Toxicologic Pathology Forum: Opinion on Performing Good Laboratory Practice Histopathology Evaluation for Nonclinical Toxicity Studies in a Remote Location.

Toxicologic/veterinary pathologists are working remotely from Good Laboratory Practice (GLP) test facilities (TFs) in increasing numbers, most commonly in home-office settings. A study pathologist (SP) generating data on GLP-compliant nonclinical studies must be keenly aware of applicable national GLP regulations and comply with TF and protocol requirements. This Toxicological Pathology Forum Opinion Piece will summarize primary areas of emphasis for the SP generating GLP data using glass slides.

Hematopoietic Tumors in a Mouse Model of X-linked Chronic Granulomatous Disease after Lentiviral Vector-Mediated Gene Therapy.

Chronic granulomatous disease (CGD) is a rare inherited disorder due to loss-of-function mutations in genes encoding the NADPH oxidase subunits. Hematopoietic stem and progenitor cell (HSPC) gene therapy (GT) using regulated lentiviral vectors (LVs) has emerged as a promising therapeutic option for CGD patients. We performed non-clinical Good Laboratory Practice (GLP) and laboratory-grade studies to assess the safety and genotoxicity of LV targeting myeloid-specific Gp91 expression in X-linked chronic granulomatous disease (XCGD) mice.

Conditioning Regimens in Long-Term Pre-Clinical Studies to Support Development of Gene Therapy: Review of Nonproliferative and Proliferative Changes.

Hematopoietic stem cell gene therapy has become a successful therapeutic strategy for some inherited genetic disorders. Pre-clinical toxicity studies performed to support the human clinical trials using viral-mediated gene transfer and autologous hematopoietic stem and progenitor cell (HSPC) transplantation are complex and the use of mouse models of human diseases makes interpretation of the results challenging. In addition, they rely on the use of conditioning agents that must induce enough myeloablation to allow engraftment of transduced and transplanted HSPC.

Phagocytosis-shielded lentiviral vectors improve liver gene therapy in nonhuman primates.

Liver-directed gene therapy for the coagulation disorder hemophilia showed safe and effective results in clinical trials using adeno-associated viral vectors to replace a functional coagulation factor, although some unmet needs remain. Lentiviral vectors (LVs) may address some of these hurdles because of their potential for stable expression and the low prevalence of preexisting viral immunity in humans. However, systemic LV administration to hemophilic dogs was associated to mild acute toxicity and low efficacy at the administered doses.

Gene Therapy: Graft-versus-host Disease (GVHD) in NSG™ (NOD.Cg-Prkdc Il2rg/SzJ) Mice Transplanted with CD34 Human Hematopoietic Stem Cells.

A therapeutic option for monogenic disorders is gene therapy with -transduced autologous hematopoietic stem cells (HSCs). Safety or efficacy studies of -modified HSCs are conducted in humanized mouse models after ablation of the murine bone marrow and transfer of human CD34 HSCs. Engrafted human CD34 cells migrate to bone marrow and differentiate into various human hematopoietic lineages.

Multiple Integrated Non-clinical Studies Predict the Safety of Lentivirus-Mediated Gene Therapy for β-Thalassemia.

Gene therapy clinical trials require rigorous non-clinical studies in the most relevant models to assess the benefit-to-risk ratio. To support the clinical development of gene therapy for β-thalassemia, we performed and studies for prediction of safety. First we developed newly GLOBE-derived vectors that were tested for their transcriptional activity and potential interference with the expression of surrounding genes.

Monocyte-derived IL-1 and IL-6 are differentially required for cytokine-release syndrome and neurotoxicity due to CAR T cells.

In the clinic, chimeric antigen receptor-modified T (CAR T) cell therapy is frequently associated with life-threatening cytokine-release syndrome (CRS) and neurotoxicity. Understanding the nature of these pathologies and developing treatments for them are hampered by the lack of appropriate animal models. Herein, we describe a mouse model recapitulating key features of CRS and neurotoxicity.

A Diagnostic Approach for Rodent Progressive Cardiomyopathy and Like Lesions in Toxicology Studies up to 28 Days in the Sprague Dawley Rat (Part 2 of 2).

To test the diagnostic approach described in part 1 of this article, 2 exercises were completed by pathologists from multiple companies/agencies. Pathologist's examination of whole slide image (WSI) heart sections from rats using personal diagnostic approaches (exercise #1) corroborated conclusions from study #1. Using the diagnostic approach described in part 1, these pathologists examined the same WSI heart sections (exercise #2) to determine whether that approach increased consistency of diagnosis of rodent progressive cardiomyopathy (PCM) lesions.

A Diagnostic Approach for Rodent Progressive Cardiomyopathy and Like Lesions in Toxicology Studies up to 28 Days in the Sprague Dawley Rat (Part 1 of 2).

Spontaneous rodent progressive cardiomyopathy (PCM) in the Sprague Dawley rat may confound identification and/or interpretation of potential test article (TA)-related cardiotoxicity. Pathologists apply diagnostic term(s) and thresholds for diagnosing and assigning severity grades for PCM and/or PCM-like (PCM/like) lesions consistently within a study, which is necessary to identify and interpret TA-related findings. Due to differences in training and/or experiences, diagnostic terms and thresholds may vary between pathologists.

Good Laboratory Practice Preclinical Safety Studies for GSK2696273 (MLV Vector-Based Ex Vivo Gene Therapy for Adenosine Deaminase Deficiency Severe Combined Immunodeficiency) in NSG Mice.

GSK2696273 (autologous CD34+ cells transduced with retroviral vector that encodes for the human adenosine deaminase [ADA] enzyme) is a gamma-retroviral ex vivo gene therapy of bone marrow-derived CD34+ cells for the treatment of adenosine deaminase deficiency severe combined immunodeficiency (ADA-SCID). ADA-SCID is a severe monogenic disease characterized by immunologic and nonimmunologic symptoms. Bone-marrow transplant from a matched related donor is the treatment of choice, but it is available for only a small proportion of patients.

Preclinical Testing of the Safety and Tolerability of Lentiviral Vector-Mediated Above-Normal Alpha-L-Iduronidase Expression in Murine and Human Hematopoietic Cells Using Toxicology and Biodistribution Good Laboratory Practice Studies.

In order to support the clinical application of hematopoietic stem cell (HSC) gene therapy for mucopolysaccharidosis I (MPS I), biosafety studies were conducted to assess the toxicity and tumorigenic potential, as well as the biodistribution of HSCs and progenitor cells (HSPCs) transduced with lentiviral vectors (LV) encoding the cDNA of the alpha-iduronidase (IDUA) gene, which is mutated in MPS I patients. To this goal, toxicology and biodistribution studies were conducted, employing Good Laboratory Practice principles. Vector integration site (IS) studies were applied in order to predict adverse consequences of vector gene transfer and to obtain HSC-related information.

Three common pathways of nephrotoxicity induced by halogenated alkenes.

Glutathione-dependent bioactivation is a common pathway in nephrotoxicity caused by haloalkanes and haloalkenes. Glutathione conjugation forms the link between halogenated hydrocarbons, based on the formation of an episulfonium ion (vicinal halomethanes) or a cysteine conjugate (haloalkenes). Herein, we review the metabolic pathways underlying the nephrotoxic effects of the three well-known haloalkenes trichloroethylene, tetrachloroethylene, and hexachloro-1:3-butadiene to emphasize the role of cysteine-conjugate β-lyase and the oxidative metabolism in renal toxicity.

Molecular biomarkers of phospholipidosis in rat blood and heart after amiodarone treatment.

Phospholipidosis (PLD) is characterized by an intracellular accumulation of phospholipids in lysosomes and concurrent development of concentric lamellar bodies. It is induced in humans and in animals by drugs with a cationic amphiphilic structure. The purpose of the present study was to identify a set of molecular biomarkers of PLD in rat blood and heart, hypotheticallya pplicable in preclinical screens within the drug development process.

Cocaine-associated increase of atrial natriuretic peptides: an early predictor of cardiac complications in cocaine users?

Objective: Cocaine is known to produce life-threatening cardiovascular complications, and the investigation of the causes of death may be challenging in forensic medicine. The increasing knowledge of the cardiac function biomarkers and the increasing sensitivity of assays provide new tools in monitoring the cardiac life-threatening pathological conditions and in the sudden death investigation in chronic abusers. In this work, cardiac dysfunction was assessed in an animal model by measuring troponin I and natriuretic peptides as biomarkers, and considering other standard endpoints used in preclinical toxicology studies.

Spontaneous cardiomyopathy in young Sprague-Dawley rats: evaluation of biological and environmental variability.

Cardiovascular safety signals in nonclinical studies remain among the main reasons for drug attrition during pharmaceutical research and development. Drug-induced changes can be functional and/or associated with morphological alterations in the normal heart histology. It is therefore crucial to understand the normal variations in histology to discriminate test article-related changes from background lesions.

Expression of the aryl hydrocarbon receptor pathway and cyclooxygenase-2 in dog tumors.

In humans, the aryl hydrocarbon receptor (AHR) gene battery constitutes a set of contaminant-responsive genes, which have been recently shown to be involved in the regulation of several patho-physiological conditions, including tumorigenesis. As the domestic dog represents a valuable animal model in comparative oncology, mRNA levels of cytochromes P450 1A1, 1A2 and 1B1 (CYP1A1, 1A2 and 1B1), AHR, AHR nuclear translocator (ARNT), AHR repressor (AHRR, whose partial sequence was here obtained) and cyclooxygenase-2 (COX2) were measured in dog control tissues (liver, skin, mammary gland and bone), in 47 mast cell tumors (MCTs), 32 mammary tumors (MTs), 5 osteosarcoma (OSA) and related surgical margins. Target genes were constitutively expressed in the dog, confirming the available human data.

Sex-related differences in renal toxicodynamics in rodents.

Introduction: An issue yet to be addressed, in the investigation of the xenobiotic toxicity, is a detailed characterization of the sex differences in toxicological responses. The 'sex issue' is particularly significant in nephrotoxicology as the kidney is a relevant target organ for xenobiotics and few studies have approached this subject in the past. There is a strong need to improve our understanding regarding the influence of sex in toxicology, given their increased requirement to establish the limits of exposure to chemicals in the environment and at work.

Atrial natriuretic peptides in Han Wistar, Sprague-Dawley and spontaneously hypertensive rats.

The atrial natriuretic peptide (ANP) and its precursor (N-terminal fragment of atrial natriuretic peptide, NT-proANP) are natriuretic peptides released into the circulation as a consequence of an acute atrial stretch. As for the brain natriuretic peptide and its N-terminal fragment, the biological significance of ANP and NT-proANP has been widely studied in humans, but the literature is lacking information about the determination of these biomarkers in veterinary medicine and, in particular, in the toxicological species used in preclinical pharmaceutical drug development. This paper describes the evaluation of ANP and NT-proANP levels in a healthy population of Han Wistar and Sprague-Dawley rats, as well as in a rodent model of hypertension (Spontaneously Hypertensive rats).

A robust experimental protocol for pharmacological fMRI in rats and mice.

Pharmacological Magnetic Resonance Imaging (phMRI) methods have significantly expanded the stimulation repertoire available to preclinical fMRI research, by allowing to selectively probe the activity of specific brain circuitries and neurotransmitter systems. However, the application of phMRI to animal models is constrained by a number of experimental factors. Firstly, in order to prevent motion artefacts and reduce restraint-induced stress, phMRI studies are typically performed under anaesthesia.

Hyaline droplet accumulation in kidney of rats treated with hexachloro-1:3-butadiene: influence of age, dose and time-course.

The present research investigates the occurrence of hyaline droplet (HD) accumulation related to age, dose and time after treatment in male Wistar rats given a single i.p. injection of hexachloro-1:3-butadiene (HCBD).

NT-proBNP as a biomarker for the assessment of a potential cardiovascular drug-induced liability in beagle dogs.

The amino-terminal pro-brain natriuretic peptide (NT-proBNP) is released into the plasma predominantly from ventricular cardiomyocytes, particularly in patients with chronic cardiac diseases, although small amounts are detectable in the plasma of healthy subjects. While NT-proBNP has been widely exploited in human medicine, limited literature is available related to its characterization in veterinary medicine (e.g.

Neuroimaging evidence of altered fronto-cortical and striatal function after prolonged cocaine self-administration in the rat.

Cocaine addiction is often modeled in experimental paradigms where rodents learn to self-administer (SA) the drug. However, the extent to which these models replicate the functional alterations observed in clinical neuroimaging studies of cocaine addiction remains unknown. We used magnetic resonance imaging (MRI) to assess basal and evoked brain function in rats subjected to a prolonged, extended-access cocaine SA scheme.

A novel and integrated approach for the identification and characterization of drug-induced cardiac toxicity in the dog.

Cardiovascular toxicity represents one of the major reasons for the termination of the development of drugs, even in late development phases. This growing issue is often not restricted to specific therapeutic areas, and it is gaining critical importance, in particular for chronically administered drugs, highlighting the limitations in terms of sensitivity of the current investigational paradigms. Furthermore, drug-related changes may become evident after long-term administration for different reasons, including accumulation of the drug in the heart.

Localization of Helicobacter spp. in the fundic mucosa of laboratory Beagle dogs: an ultrastructural study.

In dogs Helicobacter spp. are found in all gastric regions usually localized in the surface mucus, gastric glands and parietal cells. The aim of this study was to detail the distribution of Helicobacter spp.

Gold(III)-dithiocarbamato anticancer agents: activity, toxicology and histopathological studies in rodents.

Gold(III)-dithiocarbamato complexes have recently gained increasing attention as potential anticancer agents because of their strong tumor cell growth--inhibitory effects, generally achieved by exploiting non-cisplatin-like mechanisms of action. The rationale of our research work is to combine the antitumor properties of the gold(III) metal center with the potential chemoprotective function of coordinated dithiocarbamates in order to reduce toxic side effects (in particular nephrotoxicity) induced by clinically established platinum-based drugs. In this context, [Au(III) Br(2) (ESDT)] (AUL12) was proved to exert promising and outstanding antitumor activity in vitro and to overcome both acquired and intrinsic resistance showed by some types of tumors toward cisplatin.