Concentrated Supernatant Research Articles

Vitamin K2 Suppresses Proliferation and Inflammatory Cytokine Production in Mitogen-Activated Lymphocytes of Atopic Dermatitis Patients through the Inhibition of Mitogen-Activated Protein Kinases.

Vitamin K2 is suggested to have a suppressive effect on the peripheral blood mononuclear cells (PBMCs) of pediatric atopic dermatitis patients. We examined the molecular targets of vitamin K2 to suppress proliferation and cytokine production in T-cell mitogen-activated PBMCs of atopic dermatitis patients from the viewpoint of mitogen-activated protein kinase signaling molecules. The study population included 16 pediatric vitamin K2 patients and 21 healthy subjects. The effect of vitamin K2 on concanavalin A-activated PBMC proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and cell counting assays. T-helper (Th)1/Th2/Th17 cytokine profiles in plasma and PBMC-culture supernatants were analyzed by a cytometric beads array assay. Mitogen-activated protein kinase signaling molecules in concanavalin A-activated PBMCs were examined by enzyme-linked immunosorbent assay (ELISA) assays. At 10-100 µM, vitamin K2 significantly suppressed the proliferation of mitogen-activated PBMCs derived from atopic dermatitis patients and healthy subjects (p < 0.05). The interleukin (IL)-10 concentrations in plasma and the PBMC culture supernatants of atopic dermatitis patients were significantly higher than those of healthy subjects (p < 0.05). The IL-2 concentrations in the culture supernatants of atopic dermatitis PBMCs were significantly lower than those of healthy PBMCs (p < 0.05). Vitamin K2 significantly inhibited the IL-17A, IL-10, and tumor necrosis factor α (TNF-α) production (p < 0.05), and increased the IL-2 production (p < 0.01) in the culture supernatant of atopic dermatitis PBMCs. At 10-100 µM, vitamin K2 markedly decreased the of Mek1, extracellular signal-regulated kinases (ERK)1/2 mitogen-activated protein kinase, and SAPK/c-Jun N-terminal kinase (JNK) expression in atopic dermatitis PBMCs (p < 0.05). Vitamin K2 is suggested to attenuate activated T-cell immunity in atopic dermatitis patients through the inhibition of mitogen-activated protein kinase-Mek1-ERK1/2 and SAPK/JNK signaling pathways.

The potential neuroprotective roles of olive leaf extract in an epilepsy rat model induced by kainic acid.

Background and purpose:Epilepsy is recognized as a chronic neurologic disease. Increasing evidence has addressed the antioxidant and anti-inflammatory roles of olive leaf extract (OLE) in neurodegenerative diseases. So, the current study aimed to investigate the neuroprotective roles of OLE in epilepsy.Experimental approach:Forty rats were divided into 4 groups including a control group, sham group, kainic acid (KA) group, and KA + OLE group. KA (4 μg/rat) was injected intrahippocampal, and OLE (300 mg/kg) was orally administrated for 4 weeks. Animals were sacrificed, and their hippocampi were isolated. KA- induced seizure activity was recorded. Oxidative stress index was assessed by measuring its indicators including malondialdehyde (MDA), nitrite, nitrate, and glutathione (GSH) as well as the catalase (CAT) activity. The supernatant concentration of tumor necrosis factor-α (TNF-α) and the apoptosis rate in neurons were measured.Findings/Results:Treatment with OLE significantly reduced the seizure score. OLE decreased oxidative stress index by reducing the concentration of MDA, nitrite, and nitrate as well as increasing the level of GSH. OLE had a significant anti-apoptotic effect on neurons. However, CAT activity and the level of TNF-α were not affected.Conclusion and implications:Our findings indicated neuroprotective properties of OLE, which is mainly mediated by its antioxidant and anti-apoptotic effects, therefore, could be considered as a valuable therapeutic supplement for epilepsy.

Release of carboxylic acids into the exometabolome during anaerobic growth of a denitrifying bacterium with single substrates or crude oil

Anaerobic biodegradation of hydrocarbons mainly related to petroleum can be observed in numerous natural and anthropogenically overprinted environments. While microbiological and chemical tracers are used to study the development of such habitats, not much is known about the impact of particular bacteria on the system. Therefore, we used Aromatoleum sp. HxN1, an n-alkane-utilizing betaproteobacterium capable of degrading crude oil constituents under strictly anoxic conditions, as a model organism to examine its potential to release carboxylic acid metabolites into the surrounding environment. We conducted cultivation experiments with pure substrates, as well as with crude oil, and demonstrated that intermediates of catabolic pathways were highly abundant in the exometabolome. Strikingly, the metabolites found in highest concentrations in the cell-free culture supernatant of strain HxN1 grown with crude oil were associated with the transformation of potentially toxic aromatic hydrocarbons, which do not support growth of this particular bacterium. Additionally, a deficiency in coenzyme A caused by a highly productive substrate transformation may induce the excretion of a surplus of metabolites in order to regain coenzyme A. Under natural conditions, i.e., in the presence of mixed communities, such transformation products may be further degraded by other organisms, ultimately leading to mineralization.

Coordination Polymers Integrating Metalloimmunology with Immune Modulation to Elicit Robust Cancer Chemoimmunotherapy

Nanoscale coordination polymers (NCPs) constructed by metal ions and organic ligands via metalligand bonds have attracted great attention for their biomedical application. Herein, a new type of NCP...

Relationship between children physical activity, inflammatory mediators and lymphocyte activation: possible impact of social isolation (COVID-19).

ObjectiveLifestyle and body composition may be simultaneously responsible for immune response modulation. This study aimed to compare plasmatic adipokines concentration and lymphocyte cytokine production in children with different daily steps (DS) range, as well as to discuss the potential negative impact of the social isolation during COVID-19 pandemic in this context. DS can be a useful and low-cost way of monitoring children’s health status. Study designFifty children were classified into clusters based in DS measured by pedometer: Sedentary Group (DS = 9338 ± 902 steps) and Active Group (DS = 13,614 ± 1003 steps). Plasma and lymphocytes were isolated and cultured to evaluate cytokine production.ResultsSedentary group presented lower adiponectin (7573 ± 232 pg/mL), higher leptin (16,250 ± 1825 pg/mL) plasma concentration, and higher lymphocyte production of IL-17, IFN-gamma, TNF-, IL-2 in relation to active group, suggesting predominance of Th1 response. Otherwise, the active group presented higher lymphocyte supernatant concentration of IL-10 and higher regulatory T cell (Treg) percentage. ConclusionThese results indicate that lymphocytes of children performing higher DS have an anti-inflammatory profile, especially of Treg. Besides, the prolonged social isolation in children during the COVID-19 pandemic, limiting physical mobility and exercise, reduces DS and increases adiposity, which could impair the immune system function and raise the susceptibility to inflammatory diseases.

Screening of antibiofilm activity and toxicity assay of methylotrophic bacteria compounds isolated from the human mouth

Biofilm is a community consisting of one or more species of bacteria attached to a surface using a matrix. Biofilms can cause health problems in humans, such as dental plaque. Biofilm is one form of self-defense of bacteria against antibiotics, so biofilm often has implications for the rise in resistance of bacteria. Therefore, antibiofilm compounds can be used to increase the sensitivity of bacteria to the antimicrobial agent. The purpose of this study is to determine the antibiofilm activity and the potential toxicity of the methylotrophic bacteria compound isolated from the human mouth. Based on the results of the study, 21 out of 37 isolates of methylotrophic bacteria had antibiofilm activity using two different supernatant concentrations (5% and 10%). The isolate of M28L2 showed the highest anti-biofilm inhibition activity: 47.64% against EHEC and 50.97% against Pseudomonas aeruginosa ATCC 27583. Meanwhile, M30G1 isolate showed the highest anti-biofilm destruction activity: 44.062% against EHEC and 39.17% against ETEC. Toxicity test was performed on five isolates that had the highest antibiofilm activity using a lethal dose test. Based on these test results, five isolates had LC50 values greater than 1000μg/mL, indicating that the compound was not toxic. Subsequently, PCR amplification for the DNA sequencing stage was performed on one isolate, M28L2. Based on the sequencing results, M28L2 had a 99% similarity to Pseudomonas stutzeri.

MiRNA-1 promotes pyroptosis of cardiomyocytes and release of inflammatory factors by downregulating the expression level of PIK3R1 through the FoxO3a pathway.

Pyroptosis can be seen in both physiological and pathological processes, and whether it plays a positive or negative role has not been fully clarified, especially in the study of cardiology, which deserves more attention. A model of cardiomyocyte Hypoxia/Reoxygenation was established to detect MicroRNA (miRNA) expression. The C57BL/6 mice and H9c2 cells were respectively treated with Ischemic/Reperfusion (I/R) and Hypoxia/Reoxygenation (H/R). Then, the concentrations of interleukin-1β (IL-1β) and IL-18 were detected in serum and culture medium supernatant, as well as the expression levels of FOXO3, Caspase1, and PIK3R1. In I/R group, the concentrations of IL-1β and IL-18 in the supernatant of H9c2 cell culture solution were significantly higher compared with the control group. Consistent with the supernatant concentrations of IL-1β and IL-18, IL-1β, and IL-18 were also increased at a high level in I/R group at the transcriptional level. Furthermore, the in vitro experiments showed that FOXO3 and Caspase1 were significantly upregulated in myocardial cells with the treatment of I/R group compared to sham group. Concerning pyroptosis, the expression of PIK3R1 was dramatically decreased and the expression of miRNA-1 was significantly increased in H9c2 cells. MiRNA-1 promoted pyroptosis of cardiomyocytes and release of inflammatory factors by downregulating the expression level of PIK3R1.

Methyl donor supply to heat stress-challenged polymorphonuclear leukocytes from lactating Holstein cows enhances 1-carbon metabolism, immune response, and cytoprotective gene network abundance

Mechanisms controlling immune function of dairy cows are dysregulated during heat stress (HS). Methyl donor supply-methionine (Met) and choline (Chol)-positively modulates innate immune function, particularly antioxidant systems of polymorphonuclear leukocytes (PMN). The objective of this study was to investigate the effect of Met and Chol supply in vitro on mRNA abundance of genes related to 1-carbon metabolism, inflammation, and immune function in short-term cultures of PMN isolated from mid-lactating Holstein cows in response to heat challenge. Blood PMN were isolated from 5 Holstein cows (153 ± 5 d postpartum, 34.63 ± 2.73 kg/d of milk production; mean ± SD). The PMN were incubated for 2 h at thermal-neutral (37°C; TN) or heat stress (42°C; HS) temperatures with 3 levels of Chol (0, 400, or 800 μg/mL) or 3 ratios of Lys:Met (Met; 3.6:1, 2.9:1, or 2.4:1). Supernatant concentrations of IL-1β, IL-6, and tumor necrosis factor-α were measured via bovine-specific ELISA. Fold-changes in mRNA abundance were calculated separately for Chol and Met treatments to obtain the fold-change response at 42°C (HS) relative to 37°C (TN). Data were subjected to ANOVA using PROC MIXED in SAS (SAS Institute Inc., Cary, NC). Orthogonal contrasts were used to determine the linear or quadratic effect of Met and Chol for mRNA fold-change and supernatant cytokine concentrations. Compared with PMN receiving 0 μg of Chol/mL, heat-stressed PMN supplemented with Chol at 400 or 800 μg/mL had greater fold-change in abundance of CBS, CSAD, GSS, GSR, and GPX1. Among genes associated with inflammation and immune function, fold-change in abundance of TLR2, TLR4, IRAK1, IL1B, and IL10 increased with 400 and 800 μg of Chol/mL compared with PMN receiving 0 μg of Chol/mL. Fold-change in abundance of SAHH decreased linearly at increasing levels of Met supply. A linear effect was detected for MPO, NFKB1, and SOD1 due to greater fold-change in abundance when Met was increased to reach Lys:Met ratios of 2.9:1 and 2.4:1. Although increasing Chol supply upregulated BAX, BCL2, and HSP70, increased Met supply only upregulated BAX. Under HS conditions, enhancing PMN supply of Chol to 400 μg/mL effectively increased fold-change in abundance of genes involved in antioxidant production (conferring cellular processes protection from free radicals and reactive oxygen species), inflammatory signaling, and innate immunity. Although similar outcomes were obtained with Met supply at Lys:Met ratios of 2.9:1 and 2.4:1, the response was less pronounced. Both Chol and Met supply enhanced the cytoprotective characteristics of PMN through upregulation of heat shock proteins. Overall, the modulatory effects detected in the present experiment highlight an opportunity to use Met and particularly Chol supplementation during thermal stress.

Retinoic Acid Ameliorates the Severity of Food Allergy under Allergen Exposure in a Mouse Model with Food Allergy.

Effectiveness of retinoic acid (RA) in treating food allergy is not yet clear. Using an allergic mouse model, we examined the amelioration of the severity of food allergy by daily RA intake with allergen or without. Female Balb/c mice were systemically sensitized to egg white (EW) and alum by intraperitoneal injection. Sensitized mice were provided diets supplemented with 0% (non-treated group), 0.1% EW (allergen group), 0.0017% RA (RA group), or 0.1% EW plus 0.0017% RA (RA+allergen group) with 20% casein for 4 wk. Oral food challenge (OFC) and allergic biomarkers were quantified. The decrease in rectal temperature post-OFC was significantly suppressed in the RA and RA+allergen groups compared to those in the non-treated and allergen groups, respectivety. The plasma levels of ovalbumin-specific IgE, IgA and IgG1 at the study endpoint were higher in the allergen and RA+allergen groups than those in the non-treated and RA+allergen groups, respectivety. Plasma ovalbumin-specific IgG2a levels at the study endpoint were significantly higher in the RA+allergen group than those in the RA groups. The supernatant concentrations of interleukin-10 and interferon-γ in the cultured spleen lymphocytes were highest in the RA+allergen group compared to those in the other groups. Thus, continuous intake of RA under allergen exposure ameliorated the severity of food allergy in a mouse model with food allergy.

The ameliorating effects of tokishakuyakusan in a rat model of implantation failure involves endometrial gland leukemia inhibitory factor and decidualization

Ethnopharmacological relevanceTokishakuyakusan (TSS) is a Kampo medicine that is prescribed for the treatment of infertility in Japan. However, its precise mechanism of action remains unclear. Aim of the studyLeukemia inhibitory factor (LIF) in the endometrium plays an indispensable role in embryo implantation and is linked to infertility or implantation failure. Previously, we demonstrated that TSS ameliorated implantation failure induced by mifepristone (RU-486), an antagonist of progesterone, in rats. Herein, we aimed to clarify whether the ameliorating effect of TSS on implantation failure in the rat model involves endometrial LIF. Additionally, we determined whether decidualization, the dysfunction of which is linked to infertility or implantation failure similar to LIF, progesterone, and other implantation-related factors, are involved in the effect of TSS. Materials and methodsThe implantation failure rat model was developed via the subcutaneous administration of RU-486 (7 mg/kg) on day 3 post-coitus. Sesame oil was administered as the vehicle control. Rats were fed a diet containing 1% or 3% TSS or a control diet from day 13 pre-coitus. Subsequently, the implantation sites were assessed, and plasma progesterone levels were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on day 8 post-coitus. The LIF mRNA of the endometrial gland, which was segmented via laser-microdissection from the endometrial tissue, was measured, and endometrial LIF immunostaining was carried out on day 5. The gene expression of different factors related to implantation, including decidualization and progesterone-responsiveness on days 5 and 6, were measured. The human endometrial Ishikawa cell line derived from human adenocarcinoma was treated with TSS (30–300 μg/mL) for 24 h, and the LIF concentrations in the cell culture supernatants were measured. ResultsRU-486 decreased the number of implantation sites in the uterus of rats; however, the decrease was significantly alleviated by TSS (3%-diet), which tended to increase plasma progesterone. In rats with RU-486-induced implantation failure, endometrial gland LIF mRNA and endometrial LIF protein were markedly decreased while the gene expression of both decidualization-related factors such as interleukin-11, insulin-like growth factor binding protein-1, and cyclooxygenase-2, and progesterone responsive-related factors such as FK506 binding protein 5, were significantly decreased. These changes in the uterus of rats with implantation failure were significantly alleviated by TSS (3%-diet). Additionally, TSS significantly enhanced LIF protein production and LIF mRNA in Ishikawa cells. ConclusionsThe mechanism whereby TSS ameliorates RU-486-induced implantation failure in rats may involve the alleviation of decreased LIF production derived from the endometrial gland, and a dysfunction of decidualization, including lower progesterone responsiveness in the model. These findings may partly contribute to the interpretation of the beneficial effects of TSS on infertility.

Effects of tacrolimus on the TGF‑β1/SMAD signaling pathway in paraquat‑exposed rat alveolar typeII epithelial cells.

Paraquat is a highly toxic pesticide, which often causes pulmonary interstitial fibrosis after poisoning, and there is no specific antidote. At present, limited studies have reported that tacrolimus, as an immunosuppressant, can inhibit pulmonary fibrosis, but the specific mechanism remains unknown. The aim of the present study was to demonstrate the effect of tacrolimus on the TGF-β1 pathway associated with pulmonary fibrosis in paraquat exposed alveolar type II epithelial cells, and to identify the antipulmonary fibrosis mechanism of tacrolimus The rat alveolar epithelial type II RLE-6TN cell line was exposed to paraquat and treated with or without tacrolimus for 24 h, or with a TGF-β1 receptor type I/II inhibitor (LY2109761) for 1, 4, 8 or 16 h. MTT assays were used to detect the viability of rat alveolar type II epithelial cells under these different treatment conditions, while the concentrations of TGF-β1, SMAD3, SMAD7 and connective tissue growth factor (CTGF) in the cell culture supernatant were determined using ELISAs. Additionally, reverse transcription-quantitative PCR and immunofluorescence were used to analyze the mRNA and protein expression levels of TGF-β1, SMAD3, CTGF and SMAD7. The results demonstrated that the inhibition of the proliferation of RLE-6TN cells exposed to 200 nmol/l paraquat was 26.05±2.99%. The inhibition rate of 10 ng/ml tacrolimus on paraquat-exposed alveolar type II epithelial cells was 18.40±3.49%. The inhibition rate caused by 5 µmol/l LY2109761 was 26.56±4.49%. The expression levels of TGF-β1, SMAD3 and CTGF, as well as their concentrations in the culture supernatant, were significantly downregulated in the tacrolimus group compared with the paraquat group. However, both the concentration and expression levels of SMAD7 were significantly upregulated in the tacrolimus group compared with the paraquat group. In conclusion, tacrolimus can reduce the levels of TGF-β1, SMAD3 and CTGF, increase the level of SMAD7 in TGF-β1 signaling pathway and protect the development of pulmonary fibrosis in paraquat exposed alveolar epithelial cells.

Abstract 1020: Anti-oxidant amino acid L-ergothioneine exhibits immunomodulatory effect by regulating immunosuppressive cytokine production of tumor cells

Abstract Ergothioneine (ERG) is a natural antioxidant nutrient containing in edible golden oyster mushroom, Pleurotus cornucopiae (Tamogitake). In previous AACR meeting, we reported that administration of ERG rich water extracts of p. cornucopiae to tumor-bearing mice could suppress the growth of subcutaneous tumors and reduce the number of regulatory T cells (Treg) in lymph node and spleen. However, the molecular events inducing antitumor and immunomodulatory effects are still unknown. Our aim is to identify the mechanism of ERG in tumor cells which secrete various immunomodulatory cytokines. We cocultured human tumor cell lines with ERG and tested cytokine concentration in culture supernatants and expression of immune checkpoint molecules on tumors. ERG could significantly inhibit the production of VEGF, CCL2 and TGF-β1 which are important in not only angiogenesis and tumor invasion but also Treg induction. Importantly, ERG also reduced the expression of some immune checkpoint molecules on tumor cells and prolonged the survival of IL-2-activated lymphocytes in vitro. Overall, ERG rich water extract of p. citrinopileatus might inhibit the induction of Treg in tumor-bearing host via suppression of VEGF and TGF-β1, indicating a novel immunomodulatory antioxidant nutrient in mushrooms Citation Format: Kazunori Kato, Katsuhiro Kuroki, Yui Shimizu. Anti-oxidant amino acid L-ergothioneine exhibits immunomodulatory effect by regulating immunosuppressive cytokine production of tumor cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1020.

Cell cycle arrest and anti-cancer potential of probiotic Lactobacillus rhamnosus against HT-29 cancer cells.

Introduction: Nowadays, probiotic bacteria have been considered as a factor in the prevention and treatment of cancer, especially by induction of apoptosis. This study aimed to evaluate the cytotoxic, anti-proliferative, and apoptotic effects of the supernatant of probiotic Lactobacillus rhamnosus on HT-29 cell line. Methods : Molecular identification of probiotic L. rhamnosus was carried out using specific primers of 16S rRNA gene and sequencing. HT-29 cells were treated with different concentrations of bacterial supernatants at 24, 48, and 72 hours. MTT assay, Annexin V-FITC, real-time PCR, cell cycle analysis, and DAPI staining tests were conducted to evaluate the induction of apoptosis. The level of cyclin D1 protein was measured by immunocytochemistry method. Results: The supernatant of L. rhamnosus inhibited the growth of HT-29 cancer cells in a dose- and time-dependent manner. The results of flow cytometry confirmed apoptotic cell death. Probiotic bacterial supernatant caused up-regulation of pro-apoptotic genes including caspase-3, caspase-9, and Bax. In addition, they resulted in down-regulation of Bcl2 and a decrease in expression levels of cyclin D1, cyclin E, and ERBB2 genes. Cancer cells were arrested in the G0/G1 phase of the cell cycle. The results of immunocytochemistry showed significant down-regulation of cyclin D1 protein during the 48 hours treatment with bacterial supernatant compared to the untreated cells. Conclusion: The supernatant of probiotic L. rhamnosus has a great potential to inhibit the proliferation of HT-29 cells and the induction of apoptosis. L. rhamnosus might be used as a biological anti-cancer factor in the prevention and treatment of colon cancer.

Beneficial Effect of a Fermented Wheat Germ Extract in Intestinal Epithelial Cells in case of Lipopolysaccharide-Evoked Inflammation.

In this study, the protective effect of a fermented wheat germ extract (FWGE) against LPS-induced inflammation and oxidative stress in IPEC-J2 porcine intestinal epithelial cells was studied. Enterocytes were treated with LPS derived from Salmonella enterica ser. Typhimurium and Escherichia coli O55:B5, O111:B4, and O127:B8 strains. Intracellular ROS level and extracellular H2O2 level were followed up by two fluorescent assays (DCFH-DA and Amplex Red). The effect of FWGE on the intestinal barrier integrity was determined by transepithelial electric resistance measurements and using a FD4 fluorescent tracer dye. IL-6 concentration of supernatants was also measured by the ELISA method. Our data revealed that FWGE had a significant lowering effect on the inflammatory response especially related to oxidative stress. Treatment with FWGE (1-2%) significantly decreased the level of intracellular ROS compared to LPS-treated cells. Furthermore, LPS-triggered partial disruption of epithelial integrity was reduced after FWGE application.

Usefulness of neuron‐specific enolase as a serum marker of metastatic melanoma

Treatment strategies for advanced melanoma are dramatically changing, due to immune-checkpoint inhibitors and BRAF/MEK inhibitors. Nevertheless, reliable serum markers for evaluation of treatment responses and the outcome are still limited. Some previous reports suggested that serum neuron-specific enolase (sNSE) may be a useful marker for melanoma; however, its usefulness is controversial. Moreover, NSE has not been examined in vitro by using melanoma cell lines. We retrospectively evaluated sNSE and serum lactate dehydrogenase (sLDH) levels at the initial diagnosis and during therapy in 33 melanoma patients of various stages. We analyzed the NSE concentrations in cell lysates and supernatants from melanoma cell lines by enzyme-linked immunosorbent assay. The median sNSE was significantly higher in stage IV patients compared with stages I/II and III (16.3, 12.7 and 12.1ng/mL, respectively). sNSE was elevated in 20% (2/10) of stage III and 61.1% (11/18) of stage IV patients but not in stages I/II. sNSE and sLDH tended to correspond to the total tumor volume (P=0.48 and 0.58; 95% confidence intervals, 0.005-0172 and 0.776-0.836, respectively). The coincidence rate of sNSE and sLDH in stage IV at the initial diagnosis was 11 of 18 (61.1%). Of the remaining patients, elevated sNSE but not sLDH was observed in five patients (27.8%) and elevated sLDH but not sNSE was observed in two (11.1%). Four of the five patients showing elevated sNSE and normal sLDH were of the mucosal type. NSE was detected in both supernatant and cell lysate of all four melanoma cell lines (0.30-237.32ng/mL and 137-483.04ng/mg, respectively). Two cell lines with a high supernatant NSE level contained many dead cells in the supernatant. The combination of sNSE and sLDH could contribute to the early detection of distant metastasis and disease condition evaluations for advanced melanoma patients.

The effect of foal or adult horse plasma on equine monocyte-derived dendritic cell phenotype and function

Immunological and endocrine immaturity in foals increases foal morbidity and mortality from bacterial sepsis. Dendritic cells (DC) are critical in activating the adaptive immune response, but foal DC are phenotypically and functionally different than those of adult horses. Age-related variations in availability of some soluble plasma factors, such as hormones, might govern some age-related differences in DC function. Effects of exposure to plasma factors on equine DC phenotype and function have not been described. We hypothesized that exposure to plasma from foals or adult horses would differentially impact monocyte-derived DC (MoDC) phenotype and function. Eight healthy adult horses and 8 healthy foals were divided into pairs of one adult horse and one foal. Blood was collected from each pair for MoDC generation when foals were 1 and 30 days of age. MoDC from horses and foals were then exposed to killed whole-cell bacteria in the presence of their own age-matched plasma, plasma from the opposite-aged animal in the pair, and serum-free medium alone (control). Expression of DC-relevant surface markers (MHC class-II, CD86, and CD14) and endocytosis capability were measured by flow cytometry. Supernatant cytokine concentrations (IL-4, IL-17, IFN-γ, and IL-10) were quantified with a validated bead-based immunoassay. Data were analyzed using linear mixed-effects and Tobit regression models (P < 0.05). The percentage of MoDC expressing surface markers MHC class-II and CD86 was reduced in MoDC derived from 1-day-old foals in comparison to adult horse MoDC when cultured in medium alone or with either source of plasma (P = 0.0001). Foal and adult horse MoDC cultured in either source of plasma expressed more CD86 and less CD14 than cells cultured in serum-free medium alone (P ≤ 0.02). Adult horse and foal MoDC exposed to bacterial antigen in the presence of 1-day-old foal plasma secreted less IL-10 (P ≤ 0.0008) compared to those cultured in adult horse plasma. Endogenous production of IL-17 by MoDC from foals at day 1 of age cultured in adult plasma was increased compared to foal MoDC cultured in serum-free medium (P = 0.004). Phagocytosis of killed, labeled Staphylococcus aureus was reduced when MoDC generated from foals or adult horses were exposed to plasma from foals at day 1 or 30 of age (P ≤ 0.03). Age-related variation in soluble plasma factors appear to regulate equine MoDC function, but specific plasma factors capable of regulating MoDC phenotype or function were not defined in this study.

Loss of the lipopolysaccharide (LPS) inner core increases the electrocompetence of Escherichia coli.

Mutations that shorten the lipopolysaccharide (LPS) in Escherichia coli were found to significantly increase the number of transformants after electroporation. The loss of the LPS outer core increased the number of transformants with plasmid pAmCyan (3.3 kb) from 5.0 × 105 colony-forming units (CFU)/μg in the wild-type E. coli BW25113 to 3.3 × 107 CFU/μg in a ΔwaaG background, a 66.2-fold increase in efficiency. Truncation of the inner core improved this even further, with the ΔwaaF mutant exhibiting the best transformation efficiencies obtained, i.e., a 454.7-fold increase in the number of colonies over the wild-type strain. Similar results were obtained when a larger plasmid (pDA1; 11.3 kb) was used, with the ΔwaaF mutant once more giving the best transformation rates, i.e., a 73.7-fold increase. Subsequent tests proved that the enhanced transformabilities of these mutants were not due to a better survival or their surface charge properties, nor from preferential binding of these strains to the plasmid. Using N-phenyl-1-naphthylamine (NPN), we confirmed that the outer membranes of these mutant strains were more permeable. We also found that they leaked more ATP (3.4- and 2.0-fold higher for the ΔwaaF and ΔwaaG mutants, respectively, than wild-type E. coli BW25113), suggesting that the inner membrane stability is also reduced, helping to explain how the DNA enters these cells more easily. KEY POINTS: • LPS inner core gene knockouts increase the electrocompetence of E. coli. • No significant difference in survival, surface charge, or DNA binding was evident. • The LPS inner core mutants, however, exhibited higher outer membrane permeability. • Their inner membranes were also leaky, based on supernatant ATP concentrations.

Antimicrobial and Antibiofilm Activities of Probiotic Lactobacilli on Antibiotic-Resistant Proteus mirabilis.

The emergence of biofilm-forming, multi-drug-resistant (MDR) Proteus mirabilis infections is a serious threat that necessitates non-antibiotic therapies. Antibiotic susceptibility and biofilm-forming activity of P. mirabilis isolates from urine samples were assessed by disc diffusion and crystal violet assays, respectively. Antimicrobial activities of probiotic Lactobacilli were evaluated by agar diffusion. Antibiofilm and anti-adherence activities were evaluated by crystal violet assays. While most P. mirabilis isolates were antibiotic-resistant to varying degrees, isolate P14 was MDR (resistant to ceftazidime, cefotaxime, amoxicillin-clavulanic acid, imipenem, ciprofloxacin, and amikacin) and formed strong biofilms. Cultures and cell-free supernatants of Lactobacillus casei and Lactobacillus reuteri exhibited antimicrobial and antibiofilm activities. The 1/16 concentration of untreated supernatants of L. casei and L. reuteri significantly reduced mature biofilm formation and adherence of P14 by 60% and 72%, respectively (for L. casei), and by 73% each (for L. reuteri). The 1/8 concentration of pH-adjusted supernatants of L. casei and L. reuteri significantly reduced mature biofilm formation and adherence of P14 by 39% and 75%, respectively (for L. casei), and by 73% each (for L. reuteri). Scanning electron microscopy (SEM) confirmed eradication of P14’s biofilm by L. casei. L. casei and L. reuteri could be utilized to combat Proteus-associated urinary tract infections.

Exploring Mediterranean and Arctic Environments as a Novel Source of Bacteria Producing Antibacterial Compounds to be Applied in Aquaculture

The need to discover new natural compounds has become urgent as a possible alternative solution to contrast the spread of antibiotic resistance, also in the aquaculture field. Bacterium–bacterium inhibitory activity against bacterial pathogens relevant in aquaculture was evaluated on agar plates for bacteria isolated from cold Arctic (Kongsfjorden, Svalbard Islands; i.e., seawater and sediment samples) and temperate Mediterranean (Lake Faro in Messina and the Straits of Messina, Italy; i.e., brackish water and benthic filter-feeding organisms) environments. Cell-free supernatants (both pure and concentrated 10-fold) were further assayed and, in the case of a positive response, crude extracts were obtained and tested. After the pre-screening procedures, about 30% of the bacterial isolates inhibited the growth of at least one pathogen used as a target. The 10-fold concentrated supernatants of two Arctic Salinibacterium spp. strains and the Mediterranean Bacillus sp. PS62 (associated with the pennatulacean Pteroeides spinosum Ellis, 1764) resulted in being active against P. damselae subsp. piscicida. The crude extracts obtained from Bacillus sp. PS62 also showed inhibitory activity against the same pathogen. Our findings suggest that tested bacteria could represent a novel source of compounds to be applied to overcome pathogenesis in the aquaculture field.

514-P: Adaptive Downregulation of NLRP3 Inflammasome Expression in Human Renal Tubular Epithelia in the Hyperglycemic and Hyperuricemic Culture

Background: Hyperuricemia has been an independent risk factor for diabetic kidney disease. Previous studies showed that hyperuricemia may induce interstitial infiltration of macrophages and activates its NLRP3 inflammasome. Meanwhile, there has been proved that an inner NLRP3 system exists in human tubular epithelia. Here we try to find how glucose and uric acid (UA) affect NLRP3 inflammasome in cultivating tubular epithelia. Methods: HK-2 cells were cultivated. Experiment was designed according to a 2×3×2 multifactorial style. Double glucose concentrations (5.5MM and 25MM) crossed with three escalated UA concentration (0MM, 0.2MM, and 0.4MM), as well as Mcc950 (0MM and 0.1MM), an specific blocker for NLRP3 inflammasome. Morphology of HK-2 cells were observed. Supernatant concentrations and gene expressions of pivotal factors involved in NLRP3 inflammasome pathway including NLRP3, ASC, caspase-1, IL-1β and IL-18 were detected. Results: 1) Morphology: As concentrations of glucose and UA increased, HK-2 cells obviously became floating and fragmental. 2) Genes: Except for Caspase-1, gene expressions of other four components were significantly inhibited in hyperglycemic state. Expressions of five genes were all decreased in euglycemic but unchanged in hyperglycemic state when UA increased. Mcc950 treatment significantly increased gene expressions of NLRP3, ASC, IL-1β and IL-18 in euglycemic but kept them unchanged in hyperglycemic state as UA increased. 3) ELISA: Supernatant IL-1β and IL-18, two effector molecules of NLRP3 inflammasome, were significantly decreased after stimulation with increased glucose or UA cascade. Interestingly, Mcc950 treatment further decreased concentrations of two molecules when UA stepwisely increased. Conclusions: The inner NLRP3 inflammasome counter-regulates effects of hyperglycemia and hyperuricemia. Mcc950 may block the downstream points to reduce IL-1β and IL-18 production directly. Disclosure C. Ciying: None. J. Ran: None. C. Chen: None. P. Zhu: None. R. Tan: None. Y. Liu: None. Funding Guangdong Science and Technology Project Fund (2014B030303002); Guangdong Provincial Academician Workstation Fund (2017B090904027)