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Gut dysbiosis and an inflamed bowel are growing concerns in mammals, including dogs. Probiotic supplements have been used to restore the natural microbial community and improve gastrointestinal health. Biofilm formation, antimicrobial activities, and immunological responses of probiotics are crucial to improving gut health. Thus, we tested a commercial probiotic blend (LabMAX-3), a canine kibble additive comprising Lactobacillus acidophilus, Lacticaseibacillus casei, and Enterococcus faecium for their ability to inactivate common enteric pathogens; their ability to form biofilms; epithelial cell adhesion; and their anti-inflammatory response in the Madin-Darby Canine Kidney (MDCK) cell line. Probiotic LabMAX-3 blend or individual isolates showed a strong inhibitory effect against Salmonella enterica, Listeria monocytogenes, enterotoxigenic Escherichia coli, and Campylobacter jejuni. LabMAX-3 formed biofilms comparable to Staphylococcus aureus. LabMAX-3 adhesion to the MDCK cell line (with or without lipopolysaccharide (LPS) pretreatment) showed comparable adhesion and biofilm formation (p < 0.05) to L. casei ATCC 334 used as a control. LabMAX-3 had no cytotoxic effects on the MDCK cell line during 1 h exposure. The interleukin-10 (IL-10) and tumor necrosis factor alpha (TNFα) ratio of LabMAX-3, compared to the L. casei control, showed a significant increase (p < 0.05), indicating a more pronounced anti-inflammatory response. The data show that LabMAX-3, a canine kibble supplement, can improve gastrointestinal health. [ABSTRACT FROM AUTHOR]

Quantitative phase imaging (QPI) is a powerful tool for label‐free visualisation of living cells. Here, we compare two QPI microscopes – the Telight Q‐Phase microscope and the Nanolive 3D Cell Explorer‐fluo microscope. Both systems provide unbiased information about cell morphology, such as individual cell dry mass, perimeter and area. The Q‐Phase microscope uses artefact‐free, coherence‐controlled holographic imaging technology to visualise cells in real time with minimal phototoxicity. The 3D Cell Explorer‐fluo employs laser‐based holotomography to reconstruct 3D images of living cells, visualising their internal structures and dynamics. Here, we analysed the strengths and limitations of both microscopes when examining two morphologically distinct cell lines – the cuboidal epithelial MDCK cells which form multicellular clusters and solitary growing Rat2 fibroblasts. We focus mainly on the ability of the devices to generate images suitable for single‐cell segmentation by the built‐in software, and we discuss the segmentation results and quantitative data generated from the segmented images. We show that both microscopes offer slightly different advantages, and the choice between them depends on the specific requirements and goals of the user. LAY DESCRIPTION: Label‐free imaging techniques have revolutionised how we study biological samples, allowing for in‐depth analysis without staining or labelling. In this context, quantitative phase imaging (QPI) or Quantitative Phase Microscopy (QPM) has emerged as a powerful tool. Unlike traditional phase‐contrast microscopy, which provides qualitative insights, QPI offers quantitative data with information about cell dry mass or cell volume. Here, we compare two advanced QPI microscopy systems: the Telight Q‐Phase microscope and the Nanolive 3D Cell Explorer‐fluo microscope for visualisation of two morphologically distinct cell lines. Each of these instruments comes with its strengths and limitations and different software solutions for detailed cell analysis. [ABSTRACT FROM AUTHOR]

The homologous P-type copper-ATPases (Cu-ATPases) ATP7A and ATP7B are the key regulators of copper homeostasis in mammalian cells. In polarized epithelia, upon copper treatment, ATP7A and ATP7B traffic from the trans-Golgi network (TGN) to basolateral and apical membranes, respectively. We characterized the sorting pathways of Cu-ATPases between TGN and the plasma membrane and identified the machinery involved. ATP7A and ATP7B reside on distinct domains of TGN in limiting copper conditions, and in high copper, ATP7A traffics to basolateral membrane, whereas ATP7B traverses common recycling, apical sorting and apical recycling endosomes en route to apical membrane. Mass spectrometry identified regulatory partners of ATP7A and ATP7B that include the adaptor protein-1 complex. Upon knocking out pan-AP-1, sorting of both Cu-ATPases is disrupted. ATP7A loses its trafficking polarity and localizes on both apical and basolateral surfaces in high copper. By contrast, ATP7B loses TGN retention but retained its trafficking polarity to the apical domain, which became copper independent. Using isoform-specific knockouts, we found that the AP-1A complex provides directionality and TGN retention for both Cu-ATPases, whereas the AP-1B complex governs copper-independent trafficking of ATP7B solely. Trafficking phenotypes of Wilson disease-causing ATP7B mutants that disrupts putative ATP7B-AP1 interaction further substantiates the role of AP-1 in apical sorting of ATP7B. [ABSTRACT FROM AUTHOR]

The adaptation of egg-derived H7N9 candidate vaccine virus (CVV) in the mammalian cell line is an approach to developing a high-growth virus strain for the mass production of vaccine manufacturing. The adaptive mutations that occur in hemagglutinin (HA) are critical to the activity and potency of the vaccine virus. Previously, we identified a new mutation of A169S in the HA protein of an MDCK-adapted H7N9 vaccine virus (A/Anhui/2013, RG268); however, whether and how this mutation affects vaccine potency remain to be investigated. In this study, we serially passaged RG268 in MDCK cells and found that the HA titer and the TCID50 of the passaged virus RG268-M5 were 4-fold (HA units/50 μL) and 3.5-fold (log10 TCID50/mL) higher than those of the original CVV. By inspecting tandem MS spectra, we identified a new glycosylation site at N167 near the receptor binding site of the HA protein of RG268-M5. Flow cytometry results revealed that RG268-M5 could efficiently infect MDCK cells and initiate viral protein replication as well as that of RG268. Though the new glycosylation site is in the antigenic epitope of viral HA protein, the HI assay result indicated that the antigenicity of RG268-M5 was similar to RG268. Additionally, immunizing mice with RG268-M5 mixed aluminum hydroxide could induce potent antibody responses against the homologous and heterologous H7N9 viruses in vitro whereas the titers were comparable with those from the RG268 group. These results provide in-depth structural information regarding the effects of site-specific glycosylation on virus properties, which have implications for novel avian influenza vaccine development. [ABSTRACT FROM AUTHOR]

The efflux ratio (ER), determined by Caco-2/MDCK assays, is the standard in vitro metric to establish qualitatively whether a compound is a substrate of an efflux transporter. However, others have also enabled the utilisation of this metric quantitatively by deriving a relationship that expresses the ER as a function of the intrinsic membrane permeability of the membrane ( P 0 ) as well as the permeability of carrier-mediated efflux ( P p g p ). As of yet, P p g p cannot be measured directly from transport experiments or otherwise, but the ER relationship provides easy access to this value if P 0 is known. However, previous derivations of this relationship failed to consider the influence of additional transport resistances such as the aqueous boundary layers (ABLs) and the filter on which the monolayer is grown. Since single fluxes in either direction can be heavily affected by these experimental artefacts, it is crucial to consider the potential impact on the ER. We present a model that includes these factors and show both mathematically and experimentally that this simple ER relationship also holds for the more realistic scenario that does not neglect the ABLs/filter. Furthermore, we also show mathematically how paracellular transport affects the ER, and we experimentally confirm that paracellular dominance reduces the ER to unity and can mask potential efflux. [ABSTRACT FROM AUTHOR]

Influenza is a potentially fatal acute respiratory viral disease caused by the influenza virus. Influenza viruses vary in antigenicity and spread rapidly, resulting in seasonal epidemics. Vaccination is the most effective strategy for lowering the incidence and fatality rates of influenza-related disorders, and it is also an important method for reducing seasonal influenza infections. Mammalian Madin–Darby canine kidney (MDCK) cell lines are recommended for influenza virus growth, and such cell lines have been utilized in several commercial influenza vaccine productions. The limit dilution approach was used to screen ATCC-MDCK cell line subcellular strains that are especially sensitive to H1N1, H3N2, BV, and BY influenza viruses to increase virus production, and research on influenza virus culture media was performed to support influenza virus vaccine development. We also used RNA sequencing to identify differentially expressed genes and a GSEA analysis to determine the biological mechanisms underlying the various levels of susceptibility of cells to influenza viruses. MDCK cell subline 2B6 can be cultured to increase titer and the production of the H1N1, H3N2, BV, and BY influenza viruses. MDCK-2B6 has a significantly enriched and activated in ECM receptor interaction, JAK-STAT signaling, and cytokine receptor interaction signaling pathways, which may result in increased cellular susceptibility and cell proliferation activity to influenza viruses, promote viral adsorption and replication, and elevate viral production, ultimately. The study revealed that MDCK-2B6 can increase the influenza virus titer and yield in vaccine production by increasing cell sensitivity and enhancing proliferative activity. [ABSTRACT FROM AUTHOR]

Ouabain, an organic compound with the ability to strengthen the contraction of the heart muscle, was originally derived from plants. It has been observed that certain mammalian species, including humans, naturally produce ouabain, leading to its classification as a new type of hormone. When ouabain binds to Na+/K+-ATPase, it elicits various physiological effects, although these effects are not well characterized. Previous studies have demonstrated that ouabain, within the concentration range found naturally in the body (10 nmol/L), affects the polarity of epithelial cells and their intercellular contacts, such as tight junctions, adherens junctions, and gap junctional communication. This is achieved by activating signaling pathways involving cSrc and Erk1/2. To further investigate the effects of ouabain within the hormonally relevant concentration range (10 nmol/L), mRNA-seq, a high-throughput sequencing technique, was employed to identify differentially expressed transcripts. The discovery that the transcript encoding MYO9A was among the genes affected prompted an exploration of whether RhoA and its downstream effector ROCK were involved in the signaling pathways through which ouabain influences cell-to-cell contacts in epithelial cells. Supporting this hypothesis, this study reveals the following: (1) Ouabain increases the activation of RhoA. (2) Treatment with inhibitors of RhoA activation (Y27) and ROCK (C3) eliminates the enhancing effect of ouabain on the tight junction seal and intercellular communication via gap junctions. These findings further support the notion that ouabain acts as a hormone to emphasize the epithelial phenotype.

Transepithelial electrical resistance (TEER) is a widely used technique for quantifying the permeability of epithelial and endothelial cell layers. However, traditional methods of measuring TEER are limited to single timepoint measurements and can subject cells to an altered environment during the measurement. Here, we assessed the validity of TEER measurements by the ECIS TEER96 device, which is designed to take continuous TEER measurements of a cell culture system in a standard laboratory incubator. We found that the instrument accurately measures TEER across TEER values ranging from 10 to 2050 Ω*cm2 and is more accurate than the manual epithelial voltohmmeter electrode at high TEER values. Furthermore, the high-resolution measurements provided by the device allowed for a unique insight into the mechanisms and kinetics of cells in vitro. To demonstrate the continuous measurement capability of the device, we tracked the formation of an MDCKI cell monolayer until TEER plateaued. Furthermore, we treated Caco-2 monolayers with different concentrations of DMSO and the antimicrobial and surfactant compound benzethonium chloride to measure disruptions to barrier integrity. Treatment of both compounds resulted in concentration-dependent loss of barrier integrity. Our results suggest that the ECIS TEER96 device is a reliable and convenient option for measuring TEER in cell cultures and can provide valuable insights into the behavior of cells in vitro. This technology will be especially useful for increasing throughput of drug permeability assays, inflammation studies, and gaining better understanding of disease states in a cell culture system.

Introduction: The main function of the urinary tract is to form an impermeable barrier against urinary solutes and bacteria. However, this barrier can be compromised by urinary tract infections, most commonly caused by uropathogenic Escherichia coli (UPEC). This can result in damage to the epithelial barrier, leading to decreased epithelial thickness, loss of tight junctions, loss of epithelial integrity, and apoptosis. Due to the rise in antimicrobial resistance, there is worldwide interest in exploring non-antibiotic agents as alternative therapy. Methods: Using the Madin-Darby canine kidney (MDCK) cell line, a widely accepted epithelial cell model for the urinary tract, and the UPEC strain UTI89, this paper aimed to investigate the impact of UPEC on cell integrity, permeability, and barrier functions, and determine whether cranberry, D-mannose and ibuprofen could counteract the effects induced by UPEC. Furthermore, the study examined the protective potential of these agents against UPEC-induced increase in reactive oxygen species (ROS) production and programmed death-ligand 1 (PD-L1) expression. Results: The results demonstrated that UTI89 caused a marked reduction in cell viability and monolayer integrity. Cranberry (3mg/mL) was protective against these changes. In addition, cranberry exhibited protective effects against UPEC-induced damage to cell barrier integrity, escalation of oxidative stress, and UPEC/TNFa-triggered PDL1 expression. However, no effect was observed for D-mannose and ibuprofen in alleviating UPEC-induced cell damage and changes in ROS and PD-L1 levels. Conclusion: Overall, cranberry, but not D-mannose or ibuprofen, has a protective influence against UPEC associated damage in urinary epithelial cells. [ABSTRACT FROM AUTHOR]

Background: The MDCK cell line is the primary cell line used for influenza vaccine production. Using genetic engineering technology to change the expression and activity of genes that regulate virus proliferation to obtain high-yield vaccine cell lines has attracted increasing attention. A comprehensive understanding of the key genes, targets, and molecular mechanisms of viral regulation in cells is critical to achieving this goal, yet the post-transcriptional regulation mechanism involved in virus proliferation—particularly the effect of lncRNA on influenza virus proliferation—is still poorly understood. Therefore, this study used high-throughput RNA-seq technology to identify H1N1 infection-induced lncRNA and mRNA expression changes in MDCK cells and explore the regulatory relationship between these crucial lncRNAs and their target genes. Results: In response to H1N1 infection in MDCK cells 16 h post-infection (hpi) relative to uninfected controls, we used multiple gene function annotation databases and initially identified 31,501 significantly differentially expressed (DE) genes and 39,920 DE lncRNAs (|log2FC| > 1, p < 0.05). Among these, 102 lncRNAs and 577 mRNAs exhibited predicted correlations with viral response mechanisms. Based on the magnitude of significant expression differences, related research, and RT-qPCR expression validation at the transcriptional level, we further focused on 18 DE mRNAs and 32 DE lncRNAs. Among these, the differential expression of the genes RSAD2, CLDN1, HCLS1, and IFIT5 in response to influenza virus infection was further verified at the protein level using Western blot technology, which showed results consistent with the RNA-seq and RT-qPCR findings. We then developed a potential molecular regulatory network between these four genes and their six predicted lncRNAs. Conclusions: The results of this study will contribute to a more comprehensive understanding of the molecular mechanism of host cell non-coding RNA-mediated regulation of influenza virus replication. These results may also identify methods for screening target genes in the development of genetically engineered cell lines capable of high-yield artificial vaccine production. [ABSTRACT FROM AUTHOR]

IntroductionThe main function of the urinary tract is to form an impermeable barrier against urinary solutes and bacteria. However, this barrier can be compromised by urinary tract infections, most commonly caused by uropathogenic Escherichia coli (UPEC). This can result in damage to the epithelial barrier, leading to decreased epithelial thickness, loss of tight junctions, loss of epithelial integrity, and apoptosis. Due to the rise in antimicrobial resistance, there is worldwide interest in exploring non-antibiotic agents as alternative therapy.MethodsUsing the Madin-Darby canine kidney (MDCK) cell line, a widely accepted epithelial cell model for the urinary tract, and the UPEC strain UTI89, this paper aimed to investigate the impact of UPEC on cell integrity, permeability, and barrier functions, and determine whether cranberry, D-mannose and ibuprofen could counteract the effects induced by UPEC. Furthermore, the study examined the protective potential of these agents against UPEC-induced increase in reactive oxygen species (ROS) production and programmed death-ligand 1 (PD-L1) expression.ResultsThe results demonstrated that UTI89 caused a marked reduction in cell viability and monolayer integrity. Cranberry (3 mg/mL) was protective against these changes. In addition, cranberry exhibited protective effects against UPEC-induced damage to cell barrier integrity, escalation of oxidative stress, and UPEC/TNFα-triggered PD-L1 expression. However, no effect was observed for D-mannose and ibuprofen in alleviating UPEC-induced cell damage and changes in ROS and PD-L1 levels.ConclusionOverall, cranberry, but not D-mannose or ibuprofen, has a protective influence against UPEC associated damage in urinary epithelial cells.

The adaptation of egg-derived H7N9 candidate vaccine virus (CVV) in the mammalian cell line is an approach to developing a high-growth virus strain for the mass production of vaccine manufacturing. The adaptive mutations that occur in hemagglutinin (HA) are critical to the activity and potency of the vaccine virus. Previously, we identified a new mutation of A169S in the HA protein of an MDCK-adapted H7N9 vaccine virus (A/Anhui/2013, RG268); however, whether and how this mutation affects vaccine potency remain to be investigated. In this study, we serially passaged RG268 in MDCK cells and found that the HA titer and the TCID50 of the passaged virus RG268-M5 were 4-fold (HA units/50 μL) and 3.5-fold (log10 TCID50/mL) higher than those of the original CVV. By inspecting tandem MS spectra, we identified a new glycosylation site at N167 near the receptor binding site of the HA protein of RG268-M5. Flow cytometry results revealed that RG268-M5 could efficiently infect MDCK cells and initiate viral protein replication as well as that of RG268. Though the new glycosylation site is in the antigenic epitope of viral HA protein, the HI assay result indicated that the antigenicity of RG268-M5 was similar to RG268. Additionally, immunizing mice with RG268-M5 mixed aluminum hydroxide could induce potent antibody responses against the homologous and heterologous H7N9 viruses in vitro whereas the titers were comparable with those from the RG268 group. These results provide in-depth structural information regarding the effects of site-specific glycosylation on virus properties, which have implications for novel avian influenza vaccine development.

Influenza is a potentially fatal acute respiratory viral disease caused by the influenza virus. Influenza viruses vary in antigenicity and spread rapidly, resulting in seasonal epidemics. Vaccination is the most effective strategy for lowering the incidence and fatality rates of influenza-related disorders, and it is also an important method for reducing seasonal influenza infections. Mammalian Madin–Darby canine kidney (MDCK) cell lines are recommended for influenza virus growth, and such cell lines have been utilized in several commercial influenza vaccine productions. The limit dilution approach was used to screen ATCC-MDCK cell line subcellular strains that are especially sensitive to H1N1, H3N2, BV, and BY influenza viruses to increase virus production, and research on influenza virus culture media was performed to support influenza virus vaccine development. We also used RNA sequencing to identify differentially expressed genes and a GSEA analysis to determine the biological mechanisms underlying the various levels of susceptibility of cells to influenza viruses. MDCK cell subline 2B6 can be cultured to increase titer and the production of the H1N1, H3N2, BV, and BY influenza viruses. MDCK-2B6 has a significantly enriched and activated in ECM receptor interaction, JAK-STAT signaling, and cytokine receptor interaction signaling pathways, which may result in increased cellular susceptibility and cell proliferation activity to influenza viruses, promote viral adsorption and replication, and elevate viral production, ultimately. The study revealed that MDCK-2B6 can increase the influenza virus titer and yield in vaccine production by increasing cell sensitivity and enhancing proliferative activity.

The Madin-Darby canine kidney (MDCK) cell line is frequently used for permeability screening in drug discovery. It contains endogenous transporters, most prominently canine multidrug resistance P-glycoprotein (Mdr1), which can interfere with studies of P-glycoprotein substrate assessment and permeability measurements. Because MDCK wild type (WT) is genetically heterogeneous, an isolation procedure was investigated in this study to obtain the subclonal line with low P-glycoprotein expression. The best clone obtained had up to 3-fold lower amprenavir efflux and P-glycoprotein expression in comparison to WT. Of 12 standard compounds tested that exhibited active efflux in WT cells, 11 showed a decrease in efflux in the isolated clone. However, the decrease was not below the cut-off value of 2, indicating residual P--glycoprotein activity. Clone isolation via the limiting dilution method, combined with bidirectional amprenavir permeability for clone selection, successfully identified MDCK clones with substantially lower P-glycoprotein efflux and has been demonstrated as a useful tool for assessing passive permeability in early drug discovery.

Shear forces between cells occur during global changes in multicellular organization during morphogenesis and tissue growth, yet how cells sense shear forces and propagate a response across a tissue is unknown. We found that applying exogenous shear at the midline of an epithelium induced a local, short-term deformation near the shear plane, and a long-term collective oscillatory movement across the epithelium that spread from the shear-plane and gradually dampened. Inhibiting actomyosin contraction or E-cadherin trans-cell adhesion blocked oscillations, whereas stabilizing actin filaments prolonged oscillations. Combining these data with a model of epithelium mechanics supports a mechanism involving the generation of a shear-induced mechanical event at the shear plane which is then relayed across the epithelium by actomyosin contraction linked through E-cadherin. This causes an imbalance of forces in the epithelium, which is gradually dissipated through oscillatory cell movements and actin filament turnover to restore the force balance across the epithelium.

Copyright of Eurasian Journal of Applied Biotechnology is the property of National Center for Biotechnology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Etlik Veteriner Mikrobiyoloji Dergisi is the property of Veteriner Kontrol Merkez Arastirma Enstitusu and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Permeability is an important molecular property in drug discovery, as it co-determines pharmacokinetics whenever a drug crosses the phospholipid bilayer, e.g., into the cell, in the gastrointestinal tract, or across the blood-brain barrier. Many methods for the determination of permeability have been developed, including cell line assays (CACO-2 and MDCK), cell-free model systems like parallel artificial membrane permeability assay (PAMPA) mimicking, e.g., gastrointestinal epithelia or the skin, as well as the black lipid membrane (BLM) and submicrometer liposomes. Furthermore, many in silico approaches have been developed for permeability prediction: meta-analysis of publicly available databases for permeability data (MolMeDB and ChEMBL) was performed to establish their usability. Four experimental and two computational methods were evaluated. It was shown that repeatability of the reported permeability measurement is not great even for the same method. For the PAMPA method, two different permeabilities are reported: intrinsic and apparent. They can vary in degrees of magnitude; thus, we suggest being extra cautious using literature data on permeability. When we compared data for the same molecules using different methods, the best agreement was between cell-based methods and between BLM and computational methods. Existence of unstirred water layer (UWL) permeability limits the data agreement between cell-based methods (and apparent PAMPA) with data that are not limited by UWL permeability (computational methods, BLM, intrinsic PAMPA). Therefore, different methods have different limitations. Cell-based methods provide results only in a small range of permeabilities (-8 to -4 in cm/s), and computational methods can predict a wider range of permeabilities beyond physical limitations, but their precision is therefore limited. BLM with liposomes can be used for both fast and slow permeating molecules, but its usage is more complicated than standard transwell techniques. To sum up, when working with in-house measured or published permeability data, we recommend caution in interpreting and combining them.

Background: The MDCK cell line is the primary cell line used for influenza vaccine production. Using genetic engineering technology to change the expression and activity of genes that regulate virus proliferation to obtain high-yield vaccine cell lines has attracted increasing attention. A comprehensive understanding of the key genes, targets, and molecular mechanisms of viral regulation in cells is critical to achieving this goal, yet the post-transcriptional regulation mechanism involved in virus proliferation—particularly the effect of lncRNA on influenza virus proliferation—is still poorly understood. Therefore, this study used high-throughput RNA-seq technology to identify H1N1 infection-induced lncRNA and mRNA expression changes in MDCK cells and explore the regulatory relationship between these crucial lncRNAs and their target genes. Results: In response to H1N1 infection in MDCK cells 16 h post-infection (hpi) relative to uninfected controls, we used multiple gene function annotation databases and initially identified 31,501 significantly differentially expressed (DE) genes and 39,920 DE lncRNAs (|log2FC| > 1, p < 0.05). Among these, 102 lncRNAs and 577 mRNAs exhibited predicted correlations with viral response mechanisms. Based on the magnitude of significant expression differences, related research, and RT-qPCR expression validation at the transcriptional level, we further focused on 18 DE mRNAs and 32 DE lncRNAs. Among these, the differential expression of the genes RSAD2, CLDN1, HCLS1, and IFIT5 in response to influenza virus infection was further verified at the protein level using Western blot technology, which showed results consistent with the RNA-seq and RT-qPCR findings. We then developed a potential molecular regulatory network between these four genes and their six predicted lncRNAs. Conclusions: The results of this study will contribute to a more comprehensive understanding of the molecular mechanism of host cell non-coding RNA-mediated regulation of influenza virus replication. These results may also identify methods for screening target genes in the development of genetically engineered cell lines capable of high-yield artificial vaccine production.

N-acetylneuraminic acid linked to galactose by α2,6 and α2,3 linkages (Siaα2,6 and Siaα2,3) is expressed on glycoconjugates of animal tissues, where it performs multiple biological functions. In addition, these types of sialic acid residues are the main targets for the binding and entry of influenza viruses. Here we used fluorochrome-conjugated Sambuccus nigra, Maackia amurensis, and peanut lectins for the simultaneous detection of Siaα2,3 and Siaα2,6 and galactosyl residues by two-color flow cytometry on A549 cells, a human pneumocyte cell line used for in vitro studies of the infection by influenza viruses, as well as on Vero and MDCK cell lines. The dexamethasone (DEX) glucocorticoid (GC), a widely used anti-inflammatory compound, completely abrogated the expression of Siaα2,3 in A549 cells and decreased its expression in Vero and MDCK cells; in contrast, the expression of Siaα2,6 was increased in the three cell lines. These observations indicate that DEX can be used for the study of the mechanism of sialylation of cell membrane molecules. Importantly, DEX may change the tropism of avian and human/pig influenza viruses and other infectious agents to animal and human epithelial cells. [ABSTRACT FROM AUTHOR]

The influenza virus is a cause of seasonal epidemic disease and enormous economic injury. The best way to control influenza outbreaks is through vaccination. The Madin-Darby canine kidney cell line (MDCK) is currently approved to manufacture influenza vaccines. However, the viral load from cell-based production is limited by host interferons (IFN). Interferon regulating factor 7 (IRF7) is a transcription factor for type-I IFN that plays an important role in regulating the anti-viral mechanism and eliminating viruses. We developed IRF7 knock-out MDCK cells (IRF7-/- MDCK) using CRISPR/Cas9 technology. The RNA expression levels of IRF7 in the IRF7-/- MDCK cells were reduced by 94.76% and 95.22% under the uninfected and infected conditions, respectively. Furthermore, the IRF7 protein level was also significantly lower in IRF7-/- MDCK cells for both uninfected (54.85% reduction) and viral infected conditions (32.27% reduction) compared to WT MDCK. The differential expression analysis of IFN-related genes demonstrated that the IRF7-/- MDCK cell had a lower interferon response than wildtypeMDCKunder the influenza-infected condition. Gene ontology revealed down-regulation of the defense response against virus and IFN-gamma production in IRF7-/- MDCK. The evaluation of influenza viral titers by RT-qPCR and hemagglutination assay (HA) revealed IRF7-/- MDCK cells had higher viral titers in cell supernatant, including A/pH1N1 (4 to 5-fold) and B/Yamagata (2-fold). Therefore, the IRF7-/- MDCK cells could be applied to cell-based influenza vaccine production with higher capacity and efficiency. [ABSTRACT FROM AUTHOR]

The influenza virus is a cause of seasonal epidemic disease and enormous economic injury. The best way to control influenza outbreaks is through vaccination. The Madin-Darby canine kidney cell line (MDCK) is currently approved to manufacture influenza vaccines. However, the viral load from cell-based production is limited by host interferons (IFN). Interferon regulating factor 7 (IRF7) is a transcription factor for type-I IFN that plays an important role in regulating the anti-viral mechanism and eliminating viruses. We developed IRF7 knock-out MDCK cells (IRF7−/ − MDCK) using CRISPR/Cas9 technology. The RNA expression levels of IRF7 in the IRF7−/ − MDCK cells were reduced by 94.76% and 95.22% under the uninfected and infected conditions, respectively. Furthermore, the IRF7 protein level was also significantly lower in IRF7−/ − MDCK cells for both uninfected (54.85% reduction) and viral infected conditions (32.27% reduction) compared to WT MDCK. The differential expression analysis of IFN-related genes demonstrated that the IRF7−/ − MDCK cell had a lower interferon response than wildtype MDCK under the influenza-infected condition. Gene ontology revealed down-regulation of the defense response against virus and IFN-gamma production in IRF7−/ − MDCK. The evaluation of influenza viral titers by RT-qPCR and hemagglutination assay (HA) revealed IRF7−/ − MDCK cells had higher viral titers in cell supernatant, including A/pH1N1 (4 to 5-fold) and B/Yamagata (2-fold). Therefore, the IRF7−/ − MDCK cells could be applied to cell-based influenza vaccine production with higher capacity and efficiency.

Collective migration of epithelial cells is an integral part of embryonic development, wound healing, tissue renewal and carcinoma invasion. While previous studies have focused on cell-extracellular matrix adhesion as a site of migration-driving, traction force-transmission, cadherin mediated cell-cell adhesion is also capable of force-transmission. Using a soft elastomer coated with purified N-cadherin as a substrate and a Hepatocyte Growth Factor-treated, transformed MDCK epithelial cell line as a model system, we quantified traction transmitted by N-cadherin-mediated contacts. On a substrate coated with purified extracellular domain of N-cadherin, cell surface N-cadherin proteins arranged into puncta. N-cadherin mutants (either the cytoplasmic deletion or actin-binding domain chimera), however, failed to assemble into puncta, suggesting the assembly of focal adhesion like puncta requires the cytoplasmic domain of N-cadherin. Furthermore, the cytoplasmic domain deleted N-cadherin expressing cells exerted lower traction stress than the full-length or the actin binding domain chimeric N-cadherin. Our data demonstrate that N-cadherin junctions exert significant traction stress that requires the cytoplasmic domain of N-cadherin, but the loss of the cytoplasmic domain does not completely eliminate traction force transmission.

Background: The Madin-Darby canine kidney (MDCK) cell line constitutes a key component of influenza vaccine production, but its dependence on adherent growth limits cell culture density and hinders vaccine yield. There is evidence that the use of gene editing techniques to inhibit cell adhesion and establish an easily suspended cell line can improve vaccine yield; however, the mechanisms underlying MDCK cell adhesion are unclear. Methods: In this study, we used transcriptomics to analyse differentially expressed mRNAs and miRNAs in adherent and suspension cultures of MDCK cells. Results : We found that claudin-1 (CLDN1) expression was downregulated in the suspension MDCK cells and that CLDN1 promotes MDCK cell-extracellular matrix adhesion. Additionally, microRNA (miR)-175 expression was upregulated in the suspension MDCK cells. Importantly, we demonstrated that miR-175 inhibits MDCK cell adhesion by targeting the CLDN1 3'-untranslated region (UTR). These findings contribute to a more comprehensive understanding of the regulatory mechanisms modulating cell adhesion and provide a basis for establishing suspension-adapted, genetically engineered cell lines. Our work could also facilitate the identification of targets for tumour therapy.

P73, a member of p53 tumor suppressor family, plays a crucial role in tumor suppression and neural development. Due to the usage of two promoters, p73 is expressed as two isoforms, TAp73 and âNp73, with opposing functions. Here, we investigated the potential role of p73 in epithelial polarity and morphogenesis by using Madin-Darby canine kidney (MDCK) cells as a model system. We found that knockdown of TAp73 enhances, whereas knockdown of âNp73 inhibits, MDCK cell proliferation and migration in two-dimensional (2-D) culture. We also found that knockdown of TAp73, but not âNp73, disrupts cyst formation of MDCK cells in three-dimensional (3-D) culture. Interestingly, we found that p21 and PUMA, both of which are induced by TAp73 but repressed by âNp73, are required for suppressing cell proliferation and migration in 2-D culture and for MDCK ce ll morphogenesis in 3-D culture. Finally, we showed knockdown of TAp73, p21 or PUMA induces epithelial to mesenchymal transition (EMT) with a decrease in E-cadherin and an increase in EMT transcription factors. Together, our data suggest that TAp73, p21 and PUMA play a critical role in modulating MDCK cell morphogenesis by maintaining an appropriate level of the EMT.

Regulated vesicle exocytosis is a key response to extracellular stimuli in diverse physiological processes, including hormone regulated short-term urine concentration. In the renal collecting duct, the water channel aquaporin-2 (AQP2) localizes to the apical plasma membrane as well as to small, subapical vesicles. In response to stimulation with the antidiuretic hormone, arginine vasopressin, aquaporin-2-containing vesicles fuse with the plasma membrane, which increases collecting duct water reabsorption and thus, urine concentration. The nanoscale size of these vesicles has limited analysis of their three-dimensional (3D) organization. Using a cell system combined with 3D superresolution microscopy, we provide the first direct analysis of the 3D network of aquaporin-2-containing exocytic vesicles in a cell culture system. We show that aquaporin-2 vesicles are 43 ± 3 nm in diameter, a size similar to synaptic vesicles, and that one fraction of AQP2 vesicles localized with the subcortical F-actin layer and the other localized in between the F-actin layer and the plasma membrane. Aquaporin-2 vesicles associated with F-actin and this association were enhanced in a serine 256 phospho-mimic of aquaporin-2, whose phosphorylation is a key event in antidiuretic hormone-mediated aquaporin-2 vesicle exocytosis. [ABSTRACT FROM AUTHOR]

Cell competition is a phenomenon that eliminates unfit cells from cell society, a function vital for maintaining cellular and organismal homeostasis. We previously showed that Madin-Darby canine kidney (MDCK) epithelial cells expressing the active form of the transcriptional coactivator Yes-associated protein (YAP) are apically extruded when surrounded by normal MDCK cells. Although we demonstrated that the arachidonic acid (AA) cascade is involved in YAP-dependent apical extrusion, the metabolic events leading to this outcome remained unclear. Here, we present the results of metabolomic analysis that identified phosphatidylcholine (PC) biosynthesis as the most significant player in this process. Removal of the PC biosynthetic components choline and methionine from culture medium inhibited YAP-dependent apical extrusion. Inhibition of either choline uptake or metabolic cycles involving choline or methionine also decreased YAP-dependent apical extrusion. At the molecular level, active YAP induced expression of the genes encoding glycerophosphocholine phosphodiesterase 1 (GPCPD1) and lecithin–cholesterol acyltransferase (LCAT), which are involved in choline metabolism. Our results indicate that YAP-dependent cell competition depends on YAP-mediated activation of the choline metabolic cycle. • Depletion of both choline and methionine from MDCK cell culture medium inhibits YAP-dependent apical extrusion. • Inhibition of choline or methionine metabolism suppresses YAP-dependent apical extrusion. • YAP upregulates the GPCPD1 and LCAT genes during YAP-dependent apical extrusion. [ABSTRACT FROM AUTHOR]

N-acetylneuraminic acid linked to galactose by α2,6 and α2,3 linkages (Siaα2,6 and Siaα2,3) is expressed on glycoconjugates of animal tissues, where it performs multiple biological functions. In addition, these types of sialic acid residues are the main targets for the binding and entry of influenza viruses. Here we used fluorochrome-conjugated Sambuccus nigra, Maackia amurensis, and peanut lectins for the simultaneous detection of Siaα2,3 and Siaα2,6 and galactosyl residues by two-color flow cytometry on A549 cells, a human pneumocyte cell line used for in vitro studies of the infection by influenza viruses, as well as on Vero and MDCK cell lines. The dexamethasone (DEX) glucocorticoid (GC), a widely used anti-inflammatory compound, completely abrogated the expression of Siaα2,3 in A549 cells and decreased its expression in Vero and MDCK cells; in contrast, the expression of Siaα2,6 was increased in the three cell lines. These observations indicate that DEX can be used for the study of the mechanism of sialylation of cell membrane molecules. Importantly, DEX may change the tropism of avian and human/pig influenza viruses and other infectious agents to animal and human epithelial cells.

Increasingly, attention has focused on improving vaccine production in cells using gene editing technology to specifically modify key virus regulation-related genes to promote virus replication. In this study, we used DIA proteomics analysis technology to compare protein expression differences between two groups of MDCK cells: uninfected and influenza A virus (IAV) H1N1-infected cells 16 h post infection (MOI = 0.01). Initially, 266 differentially expressed proteins were detected after infection, 157 of which were upregulated and 109 were downregulated. We screened these proteins to 23 genes related to antiviral innate immunity regulation based on functional annotation database analysis and verified the mRNA expression of these genes using qPCR. Combining our results with published literature, we focused on the proteins RSAD2, KCNN4, IDO1, and ISG20; we verified their expression using western blot, which was consistent with our proteomics results. Finally, we knocked down RSAD2 using lentiviral shRNA expression vectors and found that RSAD2 inhibition significantly increased IAV NP gene expression, effectively promoting influenza virus replication with no significant effect on cell proliferation. These results indicate that RSAD2 is potentially an effective target for establishing high-yield vaccine MDCK cell lines and will help to fully understand the interaction mechanism between host cells and influenza viruses.

How cells with different genetic makeups compete in tissues is an outstanding question in developmental biology and cancer research. Studies in recent years have revealed that cell competition can either be driven by short-range biochemical signalling or by long-range mechanical stresses in the tissue. To date, cell competition has generally been characterised at the population scale, leaving the single-cell-level mechanisms of competition elusive. Here, we use high time-resolution experimental data to construct a multi-scale agent-based model for epithelial cell competition and use it to gain a conceptual understanding of the cellular factors that governs competition in cell populations within tissues. We find that a key determinant of mechanical competition is the difference in homeostatic density between winners and losers, while differences in growth rates and tissue organisation do not affect competition end result. In contrast, the outcome and kinetics of biochemical competition is strongly influenced by local tissue organisation. Indeed, when loser cells are homogenously mixed with winners at the onset of competition, they are eradicated; however, when they are spatially separated, winner and loser cells coexist for long times. These findings suggest distinct biophysical origins for mechanical and biochemical modes of cell competition.

The estimation of the extent of absorption of drug candidates intended for oral drug delivery is an important selection criteria in drug discovery. The use of cell-based transwell assays examining flux across cell-monolayers (e.g., Caco-2 or MDCK cells) usually provide satisfactory predictions of the extent of absorption in vivo. These predictions often fall short of expection for molecules outside the traditional low molecular weight property space. In this manuscript the transwell permeability assay was modified to circumvent potential issues that can be encountered when evaluating the aforementioned drug molecules. Particularly, the addition of albumin in the acceptor compartment to reduce potential binding to cells and the acceptor compartment, improved the predictive power of the assay. Cellular binding and lysosomal trapping effects are significantly reduced for larger molecules, particularly lipophilic bases under these more physiological conditions, resulting in higher recovery values and a better prediction power. The data indicate that lysosomal trapping does not impact the rate of absorption of lipophilic bases in general but is rather an exception. Finally, compounds believed to permeate by passive mechanisms were used in a calibration curve for the effective prediction of the fraction absorbed of molecules of interest in current medicinal chemistry efforts. [ABSTRACT FROM AUTHOR]

Co-infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other viruses has been reported. We evaluated cell lines commonly used to isolate viruses and diagnose related diseases for their susceptibility to SARS-CoV-2. Although multiple kidney cell lines from monkeys were susceptible to SARS-CoV-2, we found many cell types derived from humans, dogs, minks, cats, mice, and chicken were not. We analyzed MDCK cells, which are most commonly used for surveillance and study of influenza viruses, and found that they were not susceptible to SARS-CoV-2. The low expression level of the angiotensin converting enzyme 2 receptor and lower receptor affinity to SARS-CoV-2 spike, which could be overcome by overexpression of canine angiotensin converting enzyme 2 in trans, strengthened the cellular barrier to productive infection. Moreover, a D614G mutation in the spike protein did not appear to affect SARS-CoV-2 cell tropism. Our findings should help avert inadvertent propagation of SARS-CoV-2 from diagnostic cell lines. [ABSTRACT FROM AUTHOR]

Automated biomimetic systems for the preclinical testing of drugs are of great interest. Here, an in vitro testing platform for in vivo adapted drug absorption studies is presented. It has been designed with a focus on easy handling and the usability of established cell cultivation techniques in standard well plate inserts. The platform consists of a microfluidic device, which accommodates a well plate insert with pre-cultivated cells, and provides a fluid flow with dynamic drug dilution profiles. A low-cost single-board computer with a touchscreen was used as a control unit. This provides a graphical user interface, controls the syringe pump flow rates, and records the transepithelial electrical resistance. It thereby enables automated parallel testing in multiple devices at the same time. To demonstrate functionality, an MDCK cell layer was used as a model for an epithelial barrier for drug permeation testing. This confirms the possibility of performing absorption studies on barrier tissues under conditions close to those in vivo. Therefore, a further reduction in animal experiments can be expected. [ABSTRACT FROM AUTHOR]

Abstract Objective Influenza is an acute respiratory disease caused by the influenza virus which circulates annually in populations of different species. Madin-Darby Canine Kidney (MDCK) is the most widely utilized cell-line for conducting influenza research. However, the infectivity of various influenza strains in MDCK cells is not equivalent and the productivity of viral propagation is also limited. Results We tested the functional adequacy of two MDCK-lineage cell lines, conventional MDCK and MDCK/London, were evaluated by assessing their infectivity of different influenza viral strains with focus forming assays and the cellular toxicity caused by influenza infections by lactate dehydrogenase assay. Moreover, the sensitivity of cells in the presence of the antiviral agent ribavirin was assessed by MTT assay. Our results showed that MDCK/London cells efficiently propagate virus across all influenza viruses tested, are comparable to the utility of Mv1Lu cells, and are superior to conventional MDCK cells in replicating virus as indicated by an increase in virus of three to four logs, particularly in H3N2 infection. Also, the MDCK/London cells were more sensitive to the presence of antiviral drug than conventional MDCK cells. In conclusion, MDCK/London cell line could be a better platform for influenza studies and vaccine development.

Virus replication and host cell growth require host cell metabolic networks to provide energy and precursors for the synthesis of macromolecules. The aim of this study was to investigate the most direct changes in energy metabolism and small-molecule metabolism of Madin–Darby canine kidney (MDCK) cells infected with H3N2 canine influenza virus (CIV) and to determine whether small metabolites contribute to the pathogenesis of CIV. To study the metabolomics of MDCK cells infected with H3N2 CIV, we used liquid chromatography–tandem mass spectrometry combined with multivariate statistical analysis. The results showed that 798 positive ions were detected, among which 33 were upregulated and 11 were downregulated, and 406 negative ions were detected, among which 33 were upregulated and 9 were downregulated. Through Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, we found that these differentially expressed molecules were mainly concentrated in the steroid hormone biosynthesis, amino sugar and nucleotide sugar metabolism, sphingolipid metabolism, vitamin B6 metabolism, cysteine and methionine metabolism, vitamin digestion and absorption, arginine and proline metabolism, biosynthesis of amino acids, and folate biosynthesis metabolic pathways. These pathways are involved in energy metabolism and nucleic acid and protein synthesis, which are essential for virus replication. Our experimental data suggest that H3N2 CIV infection reconstitutes/influences cellular metabolic processes, which in turn may contribute to viral replication. These findings are important for the development of enzyme inhibitors or metabolites for the identification of antiviral drugs. In addition, understanding the metabolic interaction between CIV and host cells is also very important for the complex pathogenicity of CIV, providing certain guidance for the treatment of canine influenza. [ABSTRACT FROM AUTHOR]

It is widely believed that cells, derived from different species or different cell lines, behave differently. However, this study reports that a variety of forms and migration modes in isolated epithelial cells of Madin–Darby Canine Kidney type were observed, although the cells were taken from the same cell line and the experimental conditions were kept constant. To understand the diverse formation processes in such cell behavior, a simple mathematical model, namely the particle-fiber model, was constructed. In this model, a single cell is assumed to be composed of a multiple of particles, interconnected by stress fibers. The particles mimic focal adhesion biding to a substrate. The stress fibers mimic a cytoskeleton, that plays an important role in maintaining the shape and the movement of the cell. Here, a growth process was introduced, which varied the size of the particles and the thickness of the fibers in dependence on the forces exerted on the particles. Simulation of the results showed that various cell shapes can be self-organized even if the parameters, which describe cell properties and their interactions with environment, were kept constant. [ABSTRACT FROM AUTHOR]

Background: Mammalian cell culture-based technology is an innovative technique for influenza vaccine manufacturing that may be a valuable alternative to overcome some of the problems and vulnerabilities associated with conventional egg-based influenza vaccine production. Flucelvax® Quad (Seqirus, Inc.) is the first and only mammalian cell culture-based quadrivalent inactivated, subunit influenza vaccine (IIV4-cc) authorized for adult and pediatric use in Canada. The National Advisory Committee on Immunization (NACI) has not previously made a recommendation on cell culture-based influenza vaccines in any population.Objective: To review the available evidence for the efficacy, effectiveness, immunogenicity, and safety of IIV4-cc, and to summarize the NACI recommendation regarding the use of Flucelvax Quad in Canada in adults and children.Methods: A systematic literature review on the vaccine efficacy, effectiveness, immunogenicity and safety of IIV4-cc in persons four years of age and older was performed. The systematic review's methodology was specified a priori in a written protocol. The NACI evidence-based process was used to assess the quality of eligible studies, summarize and analyze the findings, and develop a recommendation regarding the use of Flucelvax Quad in adults and children. The proposed recommendation was then considered and approved by NACI in light of the available evidence.Results: Thirteen eligible studies were included in the evidence synthesis. In the four observational studies that assessed vaccine effectiveness of IIV4-cc, there were some data indicating potentially improved protection against influenza compared to conventional egg-based quadrivalent inactivated influenza vaccines (IIV4) or trivalent inactivated influenza vaccine (IIV3), particularly against A(H3N2) virus infection. There was also some evidence that IIV4-cc may be more effective than egg-based trivalent or quadrivalent influenza vaccines against non-laboratory confirmed influenza-related outcomes, but there is insufficient evidence for laboratory-confirmed outcomes. Two randomized controlled trials assessed the immunogenicity and safety of IIV4-cc compared with mammalian cell culture-based trivalent inactivated, subunit influenza vaccine (IIV3-cc). The IIV4-cc was well-tolerated and the reported solicited local and systemic adverse events were generally mild to moderate in intensity, self-limited and did not precipitate sequelae. One clinical review of cases and six peer-reviewed randomized controlled trials (four in adults and two in children) that reported on the safety of IIV3-cc were included in the review. The evidence on immunogenicity and safety was consistent across these studies and showed that there was no significant difference in adults and children four years of age and older who had received IIV3-cc or an egg-based IIV3.Conclusion: NACI concluded that there is fair evidence (Grade B Evidence) that Flucelvax Quad is effective, safe, and has non-inferior immunogenicity to comparable vaccines, based on direct evidence in adults and children nine years of age and older. NACI recommends that Flucelvax Quad may be considered among the IIV4 offered to adults and children nine years of age and older (Discretionary NACI Recommendation). [ABSTRACT FROM AUTHOR]

• Immunogenicity and efficacy of the MDCK- and egg-based LAIV against viruses of H5 and H7 subtypes was studied in ferrets. • Immunogenicity of the MDCK-based H5 and H7 LAIV was comparable to that of egg-based LAIV. • MDCK- and egg-based LAIV completely inhibited influenza virus replication in the lower respiratory tract. • Two doses of MDCK-based LAIV protected against challenge with heterologous H5N1 heterologous challenge. During a pandemic, the availability of specific pathogen free chicken eggs is a major bottleneck for up-scaling response to the demand for influenza vaccine. This has led us to explore the use of Madin-Darby Canine Kidney (MDCK) cells for the manufacture of live attenuated influenza vaccine (LAIV) that provides production flexibility and speed. The present study reports the comparison of the immunogenicity and efficacy of two MDCK-based LAIVs against two egg-based LAIVs prepared from the same pandemic potential strains of H5 and H7 subtypes after a single dose of the vaccine followed by a challenge with a homologous wild type strain. The vaccine strains have been generated by classical method of reassortment using the A/Leningrad/134/17/57 master donor strain. Additionally, a prime-boost regimen of the MDCK-based vaccine followed by a challenge with a homologous wild type strain for H5 and H7 immunized ferrets and also a heterologous wild type strain for the H5 immunized animals was studied. No difference in the hemagglutination inhibition and virus neutralization antibody titers against the homologous virus was observed following a single dose of either egg-based or MDCK-based H5 and H7 LAIV vaccine. A second dose of MDCK-based vaccine significantly boosted antibody titers in the vaccinated animals. Both a single dose or two doses of LAIV provided complete protection from lower respiratory tract infection and resulted in a significant reduction in the virus titers recovered from the throat, nasal turbinates and lungs after challenge with the homologous wild type strain. Protection from a challenge with a heterologous strain of H5 was also observed after two doses of the MDCK-based LAIVs. This data strongly supports the use of MDCK as a substrate for the manufacture of LAIV which ensures reliable quality, safety, production flexibility, speed and breadth of protection, features that are highly critical during a pandemic. [ABSTRACT FROM AUTHOR]

Abstract BACKGROUND: Canine kidney injury is characterized by the apoptosis and necrosis of renal tubular epithelial cells. Recent developments in mesenchymal stem cells and their exosomes research have shown great promise for the treatment of kidney injury in humans, rats and mice, but little research has been done on dogs. OBJECTIVE: To investigate the effects of canine adipose-derived mesenchymal stem cells and their exosomes on canine renal tubular epithelial cell injury induced by gentamicin in vitro. METHODS: Canine renal tubular epithelial cells were treated by 5 mmol/L gentamicin sulfate. Subsequently, canine adipose-derived mesenchymal stem cells and their conditional medium and exosomes were co-cultured with damaged canine renal tubular epithelial cells respectively. After 24 and 48 hours, the cell proliferation activity of each group was measured by cell counting kit-8 method, and the apoptosis rate of each group was detected by flow cytometry. Finally, Q-PCR was used to further reveal the effects of canine adipose-derived mesenchymal stem cell exosomes on PCNA, Bcl-2 and Bax genes in these cells. RESULTS AND CONCLUSION: Canine adipose-derived mesenchymal stem cells, their conditioned media, and exosomes could significantly promote proliferation and reduce apoptosis in damaged canine renal tubular epithelial cells (P < 0.05). Among them, canine adipose-derived mesenchymal stem cell exosomes worked best, which could significantly increase the expression of PCNA and Bcl-2 genes in damaged canine renal tubular epithelial cells (P < 0.05). These results suggest that canine adipose-derived mesenchymal stem cells can repair the canine renal tubular epithelial cell damage induced by gentamicin through their exosomes. [ABSTRACT FROM AUTHOR]

In vitro permeability assessment tools, like PAMPA, Caco-2, and MDCK, are frequently used to assess permeability and provide input in to various classification systems. Frequently, the measured recovery values in permeability assays are poor. Poor recovery may be a result of lysosomal trapping of compound. It was hypothesized that a relationship existed between diminished assay recovery of compound due to lysosomal trapping and underestimation of the Papp value. To examine this hypothesis, a series of experiments were conducted measuring cellular accumulation, percent recovery, and permeability in the absence or presence of an inhibitor of the V-type H+-ATPase, bafilomycin A1, to determine if a quantifiable relationship between lysosomal trapping, recovery, and permeability existed. Displacing compounds from lysosomes using bafilomycin A1 resulted in an improved compound recovery in the assay and a corresponding elevated permeability, where for each 10% loss in recovery, a Papp underestimate of ∼2.2 × 10−6 cm/s was observed. The findings highlight the potential for compound misclassification in various classification systems when assay recovery is not considered. Consideration of lysosomal trapping in the context of permeability assays may yield permeability values more reflective of the intrinsic permeability and the appropriate permeability classification. [ABSTRACT FROM AUTHOR]