Corrigendum: Progression and Regression of Hepatic Lesions in a Mouse Model of NASH Induced by Dietary Intervention and Its Implications in Pharmacotherapy.

[This corrects the article DOI: 10.3389/fphar.2018.00410.].

Progression and Regression of Hepatic Lesions in a Mouse Model of NASH Induced by Dietary Intervention and Its Implications in Pharmacotherapy.

Understanding of the temporal changes of hepatic lesions in the progression and regression of non-alcoholic steatohepatitis (NASH) is vital to elucidation of the pathogenesis of NASH, and critical to the development of a strategy for NASH pharmacotherapy. There are challenges in studying hepatic lesion progression and regression in NASH patients due to the slow development of NASH in humans, one being the requirement for multiple biopsies during the longitudinal follow-up. Here we studied lesion progression and regression in the diet-induced animal model of NASH by application or removal of the pathogenic diet for multiple time periods. Male C57BL/6 mice fed Western diet developed progressive hepatic steatosis/macrovesicular vacuolation, inflammation, and hepatocyte degeneration, as well as perisinusoidal fibrosis and occasionally portal fibrosis as early as 2 months after initiation of the Western diet. In the same period, the mice exhibited elevated ALT (alanine aminotransferase) and AST (aspartate aminotransferase) enzyme activities, CK18 (cytokeratin-18), PIIINP (N-terminal propeptide of type III collagen), and TIMP-1 (tissue inhibitor of metalloproteinase-1). Hepatic steatosis diminished rapidly when the Western diet was replaced by normal rodent chow diet and hepatic inflammation and hepatocyte degeneration were also reduced. Interestingly, perisinusoidal fibrosis and portal fibrosis regressed 8 months after chow diet replacement. To understand pharmacotherapy for NASH, mice with established NASH hepatic lesions were treated with either FXR agonist obeticholic acid (Ocaliva), or CCR2/5 antagonist Cenicriviroc. Similar to the diet replacement, metabolic modulator Ocaliva markedly reduced steatosis/macrovesicular vacuolation, hepatic inflammation, and hepatocyte degeneration effectively, but exhibited no significant effect on liver fibrosis. Anti-inflammation drug Cenicriviroc, on the other hand, markedly decreased inflammation and hepatocyte degeneration, and mildly decreased liver fibrosis, but exhibited no effect on hepatic steatosis/macrovesicular vacuolation. In conclusion, we found the progression of NASH hepatic steatosis/macrovesicular vacuolation, and inflammation eventually lead to hepatocyte death and fibrosis. Life style change and current pharmacotherapies in development may be effective in treating NASH, but their effects on NASH-induced fibrosis may be mild. Since fibrosis is known to be an independent risk for decompensated cirrhosis, cardiovascular events, and mortality, our study suggests that effective anti-fibrosis therapy should be an essential component of the combined pharmacotherapy for advanced NASH.

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

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

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

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

Best practices for veterinary toxicologic clinical pathology, with emphasis on the pharmaceutical and biotechnology industries.

The purpose of this paper by the Regulatory Affairs Committee (RAC) of the American Society for Veterinary Clinical Pathology (ASVCP) is to review the current regulatory guidances (eg, guidelines) and published recommendations for best practices in veterinary toxicologic clinical pathology, particularly in the pharmaceutical and biotechnology industries, and to utilize the combined experience of ASVCP RAC to provide updated recommendations. Discussion points include (1) instrumentation, validation, and sample collection, (2) routine laboratory variables, (3) cytologic laboratory variables, (4) data interpretation and reporting (including peer review, reference intervals and statistics), and (5) roles and responsibilities of clinical pathologists and laboratory personnel. Revision and improvement of current practices should be in alignment with evolving regulatory guidance documents, new technology, and expanding understanding and utility of clinical pathology. These recommendations provide a contemporary guide for the refinement of veterinary toxicologic clinical pathology best practices.

DFNA8/12 caused by TECTA mutations is the most identified subtype of nonsyndromic autosomal dominant hearing loss.

The prevalence of DFNA8/DFNA12 (DFNA8/12), a type of autosomal dominant nonsyndromic hearing loss (ADNSHL), is unknown as comprehensive population-based genetic screening has not been conducted. We therefore completed unbiased screening for TECTA mutations in a Spanish cohort of 372 probands from ADNSHL families. Three additional families (Spanish, Belgian, and English) known to be linked to DFNA8/12 were also included in the screening. In an additional cohort of 835 American ADNSHL families, we preselected 73 probands for TECTA screening based on audiometric data. In aggregate, we identified 23 TECTA mutations in this process. Remarkably, 20 of these mutations are novel, more than doubling the number of reported TECTA ADNSHL mutations from 13 to 33. Mutations lie in all domains of the α-tectorin protein, including those for the first time identified in the entactin domain, as well as the vWFD1, vWFD2, and vWFD3 repeats, and the D1-D2 and TIL2 connectors. Although the majority are private mutations, four of them-p.Cys1036Tyr, p.Cys1837Gly, p.Thr1866Met, and p.Arg1890Cys-were observed in more than one unrelated family. For two of these mutations founder effects were also confirmed. Our data validate previously observed genotype-phenotype correlations in DFNA8/12 and introduce new correlations. Specifically, mutations in the N-terminal region of α-tectorin (entactin domain, vWFD1, and vWFD2) lead to mid-frequency NSHL, a phenotype previously associated only with mutations in the ZP domain. Collectively, our results indicate that DFNA8/12 hearing loss is a frequent type of ADNSHL.

Post-transcriptional mechanisms contribute to the suppression of the ErbB3 negative regulator protein Nrdp1 in mammary tumors.

The ErbB2 and ErbB3 receptor tyrosine kinases act synergistically to promote cellular properties associated with tumor development. Previous studies indicate that endogenous ErbB3 protein is markedly elevated in mouse mammary tumors induced by transgenic ErbB2 overexpression. However, this occurs in the absence of elevated ErbB3 transcript, indicating that post-transcriptional regulatory mechanisms play crucial roles in suppressing ErbB3 protein in normal tissue. Our previous studies also demonstrate that protein levels of Nrdp1, an E3 ubiquitin ligase that targets ErbB3 for degradation, are markedly suppressed in tumors from ErbB2 transgenic animals relative to normal tissue. Here we demonstrate that transgenic expression of Nrdp1 cDNA in the mouse mammary gland is not sufficient to suppress elevated ErbB3 levels or tumor initiation and growth in ErbB2 transgenic mice. Unexpectedly, Nrdp1 protein is absent in tumors from Nrdp1/ErbB2 bigenic mice, and real time PCR analysis indicates that Nrdp1 protein levels are suppressed post-transcriptionally. Nrdp1 protein is more resistant to proteasome-dependent degradation when exogenously expressed in cultured MCF10A nontransformed human breast epithelial cells than in breast tumor cells. These observations indicate that mammary tumors use potent post-transcriptional mechanisms to suppress Nrdp1 protein levels and that protein destabilization may play a central role in Nrdp1 loss in tumors.

The membrane mucin MUC4 is elevated in breast tumor lymph node metastases relative to matched primary tumors and confers aggressive properties to breast cancer cells.

INTRODUCTION: Previous studies indicate that overexpression of the membrane-associated mucin MUC4 is potently anti-adhesive to cultured tumor cells, and suppresses cellular apoptotic response to a variety of insults. Such observations raise the possibility that MUC4 expression could contribute to tumor progression or metastasis, but the potential involvement of MUC4 in breast cancer has not been rigorously assessed. The present study aimed to investigate the expression of the membrane mucin MUC4 in normal breast tissue, primary breast tumors and lymph node metastases, and to evaluate the role of MUC4 in promoting the malignant properties of breast tumor cells. METHODS: MUC4 expression levels in patient-matched normal and tumor breast tissue was initially examined by immunoblotting lysates of fresh frozen tissue samples with a highly specific preparation of anti-MUC4 monoclonal antibody 1G8. Immunohistochemical analysis was then carried out using tissue microarrays encompassing patient-matched normal breast tissue and primary tumors, and patient-matched lymph node metastases and primary tumors. Finally, shRNA-mediated knockdown was employed to assess the contribution of MUC4 to the cellular growth and malignancy properties of JIMT-1 breast cancer cells. RESULTS: Immunoblotting and immunohistochemistry revealed that MUC4 levels are suppressed in the majority (58%, p < 0.001) of primary tumors relative to patient-matched normal tissue. On the other hand, lymph node metastatic lesions from 37% (p < 0.05) of patients expressed higher MUC4 protein levels than patient-matched primary tumors. MUC4-positive tumor emboli were often found in lymphovascular spaces of lymph node metastatic lesions. shRNA-mediated MUC4 knockdown compromised the migration, proliferation and anoikis resistance of JIMT-1 cells, strongly suggesting that MUC4 expression actively contributes to cellular properties associated with breast tumor metastasis. CONCLUSIONS: Our observations suggest that after an initial loss of MUC4 levels during the transition of normal breast tissue to primary tumor, the re-establishment of elevated MUC4 levels confers an advantage to metastasizing breast tumor cells by promoting the acquisition of cellular properties associated with malignancy.

The membrane mucin Muc4 inhibits apoptosis induced by multiple insults via ErbB2-dependent and ErbB2-independent mechanisms.

The aberrant expression of membrane mucins such as Muc1 and Muc4 by tumor cells has been shown to engage signaling pathways that promote cellular properties associated with tumor progression. Our previous studies have shown that Muc4 interacts with and potentiates signaling by the ErbB2 (HER2) receptor tyrosine kinase through an epidermal growth factor-like domain in its extracellular region. Here, we show that expression of Muc4 in human A375 melanoma cells and MCF7 breast cancer cells confers resistance to apoptosis induced by a variety of stimuli, including chemotherapeutic agents, the absence of serum factors, and the loss of cellular adhesion. Mapping experiments revealed that the O-glycosylation and cytosolic domains of Muc4 are dispensable for its antiapoptotic activity, and are also dispensable for the potentiation of signaling by ErbB2. Knockdown of endogenous Muc4 in JIMT-1 breast cancer cells sensitizes cells to apoptotic stimuli, and this can be rescued by Muc4 forms lacking the O-glycosylation or cytosolic domains. Surprisingly, however, the molecular mechanisms underlying Muc4 antiapoptotic activity vary among cell lines. Although Muc4 in JIMT-1 cells engages ErbB2 to promote cell survival, its antiapoptotic mechanism in MCF7 and A375 cells seems to be independent of ErbB2. However, Muc4 expression in all cell lines culminates in the phosphorylation and inactivation of the proapoptotic protein Bad and the elevation of the prosurvival protein Bcl-xL. Our observations suggest that tumor cells can exploit the versatile antiapoptotic activities of Muc4 to acquire resistance to therapeutic agents, and augment cell survival after the loss of adhesion and microenvironment-derived survival factors.

Capnocytophaga cynodegmi in a rottweiler dog with severe bronchitis and foreign-body pneumonia.

Capnocytophaga cynodegmi is a zoonotic, gram-negative, capnophilic bacterium that is usually seen in people with infections associated with dog or cat bites. The first reported case of C. cynodegmi infection in a dog is described here.

Contribution of membrane mucins to tumor progression through modulation of cellular growth signaling pathways.

Mucins are large, heavily O-glycosylated proteins expressed by epithelial tissues. The canonical function of membrane mucins is to provide protection to vulnerable epithelia by forming a steric barrier against assault, and by contributing to the formation of protective extracellular mucin gels. The aberrant overexpression of mucins is thought to contribute to tumor progression by allowing tumor cells to evade immune recognition, and by aiding in the breakdown of cell-cell and cell-matrix contacts to facilitate migration and metastasis. Recent evidence suggests that we should now modify our thinking about mucin function by considering their roles in signaling pathways leading to cellular growth control. Here we review the markedly divergent mechanisms by which membrane mucins, specifically MUC1 and MUC4, influence pathways contributing to cellular proliferation and survival. The cytoplasmic domain of MUC1 serves as a scaffold for the assembly of a variety of signaling proteins, while MUC4 influences the trafficking and localization of growth factor receptors, and hence their responses to external stimuli. We also discuss how tumor cells exploit these mechanisms to promote their own growth and metastasis.

Automated robotic liquid handling/laser-based nephelometry system for high throughput measurement of kinetic aqueous solubility.

The ability to rapidly and consistently measure aqueous solubility in a preclinical environment is critical to the successful identification of promising discovery compounds. The advantage of an early solubility screen is timely attrition of compounds likely to fail due to poor absorption or low bioavailability before more costly screens are performed. However, due to the large number of compounds and limited sample amounts, thermodynamic solubility measurements are not feasible at this stage. A kinetic solubility measurement is an alternative to thermodynamic measurements at the discovery stage that provides a rank listing of solubility values with minimal sample requirements. A kinetic solubility measurement is attractive from an automation vantage because it features rapid data acquisition and is amenable to multi-well formats. We describe the use of a robotic liquid/plate handler coupled to nephelometry detection for the measurement of kinetic solubility. We highlight the liquid handling validation, serial dilution parameters, and a comparison to the previous method. Experiments to further enhance throughput, or increase confidence in the automation steps, are described and the effects of these experiments are presented. In our integrated nephelometry method, we observe rapid liquid handling with an error of less than 10%, after a series of validation studies, and a sample throughput up to 1800 compounds per week. We compare the nephelometry method with our semi-thermodynamic flow-injection analysis (FIA) method, and find a 75% bin agreement between the methods.

Determination of acid dissociation constants by capillary electrophoresis.

Capillary electrophoresis affords a simple, automated approach for the measurement of pKa values in the range 2-11 at a throughput of less than 1 h per sample per instrument. Agreement with literature values is usually within 0.20 log units with a precision better than 0.07 log units. The attractive features of capillary electrophoresis for pKa measurements are: (1) conventional instrumentation with a high level of automation are suitable for all measurements; (2) because it is a separation method samples need not be of high purity; (3) samples of low water solubility with suitable chromophores are easily handled (detection limits in the microM range); (4) sample consumption per measurement is in the microgram range; and (5) since only mobilities are measured, exact knowledge of concentrations is not needed. The general approach can be extended to pKa measurements in aqueous-organic solvent mixtures and non-aqueous solvents with suitable calibration. The widespread use of absorbance detection in capillary electrophoresis means that the sample must have a suitable chromophore for detection. The main source of controllable error is the accuracy of buffer standardization and their stability in use, and uncontrollable error, the retentive interactions of the sample with the column wall. The latter seems to be a rare problem in practice for typical operating conditions.

Chronic lymphocytic leukemia in dogs and cats: the veterinary perspective.

Chronic lymphocytic leukemia (CLL) in dogs and cats shares many similarities with its human counterpart but also has significant differences. In marked contrast to people, CLL in dogs and cats is primarily a T-lymphocyte proliferation. Cytotoxic T-cell proliferations with granular lymphocyte morphology predominate in dogs, and T helper cell proliferations seem to be most common in cats with CLL. Immunophenotyping and assessment of clonality by molecular genetic analysis are newer adjunctive tools in veterinary medicine that are useful in the characterization and diagnosis of CLL in dogs and cats. The clinical presentation, typical hematologic findings, diagnosis, course of disease, prognosis, and therapy of CLL in dogs and cats are discussed.

Estimation of octanol-water partition coefficients for neutral and weakly acidic compounds by microemulsion electrokinetic chromatography using dynamically coated capillary columns.

Microemulsion electrokinetic chromatography (MEEKC) using dynamically coated capillary columns is shown to be suitable for estimating the octanol-water partition coefficient (log P) for neutral and weakly acidic compounds at pH 3. The solvation parameter model is used to demonstrate that the retention properties of sodium dodecyl sulfate (1.4% w/v), n-butanol (8% v/v) and n-heptane (1.2% v/v) microemulsion are strongly correlated with the octanol-water partition system. For compounds of varied structure and log P values from 0.3 to 5.15, the correlation model is able to estimate log P to better than 0.25 log units. The dynamically coated columns consisting of a bilayer of poly(vinylsulfonate) adsorbed on top of polybrene provide a suitable electroosmotic flow at pH 3 without interfering in the retention properties of the microemulsion. For automated measurements the microemulsion run buffer should be replenished after 10 runs to maintain a stable cycle time and the coated columns replaced after 40-70 runs, depending on sample properties.