Proof of Concept: The Use of Whole-Slide Images (WSI) for Peer Review of Tissues on Routine Regulatory Toxicology Studies.

This Proof of Concept (POC) study was to assess whether assessment of whole slide images (WSI) of the 2 target tissues for a contemporaneous peer review can elicit concordant results to the findings generated by the Study Pathologist from the glass slides. Well-focused WSI of liver and spleen from 4 groups of mice, that had previously been diagnosed to be the target tissues by an experienced veterinary toxicologic pathologist examining glass slides, were independently reviewed by 3 veterinary pathologists with varying experience in assessment of WSIs. Diagnostic discrepancies were then reviewed by an experienced adjudicating pathologist. Assessment of microscopic findings using WSI showed concordance with the glass slides, with only slight discrepancy in severity grades noted. None of the lesions recorded by the Study pathologist were "missed" and no lesions were added by the pathologists evaluating WSIs, thus demonstrating equivalence of the WSI to glass slides for this study.

Historical control background incidence of spontaneous pituitary gland lesions of Han-Wistar and Sprague-Dawley rats and CD-1 mice used in 104-week carcinogenicity studies.

The aim of this study was to determine the range and incidences of spontaneous microscopic lesions of the pituitary gland in control Han-Wistar and Sprague-Dawley rats and CD-1 mice from 104-week carcinogenicity studies carried out between 1998 and 2010 at Charles River Edinburgh. In both strains of rats and in CD-1 mice, non-proliferative lesions of the pituitary gland were generally uncommon, excluding cysts/pseudocysts (6.42% in Han-Wistar rats, 5.85% in Sprague-Dawley rats, and 2.08% in CD-1 mice). Primary proliferative lesions were most frequently found in the pars distalis of the pituitary gland. Adenomas and carcinomas of the pars distalis were more common in Sprague-Dawley rats (49.33% and 2.85%, respectively) than in Han-Wistar rats (27.29% and 0.21%, respectively), and adenomas in both strains of rats and CD-1 mice exhibited a marked sex predisposition, with females more commonly affected.

Historical control background incidence of spontaneous thyroid and parathyroid glands lesions of rats and CD-1 mice used in 104-week carcinogenicity studies.

The incidence and range of spontaneous thyroid and parathyroid glands findings were determined in control Han-Wistar and Sprague-Dawley rats, and CD-1 mice from 104-week carcinogenicity studies carried out between 1998 and 2010 at Charles River Edinburgh. In both strains of rats and in CD-1 mice, non-proliferative lesions of the thyroid or parathyroid glands were generally uncommon apart from some findings in CD-1 mice such as ultimobranchial duct/cyst (5.72%), follicular distension/dilatation (3.84%), and cystic follicles (3.53%). In Han-Wistar rats, thyroid proliferative lesions were slightly more frequent in males than in females, but in Sprague-Dawley rats, they were of similar incidence in both sexes. The most common findings overall in Han-Wistar and Sprague-Dawley rats were C-cell hyperplasia (48.11% and 36.56%, respectively) and adenoma (10.87% and 9.52%, respectively), follicular cell hyperplasia (4.21% and 0.91%, respectively) and adenoma (4.32% and 1.36%, respectively). Secondary neoplastic lesions either in thyroid or parathyroid gland were poorly represented.

Spontaneous and Dosing Route-related Lung Lesions in Beagle Dogs from Oral Gavage and Inhalation Toxicity Studies: Differentiation from Compound-induced Lesions.

This study was conducted to characterize lung microscopic lesions in control beagle dogs from inhalation and oral gavage toxicity studies, to determine differences associated with the route of administration, and to discuss distinguishing features from compound-induced lung lesions. Samples from 138 control dogs from oral gavage studies and 124 control dogs from inhalation (vehicle control) studies were evaluated microscopically. There was no significant sex-related difference in the incidence of all lesions. Perivascular mononuclear cell infiltration, centriacinar mixed cell infiltration, bronchopneumonia, subpleural septal fibrosis, and alveolar macrophage accumulation were the most common lesions. Aspiration pneumonia was more common in dogs from gavage studies, suggesting reflux after gavage dosing or accidental administration of test formulation as possible causes. Centriacinar mixed cell infiltration was more common in dogs from inhalation studies, suggesting mild irritation by the vehicles used. Vascular lesions, which included pulmonary arteriopathy and smooth muscle mineralization, were observed in a few animals. Some of the spontaneous lesions are similar to lesions induced by test compounds. Compared to spontaneous lesions, compound-induced lesions tend to be multifocal or diffuse, follow a pattern of distribution (e.g., centriacinar, perivascular, and interstitial), show a dose response in the incidence and severity, and may show cell-specific toxicity.

Expression of Membrane Complement Regulatory Proteins Crry and CD55 in Normal Rats.

Some anticancer therapeutic antibodies are designed to act through complement-dependent cytotoxicity (CDC). It has been reported that there are many membrane complement regulatory proteins (mCRPs) that inhibit CDC. In the present study, we examined the expression of two mCRPs, the complement receptor 1-related gene/protein Y (Crry) and the decay-accelerating factor CD55, in three normal rats by immunohistochemistry. Crry and CD55 were detected widely in rat organs and tissues. Crry was found mainly in the urinary, digestive, respiratory, immunohematopoietic, circulatory and neuroendocrine systems. CD55 was found in the urinary, digestive and neuroendocrine systems. However, the two molecules were expressed in separate cells within the same organ. These results suggest that the distribution of mCRPs is related to the strict regulation of CDC activation in these organs and tissues and that the two molecules have a nonoverlapping expression pattern, a fact indicating specific roles in CDC regulation.

Anti-Thy-1 Antibody-mediated Complement-dependent Cytotoxicity is Regulated by the Distribution of Antigen, Antibody and Membrane Complement Regulatory Proteins in Rats.

Some therapeutic antibodies as anticancer agents exert their effects through the host immune system, but the factors that predict their cytotoxicity, including complement-dependent cytotoxicity (CDC), are unclear. In the present study, we attempted to elucidate some of these factors in a preclinical model. CDC-related mesangiolysis caused by administration of the anti-Thy-1.1 antibody can be studied in the rat anti-Thy-1 glomerulonephritis model, so the model was used in this study. Three animals each were sacrificed at 0.5, 1, 8, 24 and 48 hours after i.v. administration of the anti-Thy-1.1 antibody at 1mg/kg. The distribution of the Thy-1.1 antigen and 2 membrane complement regulatory proteins (mCRPs), Crry and CD55, in three non-treated animals and the distribution of the injected antibody and C3 in the model was studied by immunohistochemistry. In the mesangial cells of the kidney, both expression of the antigen and distribution of the antibody with C3 deposition were observed with weak expression of mCRPs. There was also antigen and antibody distribution in the medullary cells of the adrenal gland and in the lymphocytes of the thymus but no C3 deposition, which was thought to be related to high expression of mCRPs. The antigen was observed in several other organs and tissues without distribution of the antibody. Cell death was only observed in the mesangial cells. These results clearly demonstrate that activation of CDC is regulated by several factors, such as distribution of the target molecule, antibody distribution and the balance among the molecules of the CDC cascade and mCRPs.

Stimulation of adrenal chromaffin cell proliferation by hypercalcemia induced by intravenous infusion of calcium gluconate in rats.

Increased incidence of adrenal pheochromocytoma is frequently encountered in rat carcinogenicity studies. In some of the studies, the finding is judged to be due to a rat-specific mechanism of carcinogenesis caused by a disturbance of calcium homeostasis. However, direct evidence that the proliferation of chromaffin cells in the adrenal medulla is induced solely by hypercalcemia is not available. In this study, calcium gluconate was intravenously infused for 7 days to rat chromaffin cells by a tail cuff method, and cumulative labeling with bromodeoxyuridine (BrdU) was carried out to evaluate the proliferative activity. The serum calcium concentration was dose-dependently increased, and a high calcium concentration was stably sustained from day 2 to 7. In the adrenal medulla, BrdU-positive chromaffin cells increased in the calcium gluconate-treated animals, and the BrdU-labeling index increased in a dose-dependent manner. In addition, an increased BrdU-labeling index of chromaffin cells was shown to correlate with the serum calcium concentration. Our results demonstrate that hypercalcemia directly enhances the proliferative activity of chromaffin cells and that the proliferative activity is correlated with the serum calcium concentration.

Cell proliferative activity in the kidney of young growing rat analyzed using flash and cumulative labeling with bromodeoxyuridine.

We examined the cell proliferation activity of kidney in young growing rats using flash and cumulative labeling with bromodeoxyuridine (BrdU). Rats were subjected to the study at the age of 6 weeks, and cumulative labeling was carried out for periods of 7 to 28 days. BrdU-positive cells were observed after flash labeling and were increased by cumulative labeling. The positive epithelia were mainly distributed in the cortex and the outer stripe of the outer medulla and were scarce in the inner stripe of the outer medulla and inner medulla throughout all labeling periods. In the tubular epithelium, the majority of positive cells were found in the proximal tubule. In the proximal tubule, positive epithelia were abundant in the medullary rays and in the outer stripe of the outer medulla. In the intermediate tubule to collecting duct, positive epithelia were rare. In the renal corpuscle, positive nuclei were mainly found in the endothelial cells and the mesangial cells and were scarce in the parietal cells of the Bowman's capsule. BrdU-positive nuclei were not observed in podocytes. These results indicate that renal tubules actively grow relative to epithelial proliferation, and that the endothelial cells, the mesangial cells and the parietal cells in the renal corpuscle also proliferate at the age of 6 to 10 weeks in rats. For assessment of renal toxicity using young growing rats, not only the morphologic and physiologic features unique to the kidney but also the growing process of the kidney should be taken into account.

Hepatic lesions of a standard poodle dog with intrahepatic portosystemic shunt.

A 1-year-and-3-month-old, male standard poodle dog with intrahepatic portosystemic shunt (PSS) was autopsied. Nineteen regions of the liver were prepared for detailed examination, and the distribution of hepatic lesions caused by PSS was studied in the liver of this dog. Histopathologically, the liver revealed a variety of hepatic lesions including lipogranulomas in the hepatic parenchyma, and a ductular reaction and microvascular proliferation in portal areas. The distribution of the lesions was not significantly different among liver regions. It is concluded that, in the present case, hepatic lesions caused by PSS are independent of shunt location, and are distributed equally in the liver.

Relation between lipogranuloma formation and fibrosis, and the origin of brown pigments in lipogranuloma of the canine liver.

BACKGROUND: In a previous study we confirmed that canine hepatic lipogranuloma, defined as lesions consisting of small round cells which contain lipid vacuoles and brown pigments in their cytoplasm, was an assembly of Kupffer cells and/or macrophages, and that the cytoplasmic brown pigments in the lesions were hemosiderin and ceroid. However, the pathogenesis of the lesion remains unclear. Kupffer cells (resident macrophages) play a key role in hepatic fibrogenesis due to the production of cytokines including TGF-beta. In the present study, we have examined 52 canine liver samples (age: newborn - 14 years; 25 males and 27 females) and investigated the correlation between lipogranuloma formation and fibrosis as well as the origin of brown pigments of lipogranulomas. RESULTS: Lipogranulomas were detected histopathologically in 23 (44.2%) of the 52 liver samples. No significant correlation was found between the density of lipogranulomas and distribution of collagen type I/III in the liver. Pigmentation of lipogranulomas showed significant correlations with that on both hepatocytes and sinusoidal cells, indicating that pigments of lipogranuloma (hemosiderin and ceroid) might be derived from hepatocytes and Kupffer cells. CONCLUSION: Lipogranulomas are not a contributing factor in hepatic fibrosis, but might be a potential indicator of the accumulation of iron and lipid inside the liver.

Histopathological characteristics of hepatic lipogranulomas with portosystemic shunt in dogs.

In the canine liver with portosystemic shunts (PSS), focal lesions consisting of cells with cytoplasmic brown pigments and lipid vacuoles are often observed in the hepatic parenchyma. Termed lipogranulomas, their histopathological characteristics have been little studied. In the present study, we examined liver biopsy samples from 144 dogs (age: 3 months-16 years; 65 PSS and 79 non-PSS cases), and investigated the histopathological characteristics, incidence, and density of lipogranulomas. Lipogranulomas were detected histopathologically in 55.4% of PSS dogs. The lesions were then grouped into 3 types according to the amount of cytoplasmic lipid vacuoles and brown pigments. The pigments were positive for Berlin blue, PAS, and Sudan black B, but negative with the Hall method. The majority of the cells were immunohistochemically positive for macrophage scavenger receptor, class A (MSR-A), while no cells were positive for hepatocyte-antigen and albumin. The cytoplasmic pigments were recognized as electron-dense microgranular materials by electron microscopy. The incidence of lipogranulomas was significantly higher in the PSS group than non-PSS group when dogs less than 1 year old were excluded. The lipogranuloma density in the liver was significantly higher in the PSS group. It is concluded that lipogranulomas are frequently observed in liver biopsies of canine PSS especially in dogs more than 1 year old. The lesions consisted of Kupffer cells and/or macrophages, and the cytoplasmic brown pigments are ceroid and hemosiderin. The pathogenesis of lipogranuloma in PSS needs to be clarified.

Histopathological characteristics of Kupffer cells and ito cells in the porcine and bovine liver.

We previously reported that no Kupffer cells reacted with the antibody against lysozyme, and Ito cells contained a large cytoplasmic vacuole in the feline liver. In this report, we further examined the characteristics of porcine and bovine hepatic non-parenchymal cells. In the liver of both animals, Kupffer cells were positive for lysozyme, and cytoplasmic vacuoles in Ito cells were small. The histopathological characteristics of porcine and bovine hepatic non-parenchymal cells were different from those of the feline liver.

Effects of olmesartan, an AT1 receptor antagonist, on hypoxia-induced activation of ERK1/2 and pro-inflammatory signals in the mouse lung.

The present study aimed to investigate the effects of olmesartan, an antagonist for angiotensin II receptor type 1(AT1), on the activation of extracellular signal-regulated kinases (ERK)1/2, tissue remodeling, and pro-inflammatory signals in the right ventricle and lung of mice during the early phase of hypobaric hypoxia. Phosphorylation of ERK1/2 in both tissue types in response to hypoxia peaked at 1-3 days, and declined rapidly in the right ventricle, whereas in the lung it was sustained for at least 8 days. Upregulation of angiotensinogen mRNA was observed in the hypoxic lung at 4-9 days, but not in the hypoxic right ventricle and pulmonary artery. Olmesartan inhibited the hypoxia-induced phosphorylation of ERK1/2 in the lung, but not in the right ventricle. Neither right ventricular hypertrophy nor the thickening of the intrapulmonary arterial wall was ameliorated by olmesartan. However, this drug inhibited the expression of the mRNA for angiotensinogen and several pro-inflammatory factors, including interleukin-6 and inducible nitric oxide synthase in the hypoxic lung. These results suggest that olmesartan blocks a potential positive feedback loop of the angiotensin II-AT1 receptor system, which may lead to attenuate pro-inflammatory signals in the mouse lung, that are associated with hypoxic pulmonary hypertension, without inducing any appreciable effects on the compensatory cardiopulmonary hypertrophy at an early phase of exposure to a hypobaric hypoxic environment.