The liver is a vulnerable target for amphetamine toxicity, but the mechanisms involved in the drug's hepatotoxicity remain poorly understood. The purpose of the current research was to characterize the mode of death elicited by four amphetamines and to evaluate whether their combination triggered similar mechanisms in immortalized human HepG2 cells. The obtained data revealed a time- and temperature-dependent mortality of HepG2 cells exposed to 3,4-methylenedioxymethamphetamine (MDMA, ecstasy; 1.3 mM), methamphetamine (3 mM), 4-methylthioamphetamine (0.5 mM) and D-amphetamine (1.7 mM), alone or combined (1.6 mM mixture). At physiological temperature (37 °C), 24-h exposures caused HepG2 death preferentially by apoptosis, while a rise to 40.5 °C favoured necrosis. ATP levels remained unaltered when the drugs where tested at normothermia, but incubation at 40.5 °C provoked marked ATP depletion for all treatments. Further investigations on the apoptotic mechanisms triggered by the drugs (alone or combined) showed a decline in BCL-2 and BCL- XL mRNA levels, with concurrent upregulation of BAX, BIM, PUMA and BID genes. Elevation of Bax, cleaved Bid, Puma, Bak and Bim protein levels was also seen. To the best of our knowledge, Puma, Bim and Bak have never been linked with the toxicity induced by amphetamines. Time-dependent caspase-3/-7 activation, but not mitochondrial membrane potential (∆ψm) disruption, also mediated amphetamine-induced apoptosis. The cell dismantling was confirmed by poly(ADP-ribose)polymerase proteolysis. Overall, for all evaluated parameters, no relevant differences were detected between individual amphetamines and the mixture (all tested at equieffective cytotoxic concentrations), suggesting that the mode of action of the amphetamines in combination does not deviate from the mode of action of the drugs individually, when eliciting HepG2 cell death.

Direct visualisation of cells for the purpose of studying their motility has typically required expensive microscopy equipment. However, recent advances in digital sensors mean that it is now possible to image cells for a fraction of the price of a standard microscope. Along with low-cost imaging there has also been a large increase in the availability of high quality, open-source analysis programs. In this study we describe the development and performance of an expandable cell motility system employing inexpensive, commercially available digital USB microscopes to image various cell types using time-lapse and perform tracking assays in proof-of-concept experiments. With this system we were able to measure and record three separate assays simultaneously on one personal computer using identical microscopes, and obtained tracking results comparable in quality to those from other studies that used standard, more expensive, equipment. The microscopes used in our system were capable of a maximum magnification of 413.6×. Although resolution was lower than that of a standard inverted microscope we found this difference to be indistinguishable at the magnification chosen for cell tracking experiments (206.8×). In preliminary cell culture experiments using our system, velocities (mean µm/min ± SE) of 0.81±0.01 (Biomphalaria glabrata hemocytes on uncoated plates), 1.17±0.004 (MDA-MB-231 breast cancer cells), 1.24±0.006 (SC5 mouse Sertoli cells) and 2.21±0.01 (B. glabrata hemocytes on Poly-L-Lysine coated plates), were measured and are consistent with previous reports. We believe that this system, coupled with open-source analysis software, demonstrates that higher throughput time-lapse imaging of cells for the purpose of studying motility can be an affordable option for all researchers.

Borrelia burgdorferi, the causative agent of Lyme disease, has a highly reduced genome and relies heavily on glycolysis for carbon metabolism. As such, established inhibitors of lactate dehydrogenase (LDH) were evaluated in cultures to determine the extent of their impacts on B. burgdorferi growth. Both racemic and enantiopure (AT-101) gossypol, as well as oxamate, galloflavin, and stiripentol, caused the dose-dependent suppression of B. burgdorferi growth in vitro. Racemic gossypol and AT-101 were shown to fully inhibit spirochetal growth at concentrations of 70.5 and 187.5 μM, respectively. Differences between racemic gossypol and AT-101 efficacy may indicate that the dextrorotatory enantiomer of gossypol is a more effective inhibitor of B. burgdorferi growth than the levorotatory enantiomer. As a whole, LDH inhibition appears to be a promising mechanism for suppressing Borrelia growth, particularly with bulky LDH inhibitors like gossypol.

Disruptions to reproductive health in wildlife species inhabiting polluted environments is often found to occur alongside compromised immunity. However, research on impacts of aquatic pollution on freshwater mollusc immune responses is limited despite their importance as vectors of disease (Schistosomiasis) in humans, cattle and wild mammals. We developed an in vitro ‘tool-kit’ of well-characterized quantitative immune tests using Biomphalaria glabrata hemocytes. We exposed hemocytes to environmentally-relevant concentrations of common aquatic pollutants (17β-estradiol, Bisphenol-A and p,p’-DDE) and measured key innate immune responses including motility, phagocytosis and encapsulation. Additionally, we tested an extract of a typical domestic tertiary treated effluent as representative of a ‘real-world’ mixture of chemicals. Encapsulation responses were stimulated by p,p’-DDE at low doses but were suppressed at higher doses. Concentrations of BPA (above 200 ng/L) and p,p’-DDE (above 500 ng/L) significantly inhibited phagocytosis compared to controls, whilst hemocyte motility was reduced by all test chemicals and the effluent extract in a dose-dependent manner. All responses occurred at chemical concentrations considered to be below the cytotoxic thresholds of hemocytes. This is the first time a suite of in vitro tests has been developed specifically in B. glabrata with the purpose of investigating the impacts of chemical pollutants and an effluent extract on immunity. Our findings indicate that common aquatic pollutants alter innate immune responses in B. glabrata, suggesting that pollutants may be a critical, yet overlooked, factor impacting disease by modulating the dynamics of parasite transmission between molluscs and humans.

Immune tolerance or learned reactivity to the non‐self ‘external’ world is largely epigenetic; acquired immune‐memory carried by a group of specialized white blood cells known as lymphocytes. Activation of lymphocytes, while an important part of normal immune function, is also linked to chronic inflammatory and autoimmune disease. These diseases occur when innate immune‐function overload causes lymphocytes to become activated against ‘self’ tissue. The Lymphocyte Response Assay (LRA) described here (LRA by ELISA/ACT) addresses the need to distinguish helpful from harmful immune memory responses. Prior LRA methods include colony formation, thymidine incorporation and cytokine release assays.In this LRA, from one ounce of whole blood, 500 separate cell cultures can be performed. In the assay, antigen pre‐coated microtiter plate wells are incubated in autologous cell‐rich plasma under standard physiologic conditions (35±2 °C in a CO2 and humidity controlled incubator), followed by direct microscopy imaging of lymphocyte‐specific responses.This LRA uses pre‐analytic techniques to avoid activation of blood clotting in the CPDA vacutainer tubes used for specimen collection and transport, with specimen temperature maintained between 4–10°C during shipment. An ex vivo brief cell culture system is followed by a cell‐membrane based, amplified signal, which is transduced into an image that can be graded for clinical interpretation. Positive and negative controls are included with each array. Assay grade is read as: non‐reactivation (<8% reactive cells compared to the internal positive control), moderate reactivation (9–50% reactive cells), and strong reactivation (>50% reactive cells). Reactive cells appear to all be T or B class lymphocytes. From reports currently unpublished, antigen‐presenting cells can be readily observed moving toward lymphocytes prior to cell reactivation. The few hours of incubation used are insufficient for lymphocyte memory to be acquired; thus reactivated cells must have learned to recognize specific antigens prior to phlebotomy.We report based on consecutive blind split samples representing 4138 items tested (Table 1).Split sample variance was less than 2.3%, qualifying this LRA as a high‐sensitivity (highly reproducible; hsLRA) clinical assay of immune‐tolerance and delayed allergy (Table 1). Since it is suitable for automation, the assay has the potential for high‐throughput screening, considerably increasing the number and rate of samples which could be analyzed. Finally, the assay needs to be compared with flow cytometry, and in clinical outcome studies, to verify its promising predictive significance as a personalized biomarker.Support or Funding InformationFunding from grant to Health Studies Collegium by ELISA/ACT BioTechnologies Performance of high‐sensitivity Lymphocyte Response Assay (hsLRA) on consecutive blind split samples. # items tested # items matched # items non‐matched % of itemsa matched % of itemsa non‐matched Test interval (year) 636 633 3 99.48±0.91 0.52±0.91 2011 1819 1778 41 97.50±3.58 2.19±3.19 2012 1156 1134 22 98.16±2.11 1.84±2.11 2013 527 505 22 95.56±3.01 3.89±2.69 2014 4138 4050 88 97.60±3.00 2.25±2.75 Total