scholarly journals Proinflammatory Effects of IL-1β Combined with IL-17A Promoted Cartilage Degradation and Suppressed Genes Associated with Cartilage Matrix Synthesis In Vitro

Molecules ◽  
2019 ◽  
Vol 24 (20) ◽  
pp. 3682 ◽  
Author(s):  
Patiwat Kongdang ◽  
Chatchadawalai Chokchaitaweesuk ◽  
Siriwan Tangyuenyong ◽  
Siriwan Ongchai
Keyword(s):  
Gene Expression ◽  
Proinflammatory Cytokines ◽  
Cartilage Degradation ◽  
In Vitro Models ◽  
Pellet Culture ◽  
Catabolic Genes ◽  
Culture Model ◽  
Safranin O ◽  
Hematoxylin Eosin

Combinations of IL-1β and other proinflammatory cytokines reportedly promote the severity of arthritis. We aimed to investigate the effects of IL-1β combined with IL-17A on cartilage degradation and synthesis in in vitro models. Cartilage explant degradation was determined using sulfated glycosaminoglycans (S-GAGs) levels, matrix metalloproteinase (MMP13) gene expression, uronic acid, and collagen contents. Cell morphology and accumulation of proteoglycans were evaluated using hematoxylin-eosin and safranin O staining, respectively. In the pellet culture model, expressions of cartilage-specific anabolic and catabolic genes were evaluated using real-time qRT-PCR. Early induction of MMP13 gene expression was found concomitantly with significant S-GAGs release. During the prolonged period, S-GAGs release was significantly elevated, while MMP-13 enzyme levels were persistently increased together with the reduction of the cartilaginous matrix molecules. The pellet culture showed anabolic gene downregulation, while expression of the proinflammatory cytokines, mediators, and MMP13 genes were elevated. After cytokine removal, these effects were restored to nearly basal levels. This study provides evidence that IL-1β combined with IL-17A promoted chronic inflammatory arthritis by activating the catabolic processes accompanied with the suppression of cartilage anabolism. These suggest that further applications, which suppress inflammatory enhancers, especially IL-17A, should be considered as a target for arthritis research and therapy.

scholarly journals An integrated framework for quantifying immune-tumour interactions in a 3D co-culture model

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Gheed Al-Hity ◽  
FengWei Yang ◽  
Eduard Campillo-Funollet ◽  
Andrew E. Greenstein ◽  
Hazel Hunt ◽  
...  
Keyword(s):  
Immune System ◽  
Immune Cell ◽  
In Vitro Models ◽  
Proof Of Concept ◽  
Culture Model ◽  
Spheroid Growth ◽  
Confocal Images ◽  
Cancer Spheroids

AbstractInvestigational in vitro models that reflect the complexity of the interaction between the immune system and tumours are limited and difficult to establish. Herein, we present a platform to study the tumour-immune interaction using a co-culture between cancer spheroids and activated immune cells. An algorithm was developed for analysis of confocal images of the co-culture to evaluate the following quantitatively; immune cell infiltration, spheroid roundness and spheroid growth. As a proof of concept, the effect of the glucocorticoid stress hormone, cortisol was tested on 66CL4 co-culture model. Results were comparable to 66CL4 syngeneic in vivo mouse model undergoing psychological stress. Furthermore, administration of glucocorticoid receptor antagonists demonstrated the use of this model to determine the effect of treatments on the immune-tumour interplay. In conclusion, we provide a method of quantifying the interaction between the immune system and cancer, which can become a screening tool in immunotherapy design.

scholarly journals A Review on In vitro Cell Culture Model for Bacterial Adhesion and Invasion: From Simple Monoculture to Co-Culture Human Intestinal Epithelium Model

2021 ◽  
pp. 97-106
Author(s):  
Nur Intan Hasbullah ◽  
Sharifah Aminah Syed Mohamad ◽  
Rashidah Iberahim ◽  
Nor'Aishah Hasan ◽  
Noorlis Ahmad ◽  
...  
Keyword(s):  
Intestinal Epithelium ◽  
Bacterial Adhesion ◽  
Complex Structure ◽  
In Vitro Models ◽  
Bacterial Pathogenicity ◽  
Culture Model ◽  
Culture Models ◽  
Human Intestinal Epithelium ◽  
Adhesion And Invasion

Aim: This paper reviews the different in vitro models of human intestinal epithelium that have been utilized for studying the adhesion and invasion properties. Problem Statement: The cell adhesion and invasion are the key mechanisms of bacterial pathogenicity that determines their possible routes of transmission. Numerous investigations related to the adhesion and invasion ability of bacterial isolates have been reported on monoculture human intestinal cells. However, the use of monoculture cells has several major disadvantages, such as the inability to reproduce the complex structure that defines the intestine and the inability to accurately predict the mechanism of bacterial adhesion and invasion. Approach: Co-culture models of human intestine have been developed as an alternative to improve the monoculture epithelial cell for adhesion and invasion studies, which provide more flexibility and overcome some of the limitations Conclusion: With the use of diverse in vitro approach, it could provide thorough information on different ability of bacterial adhesion and invasion and it could help to clarify the intricacy of host-pathogen interactions that underpin bacterial pathogenesis.

scholarly journals Chitinase 3-like 1 is a profibrogenic factor overexpressed in the aging liver and in patients with liver cirrhosis

2021 ◽  
Vol 118 (17) ◽  
pp. e2019633118
Author(s):  
Norihisa Nishimura ◽  
Davide De Battista ◽  
David R. McGivern ◽  
Ronald E. Engle ◽  
Ashley Tice ◽  
...  
Keyword(s):  
Gene Expression ◽  
Liver Fibrosis ◽  
Liver Tissue ◽  
Messenger Rna ◽  
Normal Liver ◽  
In Vitro Models ◽  
Hepatitis D ◽  
Fibrosis Progression ◽  
Increased Risk

Older age at the time of infection with hepatitis viruses is associated with an increased risk of liver fibrosis progression. We hypothesized that the pace of fibrosis progression may reflect changes in gene expression within the aging liver. We compared gene expression in liver specimens from 54 adult donors without evidence of fibrosis, including 36 over 40 y old and 18 between 18 and 40 y old. Chitinase 3-like 1 (CHI3L1), which encodes chitinase-like protein YKL-40/CHI3L1, was identified as the gene with the greatest age-dependent increase in expression in liver tissue. We investigated the cellular source of CHI3L1 in the liver and its function using liver tissue specimens and in vitro models. CHI3L1 expression was significantly higher in livers of patients with cirrhosis of diverse etiologies compared with controls represented by patients who underwent liver resection for hemangioma. The highest intrahepatic CHI3L1 expression was observed in cirrhosis due to hepatitis D virus, followed by hepatitis C virus, hepatitis B virus, and alcohol-induced cirrhosis. In situ hybridization of CHI3L1 messenger RNA (mRNA) identified hepatocytes as the major producers of CHI3L1 in normal liver and in cirrhotic tissue, wherein hepatocytes adjacent to fibrous septa showed higher CHI3L1 expression than did those in more distal areas. In vitro studies showed that recombinant CHI3L1 promotes proliferation and activation of primary human hepatic stellate cells (HSCs), the major drivers of liver fibrosis. These findings collectively demonstrate that CHI3L1 promotes liver fibrogenesis through a direct effect on HSCs and support a role for CHI3L1 in the increased susceptibility of aging livers to fibrosis progression.

scholarly journals Evolutionary insights into primate skeletal gene regulation using a comparative cell culture model

2021 ◽  
Author(s):  
Genevieve Housman ◽  
Emilie Briscoe ◽  
Yoav Gilad
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Bone Cells ◽  
Cell Types ◽  
Differentially Expressed ◽  
Functional Genomic ◽  
Cell Culture Model ◽  
Culture Model ◽  
Study Gene Regulation

AbstractThe evolution of complex skeletal traits in primates was likely influenced by both genetic and environmental factors. Because skeletal tissues are notoriously challenging to study using functional genomic approaches, they remain poorly characterized even in humans, let alone across multiple species. The challenges involved in obtaining functional genomic data from the skeleton, combined with the difficulty of obtaining such tissues from nonhuman apes, motivated us to consider an alternative in vitro system with which to comparatively study gene regulation in skeletal cell types. Specifically, we differentiated six human and six chimpanzee induced pluripotent stem cell lines (iPSCs) into mesenchymal stem cells (MSCs) and subsequently into osteogenic cells (bone cells). We validated differentiation using standard methods and collected single-cell RNA sequencing data from over 100,000 cells across multiple samples and replicates at each stage of differentiation. While most genes that we examined display conserved patterns of expression across species, hundreds of genes are differentially expressed (DE) between humans and chimpanzees within and across stages of osteogenic differentiation. Some of these interspecific DE genes show functional enrichments relevant in skeletal tissue trait development. Moreover, topic modeling indicates that interspecific gene programs become more pronounced as cells mature. Overall, we propose that this in vitro model can be used to identify interspecific regulatory differences that may have contributed to skeletal trait differences between species.Author SummaryPrimates display a range of skeletal morphologies and susceptibilities to skeletal diseases, but the molecular basis of these phenotypic differences is unclear. Studies of gene expression variation in primate skeletal tissues are extremely restricted due to the ethical and practical challenges associated with collecting samples. Nevertheless, the ability to study gene regulation in primate skeletal tissues is crucial for understanding how the primate skeleton has evolved. We therefore developed a comparative primate skeletal cell culture model that allows us to access a spectrum of human and chimpanzee cell types as they differentiate from stem cells into bone cells. While most gene expression patterns are conserved across species, we also identified hundreds of differentially expressed genes between humans and chimpanzees within and across stages of differentiation. We also classified cells by osteogenic stage and identified additional interspecific differentially expressed genes which may contribute to skeletal trait differences. We anticipate that this model will be extremely useful for exploring questions related to gene regulation variation in primate bone biology and development.

scholarly journals Regulation of Inflammatory Response in Human Osteoarthritic Chondrocytes by Novel Herbal Small Molecules

2019 ◽  
Vol 20 (22) ◽  
pp. 5745 ◽  
Author(s):  
Ziadlou ◽  
Barbero ◽  
Stoddart ◽  
Wirth ◽  
Li ◽  
...  
Keyword(s):  
Negative Control Group ◽  
Negative Control ◽  
Control Group ◽  
Catabolic Genes ◽  
Osteoarthritic Chondrocytes ◽  
Culture Model ◽  
Tnf Α ◽  
Anti Inflammatory ◽  
Safranin O ◽  
Vanilic Acid

In this study, 34 Traditional Chinese Medicine (TCM) compounds were screened for potential anabolic and anti-inflammatory properties on human osteoarthritic (OA) chondrocytes. The anabolic effects were assessed by measuring the glycosaminoglycan (GAG) relative to the DNA content using a 3D pellet culture model. The most chondrogenic compounds were tested in an inflammatory model consisting of 3 days of treatment with cytokines (IL-1β/TNF-α) with or without supplementation of TCM compounds. The anti-inflammatory effects were assessed transcriptionally, biochemically and histologically. From the 34 compounds, Vanilic acid (VA), Epimedin A (Epi A) and C (Epi C), 2′′-O-rhamnosylicariside II (2-O-rhs II), Icariin, Psoralidin (PS), Protocatechuicaldehyde (PCA), 4-Hydroxybenzoic acid (4-HBA) and 5-Hydroxymethylfurfural (5-HMF) showed the most profound anabolic effects. After induction of inflammation, pro-inflammatory and catabolic genes were upregulated, and GAG/DNA was decreased. VA, Epi C, PS, PCA, 4-HBA and 5-HMF exhibited anti-catabolic and anti-inflammatory effects and prevented the up-regulation of pro-inflammatory markers including metalloproteinases and cyclooxygenase 2. After two weeks of treatment with TCM compounds, the GAG/DNA ratio was restored compared with the negative control group. Immunohistochemistry and Safranin-O staining confirmed superior amounts of cartilaginous matrix in treated pellets. In conclusion, VA, Epi C, PS, PCA, 4-HBA and 5-HMF showed promising anabolic and anti-inflammatory effects.

Assessment of abiraterone (ABI) tumor cell activity via in vitro models of androgen (A)-responsive prostate cancer.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e15166-e15166
Author(s):  
Ian Hickson ◽  
Ann Marien ◽  
Maarten Derks ◽  
Marc Janssen
Keyword(s):  
Gene Expression ◽  
Prostate Cancer ◽  
Tumor Cells ◽  
In Vitro Models ◽  
Reporter Assay ◽  
Lncap Cells ◽  
Affymetrix Microarray ◽  
3D Cultures ◽  
2D And 3D

e15166 Background: Inhibition of CYP17 by abiraterone acetate (AA) blocks A biosynthesis, reduces A levels and results in inhibition of A-dependent tumor growth. AA has been shown to improve survival in patients with metastatic castrate-resistant prostate cancer (HR = 0.74) (Scher, ASCO 2011). AA is converted to ABI in vivo. This study investigates whether direct effects of ABI on tumor cells (ie, blockade of intratumoral A synthesis) may contribute to the clinical efficacy of AA using in vitro models. Methods: LNCaP cells (human prostate cancer cell line [HPCCL]) were grown in 2D and 3D cultures, and ABI activity was assessed by reduction of androgen receptor (AR) output in gene expression profiling (Affymetrix microarray), reporter assay, and by immunoblot for A-responsive signals. Using ABI-sensitive LNCaP cells, responses were further characterized in the presence or absence of ligand (dihydrotestosterone [DHT]) and with A-targeting agents TOK-001 and MDV3100. Results: ABI exposure resulted in dose-dependent modulation of A-dependent gene expression in Affymetrix microarray analysis of 2D and 3D cultures. Similar results were seen in an analysis of AR-dependent protein and in an AR-driven reporter assay in 2D LNCaP or VCaP (another HPCCL) cell culture. In LNCaP, AR output was inhibited by low concentrations of ABI (IC50 100-300 nM), comparable to TOK-001 (IC50 30-100 nM) and less potent than MDV3100 (IC50 10-30 nM). Growth inhibition (MTT assay) occurred at concentrations 10-fold higher for all compounds. For ABI and TOK-001, inhibition of AR output was reduced by DHT (30- to 100-fold increase in IC50); with MDV3100, the shift in IC50 was less (10-fold) with agonism of AR output at low nM concentrations. Conclusions: ABI has an inhibitory effect on AR output in LNCaP cells. Partially overcoming this inhibition through addition of DHT implies that CYP17 inhibition and the subsequent A reduction play a role in the intratumoral activity of AA. However, persistent inhibition of AR output in the presence of DHT implies additional anti-A effects of AA in prostate tumor cells. This model may provide a useful tool for the study of AR-targeted therapies, combinations of agents, and resistance to agents targeting A.

Abstract P141: Cardiac TRH Partly Mediates Angiotensin II-induced Fibrotic and Hypertrophy Effects in "in vivo" and "in vitro" Models

Hypertension ◽  
2015 ◽  
Vol 66 (suppl_1) ◽  
Author(s):  
Silvia I García ◽  
Ludmila S Peres Diaz ◽  
Maia Aisicovich ◽  
Mariano L Schuman ◽  
María S Landa
Keyword(s):  
Gene Expression ◽  
Body Weight ◽  
Cardiac Hypertrophy ◽  
Structural Changes ◽  
In Vitro Models ◽  
Heart Weight ◽  
Saline Control ◽  
Control Group

Cardiac TRH (cTRH) is overexpressed in the hypertrophied ventricle (LV) of the SHR. Additionally in vivo siRNA-TRH treatment induced downregulation of LV-TRH preventing cardiac hypertrophy and fibrosis demonstrating that TRH is involved in hypertrophic and fibrotic processes. Moreover, in a normal heart, the increase of LV TRH expression alone could induce structural changes where fibrosis and hypertrophy could be involved, independently of any other system alterations. Is well-known the cardiac hypertrophy/ fibrotic effects induced by AII, raising the question of whether specific LV cTRH inhibition might attenuates AII induced cardiac hypertrophy and fibrosis in mice. We challenged C57 mice with AII (osmotic pumps,14 days; 2 mg/kg) to induce cardiac hypertrophy vs saline. Groups were divided and , simultaneously to pump surgery, injected intracardiac with siRNA-TRH and siRNA-Con as its control. Body weight, water consume and SABP were measured daily. As expected, AII significantly increased SABP (p<0.05) in both groups treated , although cardiac hypertrophy (heart weight/body weight) was only evident in the group with the cardiac TRH system undamaged, suggesting that the cardiac TRH system function as a necessary mediator of the AII-induced hypertrophic effect. As hypothesized, we found an AII-induced increase of TRH (p<0.05) gene expression (real-t PCR) confirmed by immunofluorescence that was not observed in the group AII+siRNA-TRH demonstrating the specific siRNA treatment efficiency. Furthermore, AII significantly increase (p<0.05) BNP (hypertrophic marker), III collagen and TGFB (fibrosis markers) expressions only in the group with AII with the cardiac TRH system intact. On the contrary, the group with AII and the cTRH system inhibited, shows genes expressions similar to the saline control group. We confirmed these results by immunofluorescence. Similar fibrotic results were observed with NIH3T3 cell culture where we demonstrated that AII induced TRH gene expression (p<0.05) and its inhibition impedes AII-induced increase of TGFB and III/I collagens expressions telling us about the role of the cTRH in the AII fibrosis effects. Our results point out that the cardiac TRH is involved in the AII-induced hypertrophic and fibrotic effects.

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