Keratoacanthoma Pathobiology in Mouse Models

Recently we described skin tumors driven by skin-specific expression of Zmiz1 and here we define keratoacanthoma pathobiology in this mouse model. Similar to human keratoacanthoma development, we were able to segregate murine keratoacanthomas into three developmental phases: growth, maturation, and regression. These tumors had areas with cellular atypia, high mitotic rate, and minor local invasion in the growth phase, but with development they transitioned to maturation and regression phases with evidence of resolution. The early aggressive appearance could easily be misdiagnosed as a malignant change if the natural pathobiology was not well-defined in the model. To corroborate these findings in the Zmiz1 model, we examined squamous skin tumors from another tumor study in aging mice, and these tumors followed a similar biological progression. Lastly, we were able to evaluate the utility of the model to assess immune cell infiltration (F4/80, B220 Granzyme B, CD3 cells, arginase-1) in the regression phase; however, because inflammation was present at all phases of development, a more comprehensive approach will be needed in future investigations. Our study of keratoacanthomas in selected murine models suggests that these squamous tumors can appear histologically aggressive during early development, but with time will enter a regression phase indicating a benign biology. Importantly, studies of squamous skin tumor models should be cautious in tumor diagnosis as the early growth distinction between malignant versus benign based solely on histopathology may not be easily discerned without longitudinal studies to confirm the tumor pathobiology

A method for histopathological study of the multifocal nature of spinal cord lesions in murine experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a well-established mouse model for multiple sclerosis and is characterized by infiltration of mononuclear cells and demyelination within the central nervous system along with the clinical symptoms of paralysis. EAE is a multifocal and random disease, which sometimes makes histopathologic analysis of lesions difficult as it may not be possible to predict where lesions will occur, especially when evaluating cross sections of spinal cord. Consequently, lesions may be easily missed due to limited sampling in traditional approaches. To evaluate the entire length of the spinal cord while maintaining anatomic integrity, we have developed a method to section the cord within the decalcified spinal column, which allows for the study of the multifocal nature of this disease and also minimizes handling artifact. HE and Luxol fast blue staining of these spinal cord sections revealed a paucity of lesions in some areas, while others showed marked inflammation and demyelination. The percentage of spinal cord affected by EAE was evaluated at four separate areas of longitudinally sectioned cord and it varied greatly within each animal. Immunohistochemical staining of in situ spinal cords which had undergone decalcification was successful for key immuno-markers used in EAE research including CD3 for T cells, B220 for B cells and F4/80 for murine macrophages. This method will allow investigators to look at the entire spinal cord on a single slide and evaluate the spinal cord with and without classic EAE lesions

Immunohistochemical detection of MUC5AC and MUC5B mucins in ferrets

Abstract Objective Cystic fibrosis (CF) is a genetic condition that causes abnormal mucus secretions in affected organs. MUC5AC and MUC5B are gel-forming mucins and frequent targets for investigations in CF tissues. Our objective was to qualify MUC5AC and MUC5B immunohistochemical techniques to provide a useful tool to identify, localize and interpret mucin expression in ferret tissues. Results MUC5AC and MUC5B mucins were detected most commonly in large airways and least in small airways, consistent with reported goblet cell density in airway surface epithelia. We evaluated whether staining method affected the detection of goblet cell mucins in serial sections of bronchial surface epithelia. Significant differences between stains were not observed suggesting common co-expression MUC5AC and MUC5B proteins in goblet cells of airway surface epithelia. Gallbladder and stomach tissues are reported to have differential mucin enrichment, so we tested these tissues in wildtype ferrets. Stomach tissues were enriched in MUC5AC and gallbladder tissues enriched in MUC5B, mucin enrichment similar to human tissues. Mucin immunostaining techniques were further qualified for specificity using lung tissue from recently generated MUC5AC −/− and MUC5B −/− ferrets. Qualified techniques for MUC5AC and MUC5B immunohistochemistry will be useful tools for mucin tissue studies in CF and other ferret models

Glycogen depletion can increase the specificity of mucin detection in airway tissues

Objective: Mucin is an important parameter for detection and assessment in studies of airway disease including asthma and cystic fibrosis. Histochemical techniques are often used to evaluate mucin in tissues sections. Periodic acid Schiff (PAS) is a common technique to detect neutral mucins in tissue, but this technique also detects other tissue components including cellular glycogen. We tested whether depletion of glycogen, a common cellular constituent, could impact the detection of mucin in the surface epithelium of the trachea. Results: Normal tissues stained by PAS had significantly more staining than serial sections of glycogen-depleted tissue with PAS staining (i.e. dPAS technique) based on both quantitative analysis and semiquantitative scores. Most of the excess stain by the PAS technique was detected in ciliated cells adjacent to goblet cells. We also compared normal tissues using the Alcian blue technique, which does not have reported glycogen staining, with the dPAS technique. These groups had similar amounts of staining consistent with a high degree of mucin specificity. Our results suggest that when using PAS techniques to stain airways, the dPAS approach is preferred as it enhances the specificity for airway mucin.This article is published as Meyerholz, David K., Amanda P. Beck, J. Adam Goeken, Mariah R. Leidinger, Georgina K. Ofori-Amanfo, Hannah C. Brown, Thomas R. Businga, David A. Stoltz, Leah R. Reznikov, and Heather A. Flaherty. "Glycogen depletion can increase the specificity of mucin detection in airway tissues." BMC Research Notes 11 (2018): 1-5. DOI: 10.1186/s13104-018-3855-y. Copyright 2018 The Author(s). Attribution 4.0 International (CC BY 4.0). Posted with permission

Glycogen depletion can increase the specificity of mucin detection in airway tissues

Abstract Objective Mucin is an important parameter for detection and assessment in studies of airway disease including asthma and cystic fibrosis. Histochemical techniques are often used to evaluate mucin in tissues sections. Periodic acid Schiff (PAS) is a common technique to detect neutral mucins in tissue, but this technique also detects other tissue components including cellular glycogen. We tested whether depletion of glycogen, a common cellular constituent, could impact the detection of mucin in the surface epithelium of the trachea. Results Normal tissues stained by PAS had significantly more staining than serial sections of glycogen-depleted tissue with PAS staining (i.e. dPAS technique) based on both quantitative analysis and semiquantitative scores. Most of the excess stain by the PAS technique was detected in ciliated cells adjacent to goblet cells. We also compared normal tissues using the Alcian blue technique, which does not have reported glycogen staining, with the dPAS technique. These groups had similar amounts of staining consistent with a high degree of mucin specificity. Our results suggest that when using PAS techniques to stain airways, the dPAS approach is preferred as it enhances the specificity for airway mucin

NIAM-Deficient Mice Are Predisposed to the Development of Proliferative Lesions including B-Cell Lymphomas

<div><p>Nuclear Interactor of ARF and Mdm2 (NIAM, gene designation <i>Tbrg1</i>) is a largely unstudied inhibitor of cell proliferation that helps maintain chromosomal stability. It is a novel activator of the ARF-Mdm2-Tip60-p53 tumor suppressor pathway as well as other undefined pathways important for genome maintenance. To examine its predicted role as a tumor suppressor, we generated <i>NIAM</i> mutant (<i>NIAM^m/m</i>) mice homozygous for a β-galactosidase expressing gene-trap cassette in the endogenous gene. The mutant mice expressed significantly lower levels of NIAM protein in tissues compared to wild-type animals. Fifty percent of aged <i>NIAM</i> deficient mice (14 to 21 months) developed proliferative lesions, including a uterine hemangioma, pulmonary papillary adenoma, and a Harderian gland adenoma. No age-matched wild-type or <i>NIAM^+/m</i> heterozygous animals developed lesions. In the spleen, <i>NIAM^m/m</i> mice had prominent white pulp expansion which correlated with enhanced increased reactive lymphoid hyperplasia and evidence of systemic inflammation. Notably, 17% of <i>NIAM</i> mutant mice had splenic white pulp features indicating early B-cell lymphoma. This correlated with selective expansion of marginal zone B cells in the spleens of younger, tumor-free <i>NIAM</i>-deficient mice. Unexpectedly, basal p53 expression and activity was largely unaffected by NIAM loss in isolated splenic B cells. In sum, <i>NIAM</i> down-regulation <i>in vivo</i> results in a significant predisposition to developing benign tumors or early stage cancers. These mice represent an outstanding platform for dissecting NIAM's role in tumorigenesis and various anti-cancer pathways, including p53 signaling.</p></div

Reduced <i>NIAM</i> mRNA levels in cancer tissues.

<p>*Microarray analyses identified through ONCOMINE showing a statistically significant decrease in <i>NIAM</i> mRNA in tumors. (p<0.05).</p><p>Reduced <i>NIAM</i> mRNA levels in cancer tissues.</p

<i>NIAM^m/m</i> mice are predisposed to proliferative lesions.

<p>Histopathologic analyses of multiple mouse tissues. <b>A</b>. A hemangioma (asterisk, right panel) found in the uterus of a <i>NIAM^m/m</i> mouse compared to normal uterine tissue (left panel). (H&E, 100x) <b>B</b>. Pulmonary papillary adenoma in a homozygous <i>NIAM^m/m</i> mouse (arrow, right panel). A depiction of normal lung tissue from a control mouse is shown (left panel). (H&E, 20x) <b>C</b>. Harderian gland adenoma of a <i>NIAM^m/m</i> mouse (asterisks, right panel). A representative image of a normal Harderian gland (asterisk) from a control mouse is depicted (left panel). (H&E, 40x) <b>D</b>. A representative image of a normal liver from a control mouse (left panel) versus a focus of cellular alteration (arrows) in a <i>NIAM^m/m</i> mouse (right panel). (H&E, 40x) Controls represent tissues from similarly aged wild-type (panels B and C) and <i>NIAM^+/m</i> heterozygous (panels A and D) mice, which were found to be indistinguishable in our analyses.</p