Effects of organophosphates on precision-cut kidney slices.

Organophosphate (OP) poisoning, both accidental and with suicidal intent, is a global medical challenge. While the primary toxicity of these pesticides is based on the inhibition of acetylcholinesterase (AChE), case reports describe patients developing OP-mediated renal insufficiency. We set out to investigate possible pathomechanisms utilizing rat precision-cut kidney slices (PCKS). Depending on the method of investigation, PCKS were observed for a maximum of 10 days. PCKS exposed to OP compounds (malaoxon, malathion, paraoxon, parathion) showed a dose-dependent loss of viability and a reduction of total protein content over the course of 10 days. A concentration of 500 µM OP showed the most differences between OP compounds. After two days of incubation parathion showed a significantly lower level of viability than malathion. The respective effects of paraoxon and malaoxon were not significantly different from the control. However, effects of OP were only observed in concentrations exceeding those that were needed to achieve significant AChE inhibition in rat kidney tissue. In addition, we observed histological changes, without inducing LDH leakage. Overall, results suggest that OP exert effects in kidney tissue, that exceed those expected from the sole inhibition of AChE and vary between compounds. Without signs of necrosis, findings call for studies that address other possible pathomechanisms, including inflammatory response, oxidative stress or activation of apoptosis to further understand the nephrotoxicity of OP compounds. Monitoring oxon concentration over time, we demonstrated reduced enzyme-inhibiting properties in the presence of PCKS, suggesting interactions between OP compound and kidney tissue.

A two-decade journey in identifying high mobility group box 1 (HMGB1) and procathepsin L (pCTS-L) as potential therapeutic targets for sepsis.

INTRODUCTION: Microbial infections and resultant sepsis are leading causes of death in hospitals, representing approximately 20% of total deaths worldwide. Despite the difficulties in translating experimental insights into effective therapies for often heterogenous patient populations, an improved understanding of the pathogenic mechanisms underlying experimental sepsis is still urgently needed. Sepsis is partly attributable to dysregulated innate immune responses manifested by hyperinflammation and immunosuppression at different stages of microbial infections. AREAS COVERED: Here we review our recent progress in searching for late-acting mediators of experimental sepsis and propose high mobility group box 1 (HMGB1) and procathepsin-L (pCTS-L) as potential therapeutic targets for improving outcomes of lethal sepsis and other infectious diseases. EXPERT OPINION: It will be important to evaluate the efficacy of HMGB1- or pCTS-L-targeting agents for the clinical management of human sepsis and other infectious diseases in future studies.

Human precision-cut cystic duct and gallbladder slices: a novel method for studying cholangiopathies.

Background and aims: Precision-cut tissue slices (PCTS) are widely used as an ex vivo culture tissue culture technique to study pathogeneses of diseases and drug activities in organs in vitro. A novel application of the PCTS model may be in the field of translational research into cholangiopathies such as biliary atresia. Therefore, the aim of this study was to apply the precision-cut slice technique to human bile duct and gallbladder tissue. Methods: Cystic duct and gallbladder tissue derived from patients undergoing a cholecystectomy were collected, preserved and used for preparation of precision-cut cystic duct slices (PCCDS) and precision-cut gallbladder slices (PCGS). The PCCDS and PCGS were prepared using a mechanical tissue slicer and subsequently incubated for 24 and 48 h respectively in William's Medium E (WME) culture medium. Viability was assessed based on ATP/protein content and tissue morphology [hematoxylin and eosin (H&E) staining]. Results: It was shown that viability, assessed by the ATP/protein content and morphology, of the PCCDS (n = 8) and PCGS (n = 8) could be maintained over the 24 and 48 h incubation period respectively. ATP/protein content of the PCCDS increased significantly from 0.58 ± 0.13 pmol/µg at 0 h to 2.4 ± 0.29 pmol/µg after 24 h incubation (P = .0003). A similar significant increase from 0.94 ± 0.22 pmol/µg at 0 h to 3.7 ± 0.41 pmol/µg after 24 h (P = .0005) and 4.2 ± 0.47 pmol/µg after 48 h (P = .0002) was observed in the PCGS. Morphological assessment of the PCCDS and PCGS showed viable tissue at 0 h and after 24 and 48 h incubation respectively. Conclusion: This study is the first to report on the use of the PCTS model for human gallbladder and cystic duct tissue. PCCDS and PCGS remain viable for an incubation period of at least 24 h, which makes them suitable for research purposes in the field of cholangiopathies, including biliary atresia.

Use of Open Surface Plasmon Resonance (OpenSPR) to Characterize the Binding Affinity of Protein-Protein Interactions.

Surface Plasmon Resonance(SPR) is a label-free optical technique to assess protein-protein interaction kinetics and affinities in a real-time setting. Traditionally, Biacore SPR employs a continuous film of gold to detect any change in the angle of re-emitted light when the refractive index of a ligand conjugated to the flat gold surface is altered by its interaction with a local analyte. In contrast, the Nicoya Lifesciences' OpenSPR technology uses gold nanoparticles to detect small changes in the absorbance peak wavelength of a conjugated ligand after its engagement by an analyte. Specifically, when broadband white light is shone onto the gold nanoparticles, it produces a strong resonance absorbance peak corresponding to the refractive index of a ligand conjugated to the surface of gold nanoparticles. Upon its interaction with an analyte, however, the absorbance wavelength peak of the conjugated ligand will be changed and timely recorded as sensorgrams of dynamic ligand-analyte interactions. Thus, the improvement in the detection method (from traditional detection of changes in the angle of re-emitted light to the contemporary detection of changes in the wavelength of the absorbance peak) features OpenSPR as a cost-effective and user-friendly technique for in-depth characterization of protein-protein interactions. Here, we describe the detailed method that we used to characterize procathepsin L (pCTS-L) interactions with two putative pattern recognition receptors (TLR4 and RAGE) using the 1st generation of Nicoya Lifesciences' OpenSPR instrument with a 1-channel detection. Key features • Nicoya OpenSPR is a benchtop small-size equipment that provides in-depth label-free binding kinetics and affinity measurement for protein-protein interactions in real-time fashion. • This technology is relatively intuitive and user-friendly for scientists at any skill level. • OpenSPR sensors employ nanotechnology to reduce the cost of manufacturing complex optical hardware and Sensor Chips, and similarly reduce the consumption of precious analyte samples. • The manufacturer provides online training for OpenSPR (Catalog: TRAIN-REMOTE) and TraceDrawer (Catalog: TRAIN-TD) to customer scientists.

Precision cut tissue slices to investigate the effects of triclosan exposure in Mytilus galloprovincialis.

Precision-cut tissue slices (PCTS) are frequently used in mammalian research, but its application in the area of aquatic toxicology is still humble. This work proposes the use of PCTS to investigate the effects of the antimicrobial triclosan (TCS) in the mussel Mytilus galloprovincialis. PCTS sectioned from the digestive gland (400 µm) were exposed to 10, 100, and 500 nM TCS for 24 h, and the expression of selected genes, together with the biomarkers, carboxylesterases (CbE) and glutathione S-transferases (GST), and the analysis of lipids in PCTS and culture medium, were used to investigate the molecular initiating events of triclosan in the digestive gland of mussels. Significant dysregulation in the expression of phenylalanine-4-hydroxylase (PAH), glutamate dehydrogenase (GDH), fatty acid synthase (FASN), and 7-dehydrocholesterol reductase (DHCR7), involved in energy, phenylalanine and lipid metabolism, were detected. The analysis of lipids evidenced significant changes in cholesteryl esters (CEs) and membrane lipids in the culture medium of exposed PCTS, suggesting dysregulation of energy and lipid metabolism that can affect lipid dynamics in mussels exposed to triclosan.

Learning Health Systems, Informed Consent, and Respect for Persons.

Several pieces in the Hastings Center Report's May-June 2022 issue concern research ethics issues that arise in learning health care systems. In the lead article, Stephanie Morain and colleagues propose a new ethical framework for pragmatic clinical trials (PCTs), which are trials embedded in clinical care. Their framework consists of eight dimensions of demonstrating respect for patients enrolled in PCTs. In the second article, Robert Steel argues that patients being treated in a learning health care system can be required to participate in a clinical trial even if the risk to them is more than minimal. If they wish to refuse, they must either forgo treatment in the system or seek it elsewhere. Three commentaries explore various dimensions of Steel's argument. A third article in the issue turns in a different direction, to assumptions in bioethics about the quality of lives lived with disability. The authors, Debjani Mukherjee, Preya Tarsney, and Kristi Kirschner, find much that needs to change and offer recommendations for improvements at multiple levels.

Precision-Cut Tumor Slices (PCTS) as an Ex Vivo Model in Immunotherapy Research.

Precision-cut tumor slices (PCTS) have recently emerged as important ex vivo human tumor models, offering the opportunity to study individual patient responses to targeted immunotherapies, including CAR-T cell therapies. In this review, an outline of different human tumor models available in laboratory settings is provided, with a focus on the unique characteristics of PCTS. Standard PCTS generation and maintenance procedures are outlined, followed by an in-depth overview of PCTS utilization in preclinical research aiming to better understand the unique functional characteristics of cytotoxic T cells within human tumors. Furthermore, recent studies using PCTS as an ex vivo model for predicting patient responses to immunotherapies and other targeted therapies against solid tumors are thoroughly presented. Finally, the advantages and limitations of the PCTS models are discussed. PCTS are expected to gain momentum and be fully utilized as a significant tool towards better patient stratification and personalized medicine.

Efficacy of Artemether-Lumefantrine on various Plasmodium falciparum Kelch 13 and Pfmdr1 genes isolated in Ghana.

INTRODUCTION: Artemether-Lumefantrine (A-L) remains the drug of choice for the treatment of uncomplicated malaria in Ghana. However, the pharmaco-activity of A-L has not been assessed on various Plasmodium falciparum Kelch 13 and Pfmdr1 genes. Therefore, this study sought to determine the therapeutic efficacy of A-L on P. falciparum parasites isolated from Ghana. METHODS: The clinical study was done in Ga West Municipality, Ghana, where 78 uncomplicated malaria patients were recruited with prior consent. The patients were treated orally with A-L according to national treatment guidelines. Baseline parasitaemia was determined before treatment and 8-hourly parasitaemia posttreatment were determined till initial clearance of parasitaemia and at days 7, 14, 21, and 28. Kelch 13 and Pfmdr1 genes were genotyped by sequencing using baseline samples. Parasite clearance characteristics were determined using Parasite Clearance Estimator beta 0.9 application. RESULTS: Five Kelch 13 (F446I, S466N, R539I, A578S, and A676S) and three Pfmdr1 mutations (N86Y, Y184F and D1246Y) were identified in 78 infected samples. About 8% of the samples contained two Pfmdr1 double mutations (N86Y & D1246Y and Y184F & N86Y). Additionally, three samples (3.8%) were found to contain both Kelch 13 mutations and Pfmdr1 wild type genes. In all patients, parasitaemia persisted within the first 24 h of A-L therapy. However, at hour 40, only two patients were parasitaemic while all patients were aparasitaemic at hour 48. The genotypic profiles of the two persistent parasites at hour 40 were F446I and D1246Y, and R539I, Y184F, and N86Y. The slope half-life of the former was 6.4 h while the latter was 6.9 h and their respective PCT99 were 47.9 h and 49.2 h as well as a clearance rate constants of 0.109 and 0.092 respectively. CONCLUSION: This study reports the effectiveness of A-L on various P. falciparum mutant alleles. However, continuous surveillance of Kelch 13 mutations and Pfmdr1 gene in Ghana and regular assessment of the therapeutic efficacy of A-L and other artemisinin derivatives is recommended.

Behavioural therapy for smoking cessation: the effectiveness of different intervention types for disadvantaged and affluent smokers.

BACKGROUND: Disadvantaged smokers are less likely to be successful when trying to stop smoking than more affluent smokers. In the UK, NHS Stop Smoking Services (SSS) provide a range of pharmacotherapy and behavioural support, delivered by advisors with a range of backgrounds. Whether the types of support provided and who provides it influence differences in quit rates amongst low SES smokers compared with high SES smokers has not previously been examined. METHODS: 202,084 records of smokers in England who attended a NHS Stop Smoking Service between July 2010 and June 2011 were acquired. Smokers were followed-up by services at four weeks post quit date. Multilevel logistic regression models of CO validated quits were employed. Disadvantage was explored through the National Statistics Socio-Economic Classification (NS-SEC) and by eligibility for free prescriptions, an indicator of low income amongst adults aged between 19 and 59 in England. RESULTS: Affluent smokers were more likely to quit than disadvantaged smokers (OR 1.38 (1.35 to 1.42) for clients who paid for prescriptions compared to those eligible for free prescriptions). 80% of service clients received one-to-one counselling but open group forms of behavioural therapy were more successful (main effect OR 1.26 (1.12 to 1.41)) except amongst some of the most disadvantaged clients (long-term unemployed and prisoners). Closed groups were little deployed and they were not significantly more successful than one-to-one behavioural therapy after controls. Who delivered treatment did make a difference for some clients, with all but the most affluent less likely to be successful if they had been treated by a nurse compared with other types of advisers, including smoking cessation specialists (main effect OR 0.73 (0.65 to 0.83)). CONCLUSION: This study provides further evidence that disadvantaged smokers find quitting more difficult even when they have attended a smoking cessation programme. The findings suggest that open groups should be promoted, although they may not be as effective as other forms of behavioural therapy for the long-term unemployed or prisoners. Further research is required to explore why most groups of smokers who attended services staffed by nurses were less likely to quit than those who received treatment from other types of advisors.