including hemiepiphytes), and lichen epiphyte species occurrence and abundance; (2) describe the epiphyte distribution in the Atlantic Forest, in order to indicate future sampling efforts. Our work presents the first epiphyte data set with information on abundance and occurrence of epiphyte phorophyte species. All data compiled here come from three main sources provided by the authors: published sources (comprising peer-reviewed articles, books, and theses), unpublished data, and herbarium data. We compiled a data set composed of 2,095 species, from 89,270 holo/hemiepiphyte records, in the Atlantic Forest of Brazil, Argentina, Paraguay, and Uruguay, recorded from 1824 to early 2018. Most of the records were from qualitative data (occurrence only, 88%), well distributed throughout the Atlantic Forest. For quantitative records, the most common sampling method was individual trees (71%), followed by plot sampling (19%), and transect sampling (10%). Angiosperms (81%) were the most frequently registered group, and Bromeliaceae and Orchidaceae were the families with the greatest number of records (27,272 and 21,945, respectively). Ferns and Lycophytes presented fewer records than Angiosperms, and Polypodiaceae were the most recorded family, and more concentrated in the Southern and Southeastern regions. Data on non-vascular plants and lichens were scarce, with a few disjunct records concentrated in the Northeastern region of the Atlantic Forest. For all non-vascular plant records, Lejeuneaceae, a family of liverworts, was the most recorded family. We hope that our effort to organize scattered epiphyte data help advance the knowledge of epiphyte ecology, as well as our understanding of macroecological and biogeographical patterns in the Atlantic Forest. No copyright restrictions are associated with the data set. Please cite this Ecology Data Paper if the data are used in publication and teaching events. © 2019 The Authors. Ecology © 2019 The Ecological Society of Americ

Phenolic composition of the berry parts of hybrid grape cultivar BRS Violeta (BRS Rubea × IAC 1398-21) using HPLC–DAD–ESI-MS/MS

The grape is considered a major source of phenolic compounds when compared to other fruits and vegetables, however, there are many cultivars with distinct characteristics directly linked to phenolic profile. Thus, the present study aimed to identify and quantify, for the first time and in detail, the phenolic compounds present in the skin, flesh and seeds of BRS Violeta grape berry using combination of SPE methodologies and analytical HPLC–DAD–ESI-MS/MS. The study was extended to the different berry parts and the most important grape and wine phenolic families, and has revealed interesting features. Violeta grape has a very thick skin (46% of grape weight) that accumulated the most of grape phenolic compounds: great amount of anthocyanins (3930 mg/kg, as malvidin 3,5-diglucoside), together with also important amounts of flavonols (150 mg/kg, as quercetin 3-glucoside), hydroxycinnamic acid derivatives (HCAD; 120 mg/kg, as caftaric acid), and proanthocyanidins (670 mg/kg, as (+)-catechin); in contrast, it seems to be a low resveratrol producer. Violeta grape seeds accounted for similar proportions of low molecular weight flavan-3-ols (mainly monomers; 345 mg/kg, as (+)-catechin) and proanthocyanidins (480 mg/kg, as (+)-catechin). Violeta grape is a teinturier cultivar, but it only contained traces of anthocyanins and low amounts of all the other phenolic types in its red-colored flesh. The anthocyanin composition of Violeta grape was dominated by anthocyanidin 3,5-diglucosides (90%). Within flavonols, myricetin-type predominated and kaempferol-type was missing. In addition to expected hydroxycinnamoyl-tartaric acids, several isomeric esters of caffeic and p-coumaric acids with hexoses were tentatively identified, accounting for relevant proportions within the pool of HCAD. Although pending of further confirmation over successive vintages, the aforementioned results suggest that BRS Violeta grape cultivar could be considered an interesting candidate for the elaboration of highly colored and antioxidant-rich grape juices and wines

O espírito das leis e as leis do espírito: a evolução do pensamento legislativo brasileiro em saúde mental He spirit of the law and the laws of the spirit: the evolution of Brazilian legal thought in the realm of mental health

Examina a evolução das leis e normas da República brasileira relativas ao padecimento mental, demarcando três períodos. No período inicial (1890-1910), caracterizado pela introdução do tema da patologia mental no campo das preocupações do Estado, o interesse fundamental foi o reconhecimento e a preservação dos direitos dos portadores dessas patologias; nesse período, a alteração mental é entendida em termos de neuropatologia. No período de ampliação da ação estatal (1911-1945) permanecem os interesses observados no período anterior; a interpretação de adoecimento mental não sofre alterações substanciais, a despeito do alargamento de sua abrangência. No período do desenvolvimentismo brasileiro (1946-1982), marcado por uma descontinuidade em relação aos períodos anteriores, prevalece um espírito econômico-desenvolvimentista, expresso na vigorosa ampliação do parque hospitalar e amparado por um pensamento humanista de tons conservadores; mantém-se o interesse na proteção dos portadores de doenças mentais.<br>The article explores the evolution of the Brazilian republic's laws and norms on mental illness during three periods. The first (1890-1910) saw the topic of mental pathology introduced into the State's realm of interest, with the main concern being to recognize and preserve the rights of those displaying such pathologies. During this period, mental alterations were understood in terms of neuropathology. The following period (1911-1945) saw expansion of government initiatives, with the same concerns as the previous period. The interpretation of mental illness did not undergo any major changes, although it broadened in scope. Marked by a discontinuity in relation to previous times, Brazil's developmentalist period (1946-1982) saw an economic developmentalist spirit hold sway, underpinned by humanist thought of conservative propensity. It brought a vigorous growth in the number of Brazilian hospitals, while interest in protecting the mentally ill continued

NEOTROPICAL CARNIVORES: a data set on carnivore distribution in the Neotropics

Mammalian carnivores are considered a key group in maintaining ecological health and can indicate potential ecological integrity in landscapes where they occur. Carnivores also hold high conservation value and their habitat requirements can guide management and conservation plans. The order Carnivora has 84 species from 8 families in the Neotropical region: Canidae; Felidae; Mephitidae; Mustelidae; Otariidae; Phocidae; Procyonidae; and Ursidae. Herein, we include published and unpublished data on native terrestrial Neotropical carnivores (Canidae; Felidae; Mephitidae; Mustelidae; Procyonidae; and Ursidae). NEOTROPICAL CARNIVORES is a publicly available data set that includes 99,605 data entries from 35,511 unique georeferenced coordinates. Detection/non-detection and quantitative data were obtained from 1818 to 2018 by researchers, governmental agencies, non-governmental organizations, and private consultants. Data were collected using several methods including camera trapping, museum collections, roadkill, line transect, and opportunistic records. Literature (peer-reviewed and grey literature) from Portuguese, Spanish and English were incorporated in this compilation. Most of the data set consists of detection data entries (n = 79,343; 79.7%) but also includes non-detection data (n = 20,262; 20.3%). Of those, 43.3% also include count data (n = 43,151). The information available in NEOTROPICAL CARNIVORES will contribute to macroecological, ecological, and conservation questions in multiple spatio-temporal perspectives. As carnivores play key roles in trophic interactions, a better understanding of their distribution and habitat requirements are essential to establish conservation management plans and safeguard the future ecological health of Neotropical ecosystems. Our data paper, combined with other large-scale data sets, has great potential to clarify species distribution and related ecological processes within the Neotropics. There are no copyright restrictions and no restriction for using data from this data paper, as long as the data paper is cited as the source of the information used. We also request that users inform us of how they intend to use the data

NEOTROPICAL XENARTHRANS: a data set of occurrence of xenarthran species in the Neotropics

Xenarthrans—anteaters, sloths, and armadillos—have essential functions for ecosystem maintenance, such as insect control and nutrient cycling, playing key roles as ecosystem engineers. Because of habitat loss and fragmentation, hunting pressure, and conflicts with domestic dogs, these species have been threatened locally, regionally, or even across their full distribution ranges. The Neotropics harbor 21 species of armadillos, 10 anteaters, and 6 sloths. Our data set includes the families Chlamyphoridae (13), Dasypodidae (7), Myrmecophagidae (3), Bradypodidae (4), and Megalonychidae (2). We have no occurrence data on Dasypus pilosus (Dasypodidae). Regarding Cyclopedidae, until recently, only one species was recognized, but new genetic studies have revealed that the group is represented by seven species. In this data paper, we compiled a total of 42,528 records of 31 species, represented by occurrence and quantitative data, totaling 24,847 unique georeferenced records. The geographic range is from the southern United States, Mexico, and Caribbean countries at the northern portion of the Neotropics, to the austral distribution in Argentina, Paraguay, Chile, and Uruguay. Regarding anteaters, Myrmecophaga tridactyla has the most records (n = 5,941), and Cyclopes sp. have the fewest (n = 240). The armadillo species with the most data is Dasypus novemcinctus (n = 11,588), and the fewest data are recorded for Calyptophractus retusus (n = 33). With regard to sloth species, Bradypus variegatus has the most records (n = 962), and Bradypus pygmaeus has the fewest (n = 12). Our main objective with Neotropical Xenarthrans is to make occurrence and quantitative data available to facilitate more ecological research, particularly if we integrate the xenarthran data with other data sets of Neotropical Series that will become available very soon (i.e., Neotropical Carnivores, Neotropical Invasive Mammals, and Neotropical Hunters and Dogs). Therefore, studies on trophic cascades, hunting pressure, habitat loss, fragmentation effects, species invasion, and climate change effects will be possible with the Neotropical Xenarthrans data set. Please cite this data paper when using its data in publications. We also request that researchers and teachers inform us of how they are using these data