Small Animals Gut Microbiome and Its Relationship with Cancer

This chapter aims to discuss recent developments in understanding the small animal gut microbiome’s relationship with cancer, focusing on animals as well as a model for studying humans. Based on multidirectional interactions between the microbiome, the environment and the epigenetically/genetically vulnerable host, it intends to address the mechanisms by which microorganisms can contribute to carcinogenesis describing the roles of the microbiome directly in the pathogenesis of the disease through complex interactions between the microbiome and the host’s metabolic and immune systems. The feasibility for developing new cancer diagnostic and prognostic methodologies plus treatments based on small animals’ microbiome profiles are reviewed

Global DNA methylation in peripheral blood of healthy dogs and dogs bearing cancer

O linfoma não-Hodgkin (LNH) é bastante prevalente em cães e atualmente é aceito como modelo comparativo da doença em humanos. Padrões aberrantes de metilação de DNA parecem exercer um papel chave no desenvolvimento de tumores hematopoiéticos nos seres humanos, constituem um mecanismo especial de controle transcricional e podem ser influenciados por alterações genéticas e ambientais. Os efeitos da metilação global do DNA têm sido raramente investigados em cães, principalmente em processos neoplásicos. O objetivo do presente estudo foi quantificar a metilação global do DNA em leucócitos sanguíneos de cães portadores de LNH, comparando com a metilação global do DNA de leucócitos sanguíneos de cães sadios e identificar genes diferentemente metilados nas mesmas amostras. Para isto, utilizou-se o DNA obtido da capa leucocitária de amostras de sangue venoso periférico de 10 cães sadios e 9 cães com LNH multicêntrico. Para imunofenotipagem dos linfomas, aplicou-se o painel imunocitoquímico de anticorpos anti-CD79a, anti-PAX5 e anti-CD3. O índice de proliferação celular foi obtido por meio da contagem de núcleos positivos para Ki-67. A metilação global do DNA dos leucócitos foi quantificada pelo método High Performance Liquid Chromatography (HPLC) e visualmente (por escores) em amostras submetidas a imunocitoquímica (ICQ) com o anticorpo anti-5-metil citosina (5MetCyt). Para a identificação de genes diferentemente metilados entre ambos os grupos avaliados, utilizou-se a técnica de beadchip com o ensaio Infinium Methylation EPIC BeadChip humano (850K). Como resultados, em ambos os métodos (HPLC e ICQ), os leucócitos sanguíneos de cães portadores de LNH apresentaram metilação global do DNA significantemente inferior à dos cães sadios (HPLC: p= 0,027 / ICQ: p= 0,015). Das 853.307 ilhas CpGs investigadas no microarranjo, houve hibridização de 34.574 sondas nas amostras caninas. Desse total, observou-se diferença significante em nível de metilação de 606 sondas, e por meio da análise das similaridades homólogas e ortólogas, identificou-se 550 genes diferentemente metilados entre os dois grupos. Nosso estudo foi pioneiro em sugerir que cães com LNH apresentam hipometilação global do DNA de leucócitos circulantes quando comparados a cães sadios. Apesar de termos usado amostras caninas em um ensaio desenvolvido especificamente para o DNA humano (Infinium Methylation EPIC BeadChip) foi possível a identificação de genes diferentemente metilados e possíveis novos alvos com potencial preventivo ou terapêutico. Futuros estudos epidemiológicos são necessários para correlacionar o padrão de metilação de leucócitos com o risco de desenvolver linfomas e utilizá-lo como biomarcadorNon-Hodgkin\'s lymphoma is quite prevalent in dogs and it is currently accepted as a comparative model for the disease in humans. Aberrant patterns of DNA methylation appear to play a key role in the development of hematopoietic tumors in humans, constitute a special mechanism of transcriptional control and may be influenced by genetic and environmental variations. The effects of methylation have been rarely investigated in dogs, especially in neoplastic processes. The aim of the current study is to quantify the global DNA methylation of blood from dogs bearing non-Hodgkin\'s lymphoma, comparing them with the methylation content and pattern of healthy dogs and identify differently methylated genes in the same samples. For this, the DNA obtained from the buffy coat of peripheral venous blood samples from 10 healthy dogs and 9 dogs with multicentric non-Hodgkin\'s lymphoma were used. For immunophenotyping of lymphomas, the immunocytochemical panel of anti-CD79a, anti-PAX5 and anti-CD3 antibodies were applied. The cell proliferation index was performed by counting positive nuclei with anti-Ki-67. The global methylation of leukocyte DNA was quantified by the High Performance Liquid Chromatography (HPLC) method and visually (by scoring) in samples subjected to immunocytochemistry (ICQ) with the anti-5-methyl cytosine antibody (5MetCyt). For the identification of differently methylated genes between both groups, the bead chip technique was used with the Infinium Methylation EPIC BeadChip human assay (850K). As a result, in both methods (HPLC and ICQ), dogs with non-Hodgkin\'s lymphoma had a lower amount of methylation than healthy dogs (HPLC: p = 0.027 / ICQ: p = 0.015). Of the 853,307 CpGs investigated in the microarray, there were 34,574 probes hybridized in the canine samples. From this total, significant difference was observed in the methylation level of 606 probes and through the homologous and orthologous similarities, 550 differently methylated genes were identified between the two groups. Our study was a pioneer in suggesting that dogs bearing non-Hodgkin\'s lymphoma presented DNA global hypomethylation of circulating leukocytes when compared to healthy dogs. Although we used canine samples in an assay developed specifically for human DNA (Infinium Methylation EPIC BeadChip) it was possible to identify differently methylated genes and possible new targets with preventive or therapeutic potential. Future epidemiological studies are needed to correlate the methylation pattern of leukocytes with the risk of developing lymphomas and to use it as a biomarke

Global DNA methylation in peripheral blood of healthy dogs and dogs bearing cancer

O linfoma não-Hodgkin (LNH) é bastante prevalente em cães e atualmente é aceito como modelo comparativo da doença em humanos. Padrões aberrantes de metilação de DNA parecem exercer um papel chave no desenvolvimento de tumores hematopoiéticos nos seres humanos, constituem um mecanismo especial de controle transcricional e podem ser influenciados por alterações genéticas e ambientais. Os efeitos da metilação global do DNA têm sido raramente investigados em cães, principalmente em processos neoplásicos. O objetivo do presente estudo foi quantificar a metilação global do DNA em leucócitos sanguíneos de cães portadores de LNH, comparando com a metilação global do DNA de leucócitos sanguíneos de cães sadios e identificar genes diferentemente metilados nas mesmas amostras. Para isto, utilizou-se o DNA obtido da capa leucocitária de amostras de sangue venoso periférico de 10 cães sadios e 9 cães com LNH multicêntrico. Para imunofenotipagem dos linfomas, aplicou-se o painel imunocitoquímico de anticorpos anti-CD79a, anti-PAX5 e anti-CD3. O índice de proliferação celular foi obtido por meio da contagem de núcleos positivos para Ki-67. A metilação global do DNA dos leucócitos foi quantificada pelo método High Performance Liquid Chromatography (HPLC) e visualmente (por escores) em amostras submetidas a imunocitoquímica (ICQ) com o anticorpo anti-5-metil citosina (5MetCyt). Para a identificação de genes diferentemente metilados entre ambos os grupos avaliados, utilizou-se a técnica de beadchip com o ensaio Infinium Methylation EPIC BeadChip humano (850K). Como resultados, em ambos os métodos (HPLC e ICQ), os leucócitos sanguíneos de cães portadores de LNH apresentaram metilação global do DNA significantemente inferior à dos cães sadios (HPLC: p= 0,027 / ICQ: p= 0,015). Das 853.307 ilhas CpGs investigadas no microarranjo, houve hibridização de 34.574 sondas nas amostras caninas. Desse total, observou-se diferença significante em nível de metilação de 606 sondas, e por meio da análise das similaridades homólogas e ortólogas, identificou-se 550 genes diferentemente metilados entre os dois grupos. Nosso estudo foi pioneiro em sugerir que cães com LNH apresentam hipometilação global do DNA de leucócitos circulantes quando comparados a cães sadios. Apesar de termos usado amostras caninas em um ensaio desenvolvido especificamente para o DNA humano (Infinium Methylation EPIC BeadChip) foi possível a identificação de genes diferentemente metilados e possíveis novos alvos com potencial preventivo ou terapêutico. Futuros estudos epidemiológicos são necessários para correlacionar o padrão de metilação de leucócitos com o risco de desenvolver linfomas e utilizá-lo como biomarcadorNon-Hodgkin\'s lymphoma is quite prevalent in dogs and it is currently accepted as a comparative model for the disease in humans. Aberrant patterns of DNA methylation appear to play a key role in the development of hematopoietic tumors in humans, constitute a special mechanism of transcriptional control and may be influenced by genetic and environmental variations. The effects of methylation have been rarely investigated in dogs, especially in neoplastic processes. The aim of the current study is to quantify the global DNA methylation of blood from dogs bearing non-Hodgkin\'s lymphoma, comparing them with the methylation content and pattern of healthy dogs and identify differently methylated genes in the same samples. For this, the DNA obtained from the buffy coat of peripheral venous blood samples from 10 healthy dogs and 9 dogs with multicentric non-Hodgkin\'s lymphoma were used. For immunophenotyping of lymphomas, the immunocytochemical panel of anti-CD79a, anti-PAX5 and anti-CD3 antibodies were applied. The cell proliferation index was performed by counting positive nuclei with anti-Ki-67. The global methylation of leukocyte DNA was quantified by the High Performance Liquid Chromatography (HPLC) method and visually (by scoring) in samples subjected to immunocytochemistry (ICQ) with the anti-5-methyl cytosine antibody (5MetCyt). For the identification of differently methylated genes between both groups, the bead chip technique was used with the Infinium Methylation EPIC BeadChip human assay (850K). As a result, in both methods (HPLC and ICQ), dogs with non-Hodgkin\'s lymphoma had a lower amount of methylation than healthy dogs (HPLC: p = 0.027 / ICQ: p = 0.015). Of the 853,307 CpGs investigated in the microarray, there were 34,574 probes hybridized in the canine samples. From this total, significant difference was observed in the methylation level of 606 probes and through the homologous and orthologous similarities, 550 differently methylated genes were identified between the two groups. Our study was a pioneer in suggesting that dogs bearing non-Hodgkin\'s lymphoma presented DNA global hypomethylation of circulating leukocytes when compared to healthy dogs. Although we used canine samples in an assay developed specifically for human DNA (Infinium Methylation EPIC BeadChip) it was possible to identify differently methylated genes and possible new targets with preventive or therapeutic potential. Future epidemiological studies are needed to correlate the methylation pattern of leukocytes with the risk of developing lymphomas and to use it as a biomarke

Global DNA methylation of peripheral blood leukocytes from dogs bearing multicentric non-Hodgkin lymphomas and healthy dogs: A comparative study.

Non-Hodgkin lymphomas are among the most common types of tumors in dogs, and they are currently accepted as comparative models of the disease in humans. Aberrant patterns of DNA methylation seem to play a key role in the development of hematopoietic neoplasms in humans, constitute a special mechanism of transcriptional control, and may be influenced by genetic and environmental factors. Blood leukocyte DNA global methylation has been poorly investigated in dogs. The aim of this study is to examine whether peripheral blood global DNA methylation is associated with canine multicentric lymphomas. Peripheral venous blood samples from ten healthy dogs and nine dogs bearing multicentric lymphomas were collected, and the buffy coat was separated. Global DNA methylation was analyzed by High Performance Liquid Chromatography (HPLC) and immunocytochemistry (ICC). In both analyses, leukocytes from dogs with lymphoma presented lower global DNA methylation than in healthy dogs (HPLC: p = 0.027/ 5MeCyt immunoreactivity scores: p = 0.015). Moderate correlation was observed between the results obtained by HPLC and ICC (correlation coefficient = 0.50). For the identification of differently methylated genes between both groups, the Infinium Human Methylation (HM) EPIC BeadChip (850K) was used. Of the 853,307 CpGs investigated in the microarray, there were 34,574 probes hybridized in the canine samples. From this total, significant difference was observed in the methylation level of 8433 regions, and through the homologous and orthologous similarities 525 differently methylated genes were identified between the two groups. This study is pioneer in suggesting that dogs bearing non-Hodgkin lymphoma presented DNA global hypomethylation of circulating leukocytes compared with healthy dogs. Although canine samples were used in an assay developed specifically for human DNA, it was possible to identify differently methylated genes and our results reiterate the importance of the use of peripheral blood leukocytes in cancer research and possible new biomarkers targets