Application of LDH assay for therapeutic efficacy evaluation of ex vivo tumor models

The current standard preclinical oncology models are not able to fully recapitulate therapeutic targets and clinically relevant disease biology, evidenced by the 90% attrition rate of new therapies in clinical trials. Three-dimensional (3D) culture systems have the potential to enhance the relevance of preclinical models. However, the limitations of currently available cellular assays to accurately evaluate therapeutic efficacy in these models are hindering their widespread adoption. We assessed the compatibility of the lactate dehydrogenase (LDH) assay in 3D spheroid cultures against other commercially available readout methods. We developed a standardized protocol to apply the LDH assay to ex vivo cultures, considering the impact of culture growth dynamics. We show that accounting for growth rates and background release levels of LDH are sufficient to make the LDH assay a suitable methodology for longitudinal monitoring and endpoint assessment of therapeutic efficacy in both cell line-derived xenografts (xenospheres) and patient-derived explant cultures. This method has the added value of being non-destructive and not dependent on reagent penetration or manipulation of the parent material. The establishment of reliable readout methods for complex 3D culture systems will further the utility of these tumor models in preclinical and co-clinical drug development studies.

Dose Dependent Antimicrobial Cellular Cytotoxicity—Implications for ex vivo Diagnostics

Introduction:Ex vivo and in vitro diagnostics, such as interferon-γ (IFN-γ) release enzyme linked ImmunoSpot (ELISpot) and flow cytometry, are increasingly employed in the research and diagnostic setting for severe T-cell mediated hypersensitivity. Despite an increasing use of IFN-γ release ELISpot for drug causality assessment and utilization of a range of antimicrobial concentrations ex vivo, data regarding antimicrobial-associated cellular cytotoxicity and implications for assay performance remain scarcely described in the literature. Using the measurement of lactate dehydrogenase (LDH) and the 7-AAD cell viability staining, we aimed via an exploratory study, to determine the maximal antimicrobial concentrations required to preserve cell viability for commonly implicated antimicrobials in severe T-cell mediated hypersensitivity.Method: After an 18-h incubation of patient peripheral blood monocytes (PBMCs) and antimicrobials at varying drug concentrations, the cell cytotoxicity was measured in two ways. A colorimetric based assay that detects LDH activity and by flow cytometry using the 7-AAD cell viability staining. We used the PBMCs collected from three healthy control participants with no known history of adverse drug reaction and two patients with a rifampicin-associated drug reaction with eosinophilia and systemic symptoms (DRESS), confirmed on IFN-γ ELISpot assay. The PBMCs were stimulated for the investigated drugs at the previously published drug maximum concentration (Cmax), and concentrations 10- and 100-fold above.Results: In a human immunodeficiency virus (HIV) negative and a positive rifampicin-associated DRESS with positive ex vivo IFN-γ ELISpot assay, use of 10- and 100-fold Cmax drug concentrations decreased spot forming units/million cells by 32–100%, and this corresponded to cell cytotoxicity of more than 40 and 20% using an LDH assay and 7-AAD cell viability staining, respectively. The other antimicrobials (ceftriaxone, flucloxacillin, piperacillin/tazobactam, and isoniazid) tested in healthy controls showed similar dose-dependent increased cytotoxicity using the LDH assay, but cytotoxicity remained lower than 40% for all Cmax and 10-fold Cmax drug concentrations except flucloxacillin. All 100-fold Cmax concentrations resulted in cell death >40% (median 57%), except for isoniazid. 7-AAD cell viability staining also confirmed an increase in lymphocyte death in PBMCs incubated with 10-fold and 100-fold above Cmax for ceftriaxone, and flucloxacillin; however, piperacillin/tazobactam and isoniazid indicated no differences in percentages of viable lymphocytes across concentrations tested.Conclusion: The LDH cytotoxicity and 7-AAD cell viability staining techniques both demonstrate increased cell death corresponding to a loss in ELISpot sensitivity, with use of higher antimicrobial drug concentrations for ex vivo diagnostic IFN-γ ELISpot assays. For all the antimicrobials evaluated, the use of Cmax and 10-fold Cmax concentrations impacts cell viability and potentially affects ELISpot performance. These findings inform future approaches for ex vivo diagnostics such as IFN-γ release ELISpot.

Cratoxylum sumatranum stem bark exhibited antimalarial activity by Lactate Dehydrogenase (LDH) assay

Objectives The antimalarial drug resistance is an obstacle in the effort to overcome malaria. The new alternative antimalarial drug became in great attention of urgent need. Current antimalarial drugs were derived from plants. Therefore, the plant is considering a potential source of new drugs. Cratoxylum sumatranum belongs to the Hypericaceae family contain xanthones and phenolic compounds, which was reported for their antimalarial activities. This study aims to determine the antimalarial activities of C. sumatranum extracts and fractions. Methods Cratoxylum sumatranum stem bark (BP14-SB) collected from Balikpapan Botanical Garden in East Kalimantan, Indonesia, was extracted gradually with n-hexane, dichloromethane, and methanol by ultrasonic-assisted extraction method. All extracts were tested against Plasmodium falciparum 3D7 by lactate dehydrogenase (LDH) assay and followed by IC50 determination. The most active extract was further separated and tested for their antimalarial activities. Results The results showed that dichloromethane stem bark extract (BP14-SB-D) had the strongest inhibition of parasite growth with the IC50 value of 0.44 ± 0.05 μg/mL and moderately toxic with the CC50 value of 29.09 ± 0.05 μg/mL. Further fractionation of BP14-SB-D by open column chromatography using silica gel and gradient hexane–ethyl acetate obtained 12 fractions. LDH assay for these 12 fractions of BP14-SB-D showed that Fraction-6 (IC50 value of 0.19 ± 0.03 μg/mL) was performed the strongest inhibition of parasite growth, compared to other fractions. TLC identification showed that BP14-SB-D contains xanthone. Conclusions The dichloromethane extract of C. sumatranum stem bark (BP14-SB-D) and Fraction-6 from this extract exhibited antimalarial activity and the potential to be developed an antimalarial substance.

Hindering The Synchronization Between Mir-486-5p And H19 Lncrna By Hesperetin Halts Breast Cancer Aggressiveness Through Tuning ICAM-1

Background: Recently, a novel crosstalk between non-coding RNAs (ncRNAs) has been casted. However, this has been seldomly investigated in metastatic BC (mBC). H19 and miR-486-5p role in mBC is controversial. ICAM-1 is a recently recognized metastatic engine in mBC. Natural compounds were recently found to alter ncRNAs/target circuits. Yet, Hesperitin modulatory role in altering such circuits has never been investigated in mBC. Objective: The aim of this study is to investigate the impact of hesperitin on miR-486-5p/H19/ICAM-1 axis Methodology: BC patients (n=20) were recruited in the study. Bioinformatic analysis was performed using different prediction softwares. MDA-MB-231 and MCF-7 cells were cultured and transfected using several oligonucleotides or treated with serial dilutions of hesperitin. RNA was extracted and gene expression analysis was performed using q-RT-PCR. ICAM-1 protein levels were assessed using human ICAM-1 Elisa Kit. Cytotoxic potential of hesperitin against normal cells was assessed by LDH assay. Several functional analysis experiments were performed such as MTT, colony forming and migration assays. Results: The study showed that miR-486-5p and H19 has a paradoxical expression profiles in mBC patients. miR-486-5p mimics and H19 siRNAs repressed ICAM-1 and halted mBC hallmarks. A novel crosstalk between miR-486-5p and H19 was observed highlighting a bi-directional relationship between them. Hesperetin restored the expression of miR-486-5p, inhibited H19 lncRNA and ICAM-1 expression and selectively regressed mBC cell aggressiveness. Conclusion: miR-486-5p and H19 are inter-connected upstream regulators for ICAM-1 building up miR-486-5p/H19/ICAM-1 axis that has been successfully tuned in mBC cells by hesperitin

Investigating oxygen transport efficiencies in precision-cut liver slice-based organ-on-a-chip devices

Microfluidic ‘organ-on-a-chip’ devices hold great potential for better mimicking the continuous flow microenvironment experienced by tissue and cells in vivo, thereby ensuring realistic transport of nutrients and elimination of waste products. However, the mass transport of oxygen, which arguably is the most critical nutrient due to its inherently low solubility in water, is rarely assessed. To this aim, the suitability of various precision-cut liver slice (PCLS) microfluidic devices for the defined maintenance of oxygen mass transport were evaluated using COMSOL simulations, leading to the development of a novel, optimised design to provide defined in vivo oxygenation conditions within an organ-on-a-chip system. Simulations found that the proposed device was capable of maintaining 43% of the tissue slice volume within the physiological range of the liver against 18% for the best performing literature device. The optimal device architecture derived from the modelling was then fabricated and its operation confirmed with an LDH assay. These simulation results form the basis for a greater understanding of not just the challenges involved in designing organ-on-a-chip devices, but also highlight issues that would arise from the incorporation of additional organs, as research progresses towards complete human-on-a-chip model systems.

The Cytotoxicity Assessment of Novel Formulation Developed to Reduce Dentin Hypersensitivity Utilizing Dehydrogenase Assay

The problem of real treatment of teeth hypersensitivity is still important and unsolved. The main goal of the experiment was to calculate the possible toxic effects on the fibroblasts cells CCL-1™ (NCTC clone 929) caused by original preparation to reduce tooth surfaces’ hypersensitivity, compared to the marketable preparation Seal & Protect (Dentsply). The assessment was made through measuring lactate dehydrogenase (LDH assay). Lactate dehydrogenase releases from the cell’s cytoplasm to the culture medium as a result of cell membrane damage and lysis of the cells. The measurement is based on an assessment of the ability of LDH to oxidize lactic acid to pyruvic acid, which is dependent on the increase of the release level. The increase of LDH activity in the supernatants of cell cultures shows a relationship with the percentage of dead cells (increased cytotoxicity correlates with the increasing content of dead cells). In the LDH assay, both formulations evaluated after 24 h obtained results which were located below the control values. After seven days, the mean values obtained in cytotoxicity assay LDH are measurable and lower for the original formulation in comparison to the commercial one at the dilution of 1:5. At the dilution with 1:10 ratio, they are comparable and within the range of accepted values. At the maximal dilution of 1:15, the results are higher for the experimental formulation in comparison to the marketable formulation. The polymerization process is beneficial for the cytotoxicity test results in case of both tested preparations. Average values of cytotoxicity of both preparations attain an acceptable level of less than 22.6 ± 8.1%, reliant on the degree of dilution and the remark time. Original formulation is characterized by a greater homogeneity of results. The marketable preparation has a larger diversity of effects, dependent on the time of observation and attenuation; however, the cytotoxicity values are lower when paralleled to the experimental formulation in the test conducted after seven days. This should not have a disastrous effect on the pulp, as the values of both as the values of both preparations are within expected ranges. The obtained results allows to assume that will be possible to introduce the original formulation to the stage of clinical trials in the future.

In Vitro Effects of Bisphenol F on Antioxidant System Indicators in the Isolated Hepatocytes of Rainbow Trout (Oncorhyncus mykiss)

Bisphenol F (BPF) has been used frequently in the plastics industry and the production of daily consumer products as an alternative to bisphenol A (BPA). It was aimed herein to determine the cytotoxic effects of BPF on hepatocytes isolated from the liver of rainbow trout (Oncorhyncus mykiss) using lactate dehydrogenase (LDH) assay and antioxidant defence system indicators. The cultured hepatocytes were exposed to seven concentrations (0, 15.63, 31.25, 62.50, 125, 250, and 500 µM) of BPF for 24 h. According to the LDH assay, the percentage of cytotoxicity was increased dose dependently in the cells. The malondialdehyde content, which is indicative of lipid peroxidation, was increased significantly at BPF concentrations between 15.63 and 250 µM, whereas it remained unchanged with a concentration of 500 µM. The activities of superoxide dismutase were increased, while those of catalase were decreased with all of the BPF concentrations. Elevated levels of reduced glutathione content were determined with BPF concentrations between 15.63 and 250 µM, but decreased significantly with a concentration of 500 µM. Significant increases in the activities of the glutathione peroxidase were found in hepatocytes treated with BPF at concentrations of 31.25 to 500 µM. GST activity was only significantly increased with a BPF concentration of 250 µM. The results showed that the toxic mechanism of BPF was mainly based on cell membrane damage and oxidative stress, which have an influence on antioxidant defences. Therefore, BPF was reconsidered as a safe alternative instead of BPA in the manufacturing of industrial or daily products.

Some pharmacological properties of 4-[3-(5-bromo-2-hydroxyphenyl)-5-phenyl-3,4-dihydropyrazol-2-yl]-5H-thiazol-2-one

A series of 3,5-diaryl pyrazolyl thiazolinones were designed and synthesized as potential biologically active compounds. The study of anticancer activity of 4-[3-(5-bromo-2-hydroxyphenyl)-5-phenyl-3,4-dihydropyrazol-2-yl]-5H-thiazol-2-one (1) revealed its high antiproliferative activity against a panel of cancer cells with the lowest growth inhibition concentration (GI50) towards leukemic cell line SR (0.0351 µМ) and ovarian cancer cell line OVCAR-3 (0.248 µМ). It was also found that pyrazolyl thiazolinone 1 inhibited growth of Trypanosoma brucei brucei by 98,8% at a concentration of 10 µg/mL. The in-depth cytotoxicity study of compound 1 on human hepatocellular carcinoma HepG2 cells and non-tumorigenic murine fibroblast Balb/c 3T3 in MTT, NRU, TPC and LDH assays showed that normal cells were less sensitive to compound 1 than the cancer cells; its action had led to a disintegration of the cell membrane, inhibition of mitochondrial and lysosomal activity, and proliferation of cancer cells. The highest selectivity were detected in the LDH assay.

Iron Oxide Particles Alter Bacterial Uptake and the LPS-Induced Inflammatory Response in Macrophages

Exposure to geogenic (earth-derived) particulate matter (PM) is linked to severe bacterial infections in Australian Aboriginal communities. Experimental studies have shown that the concentration of iron in geogenic PM is associated with the magnitude of respiratory health effects, however, the mechanism is unclear. We investigated the effect of silica and iron oxide on the inflammatory response and bacterial phagocytosis in macrophages. THP-1 and peripheral blood mononuclear cell-derived macrophages were exposed to iron oxide (haematite or magnetite) or silica PM with or without exposure to lipopolysaccharide. Cytotoxicity and inflammation were assessed by LDH assay and ELISA respectively. The uptake of non-typeable Haemophilus influenzae by macrophages was quantified by flow cytometry. Iron oxide increased IL-8 production while silica also induced significant production of IL-1β. Both iron oxide and silica enhanced LPS-induced production of TNF-α, IL-1β, IL-6 and IL-8 in THP-1 cells with most of these responses replicated in PBMCs. While silica had no effect on NTHi phagocytosis, iron oxide significantly impaired this response. These data suggest that geogenic particles, particularly iron oxide PM, cause inflammatory cytokine production in macrophages and impair bacterial phagocytosis. These responses do not appear to be linked. This provides a possible mechanism for the link between exposure to these particles and severe bacterial infection.

Identification of Novel Cytotoxic T Lymphocyte Epitopes of Drug- Resistance Related Protein InhA from Mycobacterium tuberculosis

Background: Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB), especially the drug-resistant MTB, poses serious challenges to human healthcare worldwide. Cytotoxic T lymphocytes (CTLs) play a vital role in immune defense against MTB. Objective: To identify novel CTL epitopes that could induce cellular immunity against MTB infections. Methods: The HLA-A*0201 restricted CTL epitopes of the drug-resistant protein InhA from MTB were predicted by online algorisms and synthesized by the Fmoc solid phase method. The candidate peptides were used to induce CTLs from human peripheral blood mononuclear cells (PBMCs) of HLA-A*0201 healthy donors and the HLA-2.1/Kb mice. IFN-γ productions of CTLs were detected by enzyme linked immunospot assay (ELISPOT), flow cytometry and enzyme-linked immunosorbent assay (ELISA), and cytotoxicity was analyzed by lactate dehydrogenase (LDH) assay. Results: A group of 4 epitopes were screened out with high affinities to HLA-A*0201. ELISPOT and flow cytometry analysis indicated these peptides significantly induced that IFN-γ release of CTLs from the HLA-A*0201+/PPD+ donors, as the mutant analogues had more potent stimulation effects. LDH assay showed that CTLs from PPD+ donors and the immunized mice exhibited significant cytotoxicity and low cross-reactivity. ELISA analysis revealed comparative levels of IFN-γ were released by CTLs isolated from the mice spleen. Conclusion: Our study has identified 4 novel CTL epitopes of InhA that could elicit potent CTL immunity, establishing a foundation for the development of multivalent peptide vaccines against the drug-resistant MTB.