Repeat-dose and embryo-fetal developmental toxicity of zinpentraxin alfa.

Zinpentraxin alfa is a recombinant human pentraxin-2 (PTX-2) developed for the treatment of various fibrotic diseases with the hypothesis that supplementing endogenous PTX-2 levels through intravenous administration should increase its regulatory capacity in circulation and at the site of disease, thereby promoting healing and reducing fibrosis. Zinpentraxin alfa has been studied in various clinical trials, particularly in patients with idiopathic pulmonary fibrosis, where it has demonstrated efficacy in slowing decline in lung function in a phase 2 study. In the present investigation, we summarize findings from 14-day repeat-dose toxicity studies in rats and cynomolgus monkeys supporting early clinical development of zinpentraxin alfa. In addition, we also describe the findings from the embryo-fetal developmental (EFD) studies conducted in rats and rabbits, since the intended fibrosis patient population may include patients of childbearing potential. Zinpentraxin alfa was well tolerated by rats and monkeys in general toxicity studies with no treatment-related adverse effects, as well as by pregnant rats over the same dose range in a definitive EFD study. In contrast, substantial toxicity was observed in a rabbit dose-range-finder EFD study. Zinpentraxin alfa was poorly tolerated by pregnant rabbits and effects on the dams correlated with post-implantation fetal losses. The disparate effects of zinpentraxin alfa on embryo-fetal development between the two species suggests a potential unknown biological function of PTX-2 in pregnancy in the rabbit, which may be relevant to humans. Our findings warrant the consideration for highly effective contraceptive measures to avoid pregnancy in patients enrolled in clinical studies with zinpentraxin alfa.

Inflammatory ER stress responses dictate the immunopathogenic progression of systemic candidiasis.

Recognition of pathogen-associated molecular patterns can trigger the inositol-requiring enzyme 1 α (IRE1α) arm of the endoplasmic reticulum (ER) stress response in innate immune cells. This process maintains ER homeostasis and also coordinates diverse immunomodulatory programs during bacterial and viral infections. However, the role of innate IRE1α signaling in response to fungal pathogens remains elusive. Here, we report that systemic infection with the human opportunistic fungal pathogen Candida albicans induced proinflammatory IRE1α hyperactivation in myeloid cells that led to fatal kidney immunopathology. Mechanistically, simultaneous activation of the TLR/IL-1R adaptor protein MyD88 and the C-type lectin receptor dectin-1 by C. albicans induced NADPH oxidase-driven generation of ROS, which caused ER stress and IRE1α-dependent overexpression of key inflammatory mediators such as IL-1ß, IL-6, chemokine (C-C motif) ligand 5 (CCL5), prostaglandin E2 (PGE2), and TNF-α. Selective ablation of IRE1α in leukocytes, or treatment with an IRE1α pharmacological inhibitor, mitigated kidney inflammation and prolonged the survival of mice with systemic C. albicans infection. Therefore, controlling IRE1α hyperactivation may be useful for impeding the immunopathogenic progression of disseminated candidiasis.

Assessment of Skin Toxicity in an in Vitro Reconstituted Human Epidermis Model Using Deep Learning.

Skin toxicity is a common safety concern associated with drugs that inhibit epidermal growth factor receptors as well as other targets involved in epidermal growth and differentiation. Recently, the use of a three-dimensional reconstructed human epidermis model enabled large-scale drug screening and showed potential for predicting skin toxicity. Although a decrease in epidermal thickness was often observed when the three-dimensional reconstructed tissues were exposed to drugs causing skin toxicity, the thickness evaluation of epidermal layers from a pathologist was subjective and not easily reproducible or scalable. In addition, the subtle differences in thickness among tissues, as well as the large number of samples tested, made cross-study comparison difficult when a manual evaluation strategy was used. The current study used deep learning and image-processing algorithms to measure the viable epidermal thickness from multiple studies and found that the measured thickness was not only significantly correlated with a pathologist's semi-quantitative evaluation but was also in close agreement with the quantitative measurement performed by pathologists. Moreover, a sensitivity of 0.8 and a specificity of 0.75 were achieved when predicting the toxicity of 18 compounds with clinical observations with these epidermal thickness algorithms. This approach is fully automated, reproducible, and highly scalable. It not only shows reasonable accuracy in predicting skin toxicity but also enables cross-study comparison and high-throughput compound screening.

Classification and Grading of Melanocytic Lesions in a Mouse Model of NRAS-driven Melanomagenesis.

The mouse line carrying the Tg(Tyr-NRAS*Q61K)1Bee transgene is widely used to model in vivo NRAS-driven melanomagenesis. Although the pathological features of this model are well described, classification and interpretation of the resulting proliferative lesions-including their origin, evolution, grading, and pathobiological significance-are still unclear and not supported by molecular and biological evidence. Focusing on their classification and grading, this work combines histopathology and expression analysis (using both immunohistochemistry [IHC] and quantitative PCR) of selected biomarkers to study the full spectrum of cutaneous and lymph nodal melanocytic proliferations in the Tg(Tyr-NRAS*Q61K)1Bee mouse. The analysis of cutaneous and lymph nodal melanocytic proliferations has demonstrated that a linear correlation exists between tumor grade and Ki-67, microphthalmia-associated transcription factor (MITF), gp100, and nestin IHC, with a significantly increased expression in high-grade lesions compared with low-grade lesions. The accuracy of the assessment of MITF IHC in melanomas was also confirmed by quantitative PCR analysis. In conclusion, we believe the incorporation of MITF, Ki-67, gp100, and nestin analysis into the histopathological classification/grading scheme of melanocytic proliferations described for this model will help to assess with accuracy the nature and evolution of the phenotype, monitor disease progression, and predict response to experimental treatment or other preclinical manipulations.

RIP1 kinase activity is critical for skin inflammation but not for viral propagation.

Receptor interacting protein kinase 1 (RIP1) is a critical effector of inflammatory responses and cell death activation. Cell death pathways regulated by RIP1 include caspase-dependent apoptosis and caspase-independent necroptosis. The kinase activity of RIP1 has been associated with a number of inflammatory, neurodegenerative, and oncogenic diseases. In this study, we use the RIP1 kinase inhibitor GNE684 to demonstrate that RIP1 inhibition can effectively block skin inflammation and immune cell infiltrates in livers of Sharpin mutant (Cpdm; chronic proliferative dermatitis) mice in an interventional setting, after disease onset. On the other hand, genetic inactivation of RIP1 (RIP1 KD) or ablation of RIP3 (RIP3 KO) or MLKL (MLKL KO) did not affect testicular pathology of aging male mice. Likewise, infection with vaccinia virus or with mouse gammaherpesvirus MHV68 resulted in similar viral clearance in wild-type, RIP1 KD, and RIP3 KO mice. In summary, this study highlights the benefits of inhibiting RIP1 in skin inflammation, as opposed to its lack of relevance for testicular longevity and the response to certain viral infections.

Applications and considerations for the use of genetically engineered mouse models in drug development.

Considering high drug attrition rates in clinical studies and the overall complexity and challenging environment of drug development, it is increasingly important to understand the therapeutic molecule and target and how they intersect with disease biology as fully as possible. This requires one to use numerous tools and investigative approaches in combination. Genetically engineered mouse models are a critical component to the drug development toolbox as they can provide key insights across multiple steps of the drug development process. While knock-out and knock-in mice can inform questions of basic biology, genetically engineered mice can also be applied to model diseases for efficacy studies, to discriminate on-target and off-target effects of novel therapeutics, and to inform an array of biologic and pharmacologic questions, including pharmacodynamics, pharmacokinetics, and biomarker discovery. However, use of these models requires not only an understanding of their strengths and limitations but also a careful consideration of the context in which they are being used and the hypotheses being addressed by them. Additionally, they should not be used in isolation, but instead in combination with other biochemical, in vitro, and clinical data to create a broad understanding of the drug, target, and disease biology.

An Atypical Parvovirus Drives Chronic Tubulointerstitial Nephropathy and Kidney Fibrosis.

The occurrence of a spontaneous nephropathy with intranuclear inclusions in laboratory mice has puzzled pathologists for over 4 decades, because its etiology remains elusive. The condition is more severe in immunodeficient animals, suggesting an infectious cause. Using metagenomics, we identify the causative agent as an atypical virus, termed "mouse kidney parvovirus" (MKPV), belonging to a divergent genus of Parvoviridae. MKPV was identified in animal facilities in Australia and North America, is transmitted via a fecal-oral or urinary-oral route, and is controlled by the adaptive immune system. Detailed analysis of the clinical course and histopathological features demonstrated a stepwise progression of pathology ranging from sporadic tubular inclusions to tubular degeneration and interstitial fibrosis and culminating in renal failure. In summary, we identify a widely distributed pathogen in laboratory mice and establish MKPV-induced nephropathy as a new tool for elucidating mechanisms of tubulointerstitial fibrosis that shares molecular features with chronic kidney disease in humans.

Ivermectin-compounded Feed Compared with Topical Moxidectin-Imidacloprid for Eradication of Demodex musculi in Laboratory Mice.

Demodex musculi is a prostigmatid follicular mite that has rarely been reported in laboratory mice. Although prevalence of this species has not been assessed formally, we have found that many imported mouse strains from noncommercial sources harbor Demodex mites. To assess whether an acaricide can be used to eradicate this mite, infested immunocompromised mice were provided ivermectin-compounded (12 ppm) feed without restriction for 8 wk (n = 10), were treated topically with moxidectin and imidacloprid (MI; 3 and 13 mg/kg, respectively) weekly for 8 wk (n = 10), or remained untreated (n = 10). Mice were confirmed to be mite-infested before treatment and were tested after treatment by using fur plucks (FP), deep skin scrapes (DSS), and PCR analysis of fur swabs. In addition, the presence of mites was confirmed through skin biopsies at 2 study endpoints (1 wk [n = 5] and 12 wk [ n = 5] after treatment). Samples collected before treatment and from untreated mice were positive for D. musculi at all time points and by all test modalities. After treatment, all ivermectin-treated animals remained infested, whereas mice treated with MI were repeatedly negative by all test modalities. An additional shortened treatment trial revealed that 4 wk of MI (n = 7) was insufficient to eradicate mites. Neither treatment produced any evidence of adverse effects according to hematology, serum chemistry parameters, behavior, body weight, and histopathology. Of the 70 PCR assays performed in treated mice, 14 were positive when FP+DSS was negative. In 6 cases where PCR results were negative, 5 were positive by FP+DSS and a single sample was positive on skin biopsy. Although PCR analysis has a high detection rate for D. musculi, FP+DSS can further enhance the detection rate. In conclusion, topical MI administered for 8 consecutive weeks can safely eradicate D. musculi from an immunocompromised mouse strain.

Immune Relevant and Immune Deficient Mice: Options and Opportunities in Translational Research.

In 1989 ILAR published a list and description of immunodeficient rodents used in research. Since then, advances in understanding of molecular mechanisms; recognition of genetic, epigenetic microbial, and other influences on immunity; and capabilities in manipulating genomes and microbiomes have increased options and opportunities for selecting mice and designing studies to answer important mechanistic and therapeutic questions. Despite numerous scientific breakthroughs that have benefitted from research in mice, there is debate about the relevance and predictive or translational value of research in mice. Reproducibility of results obtained from mice and other research models also is a well-publicized concern. This review summarizes resources to inform the selection and use of immune relevant mouse strains and stocks, aiming to improve the utility, validity, and reproducibility of research in mice. Immune sufficient genetic variations, immune relevant spontaneous mutations, immunodeficient and autoimmune phenotypes, and selected induced conditions are emphasized.

Catheter-directed Intraportal Delivery of Endothelial Cell Therapy for Liver Regeneration: A Feasibility Study in a Large-Animal Model of Cirrhosis.

Purpose To demonstrate the feasibility of imaging-guided catheter-directed delivery of endothelial cell therapy in a porcine model of cirrhosis for liver regeneration. Materials and Methods After approval from the institutional animal care and use committee, autologous liver endothelial cells were grown from core hepatic specimens from swine. Cirrhosis was induced in swine by means of transcatheter infusion of ethanol and iodized oil into the hepatic artery. Three weeks after induction of cirrhosis, the swine were randomly assigned to receive autologous cell therapy (endothelial cells, n = 4) or control treatment (phosphate-buffered saline, n = 4) by means of imaging-guided transhepatic intraportal catheterization. Fluorescence-activated cell sorting analysis was performed on biopsy samples 1 hour after therapy. Three weeks after intraportal delivery of endothelial cells, the swine were euthanized and the explanted liver underwent quantitative pathologic examination. Statistical analysis was performed with an unpaired t test by using unequal variance. Results Liver endothelial cells were successfully isolated, cultured, and expanded from eight 20-mm, 18-gauge hepatic core samples to 50 × 106 autologous cells per pig. Intraportal delivery of endothelial cell therapy or saline was technically successful in all eight swine, with no complications. Endothelial cells were present in the liver for a minimum of 1 hour after intraportal infusion. Swine treated with endothelial cell therapy showed mean levels of surrogate markers of hepatobiliary injury that were consistent with decreases in hepatic fibrosis and biliary ductal damage relative to the control animals, although statistical significance was not met in this pilot study: The mean percentage of positive pixels at Masson trichrome staining was 7.28% vs 5.57%, respectively (P = .20), the mean proliferation index with cytokeratin wide-spectrum was 2.55 vs 1.13 (P = .06), and the mean proliferation index with Ki67 was 7.08 vs 4.96 (P = .14). Conclusion The results confirm the feasibility of imaging-guided catheter-directed endothelial cell therapy with an intraportal technique for the treatment of cirrhosis in a porcine model. A trend toward decreased liver fibrosis with endothelial cell therapy was observed. Larger animal studies and human studies are necessary to confirm significance. © RSNA, 2017.

Pathology of Aging in NOD scid gamma Female Mice.

In the past decade, NOD.Cg- Prkdcscid Il2rgtm1Wjl/SzJ (NSG, NOD scid gamma) mice have become a model of choice in several areas of biomedical research; however, comprehensive data on their spontaneous age-related pathology are not currently available in the literature. The prevalence of spontaneous morbidity affecting aged NSG female breeders enrolled in a parasitology study was documented with classification of neoplastic and non-neoplastic (inflammatory, metabolic, degenerative) lesions. Malignant mammary neoplasms were most commonly diagnosed, often accompanied by pulmonary metastases, while a low frequency of lymphoma and histiocytic sarcoma was documented. The major inflammatory conditions were suppurative pleuropneumonia and bronchopneumonia with abscess formation, from which Pasteurella pneumotropica was commonly isolated, followed by otitis media. Both inflammatory and degenerative lesions of the genital tract were identified, along with neoplasms such as endometrial yolk sac carcinomas and granulosa cell tumors. Novel conditions identified included renal tubular degeneration and necrosis associated with 2 concurrent types of intranuclear inclusions, focal or multifocal hyperostosis of the skull, and neuroendocrine tumors of the mesometrium. The majority of degenerative lesions that affected the genital tract, endocrine, and skeletal systems did not represent the actual underlying cause of death but rather were considered incidental findings. This study indicates that both inflammatory and neoplastic conditions contribute to morbidity and mortality in experimentally manipulated aged female NSG mice.

Primary angiocentric/angioinvasive T-cell lymphoma of the tympanic bulla in a feline leukaemia virus-positive cat.

Case summary A 5-year-old neutered female feline leukaemia virus (FeLV)-positive domestic shorthair cat with a 5 month history of otitis media was referred for head tilt, stertor and dyspnoea. Computed tomography scan revealed soft tissue opacities inside the right tympanic bulla, with bone remodelling, and concurrent nasopharyngeal and intracranial invasion. Endoscopically guided bioptic samples were collected from the nasopharynx and middle ear. Histology revealed dense sheets of round, large, neoplastic cells, often surrounding or invading vascular walls. Neoplastic cells expressed CD3, FeLV p27 and gp70 antigens. A middle ear angiocentric/angioinvasive T-cell lymphoma was diagnosed. After improvement of clinical conditions following radiation therapy, the cat died unexpectedly. At necropsy, hepatic and splenic spread was detected. Relevance and novel information Primary middle ear tumours are rare and their diagnosis is often delayed as clinical signs mimic more common otological conditions. Multiple bioptic specimens are pivotal for a definitive diagnosis. The young age of the cat, serology and immunohistochemistry revealed a possible transforming role of FeLV.