Bioluminescence Assay Research Articles

Naturally occurring quercetin and myricetin as potent inhibitors for human ectonucleotide pyrophosphatase/phosphodiesterase 1

Ecto-nucleotide pyrophosphatases/phosphodiesterases 1 (ENPP1) is a key enzyme in purinergic signaling pathways responsible for cell-to-cell communications and regulation of several fundamental pathophysiological processes. In this study, Kyoto Green, a rapid chemical sensor of pyrophosphate, was employed to screen for effective ENPP1 inhibitors among five representative flavonoids (quercetin, myricetin, morin, kaempferol, and quercetin-3-glucoside), five nucleosides (adenosine, guanosine, inosine, uridine, and cytidine), and five deoxynucleosides (2′- and 3′-deoxyadenosine, 2′-deoxyguanosine, 2′-deoxyinosine, and 2′-deoxyuridine). Conventional colorimetric, fluorescence, and bioluminescence assays revealed that ENPP1 was effectively inhibited by quercetin (Ki ~ 4 nM) and myricetin (Ki ~ 32 nM) when ATP was used as a substrate at pH 7.4. In silico analysis indicated that the presence of a chromone scaffold, particularly one containing a hydroxyl group at the 3′ position on the B ring, may promote binding to the active site pocket of ENPP1 and enhance inhibition. This study demonstrated that the naturally derived quercetin and myricetin could effectively inhibit ENPP1 enzymatic activity and may offer health benefits in arthritis management.

Can the adenosine triphosphate (ATP) bioluminescence assay be used as an indicator for hospital cleaning? - A pilot study.

In hospital cleaning, there is currently no standard for uniform monitoring of surface cleaning, either in Germany or internationally. One possibility for monitoring is the use of so-called objective methods for checking cleaning performance (e.g. fluorescence or adenosine triphosphate (ATP) method). The aim of the study was to monitor and evaluate the implementation of the adenosine triphosphate (ATP) bioluminescence assay as a cleaning indicator in everyday hospital cleaning, in order to verify its utility and effectiveness. In three phases, five frequently touched surfaces were examined with the ATP bioluminescence assay at different time points. 846 measurements were performed on the dermatology ward of a university hospital (phase 1), 1,350 measurements were performed on five different wards of the university hospital (phase 2), and 1,044 measurements were performed on five wards of another large hospital (phase 3). For this purpose, one structurally old and one structurally new ward as well as an intensive care unit (ICU), an outpatient clinic and a radiology department were selected for phases 2 and 3. With the ATP bioluminescence method, we were able to demonstrate a reduction in values after cleaning: before cleaning mean of ATP, 907 relative light units (RLU) (95% confidence interval [CI] 777; 1,038); after cleaning mean=286 RLU (CI=233; 495) (phase 1) and by intervention (five hours after daily cleaning mean=360 RLU (CI=303; 428); five hours after daily cleaning and two additional cleanings mean=128 RLU (CI=107; 152) (phase 3). The ATP values increased five hours after cleaning in phases 1 and 2, and eight hours after cleaning in phase 3. The structurally old wards had the highest ATP content, the ICU and the radiology department, among others, the lowest. In all phases, door handles showed both a reduction after cleaning or intervention and a subsequent increase in ATP values. Chair armrests, examination tables and door handles had high ATP values overall. The study shows ward differences both for cleaning effects and for the soiling characteristics of surfaces during the course of the day. In addition, it demonstrates the benefit of intermediate cleaning twice a day. It is noteworthy that structurally old stations and older inventory were more heavily soiled and, in some cases, more difficult to clean. The results show that the ATP bioluminescence method is suitable for detecting cleaning effects and can be used in everyday clinical practice for simple cleaning monitoring. Furthermore, it enables the detection of risk surfaces and easy-to-clean surfaces with significant re-soiling.

Structure and activity relationship analysis of xanthones from mangosteen: Identifying garcinone E as a potent dual EGFR and VEGFR2 inhibitor.

Xanthones are among the most fundamental phytochemicals in nature. The anti-cancer activities of xanthones and their derivatives have been extensively studied. Recently, we found that garcinone E (GE), an effective anti-cancer phytochemical isolated from mangosteen (Garcinia mangostanal.), showed promising anti-cancer effects in vitro and in vivo. However, little is known about its effects on epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2) activity. This study aimed to identify potent dual EGFR and VEGFR2 inhibitors from mangosteen-derived xanthones using structure-activity relationship analyses. The interaction of xanthones with EGFR and VEGFR2 was analyzed using molecular docking experiments. The kinase activities of EGFR and VEGFR2 were determined using bioluminescence assays. The rat aortic ring and Matrigel plug angiogenesis assays were used to evaluate blood vessel formation ex vivo and in vivo. A breast tumor-bearing nude mouse model was established to examine the anti-tumor effects of different xanthones. Molecular docking analysis showed that GE bound tightly to EGFR and VEGFR2, with binding energies of -9.73 and -9.56kcal/mol, respectively. Kinase activity assessment showed that GE strongly inhibited both EGFR and VEGFR2 kinase activity, with IC50 values of 315.4 and 158.2nM, respectively. Moreover, GE significantly abolished the EGF- and VEGF-induced phosphorylation of EGFR and VEGFR2, respectively. GE also showed strong inhibitory effects on cancer cell growth, endothelial cell migration, invasion, and tube formation. Ex vivo and in vivo angiogenesis assays showed that GE dose-dependently suppressed blood vessel formation in the rat aorta, Matrigel plugs, and transgenic zebrafish embryos, with the lowest effective concentration of 0.25μM. Furthermore, GE (2mg/kg) strongly inhibited tumor growth and reduced tumor weight in MDA-MB-231 breast tumor-xenografted mice. GE significantly reduced microvessel density and downregulated the expression of VEGFR2, EGFR, and Ki67 in tumor tissues. The present study demonstrated that GE was the most potent dual inhibitor of EGFR and VEGFR2 among all xanthones tested. These findings may provide valuable information for the future development of novel and effective dual inhibitors of EGFR and VEGFR2.

Bioluminescence assay for rapid detection of live Staphylococcus aureus based on the enrichment of egg yolk antibody modified magnetic metal organic framework immunobeads.

Specific and rapid detection of live Staphylococcus aureus (S.A) in environmental and food samples is critically important for protecting human health. In order to fulfill this purpose, two kinds of novel egg yolk antibody (IgY) immobilized immunomagnetic beads (IMBs; mSiO2-IgY and mMOF-IgY), with core-shell mSiO2 and mMOF as substrate, were prepared for selectively enriching S.A from samples. Furthermore, the IMBs with captured S.A were collected and re-dissolved in 0.5 mL PBS. After that, a cotton swab coated with sodium dodecylsulfate (SDS) was put in the solution to lyse S.A cells and emit ATP bioluminescence of the luciferin/luciferase system. Finally, a portable bioluminescence detector was used for quantification of ATP corresponding to S.A concentration. The results demonstrated that mMOF-IgY can enrich more S.A than mSiO2-IgY and emit a stronger signal. The reasons may be due to the higher immobilization amount of IgY on the IMBs. Under optimal conditions, the calibration line of S.A concentration was 10-105 CFU mL-1 by mMOF-IgY within 30 min. The low detection limit of S.A was 3 CFU mL-1. The results demonstrated that the assay takes much shorter time than plate counting. Its portability and excellent detection capability are suitable for rapid monitoring of specific pathogens in foods.

ADP-mediated Modulation of Intracellular Calcium Responses in Chromaffin Cells: The Role of Ectonucleoside Triphosphate Diphosphohydrolase 2 on Rat Adrenal Medulla Function.

The present study investigated the localization and the adenosine 5'-triphosphate (ATP)-degrading function of the plasma membrane-bound ecto-nucleotidase, ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2), in the rat adrenal medulla. The effect of ATP degradation product, adenosine 5'-diphosphate (ADP), on carbachol (CCh)-induced intracellular Ca2+ ([Ca2+]i) responses in adrenal chromaffin cells was examined using calcium imaging. NTPDase2-immunoreactive cells were distributed between chromaffin cells. NTPDase2-immunoreactive cells were immunoreactive for glial fibrillary acidic protein and S100B, suggesting that they were sustentacular cells. NTPDase2-immunoreactive cells surrounded chromaffin cells immunoreactive for vesicular nucleotide transporter and P2Y12 ADP-selective purinoceptors. In ATP bioluminescence assays using adrenal medullary slices, ATP was rapidly degraded and its degradation was attenuated by the NTPDase inhibitors sodium polyoxotungstate (POM-1) and 6-N, N-diethyl-d-β,γ-dibromomethylene ATP (ARL67156). ADP inhibited CCh-induced [Ca2+]i increases of chromaffin cells in adrenal medullary slices. The inhibition of CCh-induced [Ca2+]i increases by ADP was blocked by the P2Y12 purinoceptor antagonist AZD1283. CCh-induced [Ca2+]i increases were also inhibited by the P2Y1, P2Y12, and P2Y13 purinoceptor agonist 2-methylthioadenosine diphosphate trisodium (2MeSADP), in combination with the P2Y1 purinoceptor antagonist MRS2179. These results suggest that sustentacular cells express NTPDase2 to degrade ATP released from adrenal chromaffin cells, and ADP modulates the excitability of chromaffin cells via P2Y12 purinoceptors to regulate catecholamine release during preganglionic sympathetic stimuli. (J Histochem Cytochem 72: 41-60, 2024).

Assessing the genotoxic potential of wastewater effluents from three wastewater treatment plants in South Africa.

Wastewater treatment plants are mainly monitored for quality in terms of their biological oxygen demand and microbiological constituents as stipulated in the specific discharge permit. Wastewater influents and effluents were taken from three WWTPs in South Africa over the summer and winter seasons. Previous toxicity tests such as the Vibrio fischeri bioluminescence assay and the Selenastrum capricornutum algal growth inhibition test have shown that the effluents displayed acute toxicity. To further investigate the quality of the effluent, the genotoxic potential was determined using the SOS Chromosome and UMU Chromosome test. The SOS Chromotest demonstrated induction factor values of above 1.5 for influents during both seasons indicating that the influents were genotoxic (p < 0.05). Effluents discharged during winter and summer also had induction factors greater than 1.5 (p < 0.05). A range of induction factors was detected with the UMU-Chromotest for influents and effluents (1.98 ± 0.38 and 2.40 ± 0.51, respectively). Findings show point sources in the area can lead to influents and effluents that are potentially genotoxic. Designing a monitoring programme that encompasses testing of both the regulatory determinants with additional specialized tests can provide a more holistic view of wastewater quality and the efficiency of WWTP to reduce the discharge of hazards.

Effects of Liquiritin on Mitochondrial Dynamics in Cerebral Ischemia–Reperfusion Injury by Regulating Nurr1 via the YAP-INF2-Mitochondrial Fission Pathway

This study investigated whether liquiritin can alleviate cerebral ischemia–reperfusion injury by regulating Nurr1 to mediate mitochondrial homeostasis. SH-SY5Y cells were subjected to glucose deprivation and reperfusion to establish a cerebral ischemia–reperfusion injury model in vitro. Cell viability and apoptosis were then determined using a cell counting kit and flow cytometry analysis. The degree of mitochondrial swelling was evaluated using a cell mitochondria isolation kit. Reactive superoxide generation, mitochondrial membrane potential, adenosine triphosphate (ATP) content, and mitochondrial ultrastructure were analyzed using dihydroethidium, JC-1 (5,5′,6,6′-tetrachloro1,1′,3,3′-tetramethylbenzimidazolylcarbocyanine iodide), luciferase-based ATP bioluminescent assays, and transmission electron microscopy, respectively. Quantitative reverse transcription PCR and western blot assays were conducted to detect levels of mitochondrial fission-related factors. Glucose deprivation and reperfusion exposure significantly reduced the viability and induced apoptosis of SH-SY5Y cells, indicating that glucose deprivation and reperfusion exposure successfully induced cerebral ischemia–reperfusion injury. Glucose deprivation and reperfusion exposure also increased the degree of mitochondrial swelling, promoted an increase in superoxide, and decreased mitochondrial membrane potential and ATP enzyme levels. Cerebral ischemia–reperfusion injury also significantly increases Drp1 and Fis1 protein expression, reduces mitofusin-2 and optic atrophy 1 levels, increases nuclear receptor-related 1 and inverted formin-2 expression, and decreases yes-associated protein expression. Electron microscopy further revealed sparse mitochondria and broken cristae. However, these findings were reversed by liquiritin in a dose-dependent manner and were further abolished after carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone treatment. Our study suggests that the protective effects of liquiritin on cerebral ischemia–reperfusion injury are linked to nuclear receptor-related 1 upregulation, followed by the regulation of yes-associated protein-inverted formin-2-mitochondrial fission pathways. Liquiritin may represent a novel therapeutic agent for treating cerebral ischemia–reperfusion injury.Graphical

Danggui Buxue Tang improves therapeutic efficacy of doxorubicin in triple negative breast cancer via ferroptosis

Ethnopharmacological relevanceDanggui Buxue Tang (DBT) has been used for over 800 years to enhance Qi and nourish Blood, and it is particularly beneficial for cancer patients. Recent research has shown that combining DBT with chemotherapy agents leads to superior anti-cancer effects, thereby enhancing therapeutic efficacy. Aim of the studyThe aim of this study was to evaluate the effectiveness of a combination therapy involving doxorubicin (DOX) and Danggui Buxue Tang (DBT) in the treatment of triple-negative breast cancer (TNBC) and to elucidate the underlying mechanisms of action. Materials and methodsIn vitro experiments were performed using MDA-MB-231 and 4T1 cells, while in vivo experiments were carried out using MDA-MB-231 xenograft mice. The therapeutic effects of the combination therapy were evaluated using various techniques, including MTT assay, colony formation assay, flow cytometry, transwell assay, immunofluorescence, transmission electron microscopy (TEM), histological analysis, western blotting, and bioluminescence assay. ResultsDBT was found to enhance DOX's anti-TNBC activity in vitro by promoting ferroptosis, as evidenced by the observed mitochondrial morphological changes using TEM. The combination therapy was also found to reduce the expression of Nrf2, HO-1, and GPX4, which are all targets for ferroptosis induction, while simultaneously increasing ROS production. Additionally, the combination therapy reduced nuclear accumulation and constitutive activation of Nrf2, which is a significant cause of chemotherapy resistance and promotes cancer growth. In vivo experiments using an MDA-MB-231 xenograft animal model revealed that the combination therapy significantly reduced tumor cell proliferation and accelerated TNBC deaths by modulating the Nrf2/HO-1/GPX4 axis, with no evidence of tissue abnormalities. Moreover, the combination therapy exhibited a liver protective effect, and administration of Fer-1 was able to reduce the ROS formation produced by the DBT + DOX combination therapy. ConclusionThis study provides evidence that the combination therapy of DOX and DBT has the potential to treat TNBC by promoting ferroptosis through the Nrf2/HO-1/GPX4 axis.

A novel phagomagnetic separation-ATP bioluminescence (PhMS-BL) for rapid and sensitive detection of viable Vibrio parahaemolyticus in aquatic product

Detection of viable Vibrio parahaemolyticus (V. parahaemolyticus) is a major challenge due to its significant risk to food safety and human health. Herein, we developed a phagomagnetic separation-ATP bioluminescence (PhMS-BL) assay based on phage VPHZ6 for rapid and sensitive detection of viable V. parahaemolyticus. Phage as a recognition element was coupled to magnetic beads to capture and enrich V. parahaemolyticus, shortening detection time and improving method sensitivity. The intracellular ATP released by chemical lysis using CTAB was quantified using firefly fluorescein-adenosine triphosphate bioluminescence system to detect viable bacteria. So, PhMS-BL method was able to detect V. parahaemolyticus in a linear range of 2.3 × 102 to 1.3 × 107 CFU mL−1, with a detection limit of 78 CFU mL−1 within 15 min. It is successfully applied to detect V. parahaemolyticus in spiked lake water, lobster tail meat, and clam meat. The developed detection strategy can rapidly and sensitively detect viable V. parahaemolyticus in food matrixes.

Utilizing Chemotherapy to Enhance the Anti-Tumor Properties of Ex Vivo Expanded Gamma Delta (γδ) T Cells Against Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is the second most common type of blood cancer in children with a high rate of treatment failure and aggressive relapse. While allogeneic HSCT offers a cure, limitations arise in achieving remission prior to transplant. Immunotherapy-based approaches can induce remission, gaining interest in AML. Here, we explore the use of gamma delta (γδ) T cells as a promising tool to fulfill this unmet need. Unlike predominant alpha beta (αβ) T cells, γδ T cells do not require antigen presentation and identify their targets in a major histocompatibility complex (MHC) independent manner. Allogeneic γδ T cells thus have minimal risk of causing graft-versus-host disease (GvHD), creating ‘off-the-shelf’ cellular therapeutics. This would be ideal to target AML, especially given the aggressive nature of relapsed disease. Cytotoxic mechanisms used by γδ T cells include recognition of cellular stress molecules such as NKG2D and DNAM-1 receptor ligands on target cells, activation of death receptor pathway through Fas-FasL and TRAIL-R interactions, as well as γδ ΤCR mediated killing through recognition of butyrophilins (BTNs) on target cells. These stress molecules are expressed on AML cells and can be further upregulated through chemotherapeutic agents such as azacitidine (a hypomethylating agent) and venetoclax (BCL-2 antagonist), drugs currently in use to treat relapsed AML. Here, we tested these drugs in combination with γδ T cells to target AML. Methods Our studies explored four different AML cell lines (Kasumi-1, Nomo-1, MV4-11 and MOLM13). Each one was respectively treated at their corresponding inhibitory concentration 25 (IC 25) and 50 (IC 50) of azacitidine and/or venetoclax for 24 hours. After treatment, AML cells were assessed by flow cytometry for expression of NKG2D and DNAM-1 ligands, death receptor pathway molecules (CD95, TRAIL-R1/R2) as well as the butyrophillins (BTN2A and BTN3A1) to elucidate which pathways are over-expressed, aiding in γδ T cell targeting and cytotoxicity. Next, we co-cultured AML cells pre-treated with azacitidine and/or venetoclax with γδ T cells and measured cytotoxicity using a flow cytometry-based assay and a bioluminescence assay. Results: Each cell line expressed a unique pattern of markers at baseline. Cell lines treated with azacitidine had a robust expression of the NKG2D ligands ULBP1, ULBP2/5/6 and ULBP3. Azacitidine also increased CD155 and TRAIL-R2 in Nomo-1 while CD112 increased in MOLM13. Most venetoclax-treated AML cells over-expressed BTN2A and BTN3A1 along with CD95 in MOLM13. Cell lines were then treated with a combination of both drugs. Dual treatment of cells resulted in a significant increase in expression of most NKG2D markers as well the death receptor Fas (CD95). Additionally, increase in CD155 as well as BTNs was seen in several cell lines. Importantly, azacitidine and venetoclax had minimal effects on the viability of γδ Τ cells when treated for 24 hours. In vitro cytotoxicity studies indicate increased cell death across multiple AML cell lines when γδ T cells were co-cultured with cells pretreated with azacitidine and venetoclax. Conclusions: Azacitidine and venetoclax treatment resulted in upregulation of several markers involved in γδ Τ cell-mediated cytotoxicity. Ex vivo expanded γδ Τ cells combined with chemotherapy-treated AML cells resulted in increased overall cytotoxicity, which is likely both additive and synergistic. Our next steps include preclinical validation of this combinatorial approach in an in-vivo mouse study.

CD4+/CD8+ Selection of Anti-AML ENG-T Cells Affects Long-Term T-Cell Persistence in Vitro and Survival in an AML Xenograft Model

Background: Immunotherapies have shown dramatic anti-cancer efficacy in pediatric hematopoietic malignancies. Despite initial disease clearance, relapse rates are high, demanding product improvement. Manufacture of engineered cellular immunotherapies involves expansion of effector cells from patient or donor peripheral blood mononuclear cells (PBMCs) ex vivo prior to patient infusion. Expansion methods in pre-clinical studies of engineered T-cell therapies often call for activating T cells from whole PBMCs using anti-CD3 and anti-CD28 co-stimulation. However, clinical production of T-cell therapies has shifted towards selecting CD4+ and CD8+ cells prior to activation due to higher product efficacy seen in clinical trial. Many acute myeloid leukemias (AML) are CD4+, introducing the risk of blast contamination during CD4+ selection. We questioned whether CD8+ T cells alone could be used to manufacture our clinical T cell product and sought to determine how selection affected our T cells. Understanding the biological impact of manufacturing changes between pre-clinical and clinical production is imperative for clinical translation. Methods: We have engineered T cells to secrete a CD123xCD3 binding bispecific engager molecule (CD123-ENG) to treat AML. Primary T cells were isolated from PBMCs, selected with microbeads, activated, and transduced with replication incompetent retroviral vectors. Unselected T cells were compared to CD4+, CD8+, or CD4+ and CD8+ selected T cells. T-cell transduction was confirmed with flow cytometry. Expression and secretion of CD123-ENG was confirmed with ELISA. Antigen-specific T-cell activation was determined by quantifying cytokine secretion in the presence of CD123+ target cell lines. Short-term cytotoxicity was examined in vitro against CD123+ target cell lines in 18-hour bioluminescence assays. T-cell serial cytotoxicity and persistence were tracked in 10-day serial stimulation studies using an IncuCyte live cell imaging system. Cytokines present during T-cell manufacture and serial stimulation were detected and quantified with Luminex multiplex assays. Immunophenotype and exhaustion markers were analyzed using flow cytometry. In vivo anti-AML cytotoxicity and T-cell persistence were determined in an AML xenograft model. Results: In our studies, unselected, CD4+, CD8+, and CD4+/CD8+ selected CD123-ENG T cells showed no differences in activation, cytotoxicity, or immunophenotype when evaluated in short-term assays. Mice harboring human AML treated with CD4+/CD8+ selected CD123-ENG T cells had improved survival compared to mice treated with unselected and CD8+ selected CD123-ENG T cells. CD4+ T cells alone provided the best survival advantage. In addition, CD4+/CD8+ selected T cells had longer persistence when challenged by serial target cell stimulations in vitro. T cells expanded from whole PBMCs were exposed to cytokines likely to emanate from non-T cells during early manufacture. These cytokines were characteristic of those produced by myeloid cells in an inflammatory environment, and may explain functional differences observed in vivo. Conclusions: Durable anti-cancer response in patients is associated with T-cell persistence and CD4+/CD8+ pre-selection of CD123-ENG may therefore improve remission duration in patients. CD4+ and CD4+/CD8+ selected T cells provided the longest survival in vivo, dissuading us from further investigating CD8+ CD123-ENG as an AML treatment. We are actively exploring additional mechanistic determinants of the observed phenotypic differences with and without selection. Optimizing cellular immunotherapy will improve patient outcomes. It is essential that engineered cellular research products intended for clinical translation be produced by the same methods used for clinical manufacture to increase the predictive power of pre-clinical data. The use of biological correlatives to predict T-cell product function will better support successful translation. Acknowledgements: This research was funded in part by NCI Contract No. HHSN26120150003I/HHSN26100077.

Extracellular adenosine triphosphate regulates inflammatory responses of periodontal ligament cells.

Various stimuli, that is, mechanical stresses or inflammation, induce the release of adenosine triphosphate (ATP) by human periodontal ligament cells (HPDLCs). Extracellular adenosine triphosphate (eATP) affects HPDLCs' functions such as immunosuppressive action and inflammatory responses. Lipopolysaccharide (LPS) is the key factor involved in periodontal inflammation. However, the possible correlation and detailed mechanism of inflammation-mediated eATP by LPS and inflammatory cascade formation in HPDLCs is unclarified. This study aims to examine the role of eATP on the HPDLCs' responses concerning inflammatory actions after LPS treatment. HPDLCs were stimulated with Porphyromonas gingivalis LPS and polyinosinic:polycytidylic acid (poly I:C). The amount of ATP release was measured at different time points using a bioluminescence assay. HPDLCs were treated with eATP. The expression of pro-inflammatory and anti-inflammatory genes was determined. Specific P2X purinoreceptor 7 (P2X7) inhibitors (brilliant blue G [BBG] and KN62), a specific P2Y purinoreceptor 1 (P2Y1) inhibitors (MRS2179), calcium chelator (EGTA), protein kinase C (PKC) inhibitors, nuclear factor kappa-light-chain-enhancer of activated B cells (NF𝜅B) activation inhibitors, and cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) inhibitors (H89 dihydrochloride) and activators (forskolin) were used to dissect the mechanism of eATP-induced HPDLCs' inflammatory responses. LPS and poly I:C induced ATP release. A low concentration of eATP (50µM) increased pro-inflammatory genes (COX2, IL1B, IL6, IL8, IL12, and TNFA), while a high concentration (500µM) enhanced anti-inflammatory genes (IL4 and IL10). BBG, KN62, and NF𝜅B activation inhibitors impeded eATP-induced pro-inflammatory genes. MRS2179 and H89 markedly suppressed eATP-induced anti-inflammatory genes. Forskolin induced IL4 and IL10. HPDLCs respond to LPS by releasing ATP. eATP has dose-dependent dual functions on HPDLCs' inflammatory responses via different pathways. As regulation of inflammation is important in regeneration, eATP may help to limit inflammation and trigger periodontal regeneration.

Antioxidants As a Novel Treatment to Revert Impaired Angiogenic Potential Driven By Metabolic Alterations Observed in Shwachman-Diamond Syndrome Derived Mesenchymal Stromal Cells

Introduction Shwachman-Diamond Syndrome (SDS) is a rare bone marrow (BM) failure disorder. SDS BM biopsies show tortuous and collapsed vessels, highlighting angiogenic abnormalities. Our group previously demonstrated that SDS BM mesenchymal stromal cells (MSCs) have in vitro and in vivo impaired angiogenic potential compared to healthy donors (HD)-MSCs. Interestingly, recent evidence underlines that pathological angiogenesis could be accompanied by altered metabolism and that antioxidants could represent a novel strategy to target angiogenic defects. Aims We aimed to characterize the metabolic status of SDS-MSCs and to investigate if the treatment with antioxidants may restore their impaired angiogenic potential. Methods BM derived HD- and SDS-MSCs were stimulated with DMEM ± N-Acetylcysteine (NAC, 1mM) or dimethylsulfoxide (DMSO, 0.05% v/v) for 48h. Oxidative phosphorylation (OxPhos) was assayed by oximetry and bioluminescent ATP synthesis assay. Spectrophotometric analyses were performed to evaluate ATP/AMP ratio, malondialdehyde (MDA) level, and Complex IV and lactate dehydrogenase (LDH) activity. The amount of reactive oxygen species (ROS) was quantified by flow cytometry. The angiogenic capability was evaluated by performing in vitro Matrigel angiogenesis assay. Results Firstly, we evaluated OxPhos oxygen consumption and ATP production (n=6). Concerning the I-III-IV mitochondrial complexes pathway, SDS-MSCs consumed 57% less oxygen (p=0.004) and produced 64% less ATP compared to HD-MSCs (p=0.002). Accordingly, the analysis of the II-III-IV complexes pathway demonstrated that the oxygen consumption was reduced by 62% (p=0.002) and the ATP synthesis was 67% lower than HD-MSCs (p=0.002). Furthermore, the P/O ratio, index of OxPhos efficiency, was significantly reduced in SDS-MSCs in both the electron transport chain pathways (p=0.002 for both). Therefore, we demonstrated that Complex IV activity was 61% lower in SDS- vs HD-MSCs (p=0.002), highlighting its role in the SDS OxPhos defect. As for the energetic status, SDS-MSCs showed a low intracellular ATP/AMP ratio (mean=1.1, range=0.8-1.6 vs mean=3.6, range=3.1-4.0, in HDs; p=0.002) that was accompanied by 30% increase of LDH activity in SDS-MSCs (p=0.002), an expression of increased anaerobic glycolysis to compensate the mitochondrial defect. The amount of ROS in SDS-MSCs was increased by 27% compared to HD-MSCs (n=5) and also the SDS-MSCs lipid peroxidation level was significantly higher over HDs (mean=12.7 µM of MDA/mg, range=10.5-14.1µM/mg vs mean=6.6µM/mg, range=5.9-7.4µM/mg; p=0.002). Then we treated SDS-MSCs for 48h with NAC, a broad-range antioxidant (n=5), or with DMSO, which acts at very low concentrations as scavenger, specifically on lipid peroxidation products (n=6). Interestingly, NAC and DMSO stimulated SDS-MSCs increased by 75% the oxygen consumption and by 70% the ATP synthesis in both the electron transport chain pathways, thus resulting in levels comparable to HD-MSCs. The SDS OxPhos restoration was respectively associated to a 53% and 60% increase of Complex IV activity after NAC and DMSO stimulation (p&amp;lt;0.0001 for both). Moreover, antioxidants corrected the SDS energetic defect by restoring ATP/AMP ratio and LDH activity to the levels of HDs. Importantly, SDS-MSCs lipid peroxidation level was drastically reduced after NAC and DMSO treatments (mean=12.7µM/mg, range=10.5-14.1µM/mg vs NAC mean=5.1µM/mg, range=4.3-5.4µM/mg and DMSO mean=6.2µM/mg, range=5.8-6.5µM/mg; p&amp;lt;0.0001 for both). Finally, we approached SDS-MSCs angiogenic defect and showed that SDS-MSCs defective capability to recreate a defined capillary-like network under angiogenic stimuli was completely restored after antioxidant stimulations (n≥5). As shown by ImageJ Angiogenesis Analyzer several angiogenic elements, including branches and segments, were significantly increased in NAC and DMSO stimulated SDS-MSCs that become comparable to HD-MSCs. Conclusions We demonstrated that the altered OxPhos metabolism of SDS-MSCs significantly contributes to their angiogenic defect and, importantly, that antioxidants restored the metabolic alterations and the angiogenic potential in SDS-MSCs, paving the way for new therapeutic strategies.

Bispecific CAR-iNKT Immunotherapy for High Risk MLL-Rearranged Acute Lymphoblastic Leukemia

MLL-rearranged CD19+ acute lymphoblastic leukemia ( MLLr-ALL), the commonest infant leukemia, has a high mortality rate (&amp;lt;40% leukemia-free survival). While stem cell transplantation has not improved outcomes, CD19-directed immunotherapeutic approaches, including CAR-T cells, have shown promise. However, as patients can still relapse with CD19-, or lineage-switched disease, alternative strategies are needed. To enhance efficacy and mitigate against immune escape, we aimed to develop iNKT cell-based bispecific CAR immunotherapy. iNKT are rare, CD1d-restricted, glycolipid-reactive T cells that bridge adaptive and innate immunity. Furthermore, allogeneic iNKT protect from aGVHD making them ideal candidates for ‘off-the-shelf’ immunotherapy without need for TCR deletion. To target both CD19 and CD133, which is often co-expressed with CD19 on MLLr blasts and possibly also on CD19-CD133+ leukemia-initiating cells, we developed a CD19-28z/CD133-41BBz bispecific as well as CD19-28z and CD133-4-1BBz monospecific CARs and compared their anti-leukemic activity. In short- and long-term cytotoxicity assays against MLLr leukemic cell lines (SEM, RS4;11, KOPN8) we show that bispecific CAR-iNKT are more potent than their monospecific counterparts. In assays involving single or dual knock-out of CD19 and CD133 in SEM cells, both the CD19 and CD133 moieties of the bispecific CAR were reactive and specific against their respective targets. While bispecific CAR-iNKT retained killing activity against either single target-expressing leukemia cells, monospecific CAR-iNKT only killed leukemia cells expressing their cognate target. In line with the in vitro findings, in xenograft assays, a low dose of 10 6 bispecific CAR-iNKT cells significantly prolonged survival of luc-SEM leukemia-bearing mice as compared to monospecific CAR-iNKT-treated and untreated control animals (Fig 1A). Of note, in vivo as well as in vitro, bispecific CAR-iNKT very effectively eliminated high and low CD19/CD133 co-expressing, but not, as expected, CD19-CD133- disease. Next, we compared bispecific CAR-iNKT vs their CAR-T counterparts. In vitro, CAR-iNKT from three donors demonstrated higher killing activity than CAR-T, while in vivo, compared to 10 6 CAR-T, CAR-iNKT cells induced significantly improved survival of leukemia-bearing mice (Fig 1B). Of note, while CAR-T cells were not detectable in the BM and spleen at sacrifice, CAR-iNKT were readily detectable in the organs of CAR-iNKT cell-treated animals, suggesting they persist longer. Remarkably, mice treated with 5x10 6 bispecific CAR-iNKT cells survived &amp;gt;60 days without any evidence of leukemia by serial bioluminescence assay, or in the bone marrow, spleen (flow-cytometry) and leptomeninges (immunohistochemistry) upon culling. Since SEM cells do not express CD1d, we explored alternative mechanism(s) that would account for the more potent anti-leukemic activity of CAR-iNKT cells. We found that CAR-iNKT express significantly higher baseline levels of the activating receptor NKG2D than CAR-T cells. Upon co-culture with SEM cells, nearly all CAR-iNKT but only a fraction of CAR-T express NKG2D, and a blocking NKG2D Ab significantly abrogated the superior cytotoxic effect of CAR-iNKT cells. Consistent with an NKG2D-dependent mechanism, mRNA of one or more NKG2D ligands is expressed by primary MLLr blasts as well as leukemia cell lines. Since CD133 is expressed on hematopoietic stem and progenitor cells (HSPC), the potential hematopoietic toxicity of bispecific CAR-iNKT cells was tested in a series of experiments involving cord blood humanisation of NSGS mouse hematopoiesis. We found that transfer of 10 7 bispecific CAR-iNKT cells significantly but transiently depleted engrafted CB CD19+ B cells. However, phenotypic analysis showed that long term engraftment levels were similar for all mature cell lineages, CD34+ HSPC and specifically for Lin-CD34+CD38- HSC. We conclude that bispecific CAR-iNKT cell immunotherapy is a very effective treatment for pre-clinical aggressive MLLr-ALL, it outperforms CAR-T cell immunotherapy in an NKG2D-dependent manner and has the potential to protect from immune escape, leptomeningeal disease and lineage switch without discernible hematological toxicity. These findings provide the basis for clinical development of bispecific CD19/CD133 CAR-iNKT cells as an ‘off-the-shelf’ treatment for MLLr-ALL.

SAT027 Novel Obesity-Associated Melanocortin 4 Receptor Variants Modulate Signaling Differently

Abstract Disclosure: A.V. Rodriguez Rondon: None. M.S. Welling: None. E.L. van den Akker: None. E.F. van Rossum: None. P.J. Delhanty: None. J.A. Visser: None. Melanocortin-4 receptor (MC4R) is a G-protein-coupled receptor expressed in the hypothalamus regulating appetite and energy expenditure. Extensive evidence from genetic and biological studies show that MC4R is a key player in the homeostatic regulation of body weight. MC4R loss of function (LoF) variants are the most common cause of monogenic obesity. We have identified 1 homozygous and 16 heterozygous MC4R variants in patients with obesity at our academic Obesity Center CGG, Erasmus MC. The impact of these variants on MC4R signaling and the association with obesity are unknown. Here, we functionally characterized these variants by analyzing four different aspects of MC4R signaling. HEK293 cells were transiently transfected with expression plasmids encoding wild type (WT) or variant MC4R and stimulated with melanocyte stimulating hormone. Cyclic AMP response (cAMP) was measured using GloSensor cAMP bioluminescence assay, and β-arrestin-2 recruitment (βarr2) was measured using NanoBiT complementation luminescence assay. Furthermore, HiBiT Detection System was used to measure cell surface expression, and serum response element (SRE) reporter assay was used to measure extracellular signal-regulated kinase (ERK) activation. Variants were classified according to the percent of maximum response (Emax) relative to WT: complete LoF (cLoF): 0-20%; partial LoF (pLoF): 20-80%; WT: 80-120%; gain of function (GoF): &amp;gt;120%. The MC4R variants were identified by sequencing the MC4R gene of adult and pediatric patients with obesity using an obesity gene panel. Eleven of these variants have not been functionally studied previously. The median age of onset obesity for all patients is 3 years (range 0 – 6). Furthermore, the median BMI of the adult patients was 48.8 kg/m2 (range 36 - 58.8) and the median BMI-SDS of the pediatric patients was +4.2 SDS (range +3 – 7). Twelve out of 17 patients presented with hyperphagia. MC4R variants had differential effects on signaling. In 7 out of 17 variants, cLoF for cAMP was observed. This could be explained in 6 out of 7 of these variants due to lack of cell surface expression. Intriguingly, five out of 17 variants had a normal or GoF cAMP response and normal ERK activation. However, three out of these 5 variants showed cLoF or pLoF for βarr2, indicating a disrupted desensitization mechanism. In addition, the remaining five variants displayed pLoF for most studied pathways. We show that these MC4R variants identified in our patients with obesity affect signaling differently, through modulation of cAMP, βarr2, cell surface expression, and ERK pathways. This demonstrates the value of examining different aspects of MC4R signaling to understand possible biased effects of variants on these pathways. Overall, our results show the clinical importance of assessing the effect of MC4R variants on a range of molecular signaling mechanisms to determine their association with obesity. Presentation: Saturday, June 17, 2023

SAT062 Inhibition Of GIPR Expression Enhances Sulfonylurea-induced Insulin Secretion, And It Was Associated With Changes In ATP Contents

Abstract Disclosure: H. Kim: None. J. Lim: None. H. Jung: None. Introduction: Sulfonylurea (SU) might be indispensable for a subset of patients with type 2 diabetes. In our previous study, we collected some patients who were extremely sensitive to SU for optimal glycemic control, then drew several candidate single nucleotide variants (SNV) using whole exome sequencing. Among them, 3 SNV (rs550405192, rs13306403, and rs13306402) were harbored by GIPR, coding a receptor for gastric inhibitory polypeptide 1-2). Therefore, in vitro study of GIPR on insulin secretory response to SU was performed in this study. Method:Gipr silencing was done in INS-1 cells using siRNA transfection. To induce glucolipotoxicity, 15-mM glucose and 150-uM palmitic acid were treated to the cells for 24 hours. Glimepiride (50 nM) was treated for 30 min in KRBH buffer with low-/high-glucose, following low-glucose starvation. Intracellular ATP contents and released ATP were measured with a bioluminescence assay kit. Quantitative RT-PCR to assess Vdac1 expression. Results:Gipr silencing was confirmed by reduced expression of mRNA and protein. GIP-induced insulin secretion was also down-regulated. Gipr silencing did not affect either basal or glucose-stimulated insulin secretion. As for SU-induced insulin secretion, Gipr silencing significantly increased it in a low-glucose condition (2.8 mM) but not in a high-glucose (17.5 mM). However, after chronic exposure to glucolipotoxicity, Gipr silencing significantly enhanced the response to SU in a high-glucose condition, too. These effects of Gipr silencing on the SU response were associated with changes in cellular ATP contents, which is critical to SU effects. Because diabetic condition had been reported to induce Vdac1 expression and subsequent ATP release in insulin-secreting cells3), we examined them with regard to Gipr silencing. Vdac1 expression was significantly increased when exposed to glucolipotoxicity, and Gipr silencing inhibited both Vdac1 expression and ATP release in the condition. Conclusion: According to the suppression of Vdac1 expression and ATP release by Gipr silencing in INS1 cells under glucolipotoxicity, interference of GIPR signaling would enhance SU-induced insulin secretion by preservation of cellular ATP. This study supported that loss-of-function mutations in GIPR as novel candidates regarding to sensitivity to SU in type 2 diabetes.

Caffeine reduces viability, induces apoptosis, inhibits migration and modulates the CD39/CD73 axis in metastatic cutaneous melanoma cells.

We aimed to evaluate the effect of caffeine on viability, apoptosis, migration, redox profile and modulatory effect of the purinergic system of cutaneous melanoma cells. The melanoma cells SK-MEL-28 and non-tumoural CCD-1059sk cells were treated for 24 h with different concentrations of caffeine. Cell viability was evaluated by a biochemical assay and fluorescence microscopy, and flow cytometry assessed apoptosis induction. A wound-healing assay assessed cell migration. The redox profile was evaluated by the levels of markers of reactive oxygen species (ROS), nitric oxide (NOx), total thiols (PSH) and non-protein thiols (NPSH). RT-qPCR and flow cytometry assessed the expression of CD39 and CD73. ATPase/ADPase and AMPase enzyme activities were evaluated by hydrolysis of ATP, ADP and AMP nucleotides. A bioluminescent assay assessed extracellular ATP levels. Caffeine significantly reduced melanoma cell viability and migration and did not affect non-tumoural cells. Caffeine increased ROS levels and improved PSH levels in melanoma cells. Furthermore, caffeine reduced CD39 and CD73 expression, decreased ATP, ADP and AMP nucleotide hydrolysis and increased extracellular ATP levels. We have shown that caffeine reduces metastatic cutaneous melanoma cell viability and migration, induces ROS generation and improves PSH levels. In an unprecedented manner, we also showed that caffeine reduces the expression of CD39 and CD73 and, consequently, ATPase/ADPase/AMPase hydrolytic activity of ectonucleotidases, thus displacing the CD39/CD73 axis and increasing extracellular ATP levels. Therefore, caffeine may be an interesting compound for clinical trials with the CD39/CD73 axis as a therapeutic target.

The effectiveness of disinfection protocols in osteopathic family medicine offices.

Healthcare-associated infections (HAIs) pose a substantial public health threat. Despite significant strides tocurb HAIs in hospital environments, outpatient settings have not received the same degree of attention. Given their emphasis on holistic, patient-centered care, osteopathic family medicine offices are pivotal in both disease prevention and comprehensive patient treatment. The importance of simple yet effective disinfection protocols, such as thorough cleaning between patient appointments, cannot be overstated in these settings because they are integral to minimizing disease transmission. This study aims to assess the effectiveness of the current disinfection protocols in osteopathic family medicine offices. A cross-sectional study evaluating disinfection practices on 18 examination tables in an osteopathic family medicine office was conducted. Two high-touch surfaces (midtorso region and table edge) were examined. Initial swab samples were collected after morning disinfection by Environmental Services, and terminal swab samples were gathered after day's-end disinfection by the medical staff. Adenosine triphosphate (ATP) bioluminescence assays were performed utilizing AccuPoint Advanced HC Reader, which quantified ATP, indicating contamination levels in the samples. The higher the ATP levels found in a sample, the greater the amount of biological contamination. All samplers were handled and tested as per manufacturer's instructions. Apreliminary trial was conducted to confirm the internal validity of ATP bioluminescence measurements. The statistical analysis involved Shapiro-Wilk and Wilcoxon signed-rank tests, with significance set at p<0.05. Cohen's d test was utilized to calculate the effect size, identifying meaningful differences in initial and terminal swab sample relative light units (RLUs). The midtorso region demonstrated an 11.1 % increase in failure rate after terminal disinfection when compared to initial disinfection. A Wilcoxon signed-rank testrevealed a median estimated pathogen level forthemidtorso region that was higher after terminal disinfection (median, 193 RLUs; range, 1-690 RLUs; n=18) compared to initial disinfection (median, 134 RLUs; range,4-946 RLUs; n=18). However, this increase was not statistically significant, p=0.9124, with a small effect size, d=0.04. The edge showed no change in failure rate after terminal disinfection, maintaining a 100 % failure rate both before and after disinfection. However, the Wilcoxon signed-rank test revealed a slight reduction in the median estimated pathogen levels after terminal disinfection (median, 2095 RLUs; range, 891-5,540 RLUs; n=18) compared to before disinfection (median, 2,257 RLUs; range, 932-5,825 RLUs; n=18). However, this reduction wasnot statistically significant, p=0.61, with a small effect size, d=0.12. The findings from this study reveal a substantial disparity in outcomes between the two sample locations, midtorso and edge. The midtorso demonstrated arelatively low failure rate in both initial and terminal swab samples, indicating successful outcomes. In contrast, the edge consistently displayed a 100 % failure rate, emphasizing the need for more care and attention when cleaning the edge of the examination to ensure better outcomes. By prioritizing adequate disinfection protocols, including thorough cleaning between patients, osteopathic family medicine offices can more effectively prevent disease transmission and promote patient safety.

Developing a Prolamin-Based Gel for Food Packaging: In-Vitro Assessment of Cytocompatibility.

Growing environmental concerns drive efforts to reduce packaging waste by adopting biodegradable polymers, coatings, and films. However, biodegradable materials used in packaging face challenges related to barrier properties, mechanical strength, and processing compatibility. A composite gel was developed using biodegradable compounds (prolamin, d-mannose, citric acid), as a coating to increase the oxygen barrier of food packaging materials. To improve gel stability and mechanical properties, the gels were physically cross-linked with particles synthesized from tetraethyl orthosilicate and tetramethyl orthosilicate precursors. Additionally, biocompatibility assessments were performed on human keratinocytes and fibroblasts, demonstrating the safety of the gels for consumer contact. The gel properties were characterized, including molecular structure, morphology, and topography. Biocompatibility of the gels was assessed using bioluminescent ATP assay to detect cell viability, lactate dehydrogenase assay to determine cell cytotoxicity, and a leukocyte stimulation test to detect inflammatory potential. A composite gel with strong oxygen barrier properties in low-humidity environments was prepared. Increasing the silane precursor to 50 wt% during gel preparation slowed degradation in water. The addition of citric acid decreased gel solubility. However, higher precursor amounts increased surface roughness, making the gel more brittle yet mechanically resistant. The increase of precursor in the gel also increased gel viscosity. Importantly, the gels showed no cytotoxicity on human keratinocytes or fibroblasts and had no inflammatory effects on leukocytes. This composite gel holds promise for oxygen barrier food packaging and is safe for consumer contact. Further research should focus on optimizing the stability of the oxygen barrier in humid environments and investigate the potential sensitizing effects of biodegradable materials on consumers.

A novel strategy for bioaerosol rapid detection based on broad-spectrum high-efficiency magnetic enrichment and separation combined with ATP bioluminescence

Bioaerosol detection technology represented by laser-induced fluorescence (LIF) cannot effectively detect bioaerosols in the presence of interferents such as plant-derived smoke, industrial waste gas, pollen and pollen debris which can produce strong non-biological fluorescence interference. To overcome this drawback, in this study, a novel method based on broad-spectrum high-efficiency magnetic enrichment and separation combined with adenosine triphosphate (ATP) bioluminescence was proposed for Escherichia coli (E. coli) bioaerosols rapid detection. First, E. coli bioaerosols mixed with interferents were collected. Core-shell Fe3O4@Polydopamine@Polyethyleneimine magnetic particles were used as bioaerosol enrichment materials to enrich E. coli bioaerosol sampling solutions. Subsequently, an ATP bioluminescence assay was performed to determine the concentration of E. coli. A linear relationship was observed between ATP bioluminescence intensity after enrichment and the E. coli bioaerosol concentration in the range of 870–49,098 particles per liter; the bioluminescence intensity measured after enrichment was significantly higher than that before enrichment, and this enrichment method provide a 6-fold better sensitivity in bioaerosol detection. More importantly, this method efficiently enriched and detected bioaerosols in plant-derived smoke. This method can effectively improve the sensitivity of ATP bioluminescence detection, and possesses the advantages of convenient operation and strong anti-interference ability. It also provides a foundation for the effective detection of bioaerosols mixed with interfering substances, and a reference for evaluating the sensitivity and anti-interference of LIF-based instruments.