Supernatant Medium Research Articles

Store‐Operated Calcium Entry Upregulated IL6 Expression in Glomerular Mesangial Cells through NF‐κB Pathway

BackgroundIL6 is a pleotropic cytokine and functions as a pro‐ as well as anti‐inflammatory mechanism depending on the cell type or the specific pathological environment. It is unclear if a particular signaling pathway confers IL6 with this property. Immunological and inflammatory mechanisms play a significant role in the development of diabetic nephropathy (DN). Locally produced cytokines/chemokines contributes to the early changes and histological impairment in DN. Glomerular mesangial cell (MC) is a major cell type in glomerulus to produce cytokines/chemokines in response to diabetes and a major contributor to mesangial expansion in DN. We have previously demonstrated that the Orai‐1 mediated store‐operated calcium entry (SOCE) suppressed ECM protein production by MCs. The aim of this study was to determine whether and how SOCE in MCs regulated IL6 production by MCs.MethodsOrai1, the channel protein mediating SOCE in MCs was knocked down using the targeted nanoparticle‐siRNA delivery system in wild type C57BLKS/J mice at the age of 16 weeks. Immunohistochemistry was performed on the paraffin embedded kidney sections to examine glomerular IL6 expression. In cultured human MCs, the expression of IL6 was examined in culture media and whole cell lysates using ELISA and Western blot analysis, respectively in the presence of normal glucose (5 mM D‐glucose + 20 mM L‐glucose) with/without an activator (thapsigargin at 1 μM) or inhibitor (GSK‐7975A 10 μM) of SOCE for 15 hrs. To determine the role of NF‐κB in SOCE effects on IL6 production, human MCs were treated with an inhibitor of NF‐κB, helenaline or siRNA against p65 subunit of NF‐κB. Furthermore, the effect of SOCE on the nuclear translocation of p65 was examined by assessing abundance of nuclear p65 protein in response to activation and inhibition of SOCE.ResultsThe IL6 expression level was reduced in the glomeruli of the mice treated with nanoparticle/Orai1 siRNA for 2 weeks compared to that in the control mice. In cultured human MCs, thapsigargin significantly increased IL‐6 level in supernatant media and in whole cell lysates of MCs, and this effect was attenuated by GSK 7975‐A, a selective inhibitor of SOCE. However, tunicamycin, an ER stress inducer did not alter the amount of IL6 as thapsigargin did. Treatment with helenaline, an inhibitor of NF‐κB pathway, or knockdown of p65, a subunit of NF‐κB significantly blunted the thapsigargin‐induced increase in IL6 protein abundance. Moreover, thapsigargin stimulated the nuclear translocation of p65 in human MCs.ConclusionOrai1‐mediated SOCE positively regulates IL6 production by MCs through activation of NF‐κB pathway.Support or Funding InformationResearch Support: NIH/NIDDK (1RO1 DK115424); AHA Southwest Affiliate.

A Combinational Strategy for Effective Heterologous Production of Functional Human Lysozyme in Pichia pastoris.

Human lysozyme (hLYZ), known for its bacteriolytic activity, is widely applied in the food and pharmaceutical industries as an antimicrobial agent. However, its extensive application was limited by its low large-scale production efficiency. In this study, a combinational method of integrating codon optimization, multiple gene copies, and ER molecular chaperone co-expression was developed to improve the heterologous production of hLYZ in Pichia pastoris GS115. Our results showed that increasing the copy number of the optimized hLYZ gene in P. pastoris could enhance its secretory production level up to 1.57-fold. The recombinant opt-hLYZ-6C strain that contains six copies of opt-hLYZ gene exhibited the highest mRNA transcription levels, giving the highest production of 0.22 ± 0.02 mg/mL of hLYZ in the medium supernatant with a bacteriolytic activity of 14,680 ± 300 U/mL against Micrococcus lysodeikticus in the shaking flask experiment. Moreover, co-overexpression of ER retention molecular chaperones, such as Pdi1 or Ero1, in the recombinant opt-hLYZ-6C strain both presented positive effects on the secretory production of hLYZ. Our further characterization indicated that tandem co-expression of Ero1 and Pdi1 together presented an added-up effect. The secretory production of hLYZ in the medium supernatant reached 0.34 ± 0.02 mg/mL of the recombinant opt-hLYZ-6C-EP strain in the shaking flask experiment, with a bacteriolytic activity of 21,200 ± 400 U/mL. Compared to the recombinant opt-hLYZ-1C strain, these final improvements were calculated as 2.43-fold and 2.30-fold on secretory protein levels and antibacterial activity, respectively. Finally, the recombinant opt-hLYZ-6C-EP strain was applied for high-density cultivation in 5 L of fermenter, in which the secretory yield of hLYZ reached 2.34 ± 0.02 mg/mL in the medium supernatant, with a bacteriolytic activity of 1.76 ± 0.02 × 105 U/mL against M. lysodeikticus. All these numbers presented the highest heterologous production levels of hLYZ in microbial systems.

M3 receptor modulates extracellular matrix synthesis via ERK1/2 signaling pathway in human bladder smooth muscle cells

Extracellular matrix (ECM) accumulation plays a key role in the progression of bladder outlet obstruction (BOO). Muscarinic receptors have been widely reported to serve as pivotal regulators in lung tissue remodeling. However, the influence of them on human bladder smooth muscle cells (HBSMCs) and the underlying molecular mechanisms have not yet been evaluated. The purposes of the present study are to investigate the effect of muscarinic receptors on the synthesis of ECM in HBSMCs and the involvement of intracellular signal transducers. The results indicated that M1 -M5 muscarinic receptors were all encoded in HBSMCs. The expression rank order was M2 > M1 > M5 > M3 > M4 . The gene and protein expression of collagen I (COL1), TIMP-1, and TIMP-2 was carbachol (CCH) concentration-dependently enhanced. The synthesis of COL1 in the supernatant of cell culture medium was significantly elevated by exposure to CCH. The CCH-induced protein expression of COL1, TIMP-1, and TIMP-2, however, was obviously reduced by the pretreatment of muscarinic receptor antagonists, atropine, and M3 -preferring antagonist (1,1-dimethyl-4-diphenyl-acetoxypiperidinium iodide [4-DAMP]). Furthermore, ERK1/2 was activated by 100 µM CCH when compared with the control group and the pretreatment of ERK1/2 inhibitor significantly suppressed the synthesis of COL1 induced by 100 µM CCH. Besides, CCH-induced phosphorylation of ERK1/2 was remarkably restrained by the pretreatment of 4-DAMP. All in all, these findings demonstrated that M3 receptor can modulate extracellular matrix synthesis via the ERK1/2 signaling pathway, which may provide potential novel therapeutic targets for BOO.

Molecular hydrogen attenuates sepsis-induced neuroinflammation through regulation of microglia polarization through an mTOR-autophagy-dependent pathway

Sepsis-associated encephalopathy (SAE) is the cognitive impairment resulting from sepsis and is associated with increased morbidity and mortality. Hydrogen has emerged as a promising therapeutic agent to alleviate SAE. The mechanism, however, remains unclear. This research aimed to determine whether hydrogen alleviates SAE by regulating microglia polarization and whether it is mediated by the mammalian target of rapamycin (mTOR)-autophagy pathway. Septic models were established by cecal ligation and puncture (CLP) performed on mice. The Morris Water Maze was used to evaluate cognitive function. M1/M2 microglia polarization was assessed by immunofluorescence. Inflammatory cytokines were determined by ELISA. Septic cell models were established using BV-2 cells incubated with 1 μg/ml lipopolysaccharide (LPS). M1/M2 microglia polarization was assessed by flow cytometry. Inflammatory cytokines from culture medium supernatant were determined by ELISA, and associated protein expression levels of mTOR-autophagy pathway were assessed by Western blot. Hydrogen inhalation attenuated sepsis-induced cognitive impairment with improved escape latency, time spent in the target platform quadrant and number of times crossing the target platform. In both animal and cell research, hydrogen reduced TNF-α, IL-6 and HMGB1 levels and M1 polarization, but increased IL-10 and TGF-β levels and M2 polarization. Hydrogen treatment decreased the ratio of p-mTOR/mTOR and the expression of p62 and increased the ratio of p-AMPK/AMPK, LC3II/LC3I and the expression of TREM-2 and Beclin-1 in LPS-treated BV-2 cells. MHY1485, an mTOR activator, abolished the protective effects of hydrogen in vitro. Taken together, these results demonstrated that hydrogen attenuated sepsis-induced neuroinflammation by modulating microglia polarization, which was mediated by the mTOR-autophagy signaling pathway.

Differentiation of human umbilical cord mesenchymal stem cells into Leydig-like cells with defined molecular compounds.

95% of the body's testosterone is produced by the Leydig Cells (LCs) in adult testis, and LC functional degradation can cause testosterone deficiency ultimately leading towards hypogonadism. The transplantation of LCs derived from stem cells is a very promising therapy to overcome the testosterone deficiency. The isolated umbilical cord mesenchymal stem cells (UMSCs) were identified by flow cytometry and adipogenic and osteogenic differentiation. Western blotting and reverse transcription polymerase chain reaction (RT-PCR) were used for the differentiated Leydig-like cell identification. The comparisons of the testosterone levels, gene expression levels, and cyclic adenosine monophosphate (cAMP) productions were performed through radioimmunoassay, quantitative polymerase chain reaction (qPCR), and cAMP assay kit, respectively. Here, it is stated that our isolated human UMSCs, which could positively express CD29, CD44, CD59, CD90, CD105, and CD166 but negatively express CD34 as well as could be differentiated into adipocytes and osteocytes, could be differentiated into Leydig-like cells (UMSC-LCs) using a novel differentiation method based on molecular compounds. The enrichment UMSC-LCs could secrete testosterone into the medium supernatant and produce considerable cAMP at the stimulation of luteinizing hormone (LH), and positively expressed LC lineage-typical markers LHCGR, SCARB1, SATR, CYP11A1, CYP17A1, HSD3B1, HSD17B3, and SF-1 as well as negatively expressed mesenchymal stem cell typical markers CD29, CD44, and CD105. The expression levels of NR3C4, PDGFRA, and NR3A1 in UMSC-LCs were higher than those of UMSCs and were comparable with LCs. These results illuminated that UMSCs could be differentiated into Leydig-like cells using the defined molecular compounds, which might further support MSC-derived Leydig cell transplantation therapy for testosterone insufficiency.

Curcumin Suppresses Apoptosis and Inflammation in Hypoxia/Reperfusion-Exposed Neurons via Wnt Signaling Pathway.

BackgroundCerebral ischemia/reperfusion (I/R) injury contributes to mortality and morbidity in preterm infants. Curcumin has been shown to exert neuro-protective effects in the central nervous system (CNS). The aim of this study was to investigate the neuro-protective activity of curcumin and the possible underlying molecular mechanisms.Material/MethodsA hypoxia/reoxygenation (H/R) protocol was used to simulate I/R injury in vitro. Isolated neonatal neurons were pre-treated with curcumin at serially diluted concentrations and exposed to H/R injury. Cell viability and apoptosis were assessed by MTT and flow cytometry, respectively. Contents of TNFα and IL6 in supernatant of cell culture medium were detected by ELISA. Protein expression, phosphorylation, and nuclear translocation levels were studied by Western blotting.ResultsH/R reduced cell viability and increased apoptosis of neurons. H/R significantly increased Wnt5a expression, JNK1 phosphorylation, and NF-κB nuclear translocation. Moreover, expression levels of cleaved caspase3, TNFα, and IL6 were elevated in H/R-exposed neurons. Curcumin pre-treatment significantly increased cell viability and inhibited apoptosis of neurons exposed to H/R, in a concentration-dependent manner. Moreover, curcumin pre-treatment significantly decreased expression levels of Wnt5a, IL6, TNFα, and phosphorylation level of JNK1, as well as the nuclear translocation level of NF-κB in H/R-exposed neurons, in a concentration-dependent manner.ConclusionsCurcumin exerted neuro-protective effects against H/R-induced neuron apoptosis and inflammation by inhibiting activation of the Wnt/JNK1 signaling pathway.

Microbial Community Analysis of Digested Liquids Exhibiting Different Methane Production Potential in Methane Fermentation of Swine Feces.

Batch methane fermentation was conducted using seed sludge collected from six methane fermentation facilities. Swine feces were centrifuged and autoclaved, followed by its use as a substrate for methanogenesis. This "swine feces supernatant medium" facilitates the cultivation of the microbes of the seed sludge, sampling of the digested liquid using a syringe, and subculturing of the digested liquid in a subsequent medium using a syringe. Through 15 subcultures, digested liquids with high and low methane production potential were obtained, which were named "H-DS" and "L-DS," respectively. On the day 10 of cultivation, chemical oxygen demand (COD) of H-DS significantly decreased by 31% and that of L-DS did not differ significantly compared with that on the day 0 of cultivation. Acetic acid concentration of H-DS (1009mg/L) was significantly lower than that of L-DS (2686mg/L). These chemical characteristics indicate that organics decomposition in L-DS was not successful and suggest that H-DS has high relative abundance of bacteria decomposing organic matter and methanogen utilizing acetic acid compared with those in L-DS. Microbial community analysis revealed that Shannon index of H-DS was significantly higher than that of L-DS, and the relative abundance of acetogenic bacteria (e.g., Syntrophomonas) and acetic acid-utilizing methanogen (Methanosarcina) in H-DS was significantly higher than that in L-DS. Thus, the high methane production potential of H-DS might be attributable to the smooth flow from acetogenesis to methanogenesis step in the methane fermentation, compared with the case of L-DS.

Thrombin-Induced Decidual Colony-Stimulating Factor-2 Promotes Abruption-Related Preterm Birth by Weakening Fetal Membranes

Preterm premature rupture of membranes (PPROM) and thrombin generation by decidual cell-expressed tissue factor often accompany abruptions. Underlying mechanisms remain unclear. We hypothesized that thrombin-induced colony-stimulating factor-2 (CSF-2) in decidual cells triggers paracrine signaling via its receptor (CSF2R) in trophoblasts, promoting fetal membrane weakening and abruption-associated PPROM. Decidua basalis sections from term (n = 10), idiopathic preterm birth (PTB; n = 8), and abruption-complicated pregnancies (n = 8) were immunostained for CSF-2. Real-time quantitative PCR measured CSF2 and CSF2R mRNA levels. Term decidual cell (TDC) monolayers were treated with 10-8 mol/L estradiol ±10-7 mol/L medroxyprogesterone acetate (MPA)±1 IU/mL thrombin pretreatment for 4 hours, washed, and then incubated in control medium with estradiol±MPA. TDC-derived conditioned media supernatant effects on fetal membrane weakening were analyzed. Immunostaining localized CSF-2 primarily to decidual cell cytoplasm and cytotrophoblast cell membranes. CSF-2 immunoreactivity was higher in abruption-complicated or idiopathic PTB specimens versus normal term specimens (P<0.001). CSF2 mRNA was higher in TDCs versus cytotrophoblasts (P<0.05), whereas CSF2R mRNA was 1.3×104-fold higher in cytotrophoblasts versus TDCs (P<0.001). Thrombin enhanced CSF-2 secretion in TDC cultures fourfold (P<0.05); MPA reduced this effect. Thrombin-pretreated TDC-derived conditioned media supernatant weakened fetal membranes (P<0.05), which MPA inhibited. TDC-derived CSF-2, acting via trophoblast-expressed CSFR2, contributes to thrombin-induced fetal membrane weakening, eliciting abruption-related PPROM and PTB.

Tumor Cell-associated Exosomes Robustly Elicit Anti-tumor Immune Responses through Modulating Dendritic Cell Vaccines in Lung Tumor.

DC vaccine-based immunotherapy is emerging as a novel therapeutic strategy for cancer treatment, however, antitumor effect of DC vaccines based on tumor cell lysates (TCLs) remains unsatisfactory due to poor immunogenicity of tumor antigens. Although tumor-associated exosomes (TAEs) have been reported as a promising antigen for DC vaccines, it remains unclear how TAE-based DC vaccine induced antitumor immunity in lung cancer.Methods: In the present study, we extracted TAEs from the supernatant of tumor cell culture medium, and compared the effect of TAEs with TCLs on DCs. To further evaluate the therapeutic effect of DCTAE, we used immunofluorescence and flow cytometry to evaluate the apoptosis of tumor tissue, tumor-infiltrating CD8+ T cells and Tregs in TDLNs and spleen. Then the levels of cytokines of IL-12, IFN-γ, L-10 and TGF-β were quantified by ELISA assays.Results: Our data showed that TAEs were more potent than TCLs to promote DC maturation and enhance MHC cross presentation, which directly contributed to more robust tumor-specific cytotoxic T lymphocyte (CTL) response. More importantly, TAEs reduced the expression of PD-L1 of DCs, thereby led to down-regulated population of Tregs in vitro. Moreover, DCTAE remarkably suppressed the tumor growth and prolonged survival rate in vivo, due to participance of CD8+ T cells and decreased Tregs in TDLNs and spleen.Conclusion: TAEs could serve to improve vaccine-elicited immunotherapy by triggering stronger DC-mediated immune responses and decreasing Tregs in the tumor microenvironment.

The distribution pattern and growth factor level in platelet-rich fibrin incorporated skin-derived mesenchymal stem cells: An in vitro study.

Background and Aim:A skin wound in an animal must be cared for to prevent further health issues. Platelet-rich fibrin (PRF) and skin-derived mesenchymal stem cells (SMSCs) have been reported to have potential in increasing the rate of wound healing. This study aimed to analyze the distribution patterns and levels of platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), and transforming growth factor-β (TGF-β) in PRF incorporated with SMSCs.Materials and Methods:This study employed a true experiment (in vitro) design with post-test only performed in the control group alone. PRF and SMSCs were extracted from the blood and skin of 16 rabbits. SMSCs were characterized using immunocytochemistry to examine clusters of differentiation for 45, 73, 90, and 105. PRF was incorporated into the SMSCs and then divided into four groups (N=32/n=8): Group A (PRF only), Group B (PRF+SMSCs, incubated for 1 day), Group C (PRF+SMSCs, incubated for 3 days), and Group D (PRF+SMSCs, incubated for 5 days). Scanning electron microscopy was used to examine the distribution pattern of SMSCs between groups. The supernatant serum (Group A) and supernatant medium culture (Group D) were collected for the measurement of PDGF, IGF, VEGF, and TGF-β using an enzyme-linked immunosorbent assay sandwich kit. An unpaired t-test was conducted to analyze the differences between Groups A and D (p<0.01).Results:Group D had the most morphologically visible SMSCs attached to the PRF, with elongated and pseudopodia cells. There was a significant difference between the levels of growth factor in Groups A and D (p=0.0001; p<0.01).Conclusion:SMSCs were able to adhere to and distribute evenly on the surface of PRF after 5 days of incubation. The PRF incorporated SMSCs contained high levels of PDGF, IGF, VEGF, and TGF- β, which may prove to have potential in enhancing wound healing.

Expression of Thanatin in HEK293 Cells and Investigation of its Antibacterial Effects on Some Human Pathogens.

BackgroundThanatin is the smallest member of Beta-hairpin class of cationic peptide derived from insects with vast activities against various pathogens.ObjectiveIn this study, the antimicrobial activity of this peptide against some species of human bacterial pathogens as well as its toxicity on NIH cells were evaluated.MethodsThanatin DNA sequence was cloned into pcDNA3.1+ vector and transformed into a DH5α bacterial strain. Then the recombinant plasmids were transfected into HEK-293 cells by calcium phosphate co-precipitation. After applying antibiotic treatment, the supernatant medium containing thanatin was collected. The peptide quantity was estimated by SDS-PAGE and GelQuant software. The antimicrobial activity of this peptide was performed with Minimum Inhibitory Concentration (MIC) method. In addition, its toxicity on NIH cells were evaluated by MTT assay.ResultsThe peptide quantity was estimated approximately 164.21 μmolL-1. The antibacterial activity of thanatin was estimated between 0.99 and 31.58 μmolL-1 using MIC method. The result of cytotoxicity test on NIH cell line showed that the peptide toxicity up to the concentration of 394.10 μmolL-1 and for 48 hours, was not statistically significant from negative control cells (P>0.05). The antimicrobial assay demonstrated that thanatin had an antibacterial effect on some tested microorganisms. The results obtained in this study also showed that thanatin had no toxicity on mammalian cell lines including HEK293 and NIH.ConclusionAntimicrobial peptides such as thanatin are considered to be appropriate alternatives to conventional antibiotics in treating various human pathological diseases bacteria.

Connexins43 expression and its effect on chondrogenesis in the cocultures of articular chondrocytes with bone marrow mesenchymal stromal cells

Objective To investigate connexins43 (Cx43) expression and its effect on chondrogenesis in the cocultures of bone marrow mesenchymal stromal cells (BMSCs)with articular chondrocytes (ACs). Methods The second passage BMSCs and ACs from New Zealand white rabbits were cocultured at the ratio of 3∶1 (BMSCs to ACs) for 21 days in vitro. The coculture patterns included the cell-cell contact coculture in the 24-well plates as experimental group and the cell-cell noncontact coculture through a Transwell chamber as control group. Cx43 inhibitor 18-ɑ glycyrrhetnic acid (GA) 50 μmol/L was supplemented in each group and subsequently cocultured for 21 days. Levels of glycosaminoglycans (GAG) in the medium supernatants of each group were detected by enzyme linked immunosorbent assay (ELISA). Expression of Cx43 and type Ⅱ collagen a1 (COL2a1) in the each group were analyzed using immunocytochemistry staining (ICC) and Western blotting. Results GAG levels of the medium supernatants, expression of Cx43 and COL2a1 were significantly high in the experimental group compared with control group [(327.517±24.612) ng/L vs. (171.612±13.163) ng/L, t=13.683, P 0.05; 0.240±0.028 vs. 0.223±0.026, t=1.086, P>0.05], whereas these all decreased significantly (t=17.541, 6.015, P 0.05). Conclusion Expression of Cx43 increases and Cx43 promotes chondrogenesis in contact coculture of BMSCs with ACs, which plays a major role in the regulation of chondrogenic differentiation. Key words: Bone marrow mesenchymal stem cell; Chondrocyte; Coculture; Connexins 43; Chondrogenesis

All-trans-retinoic acid generation is an antidotal clearance pathway for all-trans-retinal in the retina.

The present study was designed to analyze the metabolites of all-trans-retinal (atRal) and compare the cytotoxicity of atRal versus its derivative all-trans-retinoic acid (atRA) in human retinal pigment epithelial (RPE) cells. We confirmed that atRA was produced in normal pig neural retina and RPE. The amount of all-trans-retinol (atROL) converted from atRal was about 2.7 times that of atRal-derived atRA after incubating RPE cells with 10 μmol/L atRal for 24 h, whereas atRA in medium supernatant is more plentiful (91 vs. 29 pmol/mL), suggesting that atRA conversion facilitates elimination of excess atRal in the retina. Moreover, we found that mRNA expression of retinoic acid-specific hydroxylase CYP26b1 was dose-dependently up-regulated by atRal exposure in RPE cells, indicating that atRA inactivation may be also initiated in atRal-accumulated RPE cells. Our data show that atRA-caused viability inhibition was evidently reduced compared with the equal concentration of its precursor atRal. Excess accumulation of atRal provoked intracellular reactive oxygen species (ROS) overproduction, heme oxygenase-1 (HO-1) expression, and increased cleaved poly(ADP-ribose) polymerase 1 (PARP1) expression in RPE cells. In contrast, comparable dosage of atRA-induced oxidative stress was much weaker, and it could not activate apoptosis in RPE cells. These results suggest that atRA generation is an antidotal metabolism pathway for atRal in the retina. Moreover, we found that in the eyes of ABCA4-/-RDH8-/- mice, a mouse model with atRal accumulation in the retina, the atRA content was almost the same as that in the wild type. It is possible that atRal accumulation simultaneously and equally promotes atRA synthesis and clearance in eyes of ABCA4-/-RDH8-/- mice, thus inhibiting the further increase of atRA in the retina. Our present study provides further insights into atRal clearance in the retina.

Conversion of inorganic selenium to organic form(s) by Lactobacillus acidophilus

The bioconversion of 2 forms of inorganic selenium namely selenite (SeIV) and selenate (SeVI) to organicform(s) by Lactobacillus acidophilus strain was investigated. The cultured media (MRS) was supplemented with 1,2, 5, 10, 20 ppm of Se in the form of sodium selenite (Na2SeO3, SeIV) or sodium selenate (Na2SeO4, SeVI) and incubatedat 37°C up to 24 hr Both Se forms showed no marked effect on the bacterial growth indicating no cytotoxicityat these concentrations. However, the media supplemented with 5, 10 and 20 ppm of Se(IV), but not Se(VI), becamereddish after 24 hr of incubation with increasing the red color intense with increasing the Se content in the media. Thescanning electron microscope (SEM) investigation clarified the presence of Se-nano particles (SeNPs) in the media.Se speciation of the cultured media supernatant and its corresponding cell fractions using HPLC-ICP-MS techniqueindicated that the bioconversion rate of Se to organic form(s) was extremely higher in Se(IV) than Se(VI) in both fractionshowever, the cell fractions contained the highest content. The organic Se gradually increased in both fractionswith increasing the media Se content. The inorganic Se was completely bio-converted to organic form(s) without anyresidual only in the medium contained 1 ppm Se(IV). Our results demonstrate the ability of L. acidophilus to convertSe(IV) but not Se(VI), at a limit concentration of 1 ppm and accumulate organic Se form(s) in the cell fraction. Theseresults confirm the possible bio-production of organic Se enriched fermented dairy products.

Conversion of inorganic selenium to organic form(s) by Lactobacillus acidophilus

The bioconversion of 2 forms of inorganic selenium namely selenite (SeIV) and selenate (SeVI) to organicform(s) by Lactobacillus acidophilus strain was investigated. The cultured media (MRS) was supplemented with 1,2, 5, 10, 20 ppm of Se in the form of sodium selenite (Na2SeO3, SeIV) or sodium selenate (Na2SeO4, SeVI) and incubatedat 37°C up to 24 hr Both Se forms showed no marked effect on the bacterial growth indicating no cytotoxicityat these concentrations. However, the media supplemented with 5, 10 and 20 ppm of Se(IV), but not Se(VI), becamereddish after 24 hr of incubation with increasing the red color intense with increasing the Se content in the media. Thescanning electron microscope (SEM) investigation clarified the presence of Se-nano particles (SeNPs) in the media.Se speciation of the cultured media supernatant and its corresponding cell fractions using HPLC-ICP-MS techniqueindicated that the bioconversion rate of Se to organic form(s) was extremely higher in Se(IV) than Se(VI) in both fractionshowever, the cell fractions contained the highest content. The organic Se gradually increased in both fractionswith increasing the media Se content. The inorganic Se was completely bio-converted to organic form(s) without anyresidual only in the medium contained 1 ppm Se(IV). Our results demonstrate the ability of L. acidophilus to convertSe(IV) but not Se(VI), at a limit concentration of 1 ppm and accumulate organic Se form(s) in the cell fraction. Theseresults confirm the possible bio-production of organic Se enriched fermented dairy products.

Development of a Human in Vitro Cell Model for Haemophilia a

Aim Haemophilia A is an X-chromosome linked hereditary bleeding disorder caused by loss-of-function mutations in the clotting factor VIII (FVIII) gene resulting in either a deficit or a total lack of the corresponding activity. Although the quality of the standard replacement therapy has improved significantly over the last decades, patients still require FVIII administration at regular intervals and frequently develop an immune response against the exogenous protein that interferes with FVIII function. FVIII gene therapy could provide a less intrusive and perhaps safer alternative to current FVIII replacement therapy. Here, we aimed to develop a human based cell model to facilitate future effective development and evaluation of therapeutic gene therapy approaches for haemophilia A. In order to closely model key challenges for such a development, we chose to introduce patient-specific FVIII mutations into immortalized human hepatic sinusoidal endothelial cells, which are the cells that naturally secret FVIII. Methods First, we immortalized primary human hepatic sinusoidal endothelial cells (HHSEC), the natural cell of FVIII synthesis in humans, by lentiviral transduction of a doxycycline-inducible SV40-Large T oncogene. After establishment of the HHSEC cell line, we chose from the FVIII gene variant database (European Association for Haemophilia and allied disorders) five different FVIII mutations resulting in a severe form of haemophilia A. All of the selected variants were small deletions resulting in frameshift mutations, leading to a loss-of-function FVIII gene product. The FVIII mutations were introduced by lentiviral transduction of sgRNAs causing DNA double strand breaks at the respective positions together with the RNA-guided CRISPR/Cas9 (saCas9) endonuclease. Characterization and verification of immortalized HHSEC with and without FVIII mutation were performed by sequencing, immunofluorescence and aPTT-based FVIII activity assays. Results Our first experiments resulted in the successful immortalization of primary HHSEC cells. Since the cells were conditionally immortalized, they returned to the primary cell state upon removal of doxycycline. By western blotting we could demonstrate SV40-Large T oncogene expression under doxycycline conditioning, and a decrease in expression after doxycycline withdrawal. Additionally, using cumulative proliferation assays (cell counting assay), we could estimate a doubling time of almost 2 days for the immortalized HHSEC under doxycycline treatment and strong proliferative disadvantage in absence of doxycycline. Subsequently, we examined the FVIII activityof immortalized HHSEC using immunofluorescence microscopy and an aPTT-based FVIII clotting assay. Immunohistochemical staining for FVIII in the immortalized HHSEC revealed a strong signal resembling vesicular structures as expected. Using the established aPTT-based FVIII clotting assay, which is also used in the clinic to estimate FVIII residual activities, we could demonstrate functional FVIII activity of 75% up to 99% of normal human plasma. FVIII activity was measured in medium supernatant after 24h of incubation, at least at three independent days. The FVIII activity showed always similar values and stayed constant over the time. We successfully introduced two out of five haemophilia A patient-specific mutations into the FVIII gene of the HHSEC. Sequence analysis revealed for one mutant cell line a predominant deletion of seven base pairs and for the other cell line a mixed indel generation both resulting in frameshift mutations. FVIII clotting assays showed a reduction of FVIII activity to 40%. Likewise, the immunohistochemical staining showed reduced intensity. In ongoing experiments, we currently generate a full FVIII knock-out cell line by subcloning, in order to get cell lines strongly resembling FVIII activity levels that are seen in severe haemophilia A patients (&amp;lt;1%). Conclusion By immortalization and transduction of HHSECs we generated a human haemophilia A cell line model based on mutagenesis of the endogenous genetic FVIII locus in the cell line, which mainly accounts for FVIII production in humans. This cell model will be used to study FVIII synthesis, signaling and processing and, in addition, aids in the development of new strategies for haemophilia A gene therapy or new FVIII preparations. Disclosures Miesbach: Bayer, Chugai, Novo Nordisk, Octapharma, Pfizer, Takeda/Shire, UniQure: Speakers Bureau; Bayer, BioMarin, CSL Behring, Chugai, Freeline, Novo Nordisk, Octapharma, Pfizer, Roche, Takeda/Shire, UniQure: Consultancy; Bayer, Novo Nordisk, Octapharma, Pfizer, Takeda/Shire: Research Funding.

LAG3 Inhibition Decreases AML-Induced Immunosuppression and Improves T Cell-Mediated Killing

Background: Acute Myeloid Leukemia (AML) is an aggressive hematologic malignancy with known immune dyregulation. In addition to their capacity to rapidly divide, AML cells directly inhibit the activation and proliferation of immune cells in culture. Immunosuppressive features observed in the bone marrow of AML patients include upregulation of Tregs and production of immunosuppressive cytokines (e.g., TGFβ). Irradiating AML cells diminishes their immunosuppressive capacity while maintaining antigen presentation, leading to increased activation of T cells in co-culture. We subsequently identified the immune checkpoint LAG3 as an important mediator of AML-induced immunosuppression and LAG3 modulation as potential treatment strategy. Methods: Normal PBMC were isolated from healthy donors. PBMC were co-cultured with non-irradiated and irradiated (40 Gy) human AML cell lines (Kasumi1 (K1), THP1) separately at a 1:2 ratio. On day 3 of co-culture, immunophenotypic characterization of T cells was performed on a flow cytometer using the following surface markers: CD3, CD4, CD8, CD25, CD137, CD154, PD-1, TIM3, TIGIT, and LAG3 and intracellular IFNg and FOXP3. Supernatant from co-culture media were analyzed for cytokine (IL-2, IL-6, IL-10 &amp; TGFβ) secretion by ELISA. CFSE-labeled AML cells were incubated with healthy donor PBMCs in the presence or absence of LAG3, then viability was measured by 7-ADD on flow cytometry. PBMCs were also isolated from AML patients' peripheral blood and mononuclear cells were isolated from their respective bone marrow samples. Primary AML cultures were established in RPMI complete media with 20% FBS. CFSE-7-ADD killing assay was conducted after incubation of AML cells with autologous PBMCs. Results: Healthy donor PBMC co-cultured with irradiated K1 AML cells showed higher intracellular IFNg expression (11.8% ± 3.1 v. 7% ± 3.3; n=7, P=0.012) and higher CD137 expression (9.3% ± 1.21 v. 5.7% ± 3.4; n=7, P&amp;lt;0.001) on CD8+ T cells, and higher CD154 expression on CD4+ cells (44.7% ± 20.3 v. 26.3% ± 14.2; n=5, P=0.002) when compared to the non-irradiated K1-PBMC co-cultures. There were fewer Tregs (CD4+ CD25+ FOXP3+) in the PBMC co-cultured with irradiated K1 cells (1.96% ± 0.37 v. 3.39% ± 0.58; n=4, P=0.03) compared to the non-irradiated K1-PBMC co-cultures. LAG3 expression on CD8+ T cells co-cultured with irradiated K1 was decreased (11.8% ± 2.4 v. 17.5% ± 2.5; n=4, P=0.002) compared to the PBMC co-cultured with non-irradiated K1 cells. No other changes in checkpoint expression on CD8+ T cells were observed. No changes were observed in MHCI or PDL1 expression on non-irradiated K1 AML cells before or after co-culture with PBMC. We observed similar findings with healthy donor PBMC co-cultured with a different AML cell line, THP1; CD137 expression was higher on CD8+ T cells (17.6% v. 6.5%; P=0.02, n=3). ELISA of the supernatant of culture media showed higher mean OD for secreted TGFβ in the non-irradiated AML co-cultures compared to the irradiated AML co-cultures at 6 hours (2.5 v. 2.0, P=0.03, n=3) and 72 hours (7.9 v. 5.3, P=0.04, n=3). Adding anti-LAG3 antibody (3DS223H; 100 µg/ml) to PBMC co-cultured with non-irradiated AML cells resulted in higher IFNg (16.3% v. 6.6%, P=0.01, n=4) and CD137 expression (6.5% v. 4.1%, p=.007, n=4) on CD8+ cells and fewer Tregs (1.7% v. 3.8%, P=.04, n=4) compared to no antibody added. Healthy donor PBMC (n=3) were incubated with CFSE labeled AML cells (K1 and THP1) separately at an effector:target ratio of 5:1. The addition of anti-LAG3 antibody lead to increased killing of both K1 and THP1 AML cells at 4 and 24 hours (Figure 1A). To eliminate the HLA mismatch effect, we incubated PBMC from AML patients with autologous AML cells in the presence or absence of anti-LAG3 (Figure 1B). MHC-I blocking (W6/32, 30 µg/ml) lead to inhibition of cell mediated killing in the presence of anti-LAG3 (Figure 1B). Conclusion: In this in vitro model, AML cells showed immunosuppressive features with decreased activation of CD8+ T cells, upregulation of Tregs, increased secretion of TGFβ and higher expression of LAG3 on CD8+ T cells. Antibody blocking of LAG3 mitigated this effect, resulting in increased activation of T cells, fewer T regs and improved MHC-I-mediated killing against AML cells. These results demonstrate that the immunosuppressive effects of AML cells can be modulated through inhibition of LAG-3, suggesting a potential strategy for future combination therapy in AML. Disclosures Lin: Jazz Pharmaceuticals: Honoraria; Pfizer: Membership on an entity's Board of Directors or advisory committees.

LAB-SCALE PRODUCTION OF RHAMNOLIPID BY PSEUDOMONAS AERUGINOSA A3 AND STUDY ITS SYNERGISTIC EFFECT WITH CERTAIN ANTIBIOTICS AGAINST SOME PATHOGENIC BACTERIA

This study was aimed to obtain the considerable amount of the biosurfactant (rhamnolipid) from local isolate of Pseudomonas aeruginosa on industrial-scale. A 7L laboratory bioreactor was appointed to accomplish this task and all conditions of production were set as previously by batch culture experiments. Bioreactor with 3.5 L working volume was conducted for 120 h to obtain 16 g/L at 30oC and 6.5 pH. The supernatant of production medium was used to reduce the surface tension of water from 72 to 27 mN/m, and the emulsification index was reached to 67%. The product was extracted with a mixture of solvents, purified by silica gel using a column of glass chromatography (3.5 × 30cm), and characterized by TLC and FTIR where it turn out that the biosurfactant is composed of both mono- and di-rhamnolipid. Mono-RL was tested for its antibacterial activity against some clinical isolates of Gram-positive and Gram-negative bacteria to obtain the MIC, which was about 250µg/ml, also at same concentration, the toxicity against Rat embryonic fibroblast (REF) cell line was reached to 23%. The combination effect of Mono-RL with some antibiotics was studied against pathogenic clinical isolates revealed that there is a synergistic effect with ampicillin against S. aureus and Serratia marcescens, with cefotaxime against E. coli and Kelbsiella pneumoniae, same as with tetracycline.

P2.01-91 Exosomal Analysis of ALK Rearrangements by Spin Column with Porous Glass Filter

ALK rearrangements account for about 3-5% of non-small cell lung cancer (NSCLC). ALK-tyrosin kinase inhibitors (TKI) demonstrated robust efficacy compared with cytotoxic chemotherapy in patients with ALK alterations detected in the tumor tissues. Identifying ALK rearrangement was performed using tissue samples, which are not always available. The spin column with porous glass filter has been developed by Nagoya university and AGC Inc, resulting in highly efficient and easy to use exosome isolation. The exosomes contain various molecules of their cell of origin, including proteins and RNA. The purpose of this study was to explore the spin column to capture exosome and detect ALK alterations in exosomal RNA from blood. The supernatant of cell culture medium (H3122, H2228) and plasma samples from 3 patients with ALK-positive NSCLC were passed through the filter using a conventional centrifugation. Exosome captured in the filter was lysed with reagent for RNA extraction. The total RNA was retrotranscripted by random primers. The ALK rearrangement in the exosome were determined by RT-qPCR and DNA sequencing. EML4-ALK variant 1 and 3 were detected in exosome from 500μL of culture supernatant of H3122 and H2228, respectively. In the analysis of exosome in plasma from patients with EML4-ALK determined by fluorescence in situ hybridization and immunohistochemistry, EML4-ALK variant 1 was successfully detected in all cases. Exosome remains relatively stable in the blood, making it an attractive target for liquid biopsy. Our preliminary results showed potential capability in the detection of ALK alteration in exosomes from blood. These findings require confirmation in further studies with a larger number of patients with ALK-positive NSCLC.

M81 INVESTIGATING THE FUNCTIONAL SIGNIFICANCE OF THE SCHIZOPHRENIA-ASSOCIATED DYSREGULATION OF MIR-1271-5P EXPRESSION

Sterile triploid hybrids (3n = 150) of gold fish (Carassius auratus red var., ♀, 2n = 100) and allotetroploid (♂, 2n = 100) display obviously improved disease resistance and much enhanced growth rate than their parents, which have been cultured widely in China. In this paper, one of the type I IFNs of triploid hybrid (3nIFNa2) has been cloned and characterized. The full-length cDNA of 3nIFNa2 gene consists of 715 nucleotides and the predicted 3nIFNa2 contains 183 amino acids. The transcription of 3nIFNa2 gene was detected in all the examined tissues of triploid hybrid and the mRNA level of 3nIFNa2 was obviously enhanced in response to SVCV and GCRV infection. 3nIFNa2 has been detected in the whole cell lysate of HEK293T cells transfected with plasmids expressing 3nIFNa2 but not in the supernatant media. EPC cells transfected with plasmid expressing 3nIFNa2 at 24 h before SVCV and GCRV infection showed obviously decreased cytopathic effect; and the virus titers in the supernatant media were much lower than those of the control cells. Glycosidase digestion analysis demonstrates that 3nIFNa2 is modified with N-linked glycosylation, which occurs on the asparagine (N) of residue 177 of this cytokine. The un-glycosylated mutant 3nIFNa2-N177Q shows the similar antiviral ability as that of 3nIFNa2, which suggests that the N-linked glycosylation does not contribute directly to its antiviral property. All the above data support the conclusion that 3nIFNa2 is an intracellular cytokine functioning importantly in host antiviral innate immunity.