RPMI Medium Research Articles

1681. Activity of the Novel Engineered Antimicrobial Peptide PLG0206 Against Staphylococci and Enterococci

Abstract Background PLG0206 is an investigational, engineered cationic antimicrobial peptide designed to overcome the shortcomings of other natural AMPs, such as toxicity and limited activity. PLG0206 has recently been shown to be well tolerated and safe when administered i.v. in a Phase 1 study. The initial proposed indication for this peptide is the treatment of periprosthetic joint infections via irrigation due to a broad spectrum of activity and anti-biofilm properties. This study evaluated the activity of PLG0206 and comparator antimicrobials against staphylococci and enterococci, causes of periprosthetic joint infections, from the IHMA repository of isolates collected from various world-wide locations in 2019. Methods Isolates tested included Enterococcus faecalis (77), E. faecium (75), methicillin-resistant Staphylococcus aureus (MRSA, 180), methicillin-susceptible S. aureus (MSSA, 121) and 152 coagulase-negative staphylococci (CoNS) comprised of S. epidermidis (113), S. haemolyticus (31), S. hominis (4), S. lugdunensis (1), S. saprophyticus (2) and S. simulans (1). Minimum inhibitory concentrations (MICs) were determined by CLSI broth microdilution in cation-adjusted Mueller Hinton broth (CA-MHB), except for PLG0206 which was tested in RPMI medium due to plate-reading difficulties with CA-MHB. The susceptibility of comparators was determined using the 2022 CLSI breakpoints. MDR was defined as resistance to ≥ 3 antibacterial classes. Results When tested in RPMI, 18 E. faecalis, 29 E. faecium, 9 S. aureus and 7 CoNS were unable to grow. Summary MIC and susceptibility data for PLG0206 and comparators just including those isolates able to grow in RPMI are shown in the Table. Identical MIC50 and MIC90 values were obtained for PLG0206 when tested against MDR E. faecium (38), MDR CoNS (63) and MDR MRSA (67). Insufficient numbers of MDR MSSA (7) and MDR E. faecalis (4) were tested to accurately evaluate. Conclusion PLG0206 was the most potent antimicrobial overall (based on MIC50 and MIC90) against enterococci and CoNS and compared well with the comparators against MRSA and MSSA. These data support the evaluation of this novel antimicrobial peptide as a treatment option for periprosthetic joint infections, including those caused by MDR strains. Disclosures David Huang, MD, PhD, Peptilogics: Employee Jonathan Steckbeck, PhD, Peptilogics: I am CEO of Peptilogics.

1680. Activity of the Novel Engineered Antimicrobial Peptide PLG0206 Against Enterobacterales isolates

Abstract Background PLG0206 is an investigational, engineered cationic antimicrobial peptide designed to overcome the shortcomings of other natural AMPs, such as toxicity and limited activity. PLG0206 has recently been shown to be well tolerated and safe in a phase 1 study. This study evaluated the activity of PLG0206 and comparator antimicrobials against Enterobacterales isolates from the IHMA repository collected from various world-wide locations in 2019. Methods Isolates tested included Citrobacter spp. (151), Enterobacter cloacae (152), Escherichia coli (300), Klebsiella pneumoniae (300), Morganella morganii (43), Proteus spp. (152), Providencia spp. (61) and Serratia marcescens (45). Minimum inhibitory concentrations (MICs) were determined by CLSI broth microdilution in cation-adjusted Mueller Hinton broth (CA-MHB), except for PLG0206 which was tested in RPMI medium due to plate-reading difficulties with CA-MHB. The susceptibility of comparators was determined using the 2022 CLSI breakpoints. MDR was defined as resistance to ≥ 3 antibacterial classes. Results Summary MIC and susceptibility data against Citrobacter spp., E. coli, E. cloacae, and K. pneumoniae are shown in the Table. PLG0206 retained activity against MDR isolates of Citrobacter spp. (26), E. coli (92), E. cloacae (45), and K. pneumoniae (132) with MIC50/90 values of 1/2, 1/2, 1/16 and 8/16 μg/mL, respectively. PLG0206 was essentially inactive against the Morganellaceae and Yersiniaceae isolates tested with MIC50 and MIC90 values of > 32 μg/mL (data not shown). Conclusion PLG0206 was active against isolates from the family Enterobacteriaceae, including MDR strains. The spectrum of activity of PLG0206 ranged from being most active against Citrobacter spp. and least active against K. pneumoniae. These data suggest that the novel antimicrobial peptide PLG0206 could be a potential treatment option against infections caused by Enterobacteriaceae isolates. Disclosures David Huang, MD, PhD, Peptilogics: Employee Jonathan Steckbeck, PhD, Peptilogics: I am CEO of Peptilogics.

The Influence of Different Culture Media on the Growth and Recombinant Protein Production of Iranian Lizard Leishmania Promastigote.

Leishmania is a eukaryotic protozoan parasite belonging to the Trypanosomatidae family. The Iranian Lizard Leishmania (I.L.L.), which is nonpathogenic to mammals, shows great promise to be used as an expression system for recombinant protein production. Unlike other Leishmania strains, the ideal culture medium for I.L.L. has not been established, although it is commonly cultured in the RPMI1640 medium. We investigated the growth rate of the wild and recombinant I.L.L. in BHI, RPMI1640, LB, and M199 media with and without FBS, hemin, or lyophilized rabbit serum. Subsequently, the expression rate of the recombinant protein in these media was compared. The growth rate of I.L.L. in RPMI1640 medium and LB broth was similar and supplementation with 10% FBS did not affect the growth rate. The amount of protein expression in the LB medium was higher than in the other three media. The LB broth is an appropriate medium for I.L.L. culture and recombinant protein production.

Dibutyl phthalate affects insulin synthesis and secretion by regulating the mitochondrial apoptotic pathway and oxidative stress in rat insulinoma cells

Dibutyl phthalate (DBP) is a typical phthalate (PAEs). The environmental health risks of DBP have gradually attracted attention due to the common use in the production of plastics, cosmetics and skin care products. DBP was associated with diabetes, but its mechanism is not clear. In this study, an in vitro culture system of rat insulinoma (INS-1) cells was established to explore the effect of DBP on insulin synthesis and secretion and the potential mechanisms. INS-1 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and treated with 15, 30, 60 and 120 μmol/L of DBP and dimethyl sulfoxide (vehicle, < 0.1%) for 24 h. The contents of insulin in the intracellular fluid and the extracellular fluid of the cells were measured. The results showed that insulin synthesis and secretion in INS-1 cells were significantly decreased in 120 μmol/L DBP group. The apoptosis rate and mitochondrial membrane potential of INS-1 cells were measured by flow cytometry with annexin V-FITC conjugate and PI, and JC-1, respectively. The results showed that DBP caused an increase in the apoptosis rate and a significant decrease in the mitochondrial membrane potential in INS-1 cells in 60 μmol/L and 120 μmol/L DBP group. The results of western blot showed that the expression of Bax/Bcl-2, caspase-3, caspase-9 and Cyt-C were significantly increased. Meanwhile, the level of oxidative stress in INS-1 cells was detected by fluorescent probes DCFH-DA and western blot. With the increase of DBP exposure, the oxidative stress levels (MDA, GSH/GSSG) were increased; and the antioxidant index (SOD) levels were decreased. Our experimental results provide reliable evidence that DBP induced apoptosis and functional impairment in INS-1 cells through the mitochondrial apoptotic pathway and oxidative stress. Therefore, we hypothesized that interference with these two pathways could be considered in the development of preventive protection measures.

Prolonged culture of human pancreatic islets under glucotoxic conditions changes their acute beta cell calcium and insulin secretion glucose response curves from sigmoid to bell-shaped.

The rapid remission of type 2 diabetes by a diet very low in energy correlates with a marked improvement in glucose-stimulated insulin secretion (GSIS), emphasising the role of beta cell dysfunction in the early stages of the disease. In search of novel mechanisms of beta cell dysfunction after long-term exposure to mild to severe glucotoxic conditions, we extensively characterised the alterations in insulin secretion and upstream coupling events in human islets cultured for 1-3 weeks at ~5, 8, 10 or 20 mmol/l glucose and subsequently stimulated by an acute stepwise increase in glucose concentration. Human islets from 49 non-diabetic donors (ND-islets) and six type 2 diabetic donors (T2D-islets) were obtained from five isolation centres. After shipment, the islets were precultured for 3-7 days in RPMI medium containing ~5 mmol/l glucose and 10% (vol/vol) heat-inactivated FBS with selective islet picking at each medium renewal. Islets were then cultured for 1-3 weeks in RPMI containing ~5, 8, 10 or 20 mmol/l glucose before measurement of insulin secretion during culture, islet insulin and DNA content, beta cell apoptosis and cytosolic and mitochondrial glutathione redox state, and assessment of dynamic insulin secretion and upstream coupling events during acute stepwise stimulation with glucose [NAD(P)H autofluorescence, ATP/(ATP+ADP) ratio, electrical activity, cytosolic Ca2+ concentration ([Ca2+]c)]. Culture of ND-islets for 1-3 weeks at 8, 10 or 20 vs 5 mmol/l glucose did not significantly increase beta cell apoptosis or oxidative stress but decreased insulin content in a concentration-dependent manner and increased beta cell sensitivity to subsequent acute stimulation with glucose. Islet glucose responsiveness was higher after culture at 8 or 10 vs 5 mmol/l glucose and markedly reduced after culture at 20 vs 5 mmol/l glucose. In addition, the [Ca2+]c and insulin secretion responses to acute stepwise stimulation with glucose were no longer sigmoid but bell-shaped, with maximal stimulation at 5 or 10 mmol/l glucose and rapid sustained inhibition above that concentration. Such paradoxical inhibition was, however, no longer observed when islets were acutely depolarised by 30 mmol/l extracellular K+. The glucotoxic alterations of beta cell function were fully reversible after culture at 5 mmol/l glucose and were mimicked by pharmacological activation of glucokinase during culture at 5 mmol/l glucose. Similar results to those seen in ND-islets were obtained in T2D-islets, except that their rate of insulin secretion during culture at 8 and 20 mmol/l glucose was lower, their cytosolic glutathione oxidation increased after culture at 8 and 20 mmol/l glucose, and the alterations in GSIS and upstream coupling events were greater after culture at 8 mmol/l glucose. Prolonged culture of human islets under moderate to severe glucotoxic conditions markedly increased their glucose sensitivity and revealed a bell-shaped acute glucose response curve for changes in [Ca2+]c and insulin secretion, with maximal stimulation at 5 or 10 mmol/l glucose and rapid inhibition above that concentration. This novel glucotoxic alteration may contribute to beta cell dysfunction in type 2 diabetes independently from a detectable increase in beta cell apoptosis.

Studies on intraocular transplantation of pancreatic cells in a large‐eyed preclinical animal model

AbstractPurpose: Pancreatic islet transplantation (PIT) to the anterior chamber of the eye (ACE) has been shown to correct hyperglycemia and restore/improve glycemic control in rodents and non‐human primates. These studies also suggested that the eye may be in several ways a superior host site compared to the conventional hepatic portal system. In order to bring this technology to the clinic, it is essential to establish a large‐eyed non‐primate transplantation model. This proposal aims at establishing a rabbit ACE model of allogeneic PIT. We expect to gain insight into isolated islets function as well as ACE grafting efficiency and histological morphology in the large‐eyed rabbit model.Methods: Pancreatic islets (PI) were isolated from Dutch Belted rabbits by injection of collagenase A into the pancreatic interstitium, digestion and hand picking under a stereo microscope. PI were cultured in RPMI medium overnight in a standard tissue culture incubator prior to assessment of purity. In vitro functional capacity of rabbit PI was assessed by glucose‐stimulated insulin release and intracellular Ca2+ imaging assays. PI were injected into the ACE an allogenic Dutch Belted rabbit using surgical procedures routinely applied in ophthalmology. PI engraftment and cellular content was analysed by anterior segment optical coherence tomography (ASOCT) and immunohistofluorescence (IHF).Results: Isolated PI were of high purity with an approximate yield of 600–800 islets per pancreas. Preliminary results of functional capacity demonstrate that islets are responsive to high glucose stimulus, despite some variability. Allogeneic transplantation of isolated PI into the ACE of a Dutch Belted rabbit was followed by ASOCT. Engraftment was confirmed by IHF for vimentin. IHF suggests ACE engrafted islets are viable and maintain alpha, beta and delta cells.Conclusions: The presented isolation method renders high purity and functional rabbit PI. Allogeneic transplantation of PI into the ACE was successful using clinically relevant procedures and resulted in islets engraftment and long‐term survival. These results may have implications for the clinical treatment of diabetes mellitus, particularly regarding patients with indications for PIT.

Effect of Cross-Linking Cations on In Vitro Biocompatibility of Apple Pectin Gel Beads.

The study aimed to compare the in vitro biocompatibility of pectin gels formed by different cross-linking cations. Hydrogel beads named CaPG, ZnPG, FePG, and AlPG were prepared from 4% solutions of apple pectin using ionotropic gelling with CaCl2, ZnCl2, FeCl3, and AlCl3, respectively. Cations influenced the gel strength of the wet gel beads in the following order (least strong) Ca2+ < Zn2+ < Fe3+~Al3+ (most strong). The swelling degree of the CaPG beads after 24 h of incubation in the RPMI-1640 medium was 104%, whereas the ZnPG, FePG, and AlPG beads swelled by 76, 108, and 134%, respectively. The strength of the pectin gel decreased significantly after incubation in the RPMI-1640 medium for 24 h, regardless of the cross-linking cation, although the FePG beads remained the strongest. All the pectin beads adsorbed serum proteins to a low degree, however the serum protein adsorption by the ZnPG and FePG beads (1.46 ± 0.87 and 1.35 ± 0.19 µg/mm2) was more than the CaPG and AlPG beads (0.31 ± 0.36 and 0.44 ± 0.25 µg/mm2). All the pectin beads reduced the production of TNF-α and IL-10 by hPBMCs in response to LPS stimulation. The IL-1β response of cells to LPS was significantly reduced by the CaPG, ZnPG, and FePG beads, whereas the AlPG beads enhanced it twofold. The CaPG, FePG, and AlPG beads had no cytotoxicity. The viability of hPBMCs and human fibroblasts incubated with ZnPG beads was 5.3 and 7.2%, respectively. Thus, the use of different cross-linking cations changed the properties of the pectin gel, which is important for biocompatibility.

Photoinduced Carbene for Effective Photodynamic Therapy Against Hypoxic Cancer Cells

Photoinduced Carbene for Effective Photodynamic Therapy Against Hypoxic Cancer Cells

Identification of protein profile in metacyclic and amastigote-like stages of Leishmania tropica: a proteomic approach

Leishmaniasis is a tropical disease that leads to various clinical phenotypes. This study aimed to investigate protein expression changes in metacyclic and amastigote-like stages of L. tropica isolated from Iranian cutaneous leishmaniasis patients. Isolated samples were cultured and species type identified using PCR–RFLP technique. The promastigotes were grown in RPMI1640 media and differentiated to metacyclic and amastigote-like forms, followed by the extracted proteins of both successive stages carried out for proteomics and bioinformatics analysis. Using SWATH-MS quantitative proteomics technique, a total 176 and 155 distinct proteins were identified in metacyclic and axenic amastigote stages, respectively. Of these, 65 proteins were altered significantly (p-value < 0.05 and fold change ≥ 2) between studied stages. Several gene ontology (GO) categories were enriched for biological process during conversion of metacyclic promastigotes into amastigote-like, which “metabolic process” (GO: 0044281, P-Value: 6.52e-5), and “translation” (GO: 0006412, p-value: 5.01e–14) were disclosed as the top category in up and down-regulated proteins, respectively. Also, the KEGG pathway analysis indicated “metabolic pathways” and “ribosome” term as the most important pathways in up and down-regulated proteins, respectively. According to protein interaction network analysis, enolase (ENOL) has been detected as main hub proteins during differentiation, followed by Putative NADH-dependent fumarate reductase (LmjF.35.1180) and 40S ribosomal protein S2 (LmjF.32.0450). Overall, protein changes possibly play important roles in L. tropica biology. Anabolic pathways were down-regulated, whereas catabolic pathways were up-regulated during L. tropica differentiation. These protein expression changes could provide parasite survival in host macrophages, and could use as novel potential drug and vaccine targets for leishmaniasis.Graphical

NUCKS1 promotes breast cancer cell proliferation and metastasis via PI3K/ AKT pathway

Purpose: To investigate the role of nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1) in breast cancer. &#x0D; Methods: Breast cancer cells were maintained in RPMI-1640 medium containing 10 % fetal bovine serum in a incubator at 37 °C. Cell proliferation was determined by CCK8 and colony formation assays. Flow cytometry and Transwell assays were used to determine cell cycle and metastasis, respectively. &#x0D; Results: Expression of NUCKS1 was significantly elevated in breast cancer (p &lt; 0.01). Overexpression of NUCKS1 significantly increased cell viability (p &lt; 0.01), and promoted proliferation of breast cancer cells. Knockdown of NUCKS1 inhibited cell proliferation, and induced cell cycle arrest at G1 phase. However, overexpression of NUCKS1 promoted cell cycle progression via down-regulation of p21 and up-regulation of cyclin D1 and CDK1. Cell migration and invasion were induced by overexpression of NUCKS1, and suppressed by silencing of NUCKS1. Overexpression of NUCKS1 enhanced p-AKT and p-PI3K expression, while knockdown of NUCKS1 reduced the expression of p-AKT and p-PI3K in breast cancer cells. &#x0D; Conclusion: NUCKS1 promotes breast cancer cell proliferation and metastasis via activation of PI3K/AKT signaling. The silencing of NUCKS1 can be used as a strategy to develop therapies for the management of breast cancer.

Dracocephalum kotschyi Boiss. In Vitro Efficacy on Growth and Apoptosis Induction in Leishmania major Promastigotes.

Dracocephalum kotschyi Boiss. is a plant generally used in modern medicine to treat many human illnesses. It is also used to prevent tumor cell proliferation throughout the world. This study's objective was to evaluate this plant's in vitro efficacy on growth and apoptosis induction in Leishmania major promastigotes. To do this, the essential oil is extracted for the test following the collection and identification of D. kotschyi. The essential oil was analyzed using a GC-MS analyzer. Promastigotes of L. major were cultured in RPMI-1640 media, and the MTT assay and a flow cytometry analysis were carried out on promastigotes that had entered the log phase. To differentiate between viable, necrotic, and apoptotic treated or untreated promastigotes, the flow cytometry method of double staining with annexin V-FLUOS and propidium iodide (PI) was used. Given the results obtained, 11 phytochemicals were identified in the essential oil of this plant. Copaene (22.15%), methyl geranate (16.31%), geranial (13.78%), and carvone (11.34%) were the main substances. The essential oil of D. kotschyi inhibits the proliferation of L. major promastigotes at 921 μg/mL, 252 μg/mL, and 416 μg/mL, respectively, after 24 h, 48 h, and 78 h. The cells were divided into four quadrates based on cell phases using the flow cytometry approach by double staining with annexin V-FLUOS and propidium iodide (PI): necrosis (Q1), late apoptosis (Q2), early apoptosis (Q3), and viable (Q4) quadrates. Overall, it is apparent that the different concentrations induced cell apoptosis in promastigotes. Observation under the light microscope at ×100 magnification showed that the different doses of D. kotschyi essential oil caused apparent alterations in the treated promastigotes. In this work, D. kotschyi essential oils induce programmed death on L. major promastigotes. This study opens many research perspectives, such as investigating the mechanisms of action and the production of a phytomedicine based on this plant.

Antitumor Effects of Turmeric on OVCAR-3 Ovarian Cancer Cell Lines.

Ovarian cancer is the deadliest gynecological malignancy, usually not detected until the late stages. In vitro cell culture is a method used to study the behavior of cells in a controlled environment. Turmeric has attracted the attention of scientists due to its anticancer potential. OVCAR-3 cells were cultured in RPMI medium with 100 units/mL-100 μg/mL of penicillin-streptomycin and 10% foetal bovine serum in a CO2 incubator. Turmeric extract was diluted in DMSO. Different concentrations of turmeric extract were prepared. Annexin-V staining was performed to test the translocation of phosphatidylserine to the outer side of the cell membrane as a clear indicator of apoptosis. Turmeric extract significantly reduced the viability of OVCAR-3 cells both within 24 and 48 hours of exposure. OVCAR-3 cells were treated with IC50 concentration of turmeric extract for 24 hours. 82.60% of cells were viable. The percentages of the dead, early apoptotic, and late apoptotic cells were detected to be 0.80%, 9.70%, and 6.90%, respectively. Untreated OVCAR-3 cells had migration ability. OVCAR-3 cells exposed to an IC50 concentration of turmeric extract for 24 hours did not close the scratch area. In this research, anticancer effects of turmeric have been demonstrated by different analysis methods.

COMPARISON OF DIFFERENT METHODOLOGIES FOR EXPANSION OF NATURAL KILLER CELLS FROM PERIPHERAL BLOOD AND UMBILICAL CORD BLOOD FOR IMMUNOTHERAPY APPLICATIONS

In Immunotherapy, several clinical studies with Natural Killer (NK) cells have shown promising results in the treatment of onco-hematological diseases due to their high cytotoxicity against tumor cells. There are several protocols for the expansion of these cells, but there is no consensus of a gold standard protocol that provides large numbers of functional NK cells required for conducting clinical studies. Thus, we propose the development of a protocol for the expansion of these cells with the subsequent intention of applying them as cell therapy product in the Hematology Service of the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP). Our aim was to compare different methodologies and establish a standardized protocol for the isolation and expansion of NK cells from peripheral blood (PB) and umbilical cord blood (UCB). We also characterized the expanded cells according to their functionality and cytotoxicity. The experiment was carried out at the department of Hematology and Cell Therapy of the HC-FMUSP, in the Laboratory of Medical Investigation (LIM-31), fresh PB samples were obtained from Fundação Pró-Sangue leukapheresis healthy donors and the cryopreserved UCB units were obtained from Hospital Sírio-Libanês Umbilical Cord Blood bank. Mononuclear cells were isolated by Ficoll density gradient centrifugation. The isolation and expansion of UCB-derived and PB-derived NK cells was performed with the selection of CD56+ CD3- cells by immunomagnetic separation. Cells were expanded ex vivo for 14 days in RPMI-1640 medium supplemented with different combinations of cytokines, such as interleukin IL-2 alone, IL-2 and IL-21, IL-15 and IL-18 and IL-12, co-cultured or not with CSTX002 feeder cells in a 1:2 ratio (NK:aAPC). The number of NK cells increased significantly when they were co-cultured with aAPC in comparison to conditions without feeder cells. Pre-activation conditions with IL-12, IL-15 and IL-18 for “cytokine-induced memory-like” (ML NK) cells in both PB-NK and UCB-NK expansion yielded a significantly greater fold expansion (12-fold and 8-fold) when compared to expansion with IL-2 alone or IL-2 and IL-21. Moreover, the addition of IL-21 did not have a significant impact in the expansion of CD56+ CD3- cells when compared to conditions that used IL-2 alone. Furthermore, aAPC-mediated expansion yielded a predominantly CD56bright CD16bright mature phenotype in both PB-NK and UCB-NK. To evaluate the functionality NK cells, in vitro assays against tumor target cells was performed and UCB-NK conditions with IL-2 alone or IL-2 and IL-21 showed enhanced degranulation capacity and lytic granules (Granzyme B and Perforin) and cytokine production (IFN-y and TNF-a) than PB-NK cells. ML NK cells from PB produced higher amounts of cytokines and lytic granules when compared to ML NK cells from UCB. This may be related to a lower expression of NKp46, NKG2D and CD69 activation markers in ML NK UCB, and possibly because the UCB units were cryopreserved. The results suggest that ML NK cells show good ex vivo expansion, but do not show proper functionality against tumor cells. It is possible that with the addition of IL-2, ML NK cells could improve the production of cytokines and lytic granules, as seen in the IL-2 alone and IL-2 and IL-21 conditions.

Sex-based differences in conjunctival goblet cell responses to pro-inflammatory and pro-resolving mediators

Many conjunctival inflammatory diseases differ between the sexes and altered conjunctival goblet cells (CGCs) response is often involved. Inflammation is initiated by the release of pro-inflammatory mediators and terminated by the biosynthesis of specialized pro-resolution mediators (SPMs). Herein, we determined the sex-based difference in the responses of CGCs to inflammatory stimuli or pro-resolving lipid SPMs and their interaction with sex hormones. GCs were cultured from pieces of human conjunctiva in RPMI media. CGCs were transferred 24 h before the start of experiments to phenol red-free and FBS-free media to minimize exogenous hormones. RT-PCR, immunofluorescence microscopy (IF), and Western Blot (WB) were performed to determine the presence of sex hormone receptors. Cellular response to pro-inflammatory stimuli or SPMs was studied by measuring the increase in intracellular [Ca2+] ([Ca2+]i) using fura 2/AM microscopy. Use of RT-PCR demonstrated estrogen receptor (ER) α in 4/5 males and 3/3 females; ERβ in 2/4 males and 2/3 females; and androgen receptors (AR) in 3/3 male and 3/3 female CGCs. Positive immunoreactivity by IF and protein expression by WB was detected using antibodies for the ERα and ERβ in 3/3 males and 3/3 females, while AR were only present in males. Significantly different Ca2+ responses between sexes were found with carbachol only at 10–3 M, but not with histamine or leukotriene (LT) B4 at any concentration used. Incubation with dihydrotestosterone (DHT), estrone (E1), or estradiol (E2) at 10–7 M for 30 min significantly inhibited the LTB4-stimulated [Ca2+]i increase in male and female CGCs. Incubation with DHT, E1, and E2 overnight significantly inhibited the LTB4 response in females, while DHT and E2 significantly inhibited the LTB4 response in males. The SPM lipoxin A4 (LXA4) (10–9–10−8 M), but not the resolvins D1 or D2, induced an [Ca2+]i increase that was significantly higher in males compared to females. We conclude that male and female CGCs showed differences in the expression of sex hormone receptors. Treatment with sex hormones altered pro-inflammatory mediator LTB4-induced response. Males compared to females have a higher response to the ω-6-fatty acid derived SPM LXA4, indicating males may terminate inflammation in conjunctival goblet cells faster than females.

Isolation and Quantification of Epstein-Barr Virus from the P3HR1 Cell Line.

The Epstein-Barr virus (EBV), formally designated as Human herpesvirus 4 (HHV-4), is the first isolated human tumor virus. Nearly 90-95% of the world's adult population is infected by EBV. With the recent advancements in molecular biology and immunology, the application of both in vitro and in vivo experimental models has provided deep and meaningful insight into the pathogenesis of EBV in many diseases as well as into EBV-associated tumorigenesis. The aim of this visualized experiment paper is to provide an overview of the isolation of EBV viral particles from cells of the P3HR1 cell line, followed by quantification of the viral preparation. P3HR1 cells, originally isolated from a human Burkitt lymphoma, can produce a P3HR1 virus, which is a type 2 EBV strain. The EBV lytic cycle can be induced in these P3HR1 cells by treatment with phorbol 12-myristate 13-acetate (PMA), yielding EBV viral particles. Using this protocol for the isolation of EBV particles, P3HR1 cells are cultured for 5 days at 37 °C and 5% CO2 in complete RPMI-1640 medium containing 35 ng/mL PMA. Subsequently, the culture medium is centrifuged at a speed of 120 x g for 8 min to pellet the cells. The virus-containing supernatant is then collected and spun down at a speed of 16,000 x g for 90 min to pellet the EBV particles. The viral pellet is then resuspended in a complete RPMI-1640 medium. This is followed by DNA extraction and quantitative real-time PCR to assess the concentration of EBV particles in the preparation.

In vitro effects of vitamins C and E on adipocyte function and redox status in obesity

BackgroundObesity is associated with a chronic inflammatory state and evident oxidative stress. Antioxidant supplementation may have beneficial effects on adipocyte function and on oxidative stress. Therefore, in this study, we investigated the in vitro effects of vitamins C, E on redox and metabolic parameters in adipocytes of control and obese Wistar rats. MethodsAdipocytes were isolated from abdominal adipose tissue and were cultured in RPMI medium for 24 h, in the presence or the absence of vitamins (C, E at 50 µM). Glucose consumption, lactate and glycerol release, ATP and triglyceride contents, redox balance were investigated with biochemical methods. ResultsThe results showed altered glucose consumption and lactate and glycerol efflux, high triglycerides, low ATP contents (P < 0.01), associated to an intracellular oxidative stress in adipocytes of obese rats. Vitamins C and E restored adipocyte function in obesity. Vitamin E increased lipolysis in adipocytes during obesity (P < 0.01). Redox balance was also modulated by antioxidants, including a reduction in intracellular hydroperoxides and carbonyl proteins levels and an increase in catalase and SOD activities and glutathione contents in adipocytes of obese rats. ConclusionTreatments with vitamins had beneficial effects on adipocyte function, and should be considered in therapeutic approaches for normalizing adipose function in obesity.

Induction and characterization of extracellular traps by gilthead seabream (Sparus aurata L.) head-kidney leucocytes

The aim of this study was the induction and characterization of extracellular traps (ETs) produced by gilthead seabream (Sparus aurata L.) head-kidney leucocytes. The cells were incubated several times (10, 30, 60, 120, and 180 min) with different concentrations of the stimulants diluted in RPMI-1640 culture medium: RPMI-1640 (control), β-glucan from Saccharomyces cerevisiae (BG, 0–400 μg mL−1), lipopolysaccharide from Escherichia coli (LPS, 0–10 μg mL−1), calcium ionophore A23187 (CaI, 0–5 μg mL−1), Phorbol 12-myristate 13-acetate (PMA, 0–1000 ng mL−1) and polyinosinic–polycytidylic acid sodium salt (Poly I:C, 0–200 μg mL−1). BG, LPS and CaI exerted only weak stimulatory activity, while PMA and poly I:C exerted a potent one. After stimulation of the leucocytes, ETs structures were quantified and visualised through staining of the chromatin with nucleic acid-specific dyes and immunocytochemical probing of characteristic proteins expected to decorate the structure. ETs structures had DNA and myeloperoxidase. The ETs morphology was studied by light and scanning electron microscopy. These data confirm that seabream leucocytes form ETs with different morphological properties, depending on the used stimulant. These results will be the basis for new studies to analyse the implication of this mechanism in fish immunity. All this new knowledge will have its application in fish farms when we learn to manipulate the innate immune response in order to mitigate microbial infections.

P364 Quantitative monitoring of Aspergillus fumigatus mycelial growth by optical density

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectivesFilamentous fungi generally form hyphal pellets in liquid culture. This property prevents filamentous fungi from applying the growth monitoring methods used for unicellular organisms such as yeast and bacteria. We have analyzed the biological functions of cell wall polysaccharide α-1,3-glucan and extracellular polysaccharide galactosaminogalactan (GAG) in Aspergillus species, and revealed that both α-1,3-glucan and GAG contribute to the pellet formation. Here we constructed the double disruption mutant of α-1,3-glucan and GAG biosynthesis-related genes (Δags1Δgtb3) in Aspergillus fumigatus AfS35 strain, and used the mutant for quantitative monitoring of the mycelial growth by optical density.MethodsTo measure the optical density of conidia and mycelia in shake-flask culture, conidia (final concentration, 1.0 × 10⁷/ml) of AfS35 or Δags1Δgtb3 strain were inoculated into 50 ml of Aspergillus minimal medium (AMM) medium in a 200-ml Erlenmeyer flask and rotated at 160 rpm at 37°C. At each sampling point, the culture (2 ml) was withdrawn, and 100 μL of the culture was mixed with 100 μL of 4% paraformaldehyde solution in a 96-well plate. OD₆₀₀ was measured in a microplate reader. To evaluate the susceptibility of the Δags1Δgtb3 strain to antifungal agents, conidia (final concentration, 5.0 × 10⁶/ml) were inoculated into 500 μL of RPMI medium containing an antifungal agent (voriconazole, itraconazole, amphotericin B, flucytosine, or micafungin) in a 48-well plate and rotated at 300 rpm using a microplate mixer at 35°C for 15 h, and OD₆₀₀ was measured in triplicate.ResultsIn AMM flask culture, pellets became visible in AfS35 from 9 h, and their size increased with time, whereas the Δags1Δgtb3 hyphae were continuously dispersed (Fig. 1a). The OD₆₀₀ measurements of AfS35 suggested no correlation between apparent AfS35 growth and OD₆₀₀ (Fig. 1b). In the Δags1Δgtb3 strain, the first and third quartiles were 0.633 and 0.595 at 15 h (Fig. 1b), suggesting that the measurement of OD₆₀₀ is suitable for monitoring the growth of the mutant. As an application of the monitoring method, the Δags1Δgtb3 strain was grown in an RPMI medium containing the indicating antifungal agent for 15 h in a 48-well plate (Fig. 2). Growth was repressed in the presence of antifungal agents tested, except for micafungin (Fig. 2). Growth was completely inhibited at 2 μg/ml of voriconazole, 1 μg/ml of itraconazole, 0.5 μg/ml of amphotericin B, and 256 μg/ml of flucytosine (Fig. 2), which was in agreement with MICs determined by CLSI M38-A2.ConclusionWe established a convenient strategy to monitor A. fumigatus hyphal growth. Our method can be directly applied to screening for novel antifungals against Aspergillus species.

P343 Novel hydrophobic binding surface proteins are instrumental for phagocytosis of Lichtheimia corymbifera by macrophages

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectivesPerform proteomic analysis of the spore surface alongside secretome analysis of two strains of Lichtheimia corymbifera.Evaluate interaction, recognition, and phagocytosis of Lichtheimia spores by murine alveolar macrophages.MethodsTwo strains of L. corymbifera (JMRC: FSU: 09682 and JMRC: FSU: 10164) were used in this study. For phagocytosis assay, the spores were labeled with 0.1 mg/ml Fluorescein isothiocyanate (FITC) (Sigma Aldrich Chemie) in 0.1 M Na2CO3 for 30 min 146 at 30°C. Murine alveolar macrophages MH-S (ATCC:CRL-2019) were cultivated in RPMI-1640 (Sigma, 30-2001) supplemented.Identification of the surface proteins of L. corymbifera was carried out as described before with minor modification.1 The supernatants were stored at −80°C for liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Secreted proteins of L. corymbifera were determined as described in a previous study with minor modification2.The raw files generated by the LC-MS/MS were further processed by the software Proteome Discoverer v1.4.0.288 (Thermo). Tandem mass spectra were searched against the NCBI L. corymbifera protein database.3 Approximately 10 000 MH-S cells were cultivated onto 96-well microplates (NUNC 163320) in 100 μL RPMI-1640 medium (Sigma, 30-2001). Images were acquired by the Zeiss Axio Observer 7 Spinning Disk Confocal Microscope (ZEISS, Jena, Germany) and processed with ZEN 2.1 Software (ZEISS) by 63x objective lens.ResultsAbundant surface proteins were found which serve as Candidates for secretome analysis. A total of 113 proteins were identified. Thirty proteins were confirmed to be on the spore surface based on the presence of signal peptides3. The following proteins were predominantly identified: Spore coat protein (CotH), hydrophobic surface binding protein A (HsbA), aconitase, ricin-like lectin, two transition elongation factors, multi-copper oxidase, heat shock protein 70 family (Hsp70), malate synthase, putative allergen Candidates, etc.These proteins were heterologsly overexpressed in yeast. Successful overexpression was confirmed by LC-MS/MS. The yeast mutants were subjected to phagocytosis assays (Fig. 1). The role of surface proteins in macrophages is discussed.ConclusionThe surface proteins are instrumental for recognition of L. corymbifera by macrophages and for intracellular survival in macrophages. The role of surface proteins is discussed in the light of evolution from environmental to human pathogenic fungus.Sources:Voltersen V, Blango MG, Herrmann S, et al. Proteome Analysis Reveals the Conidial Surface Protein CcpA Essential for Virulence of the Pathogenic Fungus Aspergillus fumigatus. mBio. 2018;9(5). doi:10.1128/MBIO.01557-18Thürich J, Meichsner D, Furch ACU, et al. Arabidopsis thaliana responds to colonisation of Piriformospora indica by secretion of symbiosis-specific proteins. PLOS ONE. 2018;13(12):e0209658. doi:10.1371/JOURNAL. PONE.0209658.Schwartze VU, Winter S, Shelest E, et al. Gene Expansion Shapes Genome Architecture in the Human Pathogen Lichtheimia corymbifera: An Evolutionary Genomics Analysis in the Ancient Terrestrial Mucorales (Mucoromycotina). PLOS Genetics. 2014;10(8):e1004496. doi:10.1371/JOURNAL.PGEN.1004496.

Could RPMI-1640 Medium be Used in the Diagnosis and Isolation of Cutaneous Leishmaniasis Lacking NNN Medium?

Laboratory diagnosis of leishmaniasis is based on culture, microscopic examination, serological and molecular methods. The gold standard method is to see amastigotes in microscopic examination and to grow promastigotes in Novy, MacNeal, Nicolle (NNN) medium. NNN medium is frequently used for culture all over the world. In our study, it was aimed to investigate whether the use of RPMI-1640 medium is an appropriate method in cases where the gold standard NNN medium is not available for the diagnosis of cutaneous leishmaniasis (CL). Smears were prepared from the needle aspiration fluid sample from the patient who applied to Manisa Celal Bayar University Faculty of Medicine and had lesions suspicious of CL, and were stained with Giemsa for the presence of amastigotes. The samples taken were directly inoculated into RPMI-1640 broth and incubated at 26 °C for the presence of promastigotes. On consecutive days after incubation, it was checked for promastigote growth. Genotyping of the grown isolate was performed with primers and probes specific to the internal transcribed spacer-1 (ITS-1) gene region with the help of real-time polymerase chain reaction. The amastigote form was observed in the microscopic examination of the needle aspiration fluid sample smear preparations taken from the patient. On the other hand, promastigote growth was observed in RPMI-1640 broth from the 3rd day. In addition, the isolate obtained from the CL patient was determined to be Leishmania tropica as a result of the species determination made by genotyping. It is thought that this study is important in terms of suggesting an alternative medium for the diagnosis of leishmaniasis in laboratories where the gold standard NNN medium is easily accessible. RPMI-1640 medium, which is easily obtained and prepared in parasitology laboratories, can help in the diagnosis of the disease and treatment follow-up, Leishmania spp. isolation and drug resistance studies.