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Mice from different genetic backgrounds have widely diverse responses to the same metabolic challenges. Only PWK/PhJ mice show significant mitochondrial dysfunction and progression to fibrotic NASH that resembles human NASH. The PWK/PhJ strain is a novel NASH mouse model., Non-alcoholic steatohepatitis (NASH) is a global health concern without treatment. The challenge in finding effective therapies is due to the lack of good mouse models and the complexity of the disease, characterized by gene-environment interactions. We tested the susceptibility of seven mouse strains to develop NASH. The severity of the clinical phenotypes observed varied widely across strains. PWK/PhJ mice were the most prone to develop hepatic inflammation and the only strain to progress to NASH with extensive fibrosis, while CAST/EiJ mice were completely resistant. Levels of mitochondrial transcripts and proteins as well as mitochondrial function were robustly reduced specifically in the liver of PWK/PhJ mice, suggesting a central role of mitochondrial dysfunction in NASH progression. Importantly, the NASH gene expression profile of PWK/PhJ mice had the highest overlap with the human NASH signature. Our study exposes the limitations of using a single mouse genetic background in metabolic studies and describes a novel NASH mouse model with features of the human NASH.

PURPOSE International guideline recommendations may not always be extrapolated to developing countries where access to resources is limited. In metastatic castration-sensitive prostate cancer (mCSPC), there have been successful drug and imaging advancements that were addressed in the Prostate Cancer Consensus Conference for Developing Countries for best-practice and limited-resource scenarios. METHODS A total of 24 out of 300 questions addressed staging, treatment, and follow-up for patients with mCSPC both in best-practice settings and resource-limited settings. Responses were compiled and presented in percentage of clinicians supporting each response. Questions had 4-8 options for response. RESULTS Recommendations for staging in mCSPC were split but there was consensus that chest x-ray, abdominal and pelvic computed tomography, and bone scan should be used where resources are limited. In both de novo and relapsed low-volume mCSPC, orchiectomy alone in limited resources was favored and in relapsed high-volume disease, androgen deprivation therapy plus docetaxel in limited resources and androgen deprivation therapy plus abiraterone in high-resource settings were consensus. A 3-weekly regimen of docetaxel was consensus among voters. When using abiraterone, a regimen of 1,000 mg plus prednisone 5 mg/d is optimal, but in limited-resource settings, half the panel agreed that abiraterone 250 mg with fatty foods plus prednisone 5 mg/d is acceptable. The panel recommended against the use of osteoclast-targeted therapy to prevent osseous complications. There was consensus that monitoring of patients undergoing systemic treatment should only be conducted in case of prostate-specific antigen elevation or progression-suggestive symptoms. CONCLUSION The treatment recommendations for most topics addressed differed between the best-practice setting and resource-limited setting, accentuating the need for high-quality evidence that contemplates the effect of limited resources on the management of mCSPC.

B-cell maturation antigen (BCMA) is a target for the treatment of multiple myeloma with cytolytic therapies, such as chimeric antigen receptor T-cells or T-cell redirecting antibodies. To better understand the potential for “on-target/off-tumor” toxicity caused by BCMA-targeting cytolytic therapies in the brain, we investigated normal brain BCMA expression. An immunohistochemistry (IHC) assay using the E6D7B commercial monoclonal antibody was applied to 107 formalin-fixed, paraffin-embedded brain samples (cerebrum, basal ganglia, cerebellum, brainstem; 63 unique donors). Although immunoreactivity was observed in a small number of neurons in brain regions including the striatum, thalamus, midbrain, and medulla, this immunoreactivity was considered nonspecific and not reflective of BCMA expression because it was distinct from the membranous and Golgi-like pattern seen in positive control samples, was not replicated when a different IHC antibody (D6 clone) was used, and was not corroborated by in situ hybridization data. Analysis of RNA-sequencing data from 478 donors in the GTEx and Allen BrainSpan databases demonstrated low levels of BCMA RNA expression in the striatum of young donors with levels becoming negligible beyond 30 years of age. We concluded that BCMA protein is not present in normal adult human brain, and therefore on-target toxicity in the brain is unlikely.

To further our understanding of the nonhuman primate kidney anatomy, histology, and incidences of spontaneous pathology, we retrospectively examined kidneys from a total of 505 control Cynomolgus monkeys ( Macaca fascicularis; 264 male and 241 females) aged 2 to 6 years, from toxicity studies. Kidney weights, urinalysis, and kidney-related clinical biochemistry parameters were also evaluated. Although the functional anatomy of the monkey kidney is relatively similar to that of other laboratory animals and humans, a few differences and species-specific peculiarities exist. Unlike humans, the macaque kidney is unipapillate, with a relatively underdeveloped papilla, scarce long loops of Henle, and a near-equivalent cortical to medullary ratio. The most common spontaneous microscopic findings were interstitial infiltrates or interstitial nephritis and other tubular lesions, but several forms of glomerulopathy that may be interpreted as drug-induced were occasionally observed. Common incidental findings of little pathological significance included: papillary mineralization, epithelial pigment, multinucleate cells, cuboidal metaplasia of the Bowman’s capsule, and urothelial inclusions. Kidney weights, and some clinical chemistry parameters, showed age- and sex-related variations. Taken together, these data will aid the toxicologic pathologist to better evaluate the nonhuman primate kidney and assess the species’ suitability as a model for identifying and characterizing drug-induced injury.

Two young cynomolgus macaques (Macaca fascicularis) given a small molecule kinase inhibitor ((S)-4-((2–(5-chloro-2-fluorophenyl)-5-isopropylpyrimidin-4-yl)amino)-N-(2-hydroxypropyl)nicotinamide [SCIO-120]) via nasogastric intubation gavage, once-daily for 21 days at 400 mg/kg/day, developed an unusual epithelial proliferative process in the renal parenchyma. Morphological and immunohistochemical characterization of the lesions confirmed an invasive malignant epithelial neoplasm (carcinoma). A similar renal neoplasm was seen in a third macaque after a 14-day exposure to a second kinase inhibitor in the same chemical series ((S) 4-((2–(5-chloro-2-fluorophenyl)-5-methoxypyrimidin-4-yl)amino)-N-cyclopropylnicotinamide [SCIO-974]). Despite remarkably short latency periods, exposure to these kinase inhibitors was likely causally associated with the induction of the renal tumors, as renal carcinomas are exceedingly rare spontaneously in macaques. Both SCIO-120 and SCIO-974 were designed as potent TGFβR1 inhibitors (IC50s 37 and 39 nM, respectively). SCIO-120 and SCIO-974 inhibited additional kinases, most notably closely related ALK4 (IC50 = 34 and 20 nM, respectively), c-Jun n-Terminal kinase 3 (JNK3, IC50 = 10 and 20 nM, respectively), and Fms-related tyrosine kinase 1 (29 and 76 nM, respectively). TGFβR1 has been specifically implicated in epithelial proliferative disorders, including neoplasia. Neither SCIO-120 nor SCIO-974 was genotoxic based on bacterial reverse mutation and/or clastogenicity screening assays. The rapid appearance of renal carcinomas in primates following short-term treatment with nongenotoxic kinase inhibitors is remarkable and suggests that the compounds had noteworthy tumor-enhancing effects, hypothetically linked to their TGFβR1 inhibition activity. These observations have implications for mechanisms of carcinogenesis and TGFβR1 biology.

The 2018 annual National Toxicology Program Satellite Symposium, entitled “Pathology Potpourri,” was held in Indianapolis, Indiana, at the Society of Toxicologic Pathology’s 37th annual meeting. The goal of this symposium was to present and discuss challenging diagnostic pathology and/or nomenclature issues. This article presents summaries of the speakers’ talks along with select images that were used by the audience for voting and discussion. Various lesions and other topics covered during the symposium included seminiferous tubule dysgenesis in rats, ameloblast and odontoblast degeneration/necrosis in a Sprague Dawley rat, intestinal leiomyositis in a beagle dog, gallbladder mucinous hyperplasia, focus of hepatocellular alteration and bile duct alteration in otters, renal tubule cytoplasmic vacuolation with basophilic granules in mice treated swith antisense oligonucleotide therapy, a uterine choriocarcinoma in a rhesus macaque, and rete ovarii proliferative ovarian lesions in various aged rat strains. One particularly provocative lesion was a malignant neoplastic proliferation in the renal pelvic region of a cynomolgus macaque from a 21-day study. Additional challenging lesions included thyroid proliferative lesions in zebra fish and gross findings in fish larvae during routine chemical screening. The Rabbit and Minipig International Harmonization of Nomenclature and Diagnostic Criteria Organ Working Groups also presented a series of challenging lesions.

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. In exercise #2, there was improved consistency of categorization of small borderline morphologies and mild lesions, but a decrement in consistency of categorizing minimal lesions. Exercises 1 and 2 suggest the described diagnostic approach is representative of that in use by the majority of toxicologic pathologists across companies/agencies and that application by all may improve diagnostic consistency of PCM/like lesions. Additionally, a criterion of approximately 5% heart section involvement is suggested for separating mild from moderate or greater severity. While evidence is not absolute, until further investigation shows otherwise, microscopic changes resembling PCM, but located in the epicardial and subepicardial region of the right ventricle, may be considered as part of the spectrum of PCM.

Background GPRC5D is an attractive target for treatment of multiple myeloma (MM) due to its high expression on MM cells, and favorable normal tissue expression profile that suggests a low risk for on-target/off-tumor toxicity. However, current literature and public databases report conflicting data for normal GPRC5D RNA and protein expression. For example, cerebellum is reported to have very low levels of GPRC5D mRNA by some sources whereas others report its absence. Likewise, the GTEx database has high maximum expression in the skin, but very low median expression; this is also reflected in the high variability of GPRC5D mRNA levels in skin assessed by RT-PCR reported in the literature. The protein expression data in Human Protein Atlas cannot be used to verify RNA expression because it was obtained using an unvalidated, polyclonal antibody with low specificity by protein array. Other sources of protein expression are incomplete and did not assess critical tissues. Clarification of GPRC5D expression in normal tissues is critical to understanding potential on-target/off-tumor risks to patients treated with GPRC5D-targeted therapies. Methods Key human tissues of interest for protein expression studies were identified through literature search and analysis of an internal RNAseq database. Further elucidation of normal GPRC5D expression in those tissues of interest was done using IHC and ISH on FFPE human tissue samples, including: skin, lung, tongue, tonsil, parotid gland, salivary gland, sublingual gland, submandibular gland, choroid plexus, cerebellum, and brainstem-medulla (inferior olivary nucleus, ION). The primary antibody used was a mouse anti-GPRC5D monoclonal antibody (Abcam, Ab55044, clone 6D9) qualified for its sensitivity and specificity in positive and negative control FFPE human cell pellets. The RNA-ISH GPRC5D probe for human was from ACD (Hs-GPCR5D, cat#489699). All test samples underwent QC screens for IHC and ISH, and only samples that passed were included in the definitive experiments. Stained slides were examined by a pathologist for presence and localization of IHC and ISH signals. Results By IHC and ISH, GPRC5D is expressed in epithelial cells of the hair follicles in the skin (the number of hair follicles in skin samples explains the variability in RNA expression), and at the base of the epithelial columns supporting the filiform papillae (keratinized structures in the tongue). In all other tissues assessed, GPRC5D protein was observed only in interstitial plasma cells. Mild GPRC5D RNA expression was detected by ISH in the motor neurons of the ION (without IHC correlate); its close connection with the cerebellum may explain the very low levels of GPRC5D mRNA expression that have been reported in the literature. Conclusion These data support that GPRC5D is not broadly expressed in normal tissues beyond resident plasma cell populations and hard keratinized tissues.

Inhibition of mTOR signaling using the rapalog everolimus is an FDA-approved targeted therapy for patients with lung and gastroenteropancreatic neuroendocrine tumors (NET). However, patients eventually progress on treatment, highlighting the need for additional therapies. We focused on pancreatic NETs (pNET) and reasoned that treatment of these tumors upon progression on rapalog therapy, with an mTOR kinase inhibitor (mTORKi), such as CC-223, could overcome a number of resistance mechanisms in tumors and delay cardiac carcinoid disease. We performed preclinical studies using human pNET cells in vitro and injected them subcutaneously or orthotopically to determine tumor progression and cardiac function in mice treated with either rapamycin alone or switched to CC-223 upon progression. Detailed signaling and RNA sequencing analyses were performed on tumors that were sensitive or progressed on mTOR treatment. Approximately 57% of mice bearing pNET tumors that progressed on rapalog therapy showed a significant decrease in tumor volume upon a switch to CC-223. Moreover, mice treated with an mTORKi exhibited decreased cardiac dilation and thickening of heart valves than those treated with placebo or rapamycin alone. In conclusion, in the majority of pNETs that progress on rapalogs, it is possible to reduce disease progression using an mTORKi, such as CC-223. Moreover, CC-223 had an additional transient cardiac benefit on valvular fibrosis compared with placebo- or rapalog-treated mice. These results provide the preclinical rationale to further develop mTORKi clinically upon progression on rapalog therapy and to further test their long-term cardioprotective benefit in those NET patients prone to carcinoid syndrome. Mol Cancer Ther; 16(11); 2432–41. ©2017 AACR.

Innate lymphoid cell (ILC) cytokine signatures mirror those of T helper subsets, but ILCs differ substantially as far as biological role. Tissue-resident and antigen-independent, ILCs mediate induction and resolution of inflammation, tissue homeostasis, and barrier maintenance. Although extensively character-ized in periphery, ILCs were newly identified as resident in central nervous system (CNS) meninges, and are less well-understood within this novel context. Here we show that ILC-deficient mice exhibit enhanced microglial reactivity, amplified neuroinflammatory responses and blood-brain barrier (BBB) permeability. Accordingly, transcriptomic and functional examination revealed meningeal ILC2s as vigorous interleukin (IL)-10 producers. Mechanistic relevance was demonstrated by amelioration of neuroinflammation following meningeal engraftment of adoptively-transferred wild type — but not IL-10-deficient — ILC2s. Notably, ILC2s from murine bone marrow and human blood also showed IL-10 competency, suggesting a previously-unappreciated immunoregulatory role for canonical ILC2s. Collectively, these findings reveal meningeal ILC2s as suppressors of neuroinflammation — suggesting potential for ILC2-based cell therapies in CNS pathology.

Epidemiological studies in humans and experimental work in rodents suggest that exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a persistent environmental toxicant, is associated with incidence of heart disease. Although TCDD toxicity depends by and large on the aryl hydrocarbon receptor (AHR), the role of the cardiac AHR in TCDD induced cardiovascular disease is not well defined. To determine whether the Ahr gene mediates disruption of heart function by TCDD, we generated a cardiomyocyte-specific Ahr knockout mouse by crossing Ahr(fx/fx) mice with βMhc:cre/+ mice, in which expression of Cre recombinase is driven by the promoter of the βMhc (myosin heavy chain-beta) gene. Starting at three months of age, mice with cardiomyocyte-specific Ahr ablation were exposed to 1μg/kg/week of TCDD or control vehicle by oral gavage for an additional three months. Relative to unexposed controls, TCDD-exposure induced cardiomyocyte Ahr-independent changes in males but not females, including a significant increase in body weight, blood pressure, and cardiac hypertrophy and a decrease in cardiac ejection fraction. TCDD exposure also induced cardiomyocyte Ahr-dependent changes in fibrosis and calcium signaling gene expression in both males and females. TCDD exposure appears to cause sexually dimorphic effects on heart function and induce fibrosis and changes in calcium signaling in both males and females through activation of the cardiomyocyte-specific Ahr.

Carbon nanotubes (CNTs) are emerging as important occupational and environmental toxicants owing to their increasing prevalence and potential to be inhaled as airborne particles. CNTs are a concern because of their similarities to asbestos, which include fibrous morphology, high aspect ratio, and biopersistence. Limitations in research models have made it difficult to experimentally ascertain the risk of CNT exposures to humans and whether these may lead to lung diseases classically associated with asbestos, such as mesothelioma and fibrosis. In this study, we sought to comprehensively compare profiles of lung pathology in mice following repeated exposures to multiwall CNTs or crocidolite asbestos (CA). We show that both exposures resulted in granulomatous inflammation and increased interstitial collagen; CA exposures caused predominantly bronchoalveolar hyperplasia, whereas CNT exposures caused alveolar hyperplasia of type II pneumocytes (T2Ps). T2Ps isolated from CNT-exposed lungs were found to have upregulated proinflammatory genes, including interleukin 1ß (IL-1ß), in contrast to those from CA exposed. Immunostaining in tissue showed that while both toxicants increased IL-1ß protein expression in lung cells, T2P-specific IL-1ß increases were greater following CNT exposure. These results suggest related but distinct mechanisms of action by CNTs versus asbestos which may lead to different outcomes in the 2 exposure types.

The toxicologic pathologist plays a vital role in the scientific community, using their unique blend of diagnostic and investigative skills to advance biomedical research, public health, drug discovery, or regulatory practices. But what exactly do toxicologic pathologists contribute? Where do these specialized professionals work? How can toxicologic pathologists maximize their efficiency and potential? To enlighten students and trainees, as well as early- or mid-career toxicologic pathologists, or even those approaching retirement, the Career Development and Outreach Committee of the Society of Toxicologic Pathology (STP) sponsored a career development workshop entitled “Practical Strategies for Navigating Toxicologic Pathology in One’s Early Career…and Beyond!” in conjunction with the STP 37th annual symposium. The workshop featured toxicologic pathologists from contract research organizations and the pharmaceutical industry, who provided their perspectives on career preparation, evolving veterinary pathologist roles within various sectors of toxicologic pathology, the fundamentals of safety assessment, logistics of projects involving good laboratory practices, tools for effective interpretation and communication of anatomic and clinical pathology results, and a recap of scientific resources available to support the toxicologic pathologist in his or her journey. This article provides brief summaries of the talks presented during this career development workshop.

Use of silver nanoparticles (AgNPs) for their antimicrobial properties is widespread. Much of the previous work on the toxicity of AgNPs has been conducted in vitro or following oral or intravenous administration in vivo. Intranasal (IN) instillation of AgNPs mimics inhalation exposure and allows further exploration of the toxicity of these particles via respiratory tract exposure. The present study involved 1) single-dose exposures to assess tissue distribution and toxicity and 2) repeated exposures to assess behavioral effects of IN AgNP exposure (nominally uncoated 25 nm AgNP). AgNP deposition was localized in the liver, gut-associated lymphoid tissue, and brain. Decrease cellularity in spleen follicles was observed in treated mice, along with changes in cell number and populations in the spleen. The splenic GSH:GSSG ratio was also reduced following AgNP exposure. Expression of the oxidative stress-responsive gene Hmox1 was elevated in the hippocampus, but not cortex of treated mice, as was the level of HMOX1 protein. Mice receiving 7 days of IN exposure to 50 mg/kg AgNPs exhibited similar learning- and memory-related behaviors to control mice, except that treated mice spent significantly less time in the target quadrant of the Morris Water Maze during the acquisition phase probe trial. These findings indicate systemic distribution and toxicity following IN administration of AgNPs.

Congenital heart disease (CHD) is the most common congenital abnormality and one of the leading causes of newborn death throughout the world. Despite much emerging scientific information, the precise etiology of this disease remains elusive. Here, we show that the aryl hydrocarbon receptor (AHR) regulates the expression of crucial cardiogenesis genes and that interference with endogenous AHR functions, either by gene ablation or by agonist exposure during early development, causes overlapping structural and functional cardiac abnormalities that lead to altered fetal heart physiology, including higher heart rates, right and left ventricle dilation, higher stroke volume, and reduced ejection fraction. With striking similarity between AHR knockout (Ahr � /� ) and agonist-exposed wild type (Ahr þ/þ ) embryos, in utero disruption of endogenous AHR functions converge into dysregulation of molecular mechanisms needed for attainment and maintenance of cardiac differentiation, including the pivotal signals regulated by the cardiogenic transcription factor NKH2.5, energy balance via oxidative phosphorylation and TCA cycle and global mitochondrial function and homeostasis. Our findings suggest that AHR signaling in the developing mammalian heart is central to the regulation of pathways crucial for cellular metabolism, cardiogenesis, and cardiac function, which are potential targets of environmental factors associated with CHD.

Complex mixtures of environmental agents often cause mixture-specific health effects that cannot be accounted for by a single mechanism. To study the biological effects of exposure to a mixture of chromium-VI and benzo[a]pyrene (B[a]P), often found together in the environment, we exposed mice for 60 days to 0, 55, 550, or 5500 ppb Cr(VI) in drinking water followed by 90 days of coexposure to B[a]P at 0, 1.25, 12.5, or 125 mg/kg/day and examined liver and gastrointestinal (GI) tract for exposure effects. In the liver, the mixture caused more significant histopathology than expected from the sum of effects of the individual components, while in the GI tract, Cr(VI) alone caused significant enterocyte hypertrophy and increases in cell proliferation and DNA damage that were also observed in mice coexposed to B[a]P. Expression of genes involved in drug metabolism, tumor suppression, oxidative stress, and inflammation was altered in mixed exposures relative to control and to singly exposed mice. Drug metabolism and oxidative stress genes were upregulated and tumor suppressor and inflammation genes downregulated in the proximal GI tract, whereas most markers were upregulated in the distal GI tract and downregulated in the liver. Oral exposure to Cr(VI) and B[a]P mixtures appears to have tissue-specific differential consequences in liver and GI tract that cannot be predicted from the effects of each individual toxicant. Tissue specificity may be particularly critical in cases of extended exposure to mixtures of these agents, as may happen in the occupational setting or in areas where drinking water contains elevated levels of Cr(VI).

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. Harmonized terminology and thresholds across studies will generate better historical control data, will likely enhance interpretation of study data, and may further enhance our understanding of the spontaneous change. An assessment of the diagnostic approaches of a group of 37 pathologists identified an approach that is relatively easily applied; and if adopted, it could enhance diagnostic consistency across studies. This approach uses the single “slash” term “necrosis/inflammatory cell infiltrate (NICI)” as the diagnosis for the spectrum of lesions seen in younger rats, uses no threshold for diagnosis (e.g., diagnose all lesions clearly identifiable as PCM/like), and uses aggregate lesion size of approximately ≥45% of the field of view (FOV) using a 10×/22 eyepiece and the 40× objective or approximately ≥100% of the FOV using the 60× objective as the criterion separating minimal from mild severities.

The risk of human exposure to fiber nanoparticles has risen in recent years due to increases in the manufacture and utilization of carbon nanotubes (CNTs). CNTs are present as airborne particulates in occupational settings and their hazard potential has been demonstrated in experimental lung exposure studies using inbred mouse strains. However, it is not known whether different inbred strains differ in lung responses to CNTs by virtue of their genetics. In this work, common inbred strains (BALB/c, C57Bl/6, DBA/2, and C3H/ He) were exposed to CNTs via oropharyngeal aspiration and lung histology and bronchoalveolar lavage (BAL) samples were evaluated over 28 days with the objective of evaluating sensitivity/resistance among strains. C57Bl/6 mice developed significantly mo re extensive type II pneumocyte (T2P) hyperplasia and alveolar infiltrate compared to DBA/2 mice, which were resistant. Surprisingly, DBA/2 but not C57Bl/ 6 mice were extremely sensitive to increases in leukocytes recovered in BAL fluid. Underlying global gene expression patterns in the two strains were compared using mRNA sequencing to investigate regulatory networks associated with the different effects. The impact of exposure on gene networks regulating various aspects of immune response and cell survival was limited in DBA/2 mice compared to C57Bl/6. Investigation of B6D2F1 (C57Bl/6 x DBA/2 hybrid) mice demonstrated inheritance of sensitivity to CNT exposures in regard to toxicologic lung pathology and BAL leukocyte accumulations. These findings demonstrate genetic bases of susceptibility to CNT particle exposures and both inform the use of inbred mouse models and suggest the likelihood of differences in genetic susceptibility among humans.

The cardiac regenerative capacity of MRL/MpJ mouse remains a controversy. Although the MRL mouse has been reported to exhibit minimal scarring and subsequent cardiac regeneration after cryoinjury of the right ventricle, multiple studies have been unable to replicate this cardiac regenerative capacity after both cryogenic and coronary ligation cardiac injury. Therefore, we evaluated the cardiac regenerative wound-healing response and functional recovery of MRL mice compared to C57 mice, in response to a clinically relevant left ventricular (LV) coronary ligation. Male MRL/MpJ+/+ and C57BL/6 mice underwent left coronary artery ligation followed by reperfusion. Cardiac function was evaluated by echocardiography [LV ejection fraction (LVEF), LV end-diastolic volume (LVEDV), LV mass, wall thickness] at 24 hours post-ischemia and weekly for 13 weeks thereafter. Hearts were also analyzed histologically for individual cardiomyocyte hypertrophy and cardiac fibrosis. Our results show that contrary to prior reports of cardiac regenerations, MRL mice progress to heart failure more rapidly following I/R injury as marked by a significant decrease in LVEF, increase in LVEDV, LV mass, individual myocyte size, and fibrosis in the post-ischemic myocardium. Therefore, we conclude that MRL mice do not exhibit regeneration of the LV or enhanced functional improvement in response to coronary ligation. However, unlike prior studies, we matched initial infarct size in MRL and C57 mice, used high frequency echocardiography, and histological analysis to reach this conclusion. The prospect of cardiac regeneration after ischemia in MRL mice seems to have attenuated interest, given the multiple negative studies and the promise of stem cell cardiac regeneration. However, our novel observation that MRL may possess an impaired compensated hypertrophy response makes the MRL mouse strain an interesting model in the study of cardiac hypertrophy.

Transient receptor potential cation channels have been implicated in the regulation of cardiovascular function, but only recently has our laboratory described the vanilloid-2 subtype (TRPV2) in the cardiomyocyte, though its exact mechanism of action has not yet been established. This study tests the hypothesis that TRPV2 plays an important role in regulating myocyte contractility under physiological conditions. Therefore, we measured cardiac and vascular function in wild-type and TRPV2−/− mice in vitro and in vivo and found that TRPV2 deletion resulted in a decrease in basal systolic and diastolic function without affecting loading conditions or vascular tone. TRPV2 stimulation with probenecid, a relatively selective TRPV2 agonist, caused an increase in both inotropy and lusitropy in wild-type mice that was blunted in TRPV2−/− mice. We examined the mechanism of TRPV2 inotropy/lusitropy in isolated myocytes and found that it modulates Ca2+ transients and sarcoplasmic reticulum Ca2+ loading. We show that the activity of this channel is necessary for normal cardiac function and that there is increased contractility in response to agonism of TRPV2 with probenecid.

The canine mammary tumor is the neoplasia that most commonly affects intact female dogs (not spayed) in routine veterinary practice. Canine mammary tumor therapy is a challenge because only few effective treatments have been described for high-grade tumors in veterinary medicine. One such therapeutic option that slows down tumor growth is metronomic chemotherapy (MC), a therapeutic modality that acts by decreasing tumor angiogenesis. Quantification of intratumoral microvessel density (MVD) has been proposed as a means to evaluate the effectiveness of MC in human and canine tumors. In this study, MVD is proposed as a predictive factor for the effectiveness of MC in canine mammary tumors. Twenty female dogs with mammary carcinoma were equally distributed into a mastectomy group treated only with mastectomy, and a MC group treated with MC (cyclophosphamide and piroxicam) orally and daily for 28 days followed by mastectomy. Mammary tumors were classified and graded histologically. MVD was ascertained by CD31 immunostaining. The analysis showed statistically significant difference in MVD scores between groups, corroborating a quantitative reduction in tumor microvasculature in the group treated with MC. Our findings suggest that MVD may be an important predictive factor for the selection of female dogs with malignant mammary tumors that may benefit from MC.

Background: The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates the expression of xenobiotic detoxification genes and is a critical mediator of gene–environment interactions. Many AHR target genes identified by genome-wide gene expression profiling have morphogenetic functions, suggesting that AHR may play a role in embryonic development. Objectives: To characterize the developmental functions of the AHR, we studied the consequences of AHR activation by the agonist 2,3,7,8-tetrachlorodibenzo-p-doxin (TCDD), and the result of its repression by the antagonists 6,2,4-trimethoxyflavone and CH 223191 or by short-hairpin RNA (shRNA)-mediated Ahr knockdown during spontaneous differentiation of embryonic stem (ES) cells into cardiomyocytes. Methods: We generated an AHR-positive cardiomyocyte lineage differentiated from mouse ES cells that expresses puromycin resistance and enhanced green fluorescent protein (eGFP) under the control of the Cyp1a1 (cytochrome P450 1a1) promoter. We used RNA sequencing (RNA.Seq) to analyze temporal trajectories of TCDD-dependent global gene expression in these cells during differentiation. Results: Activation, inhibition, and knockdown of Ahr significantly inhibited the formation of contractile cardiomyocyte nodes. Global expression analysis of AHR-positive cells showed that activation of the AHR/TCDD axis disrupted the concerted expression of genes that regulate multiple signaling pathways involved in cardiac and neural morphogenesis and differentiation, including dozens of genes encoding homeobox transcription factors and Polycomb and trithorax group proteins. Conclusions: Disruption of AHR expression levels resulted in gene expression changes that perturbed cardiomyocyte differentiation. The main function of the AHR during development appears to be the coordination of a complex regulatory network responsible for attainment and maintenance of cardiovascular homeostasis. Citation: Wang Q, Chen J, Ko CI, Fan Y, Carreira V, Chen Y, Xia Y, Medvedovic M, Puga A. 2013. Disruption of aryl hydrocarbon receptor homeostatic levels during embryonic stem cell differentiation alters expression of homeobox transcription factors that control cardiomyogenesis. Environ Health Perspect 121:1334–1343; http://dx.doi.org/10.1289/ehp.1307297

Embryonic eyelid closure involves forward movement and ultimate fusion of the upper and lower eyelids, an essential step of mammalian ocular surface development. Although its underlying mechanism of action is not fully understood, a functional mitogen-activated protein kinase kinase kinase 1 (MAP3K1) is required for eyelid closure. Here we investigate the molecular signatures of MAP3K1 in eyelid morphogenesis. At mouse gestational day E15.5, the developmental stage immediately prior to eyelid closure, MAP3K1 expression is predominant in the eyelid leading edge (LE) and the inner eyelid (IE) epithelium. We used laser capture microdissection (LCM) to obtain highly enriched LE and IE cells from wild type and MAP3K1-deficient fetuses and analyzed genome-wide expression profiles. The gene expression data led to the identification of three distinct developmental features of MAP3K1. First, MAP3K1 modulated Wnt and Sonic hedgehog signals, actin reorganization, and proliferation only in LE but not in IE epithelium, illustrating the temporal-spatial specificity of MAP3K1 in embryogenesis. Second, MAP3K1 potentiated AP-2α expression and SRF and AP-1 activity, but its target genes were enriched for binding motifs of AP-2α and SRF, and not AP-1, suggesting the existence of novel MAP3K1-AP-2α/SRF modules in gene regulation. Third, MAP3K1 displayed variable effects on expression of lineage specific genes in the LE and IE epithelium, revealing potential roles of MAP3K1 in differentiation and lineage specification. Using LCM and expression array, our studies have uncovered novel molecular signatures of MAP3K1 in embryonic eyelid closure.

Two of three captive adult African ostriches exhibited inappetance and weakness. In spite of treatment, the two birds were euthanized because of lack of clinical improvement. Postmortem examination demonstrated exocrine pancreatic degeneration, necrosis, and atrophy. Grossly, one ostrich had a markedly diminished pancreatic mass. Histologically, there was massive pancreatic acinar (exocrine) atrophy, marked interstitial fibrosis, and tubular complex formation in one animal, and the second ostrich had active pancreatic acinar necrosis. Toxicologic testing revealed markedly elevated liver zinc levels in the first two birds, whereas the third ostrich had normal serum levels of zinc and continues without apparent disease. This form of zinc toxicosis, while previously reported in different avian species, has been only rarely described in ratites.

Made available in DSpace on 2022-04-28T20:55:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-10-12 A four-year-old male goat with a history of neurological disorder was euthanized. It presented uncommon nodules in the brain and lungs associated with multiple abscesses, predominantly in the spleen and liver. Histological examination of brain and lung sections revealed yeast forms confirmed to be Cryptococcus gattii after a combination of isolation and polymerase chain reaction (PCR) procedures. Moreover, Corynebacterium pseudotuberculosis infection was diagnosed by PCR of samples from the lung, spleen and liver. The present report highlights the rare concurrent infection of C. gatti and C. pseudotuberculosis in an adult goat from São Paulo state, Brazil, and indicates the necessity of surveillance in the treatment of goats with atypical pulmonary infections associated with neurological disorders. Laboratory of Animal Pathology and Microbiology School of Veterinary Medicine São Paulo State University, UNESP, Araçatuba, São Paulo State Laboratory of Cellular and Molecular Genetics Federal University of Minas Gerais, UFMG, Belo Horizonte, Minas Gerais State Department of Microbiology Federal University of São Paulo, UNIFESP, São Paulo, São Paulo State Laboratório de Microbiologia Departamento de Apoio UNESP, Rua Clóvis Pestana, 793, Araçatuba, SP, 16.050-680 Laboratory of Animal Pathology and Microbiology School of Veterinary Medicine São Paulo State University, UNESP, Araçatuba, São Paulo State Laboratório de Microbiologia Departamento de Apoio UNESP, Rua Clóvis Pestana, 793, Araçatuba, SP, 16.050-680

The AHR is a ligand-activated transcription factor that mediates gene-environment interactions. Genome-wide expression profiling during differentiation of mouse ES cells into cardiomyocytes showed that AHR activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin; Dioxin (TCDD), its prototypical ligand, disrupted the expression of multiple homeobox transcription factors and inhibited cardiomyocyte contractility. Here we treated ES cells with TCDD at daily differentiation intervals to investigate whether TCDD-induced loss of contractility had a developmental window of sensitivity. Surprisingly, contractility was an AHR-dependent TCDD target solely between differentiation days 0 and 3 during the period of panmesoderm development, when TCDD also disrupted expression of genes in the TGFβ/BMP2/4 and wingless-type MMTV integration site (WNT)signaling pathways, suppressed the secretion of bone morphogenetic protein (BMP4), WNT3a, and WNT5a and elevated the secretion of Activin A, as determined by ELISA of the secreted proteins in the culture medium. Supplementing the culture medium with BMP4, WNT3a, or WNT5a during the first 3 days of differentiation successfully countered TCDD-induced impairment of contractility, while anti-WNT3a, or anti-WNT5a antibodies or continuous Noggin (a BMP4 antagonist) or Activin A treatment inhibited the contractile phenotype. In Ahr(+/+), but not in Ahr(-) (/) (-) ES cells, TCDD treatment significantly increased mitochondrial copy number, suggestive of mitochondrial stress and remodeling. Sustained AHR activation during ES cell differentiation appears to disrupt the expression of signals critical to the ontogeny of cardiac mesoderm and cause the loss of contractility in the resulting cardiomyocyte lineage.

Visceral leishmaniasis (VL), also known as kala-azar, is a disseminated protozoan infection caused by Leishmania donovani complex. Traditionally the definite diagnosis is made by amastigote detection in the tissue. The aim this study was to evaluate the PCR technique in stained slides of bone marrow and lymph nodes aspirates with suspect diagnosis for leishmaniasis. Slides were selected totaling 62 suspect cases (33 bone marrow samples and 29 lymph node samples) and 17 positive cases (8 bone marrow and 9 lymph node). From 62 suspect cases, 39 (62.90%) were confirmed to be positive being 17 (n=29) lymph node aspirates and 22 (n=33) bone marrow. This finding is in agreement with the higher sensitivity of the PCR assay compared to direct microscopic observation. In conclusion, the findings of this study supports the use of PCR on archive cytological preparation stained slides for the diagnosis of canine visceral leishmaniasis, emphasizing the higher sensitivity of this technique when compared to direct microscopic examination and mostly the use of the suspect status for the cytology samples that presents the previously mentioned particularities with focus on detecting the oligosymptomatic or assymptomatic dogs in endemic areas functioning as potential reservoirs for this disease.

The Developmental Origins of Health and Disease (DOHaD) Theory proposes that the environment encountered during fetal life and infancy permanently shapes tissue physiology and homeostasis such that damage resulting from maternal stress, poor nutrition or exposure to environmental agents may be at the heart of adult onset disease. Interference with endogenous developmental functions of the aryl hydrocarbon receptor (AHR), either by gene ablation or by exposure in utero to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent AHR ligand, causes structural, molecular and functional cardiac abnormalities and altered heart physiology in mouse embryos. To test if embryonic effects progress into an adult phenotype, we investigated whether Ahr ablation or TCDD exposure in utero resulted in cardiac abnormalities in adult mice long after removal of the agent. Ten-months old adult Ahr -/- and in utero TCDD-exposed Ahr +/+ mice showed sexually dimorphic abnormal cardiovascular phenotypes characterized by echocardiographic findings of hypertrophy, ventricular dilation and increased heart weight, resting heart rate and systolic and mean blood pressure, and decreased exercise tolerance. Underlying these effects, genes in signaling networks related to cardiac hypertrophy and mitochondrial function were differentially expressed. Cardiac dysfunction in mouse embryos resulting from AHR signaling disruption seems to progress into abnormal cardiac structure and function that predispose adults to cardiac disease, but while embryonic dysfunction is equally robust in males and females, the adult abnormalities are more prevalent in females, with the highest severity in Ahr -/- females. The findings reported here underscore the conclusion that AHR signaling in the developing heart is one potential target of environmental factors associated with cardiovascular disease.

Purpose Mammalian eye development requires temporary fusion of the upper and lower eyelids in embryogenesis. Failure of lid closure in mice leads to an eye open at birth (EOB) phenotype. Many genetic mutant strains develop this phenotype and studies of the mutants lead to a better understanding of the signaling mechanisms of morphogenesis. The present study investigates the roles of lid closure in eye development. Methods Seven mutant mouse strains were generated by different gene ablation strategies that inactivated distinct signaling pathways. These mice, including systemic ablation of Map3k1 and Dkk2, ocular surface epithelium (OSE) knockout of c-Jun and Egfr, conditional knockout of Shp2 in stratified epithelium (SE), as well as the Map3k1/Jnk1 and Map3k1/Rhoa compound mutants, all exhibited defective eyelid closure. The embryonic and postnatal eyes in these mice were characterized by histology and immunohistochemistry. Results Some eye abnormalities, such as smaller lens in the Map3k1-null mice and Harderian gland hypoplasia in the Dkk2-null mice, appeared to be mutant strain-specific, whereas other abnormalities were seen in all mutants examined. The common defects included corneal erosion/ulceration, meibomian gland hypoplasia, truncation of the eyelid tarsal muscles, failure of levator palpebrae superioris (LPS) extension into the upper eyelid and misplacement of the inferior oblique (IO) muscle and inferior rectus (IR) muscle. The muscle defects were traced to the prenatal fetuses. Conclusions In addition to providing a protective barrier for the ocular surface, eyelid closure in embryogenesis is required for the development of ocular adnexa, including eyelid and extraocular muscles.