Reduced levels of MRE11 cause disease phenotypes distinct from ataxia telangiectasia-like disorder.

The MRE11/RAD50/NBS1 (MRN) complex plays critical roles in cellular responses to DNA double-strand breaks. MRN is involved in end binding and processing, and it also induces cell cycle checkpoints by activating the ataxia-telangiectasia mutated (ATM) protein kinase. Hypomorphic pathogenic variants in the MRE11, RAD50, or NBS1 genes cause autosomal recessive genome instability syndromes featuring variable degrees of dwarfism, neurological defects, anemia, and cancer predisposition. Disease-associated MRN alleles include missense and nonsense variants, and many cause reduced protein levels of the entire MRN complex. However, the dramatic variability in the disease manifestation of MRN pathogenic variants is not understood. We sought to determine if low protein levels are a significant contributor to disease sequelae and therefore generated a transgenic murine model expressing MRE11 at low levels. These mice display dramatic phenotypes including small body size, severe anemia, and impaired DNA repair. We demonstrate that, distinct from ataxia telangiectasia-like disorder caused by MRE11 pathogenic missense or nonsense variants, mice and cultured cells expressing low MRE11 levels do not display the anticipated defects in ATM activation. Our findings indicate that ATM signaling can be supported by very low levels of the MRN complex and imply that defective ATM activation results from perturbation of MRN function caused by specific hypomorphic disease mutations. These distinct phenotypic outcomes underline the importance of understanding the impact of specific pathogenic MRE11 variants, which may help direct appropriate early surveillance for patients with these complicated disorders in a clinical setting.

High-mortality epizootic Mycobacterium ulcerans ecovar Liflandii in a colony of Zaire Dwarf Clawed Frogs (Hymenochirus boettgeri).

Mycobacterium ulcerans ecovar Liflandii (MuLiflandii) was identified as the causative agent of mycobacteriosis in a research colony of Zaire dwarf clawed frogs (Hymenochirus boettgeri) at the University of Michigan. Clinical presentation included lethargy, generalized septicemia, cutaneous granulomas, coelomic effusion, and acute mortality. Identification of the mycobacterial species was based on molecular, microbiological, and histopathologic characteristics. These findings indicate that MuLiflandii is a primary cause of morbidity and mortality in Zaire dwarf clawed frogs and should be considered in the differential diagnosis of sepsis and coelomic effusion in amphibians. Mycobacterial speciation is important given the variability in pathogenesis within the family Mycobacteriaceae and the implications for both animal and human health as potential zoonoses. The Zaire dwarf clawed frog is a species common in the pet trade, and these findings provide consideration for this pathogen as a potentially important public health concern. This is the first report of MuLiflandii infection in the genus Hymenochirus and illustrates the diagnostic challenges of differentiating among both mycolactone-producing mycobacteria and Mycobacterium marinum. Furthermore, we demonstrate the utility of environmental sampling for this pathogen within the tank system, suggesting this mode of sampling could replace the need for direct frog surveillance.

Isoflurane and Pentobarbital Anesthesia for Pulmonary Studies Requiring Prolonged Mechanical Ventilation in Mice.

Mechanical ventilation can be used in mice to support high-risk anesthesia or to create clinically relevant, intensive care models. However, the choice of anesthetic and inspired oxygen concentration for prolonged procedures may affect basic physiology and lung inflammation. To characterize the effects of anesthetics and oxygen concentration in mice experiencing mechanical ventilation, mice were anesthetized with either isoflurane or pentobarbital for tracheostomy followed by mechanical ventilation with either 100% or 21% oxygen. Body temperature, oxygen saturation, and pulse rate were monitored continuously. After 6 h, mice were euthanized for collection of blood and bronchoalveolar lavage fluid for evaluation of biomarkers of inflammation and lung injury, including cell counts and cytokine levels. Overall, both isoflurane and pentobarbital provided suitable anesthesia for 6 h of mechanical ventilation with either 21% or 100% oxygen. We found no differences in lung inflammation biomarkers attributable to either oxygen concentration or the anesthetic. However, the combination of pentobarbital and 100% oxygen resulted in a significantly higher concentration of a biomarker for lung epithelial cell injury. This study demonstrates that the combination of anesthetic agent, mechanical ventilation, and inspired oxygen concentrations can alter vital signs and lung injury biomarkers during prolonged procedures. Their combined impact may influence model development and the interpretation of research results, warranting the need for preliminary evaluation to establish the baseline effects.

Lethal eosinophilic crystalline pneumonia in mice expressing a stabilized Csf2 mRNA.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that stimulates the proliferation and differentiation of granulocyte and macrophage precursors. The mouse gene-encoding GM-CSF, Csf2, is regulated at both transcriptional and post-transcriptional levels. An adenine-uridine-rich element (ARE) within the 3'-untranslated region of Csf2 mRNA was shown in cell transfection studies to confer instability on this transcript. To explore the physiological importance of this element in an intact animal, we generated mice with a knock-in deletion of the 75-nucleotide ARE. Mice heterozygous for this ARE deletion developed severe respiratory distress and death within about 12 weeks of age. There was dense infiltration of lung alveolar spaces by crystal-containing macrophages. Increased stability of Csf2 mRNA was confirmed in bone marrow-derived macrophages, and elevated GM-CSF levels were observed in serum and lung. These mice did not exhibit notable abnormalities in blood or bone marrow, and transplantation of bone marrow from mutant mice into lethally irradiated WT mice did not confer the pulmonary phenotype. Mice with a conditional deletion of the ARE restricted to lung type II alveolar cells exhibited an essentially identical lethal lung phenotype at the same ages as the mice with the whole-body deletion. In contrast, mice with the same conditional ARE deletion in myeloid cells, including macrophages, exhibited lesser degrees of macrophage infiltration into alveolar spaces much later in life, at approximately 9 months of age. Post-transcriptional Csf2 mRNA stability regulation in pulmonary alveolar epithelial cells appears to be essential for normal physiological GM-CSF secretion and pulmonary macrophage homeostasis.

Targeting Oncogenic Wnt/ß-Catenin Signaling in Adrenocortical Carcinoma Disrupts ECM Expression and Impairs Tumor Growth.

Adrenocortical carcinoma (ACC) is a rare but highly aggressive cancer with limited treatment options and poor survival for patients with advanced disease. An improved understanding of the transcriptional programs engaged in ACC will help direct rational, targeted therapies. Whereas activating mutations in Wnt/ß-catenin signaling are frequently observed, the ß-catenin-dependent transcriptional targets that promote tumor progression are poorly understood. To address this question, we analyzed ACC transcriptome data and identified a novel Wnt/ß-catenin-associated signature in ACC enriched for the extracellular matrix (ECM) and predictive of poor survival. This suggested an oncogenic role for Wnt/ß-catenin in regulating the ACC microenvironment. We further investigated the minor fibrillar collagen, collagen XI alpha 1 (COL11A1), and found that COL11A1 expression originates specifically from cancer cells and is strongly correlated with both Wnt/ß-catenin activation and poor patient survival. Inhibition of constitutively active Wnt/ß-catenin signaling in the human ACC cell line, NCI-H295R, significantly reduced the expression of COL11A1 and other ECM components and decreased cancer cell viability. To investigate the preclinical potential of Wnt/ß-catenin inhibition in the adrenal microenvironment, we developed a minimally invasive orthotopic xenograft model of ACC and demonstrated that treatment with the newly developed Wnt/ß-catenin:TBL1 inhibitor Tegavivint significantly reduced tumor growth. Together, our data support that the inhibition of aberrantly active Wnt/ß-catenin disrupts transcriptional reprogramming of the microenvironment and reduces ACC growth and survival. Furthermore, this ß-catenin-dependent oncogenic program can be therapeutically targeted with a newly developed Wnt/ß-catenin inhibitor. These results show promise for the further clinical development of Wnt/ß-catenin inhibitors in ACC and unveil a novel Wnt/ß-catenin-regulated transcriptome.

Septicemia caused by an emerging pathogen, Elizabethkingia miricola, in a laboratory colony of African dwarf frogs (Hymenochirus curtipes).

An outbreak of morbidity and mortality in an African dwarf frog (Hymenochirus curtipes) colony was reported following arrival at an animal research facility. Animals were found dead on arrival or became moribund shortly thereafter, and additional animals showed clinical signs of lethargy, weight loss, and anorexia over the following 3 weeks. Externally, some affected animals presented with multifocal areas of hyperemia in the inguinal and axillary areas and on the limbs, and mottled tan discoloration along the ventral abdomen. Histologically, lesions were consistent with generalized septicemia, characterized by granulomatous meningitis, otitis media, peritonitis (coelomitis), myocarditis and pericarditis, nephritis, pneumonia, and arthritis. Gram staining identified gram-negative rod-shaped bacteria free within tissues and within macrophages. Culture results of coelomic swabs identified moderate to numerous Elizabethkingia miricola. Testing of water from tanks housing affected animals showed elevated levels of nitrites and ammonia, and the presence of Citrobacter, Aeromonas, Pseudomonas, and Staphylococcus spp. cultured from several tank biofilters. E miricola is a newly recognized and rapidly emerging opportunistic pathogen in anurans and has been reported as a cause of septicemia in humans. This report documents the first occurrence of E. miricola septicemia in African dwarf frogs and illustrates the importance of this potential pathogen in the laboratory setting for amphibian research colonies, as well as those individuals directly working with them.

Murine SEC24D can substitute functionally for SEC24C during embryonic development.

The COPII component SEC24 mediates the recruitment of transmembrane cargos or cargo adaptors into newly forming COPII vesicles on the ER membrane. Mammalian genomes encode four Sec24 paralogs (Sec24a-d), with two subfamilies based on sequence homology (SEC24A/B and C/D), though little is known about their comparative functions and cargo-specificities. Complete deficiency for Sec24d results in very early embryonic lethality in mice (before the 8 cell stage), with later embryonic lethality (E7.5) observed in Sec24c null mice. To test the potential overlap in function between SEC24C/D, we employed dual recombinase mediated cassette exchange to generate a Sec24cc-d allele, in which the C-terminal 90% of SEC24C has been replaced by SEC24D coding sequence. In contrast to the embryonic lethality at E7.5 of SEC24C-deficiency, Sec24cc-d/c-d pups survive to term, though dying shortly after birth. Sec24cc-d/c-d pups are smaller in size, but exhibit no other obvious developmental abnormality by pathologic evaluation. These results suggest that tissue-specific and/or stage-specific expression of the Sec24c/d genes rather than differences in cargo export function explain the early embryonic requirements for SEC24C and SEC24D.

Cobalt-induced oxidative stress contributes to alveolar/bronchiolar carcinogenesis in B6C3F1/N mice.

Rodent alveolar/bronchiolar carcinomas (ABC) that arise either spontaneously or due to chemical exposure are similar to a subtype of lung adenocarcinomas in humans. B6C3F1/N mice and F344/NTac rats exposed to cobalt metal dust (CMD) by inhalation developed ABCs in a dose dependent manner. In CMD-exposed mice, the incidence of Kras mutations in ABCs was 67% with 80% of those being G to T transversions on codon 12 suggesting a role of oxidative stress in the pathogenesis. In vitro studies, such as DMPO (5,5-dimethyl-1-pyrroline N-oxide) immune-spin trapping assay, and dihydroethidium (DHE) fluorescence assay on A549 and BEAS-2B cells demonstrated increased oxidative stress due to cobalt exposure. In addition, significantly increased 8-oxo-dG adducts were demonstrated by immunohistochemistry in lungs from mice exposed to CMD for 90 days. Furthermore, transcriptomic analysis on ABCs arising spontaneously or due to chronic CMD-exposure demonstrated significant alterations in canonical pathways related to MAPK signaling (IL-8, ErbB, Integrin, and PAK pathway) and oxidative stress (PI3K/AKT and Melatonin pathway) in ABCs from CMD-exposed mice. Oxidative stress can stimulate PI3K/AKT and MAPK signaling pathways. Nox4 was significantly upregulated only in CMD-exposed ABCs and NOX4 activation of PI3K/AKT can lead to increased ROS levels in human cancer cells. The gene encoding Ereg was markedly up-regulated in CMD-exposed mice. Oncogenic KRAS mutations have been shown to induce EREG overexpression. Collectively, all these data suggest that oxidative stress plays a significant role in CMD-induced pulmonary carcinogenesis in rodents and these findings may also be relevant in the context of human lung cancers.

Multiethnic PDX models predict a possible immune signature associated with TNBC of African ancestry.

PURPOSE: Triple-negative breast cancer (TNBC) is an aggressive subtype most prevalent among women of Western Sub-Saharan African ancestry. It accounts for 15-25% of African American (AA) breast cancers (BC) and up to 80% of Ghanaian breast cancers, thus contributing to outcome disparities in BC for black women. The aggressive biology of TNBC has been shown to be regulated partially by breast cancer stem cells (BCSC) which mediate tumor recurrence and metastasis and are more abundant in African breast tumors. METHODS: We studied the biological differences between TNBC in women with African ancestry and those of Caucasian women by comparing the gene expression of the BCSC. From low-passage patient derived xenografts (PDX) from Ghanaian (GH), AA, and Caucasian American (CA) TNBCs, we sorted for and sequenced the stem cell populations and analyzed for differential gene enrichment. RESULTS: In our cohort of TNBC tumors, we observed that the ALDH expressing stem cells display distinct ethnic specific gene expression patterns, with the largest difference existing between the GH and AA ALDH+ cells. Furthermore, the tumors from the women of African ancestry [GH/AA] had ALDH stem cell (SC) enrichment for expression of immune related genes and processes. Among the significantly upregulated genes were CD274 (PD-L1), CXCR9, CXCR10 and IFI27, which could serve as potential drug targets. CONCLUSIONS: Further exploration of the role of immune regulated genes and biological processes in BCSC may offer insight into developing novel approaches to treating TNBC to help ameliorate survival disparities in women with African ancestry.

Development of Mast Cell and Eosinophil Hyperplasia and HLH/MAS-Like Disease in NSG-SGM3 Mice Receiving Human CD34+ Hematopoietic Stem Cells or Patient-Derived Leukemia Xenografts.

Immunocompromised mouse strains expressing human transgenes are being increasingly used in biomedical research. The genetic modifications in these mice cause various cellular responses, resulting in histologic features unique to each strain. The NSG-SGM3 mouse strain is similar to the commonly used NSG (NOD scid gamma) strain but expresses human transgenes encoding stem cell factor (also known as KIT ligand), granulocyte-macrophage colony-stimulating factor, and interleukin 3. This report describes 3 histopathologic features seen in these mice when they are unmanipulated or after transplantation with human CD34+ hematopoietic stem cells (HSCs), virally transduced hCD34+ HSCs, or a leukemia patient-derived xenograft. The first feature is mast cell hyperplasia: unmanipulated, naïve mice develop periductular pancreatic aggregates of murine mast cells, whereas mice given the aforementioned human cells develop a proliferative infiltrative interstitial pancreatic mast cell hyperplasia but with human mast cells. The second feature is the predisposition of NSG-SGM3 mice given these human cells to develop eosinophil hyperplasia. The third feature, secondary hemophagocytic lymphohistiocytosis/macrophage activation syndrome (HLH/MAS)-like disease, is the most pronounced in both its clinical and histopathologic presentations. As part of this disease, a small number of mice also have histiocytic infiltration of the brain and spinal cord with subsequent neurologic or vestibular signs. The presence of any of these features can confound accurate histopathologic interpretation; therefore, it is important to recognize them as strain characteristics and to differentiate them from what may be experimentally induced in the model being studied.

Protons and High-Linear Energy Transfer Radiation Induce Genetically Similar Lymphomas With High Penetrance in a Mouse Model of the Aging Human Hematopoietic System.

PURPOSE: Humans are exposed to charged particles in different scenarios. The use of protons and high-linear energy transfer (LET) in cancer treatment is steadily growing. In outer space, astronauts will be exposed to a mixed radiation field composed of both protons and heavy ions, in particularly the long-term space missions outside of earth's magnetosphere. Thus, understanding the radiobiology and transforming potential of these types of ionizing radiation are of paramount importance. METHODS AND MATERIALS: We examined the effect of 10 or 100 cGy of whole-body doses of protons or 28Si ions on the hematopoietic system of a genetic model of aging based on recent studies that showed selective loss of the MLH1 protein in human hematopoietic stems with age. RESULTS: We found that Mlh1 deficient animals are highly prone to develop lymphomas when exposed to either low doses of protons or 28Si ions. The lymphomas that develop are genetically indistinguishable, in spite of different types of damage elicited by low- and high-LET radiation. RNA sequencing analyses reveal similar gene expression patterns, similar numbers of altered genes, similar numbers of single nucleotide variants and insertions and deletions, and similar activation of known leukemogenic loci. CONCLUSIONS: Although the incidence of malignancy is related to radiation quality, and increased due to loss of Mlh1, the phenotype of the tumors is independent of LET.

Unique microRNA alterations in hepatocellular carcinomas arising either spontaneously or due to chronic exposure to Ginkgo biloba extract (GBE) in B6C3F1/N mice.

Ginkgo biloba extract (GBE) is used in traditional Chinese medicine as a herbal supplement for improving memory. Exposure of B6C3F1/N mice to GBE in a 2-year National Toxicology Program (NTP) bioassay resulted in a dose-dependent increase in hepatocellular carcinomas (HCC). To identify key microRNAs that modulate GBE-induced hepatocarcinogenesis, we compared the global miRNA expression profiles in GBE-exposed HCC (GBE-HCC) and spontaneous HCC (SPNT-HCC) with age-matched vehicle control normal livers (CNTL) from B6C3F1/N mice. The number of differentially altered miRNAs in GBE-HCC and SPNT-HCC was 74 (52 up and 22 down) and 33 (15 up and 18 down), respectively. Among the uniquely differentially altered miRNAs in GBE-HCC, miR-31 and one of its predicted targets, Cdk1 were selected for functional validation. A potential miRNA response element (MRE) in the 3'-untranslated regions (3'-UTR) of Cdk1 mRNA was revealed by in silico analysis and confirmed by luciferase assays. In mouse hepatoma cell line HEPA-1 cells, we demonstrated an inverse correlation between miR-31 and CDK1 protein levels, but no change in Cdk1 mRNA levels, suggesting a post-transcriptional effect. Additionally, a set of miRNAs (miRs-411, 300, 127, 134, 409-3p, and 433-3p) that were altered in the GBE-HCCs were also altered in non-tumor liver samples from the 90-day GBE-exposed group compared to the vehicle control group, suggesting that some of these miRNAs could serve as potential biomarkers for GBE exposure or hepatocellular carcinogenesis. These data increase our understanding of miRNA-mediated epigenetic regulation of GBE-mediated hepatocellular carcinogenesis in B6C3F1/N mice.

Effects of Trio and Pair Breeding of Mice on Environmental Parameters and Nasal Pathology and Their Implications for Cage Change Frequency.

According to the Guide, cage change frequencies must be considered when cage density requirements are exceeded. We monitored ammonia, carbon dioxide, cage wetness, health status, and breeding parameters of trio and pair breeding cages containing CD1 mice in ventilated and static microisolation caging (4 cages per condition) daily for approximately 6 wk. Minimum cage change frequencies for each condition were determined on the basis of performance data. At 3 d after cage change, static trio and pair cages had average ammonia levels of 74 and 38 ppm. Ventilated cages remained below the 25ppm threshold reported to be potentially deleterious for mice until at least day 7 after cage change. By 7 d after cage change, ammonia levels had risen to an average of 100 ppm and 64 ppm in static trio and pair cages and to 34 ppm and 20 ppm in ventilated trio and pair cages, respectively. Ammonia levels in ventilated cages continued to rise slowly through day 14 after cage change. CO2 levels exceeded 5000 ppm in all groups at 2 d after cage change. Pair breeders in ventilated cages took the longest-10 to 14 d-to reach cage wetness threshold scores. On day 7, pups in trio static cages were noted to have decreased and squinted eyes, whereas in ventilated cages containing trios and pairs, these clinical signs were rare to absent. Histologically, there was an increasing incidence and severity of nasal lesions in weanlings with increasing housing density and decreasing ventilation, consistent with nasal epithelial toxicity. Given these parameters, we concluded that under the current husbandry conditions, it may be necessary to change breeders in static cages more frequently than every 7 d. Additional studies are necessary to evaluate the effects of more frequent cage changes on reproductive parameters, given that cage changing is stressful for mice and affects breeding results.

Histology Atlas of the Developing Mouse Urinary System With Emphasis on Prenatal Days E10.5-E18.5.

Congenital abnormalities of the urinary tract are some of the most common human developmental abnormalities. Several genetically engineered mouse models have been developed to mimic these abnormalities and aim to better understand the molecular mechanisms of disease. This atlas has been developed as an aid to pathologists and other biomedical scientists for identification of abnormalities in the developing murine urinary tract by cataloguing normal structures at each stage of development. Hematoxylin and eosin- and immunohistochemical-stained sections are provided, with a focus on E10.5-E18.5, as well as a brief discussion of postnatal events in urinary tract development. A section on abnormalities in the development of the urinary tract is also provided, and molecular mechanisms are presented as supplementary material. Additionally, overviews of the 2 key processes of kidney development, branching morphogenesis and nephrogenesis, are provided to aid in the understanding of the complex organogenesis of the kidney. One of the key findings of this atlas is the histological identification of the ureteric bud at E10.5, as previous literature has provided conflicting reports on the initial point of budding. Furthermore, attention is paid to points where murine development is significantly distinct from human development, namely, in the cessation of nephrogenesis.

Genome-wide promoter DNA methylation profiling of hepatocellular carcinomas arising either spontaneously or due to chronic exposure to Ginkgo biloba extract (GBE) in B6C3F1/N mice.

Epigenetic modifications, such as DNA methylation, play an important role in carcinogenesis. In a recent NTP study, chronic exposure of B6C3F1/N mice to Ginkgo biloba extract (GBE) resulted in a high incidence of hepatocellular carcinomas (HCC). Genome-wide promoter methylation profiling on GBE-exposed HCC (2000 mg/kg group), spontaneous HCC (vehicle-control group), and age-matched vehicle control liver was performed to identify differentially methylated genes in GBE-exposed HCC and spontaneous HCC. DNA methylation alterations were correlated to the corresponding global gene expression changes. Compared to control liver, 1296 gene promoters (719 hypermethylated, 577 hypomethylated) in GBE-exposed HCC and 738 (427 hypermethylated, 311 hypomethylated) gene promoters in spontaneous HCC were significantly differentially methylated, suggesting an impact of methylation on GBE-exposed HCC. Differential methylation of promoter regions in relevant cancer genes (cMyc, Spry2, Dusp5) and their corresponding differential gene expression was validated by quantitative pyrosequencing and qRT-PCR, respectively. In conclusion, we have identified differentially methylated promoter regions of relevant cancer genes altered in GBE-exposed HCC compared to spontaneous HCC. Further study of unique sets of differentially methylated genes in chemical-exposed mouse HCC could potentially be used to differentiate treatment-related tumors from spontaneous-tumors in cancer bioassays and provide additional understanding of the underlying epigenetic mechanisms of chemical carcinogenesis.

Long-term Effects of Hypothermic Ex Situ Perfusion on Skeletal Muscle Metabolism, Structure, and Force Generation After Transplantation.

BACKGROUND: Hypothermic ex situ perfusion (HESP) systems are used to prolong allograft survival in solid organ transplantations and have been shown to be superior to static cold storage (SCS) methods. However, the effect of this preservation method on limb allograft survival and long-term function has not yet been tested. In this study, we investigated the long-term effects of the HESP on skeletal muscle metabolism, structure, and force generation and compared it with the current standard of preservation. METHODS: Forty male Lewis rats (250 ± 25 g) were divided into 5 groups, including naive control, sciatic nerve transection or repair, immediate transplantation, SCS, and HESP. For the SCS group, limbs were preserved at 4°C for 6 hours. In the HESP group, limbs were continuously perfused with oxygenated histidine-tryptophan-ketoglutarate (HTK) solution at 10-15°C for 6 hours. Hemodynamic and biochemical parameters of perfusion were recorded throughout the experiment. At 12 weeks, electromyography and muscle force measurements (maximum twitch and tetanic forces) were obtained along with muscle samples for histology and metabolomics analysis. RESULTS: Histology demonstrated 48% myocyte injury in the HESP group compared with 49% in immediate transplantation (P = 0.96) and 74% in the SCS groups (P < 0.05). The maximum twitch force measurement revealed a significantly higher force in the HESP group compared with the SCS group (P = 0.029). Essential amino acid levels of the gastrocnemius muscle did not reach significance, with the exception of higher proline levels in the HESP group. CONCLUSIONS: HESP using HTK protects viability of the limb but fails to restore muscle force in the long term.

Do GISTs Occur in Rats and Mice? Immunohistochemical Characterization of Gastrointestinal Tumors Diagnosed as Smooth Muscle Tumors in The National Toxicology Program.

The majority of the tumors in the gastrointestinal (GI) tract of rats and mice, with spindle cell morphology, are diagnosed as smooth muscle tumors (SMTs). Similarly, several decades ago human GI tumors with spindle cell morphology were also diagnosed as SMTs. However, later investigations identified most of these tumors in humans as gastrointestinal stromal tumors (GISTs). The GISTs are considered to arise from the interstitial cells of Cajal located throughout the GI tract. Positive immunohistochemical staining with CKIT antibody is a well-accepted diagnostic marker for GISTs in humans. Since there is a considerable overlap between the histomorphology of SMTs and GISTs, it is not possible to distinguish them on hematoxylin and eosin stained sections. As a result, GISTs are not routinely diagnosed in toxicological studies. The current study was designed to evaluate the tumors diagnosed as leiomyoma or leiomyosarcoma in the National Toxicology Program's 2-year bioassays using CKIT, smooth muscle actin, and desmin immunohistochemistry. The results demonstrate that most of the mouse SMTs diagnosed as leiomyoma or leiomyosarcoma are likely GISTs, whereas in rats the tumors are likely SMTs and not GISTs.

Inflammasome activation is required for human rhinovirus-induced airway inflammation in naive and allergen-sensitized mice.

Activation of the inflammasome is a key function of the innate immune response that regulates inflammation in response to microbial substances. Inflammasome activation by human rhinovirus (RV), a major cause of asthma exacerbations, has not been well studied. We examined whether RV induces inflammasome activation in vivo, molecular mechanisms underlying RV-stimulated inflammasome priming and activation, and the contribution of inflammasome activation to RV-induced airway inflammation and exacerbation. RV infection triggered lung mRNA and protein expression of pro-IL-1ß and NLRP3, indicative of inflammasome priming, as well as cleavage of caspase-1 and pro-IL-1ß, completing inflammasome activation. Immunofluorescence staining showed IL-1ß in lung macrophages. Depletion with clodronate liposomes and adoptive transfer experiments showed macrophages to be required and sufficient for RV-induced inflammasome activation. TLR2 was required for RV-induced inflammasome priming in vivo. UV irradiation blocked inflammasome activation and RV genome was sufficient for inflammasome activation in primed cells. Naive and house dust mite-treated NLRP3-/- and IL-1ß-/- mice, as well as IL-1 receptor antagonist-treated mice, showed attenuated airway inflammation and responsiveness following RV infection. We conclude that RV-induced inflammasome activation is required for maximal airway inflammation and hyperresponsiveness in naive and allergic mice. The inflammasome represents a molecular target for RV-induced asthma exacerbations.

The Endothelin-A Receptor Antagonist Zibotentan Induces Damage to the Nasal Olfactory Epithelium Possibly Mediated in Part through Type 2 Innate Lymphoid Cells.

Zibotentan, an endothelin-A receptor antagonist, has been used in the treatment of various cardiovascular disorders and neoplasia. Castrated athymic nude mice receiving zibotentan for a preclinical xenograft efficacy study experienced weight loss, gastrointestinal bloat, and the presence of an audible respiratory click. Human side effects have been reported in the nasal cavity, so we hypothesized that the nasal cavity is a target for toxicity in mice receiving zibotentan. Lesions in the nasal cavity predominantly targeted olfactory epithelium in treated mice and were more pronounced in castrated animals. Minimal lesions were present in vehicle control animals, which suggested possible gavage-related reflux injury. The incidence, distribution, and morphology of lesions suggested direct exposure to the nasal mucosa and a possible systemic effect targeting the olfactory epithelium, driven by a type 2 immune response, with group 2 innate lymphoid cell involvement. Severe nasal lesions may have resulted in recurrent upper airway obstruction, leading to aerophagia and associated clinical morbidity. These data show the nasal cavity is a target of zibotentan when given by gavage in athymic nude mice, and such unanticipated and off-target effects could impact interpretation of research results and animal health in preclinical studies.