Morphologic and molecular analysis of 39 spontaneous feline pulmonary carcinomas.

The present study was performed to determine the morphologic change and selected molecular features of spontaneous lung tumors in cats examined at the North Carolina State University Veterinary Teaching Hospital. Thirty-nine primary lung carcinomas represented 0.69% of all feline cases admitted to the hospital. Most lung tumors were observed in aged cats (P < .0001), and no sex predilection was found (P < .4241). Persian cats with pulmonary carcinoma were overrepresented in the data set, at least 4 times more frequently than other breeds. The histologic tumor types included adenocarcinoma (64.1%), bronchioloalveolar carcinoma (20.5%), and adenosquamous carcinoma (15.4%). Metastasis was observed in about 80% of 39 cases, with decreasing order of intrapulmonary metastasis, intrathoracic carcinomatosis, regional lymph nodes, and distant organs, including digits. The size of the largest tumor mass was significantly associated with metastatic potential (P < .001). Based on immunohistochemistry, more than 80% (20 of 24) of feline lung tumors were positively labeled with either surfactant protein A or thyroid transcription factor 1. Epidermal growth factor receptor mutant and p53 proteins were detected in approximately 20% (5 of 24) and 25% (6 of 24) of the feline lung tumor cases, respectively. Limited sequencing analysis of K-ras and p53 genes in 3 selected normal and neoplastic lung tissues did not reveal any alteration. Results indicate that primary lung carcinomas are rare but aggressive tumors in cats, thereby warranting further studies on molecular carcinogenesis.

Prevalence of hepatic parasites in Korean wild rats (Rattus norvegicus) and their association with pulmonary arteriolar medial hypertrophy.

C hepatica, an important zoonotic parasite, and C fasciolaris are common parasites in rodents. In rodent livers, C hepatica causes sequential morphologic changes that are designated as early, intermediate, or late phase, and C fasciolaris forms cysts surrounded by fibroplasia and granulomatous inflammation. The present study describes the prevalence of these parasites and associated liver and lung lesions in wild rats (Rattus norvegicus) living around pig farms in South Korea. Selected parenchymal organs, including liver and lung, of 89 wild rats were examined. Of 89 rats, 28 (31.5%) were infected with either C hepatica or C fasciolaris or with both parasites. Severe medial hypertrophy of small arterioles was observed in the lungs of 11 of the 28 parasite-infected rats (P < .01). The pulmonary arteriolar hypertrophy in the rats infected with C hepatica was strongly associated with early and/or intermediate phases (88.8%) of morphologic change in the livers (P < .01). As such, this report is the first to suggest a significant association between parasite-induced hepatitis and pulmonary arteriolar hypertrophy in rodents. Further studies are warranted for the use of C hepatica-infected rats as an animal model to explore the underlying mechanisms of portopulmonary hypertension in humans.

Mechanism of action of benzene toxicity: cell cycle suppression in hemopoietic progenitor cells (CFU-GM).

The aim of this study was to clarify previously reported controversial data and hypotheses concerning the effect of benzene on the cell cycle of hemopoietic stem cells. In this study, the bromodeoxyuridine UV (BUUV) suicide assay was performed in normal C57BL/6 and p53 knockout (KO) C57BL/6 mice during and after exposure to 300 ppm of benzene for 2 weeks. Our kinetic studies revealed that the cell cycle of hemopoietic myeloid progenitor cells (colony-forming unit granulocyte-macrophage [CFU-GM]), rather than being stimulated, was suppressed by exposure to benzene. The fraction of CFU-GM in S phase was significantly depressed, from 37.1% in controls to 16.3% in normal mice. BrdUrd incorporation in both groups revealed significantly different slopes for untreated and benzene-exposed normal C57BL/6 mice. p53 appeared to induce suppression of both the number and the cycling fraction of hemopoietic progenitor cells, as demonstrated by the lack of benzene-induced suppression of these parameters in p53 KO mice. The likelihood that suppression of bone marrow cellularity and cell cycling is mediated by p53 was supported by the upregulation of p21, a cyclin-dependent kinase inhibitor. Our present study revealed the mechanism of action of benzene hematotoxicity. Benzene suppresses the cell cycle by p53-mediated overexpression of p21, a cyclin-dependent kinase inhibitor, resulting not simply in suppression of hemopoiesis but rather in a dynamic change of hemopoiesis during and after benzene exposure. Thus, the controversies raised by previously reported data are resolved by our present findings of hemopoietic stem cell kinetics.

Hemopoietic cell kinetics after intraperitoneal single injection of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widely spread environmental pollutant. Homopoietic system is one of the targets of TCDD in laboratory animals including monkeys. The present study is the hemopoietic cell kinetics in mice, from the severe depression in cellularity of bone marrow and CFU-GM, to their recovery after the intraperitoneal injection of high dosage of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). The bone-marrow cellularity and CFU-GM were severely decreased to 37.8% and 48% of the control, respectively until day 1 after exposure to TCDD. They were, however, soon recovered, even overshot the control value. Subsequently, they tended to show decrease and oscillation again to and under the control value. In conclusion, our cell kinetic study has proven the oscillation in bone-marrow cellularity and CFU-GM during the recovery period, of which the observation seems to be useful to extend our understanding in the hematotoxicity of TCDD.

Differentiation processes of oval cells into hepatocytes: proposals based on morphological and phenotypical traits in carcinogen-treated hamster liver.

Hepatic stem cells participate in the recovery process of liver with severe injury or impaired hepatocyte regeneration. Oval cells (an oval-shaped liver cell population newly emerging from the portal or periportal zones following severe hepatic cellular damage) are believed to be the progeny of liver stem cells and precursor cells of both hepatocytes and bile duct cells. An attempt was made to define the differentiation processes of hepatic oval cells into mature hepatocytes in hamsters fed a choline-deficient diet and treated with diethylnitrosamine and 2-acetyl aminofluorene, on the basis of histopathological, electron microscopical, histochemical and immunohistochemical characterization of hepatic cell components. Two putative differentiation pathways of oval cells toward mature hepatocytes are proposed, namely (1) the differentiation of ductular-like oval cells via ductular/acinar-type hepatocytes, and (2) the differentiation of individual oval cells via small hepatocytes. Those proposals were strongly supported by consistent immunoreactivity of the cells for OV-6, an oval cell marker, and differential expression patterns for CK19 and PAS-positive cytoplasmic glycogen granules.

Isolation of liver oval cells from hamsters treated with diethylnitrosamine and 2-acetyl aminofluorene.

Recently, cholangiocellular carcinoma (CCC) was successfully induced in the hamster by infecting with Clonorchis sinensis following hepatocarcinogen treatment and has been proposed as a suitable model to study the pathogenesis of human CCC. In this hamster model, oval cells are suggested to be cells of origin of CCC. More direct analysis of histogenesis of CCC would become possible if large numbers of highly purified oval cells of hamster origin are obtained. In this study, we describe successful isolation of highly purified oval cells from hamsters. Oval cells were induced by diethylnitrosamine and 2-acetylaminofluorene treatment under choline deficient diet and isolated by centrifugal elutriation method. This isolated cells were highly homogenous in size (10.9+/-1.1 microm in diameter) and had a high nuclear to cytoplasmic ratio, an oval-shaped nucleus and a few cytoplasmic organelles. Immunocytochemically, 85.4+/-1.6%, 75.1+/-2.0%, 62+/-1.5% and 25.6+/-2.7% of the isolated cells were positive for cytokeratin 19, OV-6, albumin and alpha-fetoprotein, respectively, indicating that these cells had phenotypic characteristics of both hepatocytes and bile duct epithelium. The isolated cells were therefore considered to be hamster oval cells.

Differentiation of hamster liver oval cell following Clonorchis sinensis infection.

Oval cells which appear in the liver after hepatic injuries are suspected to be progenitor cells for both hepatocytes and bile duct cells. Oval cell isolated from the livers of the hamsters treated with diethylnitrosamine and 2-acetylaminofluorene and infected with Clonorchis sinensis (CS). cultured for 2 weeks and evaluated for differentiation and plasticity by electron microscopy and immunohistochemistry. In the CS-uninfected group, glycogen granules and peroxisomes were noted in the cells that were cultured for 2 weeks. Starting at 1 week postculture, immunoreactivity of the cells to cytokeratin 19 markedly decreased but that to albumin and alpha-fetoprotein gradually increased. This means that oval cells isolated from hamsters that were not infected with CS differentiated toward hepatocyte lineage. However, in the CS-infected group, cultured cells contained numerous rough endoplasmic reticulum and showed immunoreactivity that was generally in reverse to that of CS-uninfected group, meaning that cells isolated following CS infection were primed by CS and differentiated toward bile duct cell lineage. The results of this study suggested that oval cells are indeed bipolar progenitor cells for hepatocytes and bile duct cells and can differentiate toward either lineage depending upon the priming factor.

Infectivity and pathological changes in murine clonorchiasis: comparison in immunocompetent and immunodeficient mice.

The main complications of clonorchiasis are periportal inflammation, biliary hyperplasia, periductal fibrosis, and subsequently the development of biliary tumors in the liver. This study was undertaken to compare the infectivity and histopathologic changes between in immunocompetent FVB/NJ and BALB/cA strains, and immunodeficient severe combined immunodeficient (SCID) and athymic nude mice after the metacercariae of Clonorchis (C.) sinensis were infected. The experiment showed that C. sinensis was very infective in all strains studies, but the status of worm development, infectivity, recovery rate, and morphological changes of livers were very different in each strain. FVB/NJ mice showed more worm recovery than any other strain. Histopathologically the liver of FVB/NJ mice at 4 weeks postinfection showed marked cystic and fibrotic changes, in which C. sinensis was fully developed with ovum production, severe infiltration of inflammatory cells, mostly eosinophils, and high degrees of biliary hyperplasia. In SCID and nude mice, there were few foci of inflammatory cells even at 8 weeks postinfection in periportal areas of the liver, associated with no development into adult worm with ovum production. Fibrosis occurring at 4 weeks postinfection was highly correlated with inflammatory infiltration when each strain was compared. We suggest that massive infiltration of eosinophil and plasma cells caused by the infection might initiate cystic formation and fibrosis. These data demonstrate that the infection of C. sinensis might be related to pathologic consequences of inflammatory cell infiltration, cystic formation and fibrosis which might play a role in the defense mechanism against the parasitism in the liver of each strain. The FVB/NJ mouse model might be very helpful in elucidating the mechanism for human clonorchiasis.

Mammary gland adenocarcinoma in a mandrill (Mandrillus sphinix).

A 22-year-old female mandrill (Mandrillus sphinix) with continuously growing mass at the right mammary gland area was found dead, and a postmortem examination was performed. At necropsy, an elevated firm subcutaneous mass about 5 cm in diameter was present at the right mammary gland area. Axillary, mediastinal, and tracheobronchial lymph nodes were enlarged 2 to 4 times their normal sizes. Numerous metastatic foci 2 to 5 mm in diameter were scattered in the lung. Histologically, the tumor was diagnosed as mammary gland adenocarcinoma. Metastasis to the regional lymph nodes and lung was also confirmed. This is the first reported case of a mammary gland tumor in mandrill in Asia.

Teratological effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD): induction of cleft palate in the ddY and C57BL/6 mouse.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a highly toxic halogenated aromatic hydrocarbon, is a teratogen to induce cleft palate when exposed during the pregnancy. There are inter-strain differences in the sensitivity to cleft palate induced by TCDD and other chemicals including polychlorinated terphenyls (PCTs). The C57BL/6 mouse and the ddY mouse had been shown to be different in the induction of cleft palate following the treatment of PCTs, which attempts us to evaluate the TCDD-induced cleft palate in two mouse strains to understand the mechanism through which TCDD and PCTs induce cleft palate. This study evaluated the induction of cleft palate in the fetuses of ddY and C57BL/6 mice after subcutaneous treatment of TCDD on gestation day (GD) 10.5-14.5 or oral treatment on GD 8.5-13.5. Our results clearly showed that ddY mice, a susceptible strain to PCTs-induced cleft palate, are resistant to the induction of cleft palate by TCDD comparably to the high susceptibility of C57BL/6 mice, suggesting a different teratological mechanism between TCDD and PCTs. In addition, at the low doses, our study supported the concept of "window effect" of TCDD on around GD 12 for the induction of cleft palate in C57BL/6 and ddY mice.

Overexpression of cyclin D1 and cyclin E in 1,2-dimethylhydrazine dihydrochloride-induced rat colon carcinogenesis.

Deregulation of G1 cyclins has been reported in several human and rodent tumors including colon cancer. To investigate the expression pattern of G1 cyclins in 1,2- dimethyl-hydrazine dihydrochloride (DMH)-induced rat colon carcinogenesis, we studied the expression of cyclin D1 and cyclin E by quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis and immunohistochemistry (IHC). The mRNA level of cyclin D1 was increased 1.2-fold in adenocarcinomas but not significantly in adenomas, when compared with normal rat colonic mucosa (p<0.05). The cyclin E mRNA level was increased 2.7-fold in adenomas and 3.3-fold in adenocarcinomas (p<0.05). The PCNA mRNA level was also increased 1.9-fold in adenomas and 1.8-fold in adenocarcinomas (p<0.05). Immunohistochemical staining revealed exclusive nuclear staining of the neoplastic cells for cyclin D1, cyclin E and PCNA. Cyclin D1 expression was detected in 56.3% of the adenomas and in 61.5% of the adenocarcinomas examined, whereas cyclin E expression was detected in 87.5% of the adenomas and in 92.3% of the adenocarcinomas. Overall, cyclin D1, cyclin E and PCNA expression was significantly increased at both the mRNA and protein levels in normal colonic mucosa, adenomas and adenocarcinomas, but there was no significant difference in the degree of expression of these genes in adenomas and adenocarcinomas. Our results indicate that the overexpression of cyclin D1 and cyclin E may play an important role during the multistage process of rat colon carcinogenesis, at a relatively early stage, and may disturb cell-cycle control in benign adenomas, and thereafter, participate in tumor progression.

Lymphosarcoma in a brown bear (Ursus arctos).

An example of lymphoblastic lymphosarcoma was found in a 7-year-old male brown bear (Ursus arctos) that died after having a 7-month history of depression, anorexia and watery diarrhea. Grossly the mesenteric lymph nodes were enlarged to approximately 4 to 6 times their normal size and histologically diagnosed as lymphoblastic lymphosarcoma. The small intestinal mucosa was corrugated and had severe mural thickening due to infiltrated neoplastic cells. Hepatic metastasis was also noted. This is the first reported case of lymphosarcoma in Ursidae in Korea. As an incidental finding, endogenous lipid pneumonia was noted in the lung.

Concurrent multicentric hemangiosarcoma and ovarian teratoma in an aged Père david's deer (Elaphurus davidianus).

A 20-yr-old female Père David's deer (Elaphurus davidianus) died following a 3-wk history of depression, anorexia, and progressive respiratory distress. At necropsy, numerous soft to firm, tan or blood-filled nodular structures, 1-6 cm in diameter, were noted in the lung and the visceral and parietal pleura and within the mediastinum. Similar nodules were also found in the liver, spleen, kidney, and lymph nodes. Histologically, the nodules were diagnosed as hemangiosarcoma. A benign teratoma was present in the left ovary.

Urocanic acid-modified chitosan-mediated PTEN delivery via aerosol suppressed lung tumorigenesis in K-ras(LA1) mice.

The low efficiency of conventional therapies in achieving long-term survival of lung cancer patients calls for development of novel options. Revisiting of aerosol gene delivery may provide an alternative for safe and effective treatment for lung cancer. In this study, imidazole ring-containing urocanic acid-modified chitosan (UAC) designed in the previous study was used as a gene carrier. The potential effects of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) on Akt-related signals and cell cycle regulation were evaluated. Aerosols of UAC-PTEN were delivered into K-ras(LA1) lung cancer model mice through the nose-only inhalation system twice a week for total 4 weeks. Delivered PTEN suppressed lung tumor development significantly through nuclear complex formation between PTEN and p53, suppressing Akt-related signals as well as cell cycle regulation. Together, our results suggest that aerosol delivery of UAC-PTEN may be compatible with noninvasive in vivo gene therapy.

Round cell variant of myxoid liposarcoma in a Japanese Macaque (Macaca fuscata).

A 5-year-old, female, Japanese Macaque (Macaca fuscata) was diagnosed with round cell variant of myxoid liposarcoma. At necropsy, multifocal to coalescing, reddish tan to white nodules, ranging from 0.5 to 1 cm in diameter, were noted throughout the omentum and retroperitoneum. Similar neoplastic nodules were also present in diaphragm, abdominal wall, and on hepatic capsule. Microscopically, neoplastic masses consisted of round to polyhedral cells, which had round, often eccentric nuclei and abundant eosinophilic granular and microvacuolated cytoplasm; Oil red O staining demonstrated large numbers of lipid droplets in the cytoplasm. Ultrastructurally, the cytoplasm of the tumor cells was packed with occasional lipid vacuoles and numerous enlarged mitochondria. Immunohistochemistry revealed tumor cells were positive for vimentin, while negative to cytokeratin, actin, and Factor VIII-related antigen. To the authors' knowledge, this is the first report of round-cell variant of myxoid liposarcoma in nonhuman primate.

Transgene expression of thioredoxin (TRX/ADF) protects against 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced hematotoxicity.

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) has a variety of toxic effects on a number of organs, including the hematopoietic system. The importance of TCDD-induced oxidative stress has been evaluated in several target organs. However, its role in hematotoxicity remains poorly understood, although bone marrow is known to produce reactive oxygen species. The aim of this study is to evaluate not only the contribution of oxidative stress to TCDD-induced hematotoxicity but also the protective function of TRX/ADF, a known anti-oxidative stress agent, on the hematotoxicity of TCDD in ADF wild-type (WT) and transgenic (Tg) mice. WT and Tg mice received a single intraperitoneal injection of 20 microg TCDD/kg. One day after the treatment, blood and bone marrow cellularity was measured and bone marrow levels of granulotyce/macrophage colony-forming units were determined in the in vitro colony assay. The expression of human TRX transgene by their bone marrow cells was analyzed by Western blot electrophoresis. Our results showed that overexpression of TRX/ADF protects against TCDD-induced hematotoxicity, indicating that induction of oxidative stress that results in disruption of redox regulation may be an important mechanism in TCDD-induced bone marrow toxicity. Moreover, we detected a significant decrease of AhR mRNA levels in bone marrow cells of Tg mice following TCDD treatment, suggesting a biological role of TRX/ADF in the AhR-mediated pathway through which TCDD induces oxidative stress.

Fatal measles virus infection in Japanese macaques (Macaca fuscata).

An outbreak of natural measles virus infection occurred in a group of Japanese macaques (Macaca fuscata). Over a period of 4 months, 12 of 53 Japanese macaques died following a 2-23-day history of anorexia, diarrhea, and dermatitis. The monkeys were kept in outdoor exhibits but had been moved temporarily into indoor caging and then transferred to new outdoor exhibits. Ten monkeys died while they were in temporary caging, and two monkeys died after they were moved to new outdoor exhibits. The diagnoses were made based on the results of histopathology, immunohistochemistry (IHC), in situ hybridization (ISH), and electron microscopy. Measles virus antigens were detected in the lung, stomach, skin, salivary gland, spleen, and lymph nodes. Tangled, tubular nucleocapsids compatible with paramyxovirus were noted in the lung tissue. As a result of immunosuppression following measles virus infection, various secondary infections including disseminated cytomegalovirus infection, adenoviral and bacterial pneumonia, and Candida albicans-associated gingivitis and esophagitis were noted. The primary infective source or the mode of infection could not be determined in this outbreak, but measles virus may have been transmitted to the monkeys from human visitors while the monkeys were on exhibit.

Expression profile of histone deacetylase 1 in gastric cancer tissues.

Although histone deacetylases (HDACs) appear to play a crucial role in carcinogenesis, the expression status of HDACs in primary human cancer tissues has not yet been reported. In this study, we investigated the expression level of HDAC1 in 25 paired primary human gastric cancer (GC) tissues and corresponding normal tissues through semi-quantitative RT-PCR and immunoblot analysis. The HDAC1 expression pattern was also topologically examined through immunohistochemistry. Overexpression of HDAC1 mRNA was detected in 68% of GC tissues (17 of 25), and the relative density of HDAC1 mRNA in GC tissue was increased 1.8-fold versus the normal counterpart (P < 0.01). Elevated expression of HDAC1 protein was also detected in 61% of GC samples (11 of 18), which also showed an increased mRNA level of HDAC. Immunohistochemically, overexpression of HDAC1 was predominantly localized in the nuclei of most neoplastic cells, including embolic tumor cells, whereas normal glandular epithelial cells revealed only weak HDAC1 expression that was focal in distribution. Thus, the present study clearly demonstrates that HDAC1 is overexpressed in GC and probably plays a significant role in gastric carcinogenesis.