Establishment of a multiplex RT-PCR assay for the rapid detection of fish cytokines

Kono, Tomoya; Takayama, Hiroaki; Nagamine, R.; Korenaga, Hiroki; Sakai, Masahiro

doi:10.1016/j.vetimm.2012.10.012

Cited by 22 publications

(5 citation statements)

References 66 publications

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“…We observed the strong upregulation of TGFβ1 and IL-10 genes after treatment with LPS but not polyI:C. A mechanism of feedback regulation, in which IL-10 and TGFβ1 were upregulated in a pro-inflammatory stimulation, was confirmed [41], [62]. In this study, it was suggested that Treg cells express suppressive cytokines by stimulation of LPS as an inflammatory inducer.…”

Section: Discussionsupporting

confidence: 55%

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Cytokine Gene Expression in CD4 Positive Cells of the Japanese Pufferfish, Takifugu rubripes

Kono

Korenaga

2013

PLoS ONE

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CD4+ T (Th) cells are a central component of the adaptive immune response and are divided into distinct sets based on their specific cytokine production pattern. Several reports have suggested that fish possess Th subset activity similar to that of mammals. The aim of the present study was to isolate CD4+ T cells from the blood of Japanese pufferfish, Fugu rubripes, and to characterize their cytokine expression profile. We produced a specific antibody against Fugu CD4 and performed cell sorting with the magnetic activated cell sorting system. Sorted Fugu CD4+ cells were characterized by morphology and expression analysis of cell marker genes. Fugu CD4+ cells expressed T-cell marker genes but not macrophage or B-cell marker genes. In addition, peripheral blood lymphocytes were stimulated with lipopolysaccharide (LPS), polycytidylic acid (polyI:C), concanavalin A (ConA) prior to sorting, and then Multiplex RT-PCR was used to examine the expression of Th cytokines by the stimulated Fugu CD4+ cells. LPS and polyI:C stimulation upregulated the expression of Th1, Th17 and Treg cytokines and downregulated the expression of Th2 cytokines. ConA stimulation upregulated the expression of all Th cytokines. These results suggest that fish exhibit the same upregulation of Th-specific cytokine expression as in mammals.

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Section: Discussionsupporting

confidence: 55%

“…The cytokine multiplex analysis was conducted by using the multiple assay panel as reported previously [41]. In the present study, cytokines produced by Th cells (including cytokines related to the differentiation of Th cells) were targeted.…”

Section: Methodsmentioning

confidence: 99%

Cytokine Gene Expression in CD4 Positive Cells of the Japanese Pufferfish, Takifugu rubripes

Kono

Korenaga

2013

PLoS ONE

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“…We successfully developed a multiplex RT-PCR assay for the comprehensive quantification of fish cytokine gene expression. We clarified the existence of, and cloned, various cytokine genes from the Japanese pufferfish T. rubripes (Table 2) (Kono et al 2013).…”

Section: Development Of a Multiplex Rt-pcr Assay To Quantify Fish Cytmentioning

confidence: 99%

Fish cytokines: current research and applications

2020

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Cytokines are low molecular weight glycoproteins involved in the regulation of the immune system, and more than 100 cytokines have hitherto been identified in humans. Cytokines similar to those found in humans have also been found in fish. The innate immune response of fish can be examined by using cytokines as markers. We developed a multiplex reverse transcription–polymerase chain reaction assay to analyze the expression of various cytokine genes. Using this assay, we were able to investigate the fish immune response when it had been activated by immunostimulants. The involvement of inflammatory cytokines, such as interleukin-1β, in the immune system of fish was revealed following administration of an immunostimulant.

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“…Multiplex PCR platforms have demonstrated their potential for research in fish physiology and immunology (Cleveland and Weber 2011 ; Cleveland et al 2012 ; Cleveland and Weber 2013 ; Kono and Korenaga 2013 ; Kono et al 2013 ; Cleveland and Weber 2015 ; Manor et al 2015 ). In the present study, the two multiplex panels based on previous microarray analyses were capable of detecting changes in multiple pathways and processes that are key for Atlantic salmon growth and welfare.…”

Section: Discussionmentioning

confidence: 99%

“…Multiplex PCR panels constructed with well-characterized gene biomarkers may become a useful tool for aquafeed producers in their search for superior formulations with low content in marine ingredients. Concurrently, this analytical technique could be very useful in the fields of fish nutrition and physiology research, as reflected by the body of literature published to date (e.g., Cleveland and Weber 2011 ; Cleveland et al 2012 ; Cleveland and Weber 2013 ; Kono and Korenaga 2013 ; Kono et al 2013 ; Cleveland and Weber 2015 ; Manor et al 2015 ).…”

Section: Introductionmentioning

confidence: 99%

Diet-Induced Physiological Responses in the Liver of Atlantic Salmon (Salmo salar) Inferred Using Multiplex PCR Platforms

et al. 2020

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The simultaneous quantification of several transcripts via multiplex PCR can accelerate research in fish physiological responses to diet and enable the development of superior aquafeeds for farmed fish. We designed two multiplex PCR panels that included assays for 40 biomarker genes representing key aspects of fish physiology (growth, metabolism, oxidative stress, and inflammation) and 3 normalizer genes. We used both panels to assess the physiological effects of replacing fish meal and fish oil by terrestrial alternatives on Atlantic salmon smolts. In a 14-week trial, we tested three diets based on marine ingredients (MAR), animal by-products and vegetable oil (ABP), and plant protein and vegetable oil (VEG). Dietary treatments affected the expression of genes involved in hepatic glucose and lipid metabolism (e.g., srebp1, elovl2), cell redox status (e.g., txna, prdx1b), and inflammation (e.g., pgds, 5loxa). At the multivariate level, gene expression profiles were more divergent between fish fed the marine and terrestrial diets (MAR vs. ABP/VEG) than between the two terrestrial diets (ABP vs. VEG). Liver ARA was inversely related to glucose metabolism (gck)- and growth (igfbp-5b1, htra1b)-related biomarkers and hepatosomatic index. Liver DHA and EPA levels correlated negatively with elovl2, whereas ARA levels correlated positively with fadsd5. Lower hepatic EPA/ARA in ABP-fed fish correlated with the increased expression of biomarkers related to mitochondrial function (fabp3a), oxidative stress (txna, prdx1b), and inflammation (pgds, 5loxa). The analysis of hepatic biomarker gene expression via multiplex PCR revealed potential physiological impacts and nutrient-gene interactions in Atlantic salmon fed lower levels of marine-sourced nutrients.

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Establishment of a multiplex RT-PCR assay for the rapid detection of fish cytokines

Cited by 22 publications

References 66 publications

Cytokine Gene Expression in CD4 Positive Cells of the Japanese Pufferfish, Takifugu rubripes

Cytokine Gene Expression in CD4 Positive Cells of the Japanese Pufferfish, Takifugu rubripes

Fish cytokines: current research and applications

Diet-Induced Physiological Responses in the Liver of Atlantic Salmon (Salmo salar) Inferred Using Multiplex PCR Platforms

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