Auswahl der wissenschaftlichen Literatur zum Thema „Anti-Saccharomyces cerevisiae antibody“

Background: The enhanced green fluorescent protein (EGFP) gene is a reporter gene that can be used to optimize protein isolation procedures and the functional working of a transduction protein. EGFP, with the addition of eleven arginine residues, has been engineered to functionally improve the protein transduction process, which can later be used for cell reprogramming like induced pluripotent stem cells. The addition of six histidine amino acid residues at its C-terminal is intended for the protein isolation process using the His-tag antibody. Methods: The study aimed to investigate the optimization of the EGFP polyarginine protein expression in Saccharomyces cerevisiae in sufficient quantities for the protein isolation stage. This study also analyzed EGFP expression without polyarginine to analyze the polyarginine addition effect on expression processes. Protein expression was qualitatively measured by looking at expression fluorescence and protein levels of EGFP and EGFP - PolyR proteins. Results: Bands on Western Blots with 6×His-tag monoclonal antibody (primary antibody) and Goat anti-mouse IgG HRP (secondary antibody) showed the EGFP polyarginine and EGFP proteins were expressed in Saccharomyces cerevisiae INVSc1 at relatively low levels. The lyticase incubation time modification and administration of 3-5 kDa microfilter to concentrate increased the yield of isolated protein. Conclusions: The sufficient amount of protein isolation in S. cerevisiae can be achieved by using lyticase and sonicators combination for the lysis process.

Our objective was to investigate the concentrations of IgA and IgG antibodies to Saccharomyces cerevisiae in whole saliva and serum samples from HIV-infected patients and to compare them with the corresponding antibody values of healthy controls. A cross-sectional design was used. The test group consisted of 23 HIV-infected male individuals, aged 20-41 years old, free of any other systemic disease. Twenty healthy subjects aged 27-43 years old served as controls. Whole unstimulated saliva and blood were collected from all subjects. IgA concentrations in saliva and IgA and IgG concentrations in serum were measured by solid-phase enzyme-linked immunosorbent assay. Salivary antibody concentrations were calculated by reference to a pooled standard saliva obtained from 10 healthy males with high levels of anti- S. cerevisiae antibody activity. Total IgA and IgG concentrations were measured by nephelometry/tholocymetry assay. No significant difference was observed in salivary specific IgA and serum specific IgG levels to S. cerevisiae, while serum specific IgA were significantly lower in HIV infected patients compared to control group. Opportunistic infections due to S. cerevisiae, although rare, cannot be dismissed. This yeast can show a potential virulence in debilitated patients, therefore, further extensive investigation should be considered.

Introduction. Alors que le mycobiote représente une part quantitative négligeable, en apparence, du microbiote intestinal, les preuves de son rôle dans les maladies inflammatoires chroniques de l’intestin, et notamment la maladie de Crohn (MC), sont croissantes. Ce travail avait pour objectif d’évaluer l’impact de Candida albicans et Saccharomyces cerevisiae sur l’inflammation et la fibrose intestinale (FI), mais également sur la production des anticorps anti-S. cerevisiae (ASCA), reconnaissant des séquences oligomannosidiques de faible degré de polymérisation ayant un résidu α,1-3 mannose terminal.Méthodes. Des modèles murins (C57BL/6, axénique et CEABAC10 exprimant CEACAM6 humaine) et cellulaires (fibroblaste CCD-18Co et cellule épithéliale intestinale Caco-2) d’inflammation et de FI induites (par sulfate de dextrane sodique ou TGF-β) ont été utilisés pour évaluer les réponses cellulaires, tissulaires et systémiques aux levures et à la souche bactérienne LF82, souche adhérente invasive d’Escherichia coli (AIEC) utilisée comme témoin, par analyse histologique, RT-q-PCR et détermination des ASCA. Parallèlement, une analyse métagénomique (MTG) du mycobiote fécal et la détermination des ASCA et de la calprotectine fécale étaient réalisés dans le cadre d’une étude cas-témoins, « MAGIC », incluant des sujets atteints de la MC de diagnostic récent et en rémission clinique comparés à leurs apparentés sains du premier degré, ainsi qu’à des témoins sains appariés. Cette étude était réalisée en collaboration avec d’autres équipes de recherche, analysant notamment le microbiote fécal bactérien et caractérisant les souches AIEC.Résultats. Dans le modèle murin de FI chimio-induite, LF82 aggravait l’inflammation et la FI cliniquement, microscopiquement et à l’échelle de l’expression génique (EG). C. albicans augmentait l’EG surtout des marqueurs de l’inflammation alors que S. cerevisiae n’avait pas d’effet. In vitro, seules les cellules épithéliales répondaient au TGF-β et/ou à LF82 et l’EG des marqueurs pro-fibrotiques était augmentée, tandis que les levures n’avaient pas d’effet sur la FI. Par ailleurs, les levures n’induisaient pas la synthèse d’ASCA chez les différents modèles murins étudiés. Dans l’étude clinique MAGIC, 41,7% des patients atteints de la MC étaient porteurs d’ASCA, 2,9% des apparentés sains et 1,8% des témoins sains, aucune différence n’était observée selon l’âge. L’analyse MTG bactérienne, montrait un profil spécifique chez les apparentés sains (richesse et AIEC plus élevées) vs. témoins et patients atteints de MC, liée notamment à la présence de bactéries symbiontes, mais également la présence plus élevée d’AIEC vs. témoins ; les patients atteints de MC présentaient une diversité plus basse vs. témoins et apparentés sains, ainsi que la présence d’AIEC plus élevée vs. témoins. Cependant, le mycobiote fécal était similaire entre les différents groupes, que ce soit en abondance relative (notamment pour C. albicans et S. cerevisiae), ou en alpha-, beta-diversités. Aucune association n’a été retrouvée entre micromycètes et les différents paramètres évalués (calprotectine, ASCA, AIEC).Conclusion. Dans les limites des modèles utilisés, les levures ici étudiées n’avaient pas d’impact sur la FI, contrairement à LF82, et ne semblaient pas être responsables de la synthèse d’ASCA. L’hypothèse de l’auto-anticorps reste à approfondir. L’analyse MTG fongique des selles des sujets de l’étude MAGIC fait suggérer que les modifications observées dans d’autres études seraient une conséquence de la MC. En effet, l’absence de profil fongique spécifique pourraient s’expliquer par le fait que les sujets inclus atteints de MC présentaient un diagnostic récent et donc un profil fongique peu modifié à ce stade de la maladie. Toutefois, la qualité de ces données justifie que ces résultats soient confirmés sur une large cohorte de patients atteints de MC suivis de manière séquentielle en début et au décours de la maladie
Introduction. While the mycobiota represents a seemingly negligible quantitative proportion of the intestinal microbiota, evidence of its role in chronic inflammatory bowel diseases, and in particular in Crohn's disease (CD), is growing. This work aimed to evaluate the impact of Candida albicans and Saccharomyces cerevisiae on inflammation and intestinal fibrosis (IF), but also on the production of anti-S. cerevisiae antibodies (ASCA), recognizing oligomannosidic sequences of low degree of polymerization having a terminal α, 1-3 mannose residue.Methods. Murine (C57BL/6, axenic and CEABAC10 expressing human CEACAM6) and cellular (CCD-18Co fibroblast and Caco-2 intestinal epithelial cell) models of induced inflammation and IF (by DSS or TGF-β) were used to evaluate cellular, tissue and systemic responses to yeasts and bacterial strain LF82, invasive adherent strain of E. coli (AIEC) used as control, by histological analysis, RT-q-PCR and determination of ASCA. Alongside, a metagenomic (MTG) analysis of the fecal mycobiota and the determination of ASCA and fecal calprotectin were carried out as part of a case-control study, “MAGIC”. This study included subjects with recently diagnosed CD and in clinical remission in comparison to their healthy first-degree relatives, as well as to matched healthy controls. This study was carried out in collaboration with other research teams, analyzing in particular the bacterial fecal microbiota and carrying out the characterization of AIEC strains.Results. In the mouse model of chemo-induced IF, LF82 worsened inflammation and IF, clinically, microscopically and at the level of gene expression (GE). C. albicans increased the GE especially of markers of inflammation. S. cerevisiae had no effect. In vitro, only epithelial cells responded to TGF-β and/or LF82 and the GE of pro-fibrotic markers was increased, while yeast had no effect on IF. Furthermore, yeasts did not induce the synthesis of ASCA in the different mouse models studied. In the MAGIC clinical study, 41.7% of CD patients carried ASCA, 2.9% of healthy relatives and 1.8% of healthy controls and no difference was observed according to age. Bacterial MTG analysis showed a specific profile in healthy relatives (higher richness and AIEC) vs. controls and MC patients, particularly linked to the presence of symbionts bacteria, but also the higher presence of AIEC vs. controls; patients with CD had lower diversity vs. healthy controls and relatives, as well as the presence of higher AIEC vs. controls. However, the fecal mycobiota was similar between the different groups, whether in relative abundance (notably for C. albicans and S. cerevisiae), or in alpha- beta-diversity. No association between micromycetes and the different parameters evaluated (calprotectin, ASCA, AIEC) was found.Conclusion. These results suggest, within the limits of the models used, that the studied yeasts do not have an impact on IF, unlike LF82, and do not seem responsible for the synthesis of ASCA. The autoantibody hypothesis remains to be further explored. Fecal fungal MTG analysis of the individuals included in the MAGIC study suggests that the changes observed in other studies are a consequence of CD. Indeed, the absence of a specific fungal profile could be explained by the fact that the included subjects suffering from CD had a recent diagnosis and therefore had a little modified fungal profile at this stage of the disease. However, the quality of these data warrants that these results be confirmed in a large cohort of patients with CD followed sequentially at the beginning and throughout the course of the disease

Dissertação de mestrado em Genética Molecular
Fundamental cellular processes appear to be highly conserved between Saccharomyces cerevisiae and other more complex Eukaryotic species, including humans. “Humanized yeast systems” emerged as a tool to study molecular aspects of human pathologies. The present work aimed at contributing to build and validate a large high throughput platform of yeast strains displaying phenotypes that can enable further testing galectin-related drugs and peptides. This platform was designed to consist of two types of strains, the ones expressing human galectins and the ones expressing these together with the human KRAS cDNA. The rationale behind this relates with the putative dialogue between Galectins and RAS signaling pathway in mammals. Considering that EGFR mediates KRAS signaling and that yeast also harbors a RAS signaling pathway, the “humanized yeasts” expressing KRAS were used to identify the yeast target of anti-EGFR. Furthermore, it was also used for phenotyping the most well-known biological processes known to be controlled by RAS pathway. On the other hand, considering that the deletion of GUP1 in S. cerevisiae increases the resistance to the oncological drug Imatinib, the similarities between the phenotypes associated to the deletion of RAS and GUP genes were also verified. Two Hsp70, Ssa2p and Ssb2p and one glyceraldehyde-3-phosphate dehydrogenase Tdh3p, were identified as EGFR-like proteins. The subsequent alignments analysis between EGFR and these proteins revealed that Ssb2p and its very close homologue Ssa2p present some homology with EGFR sequence, namely at the level of three EGFR conserved amino acids known to be responsible for the interaction with the anti-EGFR antibody Cetuximab used in cancer treatment. This and other lines of evidence support Ssb2p and/or Ssa2p as good candidates for EGFR homology. The phenotypic tests revealed that both the deletions of GUP and RAS genes promote a reduction in chronological life span and cell size, except in the case of Δras2 strain, whose cells were bigger than wild type control. Nutrient depletion (carbon) promoted replication stress in Δras2 cells that failed to enter into G1 arrest, and were blocked in S phase, concurring with the bigger size of Δras2 cells and their short lifespan. Moreover, the cells with GUP genes deleted, in opposition to RAS mutants, showed ability to adhere to solid nitrogen-deficient medium. Neither RAS nor GUP mutants were able to invade or filament under these conditions. With this work we were able to determine the possible homologue of EGFR, many times associated with cancer pathologies, and contributed to gain insights on RAS and GUP genes common phenotypes. In conclusion, the present work opens doors to future discovery of new pathways in yeast, in addition to showing that S. cerevisiae is a suitable model to create a platform to explore therapeutic drugs/antibodies.
Vários processos celulares fundamentais encontram-se conservados entre a levedura Saccharomyces cerevisiae e outras espécies eucariotas mais complexas, incluindo humanos. A “Levedura humanizada” surgiu como uma ferramenta de estudo sobre aspectos moleculares de patologias humanas. Com este trabalho pretendeu-se contribuir para a construção e validação de uma plataforma de estirpes de levedura que exibam determinados fenótipos, permitindo o teste de drogas e péptidos relacionados com as galectinas. Esta foi planeada para incluir duas estirpes a expressar galectinas humanas, assim como o cDNA do KRAS humano. O propósito desta plataforma advém de uma possível interação entre as Galectinas e a via de sinalização dos RAS em mamíferos. Tendo em conta que o EGFR medeia a cascata de sinalização KRAS, e que também a levedura possui uma via de sinalização Ras, usou-se as leveduras humanizadas a expressar o KRAS para identificar o alvo do anti-EGFR. Para além disso, estas foram usadas para a fenotipagem de processos biológicos controlados pela cascata RAS. Por outro lado, tendo em conta que a deleção do GUP1 aumenta a resistência à droga oncológica Imatinib, verificouse também as semelhanças fenotípicas entre as deleções RAS e GUP. Foram identificadas duas proteínas Hsp70, Ssa2p e Ssb2p, e uma gliceraldeído-3-fosfato desidrogenase Tdh3p, como sendo os alvos do anti-EGFR. Subsequentemente, a análise dos alinhamentos entre o EGFR e estas proteínas revelaram que a Ssb2p e a sua homóloga Ssa2p apresentam similaridade com a sequência do EGFR, nomeadamente ao nível de três aminoácidos responsáveis pela interação com o anticorpo anti-EGFR, Cetuximab, usado no tratamento do cancro. Esta informação suporta a hipótese das proteínas Ssb2p e/ou Ssa2p serem boas candidatas a homólogas do EGFR. Os testes fenotípicos revelaram que as deleções dos genes GUP e RAS promovem uma redução da longevidade cronológica e da área celular, com excepção para a estirpe Δras2 cujas células se revelaram maiores do que a wt. A depleção de nutrientes (carbono) induziu stress replicativo nas células Δras2, que por sua vez falharam a entrada na fase G1, ficando bloqueadas na fase S, o que está de acordo com o aumento da área celular e a baixa longevidade cronológica das células Δras2. Além disso, as células com a deleção nos genes GUP, contrariamente aos mutantes RAS, mostraram habilidade para aderir a um meio deficiente em nitrogénio. Nenhum dos mutantes RAS ou GUP foram capazes de invadir ou filamentar nas condições anteriormente descritas. Com este trabalho fomos capazes de determinar o possível homólogo do EGFR, muitas vezes associado a patologias relacionadas com o cancro, assim como contribuir para melhor compreender os fenótipos comuns associados aos genes RAS e GUP. Em conclusão, o presente trabalho abre portas para futuras descobertas de novas vias de sinalização em levedura, além de reforçar a utilização da S. cerevisiae como um bom modelo para criar uma plataforma de exploração de drogas/anticorpos.

Dissertação de mestrado em Genética Molecular
Colorectal cancer (CRC) is one of the most common malignancies affecting mankind. CRC cells over-express epidermal growth factor receptor (EGFR), which usually correlates with disease poor prognosis and reduced response to therapy. Hence, several therapeutic agents against EGFR were developed, viz. the monoclonal antibody cetuximab/Erbitux®. Such drug competes with EGFR ligands for binding to L2/III domain, which results in EGFR internalization and subsequent degradation, leading to inhibition of cell growth and angiogenesis, and induction of apoptosis. Yet, cancer patients may display or acquire resistance-inducing mutations in EGFR, as well as in its downstream effectors. These contribute to a significant degree of ineffectiveness of treatment, being one of the most prominent problems in CRC clinical assessment. Given the high degree of conservation of eukaryotic cellular processes, yeast has been a model of choice for research in many human pathologies. In this line, this work aimed at the identification of S. cerevisiae surface target of cetuximab, ultimately seeking for the possible EGFR yeast counterpart. Two different strategies were used: (1) in silico sequence and structure homology search, and (2) immune recognition in a cetuximab-based Western blot. The first approach pointed to proteins from the yeast Sporulation specific family, especially Sps2p and Sps22p. These have some structural resemblance with EGFR leucin-rich L-domains, along with cell-surface localization. Conversely, the Western blot clearly identified the Pdc1p (pyruvate decarboxylase isoform 1) as cetuximab antigen. The subsequent detailed analysis of protein features revealed that Pdc1p, as well as its close homologue Pdc5p, present some similarity with EGFR epitope sequence. Moreover, Pdc and EGFR also present some functional pathway overlapping, more evident in malignantly transformed cells. The recognition of Pdc1/5p as cetuximab antigen, combined with its extracellular localization described before, suggests that Pdc1p may have distinct functions beyond glycolytic catalysis/regulation. The double deletion of Sps and Pdc, and the use of diploid genetic background, will be needed to devise the true existence of growth phenotypes induced by cetuximab. However, this work opens a large window as to future research in novel pathways in yeast, beyond the continued exploration of yeast for the aim of generating a tool for CRC patients’ theranostics.
O cancro colo-rectal (CRC) é uma das enfermidades mais comuns no mundo. Células de CRC apresentam sobre-expressão do recetor do fator de crescimento epidérmico (EGFR), normalmente associada a um pior prognóstico e uma resposta reduzida à terapia. Desta forma, vários agentes contra EGFR foram desenvolvidos, tal como o anticorpo monoclonal cetuximab/Erbitux®. Este compete com os ligandos do EGFR para o domínio L2/III, resultando na internalização e degradação do EGFR. Isto leva à inibição da proliferação celular e angiogénese e, à indução de apoptose. No entanto, os pacientes com CRC podem ter ou adquirir mutações no EGFR, ou nos efetores da sinalização a jusante, que induzem resistência às opções terapêuticas. Estas situações contribuem para uma significativa ineficácia no tratamento do CRC, tornando-se um dos principais problemas da assistência médica a estes doentes. A elevada conservação de processos celulares eucarióticos tornou a levedura um modelo privilegiado no estudo de muitas patologias humanas. Nesse sentido, este trabalho visou a identificação do alvo do cetuximab em S. cerevisiae, em última instância, contribuindo para uma possível identificação da proteína de levedura correspondente ao EGFR. Foram usadas duas estratégias: (1) procura in silico de homologia de sequência e estrutura e, (2) imunoreconhecimento pelo cetuximab (Western blot). A primeira abordagem apontou para a família de proteínas específicas de esporulação (Sporulation specific), nomeadamente as proteínas Sps2p e Sps22p. Estas apresentam alguma semelhança estrutural com os domínios do EGFR ricos em leucinas (domínios L), além de também serem proteínas da superfície celular. Por outro lado, por Western blot identificou-se a Pdc1p (piruvato descarboxilase isoforma 1) como antigénio do cetuximab. Uma análise detalhada subsequente da composição aminoacídica revelou que a Pdc1p, bem como a sua homóloga Pdc5p, apresentam alguma similaridade com a sequência do epítopo do EGFR. Além disso, Pdc e EGFR também apresentam alguma sobreposição funcional, em particular no que diz respeito ao metabolismo das células malignas. O reconhecimento da Pdc1/5p como antigénio do cetuximab, em combinação com a descrição anterior desta proteína na superfície da célula, sugere que a Pdc1p pode ter outras funções para além do seu papel catalítico e regulador no âmbito da glicólise. Para aceder ao fenótipo que o cetuximab possa induzir no crescimento e viabilidade da levedura serão necessárias deleções duplas dos membros das famílias Sps e Pdc, bem como a utilização de estirpes diplóides de levedura. Apesar disso, e para além de permitir a prossecução do objetivo de transformar a levedura numa ferramenta para teranóstico, o presente trabalho abre uma grande janela na investigação em novas vias de sinalização em levedura.