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Tenho em mim todos os sonhos do mundo.

----Fernando Pessoa

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Olá!! Chamo-me Leonor Santos e tenho 17 anos. Frequento o 12º ano de escolaridade na Escola Secundária de Rio Tinto no curso de Ciências e Tecnologias, tendo escolhido as disciplinas de Inglês e Biologia.

Esta escolha em particular diz muito sobre mim. Sempre gostei da língua inglesa, escrevo pequenas histórias e muitos poemas. A escrita é a minha forma favorita de expressão e descobri que, quando o faço em inglês, consigo exprimir, mais detalhadamente, ideias e sentimentos. Outra área que me desperta grande interesse é a Biologia: desde que me lembro sempre tive um fascínio inexplicável por tudo o que era ciência, desde Astronomia e Física a Química e Biologia. Toda esta curiosidade e fome de informação é fruto de um hábito que tenho desde pequena de ler livros, ver séries e pesquisar notícias sobre os mais variados assuntos científicos. Vejo esta minha curiosidade como uma das minhas maiores qualidades, pois permite-me, mesmo que inconscientemente, saber sobre muitos assuntos e, acima de tudo, gostar do processo de aprendizagem.

Tenho uma enorme paixão por fotografia. Para mim, documentar o que se passa à minha volta e conseguir transpor, na forma de uma fotografia, o mundo do meu ponto de vista, é das melhores sensações que existe. Fotografar é uma maneira excelente de explorar uma cidade, a melhor forma de apreciar a beleza de uma pessoa e um meio de conexão com a comunidade de pessoas que também tem fotografia como interesse pessoal.

Sou grande apreciadora de filmes, isto porque sinto que me atingem de uma maneira profunda. Tenho uma enorme facilidade de me identificar com personagens e de criar empatia pelo seu passado, fico emocionalmente presa às histórias e isso faz com que as interprete de forma complexa e sentimental. Ouço quase todo o tipo de música sendo música clássica a minha favorita para ouvir enquanto estudo. Tenho um carinho especial pela dança e pelo desenho, embora já não os pratique tão frequentemente, e absolutamente adoro jogos de tabuleiro.

Sempre tive grandes sonhos, sempre senti que a minha vida seria destinada à perfeição. Com o passar do tempo aprendi a aceitar a imperfeição em tudo e que é nela que encontro o espaço e motivação para crescer. Até hoje convenço-me de que seria bem sucedida em qualquer carreira que escolhesse. Isso deixa-me bastante indecisa e com dificuldades no que toca a escolher o que um dia será a minha profissão. Já revirei todas as possibilidades e cheguei à conclusão que fotografia é o meu propósito. Porém, é algo que escolhi manter como passatempo e focar-me noutra área que, se tudo correr bem, me trará sucesso.

Este ano, tive a oportunidade de participar no desenvolvimento de projeto de investigação no Instituto de Investigação e Inovação em Saúde da Universidade do Porto e vou documentar todo o processo do projeto em publicações neste Padlet que partilho com a minha colega Naftali. Com isto, espero conseguir alcançar os meus objetivos no secundário e seguir os meus estudos em Biologia na Universidade do Porto.

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O primento elemento a ser utilizado foi a bactéria " E. coli ", que por sua vez é uma bactéria de Gram-negativa, encontradas no intestino humano, fermentativa, anaeróbia facultativa, cultivada, geralmente postas em placas de vidro, apartir dos meios de cultura rotinaria. Colhetar, as mesmas com ajuda da " Ansa" , colocar nas placas e pôr na estufar, esperar alguns dias para que haja mais visibilidade, favorecendo assim a técnica vindorura.

Link: https://youtu.be/UhRvu8d4Y04?si=K3i2dPLf--QQ8T5I

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Fig.2- Pesquisas relacionadas com o genoma

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Vid. 6- ELISA (Enzyme-linked Immunosorbent Assay) - consultado a 25/20/2024

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Nesta versão ELISA, a amostra experimental é "sanduíche" entre um anticorpo de captura não conjugado e um anticorpo de deteção conjugado, ambos específicos para a mesma proteína, mas em epítopos diferentes.

Utilizámos teste ELISA para quantificar as citocinas IL-8 e TNF-α nas placas.

O vídeo anexado explica, de forma resumida, os tipos de teste ELISA e como funcionam.

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Fig.6- Tubo SEPMATE com amostra de sangue + PBS após centrifugação (PL)

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O sangue utilizado foi de bovino, extraído no matadouro. Neste momento, o nosso objetivo principal era o de isolar os monócitos do sangue do resto das células.

Para isso, transferimos as amostras para falcons de 50 mL diluído em PBS (volume de PBS igual ao volume total da amostra).

O sangue foi então passado para tubos SEPMATE contendo 15 mL de Histopaque cada e foram centrifugados por 20 minutos a 1200G, sem travão.

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Fig. 17- Direcionalidade do primer de DNA

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Esta semana realizámos a técnica PCR que consiste em multiplicar pequenas porções de DNA in vitro, permitindo a sua análise.

Esta técnica utiliza variações bruscas de temperatura, juntamente com DNA polimerase. A DNA polimerase normalmente usada na PCR é chamada Taq polimerase, nome proveniente da bactéria tolerante ao calor da qual foi isolada (T hermus aq uaticus).

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Durante a semana, entre os dias 19 e 25 de janeiro, foi retirado um terço do volume de todos os poços e adicionado metade do soro de vaca que recolhemos na semana anterior.

Dia 25 tivemos a oportunidade de observar as células nos poços antes de uma nova substituição de meio (Fig.8). Para esta mudança de meio, as células foram expostas a combinações diferentes de LPS, parasita (NCT) e interferão gama (γ):

•células + meio

•células + meio + LPS

•células + meio + parasita

•células + meio + interferão γ (500 ng/mL)

•células + meio + interferão γ (100 ng/mL)

•células + meio + interferão γ (10 ng/mL)

•células + meio + parasita + interferão γ (500 ng/mL)

•células + meio + parasita + interferão γ (100 ng/mL)

•células + meio + parasita + interferão γ (10 ng/mL)

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Ao longo das próximas semanas vamos executar o protocolo de síntese de cadeias de cDNA.

O cDNA (DNA de cópia ou DNA complementar) é o DNA sintético que foi transcrito de um mRNA específico através de uma reação usando a enzima transcriptase reversa. Enquanto o DNA é composto de sequências de codificação e não-codificação, o cDNA contém apenas sequências codificantes.

Esta semana, tínhamos como objetivo extrair o RNA das células. Precisávamos de condições que permitissem que o RNA se mantivesse intacto, então, trabalhámos na hotte e com materiais livres de Ribonucleases (RNases). Isto porque, as RNases são um grande grupo de enzimas hidrolíticas que degradam as moléculas de ácido ribonucleico. Catalisam a quebra do RNA em componentes menores, operando nos níveis de transcrição e tradução.

Estas enzimas estão presentes na maioria das superfícies e é necessária a sua eliminação para este processo, para isso, utilizámos RNase AWAY, um agente de descontaminação de RNase, o que inibiu a atividade da enzima e preservou as cadeias de RNA.

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cDNA, copy DNA. If you get to spend a week in a genomics lab, it is very likely that you will hear about cDNA one way or another. That's how important this word is and for a good reason. Imagine that for one of your experiments, you need to make a protein and a lot of it. Let's say you want to create a more stable version of insulin, a diabetes drug. One way to do it is to get cells that make this protein, let's say pancreatic cells. These cells will contain a gene with instructions on how to make insulin. These cells can also transcribe this gene to temporarily store this information in the form of RNA. Using a special enzyme called traverse transcriptase, you can convert RNA back to DNA, but the resulting DNA will look a little different from the original gene. For example, it will be much shorter than the gene from where it came from. This is why scientists use another word, cDNA, copy or complementary DNA, to tell these versions apart. What you can do with the cDNA of insulin is very interesting. You can clean it up. You can make changes to the sequence. And using special molecular tools, special enzymes, you can insert it back into genome of another organism, for example, yeast. Yeast is very good at making lots of proteins. And if you insert cDNA of insulin into its genome in the right place and configuration, it can start making insulin for you. This is one way scientists can make and test new versions of insulin with properties that can be helpful in treating diabetes.

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cDNA (short for copy DNA; also called complementary DNA) is synthetic DNA that has been transcribed from a specific mRNA through a reaction using the enzyme reverse transcriptase

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A maçã orgânica possui bactérias designada por "Lactobacilios". Sendo assim um dos elementos utilizado para experimentação, durante está técnica, em princípio cada um de nós (indivíduos que executaram o procedimento) tevi a pré-viabilidade dos resultados, concernente à fruta, mas como era um projeto científico tentamos e felizmente obtivemos os resultados previstos!

Esta técnica procedeu com a ajuda da "Ansa", "Água destilada" esfregar superficialmente e profundamente, com intuito de homogenizar a água destilada e as bactérias, contida na maçã. Em que depois foram úteis, na técnica de plaqueamento, que ao longo do proejto irei de abordar.

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Olá colegas e professora Natália!

Chamo-me Naftali Makiesse, hoje falarei um pouco de mim e terão a oportunidade de conhecer quem é a Naftali e de onde surgiu.

Nasci no dia 12 de fevereiro de 2005 em Angola (atualmente tenho 18 anos de(idade).

Desde a minha infância fui sempre mimosa, curiosa, e chorona (a menina da mama), estudei em várias escolas como:a frâncesa e portuguêsa.Desta forma,tive a vantagem de aprender essas duas línguas que, posteriormente,virão a ser-me úteis.

O meu desenho animado favorito era Barbie moda, tornando-me apaixonada pelos looks e de moda, portanto sempre quis ser estilista, ter a minha própria marca como a da Barbie. Quando fiz os meus 18 anos conversei com a minha mãe sobre a minha paixão e o que realmente amava de fazer.Surprendemente fiquei após a sua reção,esperava de uma resposta negativa por sua parte sobre a minha paixão. pelo contrário sugeriu-me o curso de enfermagem, que consiste em salvar vidas dos outros. Irei de aprender, não só a cura ou salvar vidas, mais também a empatia: a capacidade de colocar-se no lugar do outro. Porque de facto é o mais importante do que estar a inventar montes de estilos vestimentários.

Foi difícil aceitar a proposta vinda pela minha mãe,desde lá então duro anos e anos em consentir-me com a situação. E no final percebi a razão dela,foi além dos meus pensamentos que no fundo queria apenas o meu bem e da sociedade. Com certeza uma mãe, não procura os seus próprios benefícios, mas o melhor para os seus filhos.

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Fig.2- Caixas de pontas, grupo de trabalho (PL)

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Começámos por encher as caixas de pontas de pipetas para, mais tarde, serem esterilizadas e estarem prontas a ser usadas. Colocar cada pontinha no seu lugar é mais difícil do que aparenta! Aprendi a valorizar as caixas já prontas e esterilizadas que nos poupam tempo e esforço..

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Inicialmente treinámos com micropipetas de monocanal, depois com as de multicanal como a da fotografia. Foi difícil ganhar o hábito de a controlar, isto porque temos que prestar atenção a mais do que uma ponta. Elas podem criar bolhas, não aspirar corretamente ou até mesmo cair da pipeta! Era questão de estarmos um pouco mais atentos a esses detalhes.

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Vid.1- Primeiro treino com a micropipeta multicanal (PD- Por Daniel)

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Fig.1- Cartão de visita (PL- Por de Leonor)

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Nesta primeira semana, ao chegar ao I3S recebemos na portaria os nossos cartões de visita. É neste momento que nos apercebemos que realmente temos muito trabalho pela frente!! Apesar de já termos visitado o edifício, não conseguimos evitar esquecer onde são as salas, cafetaria e casas de banho. Mas que lugar confuso...

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Fig.4- Controlador de pipeta elétrico (PL)

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Também aprendemos a manusear o controlador de pipeta elétrico. O meu maior problema foi tremer bastante enquanto o usava. A pressão que tinha que fazer sobre o botão de descarga acabava por desestabilizar a ponta da pipeta, fazendo com que esta tremesse um pouco. Com um pouco de treino, esse problema desapareceu e o manuseio do controlador tornou-se mais preciso e estável.

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Por fim, aprendemos a configurar a centrifugadora e a forma correta de a utilizar, visto que será necessária durante o projeto.
A técnica da centrifugação usa-se, geralmente, para separar partículas biológicas em suspensão. A alta velocidade de rotação do motor do equipamento faz com que os materiais de densidades diferentes se separem em fases: o sobrenadante que fica na parte superior e o composto que, por ter maior densidade, fica no fundo do tubo.
É bastante intuitiva e simples de usar. Temos apenas que ter em conta a forma como as amostras são dispostas no seu interior: idealmente sempre localizadas de forma oposta para que estejam em equilíbrio. No caso o número de amostras ser ímpar, é necessário criar uma tara com o mesmo peso.

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Link - Phosphate Buffered Saline - consultado a 29/01/2024

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Phosphate-buffered saline (Tampão fosfato-salino)

O PBS possui muitas aplicações, pois não é tóxico em relação às células e é isotónico (a osmolaridade e as concentrações iónicas das soluções coincidem com as do corpo humano).

Pode também ser utilizado para diluir diferentes substâncias, ou como uma solução para limpeza celular.

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Link - SepMate™ PBMC Isolation Tubes - consultado a 1/02/2024

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É um tubo que permite o isolamento de células mononucleares de sangue periférico (PBMC) consistentes e sem complicações. Permite que os usuários coloquem rapidamente o sangue sobre o meio gradiente de densidade e impede que as camadas se misturem.

Meio gradiente de densidade - Facilitam a separação das células desejadas de uma mistura complexa (ex: amostras de sangue ou tecido inteiros), com base em características ou marcadores específicos. O método de centrifugação de gradiente de densidade separa populações de células específicas de partículas biológicas com base na faixa de densidade do meio gradiente e partículas de amostra.

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SepMate™ is an innovative tube that enables consistent and hassle-free peripheral blood mononuclear cell (PBMC) isolation in just 15 minutes. The typical PBMC isolation protocol is laborious, requiring slow layering of blood on density gradient medium (e.g. Lymphoprep™, Ficoll-Paque™), lengthy 30-minute centrifugation with the brake off, and careful harvest of PBMCs using a pipette. SepMate™ simplifies and speeds up the PBMC isolation protocol to an easy 15-minute procedure. The SepMate™ insert allows users to quickly layer blood over the density gradient medium, and prevents the layers from mixing. Centrifugation is performed with the brake on, after which isolated PBMCs are simply poured off into a fresh tube. Read a SepMate™ review on Biocompare.

The Advantage of Using SepMate™ Tubes

Learn how SepMate™ tubes can simplify your density gradient centrifugation and speed up your PBMC isolation protocol.
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EASY. Say goodbye to slow and laborious sample layering over the density gradient medium.

FAST. Centrifuge for just 10 minutes with the brake on and simply pour off PBMCs into a new tube.

CONSISTENT. Minimize variability between users.

VERSATILE. Can be combined with RosetteSep™ to isolate specific cell subsets.

How to Isolate PBMCs Using SepMate™

This technical video shows the procedure for isolating peripheral blood mononuclear cells (PBMCs) from whole blood in just 15 minutes using SepMate™ tubes.
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cGMP-Manufactured

SepMate™ is manufactured following current Good Manufacturing Practices (cGMP) and tubes are sterilized by irradiation. SepMate™ is registered as an In Vitro Diagnostic (IVD) device in Australia, Canada, Europe and the United States for the isolation of mononuclear cells from whole blood or bone marrow by density gradient centrifugation. In China SepMate™ is considered a non-medical device by the China Food and Drug Administration (CFDA), and should therefore be used as general laboratory equipment. The end user is responsible for determining whether the product is suitable for their specific application. In regions outside of Australia, Canada, China, Europe and the United States SepMate™ is available as a Research Use Only (RUO) product.
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I tested the SepMate™ tubes yesterday and they worked... fantastically! I ran in parallel the same sample with my standard protocol and the cell recovery was the same (in a quarter of the time, of course).
Esperanza Perucha, PhD, King's College London

The Typical SepMate™ PBMC Isolation Protocol

I have recently completed a project isolating a rare population of NK cells ... Using SepMate™ tubes significantly reduced the ficoll-based density centrifugation process. I found it very ergonomically supportive as well because layering blood on the ficoll cushion is fast and facile so your shoulder is not stressed compared to the traditional layering. The quality of cells, measured by % of dead cells, were about 20% higher. SepMate™ is the ideal solution for laboratories having high rates of blood samples per day. It lowers the cost by reducing the tech time.
Dr. Amina Kariminia, BC Children's Hospital Research Institute

Combine SepMate™ and RosetteSep™ to Isolate Specific Cell Subsets from Whole Blood in 25 Minutes

To isolate purified cell subsets from whole blood, simply incubate your sample with the appropriate RosetteSep™ cocktail for 10 minutes prior to pipetting onto density medium in a SepMate™ tube. During centrifugation unwanted leukocytes pellet along with red blood cells, leaving untouched and highly purified target cells at the interface between the plasma and the density gradient medium above the insert. After centrifugation, simply pour highly purified target cells into a new tube. No columns or magnets are necessary, and minimal training is required. Just incubate, spin, and pour off purified cells.

The NK Cell Cytoxicity Case Study discusses a publication from Dr. Ajay Jain's lab at the University of Maryland School of Medicine, which used the RosetteSep™ and SepMate™ system to reduce NK cell isolation time from four hours to a single hour for a 450 mL unit of blood.

Watch a video showing how to combine SepMate™ with RosetteSep™ for fast cell subset isolation directly from whole blood.

Related Products

Lymphoprep™ Density Gradient Medium

A cost-effective alternative to Ficoll-Paque™ that can be used with SepMate™ for the isolation of mononuclear cells from blood.

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RosetteSep™ Immunodensity Cell Separation

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Scientific Resources

High-Throughput Immunodensity-Based Cell Subset Isolation Directly from Whole Blood

How to Isolate Specific Cell Subsets by Immunodensity Cell Separation

How to Isolate Mononuclear Cells from Whole Blood by Density Gradient Centrifugation

Explore our cell separation methods library and immunology resource center for more useful materials, including protocols, technical bulletins, and videos.

See all references using SepMate™.

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Isolate peripheral blood mononuclear cells (PBMCs) in just 15 minutes using SepMate™ tubes.

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Link - Histopaque - consultado a 1/02/2024

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Histopaque é uma solução estéril testada em endotoxina de polissucrosa e diatrizoto de sódio, ajustada a uma densidade de 1,077g/mL. Este meio facilita a recuperação rápida de linfócitos viáveis e outras células mononucleares de pequenos volumes de sangue total.

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Histopaque®-1077 sterile-filtered, density: 1.077 g/mL | Sigma-Aldrich

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Histopaque is a density gradient cell separation medium of Ficoll and sodium diatrizoate used for the separation of lymphocytes from whole blood and bone marrow.

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Link - PBMCs: The One Stop Immune Cell Shop - consultado a 29/01/2024

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Peripheral blood mononuclear cells (células mononucleadas do sangue periférico)

É qualquer célula do sangue periférico com um núcleo redondo. Essas células consistem em linfócitos (células T, células B, células NK) e monócitos, enquanto os eritrócitos e plaquetas não têm núcleos, e os granulócitos (neutrófilos, basófilos e eosinófilos) têm núcleos multilobulados.

Nos humanos, os linfócitos compõem a maioria da população de PBMC, seguida por monócitos e apenas uma pequena percentagem de células dendríticas.

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Fig.7- Processo da centrifugação do sangue nos tubos SEPMATE

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Após a centrifugação recolhemos, com uma pipeta de Pasteur, o anel de PBMCs. Estas células foram transferidas para um novo falcon de 50 mL ao qual adicionámos PBS.

15 mL células + 30 mL PBS

Estas células foram centrifugadas (lavagem) por 15 minutos, 400 G, 8ºC.

O sobrenadante foi descartado e foi adicionado MACS buffer às células que ficaram depositadas no fundo do falcon.

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Vid.2- Recolha do anel de PBMCs (PM- Por Miguel)

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Vid.3- Preparação para a contagem das células (PM)

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Em 3 eppendorfs nomeados "10", "100" e "1000" foram adicionados 90 µL de Trurks, um corante que cora leucócitos, em cada um.

Depois, no eppendorf "10" foram adicionados 10 µL de células, foi homogenizado e retirados 10 µL que seriam transferidos para o eppendorf "100". A mistura voltou a ser homogenizada e foram retirados 10 µL, transferidos para o eppendorf "1000". Só a partir deste é que são retirados os finais 10 µL que serão colocados na câmara de Neubauer para a contagem das células.

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https://www.stemcell.com/media/images/pages/blood-processing/pbmc.png - consultado a 29/01/2024

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Vid.4- Adição da suspensão das células rotuladas à coluna (PL)

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A CD14 é uma glicoproteína de membrana ancorada por glicolípido expresso nas células da linhagem mielomonocítica, incluindo monócitos, macrófagos e alguns granulócitos.

As MicroBeads CD14 são usadas para a seleção positiva ou deleção de monócitos humanos e macrófagos de sangue umbilical ou PBMCs. O antigénio CD14 pertence ao complexo recetor LPS. CD14 é fortemente expresso na maior parte dos monócitos e macrófagos e pouco presente em neutrófilos e algumas células dentríticas mieloides.

• Primeiramente, as células CD14⁺ foram magneticamente rotuladas com MicroBeads CD14. Para isto foi determinado o número de células, a suspensão de células foi centrifugada a 300 G por 10 minutos e ressuspendida em 80 µL de tampão MACS por 10⁷ células totais.

• A estas foram adicionados 20 µL de MicroBeads CD14 por 10⁷ células totais.

• Depois, foi equilibrada uma coluna com tampão MACS (coluna MIDI)- 0,5 mL tampão MACS à qual a suspensão de células foi adicionada.

• Foram feitas duas lavagens com 3 mL de tampão MACS. As células CD14⁺ rotuladas ficaram retidas na coluna enquanto as células não rotuladas passaram na lavagem.

• A coluna foi retirada do campo magnético e colocada num falcon. Foram pipetados 5 mL de tampão MACS para dentro da coluna e rapidamente empurrados, juntamente com as células magneticamente rotuladas, com o êmbolo.

Desta forma foi possível separar as células CD14⁺ das restantes.

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Vid.5- Adição de tampão MACS e descarregar com o êmbolo (PL)

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Etapa final da separação das células CD14⁺ 

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Etapa final da separação das células CD14⁺ 

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A citometria de fluxo é uma técnica que utiliza um citómetro de fluxo para medir características físicas e químicas de células ou partículas.

Cada célula ou partícula é analisada por dispersão de luz visível ou por fluorescência à medida que as células fluem rapidamente após cada laser.

Independente da análise de dispersão de luz, as medições de fluorescência são obtidas por transfeção e expressão de proteínas fluorescentes comuns e coloração com anticorpos fluorescentemente conjugados ou corantes fluorescentes.

O funcionamento deste equipamento consiste em excitar (com lasers) os fluorocromos ligados às células (ou moléculas) de interesse. A partir da difração da luz, torna-se possível adquirir dados tangíveis sobre a natureza do alvo de interesse.

Esta técnica foi utilizada ao fim do dia, com as células CD14⁺, CD14⁻ e com uma amostra total das células. Com ela foi possível analisar aspetos como quantidade de células por característica (neste caso, CD14⁺ ou CD14⁻) como forma de aprender a interpretar os resultados da citometria de fluxo, visto que a vamos utilizar novamente, mais à frente.

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As placas foram centrifugadas por pouco tempo, neste caso apenas um spin, visto que são aderentes e não é necessário um grande período de tempo sob força centrífuga.

Foram transferidos 80 µL de cada um dos poços para as placas novas de fundo redondo e adicionados 100 µL de Nyzol.

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https://blog.varsomics.com/wp-content/uploads/2022/10/citometria-de-fluxo-03-1536x1091.webp - consultado a 01/02/2024

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Em suma eu cresci,estou mais grandinha do que antes e com objectivo de fazer enfermagem,ajudar a minha família,a sociedade portuguesa ou em qualquer lugar onde eu estiver,terei orgulho de ser enfermeira,viver pelos outros (empatia)

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O falcon com as células foi centrifugado por 10 minutos, 300 G, 8ºC, para remover o tampão MACS.

Para a construção da placa foram colocados 100 µL de células + 100 µL de estímulos por poço, totalizando 200 µL por poço.

Os poços com células foram rodeados por poços com PBS para que não evaporar e perder volume.

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Foi disposto na placa um anticorpo monoclonal purificado (que não está marcado com enzimas), que foi produzido através de uma linhagem de células feita clonando um linfócito B único. Este "forra" o fundo da placa High Binding Protein.

Este anticorpo vai capturar o antigénio. As placas foram lavadas com PBS e Tween, um detergente que reduz as ligações hidrofóbicas entre as proteínas, aumentando a especificidade.

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Nesta etapa, foi adicionado um excesso de proteína BSA (Bovine Serum Abumine) que preencheu os sítios que não estavam ocupados por anticorpos. Desta forma, as citocinas apenas têm oportunidade de se ligar aos anticorpos. Foram feitas mais 3 lavagens.

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Fig. 10- Concentações de IL-8 e TNF-α (PL)

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De seguida, começámos a preparar as citocinas. Esta é uma fotografia das diferentes concentrações de TNF-α e IL-8.

Os standards servem de referência quando for feita a espetrofotometria. Esta técnica baseia-se na medida quantitativa da absorção da luz pelas soluções, onde a concentração na solução da substância absorvente é proporcional à quantidade de luz absorvida.

Foram adicionados 100 µL por poço de amostras ou standards diluídos em PBS e as placas foram incubadas por 2 horas a temperatura ambiente. Neste tempo, a citocina ligou-se ao anticorpo até todo ele estar ligado, saturando. Foram feitas mais 3 lavagens nas mesmas condições, descartando as citocinas livres.

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Vid. 7- Miguel a preparar o falcon de anti TNF-α + Buffer (PL)

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Em 2 falcons de 15 mL preparámos as soluções:

• anti TNF-α + Buffer

• anti IL-8 + Buffer

Este segundo anticorpo que vai ser adicionado, que está associado a uma biotina, vai ligar-se a outro epítopo da citocina.

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Vid. 8- Transferência das citocinas para as placas (PM)

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O conteúdo dos falcons foi disposto em 2 barcos diferentes para facilitar a transferência das citocinas para cada uma das placas utilizando uma pipeta de multicanal.

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As placas foram deixadas no agitador por 10 minutos.

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A adição de Streptavidina + HPR (horseradish peroxidase, uma enzima) faz com que a mesma se ligue à biotina que estava associada ao anticorpo secundário. A solução preparada continha 8,5 mL PBS + 8,5 µL de Streptavidina.

Foram adicionados 50 µL desta solução por poço e as placas foram incubadas por 30 minutos. Após a incubação, cada placa foi lavada 5 vezes.

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Fig. 11- Substrato TMB e ácido sulfúrico (PL)

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Foram dispostos 9 mL do substrato TMB num barco e, com uma pipeta multicanal, adicionaram-se 50 µL por poço.

O substrato vai dar início à atividade enzimática da HRP que está associada à Streptavidina e a peroxidase é metabolizada. Isto leva a que os poços adquiram uma cor azul, sendo a sua intensidade proporcional à quantidade de citocinas.

A reação foi então interrompida usando ácido sulfúrico (H₂SO₄) para termos a certeza que cada poço foi sujeito às mesmas condições e para que este mude de cor para um tom de amarelo.

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Vid. 10- Miguel a adicionar o substrato (PL)

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Vid. 11- Interrupção da atividade enzimática com ácido sulfúrico (PL)

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https://www.youtube.com/watch?v=ERk0hwqhyDw&t=2s - consultado a 25/02/2024

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Link - Lipopolysaccharide - consultado a 28/02/2024

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Os lipopolissacarídeos são componentes importantes da membrana externa das bactérias gram-negativas. A glicoproteína CD14 é essencial no reconhecimento deste componente

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Lipopolysaccharides (LPS) are important outer membrane components of gram-negative bacteria. They are large amphipathic glycoconjugates that typically consist of a lipid domain (hydrophobic) attached to a core oligosaccharide and a distal polysaccharide. These molecules are also known as lipogylcans due to the presence of lipid and sugar molecules.

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Link - Biologic functions of the IFN‐γ receptors - consultado a 28/02/2024

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O interferão gama é uma citocina que desempenha um papel importante na indução e modulação de uma série de respostas imunes. Promove a ativação dos macrófagos e a diferenciação de linfócitos T. Estimula, também, a atividade das células NK e regula positivamente moléculas que têm efeito antiviral, antiproliferativo e que favorecem a apoptose.

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Allergy. 1999 Dec; 54(12): 1233-1251.

Overview

Interferon‐gamma (IFN‐γ) is a cytokine that plays an important role in inducing and modulating an array of immune responses. Cellular responses to IFN‐γ are mediated by its heterodimeric cell‐surface receptor (IFN‐γR), which activates downstream signal transduction cascades, ultimately leading to the regulation of gene expression. In order to study the role of IFN‐γ in a number of immune responses and pathways, researchers have generated mice with altered patterns of IFN‐γR gene expression. These studies, together with analyses of naturally occurring mutations of the IFN‐γR in man, have been instrumental in elucidating the diverse functions of IFN‐γ, and are the subject of this review.

Introduction

Originally identified 30 years ago as an agent with antiviral activity, IFN‐γ has since been characterized as a homodimeric glycoprotein with pleiotropic immunologic functions ( 1, 2- 3). IFN‐γ is primarily secreted by activated T cells and natural killer (NK) cells, and can promote macrophage activation, mediate antiviral and antibacterial immunity, enhance antigen presentation, orchestrate activation of the innate immune system, coordinate lymphocyte-endothelium interaction, regulate Th1/Th2 balance, and control cellular proliferation and apoptosis ( 1, 2). It was not until 20 years after the identification of IFN‐γ that its cell‐surface receptor was discovered ( 4, 5, 6, 7, 8- 9). The α chain of the IFN‐γR, also known as IFN‐γR1 or CD119, was the first component of the receptor to be identified and cloned ( 10, 11, 12, 13- 14). Although it binds IFN‐γ with relatively high affinity, IFN‐γR1 alone is unable to mediate the biologic responses to this cytokine ( 12, 15, 16- 17). Subsequent complementation studies led to the identification and cloning of an accessory factor (AF‐1), also known as the β receptor chain or IFN‐γR2, as the protein required, in addition to IFN‐γR1, to endow a cell with the ability to respond to IFN‐γ ( 15, 18, 19, 20- 21). Specific residues within the cytoplasmic domains of both the α and β chains of the IFN‐γR are critical for transducing the IFN‐γ signal from the cell surface to the nucleus through the activation of intracellular signaling pathways ( 22, 23, 24- 25). Mutations in either component of the IFN‐γ receptor that impair or alter the ability of cells to respond to this ligand have global consequences for IFN‐γ‐mediated immunity, and therefore serve as an important tool for analyzing the pleiotropic effects of this cytokine ( 26, 27).

The biochemical and signaling properties of the IFN‐γR

The receptor complex that mediates the full biologic function of IFN‐γ consists of at least two species‐matched chains: IFN‐γR1, a 90‐kDa glycoprotein (which is 472 amino acids (aa) long in man, and 451aa in mice) encoded on human chromosome 6 and mouse chromosome 10, and IFN‐γR2, a 60-67‐kDa glycoprotein (which is 316aa in man and 314aa in mice) encoded on human chromosome 21 and mouse chromosome 16 ( 4, 18, 20, 28-35). IFN‐γR1 is the major ligand‐binding subunit, binding IFN‐γ with a K a of 10 ⁹-10 ¹⁰ M −1 and a receptor‐to‐ligand ratio of 2:1, as inferred from the crystal structure of the occupied receptor and other studies ( 4, 34, 36, 37). IFN‐γR2 increases the affinity of IFN‐γR1 for its ligand, presumably by enhancing the stability of the complex, but plays only a minor role in direct ligand binding ( 38). The β chain is, however, obligatory for transducing the IFN‐γ signal ( 21, 38, 39).

Both chains of the IFN‐γ receptor are members of the class II family of cytokine receptors that includes tissue factor, the IL‐10 ligand‐binding component, and both chains of the IFN‐α receptor (IFN‐αR) ( 40, 41). Like other family members, the IFN‐γR α and β chains lack intrinsic kinase activity. Signaling through the IFN‐γR is mediated through JAK1 and JAK2, members of the Janus family of protein tyrosine kinases, which are constitutively associated with specific membrane‐proximal residues on the cytoplasmic domains of IFN‐γR ( 25, 34, 39, 42-44). JAK1 binds the ₂₆₆LPKS ₂₆₉ motif (also known as the box 1 motif) on IFN‐γR1, while JAK2 binds the ₂₆₃PPSIPLQIEEYL ₂₇₄ motif (or the box 1, box 2 motif) on IFN‐γR2 ( 23, 25, 44-46). Ligand binding leads to receptor oligomerization, with two IFN‐γR1 chains bound to one IFN‐γ homodimer, and the subsequent recruitment of two IFN‐γR2 chains to the complex ( 25, 37, 38, 45-48). IFN‐γ‐mediated aggregation of its receptor components brings the inactive JAKs associated with the cytoplasmic tails of the α and β chains into close proximity with one another ( Fig. 1 ). Once clustered, the JAKs are reciprocally activated through sequential auto‐ and transphosphorylation events ( 42, 49). Activated JAKs phosphorylate a specific tyrosine residue near the C‐terminus of the IFN‐γR1 (Y440 in man) ( 24, 42, 45, 50) ( Fig. 1 ). This phosphorylated tyrosine residue pair (one on each IFN‐γR1 chain) is embedded within a recognition sequence ( ₄₄₀YDKPH ₄₄₄) to which STAT1 (a member of the Signal Transducers and Activators of Transcription family of latent cytoplasmic proteins) binds through its SH2 (src homology 2) domain ( 24, 45, 51, 52). The docking of STAT1 molecules at their target sequences on the IFN‐γR complex is followed by their phosphorylation on tyrosine residue Y701 by the receptor‐associated JAKs ( 53, 54- 55) ( Fig. 1 ). Once phosphorylated, two STAT1 proteins homodimerize via reciprocal SH2‐phosphotyrosine interactions, forming a protein complex first identified as GAF (gamma‐activated factor) ( 51, 56). The STAT1 homodimer then translocates to the nucleus, where it binds a nine‐nucleotide consensus sequence, TTNCNNNAA, known as a GAS (gamma‐activated site) element ( 57, 58- 59). This binding site has been identified in the regulatory regions of over 200 genes; therefore, recognition of this element by STAT1 homodimers can modulate the expression of a vast array of genes, thereby mediating the biologic functions of IFN‐γ ( 2).

Mechanistically, it has been suggested that after ligand binding, IFN‐γ signaling is initiated by JAK2 autophosphorylation, followed by phosphorylation of JAK1 ( 34, 60). Activated JAK1 is then thought to phosphorylate IFN‐γR1, providing a docking site for STAT1. After binding to its receptor site, STAT1 is believed to be activated through phosphorylation by JAK2. Studies with kinase‐negative JAK1 and JAK2 mutants have demonstrated that while the mutant JAK2 cannot support IFN‐γ‐stimulated gene transcription, the kinase activity of JAK1 is not fully required for its role in transducing the IFN‐γ signal ( 49).

Signaling through the IFN‐γ receptor may be regulated at several points along the pathway. One mechanism is the modulation of receptor expression. Control of IFN‐γR1 levels on the cell surface has been proposed to be a mechanism through which a cell alters its sensitivity to IFN‐γ ( 61, 62- 63). Attenuation of the IFN‐γ signal by this mechanism has been linked to differences in the biologic responses it elicits in cells expressing different levels of IFN‐γR1. Moreover, downmodulation of IFN‐γR2 expression in IFN‐γ‐producing T cell subsets renders these cells unresponsive to IFN‐γ ( 64, 65- 66). Cells appear to have intracellular mechanisms to regulate the IFN‐γ signal as well. Members of the recently identified SOCS ( Suppressors Of Cytokine Signaling) family of proteins may negatively regulate cytokine signaling through a number of potential mechanisms ( 67, 68- 69). For example, SOCS1 has been shown to inhibit IFN‐γ signaling, presumably by preventing JAK kinase activation ( 70, 71). In fact, the expression of SOCS‐1 is induced by IFN‐γ, suggesting that a negative feedback pathway exists in which cells may attenuate their sensitivity to this cytokine after the activation of IFN‐γ signaling. In contrast, the transcriptional activity of STAT1 (and therefore the IFN‐γ signal) has been shown to be enhanced through serine phosphorylation ( 72, 73). Additional intracellular mechanisms thought to regulate IFN‐γ‐mediated signal transduction may include the dephosphorylation of IFN‐γR1, which would prevent STAT1 docking; the dephosphorylation of STAT1, which would prevent its homodimerization; and the ubiquitination of STAT1, which would lead to its degradation by targeting it to the proteosome pathway ( 42, 51, 74, 75). These mechanisms could limit the availability of activated STAT1, although their importance in the regulation of IFN‐γ signaling is unclear.

Disruption of IFN‐γR1 expression in mice

To study the role of IFN‐γR1 in mediating IFN‐γ signaling and biologic responses, researchers have disrupted its endogenous locus to generate IFN‐γR1‐null (‐\‐) mice ( 26). These mice have not only proved to be a useful tool to study IFN‐γ signal transduction, but also constitute one of the first physiologic systems available to study IFN‐γ biology. IFN‐γR1‐deficient mice develop normally to adulthood with no phenotypic anomalies. They appear to have normal leukocyte populations in lymphoid organs and normal baseline levels of MHC class I and II molecule expression. However, cells derived from these mice are unable to initiate signaling in response to IFN‐γ. They are therefore insensitive to any of the biologic effects of IFN‐γ, including its antiviral effects and antitumor activity ( 26, 76). As described below, these mice have severe defects in their immune responses.

Macrophage function and IL‐12 in IFN‐γR1‐deficient mice

IFN‐γ has long been thought to be the most important activator of macrophages; in fact, it was, for a time, known as macrophage‐activating factor (MAF) ( 77). Nitric oxide (NO) production and MHC class II upregulation are two key effector mechanisms of macrophages, both critically dependent on IFN‐γ, and are essential for cell‐mediated and Th1‐type immune responses ( 1, 2, 77, 78). Therefore, it was not surprising that the IFN‐γR1 (‐/‐) mice exhibited severely compromised macrophage functions, such as granuloma formation ( 79, 80, 81, 82, 83, 84- 85). The IFN‐γ dependence of protective and self‐destructive immune responses mediated, in part, by macrophages was examined in several infectious and autoimmune models that are discussed in later sections of this review.

IL‐12 is a cytokine secreted primarily by activated macrophages ( 2, 86, 87- 88). It induces IFN‐γ production by NK cells and Th1 cells, while IL‐12 production itself is induced by IFN‐γ ( 89). IL‐12 also primes naive T cells (Thp) to differentiate along the Th1 pathway ( 90, 91, 92, 93- 94). It is, however, unclear whether the biologic functions of IL‐12 are mediated principally by IFN‐γ ( 95). A number of groups utilized IFN‐γR1‐deficient mice to study the dependence of the effector functions of IL‐12 on IFN‐γ.

Systemic administration of IL‐12 is known to inhibit hematopoiesis, leading to a decrease in the number of peripheral blood lymphocytes (PBLs) and a diminished bone‐marrow cellularity ( 96). Administration of IL‐12 also increases splenic cellularity, secondary to an influx of activated macrophages and NK cells. In contrast, IFN‐γR1 (‐/‐) mice injected with IL‐12 mainly develop a severe, lethal lung disorder characterized by interstitial and perivascular infiltrates and diffuse pulmonary edema. Therefore, it appears that, in the absence of IFN‐γ responses, many effects of IL‐12 are attenuated. On the other hand, IL‐12 appears to induce a different set of immune disorders that are independent of, or normally suppressed by, IFN‐γ. Other experiments also suggest that IL‐12 may have IFN‐γ‐independent effects. For example, in one study of collagen‐induced arthritis (CIA), the severity of this autoimmune disease was attenuated in IFN‐γR1‐deficient mice after treatment with anti‐IL‐12 neutralizing antibodies, which are thought to suppress inflammatory disease by, in part, indirectly inhibiting IFN‐γ secretion ( 97, 98). Therefore, while many of the effects of IL‐12 (such as cross‐regulation of type 2 inflammatory responses) are abrogated in IFN‐γR1 (‐/‐) mice, other effector functions of IL‐12 (such as direct Th1 priming, which may explain the CIA phenotype) may be independent of IFN‐γ ( 90, 97, 99, 100).

Primary CD4+ T‐cell responses in IFN‐γR1‐deficient mice

Naive CD4+ cells, or Thp cells, are believed to have the potential to develop into either of the two major subsets of CD4+ T helper cells: Th1 and Th2 cells ( 100, 101) ( Fig. 2 ). Th1 cells are primarily defined by their ability to secrete IFN‐γ, IL‐2, and tumor necrosis factor (TNF)‐β, and are clinically associated with an ability to orchestrate cell‐mediated immune responses and organ‐specific autoimmunity ( 102). Differentiation of Thp cells toward the Th1 phenotype is dependent on the presence of IL‐12 in their microenvironment during stimulation throughout the T‐cell antigen receptor (TCR) ( 92, 103). Th1‐polarizing effects similar to those of IL‐12 have been ascribed to IFN‐γ ( 104). On the other hand, Th2 cells arise when IL‐4 is present during antigenic stimulation, and produce IL‐4, IL‐5, IL‐10, and IL‐13 ( 105). These cells are clinically associated with phagocyte‐independent, antibody‐mediated host defense and allergic immune responses ( 102). Because of the potential role of IFN‐γ in generating Th1 cells, mediating their effector function and regulating Th1/Th2 balance, T‐cell responses were examined in IFN‐γR1 (‐/‐) mice ( 94, 99, 106).

In some experimental models utilizing IFN‐γR1 (‐/‐) mice, there appeared to be a shift in the Th1/Th2 balance in response to stimuli that normally induce Th1 immunity (Fig. 2). For example, as described above, a predominant Th2 response ensued in response to systemic IL‐12 administration in these mice ( 96). A similar Th2‐directed immune disorder was also seen in one model of schistosomiasis, where not only were Th1‐dependent immune responses impaired, but also aberrant Th2 responses led to a severe lung disorder ( 107). These observations support previous work that suggested that, while IFN‐γ is normally considered an effector of inflammation, it may have an important inhibitory role in certain immune responses ( 108). In another study, Th2‐mediated pulmonary inflammation was induced by rechallenge of previously sensitized mice with nasally administered OVA ( 109). This inflammatory lung disease persisted in IFN‐γR1 mutant mice long after it was resolved by wild‐type mice. Allergic pulmonary inflammatory reactions are often mediated by a type 2 response characterized by eosinophils, IgE antibodies, and cytokines such as IL‐4 and IL‐5 ( 110, 111, 112). Therefore, one explanation of the observed lung phenotype in IFN‐γR1 mutant mice is that, in the absence of the cross‐regulation normally seen between Th1 and Th2 responses (with Th1‐derived IFN‐γ presumably suppressing the Th2 response), a Th2‐mediated immune response can proceed unhindered ( 99, 102). Alternatively, there may be other mechanisms by which IFN‐γ suppresses inflammation ( 108, 113).

In another group of studies, the response to the pathogen in mutant mice was normal, without a concomitant Th2‐mediated disorder, although abnormally high levels of Th2 cytokines and/or Ig isotypes were detected (see Table 1 for a summary of all disease models in which IFN‐γR1 (‐/‐) mice were used). For instance, the outcome of mouse mammary tumor virus (MMTV) infection is similar in mutant and control mice, but is characterized by increased IL‐4 levels in the former ( 114). In other models, IFN‐γR1 (‐/‐) mice elaborated defective Th1 responses with no associated Th2 response. For example, while wild‐type mice are resistant to infection by either coronavirus or Leishmania major, IFN‐γR1 mutant mice are susceptible to these pathogens ( 115, 116). An analysis of the pathologic changes in these mice revealed no evidence of a Th2‐type immune response.

Table 1

Pathogen

Phenotype in normal mice

Phenotype in IFN‐γR1 (‐/‐) mice

In summary, some of these studies uncovered global defects in T‐cell responses, others demonstrated a specific Th1 dysfunction, and yet others revealed an altered Th1/Th2 balance in IFN‐γR1 (‐/‐) mice. However, the extent of the difference in the clinical course of disease between wild‐type and mutant mice was found to be highly variable. Nevertheless, these data suggest an interesting trend in T‐cell‐mediated immunity in IFN‐γR1 (‐/‐) mice: a Th2‐bias with impaired Th1 responses. The observed set of T‐cell phenotypes may be the consequence of a host of factors. For instance, IFN‐γ‐insensitive macrophages and dendritic cells are impaired in their ability to upregulate expression of MHC class II molecules on their cell surface ( 81). This defect in antigen presentation may alter CD4+ T‐cell activation in these mice. IFN‐γR1 (‐/‐) antigen‐presenting cells (APCs) are also unable to secrete normal amounts of IL‐12, an important Th1‐polarizing stimulus ( 120). This may lead to a bias toward Th2‐type responses. Moreover, it is believed that Th1‐derived IFN‐γ can cross‐regulate Th2 cells ( 99). Unresponsiveness of Th2 cells to IFN‐γ would therefore lead to a bias toward type 2 immune responses. It has also been suggested that in the absence of IFN‐γ responses, monocytes can become alternatively activated, and immature dendritic cells can develop into alternatively activated dendritic cells (Dc2) ( 121, 122, 123). Alternatively activated macrophages and Dc2 cells have been shown to support differentiation of Thp cells to the Th2 phenotype. The role of these unusual APCs in promoting the Th2 pathway may, in part, account for the inability of IFN‐γ‐unresponsive mice to mount type 1 inflammatory processes. Of course, it is also likely that IFN‐γR1 (‐/‐) T cells have intrinsic differences in their response to antigen. Robust type 2 responses seen in some of these immune models suggest that Th2 cells are less dependent than Th1 cells on antigen presentation by IFN‐γ‐primed APCs ( 96, 109, 121, 122). Finally, T cells must be able to migrate to their target sites in order to be effective regulators and mediators of immunity. The expression of adhesion molecules on vascular endothelial cells and on T cells, as well as the production of a number of chemokines, is regulated by IFN‐γ ( 117, 118, 119). Therefore, part of the observed T‐cell defect may be secondary to impaired migration to target sites.

CD4+ T‐cell memory in IFN‐γR1‐deficient mice

Like primary T‐cell‐mediated immunity, immunologic memory, which relies on T helper cell function, may be affected by the absence of a functional IFN‐γ system ( 124, 125, 126). To assay the role of IFN‐γ responsiveness in secondary T‐cell responses, IFN‐γR1 (‐/‐) mice were used in a number of immunization models. In every instance, mutant mice were unable to respond normally to antigenic rechallenge. Antigenic recall was impaired in these mice in response to rechallenge with Chlamydia trachomatis, pseudorabies virus, Schistosoma mansoni, and Plasmodium yoeli ( 107, 127, 128, 129). In fact, previously sensitized IFN‐γR1 (‐/‐) mice elaborate inappropriate, and therefore unproductive, T helper cell‐mediated responses upon re‐exposure to S. mansoni ( 129). While the normal response to rechallenge with this pathogen is characterized by a strong Th1 component, helping contain the infection, the response in mutant mice is Th2 in nature, and it is ineffective in controlling parasitemia. Interestingly, repeated exposure of IFN‐γR1 (‐/‐) mice to P. yoeli led to lasting immunity against this pathogen, thereby somehow bypassing the need for the IFN‐γ system ( 129). It therefore appears that T‐cell memory and, perhaps more specifically, Th1‐mediated secondary responses are usually dependent on IFN‐γ signaling.

CD8+ T‐cell function in IFN‐γR1‐deficient mice

Like CD4+ T helper cells, CD8+ cytotoxic T lymphocytes (CTLs) are activated by an antigen presented in the context of an MHC molecule. However, unlike MHC class II, MHC class I molecules (which present intracellularly derived peptides to CD8 cells) are ubiquitously expressed ( 130, 131). Interestingly, recent evidence suggests that CD8+ T cells must first be activated by dendritic cells (that, in this case, encounter and then present extracellularly derived antigen in the context of MHC class I molecules) before leaving lymphoid areas and surveying the periphery ( 132). Moreover, whereas antigen presentation through the MHC class II pathway is enhanced mainly by IFN‐γ, antigen presentation through the MHC class I pathway can be stimulated by either IFN‐γ or IFN‐α ( 2, 130, 131, 133, 134). Tc1 cells, which constitute the predominant CD8+ T‐cell subset, secrete substantial amounts of the IFN‐γ that is thought to participate in some aspects of their effector function ( 135). It was therefore of interest to examine CTL function in IFN‐γR1 (‐/‐) mice in order to clarify the relevance of IFN‐γ sensitivity to the function of these cells.

CTLs derived from IFN‐γR1 mutant mice infected with vaccinia virus lysed target cells infected with this virus normally, suggesting that IFN‐γ‐insensitive CTLs are capable of elaborating normal cytotoxic effector and memory functions ( 26). CD8+ cells in LCMV‐infected IFN‐γR1 (‐/‐) mice exhibited delayed kinetics of clonal exhaustion as compared to wild‐type mice, indicating that they are less susceptible to activation‐induced cell death (AICD), perhaps owing to inefficient activation (which may be secondary to either an antigen presentation or an intrinsic T‐cell defect) ( 136).

A number of studies suggest that CD8+ T cells are important mediators of the effector phase of contact hypersensitivity (CHS) responses to haptens ( 137, 138, 139, 140). Although tissue swelling in response to oxazolone and TNCB was comparable in IFN‐γR1 (‐/‐) mice and wild‐type mice, mutant mice had reduced dermal mononuclear infiltrates ( 141). Because of the complexity of this model of immunity, impaired CHS may be caused by defects in either the sensitization phase (dendritic cell function, homing, or antigen presentation) or elicitation phase (chemokine secretion; CD8 T‐cell activation or migration) of this response ( 142, 143, 144). Therefore, reduced dermal mononuclear infiltrates in IFN‐γR1‐deficient mice may not be caused by an intrinsic CD8+ T‐cell defect. Although CD8+ CTL also participate in graft rejection ( 145, 146, 147), models of allo‐ and xeno‐transplantation failed to uncover differences in the ability of wild‐type and IFN‐γR1 mutant mice to reject transplanted grafts ( 148, 149). These data suggest that, for the most part, intrinsic CTL function appears to be intact; CD8+ T cells can elaborate normal effector and memory functions despite their inability to transduce an IFN‐γ signal.

Resistance of IFN‐γR1 (‐/‐) mice to viral infection

Both IFN‐γR1‐ and the IFN‐αR‐deficient mice proved to be valuable model systems in elucidating the specific roles of types I and II interferons (IFN‐α/β and IFN‐γ, respectively) in fighting viral infection ( 114, 115, 128, 150, 151, 152, 153). In vitro studies have revealed that interferons must initiate signaling via their respective cell‐surface receptors in order to protect cells against the cytopathic effects of viruses ( 26, 76). In vivo studies, on the other hand, demonstrated that while type I interferons are essential for protection against viral infection, the relative importance of IFN‐γ is pathogen‐dependent, suggesting that the antiviral actions of type II interferon are partially redundant, as they duplicate those of type I interferons ( 114, 115, 128, 150, 151, 152, 153). IFN‐γR1‐deficient mice were found to be resistant to some viruses (such as vesicular stomatitis virus, LCMV, and MMTV) while susceptible to others (such as murine γ‐herpesvirus, vaccinia virus, Theiler's virus, murine cytomegalovirus, and coronavirus) ( 114, 115, 128, 150, 151, 152, 153).

Even though IFN‐γR1‐deficient mice were able to resolve some viral infections, they developed exacerbated disease in response to these pathogens as compared to control mice. For example, mutant mice could clear an infection with LCMV but exhibited delayed kinetics of viral clearance as compared to wild‐type mice ( 151). LCMV‐infected IFN‐γR1 (‐/‐) mice developed greater organ abnormality and had an increased frequency of latently infected cells relative to control mice. These studies suggest that the immune system elaborates a number of simultaneous and/or sequential, functionally complementary, antiviral responses. Some of these may be dependent on IFN‐γ, although not all may be essential for a healing or normal response.

Intracellular bacterial infection in IFN‐γR1 (‐/‐) mice

Survival and clearance of infection with intracellular bacteria are dependent upon innate and cell‐mediated immunity ( 154, 155). IFN‐γR1‐deficient mice uniformly exhibit greater susceptibility to, or increased severity of bacterial infection relative to wild‐type mice. Infection of mutant mice with Chlamydia trachomatis is characterized by a longer resolution time than in normal mice ( 156, 157). While mice normally contain and resolve infections with intracellular bacteria such as bacillus Calmette‐Guérin (an attenuated form of Mycobacterium bovis), Bordetella pertussis, and Listeria monocytogenes, these pathogens disseminate, often fatally, in mutant mice ( 82, 156, 157). As discussed earlier, IFN‐γ is an important activating stimulus for macrophages, which are involved in granuloma formation, a mechanism essential for containing and eliminating intracellular bacteria. Activated macrophages also undergo a respiratory burst, producing reactive oxygen species and NO, both of which are important for their bactericidal function ( 2). Therefore, it is likely that an underlying macrophage defect is the cause of the exquisite sensitivity of IFN‐γR1 (‐/‐) mice to infection with intracellular bacteria ( 81).

Resistance of IFN‐γR1 (‐/‐) mice to protozoan and helminth infection

Control of parasitic infection is dependent upon the development of an appropriate immune response. In general, Th1 responses are associated with healing responses to intracellular parasites, such as Leishmania major, whereas Th2 responses are associated with protection from extracellular pathogens, such as Nippostrongylus brasiliensis ( 158, 159). The innate immune system is also important in protection against parasites. The role of IFN‐γ in protection against parasitic infection and in the development of Th1‐ and Th2‐type immune responses was explored with IFN‐γR1 (‐/‐) mice.

Protection against infection with L. major depends on normal macrophage and Th1 function ( 160, 161). Infection of IFN‐γR1 (‐/‐) mice with L. major or trypanosomes is associated with significantly greater mortality than that seen in control mice in the absence of any detectable parameters of Th2‐type immunity ( 80, 83, 116). Interestingly, iNOS (inducible nitric oxide synthase)‐deficient mice display a similar phenotype in response to Trypanosoma cruzi infection to the IFN‐γR1 (‐/‐) ( 80). These studies show that the IFN‐γ system is critical for host defense against these intracellular pathogens, and indicate a defect in macrophage function as a key contributor to the observed phenotype. Studies of infection with Toxoplasma gondii suggest that the primary defect in antiprotozoan immunity in IFN‐γR1‐deficient mice may not be due to a T‐cell dysfunction ( 79). When infected with a low‐virulence form of this protozoan, normal mice are able to control infection, but develop chronic toxoplasmosis. On the other hand, IFN‐γR1 (‐/‐) mice are unable to control infection, developing a necrotizing hepatitis, and ultimately succumbing to the pathogen. T. gondii‐specific memory T cells derived from wild‐type mice are unable to confer immunity when adoptively transferred to mutant mice. Furthermore, hepatic macrophages in infected IFN‐γR1 (‐/‐) mice produced lower levels of TNF‐α, iNOS, and IL‐1‐β ( 79). These observations suggest that the specific impaired response to T. gondii infection in IFN‐γR1 (‐/‐) mice may be secondary to alteration of macrophage function.

Infection of IFN‐γR1 (‐/‐) mice with Plasmodium species recapitulates some of the immune system dysfunction seen in other infections studies ( 129, 163). Relative to normal mice, the symptoms of infections with a number of Plasmodium species of mutant mice range from prolonged convalescence to increased incidence of death. The immune response in IFN‐γR1‐deficient mice to some of these pathogens was Th2‐biased relative to the anti‐ Plasmodium immunity seen in normal mice. This, again, suggests that in the absence of IFN‐γ signaling, the elaboration of Th1 responses is impaired. In sum, mutant mice infected with parasites elaborate impaired or inappropriate immune responses that alter the clinical course of the disease, and at times lead to increased mortality as compared to normal mice.

Autoimmunity in IFN‐γR1 (‐/‐) diseases

Organ‐specific autoimmune diseases are believed to be mediated and regulated by Th1 cells and IFN‐γ ( 102, 113, 164). This paradigm, however, is not all‐encompassing since the relationship between IFN‐γ and organ‐specific autoimmune disorder is complex. IFN‐γ has been shown to have differential effects on disease progression based on the mode (local vs systemic) and timing (early or late in disease) of its administration or neutralization ( 113). To explore the IFN‐γ dependence of organ‐specific autoimmune diseases, the IFN‐γR1 (‐/‐) mice have been used in a number of model systems including CIA, nonobese diabetes (NOD), autoimmune lupus nephritis, experimental autoimmune thyroiditis (EAT), experimental autoimmune myasthenia gravis (EAMG), and experimental autoimmune encephalomyelitis (EAE) ( 165, 166, 167, 168). In most cases, the development of organ‐specific autoimmune diseases in IFN‐γR1‐deficient mice follows this Th1/Th2 paradigm. For example, IFN‐γR1 (‐/‐) mice are less susceptible to these pathologic processes and exhibit reduced penetrance, delayed onset, or attenuated severity of disorder as compared to normal mice in diseases such as diabetes (both NOD and the BDC2.5 insulinogenic TCR transgenic system), EAMG, and EAT ( 167, 168). Additionally, administration of soluble IFN‐γR1 (which neutralizes IFN‐γ) reduces the severity of NOD in wild‐type mice ( 169). These studies demonstrate the importance of the IFN‐γ system in the development of these organ‐specific autoimmune diseases.

CIA is induced in susceptible mouse strains, such as DBA/1, by immunization with type II collagen protein ( 170, 171). This autoimmune model has been used as a tool for studying the development and treatment of rheumatoid arthritis in man. The development and regulation of CIA in mice has been shown to rely on IL‐12 and, less consistently, on IFN‐γ ( 98, 172). Neutralization of IL‐12 leads to attenuation of disease, suggesting that it is Th1‐mediated, with cellular immunity and IgG2a‐type antibodies causing the abnormality. Studies with IFN‐γ‐deficient and IFN‐γR1 (‐/‐) mice corroborated this observation; these mice were more resistant to CIA induction ( 166). However, in other studies of CIA, IFN‐γR (‐/‐) mice developed a more severe disease than did normal mice, suggesting that, in some cases, IFN‐γ may have immunosuppressive functions ( 173).

IFN‐γ also appears to attenuate the severity of disease in the MOG (myelin oligodendrocyte glycoprotein) peptide‐induced EAE system ( 174). Wild‐type 129/Sv mice are resistant to EAE, whereas IFN‐γR1 (‐/‐) are susceptible. This suggests that IFN‐γ can not only promote inflammation, but also serve in an inflammation‐limiting capacity in certain cases. In fact, mechanistically, immunosuppressive properties have previously been ascribed to IFN‐γ secondary to its ability to induce NO production in macrophages, a production which has a downmodulatory effect on T‐cell activation and proliferation ( 2, 108, 162). Furthermore, adoptive transfer of splenocytes from MOG‐immunized IFN‐γR1‐deficient mice can induce disease in naive wild‐type recipients, suggesting the presence of functional memory or effector CD4+ T cells or, perhaps, a functional CD8‐mediated component in this disease ( 174).

A number of studies utilized strains of mice that are predisposed to systemic autoimmunity, in order to examine the importance of IFN‐γ in the development of autoantibodies and immune complex deposition‐induced kidney disease. Mice on the MRL‐ Fas lpr and the NZB X NZW genetic backgrounds lacking IFN‐γR1 expression develop attenuated disease relative to normal mice ( 175, 176, 177). IFN‐γR1 (‐/‐) mice in these autoimmune models produce fewer antibodies (with Th1‐ and Th2‐dependent Ig isotypes equally affected) and, therefore, develop less severe immune complex‐induced nephritis and show reduced incidence of death as compared to control mice. T‐cell activation, T:B‐cell interaction, or an intrinsic B‐cell defect may underlie impaired antibody production in the absence of IFN‐γ signaling. Like NOD, the disease phenotype in normal NZB X NZW mice can be attenuated by systemic administration of soluble IFN‐γR1 ( 178). Crescentic glomerulonephritis is experimentally induced by administration of anti‐glomerular basement membrane (GBM) Abs. IFN‐γR1‐deficient mice treated with αGBM Abs developed slightly less severe glomerular disease than wild‐type mice, suggesting that antibody production in autoantibody‐mediated immune disorder is primarily affected in the absence of an IFN‐γ signal, rather than the events that ensue after immune complex deposition ( 179).

Disrupting IFN‐γ signaling with a mutant IFN‐γR1

Tissue‐specific transgenic mouse model of IFN‐γ insensitivity

To distinguish among the effects of IFN‐γ on the various cell types whose function is thought to be particularly affected by this cytokine, it is useful to disrupt IFN‐γ signaling in only a subset of cells. Schreiber's group developed a transgenic system in which the expression of a dominant negative IFN‐γR1 is driven by a tissue‐specific promoter, thereby conferring unresponsiveness to IFN‐γ in one cell type, while leaving all others intact ( 180). The dominant negative receptor α chain (mgRΔIC) lacks the intracellular domain of the wild‐type protein; therefore, while it can participate in ligand binding and receptor complex formation, it is unable to transmit the IFN‐γ signal ( 181).

In order to generate mice in which only macrophages are unable to respond to the IFN‐γ signal, the human lysozyme promoter (hLP) was used to drive expression of the dominant‐negative transgene (hLP‐myc‐mgRΔIC) ( 180). The dominant negative α chain was indeed specifically expressed on macrophages, and inhibited IFN‐γ responses in these cells. As such, macrophages derived from this mouse were unable to produce NO in response to stimuli such as IFN‐γ and lipopolysaccharide (LPS) ( 180, 182). Macrophages derived from IFN‐γR1 (‐/‐) mice showed diminished responses to LPS as well ( 82). hLP‐myc‐mgRΔIC transgenic mice were found to be more susceptible to infection with L. monocytogenes than littermate controls. Moreover, the authors have found that neither hLP‐myc‐mgRΔIC TG macrophages nor IFN‐γR1 (‐/‐) macrophages were able to produce IL‐12 after treatment with either IFN‐γ or heat‐killed L. monocytogenes (HKLM), whereas control macrophages responded robustly. Macrophages from these two mutant mouse strains were also unable to support HKLM‐specific memory responses by wild‐type Th1 cells in vitro. Therefore, it can be inferred that IFN‐γ is pivotal in activation and function of macrophages as well as in their ability to serve as APCs to CD4+ T cells.

For determination of whether the impaired T‐cell function seen in IFN‐γR1 (‐/‐) mice was secondary to an intrinsic T‐cell defect, transgenic mice (lck‐myc‐mgRΔIC) with T‐cell‐specific expression of this mutant receptor were generated with the lck proximal promoter ( 180). In vitro, lck‐myc‐mgRΔIC TG T cells were resistant to the antiproliferative effects of IFN‐γ as compared to nontransgenic T cells. Interestingly, the transgene did not affect in vitro T helper cell differentiation under Th1‐polarizing conditions (in the presence of IL‐12), nonpolarizing conditions, or Th2‐polarizing conditions (in the presence of IL‐12‐neutralizing Abs). In contrast, polarization of transgenic T cells toward the Th2 phenotype (driven by IL‐4) may have been slightly enhanced, supporting the inhibitory role IFN‐γ may play in this differentiative process. This evidence suggests that the in vitro generation of Th1 cells can occur independently of an IFN‐γ signal in the presence of IL‐12.

The IFN‐γR signal and antitumor immunity

IFN‐γ has been shown to participate in antitumor immune responses ( 1, 2). However, the potential differential effects of this cytokine on the tumor, as opposed to the host, have not been explored. The dominant negative transgenic system described above was utilized to evaluate the role of IFN‐γ signaling by cancer cells in antitumor immune responses in a normal host. The dominant‐negative IFN‐γR1 mutant was introduced into the Meth A fibrosarcoma cell line, rendering these cells unresponsive to IFN‐γ ( 183). Modified cells were resistant to LPS‐induced rejection in a normal syngeneic host and exhibited a greater tumorigenic potential than the IFN‐γ‐responsive parental cell line. Furthermore, IFN‐γ‐insensitive cells were ineffective in priming their host to reject subsequent tumor grafts, and did not elicit antitumor immunity when grafted into a host previously sensitized by the parental cell line. One interpretation of these observations is that IFN‐γ‐resistant tumor cells are not inherently more aggressive in lesion formation per se, but are less immunogenic than their IFN‐γ‐sensitive counterparts. In a more recent study, Lee's group modified two transformed murine cell lines (SKC and K1735) to be unresponsive to IFN‐γ, utilizing a similar dominant‐negative IFN‐γR1 construct, and subsequently tested their ability to form tumors in mouse hosts ( 184). IFN‐γ‐resistant cell lines were more tumorigenic than IFN‐γ‐responsive parental lines, corroborating previous studies. IFN‐γ augments antigen presentation through both the MHC class I and class II pathways, thereby increasing the immunogenicity of tumor cells and enhancing their detection and killing by immune surveillance and effector mechanisms ( 131, 185, 186, 187). It is also believed that IFN‐γ alters the types of peptides that are presented through the MHC class I pathway by inducing expression of alternate LMP and TAP molecules. Therefore, in the absence of IFN‐γ signaling, tumor cells may be unable to present peptides that normally activate antitumor immunity, thereby evading detection by surveillance mechanisms.

The effects of disruption of IFN‐γR2 expression in mice

To study the importance of the β chain of the IFN‐γR for mediating the biologic functions of IFN‐γ, our laboratory generated mice carrying a targeted mutation in the IFN‐γR2 locus, abrogating its expression ( 27). These mice developed normally in a pathogen‐free facility, with a normal composition of lymphocyte populations in lymphoid organs. IFN‐γ signaling, however, was abrogated at all steps of the cascade. Cells derived from IFN‐γR2(‐/‐) mice were unable to activate JAK1, JAK2, and STAT1 (all expressed at normal levels) or to express IFN‐γ‐inducible genes in response to IFN‐γ treatment. When cultured on anti‐CD3 antibody‐coated plates, naive CD4+ T cells isolated from spleens of IFN‐γR2 (‐/‐) were impaired in their ability to differentiate toward the Th1 subset under Th1‐polarizing conditions (in the presence of either IFN‐γ or IL‐12), but exhibited normal Th2 differentiation ( Fig. 2 ). Interestingly, no default toward the Th2 phenotype was observed when these cells were cultured under neutral conditions.

Consistent with these in vitro findings, IFN‐γR2 (‐/‐) mice have profound defects in Th1‐mediated immunity, such as Th1 memory responses to rechallenge with protein antigen. The ability of IFN‐γR2 (‐/‐) B cells to undergo immunoglobulin heavy‐chain class switching to IFN‐γ‐dependent isotypes, such as IgG2a, was also impaired. Additionally, the inhibition of class switching to IL‐4‐dependent isotypes, such as IgG1 and IgE, normally observed when B cells are cultured in the presence of both IL‐4 and IFN‐γ, was not seen under these conditions. As in IFN‐γR1 (‐/‐) mice, impaired macrophage function may be central to the immune disorder in IFN‐γR2 (‐/‐) mice. β chain‐deficient mice are highly susceptible to infection with L. monocytogenes, a pathogen that primarily infects macrophages and requires their proper function for a healing response. Additionally, contact hypersensitivity responses were defective in these mice.

These studies not only establish the obligatory role of IFN‐γR2 in transducing the IFN‐γ signal and thus its biologic function, but also demonstrate that IFN‐γ is critical for the development of functional Th1 cells and Th1‐dependent immunity. Though Th1 cells are normally unresponsive to IFN‐γ (see below), abrogation of their ability to regulate responsiveness to this cytokine during their development may impair their generation and function. Alternatively, the T‐cell defect seen in IFN‐γR2 (‐/‐) mice may be, in part, secondary to impaired APC function, which may inefficiently activate or polarize CD4+ T cells. Furthermore, IL‐12 did not seem to have a Th1‐polarizing effect, suggesting that, in this system, this function of IL‐12 requires IFN‐γ.

In general, mice deficient in either α or β chains of the IFN‐γR had similar defects in their immune systems. Interestingly, while in vitro Th1‐polarized CD4+ T cells expressing the dominant‐negative IFN‐γR1 transgene were able to produce IFN‐γ as well as normal CD4+ cells, helper T cells derived from IFN‐γR2‐deficient mice could not. The discrepancy between these studies may be due to differences in the type of stimuli used; mgRΔIC TG T cells were stimulated with APC+ peptide whereas IFN‐γR2 (‐/‐) T cells were stimulated with αCD3. This is consistent with previous studies that have shown that the quality of the activating stimulus can affect T helper subset phenotype acquisition ( 188). It is also possible that the reason underlying the difference between the in vitro differentiation potential of T cells from these two lines of mutant mice is that the dominant‐negative IFN‐γR1 construct does not completely abolish the IFN‐γ signal in T cells but, instead, attenuates it considerably.

Obligate IFN‐γ responsiveness imparted by IFN‐γR2 transgene

Several groups have previously shown that, whereas Th2 cells are responsive to IFN‐γ, Th1 cells are unable to activate IFN‐γ signaling in response to this cytokine ( 64, 65, 66, 189, 190). These data corroborate earlier studies demonstrating differential growth arrest of Th1 and Th2 clones upon IFN‐γ treatment, with only the latter helper T‐cell subset susceptible to the antiproliferative effects of this cytokine ( 191). Further analysis of the components of the IFN‐γ response pathway in Th cells revealed that, unlike Th2 cells, Th1 cells do not express mRNA encoding IFN‐γR2 ( 65) ( Fig. 2 ). Transfecting an IFN‐γR2 construct into Th1 cells rescues their IFN‐γ‐unresponsive phenotype ( 65). These data suggest that during T helper subset differentiation, responsiveness to IFN‐γ is regulated by expression of IFN‐γR2.

Analysis of the immune functions of IFN‐γ‐insensitive mice suggests that IFN‐γ promotes Th1 cell development and function. Therefore, Th1 cells must be able to respond to this cytokine during their development, phenotype acquisition, and/or other points during their growth cycle. However, during their differentiation, Th1 cells stop expressing IFN‐γR2 and become unresponsive to IFN‐γ. We hypothesized that downmodulation of IFN‐γR2 expression, and therefore the loss of responsiveness to IFN‐γ, is a significant event in the process of Th1 phenotype acquisition and may, in fact, be important for normal Th1 cell function. To test this hypothesis, we engineered mice in which expression of IFN‐γR2 cDNA is driven by the human CD2 locus control region (LCR); thus, all CD2+ cells (TNK, and some B cells) would constitutively express this receptor ( 192). The β chain transgene rescued the IFN‐γ‐insensitive phenotype of Th1 cells, as Th1 cells cloned from transgenic (TG) mice were responsive to IFN‐γ, whereas wild‐type Th1 cells were not.

Like IFN‐γR‐deficient mice, IFN‐γR2 TG mice exhibited no gross developmental abnormalities and had normal lymphocyte numbers and composition in lymphoid organs; however, when challenged, IFN‐γR2 TG mice were found to have specific immunologic defects. IFN‐γR2 TG mice were impaired in their ability to generate Th1 cells in vitro ( Fig. 2 ). These mice were also unable to elaborate Th1‐mediated memory responses such as DTH (delayed‐type hypersensitivity) in vivo, and responses to protein and bacterial antigens (such as keyhole limpet hemocyanin and heat‐killed L. monocytogenes, respectively) in vitro ( 192, 193).

Unlike T‐cell function, antigen presentation in IFN‐γR2 TG appears to be normal. In vitro, TG APCs are able to support normal T‐cell activation, whereas normal APCs were unable to remedy the T‐cell defect in TG T cells ( 192, 193). Survival of primary listeriosis is dependent on innate immunity including leukocytes such as neutrophils, NK cells, and especially macrophages ( 154, 155). Naive IFN‐γR2 TG mice withstood challenge with L. monocytogenes, indicating that macrophage function is intact in these mice ( 193). It is possible that the IFN‐γ necessary for normal macrophage activation is secreted by NK cells in these mice. While host defense against L. monocytogenes depends primarily on the innate immune system, survival of infection with Leishmania major also requires normal Th1 function ( 160). TG mice are susceptible to infection with L. major, and develop nonhealing lesions, suggesting, once again, that Th1 responses in these mice are impaired ( 192).

CNS pathology in EAE (a model of autoimmune demyelinating disease) is believed to be mediated by Th1 cells ( 164). We examined the effect of forced responsiveness to IFN‐γ in Th1 cells on the induction of this organ‐specific autoimmune disease ( 193). While control mice developed severe and often fatal paralysis, IFN‐γR2 TG mice were generally resistant to EAE induction. The ability to modulate IFN‐γR2 expression in Th cells is therefore required for a broad range of Th1 effector functions including autoimmunity.

Like helper T cells, CD8+ T cells can be grouped into an IFN‐γ‐producing subset (Tc1) and an IL‐4‐producing subset (Tc2) ( 194, 195). We have recently examined patterns of IFN‐γ responsiveness in Tc subsets and found that, like Th1 cells, Tc1 cells lack IFN‐γR2 and are therefore unable to activate STAT1 or upregulate cell‐surface expression of MHC class I molecules in response to IFN‐γ ( 193). Tc1 clones generated from IFN‐γR2 TG mice are responsive to IFN‐γ, whereas those generated from littermate controls do not respond to IFN‐γ. TG Tc1 clones have profound defects in their cytotoxicity (measured by their ability to lyse target cells) as compared to wild‐type CTLs. TG Tc1 clones, however, do not show impaired activation, proliferation, or IFN‐γ secretion, indicating that the cytolytic effector functions of these cells are not necessarily dependent on IFN‐γ. It is possible that other effector mechanisms of Tc1 cells, such as FasL expression or perforin production, may be impaired. Contact hypersensitivity responses to haptens in IFN‐γR2 TG mice are markedly diminished ( 192). As discussed earlier, CHS is dependent on normal APC and CD8⁺ T‐cell function. Since antigen presentation appears normal in these mice, the defect in this in vivo model of CTL function may lie in the CD8⁺ T‐cell compartment. Interestingly, earlier studies demonstrated that although exogenous IFN‐γ can augment CTL activity and proliferation in vitro, this cytokine is not required for their effector activity ( 196, 197).

Taken together, these studies demonstrate that IFN‐γR2 is obligatory in transducing the IFN‐γ signal and in mediating its immunologic function. Furthermore, forced responsiveness to IFN‐γ results in severe Th1 and CTL dysfunction that is intrinsic to these cells and is unlikely to be related to impaired antigen presentation. Therefore, modulation of responsiveness to IFN‐γ (by regulating expression of the IFN‐γR2 gene) during Th1 and Tc1 differentiation, and probably in the mature Th1 and Tc1 states, is essential for the generation of these cells and their proper function.

Naturally occurring IFN‐γR mutations in man

Man is normally resistant to nontuberculous mycobacteria (NTM) and BCG. Nevertheless, cases of disseminated atypical mycobacterial infection in children have been reported ( 198, 199, 200). The underlying causes in most of these cases were found to be classical immunodeficiencies such as severe combined immunodeficiency (SCID) or chronic granulomatous disease. Genetic mapping and Mendelian analysis of idiopathic cases have revealed mutations in either IFN‐γR1 or IFN‐γR2 that are transmitted in an autosomal recessive pattern. Interestingly, children unresponsive to IFN‐γ were found to be resistant to most common viral and microbial infections or fungi, and presented solely with disseminated mycobacterial infection ( Mycobacterium fortuitum, M. avium, M. bovis, BCG, M. chelonei, M. smegmatis, and M. tuberculosis), Salmonella enteritidis, or L. monocytogenes ( 201, 202, 203, 204, 205, 206, 207). A genetically heterogeneous array of alterations (missense, nonsense, insertions, deletions, and splice mutations) in the IFN‐γR1 locus were identified. In contrast, only one case in which the mutation lay in the IFN‐γR2 locus has been reported ( 202).

These cases of idiopathic, disseminated mycobacterial infection can be grouped into two phenotypic categories by their clinical presentation and outcome: complete and partial IFN‐γ insensitivity. Children with complete IFN‐γ insensitivity respond poorly to antimycobacterial treatment, either succumbing to the pathogen or developing chronic infection. These patients form poorly differentiated and poorly circumscribed granulomas and are therefore unable to control effectively their mycobacterial infection ( 204). Mutations underlying complete IFN‐γ insensitivity result in a truncated IFN‐γR1 or IFN‐γR2 peptide that is not expressed on the cell surface.

In contrast, the two siblings with partial IFN‐γ insensitivity were found to have a missense mutation in the IFN‐γR1 extracellular domain (I187T) ( 207). Although it is expressed, this IFN‐γR1 mutant binds IFN‐γR with very low affinity, and cells carrying this mutation respond only to very high doses of IFN‐γ. In vitro analysis of T cells isolated from these kindred and their parents show diminished parameters of antigenic recall such as T‐cell proliferative responses and IFN‐γ secretion. Granulomas in mycobacteria‐infected partially IFN‐γ‐insensitive individuals are well defined with fully differentiated, multinucleated epithelioid cells, and are circumscribed by lymphocytes. These patients respond to antimycobacterial treatment and can survive pathogen‐free without prophylaxis.

Cases of idiopathic mycobacterial infection arising from mutations in either of the IFN‐γR chains are helpful for defining the subset of nonredundant immunologic function of IFN‐γ essential for survival in a natural environment. All such kindred presented with cases of mycobacterial infection either secondary to immunization with BCG or naturally acquired, indicating that IFN‐γ plays a critical, nonredundant role in mediating antimycobacterial immunity ( 208). However, the absence of consistent presentation with other infections does not preclude the participation of IFN‐γ in immunity to other pathogens, but does suggest that IFN‐γ is redundant in other immune mechanisms. These findings suggest that responsiveness to IFN‐γ, and therefore IFN‐γR, is required for host defense against these intracellular pathogens whose clearance is mediated by macrophages. Impaired antigenic recall in these individuals suggests that IFN‐γR is important in the development of Th1‐mediated immunologic memory, although this defect may be, in part, caused by APC dysfunction rather than fully by T‐cell‐intrinsic mechanisms ( 209).

Commentary

The study of mice and human subjects with altered IFN‐γR expression pattern has told us a great deal about the diverse roles IFN‐γ plays in inducing and modulating immune responses. First and foremost, both the α and β chains of IFN‐γR are obligatory in initiating the known IFN‐γ signaling pathway through JAK1, JAK2, and STAT1 and in mediating the cellular effects of this cytokine. IFN‐γ responsiveness has been shown to be essential to, or at least to participate in, host defense against a wide array of pathogens including viruses, bacteria, and protozoa. Much of the phenotype in IFN‐γR‐deficient animals can be attributed to a defect in macrophage function. IFN‐γ responsiveness is critical for macrophage activation, function, and antigen presentation. These cells play a pivotal role in both innate and acquired immune responses. They must be activated for clearance of intracellular pathogens such as Listeria and mycobacteria and the development of their phagocytic functions. They are also important in further activating NK cells by secreting IL‐12. Neutrophils are also activated by IFN‐γ ( 182). Therefore, in infections such as listeriosis, where the first leukocyte line of defense is mediated by neutrophils, impaired function of these cells undoubtedly enhances the susceptibility of IFN‐γ‐unresponsive mice to such infections.

Unlike the IFN‐γR (‐/‐) mice, in which a macrophage defect supersedes the T‐cell defect, T‐cell function is clearly impaired in IFN‐γR2 TG mice. These animals have allowed us to explore the functional role of IFN‐γ‐responsiveness and the ability of T cells to modulate it. We have demonstrated that Th1 and Tc1 cells that are constitutively responsive to IFN‐γ are unable to elaborate their respective effector functions. It is possible that constitutive signaling through the IFN‐γR (such as in TG Th1 and Tc1 cells which are continuously bathed in an IFN‐γ‐rich milieu) activates inhibitory pathways, or inactivates an effector mechanism in these cells, thereby causing the observed defect. Therefore, not only must cells be responsive to IFN‐γ for normal innate, acquired, and antitumor immunity, but IFN‐γ‐secreting T cells also must be able to regulate their ability to respond to IFN‐γ for their normal function. It appears that for IFN‐γ, as for life in general, timing is everything.

Acknowledgments

We thank Miera Harris for her critical reading of this paper and Ned Braunstein for insightful suggestions and valuable discussion.

References

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Articles from Allergy are provided here courtesy of Wiley-Blackwell

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Biologic functions of the IFN‐γ receptors

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Allergy. 1999 Dec; 54(12): 1233-1251. Interferon‐gamma (IFN‐γ) is a cytokine that plays an important role in inducing and modulating an array of immune responses. Cellular responses to IFN‐γ are mediated by its heterodimeric cell‐surface receptor (IFN‐γR), which activates downstream signal transduction cascades, ultimately leading to the regulation of gene expression.

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O espetofotometro está ligado a um computador e é nele que vamos observamos os resultados. Os valores de irradiação de cada poço são automaticamente traduzidos para um Exel de forma a obtermos uma curva de irradiação por concentração. Esta serve como indicação da quantidade de cada uma das citocinas, pois a quantidade presente por poço é proporcional à irradiação medida.

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Acessar e isolar o RNA; o que é o TRIzol?

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Acessar e isolar o RNA; o que é o TRIzol?

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Para acessar e isolar o RNA é necessário destruir a membrana celular, bem como o separar do DNA e das proteínas da célula. Foi usado TRIzol para extrair o RNA e clorofórmio para o separar dos outros componentes da célula a descartar.

O TRIzol é um reagente à base de ácido-guanidínio-fenol idealmente projetado para a extração de RNA. O sal de guanidínio serve para desnaturar proteínas e o fenol é um composto orgânico também utilizado para extrair ácidos nucleicos e proteínas.

Este link foi resultado de uma pequena pesquisa de preparação para esta sessão com o objetivo de entender melhor o porquê de alguns passos do protocolo de purificação de RNA usando TRIzol:

https://pubmed.ncbi.nlm.nih.gov/20516177/ - consultado a 28/02/2024

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Vid. 12- Miguel a adicionar o TRIzol em cada eppendorf (PL)

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Começámos por ir buscar as nossas amostras ao congelador onde foram mantidas durante a semana a uma temperatura por volta dos -80ºC. Enquanto estas descongelavam, todos os materiais foram descontaminados de RNases antes de serem utilizados.

O conteúdo dos 25 diferentes poços da placa foi transferido para 25 eppendorfs numerados de 1 a 25, aos quais adicionámos 100 µL de TRIzol em cada um.

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Estás no bom caminho. Podes, ainda, fazer melhorias em algumas publicações, para as tornar mais claras e compreensíveis quanto ao objetivo de determinados procedimentos. Lê os comentários. Continua a investir.

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Fig. 14-Separação convencional da fase fenol-clorofórmio demonstrando as três camadas. A fase aquosa superior (RNA), a interfase e a fase orgânica (DNA e proteína). Posteriormente, o RNA é extraído da fase aquosa por precipitação isopropanol. - Consultado a 28/02/2024

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Após a solubilização e homogeneização das amostras em TRIzol, o RNA, o DNA e a proteína são diferencialmente extraídos pela adição de clorofórmio que provoca a separação por fases, em diferentes camadas (Fig. 14). A fase aquosa superior contém, principalmente, RNA, enquanto a fase orgânica e a interfase contêm DNA, proteínas e lípidios.

Adicionámos 80 µL de cromofórmio por eppendorf, tendo estes sido vortexados por 15 segundos, cada um. Posteriormente, foram deixados a incubar durante 15 minutos em gelo e, logo de seguida, centrifugados a 13000 rpm (rotações por minuto), durante 15 minutos e a 4ºC. Esta centrifugação permitiu-nos ter a certeza que o cromofórmio teve a possibilidade de atuar sobre todas as células, provocando a separação por fases e originando as diferentes camadas.

O RNA presente na fase aquosa superior tem que ser recolhido por precipitação à base de álcool. Para isso, a fase aquosa de cada eppendorf foi transferida para novos eppendorfs e foram adicionados 150 µL de isopropanol por eppendorf. O isopropanol vai promover a precipitação do RNA.

Para precipitar o RNA, os tubos foram incubados até à semana seguinte a -80ºC.

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Continuação do protocolo para isolar o RNA

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Continuação do protocolo para isolar o RNA

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Neste dia, começámos por retirar os nossos eppendorfs do congelador e, novamente, descontaminar a hotte e os instrumentos a usar com RNase AWAY.
Os eppendorfs foram centrifugados novamente a 13000 rpm, durante 15 minutos a 4ºC. Enquanto isso, preparámos um falcon de Etanol 70% (35 mL de Etanol 100% + 15 mL de H₂O ultrapura).

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Os próximos passos consistem numa lavagem em etanol e a sua evaporação (secagem):

Após a centrifugação dos eppendorfs, o sobrenadante foi descartado com pipetas de filtro e foram adicionados 400 µL de Etanol 70% por eppendorf e vortexou-se.

Como o nosso pellet seria pequeno, centrifugamos os eppendorfs a 10000 rpm, durante 10 minutos e a 4ºC. Para secar o pellet, retirou-se a maior parte do etanol com uma pipeta e deixou-se secar ao ar por alguns minutos. Acabámos por ter que esperar por esta secagem mais que o normal por não termos retirado etanol suficiente logo numa primeira fase.

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Link do artigo científico "Review of Neospora caninum and neosporosis in animals" - consultado a 27/02/2024

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O nosso projeto vai ser realizado tendo por base no parasita Neospora caninum. Este é intracelular obrigatório e é um dos parasitas mais transmitidos no gado, estando até 90% do gado de alguns rebanhos infetado.

N. caninum é também uma das principais causas de aborto em bovinos em muitos países sendo a principal via de transmissão a transplacentária.

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Abstract

Neospora caninum is a coccidian parasite of animals. It is a major pathogen for cattle and dogs and it occasionally causes clinical infections in horses, goats, sheep, and deer. Domestic dogs are the only known definitive hosts for N. caninum. It is one of the most efficiently transmitted parasite of cattle and up to 90% of cattle in some herds are infected. Transplacental transmission is considered the major route of transmission of N. caninum in cattle. Neospora caninum is a major cause of abortion in cattle in many countries. To elicit protective immunity against abortion in cows that already harbor a latent infection is a major problem. This paper reviews information on biology, diagnosis, epidemiology and control of neosporosis in animals.

Keywords: Neospora caninum, neosporosis, animals, cattle, dogs

INTRODUCTION

Since its first recognition in 1984 in dogs in Norway ( Bjerkås et al., 1984) and the description of a new genus and species Neospora caninum ( Dubey et al., 1988a), neosporosis has emerged as a serious disease of cattle and dogs worldwide. Additionally, clinical neosporosis has been reported in sheep, goats, deer, a rhinocerus, and horses and antibodies to N. caninum have been found in the sera of water buffaloes, red and gray foxes, coyotes, and camels, and felids.

BIOLOGY

Life Cycle

Dogs are both the intermediate and definitive host for N. caninum ( McAllister et al., 1998; Lindsay et al., 1999a, 1990b, 2001a; Basso et al., 2001a; Dubey et al., 2002). The life cycle is typified by 3 infectious stages: tachyzoites, tissue cysts, and oocysts ( Fig. 1 and Fig. 2 ). Tachyzoites and tissue cysts are the stages found in the intermediate hosts and they occur intracellularly ( Dubey et al., 2002). Tachyzoites are approximately 6 × 2 μm ( Fig. 1 ). Tissue cysts are often round or oval in shape, up to 107 μm long and are found primarily in the central nervous system (CNS). The tissue cyst wall is up to 4 μm thick and the enclosed bradyzoites are 7-8 × 2 μm. Thin-walled (0.3-1.0 μm) tissue cysts have been recently reported in muscles of cattle and dogs naturally-infected with a N. caninum-like parasite ( Peters et al., 2001a). These intramuscular tissue cysts have not been yet found in experimentally-infected animals ( Dubey et al., 2002).

Domestic dogs are the only known definitive host for N. caninum. N. caninum unsporulated oocysts from experimentally-infected dogs were 11.7 × 11.3 (10.6-12.4 × 10.6-12.0) μm in size ( Lindsay et al., 1999a). N. caninum oocysts sporulate outside the host. N. caninum oocysts are morphologically similar to Toxoplasma gondii and Hammondia hammondi oocysts in cat feces and broadly resemble oocysts of Hammondia heydorni-like parasite in dog feces ( Dubey et al., 2002). At the present, nothing is known regarding the frequency of shedding of oocysts, the survival of the oocysts in the environment, and whether other canids are also definitive hosts for N. caninum.

Transmission

Little is known about the mode of spread and tissue distribution of N. caninum in animals using the natural routes of transmission. The parasite can be transmitted transplacentally in several hosts and vertical route is the major mode of its transmission in cattle. Carnivores can acquire infection by ingestion of infected tissues ( McAllister et al., 1998; Lindsay et al., 1999a, 1999b; Dijkstra et al. 2001; Schares et al., 2001; Gondim et al., 2002).

It is epidemiologically important to be able to identify N. caninum oocysts in dog feces. Microscopic examination alone will not be enough to identify N. caninum oocysts in dog feces. Methods have also been developed to genetically distinguish N. caninum oocysts from H. heydorni oocysts ( Hill et al., 2001; Šlapeta et al., 2002). Neospora caninum oocysts have been identified feces of only two naturally-infected dogs ( Basso et al., 2001a; Šlapeta et al., 2002).

Animal models

There are no suitable animal models at the present to perform bioassay to detect N. caninum oocysts in dog feces. Although interferon-gamma gene knockout (KO) mice are highly susceptible to parenteral inoculation with N. caninum tachyzoites and tissue cysts ( Dubey and Lindsay, 1996; Dubey et al., 1998a), they are less susceptible to parenteral or oral inoculation with oocysts. Gerbils ( Meriones unguiculatus) were susceptible to N. caninum infection with oocysts ( Dubey and Lindsay, 2000; Basso et al., 2001a; Schares et al., 2001). Another species of gerbils, Meriones tristrami and sand rats ( Psammoomys ubesus) also susceptible to tachyzoites infection ( Pipano et al., 2002).

Comparison between neosporosis and toxoplasmosis

A large body of knowledge has been generated concerning biology of N. caninum because of previous experience with the related parasite, Toxoplasma gondii. Although N. caninum and T. gondii are very closely related parasites structurally, genetically, and immunologically, caution should be used in making generalizations about N. caninum based on the biology of T. gondii because neosporosis and toxoplasmosis are biologically distinct diseases. Toxoplasma gondii is a major disease of sheep and humans and not of cattle, whereas neosporosis is a major disease in cattle, not of sheep and there is no evidence for human infection.

NEOSPOROSIS IN CATTLE

Bovine neosporosis has been reviewed in several papers ( Dubey and Lindsay, 1996; Wouda, 1998; Dubey, 1999; Anderson et al., 2000; Buxton et al., 2002; Dijkstra, 2002; Innes et al., 2002; Jenkins et al., 2002; Dubey, 2003). Therefore, most references on bovine neosporosis were omitted from this review.

Clinical signs

Neospora caninum causes abortion both dairy and beef cattle. Cows of any age may abort from 3 month gestation to term. Most neosporosis-induced abortions occur at 5-6 month gestation. Fetuses may die in utero, be resorbed, mummified, autolyzed, stillborn, born alive with clinical signs, or born clinically normal but chronically infected. Neosporosis-induced abortions occur year-round. Cows with N. caninum antibodies (seropositive) are more likely to abort than seronegative cows and this applies to both dairy and beef cattle. However, up to 95% of calves born congenitally-infected from seropositive dams remain clinically normal. The age of dam, lactation number, and history of abortion generally do not affect rate of congenital infection but there are reports indicating that in persistently infected cattle vertical transmission is more efficient in younger than older cows.

Clinical signs have only been reported in cattle younger than 2 month of age. Neospora caninum-infected calves may have neurologic signs, be underweight, unable to rise, or be born without clinical signs of disease. Hind limbs or forelimbs or both may be flexed or hyperextended. Neurologic examination may reveal ataxia, decreased patellar reflexes, and loss of conscious proprioception. Calves may have exophthalmia or asymmetrical appearance in the eyes. Neospora caninum occasionally causes birth defects including hydrocephalus and narrowing of the spinal cord.

Abortions may be epidemic or endemic ( Wouda et al., 1999a). In infected areas, as many as 33% of dairy cow fetuses have been reported to abort within a few months. Abortions were considered epidemic if more than 10% of cows at risk aborted within 6-8 weeks. A small proportion (< 5%) of cows have been reported to have repeated abortion due to neosporosis ( Anderson et al., 1995).

Prevalence

Neospora caninum infections have been reported from most parts of the world including Australia, New Zealand, Europe, Korea, Japan, Thailand, and the Americas. Neosporosis-associated bovine abortion and neonatal mortality has been reported from Argentina, Australia, Belgium, Brazil, Canada, Costa Rica, Denmark, France, Germany, Hungary, Ireland, Israel, Italy, Japan, Korea, Mexico, the Netherlands, New Zealand, Poland, Portugal, Spain, South Africa, Sweden, United Kingdom, USA, and Zimbabwe. Quantitative studies in the USA, New Zealand, the Netherlands, and Germany indicate that 12 to 42% of aborted fetuses from dairy cattle are infected with N. caninum ( Table 1 ).

Table 1

Serologic prevalence in cattle varies, depending on the country, region, type of serologic test used, and cut-off level used to determine the exposure. In some dairies up to 87% of cows are seropositive.

Diagnosis

Examination of the serum from an aborting cow is only indicative of exposure to N. caninum and histologic examination of the fetus is necessary for a definitive diagnosis of neosporosis. The brain, heart, liver, placenta, and body fluids or blood serum are the best specimens for diagnosis and diagnostic rates are higher if multiple tissues are examined. Although lesions of neosporosis are found in several organs, fetal brain is the most consistently affected organ. Because most aborted fetuses are likely to be autolyzed, even semi-liquid brain tissue should be fixed in 10% buffered neutral formalin for histologic examination of hematoxylin and eosin (HE) stained sections. Immunohistochemistry is necessary because there are generally only a few N. caninum present in autolyzed tissues and these are often not visible in H and E stained sections. The most characteristic lesion of neosporosis is focal encephalitis characterized by necrosis and nonsuppurative inflammation ( Barr et al., 1991). Hepatitis is more common in epizootic than sporadic abortions. Lesions are also present in placenta but protozoa are difficult to find.

The efficiency of the diagnosis by PCR is dependent on the laboratory, stage of the autolysis of the fetus, and sampling procedures. Although immunohistochemical demonstration of N. caninum in lesions is the best evidence for etiology of abortion at the present time, it is very insensitive. Neospora caninum DNA can be detected by PCR in formalin-fixed, paraffin-embedded bovine aborted brain tissue.

Several serologic tests can be used to detect N. caninum antibodies including various ELISAs, the indirect fluorescent antibody test (IFAT), and the Neospora agglutination test (NAT). Immunoblots are useful in detecting N. caninum-specific antibodies. Avidity-ELISAs designed to distinguish recent and chronic infections in cattle appear promising to distinguish endemic and epidemic abortion.

Finding N. caninum antibody in serum from the fetus can establish N. caninum infection, but a negative result is not informative because antibody synthesis in the fetus is dependent on the stage of gestation, level of exposure, and the time between infection and abortion. Even a low IFAT titer of 1:25 should be regarded as specific for N. caninum infection, especially in fetuses. Immunoblotting using N. caninum specific antigen improves diagnosis. Although blood serum or any body fluid from the fetus may be used for serologic diagnosis, peritoneal fluid is better than other body fluids. In calves, presuckling serum can be submitted for diagnosis of congenital infection.

The definitive antibody level that should be considered diagnostic for neosporosis has not been established for bovines because of the uncertainty of serologic diagnosis in chronically infected animals and the availability of sera from noninfected cattle. In serological assays, titer and absorbance values are dependant on antigen composition, secondary antibodies and other reagents. Further, cut-off levels can be arbitrarily selected to provide sensitivity and specificity requested for a particular application. The age and class of an animal may also affect selection of a cut-off level. Although N. caninum is closely related to Toxoplasma gondii, Sarcocystis species, and other apicomplexans, cross reactivity has not been a major issue. Antibody titers in general are higher in cattle that have aborted due to neosporosis than those with normal pregnancy; however, titers in individual cows cannot determine etiology of abortions.

Toxoplasma gondii and Sarcocystis cruzi are 2 other protozoans that should be considered in the differential diagnosis of protozoal abortion in cattle. Immunohistochemical and detection of parasite DNA by PCR can distinguish them from N. caninum. Sarcocystis cruzi forms schizonts in vascular endothelium and is rarely (< 0.1%) found in aborted fetal brains, whereas N. caninum is usually located in extravascular tissues. Additionally, there are no immature schizonts in N. caninum infection in contrast with S. cruzi infections. Infection by T. gondii in bovine fetuses is rare ( Canada et al., 2002b).

Attempts at isolation of viable N. caninum ( Table 2 ) by bioassay in mice or cell culture have been largely unsuccessful and little is known of the antigenic variability among isolates of N. caninum, especially isolates from healthy animals. Many attempts to isolate viable N. caninum were unsuccessful because most N. caninum in fetuses die with the host. It is easier to isolate N. caninum from neural tissues of congenitally-infected full term calves ( Table 2 ) because tissue cysts are likely to be present and tissue cysts are relatively more resistant to autolysis than tachyzoites.

Table 2

Pathogenesis of abortion

Cows with N. caninum antibodies (seropositive) are more likely to abort than seronegative cows. There is a rise in antibody titers 4 to 5 months before parturition. These observations strongly suggest reactivation of latent infection. Little is known of the mechanism of reactivation. It is likely that there is parasitemia during pregnancy leading to fetal infection. However, N. caninum has never been identified in histologic sections of adult cows and there is a single report of isolation of viable N. caninum from the brain of an adult cow ( Sawada et al., 2000). Although it is reasonable to speculate that pregnancy-induced immune-suppression may reactivate latent tissue cysts of N. caninum, such a mechanism has not been demonstrated for neosporosis.

Little is known at the present time concerning the pathogenesis of infection in cows following infection with a natural route (oral) and using the naturally transmitted stage of the parasite (oocyst) although oocysts are infective to cattle by the oral route. Results of experiments with dairy or beef cows inoculated parenterally with N. caninum tachyzoites indicate that the fetus can become infected and sometimes diseased by 4 weeks after inoculation of the parasite. Even at the early stage, there were lesions in placenta and the CNS and encephalitis was the predominent lesion. The extent of damage to placenta directly due to multiplication of N. caninum and by immune response is not known ( Buxton et al., 2002). Gestational age may determine the outcome of infection. Fetuses infected early in pregnancy are likely to die.

Neospora caninum is a primary pathogen in cattle

Neospora caninum is one of the most efficiently transplacentally-transmitted organisms in cattle. In some herds up to 90% of cattle are infected, and most calves born congenitally-infected with N. caninum remain healthy. Therefore, there is a debate concerning whether N. caninum causes abortion in cattle or whether it is a bystander ( Thurmond et al., 1999). All evidence at the present time indicates that N. caninum is a primary pathogen. However, there is a need to distinguish clinical and nonclinical N. caninum infections. In the initial reports by Anderson et al. ( 1991) and Barr et al. ( 1991), the following criteria were established to diagnose N. caninum abortion. First, the fetuses had lesions, mostly encephalitis. Second, N. caninum was found in lesions. In my opinion, identification of organisms in lesions is a good evidence for the etiology of the lesions. Additionally, abortion has been induced in experimentally-infected cattle. The percentage of fetuses that have histologically detectable N. caninum and no lesions is unknown. Therefore, detection of N. caninum DNA alone in aborted fetuses may not be enough to establish cause and effect relationship. Host factors (cytokines, hormones) that may contribute to pathogenesis of N. caninum infection have not yet been established ( Buxton et al., 2002; Innes et al., 2002).

Transmission

There is no cow to cow transmission of N. caninum. In the study by Anderson et al. ( 1997), 25 seronegative heifers were housed with 25 seropositive heifers since birth and their progeny were evaluated for N. caninum infection. The seronegative heifers remained seronegative and gave birth to calves not infected with N. caninum. Seropositive heifers remained clinically normal but gave birth to congenitally-infected calves. Seven of these congenitally-infected calves were necropsied; all had histologic evidence of N. caninum infection and 4 were recumbent. Although most N. caninum infections in cattle are transmitted transplacentally, postnatal rates have been variable depending on the region of the country, type of test used and cut-off values used.

It is unlikely that N. caninum is transmitted venereally or by embryo transfer in cattle and embryo transfer is even recommended as a method of control to prevent vertical transmission ( Baillargeon et al., 2001). These researchers conducted an important study on embryo transfer and N. caninum infection involving 87 recipient cows or heifers. Neospora caninum infection was not demonstrable in any of the 70 fetuses or calves born to seronegative cows transplanted embryos from seropositive donors whereas 5 of 6 calves resulting from embryo transfer from seronegative donors to seropositive recipients were infected with N. caninum. Landmann et al. ( 2002) confirmed these findings and showed that commercially used embryo transfer procedures also prevented transfer of N. caninum from seropositive cows to seronegative recipients. Additionally, preimplanted bovine embryos are resistant to N. caninum invasion ( Bielanski et al., 2002).

Lactogenic transmission of N. caninum has been demonstrated experimentally in newborn calves fed colostrum spiked with tachyzoites, but there is no evidence that it occurs naturally ( Davison et al., 2001); dogs fed milk spiked with N. caninum tachyzoites did not shed oocysts ( Dijkstra et al., 2001).

Economic impact

There are no firm data on the economic losses due to neosporosis to cattle industry anywhere in the world but losses are estimated in millions of dollars. As many as 42% of cows may abort due to neosporosis, the economic impact will depend on the direct cost and value of fetuses lost. Indirect costs include professional help and costs associated with establishing diagnosis, rebreeding, possible loss of milk yield, and replacement costs if aborted cows are culled ( Thurmond and Hietala, 1996, 1997; Hernandez et al., 2001).

In general, less is known of the causes of abortion in beef cattle than in dairy cattle because of the difficulty of finding small fetuses expelled in the first trimester. Therefore, there are no accurate assessments of Neospora-induced losses in beef cattle. Because clinical disease has not been reported in calves older than 2 months of age, there is no direct evidence of N. caninum-associated morbidity in adult cattle. However, in a sero-epidemiological study, Barling et al. ( 2000) found a positive association between N. caninum antibody status of the calf and weight gain and projected a $15.62 loss per calf.

Epidemiology and control

As stated earlier, N. caninum is efficiently transmitted vertically in cattle, perhaps for several generations. Therefore, culling is the only way at present to prevent this transmission from cow to heifer. Until the discovery of the resistant stage (oocyst) of N. caninum, horizontal transmission was unexplained. Evidence is accumulating that transmission by oocysts may be more prevalent than initially realized, at least in some parts of the world. Storms of abortions, abortion in recently infected cattle as evidenced by low avidity antibodies, and seroconversion in a high portion of cattle, indicate transmission of N. caninum by fecal-oral route. Postnatal transmission also occurs in herds not experiencing storms of abortions and seroepidemiological data support the role of the dog in the life cycle of N. caninum.

How dogs become infected in nature is not known. Small animals that dogs might prey or consume naturally have not been identified as a natural host for N. caninum. Consumption of aborted bovine fetuses does not appear to be an important source of N. caninum infection in dogs. The consumption of placental membranes may be a source of N. caninum infection in dogs because the parasite has been found in naturally-infected placentas and dogs fed placentas shed N. caninum oocysts. Little is known at present regarding the frequency of shedding of N. caninum oocysts by canids in nature, the resistance of the oocysts, and whether dogs shed oocysts more than once. Until more definitive hosts of N. caninum are found, dogs should not be allowed to eat aborted fetuses, fetal membranes or dead calves. Drugs that will prevent transmission of the parasite for the dam to the fetus are unknown but research is continuing in this area.

There is no proven vaccine to prevent N. caninum abortion in cattle ( Innes et al., 2002). However, encouraging results have been obtained in mice vaccinated with killed parasites or their products. Vaccination of mice before pregnancy with killed N. caninum tachyzoites blocked transplacental transfer of N. caninum when mice were challenged during pregnancy demonstrated protective immunity to congenital transfer of N. caninum infection has been demonstrated in experimentally-infected cattle ( Innes et al., 2001b). As mentioned earlier, protective immunity appears to develop which reduces or prevents subsequent abortion after a primary infection and resultant abortion in cattle ( Anderson et al., 1995; McAllister et al., 2000). This protective immunity appears to be more effective in cows that are infected again from an exogenous source (oocysts) than in cows that relapse from an endogenous infection ( Trees and Williams, 2003). Therefore, inducing protective immunity in cows that are already infected naturally is a problem.

NEOSPOROSIS IN DOGS

Neosporosis was first recognized in dogs in Norway by Bjerkås et al. ( 1984). Retrospectively, the earliest record of neosporosis in dogs was an outbreak in 1957 in the U.S. ( Dubey et al., 1990a). Worldwide reports of clinical and subclinical infections were summarized by Dubey and Lindsay ( 1996) and Lindsay and Dubey ( 2000) and these references are not repeated here. Since then, antibodies to N. caninum were reported in 121 of 320 (37.8%) dogs from Argentina ( Basso et al., 2001b), 22% of 200 dogs from New Zealand ( Reichel et al., 1998), 10% of 150 dogs from Turkey ( Coškun et al., 2000), 6.7% of 163 dogs from Brazil ( Mineo et al., 2001), 10% of 500 pet dogs and 25% of 611 stray dogs from Brazil ( Gennari et al., 2002), 6.4% of 1,058 dogs from Italy ( Cringoli et al., 2002), and in 12.% of 120 urban and 26% of 81 rural dogs from Chile ( Patitucci et al., 2001). Klein and Müller ( 2001) reported N. caninum antibodies in 4% of 50 dogs in Germany without clinical signs and 13% of 200 dogs with clinical signs. Antibodies to N. caninum were found in 21.6% of 134 dogs from cattle farms in Parana, Brazil ( de Souza et al., 2002).

Three surveys compared the prevalence of N. caninum in urban and rural dogs. Sawada et al. ( 1998) reported N. caninum antibodies in 31% of 48 dogs from dairy farms and 7% of 198 dogs from urban areas in Japan. Wouda et al. ( 1999b) reported a higher prevalence in farm dogs (23.6% of 152) versus urban dogs (5.5% of 344) from the Netherlands. Basso et al. ( 2001b) reported higher seroprevalence in dogs from dairy farms (48% of 125) and beef farms (54.2% of 35) than in dogs from urban areas (22.2% of 160) of Argentina.

In addition to clinical infections summarized by Dubey and Lindsay ( 1996) and Lindsay and Dubey ( 2000), Boydell and Brogan ( 2000), Cantile and Arispici ( 2002) reported clinical neurologic neosporosis in dogs. Four cases of cutaneous neosporosis were recently reported, including mixed infection with Leishmania sp. in 1 dog ( Tarantino et al., 2001; Perlé et al., 2001; Ordeix et al., 2002).

Barber and Trees ( 1998) reported an interesting seroepidemiologic study of N. caninum infections in 373 breeding dogs in U.K. Fifty (13.4%) of 373 bitches had an IFAT titer of > 1:50. Initially, approximately 50% pups born were seropositive and approximately 25% of pups born from seropositive dams developed neosporosis-like illness. Three bitches produced successive litters of N. caninum infected pups. In subsequent breeding only 3 of 118 pups from seronegative dams were seropositive. Thus, vertical transmission alone could not maintain the parasite in dogs ( Barber and Trees, 1998).

The most severe cases of neosporosis occur in young, congenitally infected pups. Young dogs develop hind limb paresis that develops into a progressive paralysis. Neurologic signs are dependent on the site parasitized. The hind limbs are more severely affected than the front limbs and often in rigid hyperextention. Other dysfunctions which occur include difficulty in swallowing, paralysis of the jaw, muscle flaccidity, muscle atrophy and even heart failure. Dogs with hind limb paralysis may be alert and survive for months.

The disease may be localized or generalized and virtually all organs may be involved including the skin. Dermatitis may be severe involving enormous numbers of N. caninum. Dogs of any age can be affected. Fatal neosporosis has been reported in eight to 15-year-old dogs.

Subclinically infected bitches can transmit the parasite to their fetuses, and successive litters from the same bitch may be born infected. Whether there is breed predisposition and differential sex susceptibility to neosporosis in dogs is not known. Most described cases have been in Labrador retrievers, Boxers, Greyhounds, Golden retrievers, and Basset hounds.

Neospora caninum has been isolated several times from dogs, mostly from dogs with neuromuscular signs ( Table 3 ). Nothing is known of the isolates of N. caninum from dogs without any clinical signs.

Table 3

NEOSPOROSIS IN WATER BUFFALOES

Water buffaloes ( Bubalus bubalis) are important to the economy of several countries including Brazil, India, Italy, and Vietnam. Prevalence of antibodies indicates that buffaloes in four countries were exposed to N. caninum ( Table 4 ).

Table 4

Little is known of the role of N. caninum in causing abortion in buffaloes. Guarino et al. ( 2000) reported lesions and Neospora-like tissue cysts in 2 of 4 fetuses aborted from 2 dairy farms in Italy; this report needs confirmation.

NEOSPOROSIS IN SHEEP

Neospora caninum was first diagnosed in a congenitally-infected lamb in England, ( Dubey et al., 1990b). Historically, this was the first record of N. caninum-like infection in a ruminant ( Hartley and Bridge, 1975). The lamb was born weak, partially ataxic, and died at one week of age. There was unilateral reduction of gray matter in the ventral horn and local cavitation. This case was published because congenital deformities due to toxoplasmosis had not been seen in any animal (other than humans), only tissue cysts and not tachyzoites were seen. I had known of this case even before it was published. After the discovery of the N. caninum in dogs in 1988, I contacted William Hartley and retrospectively examined tissues of this lamb and an identical case in a calf that was added as an addendum to the case in lamb ( Hartley and Bridge, 1975); the calf was from Australia. Subsequently, clinical ovine neosporosis was reported from Japan. Kobayashi et al. ( 2001) found naturally-infected neosporosis in ewe and her twin fetus in Japan. Focal encephalitis and thick-walled N. caninum tissue cysts were seen in all three animals. Koyama et al. ( 2001) obtained the first isolate of N. caninum from another adult ewe.

Little is known of the seroprevalence of N. caninum in naturally exposed sheep and evidence for N. caninum as an ovine abortifacient in the field has not been found ( Otter et al., 1997). In a recent survey study from U.K., only 3 of 660 ewes that had aborted had antibodies to N. caninum ( Helmick et al., 2002). Pregnant sheep, however, are highly susceptible to experimental infection with N. caninum tachyzoites ( Dubey and Lindsay, 1990; McAllister et al., 1996; Buxton et al., 1997b, 1998; Jolley et al., 1999; Innes et al., 2001a) and are an alternative ruminant model for bovine neosporosis. Sheep can also be infected orally with N. caninum oocysts ( O'Handley et al., 2002).

NEOSPOROSIS IN GOATS

Abortion and neonatal mortality associated with N. caninum was reported in pygmy goats in the U.S. ( Barr et al., 1992; Dubey et al., 1992), in a dairy goat herd in Costa Rica ( Dubey et al., 1996a), and in a 3-day-old dairy goat from Brazil ( Corbellini et al., 2001). Antibodies to N. caninum were found in 5 of 77 dairy goats in Costa Rica that had aborted. Little is known of the seroprevalence of N. caninum antibodies in goats. Ooi et al. ( 2000) did not find N. caninum antibodies in 24 goats from Taiwan.

Pygmy goats, however, are quite susceptible to experimental N. caninum infection. Goats inoculated N. caninum during pregnancy aborted N. caninum-infected fetuses ( Lindsay et al., 1995).

NEOSPOROSIS IN HORSES

Neospora-like parasites have been found in tissues of two aborted foals ( Dubey and Porterfield, 1990; Pronost et al., 1999), a congenitally-infected foal ( Lindsay et al., 1996b) and five adult horses ( Gray et al., 1996; Daft et al., 1996; Marsh et al., 1996; Hamir et al., 1998; Cheadle et al., 1999b). Marsh et al. ( 1998) proposed a new name, Neospora hughesi, for the parasite in the horse they reported in 1996. Three isolates of N. hughesi from adult horses have been reported ( Marsh et al., 1998; Cheadle et al., 1999b; Dubey et al., 2001). Molecular and biological characteristics of these three isolates have been reported ( Cheadle et al., 1999a; Walsh et al., 2000; Dubey et al., 2001; Marsh et al., 2001).

Tissue cysts of N. hughesi were smaller than N. caninum with thinner cyst walls (less than 1.0 µm thick), and bradyzoites were smaller than those of N. caninum ( Marsh et al., 1998). It is, however, not clear at the present time whether N. hughesi is the sole species of Neospora that infects horses. Thick walled tissue cysts, characteristics of N. caninum were reported from a horse from California ( Daft et al., 1996) and a congenitally infected foal from Wisconsin ( Lindsay et al., 1996b).

Seroprevalence of Neospora infections in horses is summarized in Table 5 . In two surveys or horses in France and the U.S. the seroprevalence by NAT was 23%.

Table 5

NEOSPORA INFECTIONS IN HUMANS

Because two rhesus monkeys ( Macaca mulata) have been successfully infected with N. caninum ( Barr et al., 1994), there is a concern about the zoonotic potential of N. caninum. However, at present there is no evidence that N. caninum successfully infects humans. Graham et al. ( 1999) did not find N. caninum antibodies in 1:160 dilution of sera from 199 blood donors and 48 agricultural workers in Northern Ireland. Petersen et al. ( 1999) did not find antibodies to N. caninum in 76 women with history of abortion using ELISA, immunoblotting, and IFAT. Low level IFAT (1:100) antibodies were reported by Tranas et al. ( 1999) in 69 (6.7%) of 1,029 sera from blood donors in California, however, all sera were negative at 1:200 dilution. Nam et al. ( 1998) reported antibodies to N. caninum in human sera using the immunoblots; however, these sera were negative by NAT. Trees and Williams. ( 2000) found low (IFAT < 1:200) in 2 of 500 (400 farm workers and 100 women with recurrent abortions) people in England. In conclusion, there is no evidence that N. caninum infection is zoonotic.

NEOSPOROSIS IN WILDLIFE

Neosporosis was diagnosed at necropsy in two black-tailed deer ( Odocoileus hemionus columbianus) found dead in the wild in California ( Woods et al., 1994), in a captive deer in zoos in France ( Dubey et al., 1996b, and Germany ( Peters et al., 2001b. Neospora caninum tissue cysts were found in the brain of a full term stillborn deer ( Cervus eldi siamensis) in a Paris Zoo ( Dubey et al., 1996b. Peters et al. ( 2001b) reported antibodies to N. caninum in fetal fluid and N. caninum DNA by PCR in the brain, heart, lung, liver and spleen of 2 full term twin calves of antelope ( Tragelaphus imberberis) in a zoo in Hannover, Germany.

Neosporosis was reported in a rhinoceros ( Ceratotherium simum) calf ( Williams et al., 2002). The animal died suddenly, considered to be due to myocarditis. Rhinoceroses are related to equids, all belonging to the order Perissodactyla.

Finding of antibodies in 40% of wild deer (Table 9) indicates there may be a sylvatic cycle of N. caninum. A large percentage of these deer had high titered (> 1:1,600) N. caninum antibodies and the prevalence did not increase with age indicating that congenital transmission ( Dubey et al., 1999a). Similarly, red foxes in Belgium had a high seroprevalence of N. caninum. Schares et al. ( 2001) presented evidence of congenital infection of N. caninum in foxes.

​

Table 6

References

41. Dijkstra T. Horizontal and vertical transmission of [ Neospora caninum. the Netherlands: Univ. Utrecht; 2002. pp. 1-140. PhD Thesis. Google Scholar]

86. Klein BU, Muller E. Seroprävalenz von antikörpern gegen [ Neospora caninum bei Hunden mit und ohne klinischem Neosporoseverdacht in Deutschland. Der praktische Tierarzt. 2001;82:437-440. Google Scholar]

91. Lindsay DS, Kelly EJ, McKown R, et al. Prevalence of [ Neospora caninum and Toxoplasma gondii antibodies in coyotes ( Canis latrans) and experimental infections of coyotes with Neospora caninum. J Parasitol. 1996a;82:657-659. [ PubMed] Google Scholar]

112. Nam HW, Kang SW, Choi WY. Antibody reaction of human anti-[[ Toxoplasma gondii positive and negative sera with Neospora caninum antigens. Korean J Parasitol. 1998;36:269-275. PMC free article] [ PubMed] Google Scholar]

118. Patitucci AN, Phil M, Pérez MJ, Rozas MA, Israel KF. [ Neosporosis canina: presencia de anticuerpos séricos en poblaciones caninas rurales y urbanas de Chile. Arch Med Vet. 2001;33:227-232. Google Scholar]

123. Petersen E, Lebech M, Jensen L, et al. [[ Neospora caninum infection and repeated abortions in humans. Emerging Infectious Diseases. 1999;5:278-280. PMC free article] [ PubMed] Google Scholar]

126. Pronost S, Pitel PH, Romand S, Thulliez P, Collobert C, Fortier G. [ Neospora caninum: premiére mise en évidence in France sur un avorton équin, analyse et perspectives. Prat Vét Equine. 1999;31:31-34. Google Scholar]

140. Thurmond MC, Hietala SK. 4. 1999. [ Neospora caninum infection and abortion in cattle; pp. 425-431. Google Scholar]

141. Tranas J, Heinzen RA, Weiss LM, McAllister MM. Serological evidence of human infection with the protozoan [[ Neospora caninum. Clin Diagn Lab Immunol. 1999;6:765-767. PMC free article] [ PubMed] Google Scholar]

150. Wouda W. [ Neospora abortion in cattle, aspects of diagnosis and epidemiology. the Netherlands: Univ. Utrecht; 1998. pp. 1-176. PhD Thesis. Google Scholar]

Articles from The Korean Journal of Parasitology are provided here courtesy of The Korean Society for Parasitology and Tropical Medicine

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Review of Neospora caninum and neosporosis in animals

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Neospora caninum is a coccidian parasite of animals. It is a major pathogen for cattle and dogs and it occasionally causes clinical infections in horses, goats, sheep, and deer. Domestic dogs are the only known definitive hosts for N. caninum. It is one ...

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" Após a técnica de Gram e a visão microscópica " 

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" Após a técnica de Gram e a visão microscópica " 

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Os resultados apresentado nesta figura evidencia a distinção entre as bactérias de Gram-porisitiva e Gram-negativa, através da cor.

A cor vermelha: são bactérias de Gram negativas e causam doenças.

A cor roxa: são bactérias de Gram positivas, beneficiam o organismo, por sua vez não causam doença.

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https://cdn.kastatic.org/ka-perseus-images/10bfd1ddbb384800fba4021760d503ffa3287576.png - consultado a 17/03/2024

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Colheta de "Bactérias" mantidas no "Iorgurte"

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Colheta de "Bactérias" mantidas no "Iorgurte"

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No mesmo dia, após a colheta da maçã, executar o de "iorgurte". E consequentemente, colhetar o soro que nela contém com algumas porções do próprio elemento. Ao findar disto, colocar na placa, com ajuda da "Ansa", para fixar através, do parafilme. Feito isso, deixar na estufa e esperar alguns dias, com intuíto que as bactérias se manifestem com facilidade e prever o êxito da próxima técnica, que é o plaquamento. Em suma foi um dia de preparar os elementos, para efeituar posteriomente.

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Após a secagem, dissolveu-se o pellet de cada eppendorf em 8 µL de H₂O livre de RNase e o RNA foi transferido para eppendorfs mais pequenos de 200 µL, visto que era uma quantidade bastante reduzida.
Estes eppendorfs mais pequenos foram numerados de acordo com os números dos eppendorfs anteriores, respetivamente. Além destes, acrescentámos 2 eppendorfs de controlo titulados RNA e H2O.

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Num eppendorf, preparámos uma solução contendo 286 µL de Master Mix (solução contendo primers de RNA e nutrientes) e 57,2 µL de Enzyme Mix (solução contendo a enzima transcriptase reversa, responsável pela polimerização do cDNA).

Em cada eppendorf numerado de 1 a 24 adicionámos:

12 µL desta solução;

2 µL de RNA;

8 µL de água.

No eppendorf "RNA" adicionámos :

2 µL de RNA do eppendorf 25;

20 µL de água.

Este eppendorf vai servir como controlo pois, no PCR, apenas será amplificado DNA genómico, uma vez que o RNA não será polimerizado em DNA pela transcriptase reversa. Este eppendorf vai comprovar que todo o DNA analisado terá origem em transcritos e não em DNA genómico. Desta forma, conseguimos comprovar a não contaminação das amostras por qualquer tipo de DNA genómico.

No eppendorf "H2O" adicionámos:

12 µL da solução preparada anteriormente;

10 µLde água.

Este eppendorf será utilizado como controlo. Isto porque, como não apresenta RNA, todo o DNA que possa ser encontrado nele terá origem na contaminação da água por algum DNA. Desta forma, podemos afirmar que a água utilizada não contém qualquer DNA ou RNA contaminante.

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Vid. 13- Programação do termociclador (PL)

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O termociclador eleva e reduz a temperatura do bloco em ciclos de tempo pré-programados, permitindo a multiplicação em massa de moléculas de DNA. Desta forma podemos colocar os eppendorfs durante um longo período de tempo e ir variando a temperatura consoante o protocolo.
Colocámos os eppendorfs no termociclador programado para ficar 10 minutos a 25ºC, posteriormente ficar 10 minutos a 50ºC, a seguir ficar 5 horas a 85ºC e, no final, ficar a 4ºC até alguém abrir o aparelho.
Os eppendorfs foram deixados no frigorífico até à sessão seguinte.

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Fig. 16- Gráfico resultante da espectrofotometria (PL) 

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A espectrofotometria é um método utilizado para medir o quanto uma substância química absorve a luz, medindo a intensidade quando um feixe de luz passa através da solução da amostra. O princípio básico é que cada composto absorve ou transmite luz numa certa amplitude de comprimento de onda, desta forma, o espectofotometro permite determinar a concentração dos compostos em solução.

A máquina envia um feixe luminoso que irá atravessar a solução e incidirá num recetor. Consoante a concentração e composição das soluções certos comprimentos de onda não atingirão o sensor.

No nosso caso, visto que o RNA e a proteína absorvem comprimentos de onda diferentes, o feixe lido no recetor terá falhas em dois comprimentos de onda. Desta forma, poderemos determinar a presença de proteína nas amostras e a razão entre a concentração de proteína e a concentração de RNA. Nas nossas amostras pretendemos encontrar o máximo de RNA e o mínimo de proteína possível.

O protocolo de utilização do espectofotometro é bastante simples, apenas temos que o abrir e limpar a fonte de luz e o sensor, pipetar 2 µL da solução no sensor, fechar o aparelho e ler os resultados no ecrã.

Analisando a fig. 16, podemos notar o primeiro pico, que corresponde ao RNA, (mais especificamente aos compostos fenólicos a ele associado) e o segundo pico, correspondente à proteína presente na amostra.

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Os passos do PCR são os seguintes:

Desaturação (96ºC)

‍  Aqueçer a reação para desnaturar os filamentos de DNA, separando a cadeia dupla em duas cadeias simples.

Recozimento (55-65ºC)

Arrefecer a reação para que os primers possam ligar-se às suas sequências complementares no DNA do modelo de cadeia única.

Extensão (72ºC)

Aumentar as temperaturas de reação para que a Taq polimerase estenda os primers, sintetizando novos filamentos de DNA.

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Fig: (19)  Reagentes utilizado para a técnica de coloração de Gram

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" Processo da técnica de Gram "

a) Cobrir o esfregaço com o corante violeta de genciana (corante primário) e deixar o corante agir por um minuto;

b) Escorrer o corante e lavar rapidamente a lâmina com água destilada;

c) Cobrir o esfregaço com solução de Iodo (mordente) e deixar agir por um minuto;

d) Escorrer o mordente e lavar rapidamente a lâmina com água destilada;

e) Inclinar a lâmina e gotejar a solução de álcool-acetona (descorante) por aproximadamente 15 segundos, até perceber que não está mais havendo descoloração;

f) Em seguida, lavar a lâmina em água destilada;

g) Cobrir o esfregaço com fucsina ou safranina (contracorante) e deixar agir por 30 segundos;

h) Escorrer o corante e lavar a lâmina em água destilada;

i) Ápos a técnica, levar ao microscópio onde será observado os resultados

Linck: https://www.ufrgs.br/aulaspraticasdemip/?page_id=134

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Sabendo que a resposta das células se vai refletir numa série de transcritos, será necessário avaliar, individualmente, a quantidade de cada um deles. Uma vez que a partícula utilizada se liga a todo o cDNA, conferindo-lhe fluorescência, não é possível diferenciar genes distintos no mesmo poço, por isso, é necessário que identificá-los individualmente. Estes genes têm origem no cDNA.

Genes analisados:

B2M (b-2-microglobulina) - responsável pela polimerização de glicoproteínas associadas ao MHC I;

MARVEL d1 - responsável pela produção de proteínas associadas aos microtúbulos (filamentos de actina, também chamados de microfilamentos, e filamentos intermediários) que constituem o citoescleto das células;

GBP4 (guanylate binding protein 4) - proteína associada à imunidade inata contra uma gama diversificada de patógenos bacterianos, virais e protozoários.

Sabendo que os dois primeiros genes apresentam uma expressão constante independentemente do estímulo a que são sujeitos, a quantidade de GBP4 foi comparada com as quantidades dos outros genes.

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Fig. 19- Eppendorfs nomeados para cada um dos genes (PH)

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Começámos por nomear 3 eppendorfs, cada um deles com o nome do gene que será detetado para cada uma das soluções.
Como temos 26 amostras para cada gene e é aconselhado que as quantidades usadas sejam calculadas para 26+10%, ou seja 28,6 amostras (para termos a certeza que são suficientes), em cada um dos 3 eppendorfs colocámos:

143 μL de Sybr Green Mix - contém bases e nutrientes necessários à polimerização e partículas que, quando integradas na cadeia dupla de DNA, emitem fluorescência. (quanto maior a quantidade de DNA maior será a fluorescência observada)

5,72 μL de Primer Forward - inicia a replicação de uma das cadeias de DNA

5,75 μL Primer Reverse - realiza a replicação da cadeia complementar

74,4 μL de água

Isto porque, idealmente, 1 amostra contém:

5 μL de Mix Sybr Green

0.2 μL de Primer Forward

0.2 μL de Primer Reverse

2.6 μL de água

2 μL de cDNA

De seguida colocámos os eppendorfs no vórtex para homogeneizar a solução.

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https://cdn.kastatic.org/ka-perseus-images/a60b6fd1cd6918686c67e7d1559d070e7b154e20.png - consultado a 17/03/2024

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Fig. 20- Esquema de organização da placa PCR (PH)

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Numa placa própria para testes PCR, adicionámos 2 μL de cDNA de cada um dos eppendorfs por poço, de acordo com a numeração dos esquemas preparados anteriormente.

Ao cDNA, acrescentámos 8 μL de cada uma das soluções nos poços correspondentes, vedámos a placa e indicámos, num papel, o protocolo de PCR desejado.

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Fig. 21- Programação do termociclador (PH)

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A placa foi levada para a sala de PCR e deixada no frigorífico. O protocolo escrito na placa será realizado por especialistas: 40 ciclos de 5 segundos a 95ºC seguidos de 20 segundos a 65ºC.

A informação correspondente à fluorescência emitida no início de cada um dos ciclos, será gravada (como já sabemos, quanto maior a fluorescência maior a quantidade de DNA).

No final do PCR será traçada uma melt curve pois a temperatura será aumentada até 95ºC. Com a elevação da temperatura, as cadeias de DNA vão desnaturar, libertando por isso menos radiação. Tendo em conta que, a temperatura de desnaturação varia entre os genes consoante a percentagem de pares GC e AT, ao fazer a melting curve, podemos confirmar que o gene que foi amplificado foi sempre o mesmo.

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Para a análise dos resultados do teste PCR utilizámos o programa CFX Maestro, da BioRad. Com ele, analisámos os gráficos da radiação emitida em função do número de ciclos no termociclador. O aumento das quantidades de DNA segue uma função exponencial. Desta forma, quanto maior for a quantidade inicial de DNA, mais cedo se dará o crescimento exponencial.

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Fig.4- Informações importantes à cerca do DNA mitocondrial

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Fig.5- Curiosidade sobre a Síndrome de Leigh

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Fig:(8) "Breve resumo sobre ativadades que serão abordadas ao longo do projeto"

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Neste dia foram-nos dados papéis para escrevermos o nome e o nosso email, que posteriormente serão úteis na compartilhação de informações inseridas pelos investigadores.

Ao fim da sessão cada aluno apresentou-se pelo seu nome e sua turma. Em suma foi um dia especificamente para esclarecimento do funcionamento do projeto e partilhas de identificações dos investigadores e de cada aluno.

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Mitocôndria (Fig.1): https://pt.wikipedia.org/wiki/Mitoc%C3%B4ndria (consultado a 16/04/2024)

Doenças das mitocôndrias (Fig.2):

https://www.tuasaude.com/doenca-mitocondrial/#google_vignette

(consultado a 16/03/2024)

Lebre-cantábrica (Fig.3):

https://animalia.bio/pt/broom-hare (consultado a 16/03/2024)

https://eol.org/pages/133005

(consultado a 16/03/2024)

Informações importantes à cerca do DNA mitocondrial (Fig.4):

https://www.infoescola.com/genetica/dna-mitocondrial/ (consultado a 16/03/2024)

https://images.app.goo.gl/jsTrwasipbPFXB1g7 (consultado a 16/03/2024)

Curiosidade a Doença de Leigh (Fig.5):

https://www.casahunter.org.br/doencas-raras/sindrome-leigh.php (consultado a 16/03/2024)

https://images.app.goo.gl/JcHdShkwG8ggnrv8A (consultado a 16/03/2024)

Efeito da temperatura em relação as alteraões climaticas nas mitocôndrias (Fig.6):

cplp.sef.https://ccnh.ufabc.edu.br/arquivos/CENTRAL/4.Ensino/1.Graduacao/TCCs/Marcela_Simoes_Teruel_versao_final_BacBio-QS.2021.1.pdf

(consultado a 16/03/2024)

Hereditariedade do DNA mitocondrial em relação com as alterações climaticas (Fig.6): ttps://www.igenomix.com.br/blog/mitocondrias-e-doencas-de-heranca-mitocondrial/

(consultado a 16/03/2024)

Informações úteis sobre as alterações climaticas (Fig.6): https://www.igenomix.com.br/blog/mitocondrias-e-doencas-de-heranca-mitocondrial/

(consultado a 16/03/2024)

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Fig.6- Relação das Alterações climáticas nas mitocôndrias

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Relação das mitocôndrias e as alterações climáticas

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Relação das mitocôndrias e as alterações climáticas

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A temperatura pode afetar várias etapas do funcionamento mitocondrial. Como taxa de respiração celular, a produção de calor, de radicais livres, e até mesmo os mecanismos mitocondriais envolvidos com a morte celular.

Podem incluir mutações genéticas que afetam as proteínas mitocondriais, danos causados por radicais livres (stresse oxidativo), bem como exposições a certas drogas e toxinas. Além disso, doenças crónicas como: diabetes e obesidade têm sido associadas a alterações na função mitocondrial.

Alterações que envolvem herança de DNA mitocondrial podem causar problemas de crescimento, desenvolvimento e funções dos sistemas do corpo.

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Fig. 22- Legenda colorimetrica para interpretação dos gráficos

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Legenda para a interpretação dos dados dos gráficos

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Legenda para a interpretação dos dados dos gráficos

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Fig. 23- Gráfico quantidade de B2M em função do número de ciclos

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Este é um gráfico da quantidade de B2M em função do número de ciclos. Não tivemos os resultados esperados neste PCR, isto porque apenas 6 de todos os poços apresentaram um aumento da quantidade de DNA, tendo sido significativo em apenas 3 destes.

Os poços H2O e RNA não apresentaram crescimento, comprovando a não contaminação das amostras.

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Fig. 24- Gráfico quantidade de MARVEL d1 em função do número de ciclos

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Este é o gráfico quantidade de MARVEL d1 em função do número de ciclos. Não foi identificado aumento da quantidade de DNA em cerca de metade dos poços.

Conseguimos apenas uma as réplicas de cada um dos estímulos, exceto LPS e parasita + interferão (100 pg/mL).

Os poços H2O e RNA voltaram a não apresentar crescimento, comprovando a não contaminação das amostras.

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Fig. 25- Gráfico quantidade de GBP4 em função do número de ciclos

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E este é o gráfico da quantidade de GBP4 em função do número de ciclos. Verificámos, novamente, que não foi identificado aumento da quantidade de DNA em cerca de metade dos poços.

Voltámos a conseguir apenas uma as réplicas de cada um dos estímulos, exceto LPS e parasita + interferão (100 pg/mL).

Não foi verificado crescimento no poço H2O, mas o poço RNA apresentou um ligeiro aumento (curva azul mais grossa), indicando a presença de DNA genómico, ou seja, estava contaminado.

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Fig.3- Informações sobre a lebre-cantábrica

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Após alguns dias obtivemos este resultado, para cada placa colocada na estufa.

Com isso iniciamos o processo chamado de" coloração de Gram". Que consiste em distiguir as "Bactérias"de Gram-positivo e Gram-negativo

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É importante analisar a melt curve já que houve ampliação de DNA genómico. Esta análise é necessária para identificarmos os poços onde houve ampliação de DNA genómico e onde houve ampliação do gene em estudo.

No gráfico da Fig. 26 podemos observar que a fluorescência de todas as funções diminui no mesmo ponto, exceto a função azul (RNA) e a função laranja (parasita). Isto pode indicar que, no poço contendo parasita não ocorreu amplificação do gene GBP4, mas ocorreu amplificação de DNA genómico.

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Negativo da primeira derivada da melting curve (Melt Peak)

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Negativo da primeira derivada da melting curve (Melt Peak)

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Esta função já é bem mais fácil de analisar, visto que apresenta picos nos pontos onde 50% do DNA se encontra desnaturado. A análise deste gráfico permite-nos concluir que todas as funções apresentam picos correspondentes à mesma temperatura (aproximadamente 84ºC), à exceção da azul e da laranja. Assim, retiramos os dados provenientes deste poço do estudo.

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Devido a incompstibilidade do projeto anterior, foi-me sugerida de alterar o meu projeto pois não me compateceu. No entando agradou-me e aceitei.

Agora em diante, apresentarei um novo projeto com objectivo de conhcer melhor como são e funcionam as " Bactérias ".

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O que são Bactérias ?

As bactérias são seres procariontes, ou seja, não possuem núcleo definido, e seu material genético está concentrado em uma região que não é envolta por membrana. Essa região é denominada  nucleoide. Além do material genético presente no nucleoide, nas bactérias podem ser observadas moléculas circulares de DNA pequenas — chamadas de plasmídeos —, as quais se replicam independentemente. https://brasilescola.uol.com.br/biologia/bacterias.htm

1- Qual é a importância de se identificar bactérias:

A detecção e identificação de micro-organismos em qualquer ambiente é essencial para controle de qualidade, controle de processos de fabricação e manipulação, garantia da segurança em alimentos e qualidade microbiológica em ambientes hospitalares.

https://blog.neoprospecta.com/como-fazer-identificacao-de-micro-organismos/

2-Como se classificam-as : Monera, procarionte:

O reino Monera engloba todos os organismos procariontes existentes, compreendendo as bactérias, cianobactérias (algas azuis) e arqueias (procariontes primitivos). Atualmente o termo monera encontra-se em desuso, e seus integrantes foram divididos em outra forma de agrupamento, formando o sistema de domínios.

https://querobolsa.com.br/enem/biologia/reino-monera

3- Tipos de bactérias e as mais resistentes

"Classificação das bactérias

As bactérias são classificadas de diferentes formas, sendo uma delas o formato de suas células.

Observe os principais formatos apresentados pelas bactérias.

Cocos: bactérias que apresentam formato esférico. Podem ocorrer isoladas ou em agrupamentos. Quando ocorrem aos pares, são denominadas diplococos; quando formam uma cadeia, são denominadas estreptococos; quando se agrupam como um cacho de uva, são chamadas de estafilococos.

Bacilos: bactérias que apresentam formato de bastão. Podem ocorrer isoladamente, aos pares (diplobacilos) ou em cadeias (estreptobacilos).

Espirilos: bactérias com formato helicoidal e rígidas.

Espiroquetas: bactérias com formato helicoidal e flexíveis.

Vibrião: bactérias que apresentam formato de vírgula.

https://brasilescola.uol.com.br/biologia/bacterias.htm

4-"Bactérias gram-positivas e gram-negativas

Uma técnica denominada coloração de Gram permite classificar as bactérias em dois grupos, por meio da análise das diferenças existentes na composição da parede celular. Quando submetida à coloração de Gram, a parede celular das bactérias pode adquirir coloração roxa ou vermelha. As bactérias coradas de violeta são chamadas de gram-positivas e se caracterizam por apresentar uma parede celular mais simples e rica em peptideoglicano. As bactérias gram-negativas, por sua vez,  coram-se em vermelho e apresentam uma estrutura mais complexa, com menos peptideoglicano.

Determinar se uma bactéria é gram-positiva ou gram-negativa é importante para indicar, por exemplo, que tratamento deve ser adotado em caso de infecções. As bactérias gram-negativas, por exemplo, são, em geral, mais resistentes aos antibióticos que as gram-positivas.

5- "Importância das bactérias.

As bactérias, diferentemente do que muitos pensam, não são responsáveis apenas por causar prejuízos aos seres humanos, havendo muitas espécies importantes para a nossa saúde. No nosso intestino, por exemplo, há várias espécies de bactérias, as quais são fundamentais para garantir o funcionamento normal do órgão. A nossa microbiota intestinal auxilia na absorção de nutrientes, produz vitaminas e auxilia a evitar a proliferação de agentes patogênicos.

Nosso intestino é repleto de bactérias benéficas, que auxiliam no funcionamento adequado do órgão.

Economicamente, as bactérias são importantes, por exemplo, por serem usadas na fabricação de vinagre e iogurte. Não podemos nos esquecer também da toxina botulínica, produzida pela espécie Clostridium botulinum. Essa toxina é bastante utilizada para amenizar rugas e linhas de expressão. Alguns antibióticos também são produzidos por bactérias.

As bactérias também apresentam importância ecológica, atuando, por exemplo, na decomposição da matéria orgânica, junto com os fungos. As bactérias também participam do ciclo do nitrogênio."

https://brasilescola.uol.com.br/biologia/bacterias.htm

6-O QUE SÃO SUPERBACTÉRIAS E COMO ELAS RESISTEM AOS ANTIBIÓTICOS?

As superbactérias são cepas de bactérias que se tornaram resistentes aos antibióticos conhecidos, muitas vezes incluindo os mais modernos. As bactérias, como qualquer outro organismo, tentam sobreviver diante das tensões externas e têm a vantagem adicional de terem uma grande facilidade para sofrer mutações, assim como para se reproduzir; em condições adequadas, centenas de milhões podem aparecer em poucas horas. Isso permite que suas mutações de DNA sejam relativamente rápidas e, se estas modificações se mostrarem úteis e sobreviverem, essa mutação pode ser ainda mais refinada.

Porque que elas aparecem?

viver ou trabalhar em condições sujas

uso indevido de antibióticos

Manuseio incorreto de alimentos

práticas inadequadas de prevenção e controle de infecções

As superbactérias são cepas de bactérias, vírus, parasitas e fungos que são resistentes à maioria dos antibióticos e outros medicamentos normalmente usados para tratar infecções que elas causam.

https://www.iberdrola.com/inovacao/superbacterias

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7-Que técnica nos permite identificar as Bactérias

A técnica de coloração de Gram, desenvolvida pelo médico dinamarquês Hans Christian Joachim Gram em 1884, é um método amplamente reconhecido para diferenciar bactérias com diferentes estruturas de parede celular.05/10/2023

https://pt.linkedin.com/pulse/colora%C3%A7%C3%A3o-de-gram-princ%C3%ADpios-e-procedimentos-fermentec-1f

8-Objetivo da técnica de coloração de Gram

Em 1884, Gram observou que as bactérias adquiriram cores diferentes, quando tratadas com diferentes corantes. Isso permitiu classificá-las em dois grupos distintos: as que ficavam roxas, que foram chamadas de Gram- positivas, e as que ficavam vermelhas, chamadas de Gram-negativas.

https://bvsms.saude.gov.br/bvs/publicacoes/115_03gram.pdf

9- Qual é a diferença entre Gram-posistivo e Gram- negativo

As bactérias Gram-positivas são classificadas pela cor que adquirem após aplicação de um processo químico denominado coloração de Gram. As bactérias Gram-positivas adquirem coloração azul quando essa coloração é aplicada a elas. (Outras bactérias se coram de vermelho. Elas são chamadas de Gram‑negativas.)

https://www.msdmanuals.com/pt-br/casa/infec%C3%A7%C3%B5es/infec%C3%A7%C3%B5es-bacterianas-bact%C3%A9rias-gram-positivas/considera%C3%A7%C3%B5es-gerais-sobre-bact%C3%A9rias-gram-positivas

10- Doençcas causadas pelo Gram positivo

Essas bactérias Gram-positivas, em forma de esferas (cocos) causam muitos distúrbios, incluindo faringite estreptocócica, pneumonia e infecções em feridas, na pele, em válvulas cardíacas e na corrente sanguínea.

https://www.msdmanuals.com/pt-pt/casa/infec%C3%A7%C3%B5es/infec%C3%A7%C3%B5es-bacterianas-bact%C3%A9rias-gram-positivas/infec%C3%A7%C3%B5es-estreptoc%C3%B3cicas

Os bacilos Gram-positivos causam alguns tipos de infecção, incluindo:

Carbúnculo.

Difteria.

Erisipeloide.

Listeriose.

11- Doenças causada pelo Gram-negativo

As bactérias Gram-negativas causam peste, cólera e febre tifoide. Essas infecções são raras em Portugal, mas são mais comuns em regiões do mundo com condições sanitárias precárias e/ou água e suprimento de alimentos inseguros.

https://www.msdmanuals.com/pt-pt/profissional/doen%C3%A7as-infecciosas/bacilos-gram-negativos/introdu%C3%A7%C3%A3o-a-bacilos-gram-negativos

12- Qual é o nome da bactéria utilizada em " Iorgurte"

O iogurte é produzido a partir da ação de uma cultura mista dos microorganismos Lactobacillus bulgaricus e do Streptococcus thermophilus.

Essas bactérias se mantêm em crescimento associado ou culturas separadas que são inoculadas no leite em proporções definidas. Geralmente, utiliza-se a proporção de 1:1 (cocos para bacilos), que definem as características reológicas e aromáticas ideais. As bactérias lácticas, em convívio simbiótico, estimulam-se mutuamente, complementando o crescimento uma da outra

https://www.einstein.br/noticias/noticia/iogurte

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Decidimos repetir o testes nos eppendorfs relativos à influência do LPS, à influência do parasita e de parasita juntamente com interferão (100pg/mL).

Desta vez utilizámos uma quantidade maior de DNA, neste caso 3 µL.

Para além disso, decidimos testar novos genes nos eppendorfs onde o teste PCR correu bem e forneceu os melhores resultados.

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Fig. 28-Esquema utilizado para a organização da placa PCR (PH)

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Primeiramente preparámos as soluções para testar MARVEL d1 e e GBP4 em 5 poços. Pipetámos, para dois eppendorfs:

27,5 µL Sybr Green Master Mix;

1,1 µL de primer forward e primer reverse (correspondente a cada um dos genes);

8,8 µL de água.

Pipetámos 7 µL da solução para cada um dos poços e 3 µL de DNA dos eppendorfs 4,5,6,16,17.

Preparar as soluções para testar os genes TNF-a (gene responsável pela tradução da citocina TNF-a), iNOS (Sintase do óxido nítrico induzida - enzima que produzem óxido nítrico a partir da L-arginina. O óxido nítrico é extremamente tóxico para os parasitas, promovendo assim a sua morte), e GBP5 (guanylate binding protein 5 - proteína relacionada com a resposa imunitária das células e com a produção de citocinas).

Pipetámos também, para 3 eppendorfs, 60,5 µL de Sybr Green Master Mix, 2,42 µL de primer forward e reverse, correspondente a cada um dos genes e 31,46 microlitos de água.

Foram adicionados 8 µL da solução em cada um dos poços.

Adicionámos 3 µL de DNA dos eppendorfs para os poços correspondentes e de seguida vedamos a placa e escrevemos num papel o protocolo de PCR desejado. As placas foram deixadas no frigoríficos da sala PCR e o protocolo descrito e seguido foi o mesmo da semana anterior.

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No dia seguinte, exercer a técnica de " plaquamento ", que consiste em meios de cultura, requerem cuidados para sua estocagem no laboratório após seu preparo, pois a função de cada um seus constituintes deve ser preservada, a fim de se garantir a obtenção de resultados confiáveis.

Linck: https://brqualityconsultoria.com.br/cuidados-importantes-com-meios-de-cultura-em-seu-laboratorio-de-microbiologia/

As bactérias aprofundadas são:

1-" Esherichia coli", encontrada no intestino humano , pertencem no grupo do Gram negativo.

2- " lactobacillus bulgaricus", encontradas no iorgurte, pertencem no grupo do Gram positivo

3- " "Lactobacilos", encontradas nas maçãs orgânicas, que beneficiam o organismo dos seres vivos e não causam doenças.

     "Procedimento de prevenção"

- Desinfetar o local antes da utilização
- Pôr lâmpada para esterilizar
- Usar: bata, luva e mascara
- E por fim desinfetar o local após a utilização

         "Técnica"
- Meio de cultura de placas com bactérias mais água destilada com ajuda da Ansa, colocamos nos tubinhos e misturamos até homogenização, durante este todo passo não devemos esquecer da lâmpada em volta de nos em que a cada momento usamos para desinfetar a ansa.

- Duas placas para cada tipo de bactéria em total foram 6 placas de bactérias

- Após da homogenização das bactérias e da água destilada, entramos no processo de colocação da mistura homogenicas nas placas utilisando dois elementos fundamentais para este processo:

- Parafilme, ansa, lâmpadas desinfetante

1- parafilme:serve para prender a placae evitar a evaporação ou a penetração de bactérias desconhecidas que por consequência alterar os resultados esperados.

2- Ansa: serve para colocar a mistura homogenica na placa, sem esquecer de desinfetar a cada momento.


 3- lâmpada desinfetante: servi para esterilizar a ansa e desinfetar o local onde está sendo feita o procedimento.

Procedimento da técnica de plaquamento

Homogenizar a solução de bactérias com água destilado, se for preciso

utilizar a " Ansa" para a recolhar das mesma, sem esquecer da " lâmpada desinfetante, pois permite que a solução ão ser invadida por agentes desnecessário.

Colocar na placa petri a mistura homogenizada, feito isso, esperar alguns dias enquanto estiver dentro da estufa. Após o tempo de espera, concede acesso para a próxima técnica denominada "Coloração de Gram"

Este projeto teve como finalidade de: identificar bactérias e aprender a técnica de" Coloração de Gram ", pois durante o dia a dia, temos como agente acompanhante as " Bácterias ", que são benéficas e patogenicas para os seres vivos e ao meio ambiente.

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Elementos essenciais para a "técnica  de plaqueamento"

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Elementos essenciais para a "técnica  de plaqueamento"

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Parafilme: serve para fixar a placa petri, prevenindo assim a penetração de agentes improviso.

Lâmpada desinfetante: Como o próprio nome diz, desinfetar o local onde está ser feita a técnica

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A água destilada é uma das ferramentas mais importantes em um laboratório. É usada como solvente, lubrificante e reagente, e também pode ser usada para limpar equipamentos e vidrarias. Além de ser importante para os processos químicos, a água destilada é essencial para manter o ambiente do laboratório limpo e seguro.

Linck: https://www.splabor.com.br/blog/destilador-2/o-que-e-agua-destilada-conhecendo-os-beneficios/

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Neste dia abordamos sobre como será procedido o nosso projeto, fizemos uma breve planificação de tudo que iremos de inferir ao longo deste novo projeto intitulado " Bactérias", que durante as minhas publicações entenderão.

Planeou-se sobre a "Importância das Bactérias no dia-dia", os seus benefícios e os prejuízos,que podem causar no meio ambiente e no organismo de um ser vivo. Sendo assim, darei a iniciativa e a contextualização do projeto em sim. como todos nós sabemos que as "Bactérias" são células biológicas encontradas no meio ambiente, e no organismo de um ser vivos, em que são benéficas e patogénicas.

O objetivo crucial deste projeto foi de, identifcar bactérias e aprender sobre o procedimento denominada "coloração de Gram", que é baseada em descobrir bactérias de Gram-positivo e Gram-negativo.

A técnica denominada coloração de Gram permite classificar as bactérias em dois grupos, por meio da análise das diferenças existentes na composição da parede celular.

Quando submetida à coloração de Gram, a parede celular das bactérias pode adquirir coloração roxa ou vermelha.

As bactérias coradas de roxa são chamadas de gram-positivas e se caracterizam por apresentar uma parede celular mais simples e rica em peptideoglicano.

As bactérias gram-negativas, por sua vez,  coram-se em vermelho e apresentam uma estrutura mais complexa, com menos peptideoglicano.

Determinar se uma bactéria é gram-positiva ou gram-negativa é importante para indicar, por exemplo, que tratamento deve ser adotado em caso de infecções. As bactérias gram-negativas, por exemplo, são, em geral, mais resistentes aos antibióticos que as gram-positivas.

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Fig: (9) Conhecimento do site " European Galaxy Team "

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Neste dia os investigadores ensinaram-nos a manusear corretamente este site "European Galaxy Team" pois tivemos muitas dificuldade de aceder-lo, todavia contém informações pertinentes sobre tudo que se trata das mitocôndrias e da doença em que os meus colegas estão a estudar.

Houve partilhas de espécie, onde cada aluno tevi o direito de estudar uma, pois devemos distinguir-nos, com intuito de obter resultados diferentes.

Por exemplo: a min foi-me dado a espécie " Lepus castroviejoi", que abordei no início do projeto.

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Nesta sessão focámo-nos na análise e construção de gráficos com os resultados obtidos no teste ELISA realizado na quarta semana.

Inicialmente trabalhámos no Excel que, apesar de apresentar certas limitações já que certas funcionalidades apenas estão disponíveis na versão paga, foi bastante útil na parte inicial da nossa organização de dados.

Nele introduzimos todos os nossos dados obtidos pelo ELISA Reader, na quarta semana, numa tabela. Estes dados consistiam na leitura de dois comprimentos de onda diferentes (450 nm e 570 nm). Uma das tabelas tinha como valor da absorvância 570 nm (comprimento de onda que corresponde às interferências que possam existir na placa utilizada no ELISA Reader), já a outra tabela tem o valor da absorvância a 450 nm (comprimento de onda que correspondente a amarelo).

De seguida, criámos uma tabela correspondente à subtração dos valores da tabela de 570 nm à de 450 nm (450-570). Isto minimiza a influência de interferências, contribuindo para uma maior precisão dos resultados.

Os dados correspondentes ao grupo de controlo foram assinalados e criámos duas tabelas, uma para cada conjunto de dados, cada uma correspondente a uma das placas colocadas no ELISA Reader.

Estes dados inseridos em cada tabela possibilitaram a construção de dois gráficos da absorvância em função das citocinas. Para isto os eixos x e y foram colocados em logaritmo o que facilita a interpretação dos dados dos gráficos. Após comparação dos dois gráficos, foi selecionado aquele apresentava menos desvios.

Uma vez com os valores de absorvância, determinámos a equação do gráfico e definimos uma equação que corresponde ao seu inverso. Assim, poderemos determinar a concentração de citocinas em função da absorvância.

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Inicialmente trabalhámos no Excel que, apesar de apresentar certas limitações já que certas funcionalidades apenas estão disponíveis na versão paga, foi bastante útil na parte inicial da nossa organização de dados.

Nele introduzimos todos os nossos dados obtidos pelo ELISA Reader, na quarta semana, numa tabela. Estes dados consistiam na leitura de dois comprimentos de onda diferentes (450 nm e 570 nm) - Tabelas A.

Uma das tabelas tinha como valor da absorvância 570 nm (comprimento de onda que corresponde às interferências que possam existir na placa utilizada no ELISA Reader), já a outra tabela tem o valor da absorvância a 450 nm (comprimento de onda que correspondente a amarelo).

De seguida, criámos tabelas correspondentes à subtração dos valores da tabela de 570 nm à de 450 nm (450-570) para cada uma das citocinas (TNF-α e IL-8) - Tabelas B. Isto minimiza a influência de interferências, contribuindo para uma maior precisão dos resultados.

Os dados correspondentes ao grupo de controlo foram assinalados e criámos duas tabelas, uma para cada conjunto de dados, cada uma correspondente a uma das placas colocadas no ELISA Reader.

Estes dados inseridos em cada tabela possibilitaram a construção de dois gráficos da absorvância em função das citocinas. Para isto os eixos x e y foram colocados em logaritmo o que facilita a interpretação dos dados dos gráficos. Após comparação dos dois gráficos, foi selecionado aquele apresentava menos desvios.

Uma vez obtidos os valores de absorvância, determinámos a equação do gráfico e definimos uma equação que corresponde ao seu inverso. Assim, poderemos determinar a concentração de citocinas em função da absorvância.

De seguida, criámos uma tabela como os valores de concentração obtidos através da equação e, seguindo o esquema que tínhamos feito na quarta semana, associámos cada valor de concentração ao respetivo estímulo. Como os valores dos dois animais apresentavam grandes diferenças entre si (cada uma das placas correspondia ao sangue de um animal diferente) optamos por fazer duas tabelas distintas para cada um deles.

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Para esta segunda etapa, a nossa mentora sugeriu-nos uma plataforma chamada Gaph Pad Prism 10 que combina gráficos científicos, ajuste abrangente de curvas, estatísticas compreensíveis e organização de dados. Apesar do software desta aplicação ser pago, conseguimos ter acesso ao mesmo devido à oferta de 30 dias grátis para teste do mesmo.

Com esta aplicação fizemos as médias dos valores selecionados, organizando os valores em tabelas que nos permitem ver o desvio padrão. Nestas tabelas, também é necessário colocar a escala y em logaritmo.

Inicialmente, transferimos para a plataforma os dados das duas tabelas construídas anteriormente no Excel. Com eles, fizemos as médias dos valores e organizamos os valores em tabelas que nos permitiram ver o desvio padrão. Nestas tabelas também colocamos a escala y em logaritmo para uma leitura mais fácil dos resultados.

Dado isto, aplicámos o protocolo ANOVA que comparou os valores obtidos e nos indicou quais dos estímulos tiveram a maior diferença de valores.

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Fig. 29- Dados do teste Elisa para a citocina TNF-α.

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TNF-α: Tabela com valores de 450 nm e tabela com valores de 570 nm.

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Fig. 30- Tabela com os valores correspondente ao cálculo da diferença dos valores das absorvâncias da citocina TNF-α.

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Tabela com os valores correspondente ao cálculo da diferença dos valores das absorvâncias, tabela superior menos a tabela inferior (450 nm-570 nm) para a citocina TNF-α.

Legenda das cores:

Grupos de controlo: amarelo

Poço vazio: cinzento

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Fig. 31- Dados do teste Elisa para a citocina IL-8.

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IL-8: Tabela com valores de 450 nm e tabela com valores de 570 nm.