Multicellular ovarian cancer spheroids: novel 3D model to mimic tumour complexity.

In vitro, spheroid models have become well established in cancer research because they can better mimic certain characteristics of in vivo tumours. However, interaction with the tumour microenvironment, such as cancer-associated fibroblasts, plays a key role in tumour progression. We initially focused on the interaction of tumour cells with fibroblasts. To model this interaction, we developed a spheroid model of ovarian cancer and fibroblasts. To this end, ovarian cancer cell lines and ex vivo primary cells were simultaneously and sequentially seeded with fibroblasts in a scaffold-free system at different ratios and subsequently characterized with respect to changes in morphology, proliferation, and viability. We demonstrated that co-cultures are able to form by far more compact spheroids, especially in cells that form aggregates in mono-culture. In addition, the co-cultures were able to increase proliferation and sensitivity to cisplatin. Simultaneous seeding led fibroblasts invade the core in both cell lines and primary cells. These results show differences in formation, firmness, and size between co-culture and mono-culture. Our model is designed to better represent and characterize the mutual influencing factors of fibroblasts and tumour cells. Fibroblast-supplemented multicellular spheroids are a valuable tool for tumour microenvironment interaction and new drug discovery.

Global and single-cell proteomics view of the co-evolution between neural progenitors and breast cancer cells in a co-culture model.

BACKGROUND: Presence of nerves in tumours, by axonogenesis and neurogenesis, is gaining increased attention for its impact on cancer initiation and development, and the new field of cancer neuroscience is emerging. A recent study in prostate cancer suggested that the tumour microenvironment may influence cancer progression by recruitment of Doublecortin (DCX)-expressing neural progenitor cells (NPCs). However, the presence of such cells in human breast tumours has not been comprehensively explored. METHODS: Here, we investigate the presence of DCX-expressing cells in breast cancer stromal tissue from patients using Imaging Mass Cytometry. Single-cell analysis of 372,468 cells across histopathological images of 107 breast cancers enabled spatial resolution of neural elements in the stromal compartment in correlation with clinicopathological features of these tumours. In parallel, we established a 3D in vitro model mimicking breast cancer neural progenitor-innervation and examined the two cell types as they co-evolved in co-culture by using mass spectrometry-based global proteomics. FINDINGS: Stromal presence of DCX + cells is associated with tumours of higher histological grade, a basal-like phenotype, and shorter patient survival in tumour tissue from patients with breast cancer. Global proteomics analysis revealed significant changes in the proteomic landscape of both breast cancer cells and neural progenitors in co-culture. INTERPRETATION: These results support that neural involvement plays an active role in breast cancer and warrants further studies on the relevance of nerve elements for tumour progression. FUNDING: This work was supported by the Research Council of Norway through its Centre of Excellence funding scheme, project number 223250 (to L.A.A), the Norwegian Cancer Society (to L.A.A. and H.V.), the Regional Health Trust Western Norway (Helse Vest) (to L.A.A.), the Meltzer Research Fund (to H.V.) and the National Institutes of Health (NIH)/NIGMS grant R01 GM132129 (to J.A.P.).

Pasteurized form of a potential probiotic lactobacillus brevis IBRC-M10790 exerts anti-inflammatory effects on inflammatory bowel disease in vitro.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic, relapsing inflammatory disorder of the gastrointestinal system. So far, no treatment has been identified that can completely cure IBD. Lactobacillus brevis is hypothesized to be beneficial in preventing inflammation. This study aimed to evaluate the potential probiotic effects of live and pasteurized L. brevis IBRC-M10790 on the in vitro cell co-culture model of IBD. METHODS: An in vitro intestinal model was established using a transwell co-culture system of Caco-2 intestinal epithelial cells and RAW264.7 macrophages. Inflammatory conditions were induced in RAW264.7 cells using lipopolysaccharide. The effects of live and pasteurized L. brevis IBRC-M10790 on inflammatory mediators and epithelial barrier markers were investigated. RESULTS: L. brevis IBRC-M10790 was able to significantly decrease the proinflammatory cytokines (IL-6, IL-1ß, and TNF-α) and increase the anti-inflammatory cytokine (IL-10) in the in vitro co-culture system. In addition, L. brevis increased adherens and tight junction (TJ) markers (ZO-1, E-cadherin, and Occludin) in Caco-2 intestinal epithelial cells. Based on the results, pasteurized L. brevis showed a higher protective effect than live L. brevis. CONCLUSIONS: Our findings suggest that live and pasteurized forms of L. brevis possess probiotic properties and can mitigate inflammatory conditions in IBD.

An evaluation of a hepatotoxicity risk induced by the microplastic polymethyl methacrylate (PMMA) using HepG2/THP-1 co-culture model.

Inappropriate disposal of plastic wastes and their durability in nature cause uncontrolled accumulation of plastic in land/marine ecosystems, also causing destructive effects by bioaccumulating along the food chain. Microplastics may cause chronic inflammation in relation to their permanent structures, especially through oxidative stress and cytotoxic cellular damage, which could increase the risk of cancer development. The accumulation of microplastics in the liver is a major concern, and therefore, the identification of the mechanisms of their hepatotoxic effects is of great importance. Polymethyl methacrylate (PMMA) is a widely used thermoplastic. It has been determined that PMMA disrupts lipid metabolism in the liver in various aquatic organisms and causes reproductive and developmental toxicity. PMMA-induced hepatotoxic effects in humans have not yet been clarified. In our study, the toxic effects of PMMA (in the range of 3-10 µm) on the human liver were investigated using the HepG2/THP-1 macrophage co-culture model, which is a sensitive immune-mediated liver injury model. Cellular uptake of micro-sized PMMA in the cells was done by transmission electron microscopy. Determination of its effects on cell viability and inflammatory response, oxidative stress, along with gene and protein expression levels that play a role in the mechanism pathways underlying the effects were investigated. The results concluded that inflammation, oxidative stress, and disruptions in lipid metabolism should be the focus of attention as important underlying causes of PMMA-induced hepatotoxicity. Our study, which points out the potential adverse effects of microplastics on human health, supports the literature information on the subject.

An autologous ex vivo model for exploring patient-specific responses to viro-immunotherapy in glioblastoma.

Oncolytic virus (OV) clinical trials have demonstrated remarkable efficacy in subsets of patients with glioblastoma (GBM). However, the lack of tools to predict this response hinders the advancement of a more personalized application of OV therapy. In this study, we characterize an ex vivo co-culture system designed to examine the immune response to OV infection of patient-derived GBM neurospheres in the presence of autologous peripheral blood mononuclear cells (PBMCs). Co-culture conditions were optimized to retain viability and functionality of both tumor cells and PBMCs, effectively recapitulating the well-recognized immunosuppressive effects of GBM. Following OV infection, we observed elevated secretion of pro-inflammatory cytokines and chemokines, including interferon γ, tumor necrosis factor α, CXCL9, and CXCL10, and marked changes in immune cell activation markers. Importantly, OV treatment induced unique patient-specific immune responses. In summary, our co-culture platform presents an avenue for personalized screening of viro-immunotherapies in GBM, offering promise as a potential tool for future patient stratification in OV therapy.

Chrysophanol accelerates astrocytic mitochondria transfer to neurons and attenuates the cerebral ischemia-reperfusion injury in rats.

Astrocytes transfer extracellular functional mitochondria into neurons to rescue injured neurons after a stroke. However, there are no reports on drugs that interfere with intercellular mitochondrial transfer. Chrysophanol (CHR) was an effective drug for the treatment of cerebral ischemia-reperfusion injury (CIRI) and was selected as the test drug. The oxygen-glucose deprivation/reoxygenation (OGD/R) cell model and the middle cerebral artery occlusion animal model were established to investigate the effect of CHR on CIRI. The result showed that astrocytes could act as mitochondrial donors to ameliorate neuronal injury. Additionally, the neuroprotective effect of astrocytes was enhanced by CHR, the CHR improved the neuronal mitochondrial function, decreased the neurological deficit score and infarction volume, recovered cell morphology in ischemic penumbra. The mitochondrial fluorescence probe labeling technique has shown that the protective effect of CHR is associated with accelerated astrocytic mitochondrial transfer to neurons. The intercellular mitochondrial transfer may be an important way to ameliorate ischemic brain injury and be used as a key target for drug treatment.

Mutual interaction of neurons and astrocytes derived from iPSCs with APP V717L mutation developed the astrocytic phenotypes of Alzheimer's disease.

BACKGROUND: The development of induced pluripotent stem cells (iPSCs) technology has enabled human cellular disease modeling for inaccessible cell types, such as neural cells in the brain. However, many of the iPSC-derived disease models established to date typically involve only a single cell type. These monoculture models are inadequate for accurately simulating the brain environment, where multiple cell types interact. The limited cell type diversity in monoculture models hinders the accurate recapitulation of disease phenotypes resulting from interactions between different cell types. Therefore, our goal was to create cell models that include multiple interacting cell types to better recapitulate disease phenotypes. METHODS: To establish a co-culture model of neurons and astrocytes, we individually induced neurons and astrocytes from the same iPSCs using our novel differentiation methods, and then co-cultured them. We evaluated the effects of co-culture on neurons and astrocytes using immunocytochemistry, immuno-electron microscopy, and Ca2+ imaging. We also developed a co-culture model using iPSCs from a patient with familial Alzheimer's disease (AD) patient (APP V717L mutation) to investigate whether this model would manifest disease phenotypes not seen in the monoculture models. RESULTS: The co-culture of the neurons and astrocytes increased the branching of astrocyte processes, the number of GFAP-positive cells, neuronal activities, the number of synapses, and the density of presynaptic vesicles. In addition, immuno-electron microscopy confirmed the formation of a tripartite synaptic structure in the co-culture model, and inhibition of glutamate transporters increased neuronal activity. Compared to the co-culture model of the control iPSCs, the co-culture model of familial AD developed astrogliosis-like phenotype, which was not observed in the monoculture model of astrocytes. CONCLUSIONS: Co-culture of iPSC-derived neurons and astrocytes enhanced the morphological changes mimicking the in vivo condition of both cell types. The formation of the functional tripartite synaptic structures in the co-culture model suggested the mutual interaction between the cells. Furthermore, the co-culture model with the APP V717L mutation expressed in neurons exhibited an astrocytic phenotype reminiscent of AD brain pathology. These results suggest that our co-culture model is a valuable tool for disease modeling of neurodegenerative diseases.

Rice-derived arabinoxylan fibers are particle size-dependent inducers of trained immunity in a human macrophage-intestinal epithelial cell co-culture model.

Arabinoxylans have been identified for a wide range of purported health-promoting applications, primarily attributed to its immunomodulatory effects. Previously, we have reported the ability of arabinoxylans to induce non-specific memory in innate immune cells, commonly referred to as "trained innate immunity". In the present study, we investigated the effect of particle size on innate immune training and resilience in primary human macrophages as well as in a more physiologically relevant macrophage-intestinal epithelial cell co-culture model. We demonstrated that smaller (>45 & < 90 µm) compared to larger (>90 µm) particle size fractions of rice bran-derived arabinoxylan preparations have a higher enhancing effect on training and resilience in both models. Smaller particle size fractions elevated TNF-α production in primary macrophages and enhanced Dectin-1 receptor activation in reporter cell lines compared to larger particles. Responses were arabinoxylan source specific as only the rice-derived arabinoxylans showed these immune-supportive effects. This particle size-dependent induction of trained immunity was confirmed in the established co-culture model. These findings demonstrate the influence of particle size on the immunomodulatory potential of arabinoxylans, provide further insight into the structure-activity relationship, and offer new opportunities to optimize the immune-enhancing effects of these dietary fibers.

The cyclin-dependent kinase inhibitor abemaciclib-induced hepatotoxicity: Insight on the molecular mechanisms in HepG2/THP-1 co-culture model.

Drug-induced liver injury (DILI) is one of the widespread causes of liver injury and immune system plays important role. Abemaciclib (ABE) is a cyclin-dependent kinase inhibitor used as monotherapy or combination therapy in the treatment of breast cancer. Like other kinase inhibitors, the underlying mechanisms of ABE-induced hepatotoxicity are not completely known yet. In the current study, hepatotoxicity of ABE was evaluated with HepG2/THP-1 co-culture model which has been developed in recent years for the evaluation of DILI potential. Following ABE treatment, oxidative stress, mitochondrial damage, cytokine secretion levels, apoptotic/necrotic cell death were determined. According to our results, ROS production along with GSH depletion was observed in HepG2 cells after ABE treatment. ABE promoted secretion of pro-inflammatory mediators (TNF-α and MCP-1) and declined anti-inflammatory cytokine IL-10 release. Besides, NFKß and JNK1 protein expression levels increased following ABE treatment. ABE enhanced intracellular calcium levels, induced early apoptotic and necrotic cell deaths in HepG2 cells. Furthermore, the changes in some mitochondrial parameters including a reducement in intracellular ATP levels and complex V activity; hyperpolarized mitochondrial membrane potential and enhanced mitochondrial ROS levels were observed, whereas mitochondrial mass did not show any differences after ABE treatments. Therefore, ABE-induced hepatotoxic effects is probably via oxidative stress, inflammatory response and necrotic cell death rather than direct mitochondrial toxicity. In conclusion; the study makes a significant contribution to strengthening the infrastructure we have on in vitro toxicity mechanism evaluations, which are the basis of preclinical toxicity studies.

Simultaneous isolation of hormone receptor-positive breast cancer organoids and fibroblasts reveals stroma-mediated resistance mechanisms.

Recurrent hormone receptor-positive (HR+) breast cancer kills more than 600,000 women annually. Although HR+ breast cancers typically respond well to therapies, approximately 30% of patients relapse. At this stage, the tumors are usually metastatic and incurable. Resistance to therapy, particularly endocrine therapy is typically thought to be tumor intrinsic (e.g., estrogen receptor mutations). However, tumor-extrinsic factors also contribute to resistance. For example, stromal cells, such as cancer-associated fibroblasts (CAFs), residing in the tumor microenvironment, are known to stimulate resistance and disease recurrence. Recurrence in HR+ disease has been difficult to study due to the prolonged clinical course, complex nature of resistance, and lack of appropriate model systems. Existing HR+ models are limited to HR+ cell lines, a few HR+ organoid models, and xenograft models that all lack components of the human stroma. Therefore, there is an urgent need for more clinically relevant models to study the complex nature of recurrent HR+ breast cancer, and the factors contributing to treatment relapse. Here, we present an optimized protocol that allows a high take-rate, and simultaneous propagation of patient-derived organoids (PDOs) and matching CAFs, from primary and metastatic HR+ breast cancers. Our protocol allows for long-term culturing of HR+ PDOs that retain estrogen receptor expression and show responsiveness to hormone therapy. We further show the functional utility of this system by identifying CAF-secreted cytokines, such as growth-regulated oncogene α , as stroma-derived resistance drivers to endocrine therapy in HR+ PDOs.

In vitro acne disease model from inertial focusing effect for studying the interactions between sebocyte glands and macrophages.

Acne is one of the most widespread skin diseases. The acne mechanism is intricate, involving interactions between different types of cells (i.e., sebocytes and macrophages). One of the challenges in studying the mechanism of acne is that current in vitro culture methods cannot reflect the 3D cellular environment in the tissue, including inflammatory stimuli and cellular interactions especially the interactions between sebocytes and immune cells. To solve this issue, we generated an in vitro acne disease model consisting of 3D artificial sebocyte glands and macrophages through the inertial focusing effect method. Using this model, we produced a controllable inflammatory environment similar to the acne pathogenetic process in the skin. The 3D artificial sebocyte glands and macrophages can be separated for analyzing each cell type, assisting the in-depth understanding of the acne mechanism. This study indicates that proinflammatory macrophages promote lipid accumulation and induce oxidative stress in sebocyte glands. Additionally, in an inflammatory environment, sebocyte glands induce macrophage polarization into the M1 phenotype. Employing this model for drug screening, we also demonstrated that, cannabidiol (CBD), a clinically investigated drug, is effective in restoring lipid accumulation, oxidative stress, inflammatory cytokines and macrophage polarization in the acne disease.

Anti-cancer effects of metformin in a 3D co-culture model of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) presents with condensed stroma that contributes to its high invasive capability. Although metformin adjuvant treatment has been suggested to improve the survival times of patients with PDAC, the mechanism responsible for that benefit has been investigated only in two-dimensional cell lines. We assessed the anti-cancer effect of metformin in a three-dimensional (3D) co-culture model to quantify the migration behavior of patient-derived PDAC organoids and primary pancreatic stellate cells (PSCs). At a concentration of 10 µM, metformin reduced the migratory ability of the PSCs by downregulating the expression of matrix metalloproteinase-2 (MMP2). In the 3D direct co-cultivation of PDAC organoids and PSCs, metformin attenuated the transcription of cancer stemness-related genes. The reduced stromal migratory ability of PSCs was associated with the downregulation of MMP2, and MMP2 knockdown in PSCs reproduced their attenuated migratory ability. The anti-migration effect of a clinically relevant concentration of metformin was demonstrable in a 3D indirect co-culture model of PDAC consisting of patient-derived PDAC organoids and primary human PSCs. The metformin suppressed PSC migration via MMP2 downregulation and attenuated cancer stemness factors. Furthermore, oral administration of metformin (30 mg/kg) strikingly suppressed the growth of PDAC organoids xenograft in immunosuppressed mice. These results indicate metformin could offer the potential approach as an effective therapeutic drug for PDAC.

Cytokine signaling converging on IL11 in ILD fibroblasts provokes aberrant epithelial differentiation signatures.

Introduction: Interstitial lung disease (ILD) is a heterogenous group of lung disorders where destruction and incomplete regeneration of the lung parenchyma often results in persistent architectural distortion of the pulmonary scaffold. Continuous mesenchyme-centered, disease-relevant signaling likely initiates and perpetuates the fibrotic remodeling process, specifically targeting the epithelial cell compartment, thereby destroying the gas exchange area. Methods: With the aim of identifying functional mediators of the lung mesenchymal-epithelial crosstalk with potential as new targets for therapeutic strategies, we developed a 3D organoid co-culture model based on human induced pluripotent stem cell-derived alveolar epithelial type 2 cells that form alveolar organoids in presence of lung fibroblasts from fibrotic-ILD patients, in our study referring to cases of pulmonary fibrosis, as well as control cell line (IMR-90). Results: While organoid formation capacity and size was comparable in the presence of fibrotic-ILD or control lung fibroblasts, metabolic activity was significantly increased in fibrotic-ILD co-cultures. Alveolar organoids cultured with fibrotic-ILD fibroblasts further demonstrated reduced stem cell function as reflected by reduced Surfactant Protein C gene expression together with an aberrant basaloid-prone differentiation program indicated by elevated Cadherin 2, Bone Morphogenic Protein 4 and Vimentin transcription. To screen for key mediators of the misguided mesenchymal-to-epithelial crosstalk with a focus on disease-relevant inflammatory processes, we used mass spectrometry and characterized the secretome of end stage fibrotic-ILD lung fibroblasts in comparison to non-chronic lung disease (CLD) patient fibroblasts. Out of the over 2000 proteins detected by this experimental approach, 47 proteins were differentially abundant comparing fibrotic-ILD and non-CLD fibroblast secretome. The fibrotic-ILD secretome profile was dominated by chemokines, including CXCL1, CXCL3, and CXCL8, interfering with growth factor signaling orchestrated by Interleukin 11 (IL11), steering fibrogenic cell-cell communication, and proteins regulating extracellular matrix remodeling including epithelial-to-mesenchymal transition. When in turn treating alveolar organoids with IL11, we recapitulated the co-culture results obtained with primary fibrotic-ILD fibroblasts including changes in metabolic activity. Conclusion: We identified mediators likely contributing to the disease-perpetuating mesenchymal-to-epithelial crosstalk in ILD. In our alveolar organoid co-cultures, we were able to highlight the importance of fibroblast-initiated aberrant epithelial differentiation and confirmed IL11 as a key player in fibrotic-ILD pathogenesis by unbiased fibroblast secretome analysis.

Tiagabine and zonisamide differentially regulate the glial properties in an astrocyte-microglia co-culture model of inflammation.

Due to the role of astrocytes and microglia in the pathophysiology of epilepsy and limited studies of antiseizure medication (ASM) effects on glial cells, we studied tiagabine (TGB) and zonisamide (ZNS) in an astrocyte-microglia co-culture model of inflammation. Different concentrations of ZNS (10, 20, 40, 100 µg/ml) or TGB (1, 10, 20, 50 µg/ml) were added to primary rat astrocytes co-cultures with 5-10% (M5, physiological conditions) or 30-40% (M30, pathological inflammatory conditions) microglia for 24 h, aiming to study glial viability, microglial activation, connexin 43 (Cx43) expression and gap-junctional coupling. ZNS led to the reduction of glial viability by only 100 µg/ml under physiological conditions. By contrast, TGB revealed toxic effects with a significant, concentration-dependent reduction of glial viability under physiological and pathological conditions. After the incubation of M30 co-cultures with 20 µg/ml TGB, the microglial activation was significantly decreased and resting microglia slightly increased, suggesting possible anti-inflammatory features of TGB under inflammatory conditions. Otherwise, ZNS caused no significant changes of microglial phenotypes. The gap-junctional coupling was significantly decreased after the incubation of M5 co-cultures with 20 and 50 µg/ml TGB, which can be related to its anti-epileptic activity under noninflammatory conditions. A significant decrease of Cx43 expression and cell-cell coupling was found after the incubation of M30 co-cultures with 10 µg/ml ZNS, suggesting additional anti-seizure effects of ZNS with the disruption of glial gap-junctional communication under inflammatory conditions. TGB and ZNS differentially regulated the glial properties. Developing novel ASMs targeting glial cells may have future potential as an "add-on" therapy to classical ASMs targeting neurons.

Influence of bacterial and alveolar cell co-culture on microbial VOC production using HS-GC/MS.

Volatile organic compounds (VOCs) found in exhaled breath continue to garner interest as an alternative diagnostic tool in pulmonary infections yet, their clinical integration remains a challenge with difficulties in translating identified biomarkers. Alterations in bacterial metabolism secondary to host nutritional availability may explain this but is often inadequately modelled in vitro. The influence of more clinically relevant nutrients on VOC production for two common respiratory pathogens was investigated. VOCs from Staphylococcus aureus (S.aureus) and Pseudomonas aeruginosa (P.aeruginosa) cultured with and without human alveolar A549 epithelial cells were analyzed using headspace extraction coupled with gas chromatography-mass spectrometry. Untargeted and targeted analyses were performed, volatile molecules identified from published data, and the differences in VOC production evaluated. Principal component analysis (PCA) could differentiate alveolar cells from either S. aureus or P. aeruginosa when cultured in isolation based on PC1 (p = 0.0017 and 0.0498, respectively). However, this separation was lost for S. aureus (p = 0.31) but not for P. aeruginosa (p = 0.028) when they were cultured with alveolar cells. S. aureus cultured with alveolar cells led to higher concentrations of two candidate biomarkers, 3-methyl-1-butanol (p = 0.001) and 3-methylbutanal (p = 0.002) when compared to S. aureus, alone. P. aeruginosa metabolism resulted in less generation of pathogen-associated VOCs when co-cultured with alveolar cells compared to culturing in isolation. VOC biomarkers previously considered indicative of bacterial presence are influenced by the local nutritional environment and this should be considered when evaluating their biochemical origin.

Advanced co-culture model: Soft tissue cell and bacteria interactions at the transgingival dental implant interface.

OBJECTIVES: To better simulate and understand the clinical situation in which tissue cells and bacteria compete for settlement on an implant surface, the aim was to develop an improved transgingival co-culture model. METHODS: For this model human gingival fibroblasts (HGF) were seeded on different titanium surfaces in the presence of the early colonizer Streptococcus gordonii or mixed oral bacteria. Subsequently adhesion and viability of HGF cells was analyzed. RESULTS: Simultaneous co-culture showed no decrease in the viability of HGF cells at early stages compared to the control group. However, a moderate impact on HGF viability (76 ± 23 %) was observed after 4 h of co-culture, which then significantly decreased after 5 h (21 ± 2 %) of co-cultivation, resulting in cell death and detachment from the surface. Further experiments including saliva pre-treatment of smooth and structured titanium surfaces with Streptococcus gordonii or mixed oral bacteria suggested a cell-protective property of saliva. SIGNIFICANCE: Our study revealed that during simultaneous co-culture of cells and bacteria, which resembles the clinical situation the closest, the viability of gingival cells is considerably high in the early phase, suggesting that increasing initial cell adhesion rather than antibacterial functionality is a major goal and a relevant aspect in the development and testing of transgingival implant and abutment surface modifications.

Inhibition of Microglial Activation by Amitriptyline and Doxepin in Interferon-ß Pre-Treated Astrocyte-Microglia Co-Culture Model of Inflammation.

Depression may occur in patients with multiple sclerosis, especially during interferon-ß (IFN-ß) treatment, and therapy with antidepressants may be necessary. Interactions of IFN-ß with antidepressants concerning glia-mediated inflammation have not yet been studied. Primary rat co-cultures of astrocytes containing 5% (M5, consistent with "physiological" conditions) or 30% (M30, consistent with "pathological, inflammatory" conditions) of microglia were incubated with 10 ng/mL amitriptyline or doxepin for 2 h, or with 2000 U/mL IFN-ß for 22 h. To investigate the effects of antidepressants on IFN-ß treatment, amitriptyline or doxepin was added to IFN-ß pre-treated co-cultures. An MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to measure the glial cell viability, immunocytochemistry was performed to evaluate the microglial activation state, and ELISA was performed to measure pro-inflammatory TNF-α and IL-6 cytokine concentrations. Incubation of inflammatory astrocyte-microglia co-cultures with amitriptyline, doxepin or IFN-ß alone, or co-incubation of IFN-ß pre-treated co-cultures with both antidepressants, significantly reduced the extent of inflammation, with the inhibition of microglial activation. TNF-α and IL-6 levels were not affected. Accordingly, the two antidepressants did not interfere with the anti-inflammatory effect of IFN-ß on astrocytes and microglia. Furthermore, no cytotoxic effects on glial cells were observed. This is the first in vitro study offering novel perspectives in IFN-ß treatment and accompanying depression regarding glia.

Experimental Investigations of Monomethyl and Dimethyl Fumarate in an Astrocyte-Microglia Co-Culture Model of Inflammation.

INTRODUCTION: Multiple sclerosis (MS) is the most common chronic inflammatory, demyelinating disease of the central nervous system. Dimethyl fumarate (DMF) and monomethyl fumarate (MMF) belong to the disease-modifying drugs in treatment of MS. There is evidence that astrocytes and microglia are involved in MS pathology, but few studies are available about MMF and DMF effects on astrocytes and microglia. The aim of this study was to investigate the effects of MMF and DMF on microglial activation and morphology as well as potential effects on glial viability, Cx43, and AQP4 expressions in different set-ups of an in vitro astrocyte-microglia co-culture model of inflammation. METHODS: Primary rat glial co-cultures of astrocytes containing 5% (M5, mimicking "physiological" conditions) or 30% (M30, mimicking "pathological, inflammatory" conditions) of microglia were treated with different concentrations of MMF (0.1, 0.5, and 2 µg/mL) or DMF (1.5, 5, and 15 µM) for 24 h. Viability, proliferation, and cytotoxicity of glial cells were examined using MTT assay. Immunocytochemistry was performed to analyze the microglial phenotypes. Connexin 43 (Cx43) and aquaporin 4 (AQP4) expressions were quantified by immunoblot analysis. RESULTS: Treatment with different concentrations of MMF or DMF for 24 h did not change the glial cell viability in M5 and M30 co-cultures. Microglial phenotypes were not altered by DMF under physiological M5 conditions, but treatment with higher concentration of DMF (15 µM) induced microglial activation under inflammatory M30 conditions. Incubation with different concentrations of MMF had no effects on microglial phenotypes. The Cx43 expression in M5 and M30 co-cultures was not changed significantly by immunoblot analysis after incubation with different concentrations of DMF or MMF for 24 h. The AQP4 expression was significantly increased in M5 co-cultures after incubation with 5 µm DMF. Under the other conditions, AQP4 expression was not affected by DMF or MMF. DISCUSSION: In different set-ups of the astrocyte-microglia co-culture model of inflammation, MMF has not shown significant effects. DMF had only limited effects on microglia phenotypes and AQP4 expression. In summary, mechanisms of action of fumarates probably do not involve direct effects on microglia phenotypes as well as Cx43 and AQP4 expression.

The Crosstalk between Intestinal Epithelial Cells and Mast Cells Is Modulated by the Probiotic Supplementation in Co-Culture Models.

The intestinal epithelium constitutes a selectively permeable barrier between the internal and external environment that allows the absorption of nutrients, electrolytes, and water, as well as an effective defense against intraluminal bacteria, toxins, and potentially antigenic material. Experimental evidence suggest that intestinal inflammation is critically dependent on an imbalance of homeostasis between the gut microbiota and the mucosal immune system. In this context, mast cells play a crucial role. The intake of specific probiotic strains can prevent the development of gut inflammatory markers and activation of the immune system. Here, the effect of a probiotic formulation containing L. rhamnosus LR 32, B. lactis BL04, and B. longum BB 536 on intestinal epithelial cells and mast cells was investigated. To mimic the natural host compartmentalization, Transwell co-culture models were set up. Co-cultures of intestinal epithelial cells interfaced with the human mast cell line HMC-1.2 in the basolateral chamber were challenged with lipopolysaccharide (LPS), and then treated with probiotics. In the HT29/HMC-1.2 co-culture, the probiotic formulation was able to counteract the LPS-induced release of interleukin 6 from HMC-1.2, and was effective in preserving the epithelial barrier integrity in the HT29/Caco-2/ HMC-1.2 co-culture. The results suggest the potential therapeutic effect of the probiotic formulation.

Potential toxicity and dietary risk of tricyclazole to Chinese mitten crab (Eriocheir sinensis) in the rice-crab co-culture model.

Tricyclazole is used as a common fungicide to control rice blast. However, studies on the toxicity of tricyclazole to crabs in the rice-crab co-culture system are still extremely rare. Here, the environmental dissipation of tricyclazole was monitored in this model, and the potential toxicity of tricyclazole to E. sinensis at environmental concentrations as well as the dietary risk was evaluated. The results showed that tricyclazole had no significant acute toxicity to E. sinensis (LC50 > 100 mg/L), while it promoted body weight gain. Tricyclazole in the hepatopancreas had a higher persistent bioaccumulation risk than in the muscle. Tricyclazole suppressed the immune response of E. sinensis under prolonged exposure and there should be gender differences, with females being more sensitive. Lipid metabolism enzymes were also significantly inhibited. While tricyclazole stimulated males molting but prolonged molting duration, both molting and duration of females were also disturbed. The dietary risk assessment indicated that tricyclazole intake from current crab consumption was low risk. This evidence demonstrated that tricyclazole may have potential risks to individual development, nutritional quality, and economic value on E. sinensis and should be used with caution in rice-crab co-culture system whenever possible.