Mixed Cell Cultures Research Articles

A mixed culture of bacterial cells enables an economic DNA storage on a large scale

DNA emerged as a novel potential material for mass data storage, offering the possibility to cheaply solve a great data storage problem. Large oligonucleotide pools demonstrated high potential of large-scale data storage in test tube, meanwhile, living cell with high fidelity in information replication. Here we show a mixed culture of bacterial cells carrying a large oligo pool that was assembled in a high-copy-number plasmid was presented as a stable material for large-scale data storage. The underlying principle was explored by deep bioinformatic analysis. Although homology assembly showed sequence context dependent bias, the large oligonucleotide pools in the mixed culture were constant over multiple successive passages. Finally, over ten thousand distinct oligos encompassing 2304 Kbps encoding 445 KB digital data, were stored in cells, the largest storage in living cells reported so far and present a previously unreported approach for bridging the gap between in vitro and in vivo systems.

Gas6 Induces Myelination through Anti-Inflammatory IL-10 and TGF-β Upregulation in White Matter and Glia.

The Gas6–TAM (Tyro3, Axl, Mer) ligand–receptor system is believed to promote central nervous system (CNS) (re)myelination and glial cell development. An additional important function of Gas6–TAM signalling appears to be the regulation of immunity and inflammation, which remains to be fully elucidated in the CNS. Here, we characterised the expression of TAM receptors and ligands in individual CNS glial cell types, observing high expression of Gas6 and the TAM receptors, Mer and Axl, in microglia, and high expression of Tyro3 in astrocytes. We also investigated the effect of Gas6 on the inflammatory cytokine response in the optic nerve and in mixed glial cell cultures from wildtype and single TAM receptor knockout mice. In wildtype and Mer-deficient cultures, Gas6 significantly stimulated the expression of the anti-inflammatory/pro-repair cytokines interleukin 10 (IL-10) and transforming growth factor β (TGF-β), whereas this effect was absent in either Tyro3 or Axl knockout cultures. Furthermore, Gas6 caused upregulation of myelin basic protein (MBP) expression in optic nerves, which was blocked by a neutralising antibody against IL-10. In conclusion, our data show that microglia are both a major source of Gas6 as well as an effector of Gas6 action in the CNS through the upregulation of anti-inflammatory and pro-repair mediators. Furthermore, the presence of both Axl and Tyro3 receptors appears to be necessary for these effects of Gas6. In addition, IL-10, alongside suppressing inflammation and immunity, mediates the pro-myelinating mechanism of Gas6 action in the optic nerve. Therefore, Gas6 may present an attractive target for novel therapeutic interventions for demyelinating as well as neuroinflammatory disorders of the CNS.

Cytogenetic effects in mixed culture of blood cells from patients with chronic lymphocytic leukemia with blood lymphocytes of healthy individuals

Cytogenetic effects in mixed culture of blood cells from patients with chronic lymphocytic leukemia with blood lymphocytes of healthy individuals

Antenatal PPAR-γ agonist pioglitazone stimulates fetal lung maturation equally in males and females.

Antenatal steroids (ANS) accelerate fetal lung maturation and reduce the incidence of respiratory distress syndrome. However, sex specificity, i.e., being less effective in males, and potential long-term neurodevelopmental sequelae, particularly with repeated courses, remain significant limitations. The differential sex response to ANS is likely mediated via the inhibitory effect of fetal androgens on steroid's stimulatory effect on alveolar epithelial-mesenchymal interactions. Since peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists accelerate lung maturation by stimulating alveolar epithelial-mesenchymal interactions, independent of fetal sex, we hypothesized that the effect of PPAR-γ agonist pioglitazone (PGZ) would be sex-independent. Pregnant Sprague-Dawley rat dams were intraperitoneally administered dexamethasone (DEX) or PGZ on embryonic day (e) 18 and e19. At e20, pups were delivered by cesarean section, and fetal lungs and brains were examined for markers of lung maturation and apoptosis, respectively. Mixed epithelial-fibroblast cell cultures were examined to gain mechanistic insights. Antenatal PGZ increased alveolar epithelial and mesenchymal maturation markers equally in males and females; in contrast, antenatal DEX had sex-specific effects. Additionally, unlike DEX, antenatal PGZ did not increase hippocampal apoptosis. We conclude that PPAR-γ agonist administration is an effective, and probably even a superior, alternative to ANS for accelerating fetal lung maturity equally in both males and females.

Exploiting collateral sensitivity controls growth of mixed culture of sensitive and resistant cells and decreases selection for resistant cells in a cell line model

BackgroundCDK4/6 inhibitors such as ribociclib are becoming widely used targeted therapies in hormone-receptor-positive (HR+) human epidermal growth factor receptor 2-negative (HER2−) breast cancer. However, cancers can advance due to drug resistance, a problem in which tumor heterogeneity and evolution are key features.MethodsRibociclib-resistant HR+/HER2− CAMA-1 breast cancer cells were generated through long-term ribociclib treatment. Characterization of sensitive and resistant cells were performed using RNA sequencing and whole exome sequencing. Lentiviral labeling with different fluorescent proteins enabled us to track the proliferation of sensitive and resistant cells under different treatments in a heterogeneous, 3D spheroid coculture system using imaging microscopy and flow cytometry.ResultsTranscriptional profiling of sensitive and resistant cells revealed the downregulation of the G2/M checkpoint in the resistant cells. Exploiting this acquired vulnerability; resistant cells exhibited collateral sensitivity for the Wee-1 inhibitor, adavosertib (AZD1775). The combination of ribociclib and adavosertib achieved additional antiproliferative effect exclusively in the cocultures compared to monocultures, while decreasing the selection for resistant cells.ConclusionsOur results suggest that optimal antiproliferative effects in heterogeneous cancers can be achieved via an integrative therapeutic approach targeting sensitive and resistant cancer cell populations within a tumor, respectively.

Pt-S Bond-Mediated Nanoflares for High-Fidelity Intracellular Applications by Avoiding Thiol Cleavage.

The Au-S bond is the classic way to functionalize gold nanoparticles (AuNPs). However, cleavage of the bond by biothiols and other chemicals is a long-standing problem hindering practical applications, especially in cells. Instead of replacing the thiol by a carbene or selenol for stronger adsorption, it is now shown that the Pt-S bond is much more stable, fully avoiding cleavage by biothiols. AuNPs were deposited with a thin layer of platinum, and an AuNP@Pt-S nanoflare was constructed to detect the miRNA-21 microRNA in living cells. This design retained the optical and cellular uptake properties of DNA-functionalized AuNPs, while showing high-fidelity signaling. It discriminated target cancer cells even in a mixed-cell culture system, where the Au-S based nanoflare was less sensitive. Compared to previous methods of changing the ligand chemistry, coating a Pt shell is more accessible, and previously developed methods for AuNPs can be directly adapted.

HSV1 VP1-2 deubiquitinates STING to block type I interferon expression and promote brain infection.

Herpes simplex virus (HSV) is the main cause of viral encephalitis in the Western world, and the type I interferon (IFN) system is important for antiviral control in the brain. Here, we have compared Ifnb induction in mixed murine brain cell cultures by a panel of HSV1 mutants, each devoid of one mechanism to counteract the IFN-stimulating cGAS-STING pathway. We found that a mutant lacking the deubiquitinase (DUB) activity of the VP1-2 protein induced particularly strong expression of Ifnb and IFN-stimulated genes. HSV1 ΔDUB also induced elevated IFN expression in murine and human microglia and exhibited reduced viral replication in the brain. This was associated with increased ubiquitination of STING and elevated phosphorylation of STING, TBK1, and IRF3. VP1-2 associated directly with STING, leading to its deubiquitination. Recruitment of VP1-2 to STING was dependent on K150 of STING, which was ubiquitinated by TRIM32. Thus, the DUB activity of HSV1 VP1-2 is a major viral immune-evasion mechanism in the brain.

Neuroinflammation Mediated by Glia Maturation Factor Exacerbates Neuronal Injury in an in vitro Model of Traumatic Brain Injury.

Traumatic brain injury (TBI) is the primary cause of death and disability affecting over 10 million people in the industrialized world. TBI causes a wide spectrum of secondary molecular and cellular complications in the brain. However, the pathological events are still not yet fully understood. Previously, we have shown that the glia maturation factor (GMF) is a mediator of neuroinflammation in neurodegenerative diseases. To identify the potential molecular pathways accompanying TBI, we used an in vitro cell culture model of TBI. A standardized injury was induced by scalpel cut through a mixed primary cell culture of astrocytes, microglia and neurons obtained from both wild type (WT) and GMF-deficient (GMF-KO) mice. Cell culture medium and whole-cell lysates were collected at 24, 48, and 72 h after the scalpel cuts injury and probed for oxidative stress using immunofluorescence analysis. Results showed that oxidative stress markers such as glutathione and glutathione peroxidase were significantly reduced, while release of cytosolic enzyme lactate dehydrogenase along with nitric oxide and prostaglandin E2 were significantly increased in injured WT cells compared with injured GMF-KO cells. In addition, injured WT cells showed increased levels of oxidation product 4-hydroxynonenal and 8-oxo-2'-deoxyguanosine compared with injured GMF-KO cells. Further, we found that injured WT cells showed a significantly increased expression of glial fibrillary acidic protein, ionized calcium binding adaptor molecule 1, and phosphorylated ezrin/radixin/moesin proteins, and reduced microtubule associated protein expression compared with injured GMF-KO cells after injury. Collectively, our results demonstrate that GMF exacerbates the oxidative stress-mediated neuroinflammation that could be brought about by TBI-induced astroglial activation.

Neutrophil Extracellular Trap Induced Dendritic Cell Activation Leads to Th1 Polarization in Type 1 Diabetes.

Neutrophils releasing neutrophil extracellular traps (NETs) infiltrate the pancreas prior to type 1 diabetes (T1D) onset; however, the precise nature of their contribution to disease remains poorly defined. To examine how NETs affect immune functions in T1D, we investigated NET composition and their effect on dendritic cells (DCs) and T lymphocytes in T1D children. We showed that T1D patient NET composition differs substantially from that of healthy donors and that the presence of T1D-NETs in a mixed peripheral blood mononuclear cell culture caused a strong shift toward IFNγ-producing T lymphocytes, mediated through activation of innate immunity cells in T1D samples. Importantly, in a monocyte-derived DC (moDC) culture, NETs induced cytokine production, phenotypic change and IFNγ-producing T cells only in samples from T1D patients but not in those from healthy donors. RNA-seq analysis revealed that T1D-NETs presence causes TGFβ downregulation and IFNα upregulation and creates pro-T1D signature in healthy moDCs.

A Nonperfusion‐Based Method of Hepatic Cell Isolation and Development of Fatty Liver Disease Model for Dairy Cattle

Fatty liver disease (FLD) is a major metabolic disorder of over‐conditioned periparturient dairy cattle and can precede many costly comorbidities, such as mastitis and ketosis. While management practices can decrease the incidence rate of FLD, the etiology is not fully understood. In vivo studies using large animals are expensive, difficult to perform, and time‐consuming; so, an in vitro model of FLD would be valuable for probing the biochemical mechanisms of the disease. Available methods for the isolation of bovine liver cells are mostly perfusion‐based and are often time‐consuming, require specialized equipment, or tend to favor the isolation of vascular epithelial cells rather than parenchymal cells. With these considerations in mind, our objective was to develop an in vitro model of FLD, optimizing a non‐perfusion method of hepatic cell isolation from adult cows. Hepatic tissues (~10 g) of cows from the abattoir were immediately collected following slaughter and transported to the laboratory on ice. After several washings with PBS, tissues were chopped, washed, and digested in 200 U/mL of collagenase II for ~30 min with shaking at 37 °C. When hepatic cells were grown, an overnight serum deprivation was done before the treatment with 0.4 mM palmitate, 20 ng/mL TNFα, and a cocktail of 0.4 mM palmitate and 20 ng/mL TNFα. Control cells were grown in growth medium alone. Identification of hepatic cells in culture was done by using immunolocalization of two epithelial cell markers namely, e‐cadherin and mesenchymal marker alpha smooth muscle actin (αSMA). Cell viability, apoptosis, and lipid accumulation were estimated by using lactate dehydrogenase (LDH) assay, caspase 3/7 assay, and oil red O staining, respectively. Hepatic cells were grown after 7–10 days of culture. Mixed cultures of hepatic cells showed presence of e‐cadherin positive hepatocytes and αSMA‐positive stellate cells. Consistent with FLD in vivo, presence of palmitate and palmitate along with TNFα increased (p < 0.05) LDH leakage in comparison to control culture. Likewise, TNFα and palmitate increased (p < 0.05) accumulation of lipid within the cell. In comparison to control cells, presence of palmitate and TNFA enhanced (3–3.5‐fold; p < 0.05) expression of caspase 3/7. Based on preliminary data presented herein, it appears that the non‐perfusion method of hepatic cell isolation and treating primary hepatic cells with palmitate and TNFα is an in vitro model for FLD.

Comparative analysis of chondrogenic and epigenetic differences in fore or hind limb bud-derived chicken micromass cell cultures

Purpose: Chicken embryos are often-used experimental models in developmental biology researches. Micromass cell culture system derived from mesenchymal cells of the embryo limb buds can serve as a reliable in vitro model for cartilage differentiation. In order to harvest the required high number of cells, these cell cultures are established from a mixture of fore and hind limb buds. However, the epigenetic regulation of the two limb bud types may vary. Our objective was to investigate the different chondrogenic and epigenetic regulation of fore or hind limb bud-derived high density cultures. Methods: Chicken embryos of Hamburger-Hamilton 22-24 stages were used to establish primary micromass cell cultures of chondrifying mesenchymal cells. Fore or hind limb buds were collected separately. Cell cultures established from a mixture of fore and hind limb buds served as control groups. Cultures were harvested on the 3rd and 6th day of culturing (according to the major steps of in vitro chondrogenesis). Changes in the quantity of cartilage-specific extracellular matrix were examined by dimethyl-methylene blue and toluidine blue metachromatic staining methods. Real-time quantitative PCR reactions were carried out to study the alterations of cartilage-specific (Sox9: SRY-box 9, Col2a1: collagen type II alpha 1 chain, Col1a1: collagen type I alpha 1 chain, Acan: aggrecan, Has2: hyaluronan synthase 2) and epigenetic marker gene expression levels (Dnmt3a: DNA methyltransferase 3 alpha, Tet1: tet methylcytosine dioxygenase 1, Ogt: O-linked N-acetylglucosamine (GlcNAc) transferase, Hdac3: histone deacetylase 3, Hdac4: histone deacetylase 4). Protein expressions of specific chondrogenic and epigenetic markers were examined by Western Blot technique. Measurements of mitochondrial activity with MTT-assay were also carried out. Results: Fore limb bud-derived micromass cell cultures showed a significantly pronounced and more advanced cartilage formation in comparison with hind limb bud- as well as mixed limb bud-derived micromass cell cultures. This phenomenon was verified not only with specific histological staining methods, but also with modern molecular biological techniques on mRNA and protein levels. Interestingly, our results indicated that the epigenetic regulatory pathway was altered in a negative way in the two examined cell culture types compared to the control cell culture. Results of the MTT-assay showed that the mitochondrial activity of hind limb bud-derived high density cultures was significantly lower compared to the other two experimental groups. Conclusions: Chicken limb bud-derived micromass cell culture system is a popular experimental model for chondrogenic researches. However, no modern scientific results can be found on specific molecular characteristics of high density cultures established from fore or hind limb buds separately. Our research aims to optimise this cell culture system for later scientific investigations, with a special regard to epigenetic analysis. Our experiments were supported by the EFOP-3.6.1-16-2016-00022, Debrecen Venture Catapult program.

Neuroprotective Effects of Ethyl Pyruvate against Aluminum Chloride-Induced Alzheimer's Disease in Rats via Inhibiting Toll-Like Receptor 4.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by the formation of insoluble deposits of β-amyloid (Aβ) plaques within the parenchyma of the brain. The present study aimed to investigate the neuroprotective role of ethyl pyruvate against in vitro and in vivo model of aluminum chloride (AlCl3)-induced AD. Effect of ethyl pyruvate (5, 10, 20, 40mM) against AlCl3 (1250μM)-induced neurotoxicity in primary neuron-glial mixed cell culture was evaluated using cell viability assays (MTT assay as well as calcein-AM/propidium iodide fluorescent dyes). In vivo model, AlCl3 (50mg/kg) were given through intraperitoneal route (i.p.) once daily for 4weeks in rats and after 2weeks, ethyl pyruvate (50, 100, 200mg/kg/day) was co-administered with AlCl3 once daily via the oral route. The present study, in addition to perform histopathology of the brain, also estimated oxidant and antioxidant parameters as well as memory impairment using pole test, plus maze, and Morris water maze test. The binding mode of ethyl pyruvate in the hMD-2 was also studied. Results of in vitro studies showed that the AlCl3 administration resulted in neuronal cell death. AlCl3 administration in rats resulted in memory loss, oxidative stress (increasedlipid peroxide and nitric oxide), impairment of antioxidant mechanisms (superoxide dismutase, catalase, and reduced glutathione), and deposition of amyloid plaques in cerebral cortex region of the brain. AlCl3 also resulted in the overexpression of the TLR4 receptors in the brain tissues. Administration of ethyl pyruvate ameliorated the AlCl3-induced neurotoxicity in neuron-glial mixed cell culture as well as histopathological, neurochemical, and behavioral consequences of chronic administration of AlCl3 in the rat. Ethyl pyruvate showed a docking score of 4.048. Thus, ethyl pyruvate is effective against in vitro and in vivo models of AlCl3-induced AD.

Role of zinc dyshomeostasis in inflammasome formation in cultured cortical cells following lipopolysaccharide or oxygen-glucose deprivation/reperfusion exposure

Exposure of mouse mixed cortical cell cultures to lipopolysaccharide (LPS) resulted in inflammasome formation in neurons and astrocytes, as indicated by increases in the levels of NLRP3, ASC, caspase-1, and IL-1β. LPS exposure concurrently increased the level of free zinc in the cytosol of both cell types. Addition of the membrane-permeant zinc chelator TPEN blocked the increases in the levels of NLRP3 and caspase-1 as well as the release of inflammatory cytokines, indicating a role for increased zinc in LPS-induced inflammasome formation. Oxygen-glucose deprivation (OGD), a cellular model of hypoxia, also induced inflammasome formation and zinc dyshomeostasis in cortical cells, effects that were abolished upon zinc chelation with TPEN. A similar mechanism appeared to be at work in vivo. Whereas intraperitoneal injection of LPS in mice resulted in inflammasome formation and microglial activation in the brain, it caused little induction of inflammasome formation in ZnT3-null mice, which lack synaptic zinc, suggesting a specific role for synaptic zinc in LPS-induced formation of inflammasomes in the mouse brain.

Coupling delay controls synchronized oscillation in the segmentation clock.

Individual cellular activities fluctuate but are constantly coordinated at the population level via cell-cell coupling. A notable example is the somite segmentation clock, in which the expression of clock genes (such as Hes7) oscillates in synchrony between the cells that comprise the presomitic mesoderm (PSM)1,2. This synchronization depends on the Notch signalling pathway; inhibiting this pathway desynchronizes oscillations, leading to somite fusion3-7. However, how Notch signalling regulates the synchronicity of HES7 oscillations is unknown. Here we establish a live-imaging system using a new fluorescent reporter (Achilles), which we fuse with HES7 to monitor synchronous oscillations in HES7 expression in the mouse PSM at a single-cell resolution. Wild-type cells can rapidly correct for phase fluctuations in HES7 oscillations, whereas the absence of the Notch modulator gene lunatic fringe (Lfng) leads to a loss of synchrony between PSM cells. Furthermore, HES7 oscillations are severely dampened in individual cells of Lfng-null PSM. However, when Lfng-null PSM cells were completely dissociated, the amplitude and periodicity of HES7 oscillations were almost normal, which suggests that LFNG is involved mostly in cell-cell coupling. Mixed cultures of control and Lfng-null PSM cells, and an optogenetic Notch signalling reporter assay, revealed that LFNG delays the signal-sending process of intercellular Notch signalling transmission. These results-together with mathematical modelling-raised the possibility that Lfng-null PSM cells shorten the coupling delay, thereby approaching a condition known as the oscillation or amplitude death of coupled oscillators8. Indeed, a small compound that lengthens the coupling delay partially rescues the amplitude and synchrony of HES7 oscillations in Lfng-null PSM cells. Our study reveals a delay control mechanism of the oscillatory networks involved in somite segmentation, and indicates that intercellular coupling with the correct delay is essential for synchronized oscillation.

NF155-overexpression promotes remyelination and functional restoration in a hypoxic-ischemic mixed neonatal rat forebrain cell culture system

White matter injury caused by perinatal hypoxia-ischemia is characterized by myelination disorders; however, its pathophysiological mechanisms are not fully elucidated. The neurofascin 155 (NF155) protein, expressed in oligodendrocytes, is critical for myelination. Previous findings suggest that NF155 participates in the pathological mechanisms of developmental myelination disorders in hypoxic-ischemic cerebral white matter lesions, and it might regulate cytoskeletal changes. Therefore, we hypothesized that increased NF155 expression during the early stages of hypoxic oligodendrocyte injury helps normalize myelin sheath development and consequently improves neural function by repairing paranodal structures of myelin sheaths and regulating cytoskeletal changes. To test this hypothesis, we established a hypoxic-ischemic, mixed neonatal rat forebrain cell culture model. When NF155 expression was upregulated, synergistic effects occurred between this protein and the paranodal proteins CASPR and contactin. In addition, the expression of Rho GTPase family proteins that regulate key cytoskeletal pathways, myelin sheath structures, and functions were restored, and axonal structures acquired a clear and transparent appearance. These results suggest that NF155 may enable myelin sheath repair by repairing paranodal region structures and regulating oligodendrocyte cytoskeletal mechanisms. Overall, the present study provides new insights into the pathogenesis of hypoxic-ischemic cerebral white matter lesions.

Selective Regulation of Neurons, Glial Cells, and Neural Stem/Precursor Cells by Poly(allylguanidine)-Coated Surfaces.

Poly(allylguanidine) (PAG) was synthesized and characterized as a polycationic coating material for culturing neurons, glial cells, and neural stem/precursor cells (NSPCs) to apply PAG for neural tissue engineering. For comparison, poly-d-lysine (PDL), the golden benchmark of the neuron cell culture system, was also used in this study. When PAG was subjected to a mixed culture of neurons and glial cells, cell adhesion and neurite extension of neuronal cells were clearly observed but only few glial cells could be found alongside the neurons. Compared to PDL, the significantly lower density of the glial fibrillary acidic protein-positive cells implied that PAG suppressed the glial cell development. Likewise, PAG was demonstrated to dominate the differentiation of NSPCs principally into neurons. To investigate whether the different effects of PAG and PDL on neuron and glial cell behaviors resulted from the difference of guanidinium cations and ammonium cations, poly-l-arginine (PLA) was included and compared in this study. Similar to PDL, PLA supported high neuron and glial cell viability simultaneously. Consequently, glial cell growth and viability restrained on PAG was not only affected by the side-chain guanidino groups but also by the backbone structure property. The absence of the peptide structure in the backbone of PAG and the conformation of coated PAG on tissue culture polystyrene possibly determined the polycationic biomaterial to limit the growth of glial cells.

Design, synthesis and cytotoxicity of the antitumor agent 1-azabicycles for chemoresistant glioblastoma cells.

Twelve multi-functional pyrrolizidinones, indolizidinones and pyrroliazepinones were prepared from formal aza-[3 + 2] and aza-[3 + 3] cycloadditions of five- to seven-membered heterocyclic enaminones as diverse ambident electrophiles. The antitumor activity of these alkaloid-like compounds was investigated through an initial screening performed on human glioblastoma multiform (GBM) cell lines (GL-15, U251), on murine glioma cells line (C6) and on normal glial cells. Of the compounds tested, the new pyrrolo[1,2a]azepinone, [ethyl (3-oxo-1,2-diphenyl-6,7,8,9-tetrahydro-3H-pyrrolo[1,2a]azepin-9a(5H)-yl)acetate] or (Compound-13) exhibited selective cytotoxic effects on GBM-temozolomide resistant cells. Compound-13 exerted dose-dependent cytotoxic activity by promoting arrest of cells in the G0/G1 phase of the cell cycle in the first 24h. The apoptotic effect observed was in a time-dependent manner. Anti-migratory effect promoted by the treatment with compound-13 was also observed. Moreover, healthy mixed glial cell cultures from rat brain exhibited no cytotoxicity effect upon exposure to compound-13. Thus, the present study paves the way for the use of compound-13 as novel antitumor scaffold candidate for glioma cell therapy.

Comparison of the effectiveness of available sources of autologous colony-forming endothelial cells

Endothelial Colony-forming cells (ECFCs) are valuable material for tissue vascular engineering and cell therapy of ischemic tissues. Difficult isolation is the main problem for using of ECFCs. ECFCs isolation from peripheral blood and adipose tissue has been previously described. In the presented research we compared effectiveness of peripheral blood, subcutaneous and epicardial adipose tissue for the ECFCs isolation without cell sorting. ECFCs was isolated from peripheral blood, subcutaneous and epicardial adipose tissue obtained from coronary heart disease patients (males, n=8) undergoing elective coronary artery bypass surgery. The stromal-vascular fraction of subcutaneous (SVF-ST) and epicardial (SVF-ET) adipose tissue as well as the mononuclear blood fraction (MNF) were cultivated in the complied EGM-2 medium. Cell cultures phenotyping was performed by flow cytometry and confocal microscopy. Their angiogenic (Matrigel) and proliferative activity (xCELLigence analyzer) in vitro were studied. ECFCs were isolated from MNF in 50% of cases, from SVF-ST in 12.5% and SVF-ET in 37.5%. The proliferative activity of ECFCs isolated from adipose tissue was low while cultures from MNF showed high and medium activity in 75% of cases. Pure ECFCs (more 99%) were obtained from MNF to third passage without cell sorting. Cultures from adipose tissue were contaminated by mesenchymal-stromal cells (MSCs) and contained ECFCs and MSCs. Thus, peripheral blood is the most effective source of autologous ECFCs compared with adipose tissue for this isolation method. However, adipose tissue is a suitable source of MSC and mixed cultures of MSC and endothelial cells.

Oregano powder reduces Streptococcus and increases SCFA concentration in a mixed bacterial culture assay.

Food borne illnesses have a world-wide economic impact and industries are continuously developing technologies to reduce the spread of disease caused by microorganisms. Antimicrobial growth promoters (AGPs) have been used to decrease microbiological infections in animals and their potential transfer to humans. In recent years there has been a global trend to remove AGPs from animal feed in an attempt to reduce the spread of antimicrobial resistant genes into the human population. Phytobiotics, such as oregano powder, are one of the potential replacements for AGPs due to their well-established antimicrobial components. 16S rRNA gene amplicons were used to determine the effect of oregano powder (1% w/v) on the microbiota of mixed bacterial cell cultures, which were obtained from the ceca of traditionally grown meat chickens (broilers). Oregano powder had a mild effect on the microbial cell cultures increasing Enterococcus faecium, rearranging ratios of members in the genus Lactobacillus and significantly reducing the genus Streptococcus (p = 1.6e-3). Beneficial short chain fatty acids (SCFA), acetic and butyric acid, were also significantly increased in oregano powder supplemented cultures. These results suggest that oregano powder at a concentration of 1% (w/v) may have beneficial influences on mixed microbial communities and SCFA production.

Toxoplasma ROP16I/III ameliorated inflammatory bowel diseases via inducing M2 phenotype of macrophages.

BACKGROUNDInflammatory bowel disease (IBD) is characterized by chronic and non-specific inflammation of the intestinal mucosa and mainly includes ulcerative colitis and Crohn's disease.AIMTo explore the beneficial effect of ToxoROP16I/III-induced M2 phynotype macrophages in homeostasis of IBDs through downregulation of M1 inflammatory cells.METHODSRAW264.7 macrophages stimulated by lipopolysaccharide (LPS) (M1 cells) were co-cultured with Caco-2 cells as an inflammatory model of IBD in vitro. The expression of ToxoROP16I/III was observed in RAW264.7 macrophages that were transfected with pEGFP-rop16I/III. The phenotypes of M2 and M1 macrophage cells were assessed by quantitative real-time reverse transcriptase polymerase chain reaction and the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, transforming growth factor (TGF)-β1, IL-10, inducible nitric oxide synthase (iNOS), and arginase-1 (Arg-1) was detected. The expression of iNOS, Arg-1, signal transducer and activator of transcription 3 (Stat3), p-Stat3, Stat6, p-Stat6, programmed death ligand-2 (PD-L2), caspase-3, -8, and -9 was analyzed by Western blotting, and Griess assays were performed to detect nitric oxide (NO). TNF-α, IL-1β, IL-6, TGF-β1, and IL-10 expression in the supernatants was detected by enzyme-linked immunosorbent assay, and Caco-2 cell apoptosis was determined by flow cytometry after mixing M1 cells with M2 cells in a Caco-2 cell co-culture system.RESULTSM1 cells exhibited significantly increased production of iNOS, NO, TNF-α, IL-1β, and IL-6, while ToxoROP16I/III induced macrophage bias to M2 cells in vitro, showing increased expression of Arg-1, IL-10 and TGF-β1 and elevated production of p-Stat3 and p-Stat6. The mixed M1 and M2 cell culture induced by ToxoROP16I/III exhibited decreased production of NO and iNOS and upregulated expression of Arg-1 and PD-L2. Accordingly, Caco-2 cells became apoptotic, and apoptosis-associated proteins such as caspase-3, -8 and -9 were dampened during co-culture of M1 and M2 cells. Flow cytometry analysis showed that co-culture of M1 cells with Caco-2 cells facilitated the apoptosis of Caco-2 cells, but co-culture of M1 and M2 cells alleviated Caco-2 cell apoptosis.CONCLUSIONToxoROP16I/III-induced M2 macrophages inhibited apoptosis of Caco-2 cells caused by M1 macrophages. This finding may help gain a better understanding of the underlying mechanism and represent a promising therapeutic strategy for IBDs.