Advances in Animal Models and Cutting-Edge Research in Alternatives: Proceedings of the Second International Conference on 3Rs Research and Progress, Hyderabad, 2021.

The fact that animal models fail to replicate human disease faithfully is now being widely accepted by researchers across the globe. As a result, they are exploring the use of alternatives to animal models. The time has come to refine our experimental practices, reduce the numbers and eventually replace the animals used in research with human-derived and human-relevant 3-D disease models.

A Systematic Comparative Assessment of the Response of Ovarian Cancer Cells to the Chemotherapeutic Cisplatin in 3D Models of Various Structural and Biochemical Configurations-Does One Model Type Fit All?

Epithelial Ovarian Cancer (EOC) is a silent, deadly and aggressive gynaecological disease with a relatively low survival rate. This has been attributed, to some extent, to EOC's high recurrence rate and resistance to currently available platinum-based chemotherapeutic treatment methods. Multiple groups have studied and reported the effect of chemotherapeutic agents on various EOC 3D in vitro models.

Development and Characterization of a Porcine Liver Scaffold.

The growing number of patients requiring liver transplantation for chronic liver disease cannot be currently met due to a shortage in donor tissue. As such, alternative tissue engineering approaches combining the use of acellular biological scaffolds and different cell populations (hepatic or progenitor) are being explored to augment the demand for functional organs. Our goal was to produce a clinically relevant sized scaffold from a sustainable source within 24 h, while preserving the extracellular matrix (ECM) to facilitate cell repopulation at a later stage.

Application of porcine gastrointestinal organoid units as a potential in vitro tool for drug discovery and development.

The gastrointestinal tract (GI) is a crucial part of the body for growth and development and its dysregulation can lead to several diseases with detrimental effects. Most of these diseases lack effective treatment, occurring as a result of inappropriate models to develop safe and potent therapies. Organoids are three-dimensional self-organizing and self-renewing structures that are composed of a cluster of different cells in vitro that resemble their organ of origin in architecture and function.

Effect of Repeated Daily Dosing with 2,4-Dinitrochlorobenzene on Glutathione Biosynthesis and Nrf2 Activation in Reconstructed Human Epidermis.

Glutathione (GSH) plays a major role in skin detoxification processes due to its ability to conjugate electrophilic exogenous compounds with, and sometimes without, catalysis by glutathione-s-transferase (GST). GST activity has been demonstrated both in skin and in most in vitro skin equivalents but so far studies have focussed on chemical clearance (conjugate identification and rate of conjugation) and did not consider the GSH lifecycle (conjugation, recycling, synthesis). We used the model skin sensitizer 2,4-dinitrochlorobenzene (DNCB) to investigate the effects of chemical exposure on GSH lifecycle in reconstructed human epidermis (RHE).

UHRF1 regulation of the Keap1-Nrf2 pathway in pancreatic cancer contributes to oncogenesis.

The cellular defence protein Nrf2 is a mediator of oncogenesis in pancreatic ductal adenocarcinoma (PDAC) and other cancers. However, the control of Nrf2 expression and activity in cancer is not fully understood. We previously reported the absence of Keap1, a pivotal regulator of Nrf2, in ∼70% of PDAC cases.

Glutathione metabolism in the HaCaT cell line as a model for the detoxification of the model sensitisers 2,4-dinitrohalobenzenes in human skin.

Glutathione (GSH) is the most prominent antioxidant in cells and the co-factor of an important set of enzymes involved in the skin metabolic clearance system, glutathione S-transferases (GST). Here, we describe an LC-MS (liquid chromatography-mass spectroscopy) method to measure GSH and its disulfide form (GSSG) in HaCaT cells and a 3D Reconstructed Human Epidermis (RHE) model. In our assay, the basal level of GSH in both systems was in the low nmol/mg soluble protein range, while the level of GSSG was systematically below our limit of quantification (0.

Failure to upregulate cell surface PD-1 is associated with dysregulated stimulation of T cells by TGN1412-like CD28 superagonist.

The CD28 superagonist (CD28SA) TGN1412 was administered to humans as an agent that can selectively activate and expand regulatory T cells but resulted in uncontrolled T cell activation accompanied by cytokine storm. The molecular mechanisms that underlie this uncontrolled T cell activation are unclear. Physiological activation of T cells leads to upregulation of not only activation molecules but also inhibitory receptors such as PD-1.

Brusatol provokes a rapid and transient inhibition of Nrf2 signaling and sensitizes mammalian cells to chemical toxicity-implications for therapeutic targeting of Nrf2.

The transcription factor Nrf2 regulates the basal and inducible expression of a battery of cytoprotective genes. Whereas numerous Nrf2-inducing small molecules have been reported, very few chemical inhibitors of Nrf2 have been identified to date. The quassinoid brusatol has recently been shown to inhibit Nrf2 and ameliorate chemoresistance in vitro and in vivo.

Identification and quantification of the basal and inducible Nrf2-dependent proteomes in mouse liver: biochemical, pharmacological and toxicological implications.

Unlabelled: The transcription factor Nrf2 is a master regulator of cellular defence: Nrf2 null mice (Nrf2((-/-))) are highly susceptible to chemically induced toxicities. We report a comparative iTRAQ-based study in Nrf2((-/-)) mice treated with a potent inducer, methyl-2-cyano-3,12-dioxooleana-1,9(11)dien-28-oate (CDDO-me; bardoxolone -methyl), to define both the Nrf2-dependent basal and inducible hepatoproteomes. One thousand five hundred twenty-one proteins were fully quantified (FDR <1%).

Safety pharmacology--current and emerging concepts.

Safety pharmacology (SP) is an essential part of the drug development process that aims to identify and predict adverse effects prior to clinical trials. SP studies are described in the International Conference on Harmonisation (ICH) S7A and S7B guidelines. The core battery and supplemental SP studies evaluate effects of a new chemical entity (NCE) at both anticipated therapeutic and supra-therapeutic exposures on major organ systems, including cardiovascular, central nervous, respiratory, renal and gastrointestinal.

The Nrf2 cell defence pathway: Keap1-dependent and -independent mechanisms of regulation.

The transcription factor Nrf2 (NF-E2-related factor 2) plays a vital role in maintaining cellular homeostasis, especially upon the exposure of cells to chemical or oxidative stress, through its ability to regulate the basal and inducible expression of a multitude of antioxidant proteins, detoxification enzymes and xenobiotic transporters. In addition, Nrf2 contributes to diverse cellular functions including differentiation, proliferation, inflammation and lipid synthesis and there is an increasing association of aberrant expression and/or function of Nrf2 with pathologies including cancer, neurodegeneration and cardiovascular disease. The activity of Nrf2 is primarily regulated via its interaction with Keap1 (Kelch-like ECH-associated protein 1), which directs the transcription factor for proteasomal degradation.