scholarly journals Two Sides to Every Question: Attempts to Activate Chicken Innate Immunity in 2D and 3D Hepatic Cell Cultures

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1910
Author(s):  
Csilla Sebők ◽  
Patrik Tráj ◽  
Júlia Vörösházi ◽  
Máté Mackei ◽  
Márton Papp ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Culture ◽  
Innate Immunity ◽  
Cell Cultures ◽  
Metabolic Activity ◽  
Hepatic Cell ◽  
Poly I:C ◽  
3D Cultures ◽  
Bacterial Endotoxins ◽  
2D And 3D

The liver with resident tissue macrophages is the site of vivid innate immunity, activated also by pathogen-associated molecular patterns (PAMPs) leaking through the intestinal barrier. As gut-derived inflammatory diseases are of outstanding importance in broiler chickens, the present study aimed to establish a proper hepatic inflammatory model by comparing the action of different PAMPs from poultry pathogens on chicken 2D and 3D primary hepatocyte—non-parenchymal cell co-cultures, the latter newly developed with a magnetic bioprinting method. The cultures were challenged by the bacterial endotoxins lipopolysaccharide (LPS) from Escherichia coli, lipoteichoic acid (LTA) from Staphylococcus aureus and by enterotoxin (ETxB) from Escherichia coli, Salmonella Typhimurium derived flagellin, phorbol myristate acetate (PMA) as a model proinflammatory agent and polyinosinic polycytidylic acid (poly I:C) for mimicking viral RNA exposure. Cellular metabolic activity was assessed with the CCK-8 test, membrane damage was monitored with the lactate dehydrogenase (LDH) leakage assay and interleukin-6 and -8 (Il-6 and -8) concentrations were measured in cell culture medium with a chicken specific ELISA. Both LPS and LTA increased the metabolic activity of the 3D cultures, concomitantly decreasing the LDH leakage, while in 2D cultures ETxB stimulated, PMA and poly I:C depressed the metabolic activity. Based on the moderately increased extracellular LDH activity, LTA seemed to diminish cell membrane integrity in 2D and poly I:C in both cell culture models. The applied endotoxins remarkably reduced the IL-8 release of 3D cultured cells, suggesting the effective metabolic adaptation and the presumably initiated anti-inflammatory mechanisms of the 3D spheroids. Notwithstanding that the IL-6 and IL-8 production of 2D cells was mostly not influenced by the endotoxins used, only the higher LTA dose was capable to evoke an IL-8 surge. Flagellin, PMA and poly I:C exerted proinflammatory action in certain concentrations in both 2D and 3D cultures, reflected by the increased cellular IL-6 release. Based on these data, LTA, flagellin, PMA and poly I:C can be considered as potent candidates to induce inflammation in chicken primary hepatic cell cultures, while LPS failed to trigger proinflammatory cytokine production, suggesting the relatively high tolerance of avian liver cells to certain bacterial endotoxins. These results substantiate that the established 3D co-cultures seemed to be proper tools for testing potential proinflammatory molecules; however, the remarkable differences between 2D and 3D models should be addressed and further studied.

Mechanisms involved in effects of antimicrobial peptides, bactenectins ChBac3.4, ChBac5, and mini-ChBac7.5Nb, on bacterial cells

2017 ◽  
pp. 43-50
Author(s):  
О.В. Шамова ◽  
М.С. Жаркова ◽  
П.М. Копейкин ◽  
Д.С. Орлов ◽  
Е.А. Корнева
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Activity ◽  
Innate Immunity ◽  
Infectious Diseases ◽  
Metabolic Activity ◽  
Capra Hircus ◽  
Bacterial Cells ◽  
Antibiotic Resistant ◽  
Resistant Strains

Антимикробные пептиды (АМП) системы врожденного иммунитета - соединения, играющие важную роль в патогенезе инфекционных заболеваний, так как обладают свойством инактивировать широкий спектр патогенных бактерий, обеспечивая противомикробную защиту живых организмов. В настоящее время АМП рассматриваются как потенциальные соединения-корректоры инфекционной патологии, вызываемой антибиотикорезистентными бактериями (АБР). Цель данной работы состояла в изученим механизмов антибактериального действия трех пептидов, принадлежащих к семейству бактенецинов - ChBac3.4, ChBac5 и mini-ChBac7.5Nb. Эти химически синтезированные пептиды являются аналогами природных пролин-богатых АМП, обнаруженных в лейкоцитах домашней козы Capra hircus и проявляющих высокую антимикробную активность, в том числе и в отношении грамотрицательных АБР. Методы. Минимальные ингибирующие и минимальные бактерицидные концентрации пептидов (МИК и МБК) определяли методом серийных разведений в жидкой питательной среде с последующим высевом на плотную питательную среду. Эффекты пептидов на проницаемость цитоплазматической мембраны бактерий для хромогенного маркера исследовали с использованием генетически модифицированного штамма Escherichia coli ML35p. Действие бактенецинов на метаболическую активность бактерий изучали с применением маркера резазурина. Результаты. Показано, что все исследованные пептиды проявляют высокую антимикробную активность в отношении Escherichia coli ML35p и антибиотикоустойчивых штаммов Escherichia coli ESBL и Acinetobacter baumannii in vitro, но их действие на бактериальные клетки разное. Использован комплекс методик, позволяющих наблюдать в режиме реального времени динамику действия бактенецинов в различных концентрациях (включая их МИК и МБК) на барьерную функцию цитоплазматической мембраны и на интенсивность метаболизма бактериальных клеток, что дало возможность выявить различия в характере воздействия бактенецинов, отличающихся по структуре молекулы, на исследуемые микроорганизмы. Установлено, что действие каждого из трех исследованных бактенецинов в бактерицидных концентрациях отличается по эффективности нарушения целостности бактериальных мембран и в скорости подавления метаболизма клеток. Заключение. Полученная информация дополнит существующие фундаментальные представления о механизмах действия пролин-богатых пептидов врожденного иммунитета, а также послужит основой для биотехнологических исследований, направленных на разработку на базе этих соединений новых антибиотических препаратов для коррекции инфекционных заболеваний, вызываемых АБР и являющимися причинами тяжелых внутрибольничных инфекций. Antimicrobial peptides (AMPs) of the innate immunity are compounds that play an important role in pathogenesis of infectious diseases due to their ability to inactivate a broad array of pathogenic bacteria, thereby providing anti-microbial host defense. AMPs are currently considered promising compounds for treatment of infectious diseases caused by antibiotic-resistant bacteria. The aim of this study was to investigate molecular mechanisms of the antibacterial action of three peptides from the bactenecin family, ChBac3.4, ChBac5, and mini-ChBac7.5Nb. These chemically synthesized peptides are analogues of natural proline-rich AMPs previously discovered by the authors of the present study in leukocytes of the domestic goat, Capra hircus. These peptides exhibit a high antimicrobial activity, in particular, against antibiotic-resistant gram-negative bacteria. Methods. Minimum inhibitory and minimum bactericidal concentrations of the peptides (MIC and MBC) were determined using the broth microdilution assay followed by subculturing on agar plates. Effects of the AMPs on bacterial cytoplasmic membrane permeability for a chromogenic marker were explored using a genetically modified strain, Escherichia coli ML35p. The effect of bactenecins on bacterial metabolic activity was studied using a resazurin marker. Results. All the studied peptides showed a high in vitro antimicrobial activity against Escherichia coli ML35p and antibiotic-resistant strains, Escherichia coli ESBL and Acinetobacter baumannii, but differed in features of their action on bacterial cells. The used combination of techniques allowed the real-time monitoring of effects of bactenecin at different concentrations (including their MIC and MBC) on the cell membrane barrier function and metabolic activity of bacteria. The differences in effects of these three structurally different bactenecins on the studied microorganisms implied that these peptides at bactericidal concentrations differed in their capability for disintegrating bacterial cell membranes and rate of inhibiting bacterial metabolism. Conclusion. The obtained information will supplement the existing basic concepts on mechanisms involved in effects of proline-rich peptides of the innate immunity. This information will also stimulate biotechnological research aimed at development of new antibiotics for treatment of infectious diseases, such as severe in-hospital infections, caused by antibiotic-resistant strains.

Investigating the effect of adrenomedullin on hepatic NF-kB activation by 2D and 3D hepatic cell cultures

2018 ◽  
Vol 68 ◽  
pp. S134
Author(s):  
S.D. Martin ◽  
E. Caon ◽  
D. Gabbia ◽  
G. Zigiotto ◽  
Z. Zhang ◽  
...  
Keyword(s):  
Cell Cultures ◽  
Hepatic Cell ◽  
2D And 3D

scholarly journals Cellular Effects of T-2 Toxin on Primary Hepatic Cell Culture Models of Chickens

Toxins ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 46 ◽  
Author(s):  
Máté Mackei ◽  
Kata Orbán ◽  
Andor Molnár ◽  
László Pál ◽  
Károly Dublecz ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Cell Culture ◽  
Cell Cultures ◽  
Metabolic Activity ◽  
Incubation Time ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Cellular Effects ◽  
Culture Models ◽  
Cell Culture Models

Trichothecene mycotoxins such as T-2 toxin cause severe problems for agriculture, as well as for veterinary medicine. As liver is one of the key organs in metabolism, the main aim of our study was to investigate the immunomodulatory and cytotoxic effects of T-2 toxin, using primary hepatocyte mono-culture and hepatocyte—nonparenchymal cell (predominantly Kupffer cell) co-culture models of chicken. Cultures were exposed to 10 (T10 group), 100 (T100 group) and 1000 (T1000 group) nmol/L T-2 toxin treatment for 8 or 24 h. Alterations of cellular metabolic activity, the production of reactive oxygen species (extracellular H2O2), heat shock protein 70 (HSP70), and the concentration of different inflammatory cytokines such as interleukin (IL-)6 and IL-8 were investigated. Metabolic activity was intensely decreased by T-2 toxin administration in all of the cell culture models, in every applied concentration and incubation time. Concentrations of HSP70 and IL-8 were significantly increased in hepatocyte mono-cultures exposed to higher T-2 toxin levels (both in T100 and T1000 groups for HSP70 and in T1000 group for IL-8, respectively) compared to controls after 24 h incubation. Similarly, IL-6 levels were also significantly elevated in the T100 and T1000 groups in both of mono- and co-cultures, but only after 8 h of incubation time. In spite of the general harmful effects of T-2 toxin treatment, no significant differences were observed on reactive oxygen species production. Furthermore, the two cell culture models showed different levels of H2O2, HSP70, and IL-8 concentrations independently of T-2 toxin supplementation. In conclusion, the established primary cell cultures derived from chicken proved to be proper models to study the specific molecular effects caused by T-2 toxin. Metabolic activity and immune status of the different examined cell cultures were intensively affected; however, no changes were found in H2O2 levels.

scholarly journals Comparison of antioxidant activity between cyanidin-3-O-glucoside (C3G) liposome and cyanidin-3-O-glucoside (C3G) in 2D and 3D cell cultures

2018 ◽  
Author(s):  
Tisong Liang ◽  
Rongfa Guan ◽  
Guozhou Cao ◽  
Haitao Shen ◽  
Zhenfeng Liu ◽  
...  
Keyword(s):  
Cell Culture ◽  
Antioxidant Activity ◽  
Cell Cultures ◽  
Cell Model ◽  
3D Cell Culture ◽  
Culture Model ◽  
Mda Content ◽  
2D And 3D ◽  
Cells Cultured

ABSTRACTThe 2D cell culture is the predominant in vitro model for numerous studies. However, 2D cell cultures may not accurately reflect the functions of three-dimensional (3D) tissues, which have extensive cell–cell and cell–matrix interactions; thus, using 2D cell cultures may lead to inaccurate experimental results. Therefore, to obtain adequate and detailed information about the antioxidant activity of cyanidin-3-O-glucoside (C3G) and C3G liposomes in the 2D and 3D cell culture models, we used in this study H2O2to construct the cell damage model and assess the antioxidant activity of C3G and C3G liposomes on Caco-2 cells cultured in the 3D model. We also measured the cell viability, cell morphology, and activity of glutathione (GSH), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) content of Caco-2 cells treated with H2O2, C3G, and C3G liposomes. Results showed that cells cultured in the 3D culture model formed a 3D structure and tight spheroids and showed increased cell activity and IC50. The C3G and C3G liposomes can enhance the activity of GSH, SOD, and T-AOC but decrease the MDA content. At the same time, the effect was more obvious in the 3D cell culture model than in the cells cultured in the 2D model. This study revealed that the results obtained from the 2D cell model may be inaccurate compared with the results obtained from the 3D cell model. A realistic mechanism study of antioxidant activity of C3G and C3G liposomes in the 3D cell model, which acts as an intermediate stage bridging the in vitro 2D and in vivo models, was observed.

scholarly journals Functional analysis of N-acetylglucosaminyltransferase-I knockdown in 2D and 3D neuroblastoma cell cultures

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0259743
Author(s):  
M. Kristen Hall ◽  
Adam P. Burch ◽  
Ruth A. Schwalbe
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Cell Cultures ◽  
Lectin Binding ◽  
Mutant Cell ◽  
Parental Cell Line ◽  
Binding Studies ◽  
3D Cultures ◽  
Cell Invasiveness ◽  
2D And 3D

Tumor development can be promoted/suppressed by certain N-glycans attached to proteins at the cell surface. Here we examined aberrant neuronal properties in 2D and 3D rat neuroblastoma (NB) cell cultures with different N-glycan populations. Lectin binding studies revealed that the engineered N-glycosylation mutant cell line, NB_1(-Mgat1), expressed solely oligomannose N-glycans, and verified that the parental cell line, NB_1, and a previous engineered N-glycosylation mutant, NB_1(-Mgat2), expressed significant levels of higher order N-glycans, complex and hybrid N-glycans, respectively. NB_1 grew faster than mutant cell lines in monolayer and spheroid cell cultures. A 2-fold difference in growth between NB_1 and mutants occurred much sooner in 2D cultures relative to that observed in 3D cultures. Neurites and spheroid cell sizes were reduced in mutant NB cells of 2D and 3D cultures, respectively. Cell invasiveness was highest in 2D cultures of NB_1 cells compared to that of NB_1(-Mgat1). In contrast, NB_1 spheroid cells were much less invasive relative to NB_1(-Mgat1) spheroid cells while they were more invasive than NB_1(-Mgat2). Gelatinase activities supported the ranking of cell invasiveness in various cell lines. Both palladin and HK2 were more abundant in 3D than 2D cultures. Levels of palladin, vimentin and EGFR were modified in a different manner under 2D and 3D cultures. Thus, our results support variations in the N-glycosylation pathway and in cell culturing to more resemble in vivo tumor environments can impact the aberrant cellular properties, particularly cell invasiveness, of NB.

scholarly journals Three dimensional cultivation increases chemo- and radioresistance of colorectal cancer cell lines

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0244513
Author(s):  
Jana Koch ◽  
Dina Mönch ◽  
Annika Maaß ◽  
Christian Gromoll ◽  
Thomas Hehr ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Lines ◽  
Cell Cultures ◽  
Therapy Response ◽  
Fold Increase ◽  
3D Cultures ◽  
2D And 3D ◽  
3D Spheroids

Although 2D cell cultures are commonly used to predict therapy response, it has become clear that 3D cultures may better mimic the in vivo situation and offer the possibility of tailoring translational clinical approaches. Here, we compared the response of 2D and 3D colorectal cancer (CRC) cell lines to irradiation and chemotherapy. Classic 2D cultures and 3D spheroids of CRC cell lines (CaCo2, Colo205, HCT116, SW480) were thoroughly established, then irradiated with doses of 1, 4, or 10 Gy, using a clinical-grade linear accelerator. The response was assessed by immunohistochemistry, flow cytometry, and TUNEL assays. Upon irradiation, CRC 3D spheroids were morphologically altered. After irradiation with 10 Gy, annexin V/PI staining revealed a 1.8- to 4-fold increase in the apoptosis rate in the 2D cell cultures (95% CI 3.24±0.96), and a 1.5- to 2.4-fold increase in the 3D spheroids (95% CI 1.56±0.41). Irradiation with 1 Gy caused 3- and 4-fold increases in TUNEL positive cells in the CaCo2 and HCT116 (p = 0.01) 2D cultures, respectively, compared with a 2-fold increase in the 3D spheroids. Furthermore, the 2D and 3D cultures responded differently to chemotherapy; the 3D cultures were more resistant to 5-FU and cisplatin, but not to doxorubicin and mitomycin C, than the 2D cultures. Taken together, CRC cells cultured as 3D spheroids displayed markedly higher resistance to irradiation therapy and selected chemotherapeutic drugs than 2D cultures. This in vitro difference must be considered in future approaches for determining the ideal in vitro systems that mimic human disease.

Fluorination on polyethylenimine allows efficient 2D and 3D cell culture gene delivery

2015 ◽  
Vol 3 (4) ◽  
pp. 642-650 ◽  
Author(s):  
Jia Lv ◽  
Hong Chang ◽  
Yu Wang ◽  
Mingming Wang ◽  
Jianru Xiao ◽  
...  
Keyword(s):  
Cell Culture ◽  
Gene Delivery ◽  
Cell Cultures ◽  
Gene Transfection ◽  
3D Cell Culture ◽  
Transfection Efficacy ◽  
3D Cell Cultures ◽  
2D And 3D

Fluorinationviaanhydride and oxirane reactions enhances the gene transfection efficacy of PEI on 3D cell cultures.

The inhibitory effect of ADM on hepatic NF-κB activation in 2D and 3D hepatic cell cultures

2018 ◽  
Vol 50 (1) ◽  
pp. 24
Author(s):  
S. De Martin ◽  
E. Caon ◽  
D. Gabbia ◽  
A. Floreani ◽  
G. Zigiotto ◽  
...  
Keyword(s):  
Cell Cultures ◽  
Inhibitory Effect ◽  
Hepatic Cell ◽  
2D And 3D

MCPIP1 mediates silica-induced cell migration in human pulmonary fibroblasts

2016 ◽  
Vol 310 (2) ◽  
pp. L121-L132 ◽  
Author(s):  
Haijun Liu ◽  
Xiaoniu Dai ◽  
Yusi Cheng ◽  
Shencun Fang ◽  
Yingming Zhang ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Migration ◽  
Dependent Manner ◽  
Fibroblast Proliferation ◽  
Pulmonary Fibroblasts ◽  
3D Cultures ◽  
Fibroblast Migration ◽  
And Migration ◽  
2D And 3D ◽  
Proliferation And Migration

Silicosis is a systemic disease caused by inhaling silicon dioxide (SiO2). Phagocytosis of SiO2 in the lungs initiates an inflammatory cascade that results in fibroblast proliferation and migration followed by fibrosis. According to previous data from our laboratory, monocyte chemotactic protein-1 (MCP-1) plays a critical role in fibroblast proliferation and migration in conventional two-dimensional (2D) monolayer cultures. The present study aimed to explore the downstream cascade of MCP-1 in both 2D and three-dimensional (3D) cell culture models of silicosis. Experiments using primary cultured adult human pulmonary fibroblasts (HPF-a) demonstrated the following: 1) SiO2 treatment induces expression of MCP-1-induced protein (MCPIP1) in a time- and dose-dependent manner in both 2D and 3D cultures; 2) the MAPK and phosphatidylinositol-3-kinase (PI3K)/Akt pathways are involved in SiO2-induced MCPIP1 expression; and 3) MCPIP1 induction mediates the SiO2-induced increase in cell migration in both 2D and 3D cultures. The effect of MCP-1 in silicosis occurs mainly through MCPIP1, which, in turn, mediates the observed SiO2-induced increase in pulmonary fibroblast migration. However, the time frame for MCPIP1 induction differed between 2D and 3D cultures, indicating that, compared with conventional 2D cell culture systems, 3D culture may be useful for analyses of fibroblast physiology under conditions that more closely resemble in vivo environments. Our study determined the link between fibroblast-derived MCPIP1 and SiO2-induced cell migration, and this finding provides novel evidence of the potential of MCPIP1 in the development of novel therapeutic strategies for silicosis.

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