scholarly journals The transcriptional landscape of the murine middle ear epithelium in vitro

2019 ◽  
Author(s):  
Apoorva Mulay ◽  
Md Miraj K Chowdhury ◽  
Cameron James ◽  
Lynne Bingle ◽  
Colin D Bingle
Keyword(s):  
Gene Expression ◽  
Middle Ear ◽  
In Vitro Model ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Inflammatory Cells ◽  
Differentially Expressed ◽  
Vitro Model ◽  
Transcriptional Landscape

AbstractOtitis media (OM) is the most common paediatric disease and leads to significant morbidity. Although understanding of underlying disease mechanisms is hampered by complex pathophysiology, it is clear that epithelial abnormalities underpin the disease. The mechanisms underpinning epithelial remodelling in OM remain unclear. We recently described a novel in vitro model of mouse middle ear epithelial cells (mMEECs) that undergoes mucociliary differentiation into the varied epithelial cell populations seen in the middle ear cavity. We now describe genome wide gene expression profiles of mMEECs as they undergo differentiation. We compared the gene expression profiles of original (uncultured) middle ear cells, confluent cultures of undifferentiated cells (day 0 of ALI) and cells that had been differentiated for 7 days at an ALI. >5000 genes were differentially expressed among the three groups of cells. Approximately 4000 genes were differentially expressed between the original cells and day 0 of ALI culture. The original cell population was shown to contain a mix of cell types, including contaminating inflammatory cells that were lost on culture. Approximately 500 genes were upregulated during ALI induced differentiation. These included some secretory genes and some enzymes but most were associated with the process of ciliogenesis. Our in vitro model of differentiated murine middle ear epithelium exhibits a transcriptional profile consistent with the mucociliary epithelium seen within the middle ear. Knowledge of the transcriptional landscape of this epithelium will provide a basis for understanding the phenotypic changes seen in murine models of OM.

scholarly journals The transcriptional landscape of the cultured murine middle ear epithelium in vitro

Biology Open ◽  
2021 ◽  
Vol 10 (4) ◽  
Author(s):  
Apoorva Mulay ◽  
Md Miraj K. Chowdhury ◽  
Cameron T. James ◽  
Lynne Bingle ◽  
Colin D. Bingle
Keyword(s):  
Gene Expression ◽  
Middle Ear ◽  
In Vitro Model ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Inflammatory Cells ◽  
Differentially Expressed ◽  
Vitro Model ◽  
Transcriptional Landscape

ABSTRACT Otitis media (OM) is the most common paediatric disease and leads to significant morbidity. Although understanding of underlying disease mechanisms is hampered by complex pathophysiology, it is clear that epithelial abnormalities underpin the disease. The mechanisms underpinning epithelial remodelling in OM remain unclear. We recently described a novel in vitro model of mouse middle ear epithelial cells (mMEECs) that undergoes mucociliary differentiation into the varied epithelial cell populations seen in the middle ear cavity. We now describe genome wide gene expression profiles of mMEECs as they undergo differentiation. We compared the gene expression profiles of original (uncultured) middle ear cells, confluent cultures of undifferentiated cells and cells that had been differentiated for 7 days at an air liquid interface (ALI). >5000 genes were differentially expressed among the three groups of cells. Approximately 4000 genes were differentially expressed between the original cells and day 0 of ALI culture. The original cell population was shown to contain a mix of cell types, including contaminating inflammatory cells that were lost on culture. Approximately 500 genes were upregulated during ALI induced differentiation. These included some secretory genes and some enzymes but most were associated with the process of ciliogenesis. The data suggest that the in vitro model of differentiated murine middle ear epithelium exhibits a transcriptional profile consistent with the mucociliary epithelium seen within the middle ear. Knowledge of the transcriptional landscape of this epithelium will provide a basis for understanding the phenotypic changes seen in murine models of OM.

171 THE EFFECT OF TRICHOSTATIN A ON THE DEVELOPMENT AND THE GLOBAL GENE EXPRESSION PATTERNS IN MOUSE EMBRYOS DEVELOPING IN VITRO

2008 ◽  
Vol 20 (1) ◽  
pp. 165
Author(s):  
X. S. Cui ◽  
X. Y. Li ◽  
T. Kim ◽  
N.-H. Kim
Keyword(s):  
Gene Expression ◽  
Trichostatin A ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Gene Expression Profiles ◽  
Mouse Embryos ◽  
Differentially Expressed ◽  
Gene Expression Patterns ◽  
Cell Stage

Trichostatin A (TSA) is an inhibitor of histone deacetylase and is able to alter gene expression patterns by interfering with the removal of acetyl groups from histones. The aim of this study was to determine the effect of TSA treatment on the development and gene expression patterns of mouse zygotes developing in vitro. The addition of 100 nm TSA to the culture medium did not affect the cleavage of mouse embryos (TSA treatment, 148/150 (99%) v. control, 107/107 (100%)); however, embryos that were treated with TSA arrested at the 2-cell stage (145/148, 98%). We estimated the number of nuclei in control and TSA-treated embryos by propidium iodide staining, taking into account the presence of any cells with two or more nuclei. At 62–63 h post-hCG stimulation, control zygotes had developed to the 4-cell stage and exhibited one nucleus in each blastomere, indicative of normal development. In contrast, we observed tetraploid nuclei in at least one blastomere in 20.8% (11/53) of the embryos that had been treated with TSA. At 28–29 h post-hCG stimulation (metaphase of the 1-cell stage), there was no difference in the mitotic index (as determined by analyzing the microtubule configuration) in the TSA group compared to the control group. At the 2-cell stage, however, we did not observe mitotic spindles and metaphase chromatin in embryos in the TSA treatment group compared to the controls. Interestingly, when embryos were cultured in TSA-free medium from 35 h post-hCG stimulation (S- or early G2-phase of the 2-cell stage) onward, almost all of them (47/50) developed to the blastocyst stage. In contrast, when embryos were cultured in TSA-free medium from 42 h post-hCG stimulation (middle G2-phase of the 2-cell stage) onward, they did not develop to the 4-cell stage. We used Illumina microarray technology to analyze the gene expression profiles in control and TSA-treated late 2-cell-stage embryos. Applied Biosystems Expression System software was used to extract assay signals and assay signal-to-noise ratio values from the microarray images. Our data showed that 897 genes were significantly (P < 0.05; 2-sample t-test) up- or down-regulated by TSA treatment compared to controls. Analysis using the PANTHER classification system (https://panther.appliedbiosystems.com) revealed that the 575 genes that were differentially expressed in the TSA group compared to the control were classified as being associated with putative biological processes or molecular function. Overall, in terms of putative biological processes, more nucleoside, nucleotide, and nucleic acid metabolism, protein metabolism and modification, signal transduction, developmental process, and cell cycle genes were differentially expressed between the TSA and control groups. In terms of putative molecular function, more nucleic acid-binding transcription factor and transferase genes were differentially expressed between the groups. The results collectively suggest that inhibition of histone acetylation in mouse embryos affects gene expression profiles at the time of zygotic genome activation, and this subsequently affects further development.

scholarly journals An in Vitro Model of Differentiation of Memory B Cells Into Plasmablasts and Plasma Cells Including Detailed Phenotypic and Molecular Characterization.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1636-1636
Author(s):  
Michel Jourdan ◽  
Anouk Caraux ◽  
John De Vos ◽  
Geneviève Fiol ◽  
Marion Larroque ◽  
...  
Keyword(s):  
B Cells ◽  
In Vitro Model ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Memory B Cells ◽  
Vitro Model ◽  
Intermediate Cells

Abstract Abstract 1636 Poster Board I-662 Human plasma cells (PCs) and their precursors play an essential role in humoral immune response, but are rare and difficult to harvest. We report here i) the generation of human syndecan-1+ and immunoglobulin secreting PCs starting from memory B cells (MBCs) in a 3-step- and 10-day (D) culture, including a 6-fold cell amplification. ii) We report the detailed phenotypic and Affymetrix gene expression profiles of these in vitro PCs as well as of intermediate cells - activated B cells (actBCs) and plasmablasts (PBs) - compared to MBCs and bone marrow PCs, which is accessible through an open web ATLAS (http://amazonia.transcriptome.eu/). iii) We show this B cell to PC differentiation to involve IRF4 and AICDA expression in D4 actBCs, decrease of PAX5 and BCL6 expressions and increase in PRDM1 and XBP1 expressions in D7 PBs and D10 PCs. It involves downregulation of genes controlled by Pax5, induction of genes controlled by Blimp-1 and XBP1 (unfold protein response). iv) The phenotype of D10 PCs resembles that of peripheral blood PCs detected after immunization of healthy donors. This in vitro model will facilitate further studies in PC biology. It will likewise be helpful to study plasma-cell dyscrasias, including Multiple Myeloma. Disclosures No relevant conflicts of interest to declare.

scholarly journals An in vitro model of differentiation of memory B cells into plasmablasts and plasma cells including detailed phenotypic and molecular characterization

Blood ◽  
2009 ◽  
Vol 114 (25) ◽  
pp. 5173-5181 ◽  
Author(s):  
Michel Jourdan ◽  
Anouk Caraux ◽  
John De Vos ◽  
Geneviève Fiol ◽  
Marion Larroque ◽  
...  
Keyword(s):  
B Cells ◽  
In Vitro Model ◽  
Plasma Cells ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Memory B Cells ◽  
Vitro Model ◽  
Intermediate Cells ◽  
Activated B Cells

Abstract Human plasma cells (PCs) and their precursors play an essential role in humoral immune response but are rare and difficult to harvest. We report the generation of human syndecan-1+ and immunoglobulin secreting PCs starting from memory B cells in a 3-step and 10-day (D) culture, including a 6-fold cell amplification. We report the detailed phenotypic and Affymetrix gene expression profiles of these in vitro PCs as well as of intermediate cells (activated B cells and plasmablasts) compared with memory B cells and bone marrow PCs, which is accessible through an open web ATLAS (http://amazonia.transcriptome.eu/). We show this B cell–to-PC differentiation to involve IRF4 and AICDA expressions in D4 activated B cells, decrease of PAX5 and BCL6 expressions, and increase in PRDM1 and XBP1 expressions in D7 plasmablasts and D10 PCs. It involves down-regulation of genes controlled by Pax5 and induction of genes controlled by Blimp-1 and XBP1 (unfold protein response). The detailed phenotype of D10 PCs resembles that of peripheral blood PCs detected after immunization of healthy donors. This in vitro model will facilitate further studies in PC biology. It will likewise be helpful to study PC dyscrasias, including multiple myeloma.

Gene expression profiles of RAW264.7 macrophages stimulated with preparations of LPS differing in isolation and purity

2011 ◽  
Vol 18 (1) ◽  
pp. 80-88 ◽  
Author(s):  
Holly R Rutledge ◽  
Weiwen Jiang ◽  
Jun Yang ◽  
Laura A Warg ◽  
David A Schwartz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Differentially Expressed ◽  
Transcriptional Networks ◽  
Molecular Pathways ◽  
Raw264.7 Macrophages ◽  
Lps Treatment

Lipopolysaccharide is a major component of the cell wall of Gram-negative bacteria and a potent stimulator of innate immune response via TLR4. Studies on the LPS action both in vivo and in vitro have used different preparations of LPS, including ultra-pure LPS (LIST) and a less pure but less expensive form (Sigma) isolated from Escherichia coli serotype O111:B4. The difference between the effects of these compounds has not been well studied although this information is important in understanding TLR stimulation. In this study, we compared response of RAW264.7 macrophage cells treated LIST or Sigma LPS for 6 h and 24 h. Gene expression data were analyzed to identify specific genes and pathways that are in common and unique to the two LPS preparations. Seven hundred fifty-five genes were differentially expressed at 6 h in response to Sigma LPS and 973 were differentially expressed following LIST LPS treatment, with 503 in common. At 24 h, Sigma LPS induced or repressed 901 genes while 1646 genes were differentially regulated by LIST LPS treatment; 701 genes were shared by two forms of LPS. Although considerably more genes were differentially expressed in response to LIST LPS, similar molecular pathways and transcriptional networks were activated by the two LPS preparations. We also treated bone marrow-derived macrophages (BMMs) from three strains of mice with different concentrations of LIST and Sigma LPS and showed that BMMs produced more IL-6 and TNF-α in response to LIST LPS at low LPS concentrations but, at higher LPS concentrations, more cytokines were produced in response to stimulation by Sigma LPS. Together, these findings suggest that, despite activation of similar molecular pathways by LIST and Sigma LPS preparations, residual protein impurities in the Sigma LPS preparation may nevertheless influence the transcriptional profile attributed to TLR4 stimulation.

Global Gene Expression Profiles of Activated CD4+CD25high (Regulatory) and CD4+CD25− (Non-Regulatory) T Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3308-3308
Author(s):  
ChaoYan Liu ◽  
Qi-Hong Sun ◽  
Gian Paolo Visentin
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Cd4 T Cells ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Global Gene Expression ◽  
Differentially Expressed ◽  
Platelet Factor ◽  
Homogeneous Population

Abstract Autoreactive T and B cells can be detected in healthy individuals but are normally kept in check by regulatory mechanisms. Among those is an active suppression of naïve T cells by endogenous T regulatory (Tr) cells. Several types of Tr cells exist, including CD4+ T cells which constitutively express the IL-2 receptor α chain (CD25), do not secrete IL-10, and suppress immune responses via direct cell-to-cell interactions. CD4+CD25+ T regulatory cells represent 5%–10% of the endogenous CD4+ T cells subset and are able to suppress CD4+ and CD8+ T cell responses in vitro and in vivo upon TCR ligation. Our recent observation that human platelet factor 4 (PF4; CXCL4) inhibits the proliferative response of human CD4+CD25− T cells, while inducing expansion of CD4+CD25+ Tr cells, and that PF4-induced CD4+CD25+ Tr cells lose their potent suppressor function in vitro, suggests a previously unrecognized role of PF4 in the regulation of immune responsiveness (Liu, et al. J Immunol174:2680–86, 2005). A large body of evidence suggests that human CD4+CD25+ Tr cells share many of the characteristics of murine CD4+CD25+ Tr cells. McHugh et al. (Immunity16:311–23, 2002), have successfully used the microarray approach to identify genes differentially expressed in resting CD4+CD25+ and CD4+CD25− mouse T cells, but with the only exception of a small preliminary report (Pati et al. Ann N Y Acad Sci. 1005:279–83, 2003), little information is available on the gene expression profile of human CD4+CD25+ and CD4+CD25− T cells. We performed global gene expression analysis using oligo-DNA microarrays (CodeLink, Amersham Biosciences) that monitor the expression of whole human genome, to define the gene expression profiles in CD4+CD25+ Tr cells stimulated by anti-CD3 mAb and exposed to PF4. CD4+ T cells were isolated from normal donor’s peripheral blood mononuclear cells by positive selection on magnetic beads (Miltenyi Biotec, Auburn, CA), then labeled with PE-conjugated anti-CD4 and FITC-conjugated anti-CD25 and sorted on a FACStar (BD Biosciences, San Jose, CA) to obtain a homogeneous population of T cells consisting of CD4+CD25+ Tr cells expressing CD25 at high levels (CD4+CD25high) and CD4+CD25− T cells (non-regulatory). Total RNA was extracted from the freshly isolated CD4+CD25high and CD4+CD25− T cells subsets, stimulated with anti-CD3 mAb in the presence or the absence of PF4 for 24 hours. Using this approach, we have identified a little over 100 genes that are differentially expressed, in the presence of PF4, in CD4+CD25+ Tr cells following activation with anti-CD3 mAb. We have focused our attention on about 40 target genes whose increased expression has been validated using real time PCR and, were appropriate, at the protein levels, by flow cytometry or Luminex 100 multiplex cytokine quantification (Table 1). Our data suggest that PF4 modulates proliferation and function of CD4+CD25+ Tr cells by the coordinate increasing expression of a relatively large number of genes, coupled with a further enhanced expression of a limited number of growth promoting genes and the specific silencing of a small subset of negative growth regulatory genes.

scholarly journals Transcriptome Analysis Reveals Dynamic Gene Expression Profiles in Porcine Alveolar Macrophages in Response to the Chinese Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

2018 ◽  
Vol 2018 ◽  
pp. 1-23 ◽  
Author(s):  
Nanfang Zeng ◽  
Cong Wang ◽  
Siyu Liu ◽  
Qi Miao ◽  
Lei Zhou ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alveolar Macrophages ◽  
Transcriptome Analysis ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Growing Pigs ◽  
Differentially Expressed ◽  
Respiratory Syndrome Virus ◽  
Neighboring Gene

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important swine pathogens and causes reproductive failure in sows and respiratory disease in growing pigs. PRRSV mainly infects porcine alveolar macrophages (PAMs), leading to the subversion of innate and adaptive immunity of pigs. The transcriptome analysis of gene expression profiles in PRRSV-infected PAMs is essential for understanding the pathogenesis of PRRSV. Here we performed next-generation RNA sequencing and a comprehensive bioinformatics analysis to characterize the dynamic transcriptome landscapes in PAMs following PRRSV infection. Totally 38222 annotated mRNAs, 12987 annotated long noncoding RNAs (lncRNAs), and 17624 novel lncRNAs in PRRSV-infected PAMs were identified through a transcripts computational identification pipeline. The differentially expressed mRNAs and lncRNAs during PRRSV infection were characterized. Several differentially expressed transcripts were validated using qRT-PCR. Analyses on dynamic overrepresented GO terms and KEGG pathways in PRRSV-infected PAMs at different time points were performed. Meanwhile the genes involved in IFN-related signaling pathways, proinflammatory cytokines and chemokines, phagocytosis, and antigen presentation and processing were significantly downregulated, indicating the aberrant function of PAMs during PRRSV infection. Moreover, the differentially and highly expressed lncRNA XR_297549.1 was predicted to both cis-regulate and trans-regulate its neighboring gene, prostaglandin-endoperoxide synthase 2 (PTGS2), indicating its role in inflammatory response. Our findings reveal the transcriptome profiles and differentially expressed mRNAs and lncRNAs in PRRSV-infected PAMsin vitro, providing valuable information for further exploration of PRRSV pathogenesis.

scholarly journals Transcriptome analysis of gravitational effects on mouse skeletal muscles under microgravity and artificial 1 g onboard environment

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Risa Okada ◽  
Shin-ichiro Fujita ◽  
Riku Suzuki ◽  
Takuto Hayashi ◽  
Hirona Tsubouchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Muscle Mass ◽  
Muscle Atrophy ◽  
Transcriptome Analysis ◽  
Fiber Type ◽  
Expression Profiles ◽  
Space Station ◽  
Gene Expression Profiles

AbstractSpaceflight causes a decrease in skeletal muscle mass and strength. We set two murine experimental groups in orbit for 35 days aboard the International Space Station, under artificial earth-gravity (artificial 1 g; AG) and microgravity (μg; MG), to investigate whether artificial 1 g exposure prevents muscle atrophy at the molecular level. Our main findings indicated that AG onboard environment prevented changes under microgravity in soleus muscle not only in muscle mass and fiber type composition but also in the alteration of gene expression profiles. In particular, transcriptome analysis suggested that AG condition could prevent the alterations of some atrophy-related genes. We further screened novel candidate genes to reveal the muscle atrophy mechanism from these gene expression profiles. We suggest the potential role of Cacng1 in the atrophy of myotubes using in vitro and in vivo gene transductions. This critical project may accelerate the elucidation of muscle atrophy mechanisms.

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