Control Supernatants Research Articles

In Vitro FVIII Encoding Transgenic Mesenchymal Stem Cells Maintain A Successful Coagulation in FVIII- Deficient Plasma Mimicking Hemophilia A.

Hemophilia A is an X-linked recessive bleeding disorder caused by deficiency of plasma coagulation factor VIII (FVIII), and accounts for about 80-85% of patients with hemophilia. Plasma-derived therapies or recombinant factor VIII concentrates and also FVIII mimicking antibodies are used to prevent and treat the bleeding symptoms. Recently, EMA granted a conditional marketing approval for the first gene therapy of Hemophilia A. Aim of this study was to determine the effectiveness of coagulation in correcting FVIII deficiency with FVIII secreting transgenic mesenchymal stem cells (MSC). A lentiviral vector encoding B-domain-deleted Factor VIII cDNA sequence with CD45 R0 truncated (CD45R0t) surface marker was designed to develop a transgenic FVIII expressing primary cell line by transducing MSC. Efficacy and functionality of secreted FVIII from MSCs was assessed with anti- FVIII ELISA, CD45R0t flow cytometry, FVIII Western Blot and Mixing Test analysis in vitro. Results of this study showed that the transgenic MSCs maintain a persistent FVIII secretion. There was no significant difference in FVIII secretion over time, suggesting stable FVIII expression from the MSCs. Functionality of FVIII protein secreted in MSC supernatant was demonstrated by using Mixing Test in coagulation analysis. In the Mixing Test analysis, FVIIIdeficient human plasma products were mixed either with saline control or FVIII-secreted MSC supernatant. Mean FVIII levels of saline control group were 0.41±0.03 IU/dL whereas mean FVIII levels were 25.41±33.38 IU/dL (p<0.01) in FVIII-secreting MSC supernatant mixed group. Mean activated partial thromboplastin time (aPTT) of saline control group was 92.69±11.38 seconds whereas in FVIII-secreting MSC supernatant mixed group, mean aPTT levels were decreased to 38.60±13.38 seconds (p<0.001). Findings of this in vitro study shows a promising new method applicable to treat Hemophilia A. Accordingly, a FVIII secreting transgenic mesenchymal stem cell study will be initiated in FVIII knock-out animal model.

In Vitro FVIII-Encoding Transgenic Mesenchymal Stem Cells Maintain Successful Coagulation in FVIII-Deficient Plasma Mimicking Hemophilia A

Hemophilia A is an X-linked recessive bleeding disorder caused by a deficiency of plasma coagulation factor VIII (FVIII), and it accounts for about 80%-85% of all cases of hemophilia. Plasma-derived therapies or recombinant FVIII concentrates are used to prevent and treat the bleeding symptoms along with FVIII-mimicking antibodies. Recently, the European Medicines Agency granted conditional marketing approval for the first gene therapy for hemophilia A. The aim of this study was to determine the effectiveness of coagulation in correcting FVIII deficiency with FVIII-secreting transgenic mesenchymal stem cells (MSCs). A lentiviral vector encoding a B domain-deleted FVIII cDNA sequence with CD45R0 truncated (CD45R0t) surface marker was designed to develop a transgenic FVIII-expressing primary cell line by transducing MSCs. The efficacy and functionality of the FVIII secreted from the MSCs was assessed with anti-FVIII ELISA, CD45R0t flow cytometry, FVIII western blot, and mixing test analysis in vitro. The findings of this study showed that the transgenic MSCs maintained persistent FVIII secretion. There was no significant difference in FVIII secretion over time, suggesting stable FVIII expression from the MSCs. The functionality of the FVIII protein secreted in the MSC supernatant was demonstrated by applying a mixing test in coagulation analysis. In the mixing test analysis, FVIII-deficient human plasma products were mixed with either a saline control or FVIII-secreted MSC supernatant. The mean FVIII level of the saline control group was 0.41±0.03 IU/dL, whereas the mean level was 25.41±33.38 IU/dL in the FVIII-secreting MSC supernatant mixed group (p<0.01). The mean activated partial thromboplastin time (aPTT) of the saline control group was 92.69±11.38 s, while in the FVIII-secreting MSC supernatant mixed group, the mean aPTT level decreased to 38.60±13.38 s (p<0.001). The findings of this in vitro study suggest that the new method presented here is promising as a possible treatment for hemophilia A. Accordingly, a study of FVIII-secreting transgenic MSCs will next be initiated in a FVIII-knockout animal model.

Evaluation of corn steep liquor as fermentation media for recombinant Lactococcus lactis producing antifreeze proteins.

Corn processing byproducts corn steep liquor (CSL), and thin stillage were evaluated as growth media for recombinant Lactococcus lactis modified to produce antifreeze proteins (AFPs) that could have important food and non-food applications. The AFP III sequence from ocean pout was cloned into a shuttle vector to make an expression vector that facilitated the production of recombinant AFP III in Lactococcus lactis. Light CSL from yellow dent corn and thin stillage from the industrial corn bioethanol process were optimized as fermentation media with a combination of the following additives and trace elements: disodium-β-glycerophosphate (DG), tryptone (T), ascorbic acid (AA), iron (Fe), zinc (Zn), and magnesium (Mg). The growth of wild-type and recombinant Lactococcus lactis strains were compared over a 72 h period in 96-well plates and 250 mL shake flasks. The corn coproducts media consisting of 50% (v/v) light steep in water supplemented with DG-5g L-1 , T-5g L-1 , AA-0.5g L-1 , and Zn-4ppm resulted in best growth and was considered as the best-optimized media. The addition of additives and trace elements better supported the growth of both wild-type and recombinant Lactococcus lactis strains compared to control media without any additives. Respective fermentation supernatants were frozen to -20 °C, and the time to supercool and freeze was compared. A distinct supercooling effect was observed for the supernatants from recombinant strains thus, extending the time and temperature of supercooling and freezing. The maximum time of supercooling extended was 17.55 ± 4.45 min for thin stillage followed by M17 media (17.25 ± 4.45 min), Kent Corporation CSL (10.80 ± 2.12 min), and yellow dent CSL (6.9 ± 0.85 min) when fermented with recombinant Lactococcus lactis strains. The supplemented corn coproduct-based media enhanced the growth of both wild-type and recombinant Lactococcus lactis strains. These optimized media can replace or supplement more expensive media (e.g. M17), potentially reducing cost. The fermentation supernatants exhibited longer times to supercool, and freeze compared to control supernatants, indicating potential use as antifreeze compounds in frozen food and non-food applications. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Mesenchymal stromal cells pretreated with proinflammatory cytokines enhance skin wound healing via IL-6-dependent M2 polarization

BackgroundNumerous studies have shown that mesenchymal stromal cells (MSCs) promote cutaneous wound healing via paracrine signaling. Our previous study found that the secretome of MSCs was significantly amplified by treatment with IFN-γ and TNF-α (IT). It has been known that macrophages are involved in the initiation and termination of inflammation, secretion of growth factors, phagocytosis, cell proliferation, and collagen deposition in wound, which is the key factor during wound healing. In this study, we aim to test whether the supernatant of MSCs pretreated with IT (S-IT MSCs) possesses a more pronounced effect on improving wound healing and describe the interplay between S-IT MSCs and macrophages as well as the potential mechanism in skin wound healing.MethodsIn the present study, we used a unique supernatant of MSCs from human umbilical cord-derived MSCs (UC-MSCs) pretreated with IT, designated S-IT MSCs, subcutaneously injected into a mice total skin excision. We evaluated the effect of S-IT MSCs on the speed and quality of wound repair via IT MSCs-derived IL-6-dependent M2 polarization in vivo by hematoxylin–eosin staining (H&E), immunohistochemistry (IHC), immunofluorescence (IF), Masson’s trichrome staining, Sirius red staining, quantitative real-time PCR (qPCR). In addition, the effect of S-IT MSCs on the polarization of macrophages toward M2 phenotype and the potential mechanism of it were also investigated in vitro by flow cytometry (FCM), enzyme-linked immunosorbent assay (ELISA), tube formation assay, and western blot analysis.ResultsCompared with control supernatant (S-MSCs), our H&E and IF results showed that S-IT MSCs were more effectively in promoting macrophages convert to the M2 phenotype and enhancing phagocytosis of M2 macrophages. Meanwhile, the results of tube formation assay, IHC, Masson’s trichrome staining, Sirius red staining showed that the abilities of M2 phenotype to promote vascularization and collagen deposition were significantly enhanced by S-IT MSCs-treated, thereby accelerating higher quality wound healing. Further, our ELISA, FCM, qPCR and western blot results showed that IL-6 was highly enriched in S-IT MSCs and acted as a key regulator to induce macrophages convert to the M2 phenotype through IL-6-dependent signaling pathways, ultimately achieving the above function of promoting wound repair.ConclusionsThese findings provide the first evidence that the S-IT MSCs is more capable of eliciting M2 polarization of macrophages via IL-6-dependent signaling pathways and accelerating wound healing, which may represent a new strategy for optimizing the therapeutic effect of MSCs on wound healing.

Mesenchymal stem cells pretreated with proinflammatory cytokines accelerate skin wound healing by promoting macrophages migration and M2 polarization.

IntroductionNumerous studies have shown that mesenchymal stem cells (MSCs) promote cutaneous wound healing via paracrine signaling. Our previous study found that the secretome of MSCs was significantly amplified by treatment with IFN-γ and TNF-α (IT). It has been known that macrophages are involved in the initiation and termination of inflammation, secretion of growth factors, phagocytosis, cell proliferation and collagen deposition in wound, which is the key factor during wound healing. In the present study, we used a unique supernatant of MSCs from human umbilical cord-derived MSCs (UC-MSCs) pretreated with IT, designated S-IT MSCs, to explore whether S-IT MSCs have a better effect on improving wound healing by improving the biological function of macrophages than the control supernatant of MSCs (S-MSCs).MethodsIn the present study, we used a unique supernatant of MSCs pretreated with IT subcutaneously injected into a mice total skin excision. We evaluated the effect of S-IT MSCs on wound healing and the quality of wound repair via promoting macrophages migration and M2 polarization in vivo. In addition, the effect of S-IT MSCs on macrophages migration, converting toward M2 phenotype and phagocytosis were also investigated in vitro.ResultsIndeed, S-IT MSCs were found to be more potent in promoting macrophage migration, M2 polarization, phagocytosis, and promoting wound closure than S-MSCs during the wound repair. High levels of CCL2 and IL-6 were found in S-IT MSCs, which indicated that the optimization of macrophage function by S-IT MSCs may be achieved through their high expression of CCL2 and IL-6.ConclusionsOur results suggest that the beneficial paracrine effect of MSCs on wound healing can be amplified by pretreatment with IT, which may represent a new strategy for optimizing the therapeutic effect of MSCs on wound healing.

An Immunological Axis Involving Interleukin 1β and Leucine-Rich-α2-Glycoprotein Reflects Therapeutic Response of Children with Kawasaki Disease: Implications from the KAWAKINRA Trial

PurposeA recent phase II open-label study of the interleukin 1 (IL-1) receptor antagonist (IL-1Ra) anakinra in treating IVIG-resistant Kawasaki disease (KD) patients reported promising results. Here, we aimed to characterize the immunological impact of IL-1 blockade in this unique study population.MethodsPatients’ and control sera and supernatants of cells (whole blood, neutrophils, coronary artery endothelial cells) stimulated with recombinant IL-1β were analyzed for single or multiple marker (n = 22) expression by ELISA or multiplexed bead array assay. Data were analyzed using unsupervised hierarchical clustering, multiple correlation, and multi-comparison statistics and were compared to retrospective analyses of KD transcriptomics.ResultsInflammation in IVIG-resistant KD (n = 16) is hallmarked by over-expression of innate immune mediators (particularly IL-6 > CXCL10 > S100A12 > IL-1Ra). Those as well as levels of immune or endothelial cell activation markers (sICAM-1, sVCAM-1) declined most significantly in course of anakinra treatment. Prior as well as following IL-1R blockade, over-expression of leucine-rich-α2-glycoprotein 1 (LRG1) associated best with remnant inflammatory activity and the necessity to escalate anakinra dosage and separated inflammatory KD patients from sJIA-MAS (n = 13) and MIS-C (n = 4). Protein as well as retrospective gene expression analyses indicated tight association of LRG1 with IL-1β signaling and neutrophilia, while particularly neutrophil stimulation with recombinant IL-1β resulted in concentration-dependent LRG1 release.ConclusionOur study identifies LRG1 as known trigger of endothelial activation and cardiac re-modeling to associate with IL-1β signaling in KD. Besides a potential patho-mechanistic implication of these findings, our data suggest blood leukocyte and neutrophil counts to best predict response to IL-1Ra treatment in IVIG-resistant KD.

SHED-derived exosomes improve the repair capacity and osteogenesis potential of hPDLCs.

Exosomes secreted by stem cells are recognized as a critical component in tissue regeneration during stem cell-based therapy. Considering the limited sources and bone regeneration efficiency of human periodontal ligament cells (hPDLCs), we explored whether exosomes secreted by stem cells from human exfoliated deciduous teeth (SHED-exo) could improve the pluripotency and regenerative potential of hPDLCs. In hPDLCs, cell proliferation, migration, cell cycle, apoptosis, and osteogenic differentiation were detected after cells were exposed to SHED-exo (SHED-exo group), blank (control group), or control supernatant without exo (Csup group), via CCK-8, scratch analysis, flow cytometric, real-time PCR, and so on. Exosomes sequencing was performed to compare and analyze miRNAs contented in SHED-exo and hPDLC-exo. As compared to control or Csup, SHED-exo significantly increased migration, apoptosis, and proliferation, promoted cell cycle transition from G1 to S phase in hPDLCs, and enhanced Runx2 expression and mineralization. In addition, it may be explained by the significant differences in miRNA contented in SHED-exo and hPDLC-exo. Exosomes from SHED can improve cell proliferation, migration, cell cycle, apoptosis, and osteogenic differentiation of hPDLCs, which highlights the therapeutic value of this bioactive component in the regeneration of periodontal tissues using hPDLCs in clinical practice.

Effect of Dairy Pond Sludge/Supernatant Application on Ryegrass Dry Matter Yield and Phosphorus Fractions in Soil

Pasture yield in dairy grazing systems is critical to supplying sufficient feed for milking cows and maintaining productivity. In the Australian dairy industry, ryegrass and clover are common grasses used in grazed pastures. Dairy shed effluent (DSE), the wastewater produced from washing down the dairy holding yards during and after milking, is generally managed through application to pasture as a fertilizer substitute/supplement following partial treatment in stabilization ponds. The aim of this study is to assess the benefits of applying sludge and supernatant collected from two-stage DSE pond systems to ryegrass pasture. A pot experiment was conducted which involved applying pond sludges and supernatant to soil seeded with ryegrass. The application rates of the pond by-products were set according to their labile (plant available) phosphorus content. Ryegrass yield and leachate generated from each of the pots were recorded, and samples were collected for analysis of nutrients and other parameters. The ryegrass grown in soil treated with pond sludge and supernatant yielded greater dry matter (DM) with higher nutrient content than untreated control pots. In addition, pots treated with pond sludge exhibited lower rates of phosphorus leaching from the soil compared with pots treated with supernatant. Thus, pond sludge retained more plant available phosphorus in soil than both the control and pond supernatant treatment. The potassium to calcium/magnesium ratios in the ryegrass in the pots treated with pond sludge and supernatant were below the recommended upper limit for grazing. Therefore, the application of pond sludges on the dairy paddocks was found to be superior to applying supernatant in terms of utilization and conservation of phosphorus within the dairy farm and presents low risks of groundwater pollution and grass tetany.

A234 EVALUATING THE EFFECT OF STOOL SUPERNATANTS FROM IBS PATIENTS AND HEALTHY CONTROLS ON THE EXCITABILITY OF DRG NEURONS

Abstract Background Abdominal pain is commonly described in chronic disorders such as irritable bowel syndrome (IBS), but the underlying mechanisms are currently unclear. The stool metabolomic and microbiota profiles of IBS and healthy patients have shown distinct differences. Additionally, IBS stool supernatants have previously been demonstrated to induce hypersensitivity of nociceptive nerves in the ex vivo mouse colon, suggesting that mediators in the stool can sensitize nociceptors. However, the effects of healthy control (HC) or IBS patient stool supernatants on the excitability of DRG neurons have not been clarified. Aims To evaluate the effect of HC and IBS supernatant on DRG neurons. Methods HC (n=8 patients) or IBS (n=10 patients) stool was collected, dissolved and homogenized with bicarbonate-buffered Krebs solution at 37°C in a 1/10 dilution. DRG neurons from C57BL/6 mice were dissociated and incubated overnight with HC or IBS supernatant in a Krebs dissolution. Changes in DRG neuronal excitability were recorded using perforated patch-clamp techniques to measure the rheobase (amount of current needed to elicit an action potential). The effect of the IBS and HC stool supernatants on the resting membrane potential (RMP) was also recorded. Results Overnight incubations with supernatant of HC stool diluted in Krebs solution (n=28 neurons) did not significantly decrease the rheobase compared to control neurons (n=22) (62.7 ± 3.9 pA vs 64.2 ± 2.7 pA). In a parallel experiment, we evaluated the effect of IBS stool supernatants diluted in Krebs (n=52 neurons) and found that they significantly decreased the rheobase compared to the supernatant of HC diluted in Krebs and control neurons (52.3 ± 2.3; p&amp;lt;0.05). The data were analyzed with a one-way ANOVA and Tukey’s test. Incubations with IBS supernatant decreased the RMP compared to HC supernatant (-42.6 ± 0.6 mV vs. -46.0 ± 0.9 mV; p&amp;lt;0.01), which was calculated with an unpaired t-test. Conclusions These findings suggest that mediators in IBS stool increase the excitability of DRG neurons compared to HC stool supernatant, and thus may contribute to pain signaling in IBS patients. Funding Agencies CIHR

Effects of vitamin D-induced supernatant of placental explants from preeclamptic women on oxidative stress and nitric oxide bioavailability in human umbilical vein endothelial cells.

The study evaluated the effect of the supernatant of placental explants from preeclamptic (PE) and normotensive (NT) pregnant women after tissue treatment with or without vitamin D (VD) on oxidative stress and nitric oxide (NO) bioavailability in human umbilical vein endothelial cells (HUVEC). Placental explants were prepared from eight NT and eight PE women, and supernatants were obtained after incubation with or without hydrogen peroxide (H2O2) and/or VD. HUVEC were cultured for 24 h with supernatants, and the following parameters were analyzed in HUVEC cultures: NO, nitrate (NO3 -), and nitrite (NO2 -) levels, lipid peroxidation, and intracellular reactive oxygen species (ROS). Results showed that the production of NO3 -, NO2 -, malondialdehyde (MDA), and ROS were significantly higher in HUVEC treated with explant supernatant from PE compared to NT pregnant women, while the supernatant of PE explants treated with VD led to a decrease in these parameters. A significantly high production of NO was detected in HUVEC cultured with control supernatant of NT group, and in cultures treated with supernatant of PE explants treated with VD. Taken together, these results demonstrated that cultures of placental explants from PE women with VD treatment generated a supernatant that decreased oxidative stress and increased the bioavailability of NO in endothelial cells.

Expression profile of macrophage migration inhibitory factor in periodontitis

ObjectiveMacrophage migration inhibitory factor (MIF) is a pivotal mediator of host innate immunity and influences the development of several inflammatory diseases. The role of MIF in periodontitis is unclear. MethodsEighteen periodontally healthy volunteers and 18 patients with stage III or IV periodontitis were enrolled. Blood samples and gingival tissues were collected from all individuals. The serum concentrations of MIF and MCP-1 were measured by ELISA. The protein and mRNA levels of MIF and MCP-1 in gingival tissue were evaluated by immunohistochemical staining and quantitative PCR. The levels of secreted MIF and MCP-1, as well as their mRNA levels, were determined by ELISA and quantitative PCR in oral epithelial cells infected with Porphyromonas gingivalis. ResultsAfter adjusting for age, the level of MCP-1 was significantly higher in the serum and gingival tissue of periodontitis patients, as well as in infected epithelial cells. The serum concentration of MIF was increased in periodontitis patients (15.25 ± 2.16 ng/mL, P < 0.05) compared to healthy controls (10.43 ± 1.02 ng/mL). Increased MIF immunoreactivity was found in gingival epithelial tissue but not in the gingival connective tissue of periodontitis patients. The secretion of MIF was 3.82-fold higher in the supernatant of infected cells than in the supernatant of control (P < 0.01). No increase in the MIF mRNA level was found in either gingival tissue or epithelial cells. ConclusionsBased on our limited evidence, we showed the level of MIF was related to periodontal conditions. P. gingivalis may contribute to the development and progression of periodontitis through MIF.

Mesenchymal stromal cells pretreated with pro-inflammatory cytokines promote skin wound healing through VEGFC-mediated angiogenesis.

Skin is the largest organ of the human body. Skin wound is one of the most common forms of wound. Mesenchymal stromal cells (MSCs) have been used to aid skin wound healing via their paracrine factors. Because the secretome of MSCs can be greatly enriched and amplified by treatment with IFN‐γ and TNF‐α (IT), we here tested whether supernatant derived from MSCs pretreated with IT, designated as S‐MSCs‐IT, possesses improved wound healing effect by using a murine model of cutaneous excision, S‐MSCs‐IT was found to be more potent in promoting angiogenesis, constricting collagen deposition and accelerating wound closure than control supernatant (S‐MSCs) during the healing of skin wound. VEGFC, but not VEGFA, was greatly upregulated by IT and was found to be a key factor in mediating the improved wound healing effect of S‐MSCs‐IT. Our results indicate that the beneficial paracrine effect of MSCs on wound healing can be enhanced by pretreatment with inflammatory cytokines. IT treatment may represent a new strategy for optimizing the therapeutic effect of MSCs on skin injuries.

Tocotrienols Modulate Breast Cancer Secretomes and Affect Cancer-Signaling Pathways in MDA-MB-231 Cells: A Label-Free Quantitative Proteomic Analysis

Tocotrienols (T3), a family of vitamin E, are reported to possess potent anti-cancer effects but the molecular mechanisms behind these effects still remain unclear. The aim of this study was to investigate how T3 exert anti-cancer effects on MDA-MB-231 human breast cancer cells. The MDA-MB-231 cells were chosen for this study as they are triple-negative and highly metastatic cells, which form aggressive tumors in experimental models. The MDA-MB-231 cells were treated with varying concentrations (0–20 µg mL−1) of gamma (γ) or delta (δ) T3 and the secretome profiles of these cells treated with half maximal inhibitory concentration (IC50) of γT3 (5.8 µg mL−1) or δT3 (4.0 µg mL−1) were determined using label-free quantitative proteomic strategy. A total of 103, 174 and 141 proteins were identified with ProteinLynx Global Server (PLGS) score of more than 200 and above 25% sequence coverage in the untreated control and T3-treated cell culture supernatant respectively. A total of 18 proteins were dysregulated between untreated control and T3 (δT3 or γT3) treated conditions. The results showed that T3 treatment downregulated the exogenous Cathepsin D and Serpine1 proteins but upregulated Profilin-1 protein, which play a key role in breast cancer in the MDA-MB-231 cells. These findings strongly suggest that T3 may induce differential expression of secreted proteins involved in the cytoskeletal regulation of RHO GTPase signaling pathway.

A304 FOOD ANTIGEN-STRESS INTERACTION INCREASES PERIPHERAL PAIN SIGNALING IN A MOUSE MODEL OF IBS

Abstract Background Food and stress are common triggers of symptoms in irritable bowel syndrome (IBS). However, it is unknown whether a specific food antigen alone can induce symptoms such as abdominal pain or if a second trigger, such as stress, is required for a food antigen to increase pain signaling. Aims To determine if a food antigen (ovalbumin) can induce hyperexcitabiity in nociceptive neurons alone and/or in combination with stress. Methods Mice were exposed to water avoidance stress (WAS; 1 hr for 6 days). On day 2, mice were gavaged ovalbumin (20mg) daily after completing WAS (WAS/OVA). These were compared to 3 groups: no WAS and no ovalbumin (control); WAS but no ovalbumin (WAS); and ovalbumin (OVA) alone. On day 8, WAS, OVA and WAS/OVA mice were given ovalbumin (50 μg) subcutaneously; 3 hours later euthanized. Colons were removed and incubated with ovalbumin (100 μg/ml) for 4 hrs. Supernatants were collected and DRG neurons from control mice were incubated overnight with supernatants from each of the four groups. Changes in neuronal excitability were examined by measuring the rheobase (minimal current to evoke an action potential) using perforated patch clamp recording techniques. Stress effects on intestinal permeability in the ileum and colon were assessed in Ussing chambers. One way ANOVA and Bonferroni post hoc test or unpaired t test were used to analyze the data. Results Incubation with supernatants obtained from WAS/OVA mice evoked hyperexcitability in DRG neurons compared to incubation with control supernatant (rheobase: control = 82 ± 10 pA vs WAS/OVA = 56 ± 4 pA, p &amp;lt; 0.05). Similarly, WAS/OVA supernatant decreased the rheobase compared to both OVA mice (OVA = 80 ± 8 pA, p&amp;lt;0.05) and WAS mice (WAS = 81 ± 9 pA, p&amp;lt;0.05). The effect of supernatants from OVA mice and WAS mice did not differ from controls. In a separate series of experiments, the PAR2 antagonist GB83 inhibited the effect of WAS/OVA supernatant (p &amp;lt; 0.01). There was no effect of GB83 on WAS or OVA supernatants. Stress decreased tissue resistance in ileum (p&amp;lt;0.05) but not in colon. Conclusions The food antigen ovalbumin induced hyperexcitability in DRG nociceptive neurons only when combined with stress. This action was PAR2 dependent suggesting a role of tissue proteases. Stress may cause a loss of oral tolerance to ovalbumin by increasing mucosal permeability in the small intestine. The interaction of food antigens and stress may be a mechanism of meal induced increase in abdominal pain in IBS. Funding Agencies CIHR

Decreased Expression of Thrombomodulin in Endothelial Cells by Fibroblast Growth Factor-23/α-Klotho.

Chronic kidney disease (CKD) has been known to be a state of excessive fibroblast growth factor-23 (FGF23) and α-Klotho deficiency. Patients undergoing hemodialysis have an increased mortality risk associated with cardiovascular disease and endothelial dysfunction. The mechanism responsible for the relationship of FGF23 to endothelial damage in these patients has been unclear. On the other hands, increasing evidences have demonstrated that thrombomodulin (TM) plays an important role in the endothelial barrier. Here, we report the suppression of membrane TM, in a dose-dependent manner, in human umbilical vein endothelial cells after FGF23 and FGF23/α-Klotho stimulation. In addition, the levels of soluble TM, which reflect endothelial cell injury, were much higher in cell supernatants after FGF23 and FGF23/α-Klotho stimulation than in the control supernatant. This study indicates a possible mechanism by which excessive levels of FGF23 are involved in endothelial TM disruption, which has been implicated as a potential cardiovascular risk factor in patients with CKD, especially in HD patients.

Systematic Analysis of Cell-Type Differences in the Epithelial Secretome Reveals Insights into the Pathogenesis of Respiratory Syncytial Virus-Induced Lower Respiratory Tract Infections.

Lower respiratory tract infections from respiratory syncytial virus (RSV) are due, in part, to secreted signals from lower airway cells that modify the immune response and trigger airway remodeling. To understand this process, we applied an unbiased quantitative proteomics analysis of the RSV-induced epithelial secretory response in cells representative of the trachea versus small airway bronchiolar cells. A workflow was established using telomerase-immortalized human epithelial cells that revealed highly reproducible cell type-specific differences in secreted proteins and nanoparticles (exosomes). Approximately one third of secretome proteins are exosomal; the remainder are from lysosomal and vacuolar compartments. We applied this workflow to three independently derived primary human cultures from trachea versus bronchioles. A total of 577 differentially expressed proteins from control supernatants and 966 differentially expressed proteins from RSV-infected cell supernatants were identified at a 1% false discovery rate. Fifteen proteins unique to RSV-infected primary human cultures from trachea were regulated by epithelial-specific ets homologous factor. A total of 106 proteins unique to RSV-infected human small airway epithelial cells was regulated by the transcription factor NF-κB. In this latter group, we validated the differential expression of CCL20/macrophage-inducible protein 3α, thymic stromal lymphopoietin, and CCL3-like 1 because of their roles in Th2 polarization. CCL20/macrophage-inducible protein 3α was the most active mucin-inducing factor in the RSV-infected human small airway epithelial cell secretome and was differentially expressed in smaller airways in a mouse model of RSV infection. These studies provide insights into the complexity of innate responses and regional differences in the epithelial secretome participating in RSV lower respiratory tract infection-induced airway remodeling.

Action mechanisms involved in the bioprotective effect of Lactobacillus harbinensis K.V9.3.1.Np against Yarrowia lipolytica in fermented milk

The use of lactic acid bacteria (LAB) as bioprotective cultures can be an alternative to chemical preservatives or antibiotic to prevent fungal spoilage in dairy products. Among antifungal LAB, Lactobacillus harbinensis K.V9.3.1Np showed a remarkable antifungal activity for the bioprotection of fermented milk without modifying their organoleptic properties (Delavenne et al., 2015). The aim of the present study was to elucidate the action mechanism of this bioprotective strain against the spoilage yeast Yarrowia lipolytica. To do so, yeast viability, membrane potential, intracellular pH (pHi) and reactive oxygen species (ROS) production were assessed using flow cytometry analyses after 3, 6 and 10days incubation in cell-free supernatants. The tested supernatants were obtained after milk fermentation with yogurt starter cultures either in co-culture with L. harbinensis K.V9.3.1Np (active supernatant) or not (control supernatant). Scanning-electron microscopy (SEM) was used to monitor yeast cell morphology and 9 known antifungal organic acids were quantified in both yogurt supernatants using high-performance liquid chromatograph (HPLC). Yeast growth occurred within 3days incubation in control supernatant, while it was prevented for up to 10days by the active supernatant. Interestingly, between 66 and 99% of yeast cells were under a viable but non-cultivable (VNC) state despite an absence of membrane integrity loss. While ROS production was not increased in active supernatant, cell physiological changes including membrane depolarization and pHi decrease were highlighted. Moreover, morphological changes including membrane collapsing and cell lysis were observed. These effects could be attributed to the synergistic action of organic acids. Indeed, among the 8 organic acids quantified in active supernatant, five of them (acetic, lactic, 2-pyrrolidone-5-carboxylic, hexanoic and 2-hydroxybenzoic acids) were at significantly higher concentrations in the active supernatant than in the control one. In conclusion, this study has provided new information on the physiological mechanisms induced by an antifungal LAB that could be used as part of the hurdle technology to prevent fungal spoilage in dairy products.

Effects of zinc supplementation on Shiga toxin 2e-producing Escherichia coli in vitro.

Swine edema disease is caused by Shiga toxin (Stx) 2e–producing Escherichia coli (STEC). Addition of highly concentrated zinc formulations to feed has been used to treat and prevent the disease, but the mechanism of the beneficial effect is unknown. The purpose of the present study was to investigate the effects of highly concentrated zinc formulations on bacterial growth, hemolysin production, and an Stx2e release by STEC in vitro. STEC strain MVH269 isolated from a piglet with edema disease was cultured with zinc oxide (ZnO) or with zinc carbonate (ZnCO3), each at up to 3,000 ppm. There was no effect of zinc addition on bacterial growth. Nonetheless, the cytotoxic activity of Stx2e released into the supernatant was significantly attenuated in the zinc-supplemented media compared to that in the control, with the 50% cytotoxic dose values of 163.2 ± 12.7, 211.6 ± 33.1 and 659.9 ± 84.2 after 24 hr of growth in the presence of ZnO, ZnCO3, or no supplemental zinc, respectively. The hemolytic zones around colonies grown on sheep blood agar supplemented with zinc were significantly smaller than those of colonies grown on control agar. Similarly, hemoglobin absorbance after exposure to the supernatants of STEC cultures incubated in sheep blood broth supplemented with zinc was significantly lower than that resulting from exposure to the control supernatant. These in vitro findings indicated that zinc formulations directly impair the factors associated with the virulence of STEC, suggesting a mechanism by which zinc supplementation prevents swine edema disease.

MFHAS1 promotes colorectal cancer progress by regulating polarization of tumor-associated macrophages via STAT6 signaling pathway.

Malignant fibrous histiocytoma amplified sequence 1 (MFHAS1) is a predicted oncoprotein that demonstrates tumorigenic activity in vivo; however, the mechanisms involved are unknown. Macrophages are divided into the pro-inflammatory M1 and anti-inflammatory/protumoral M2 subtypes. Tumor cells can induce M2 polarization of tumor-associated macrophages (TAMs) to promote metastasis; but the underlying pathways require to be elucidated. In this study, we detected a positive association between MFHAS1 expression in TAMs and human colorectal cancer (CRC) TNM stage. Supernatant of CT26 murine CRC cells induced MFHAS1 expression in RAW264.7 murine macrophages. Additionally, CT26 supernatant induced the M2 marker CD206 and activated the pro-M2 STAT6 and KLF4 signaling in control but not MFHAS1-silenced RAW264.7 macrophages. Moreover, supernatant of control, but not MFHAS1-silenced macrophages promoted CT26 cell proliferation, migration and epithelial-mesenchymal transition. Compared with control macrophages, MFHAS1-silenced macrophages showed significantly reduced protumoral effects in vivo. Together, these results suggested that CRC cells induce M2 polarization of TAMs through MFHAS1 induction and subsequent STAT6 and KLF4 activation to promote CRC progress. Finally, similar to CT26 supernatant stimulation, peroxisome proliferator-activated receptor-γ (PPARγ) activation by rosiglitazone induced M2 polarization of RAW264.7 macrophages through MFHAS1-dependent pathway. Our results highlight the role of MFHAS1 as a regulator of macrophages polarization and CRC progress.

Supernatants of irritable bowel syndrome mucosal biopsies impair human colonic smooth muscle contractility.

Changes in intestinal motility are likely to contribute to irritable bowel syndrome (IBS) pathophysiology. The aim of the study was to investigate the effects of IBS mucosal supernatants on human colonic muscle contractility. Supernatants were obtained from biopsies of 18 IBS patients-nine with constipation (IBS-C) and nine with diarrhea-predominant IBS (IBS-D)-and nine asymptomatic subjects, used as controls. Colonic circular smooth muscle strips or isolated cells (SMC) were exposed to control or IBS supernatants. Spontaneous phasic contractions on strips and morphofunctional parameters on cells were evaluated in basal conditions and in response to acetylcholine (Ach). Incubation with IBS supernatants was also conducted in the presence of antagonists and inhibitors (namely histamine, protease and prostaglandin antagonists, nuclear factor-kappa B inhibitor, catalase, NADPH oxidase inhibitor, and the cAMP- and/or cGMP-cyclase inhibitors). Exposure to IBS-C and IBS-D supernatants induced a significant reduction in basal tone and Ach-elicited contraction of muscle strips and a significant shortening and impairment of Ach contraction of SMCs. The NADPH oxidase inhibitor prevented the effect of supernatants, while the protease antagonist only IBS-C effect. No effect was observed with the other antagonists and inhibitors. Dilution of IBS-D supernatants partially restored the effects only on SMCs, whereas dilution of IBS-C supernatants significantly reverted the effects on muscle strips and Ach-elicited response on SMC. Supernatants from mucosal biopsies of IBS patients reduce colonic contractility. The observed impairment was concentration dependent, likely occurring through intracellular oxidative stress damage, involving different neuromotor mechanisms depending on the IBS subtype.