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.

Allograft Inflammatory Factor 1 as an Immunohistochemical Marker for Macrophages in Multiple Tissues and Laboratory Animal Species.

Allograft inflammatory factor 1 (AIF1) is a commonly used marker for microglia in the brains of humans and some animal models but has had limited applications elsewhere. We sought to determine whether AIF1 can be used as a macrophage marker across common laboratory animal species and tissues. We studied tissues (that is, spleen, liver, and lung) with defined macrophage populations by using an AIF1 immunostaining technique previously validated in human tissue. Tissues were collected from various mouse strains (n = 20), rat strains (n = 15), pigs (n = 4), ferrets (n = 4), and humans (n = 4, lung only). All samples of liver had scattered immunostaining in interstitial cells, consistent with resident tissue macrophages (Kupffer cells). Spleen samples had cellular immunostaining of macrophages in both the red and white pulp compartments, but the red pulp had more immunostained cellular aggregates and, in some species, increased immunostaining intensity compared with white pulp. In lung, alveolar macrophages had weak to moderate staining, whereas interstitial and perivascular macrophages demonstrated moderate to robust staining. Incidental lesions and tissue changes were detected in some sections, including a tumor, inducible bronchus-associated lymphoid tissue, and inflammatory lesions that demonstrated AIF1 immunostaining of macrophages. Finally, we compared AIF1 immunostaining of alveolar macrophages between a hypertensive rat model (SHR strain) and a normotensive model (WKY strain). SHR lungs had altered intensity and distribution of immunostaining in activated macrophages compared with macrophages of WKY lungs. Overall, AIF1 immunostaining demonstrated reproducible macrophage staining across multiple species and tissue types. Given the increasing breadth of model species used to study human disease, the use of cross-species markers and techniques can reduce some of the inherent variability within translational research.

Approaches to Evaluate Lung Inflammation in Translational Research.

Inflammation is a common feature in several types of lung disease and is a frequent end point to validate lung disease models, evaluate genetic or environmental impact on disease severity, or test the efficacy of new therapies. Questions relevant to a study should be defined during experimental design and techniques selected to specifically address these scientific queries. In this review, the authors focus primarily on the breadth of techniques to evaluate lung inflammation that have both clinical and preclinical applications. Stratification of approaches to assess lung inflammation can diminish weaknesses inherent to each technique, provide data validation, and increase the reproducibility of a study. Specialized techniques (eg, imaging, pathology) often require experienced personnel to collect, evaluate, and interpret the data; these experts should be active contributors to the research team through reporting of the data. Scoring of tissue lesions is a useful method to transform observational pathologic data into semiquantitative or quantitative data for statistical analysis and enhanced rigor. Each technique to evaluate lung inflammation has advantages and limitations; understanding these parameters can help identify approaches that best complement one another to increase the rigor and translational significance of data.

Essentiality of Regulator of G Protein Signaling 6 and Oxidized Ca2+/Calmodulin-Dependent Protein Kinase II in Notch Signaling and Cardiovascular Development.

BACKGROUND: Congenital heart defects are the most common birth defects worldwide. Although defective Notch signaling is the major cause of mouse embryonic death from cardiovascular defects, how Notch signaling is regulated during embryonic vasculogenesis and heart development is poorly understood. METHODS AND RESULTS: Regulator of G protein signaling 6 (RGS6)-/-/Ca2+/calmodulin-dependent protein kinase II (CaMKII)VV double mutant mice were developed by crossing RGS6-/- mice with mice expressing an oxidation-resistant CaMKIIδ (CaMKIIVV), and the resulting embryonic defects/lethality were investigated using E7.5 to E15.5 embryos. While loss of either RGS6 or oxidized CaMKIIδ does not alter embryogenesis, their combined loss causes defective Notch signaling, severe cardiovascular defects, and embryonic lethality (≈E10.5-11.5). Embryos lacking RGS6 and expressing oxidation-resistant CaMKIIδ exhibit reduced myocardial wall thickness, abnormal trabeculation, and arterial specification defects. Double mutants show vascular remodeling defects, including reduced neurovascularization, delayed neural tube maturation, and small dorsal aortae. These striking cardiovascular defects were accompanied by placental and yolk sac defects in angiogenesis, hematopoiesis, and vascular remodeling similar to what is seen with defective Notch1 signaling. Double mutant hearts, embryos, and yolk sacs exhibit profound downregulation of Notch1, Jagged 1, and Notch downstream target genes Hey1, Hey2, and Hey1L as well as impaired Notch1 signaling in embryos/hearts. CONCLUSIONS: RGS6 and oxidized CaMKIIδ together function as novel critical upstream modulators of Notch signaling required for normal cardiovascular development and embryo survival. Their combined need indicates that they function in parallel pathways needed for Notch1 signaling in yolk sac, placenta and embryos. Thus, dysregulated embryonic RGS6 expression and oxidative activation of CaMKII may potentially contribute to congenital heart defects.

Mouse-adapted MERS coronavirus causes lethal lung disease in human DPP4 knockin mice.

The Middle East respiratory syndrome (MERS) emerged in Saudi Arabia in 2012, caused by a zoonotically transmitted coronavirus (CoV). Over 1,900 cases have been reported to date, with ∼36% fatality rate. Lack of autopsies from MERS cases has hindered understanding of MERS-CoV pathogenesis. A small animal model that develops progressive pulmonary manifestations when infected with MERS-CoV would advance the field. As mice are restricted to infection at the level of DPP4, the MERS-CoV receptor, we generated mice with humanized exons 10-12 of the mouse Dpp4 locus. Upon inoculation with MERS-CoV, human DPP4 knockin (KI) mice supported virus replication in the lungs, but developed no illness. After 30 serial passages through the lungs of KI mice, a mouse-adapted virus emerged (MERSMA) that grew in lungs to over 100 times higher titers than the starting virus. A plaque-purified MERSMA clone caused weight loss and fatal infection. Virus antigen was observed in airway epithelia, pneumocytes, and macrophages. Pathologic findings included diffuse alveolar damage with pulmonary edema and hyaline membrane formation associated with accumulation of activated inflammatory monocyte-macrophages and neutrophils in the lungs. Relative to the parental MERS-CoV, MERSMA viruses contained 13-22 mutations, including several within the spike (S) glycoprotein gene. S-protein mutations sensitized viruses to entry-activating serine proteases and conferred more rapid entry kinetics. Recombinant MERSMA bearing mutant S proteins were more virulent than the parental virus in hDPP4 KI mice. The hDPP4 KI mouse and the MERSMA provide tools to investigate disease causes and develop new therapies.

Use of terbinafine in the treatment protocol of intestinal Cryptococcus neoformans in a dog.

A 2.5 yr old sexually intact male vizsla was admitted to the Iowa State University Veterinary Teaching Hospital for persistent diarrhea, weight loss, and panhypoproteinemia. Examination revealed an emaciated condition and melena. Two masses were palpated in the cranial abdomen. Hematology and serum biochemistry exhibited a regenerative anemia and confirmed the presence of panhypoproteinemia, suggestive of a protein-losing eneteropathy. Distinct areas of thickened intestinal wall and enlarged mesenteric lymph nodes were found on abdominal ultrasound. Cytology from those nodes showed the presence of suspected Cryptococcus spp., and infection was confirmed utilizing a cryptococcal antigen titer. Medical therapy with lipid-complexed amphotericin B and fluconazole was unsuccessful. Two surgical procedures were performed to remove the affected areas of intestine and lymph nodes, but the disease persisted as evidenced by a persistently elevated cryptococcal antigen titer. Terbinafine was prescribed, which resulted in complete resolution of clinical signs and a steadily decreasing cryptococcal antigen titer. Very few cases of intestinal cryptococcosis have been reported. In this case, infection resulted in a protein-losing enteropathy. In addition, this article describes the use of terbinafine in the treatment of intestinal cryptococcal infection in the dog, which has not been previously reported.

Idiopathic eosinophilic meningoencephalomyelitis in a Rottweiler dog.

A 6-month-old, female, intact Rottweiler dog was presented to the Iowa State University Veterinary Teaching Hospital for a progressive history of abnormal behavior and generalized ataxia. At necropsy, there was eosinophilic infiltration of the brain and spinal cord, most severe in the medulla oblongata, cerebellum, and cervical spinal cord. Infiltrates of eosinophils were also present in the liver and small intestines. The dog was diagnosed with idiopathic eosinophilic meningoencephalomyelitis based on cerebrospinal fluid analysis, histopathology, and special stains to exclude etiologic agents.