Proliferative changes in the adrenal medulla of aged Chinese native pigs.

Four aged retired Chinese native pigs, three females and one male, estimated as over 10-year-old, were subjected to autopsy because of infertility due to aging. Grossly, nodular lesions were found bilaterally in the adrenal medulla of all four pigs. Based on the gross and the histopathological findings, they were diagnosed as either medullary nodular hyperplasia or pheochromocytoma. Immunohistochemically, proliferating cells of all these lesions were immuno-positive for chromogranin-A, indicating adrenal medulla-derived. Ultrastructurally, cytoplasmic neurosecretory granules suggestive of secretion were observed in these proliferating cells. There have been only limited numbers of reports on adrenal medullar proliferative changes including pheochromocytoma in pigs. The present cases will provide a valuable information for the characterization of similar changes in animals and human.

Experimental induction of necrotic enteritis in chickens by a netB-positive Japanese isolate of Clostridium perfringens.

Necrotic enteritis (NE) is one of the most important bacterial diseases in terms of economic losses. Clostridium perfringens necrotic enteritis toxin B, NetB, was recently proposed as a new key virulent factor for the development of NE. The goal of this work was to develop a necrotic enteritis model in chickens by using a Japanese isolate of C. perfringens. The Japanese isolate has been found to contain netB gene, which had the same nucleotide and deduced amino acid sequences as those of prototype gene characterized in Australian strain EHE-NE18, and also expressed in vitro a 33-kDa protein identified as NetB toxin by nano-scale liquid chromatographic tandem mass spectrometry. In the challenge experiment, broiler chickens fed a commercial chicken starter diet for 14 days post-hatch were changed to a high protein feed mixed 50:50 with fishmeal for 6 days. At day 21 of age, feed was withheld for 24 hr, and each chicken was orally challenged twice daily with 2 ml each of C. perfringens culture (109 to 1010 CFU) on 5 consecutive days. The gross necrotic lesions were observed in 90 and 12.5% of challenged and control chickens, respectively. To our knowledge, this is the first study that demonstrated that a netB-positive Japanese isolate of C. perfringens is able to induce the clinical signs and lesions characteristic of NE in the experimental model, which may be useful for evaluating the pathogenicity of field isolates, the efficacy of a vaccine or a specific drug against NE.

Tumor-Free Transplantation of Patient-Derived Induced Pluripotent Stem Cell Progeny for Customized Islet Regeneration.

UNLABELLED: Human induced pluripotent stem cells (iPSCs) and derived progeny provide invaluable regenerative platforms, yet their clinical translation has been compromised by their biosafety concern. Here, we assessed the safety of transplanting patient-derived iPSC-generated pancreatic endoderm/progenitor cells. Transplantation of progenitors from iPSCs reprogrammed by lentiviral vectors (LV-iPSCs) led to the formation of invasive teratocarcinoma-like tumors in more than 90% of immunodeficient mice. Moreover, removal of primary tumors from LV-iPSC progeny-transplanted hosts generated secondary and metastatic tumors. Combined transgene-free (TGF) reprogramming and elimination of residual pluripotent cells by enzymatic dissociation ensured tumor-free transplantation, ultimately enabling regeneration of type 1 diabetes-specific human islet structures in vivo. The incidence of tumor formation in TGF-iPSCs was titratable, depending on the oncogenic load, with reintegration of the cMYC expressing vector abolishing tumor-free transplantation. Thus, transgene-free cMYC-independent reprogramming and elimination of residual pluripotent cells are mandatory steps in achieving transplantation of iPSC progeny for customized and safe islet regeneration in vivo. SIGNIFICANCE: Pluripotent stem cell therapy for diabetes relies on the safety as well as the quality of derived insulin-producing cells. Data from this study highlight prominent tumorigenic risks of induced pluripotent stem cell (iPSC) products, especially when reprogrammed with integrating vectors. Two major underlying mechanisms in iPSC tumorigenicity are residual pluripotent cells and cMYC overload by vector integration. This study also demonstrated that combined transgene-free reprogramming and enzymatic dissociation allows teratoma-free transplantation of iPSC progeny in the mouse model in testing the tumorigenicity of iPSC products. Further safety assessment and improvement in iPSC specification into a mature ß cell phenotype would lead to safe islet replacement therapy for diabetes.

Mechanisms of Mycotoxin-induced Dermal Toxicity and Tumorigenesis Through Oxidative Stress-related Pathways.

Among the many mycotoxins, T-2 toxin, citrinin (CTN), patulin (PAT), aflatoxin B1 (AFB1) and ochratoxin A (OTA) are known to have the potential to induce dermal toxicity and/or tumorigenesis in rodent models. T-2 toxin, CTN, PAT and OTA induce apoptosis in mouse or rat skin. PAT, AFB1 and OTA have tumor initiating properties, and OTA is also a tumor promoter in mouse skin. This paper reviews the molecular mechanisms of dermal toxicity and tumorigenesis induced in rodent models by these mycotoxins especially from the viewpoint of oxidative stress-mediated pathways.

A case of congenital hyperostosis in a newborn piglet.

One female newborn piglet showed prominent thickening of both forelimbs and died soon after birth. Histopathologically, thin and woven trabeculae of bone was extending out from the edge of cortical bone in the affected forelimbs, and diagnosed as congenital hyperostosis. The extent of radially proliferated trabeculae was most prominent in radioulna. Many round- to spindle-shaped cells were observed in periosteum, which were considered to be osteoblasts. Around the periosteum, the mesenchymal proliferation was extensive with abundant mucus, and cartilaginous metaplastic changes were observed mainly around the radioulna and humerus. Dilatation of vessels with fibrin deposition in vessel walls was often observed, which were considered to reflect the localized circulatory disturbance.

Malignant sertoli cell tumor in a goose (Anser cygnoides domesticus).

This paper describes the pathologic features of a malignant Sertoli cell tumor found in an adult goose (Anser cygnoides domesticus). At necropsy, in addition to one large tumor mass (15 cm in diameter), multiple small tumor masses were observed over the peritoneum and mesenterium in the coelomic cavity. The large tumor mass was composed of sheets, lobules, and small islands of tumor cells, and elongated tumor cells lying perpendicular to fibrous connective tissue were characteristic. Such histopathologic characteristics were common to all the tumors. The tumor cells were immunohistochemically positive for neuron-specific enolase and S-100, and some tumor cells contained fine intracytoplasmic pigments that stained red by oil red O staining. These findings, taken together with the fact that one testis was markedly atrophied and bore no tumor cells and the other testis was not discernible, the present case was diagnosed as unilateral malignant Sertoli cell tumor arising from the unilateral testis. To our knowledge, this is the first report of Sertoli cell tumor in the goose.

A case of renal oxalosis in a 3-month-old cat raised under controlled conditions.

The kidneys of a 3-month-old female cat were examined. The cat which had been raised under controlled conditions with no history of any poisoning showed progressive weight loss with increases in blood BUN and creatinine concentrations. At necropsy, both kidneys were firm in consistency with formation of focal scars. Histopathologically, widespread deposition of crystals was observed in the renal tubules (in both dilated lumina and degenerative epithelia) accompanying mild interstitial fibrosis with lymphocyte infiltration. The crystals were colorless or basophilic on the hematoxilin and eosin-stained section and could be visualized with polarized light as doubly fractile crystals. The crystals were identified as calcium oxalate crystals by histochemical examinations using von Kossa stain and alizarin red S stain under different conditions and by ultrastructural examination. Judging from the above-mentioned findings, the present renal lesion detected in an infant cat was diagnosed as renal oxalosis which was suspected to be hereditary in nature.

Mechanisms of mycotoxin-induced neurotoxicity through oxidative stress-associated pathways.

Among many mycotoxins, T-2 toxin, macrocyclic trichothecenes, fumonisin B(1) (FB(1)) and ochratochin A (OTA) are known to have the potential to induce neurotoxicity in rodent models. T-2 toxin induces neuronal cell apoptosis in the fetal and adult brain. Macrocyclic trichothecenes bring about neuronal cell apoptosis and inflammation in the olfactory epithelium and olfactory bulb. FB(1) induces neuronal degeneration in the cerebral cortex, concurrent with disruption of de novo ceramide synthesis. OTA causes acute depletion of striatal dopamine and its metabolites, accompanying evidence of neuronal cell apoptosis in the substantia nigra, striatum and hippocampus. This paper reviews the mechanisms of neurotoxicity induced by these mycotoxins especially from the viewpoint of oxidative stress-associated pathways.

Proventricular dilatation disease associated with avian bornavirus infection in a Citron-crested Cockatoo that was born and hand-reared in Japan.

A 5-month-old female Citron-crested Cockatoo (Cacatua sulphurea citrinocristata) that was born and hand-reared in Japan died with suspected proventricular dilatation disease (PDD). Macroscopic and microscopic examinations of the bird revealed characteristic features of PDD, i.e., distention of the proventriculus and infiltration of lymphocytes and plasma cells in ganglia of various organs and in central and peripheral nerves. A linkage of this PDD case to infection with avian bornavirus (ABV) was documented by RT-PCR amplification of the virus genomes from the affected bird. Phylogenetic analysis revealed that the ABV identified in this study clustered into the genotype 2, which is one of the dominant ABV genotypes worldwide. To the best of our knowledge, this is the first report of a natural case of PDD associated with ABV infection in Japan.

Disseminated histiocytic sarcoma with excessive hemophagocytosis in a cat.

A 10-year-old Japanese domestic cat was presented with anorexia and weight loss. Severe anemia and thrombocytopenia were detected. Abdominal radiography and ultrasonography revealed the presence of multiple masses in the spleen. Cytological analyses of the masses revealed several atypical histiocytic cells and considerable hemophagocytosis. A splenectomy was performed, and the mass was diagnosed as histiocytic sarcoma on the basis of histopathological, cytochemical and immunohistochemical analyses. Further, abnormal hemophagocytosis was observed in the bone marrow. The cat was administered prednisolone and lomustine, and it survived for 107 days after admission. An autopsy revealed the presence of neoplastic histiocytic cells in the bone marrow, liver, pancreatic lymph node and glomeruli. This is the first case of histiocytic sarcoma in a cat to be reported in Japan.

Spontaneous cutaneous mast cell tumor with lymph node metastasis in a Richardson's ground squirrel (Spermophilus richardsonii).

A 4-year-old female Richardson's ground squirrel (Spermophilus richardsonii) presented with multicentric nodules arising from the skin of the middle of the tail and lumbosacral regions. Histologically, the nodules were composed of a proliferation of spindloid to pleomorphic cells that sometimes formed sheets and fascicular to storiform patterns. Diffuse infiltration of eosinophils was also noted. The results of immunohistochemistry indicated positive labeling for vimentin, mast cell tryptase, c-kit, and Ki-67. Toluidine blue stain revealed fine, metachromatic, cytoplasmic granules. The histologic diagnosis was mast cell tumor. The neoplasm recurred and metastasized to the right lumbar lymph node 1 month later.

Etoposide induces TRP53-dependent apoptosis and TRP53-independent cell cycle arrest in trophoblasts of the developing mouse placenta.

Abnormal regulation of placental apoptosis and proliferation has been implicated in placental disorders. Recently, several DNA-damaging agents were reported to induce excessive apoptosis and reduce cell proliferation in the placenta; however, the molecular pathways of these toxic effects on the placenta are unclear. The aim of the present study was to determine the involvement of TRP53, a tumor suppressor that mediates cellular responses to DNA damage, in the induction of apoptosis and cell cycle arrest in the developing placenta. For this purpose, we treated pregnant mice on Day 12 of gestation with 10 mg/kg of etoposide and 5-Gy gamma irradiation, potent inducers of DNA damage. We found an increase in the number of trophoblastic apoptoses 8 and 24 h after etoposide injection and 6 and 24 h after irradiation in the placental labyrinth zone. The number of mitoses and DNA syntheses in trophoblasts decreased after treatment. The accumulation and phosphorylation of TRP53 protein were detected 8 and 6 h after etoposide injection and irradiation, respectively. In Trp53-deficient placentas, the induction of etoposide-induced trophoblastic apoptosis is abrogated, while the reduction of proliferation occurred similarly as in wild-type placentas. CDC2A, a regulator of G2/M progression, was inactivated by phosphorylation after etoposide injection and irradiation, suggesting that the cell cycle was arrested at the G2/M border by treatment. Our study demonstrated that etoposide injection induced TRP53-dependent apoptosis and TRP53-independent cell cycle arrest in labyrinthine trophoblasts, providing insights into the molecular pathway of placental disorders.

Hypereosinophilic syndrome in two cats.

Two cats showing chronic vomiting, diarrhea and weight loss were found to have leukocytosis with marked eosinophilia. Both cats were diagnosed with hypereosinophilic syndrome by the findings of increased eosinophils and their precursors in the bone marrow, eosinophilic infiltration into multiple organs, and exclusion of other causes for eosinophilia. Although cytoreductive chemotherapy with hydroxycarbamide and prednisolone was performed, these two cats died 48 days and 91 days after the initial presentation.

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.

The effect of fasting on hepatic lipid accumulation and transcriptional regulation of lipid metabolism differs between C57BL/6J and BALB/cA mice fed a high-fat diet.

The effects of fasting on hepatic lipid metabolism in mice fed a high-fat diet (HFD) are still unclear. After fasting, the degree of hepatic lipid accumulation differs between HFD-fed C57BL/6J (B6) and BALB/cA (BALB/c) mice. It is not clear whether this difference is due to sensitivity to fasting or HFD. The aim of this study is to elucidate this difference among strains. After nine weeks of HFD feeding, both B6 and BALB/c mice showed moderate hepatic steatosis. However, after a subsequent twenty-hour fast, the hepatic lipid accumulation was markedly decreased in B6 but not in BALB/c mice. Moreover, the mRNA expression of a transcription factor, Srebp1(regulates hepatic lipid metabolism), and its target genes-malic enzyme, acetyl-CoA carboxylase, fatty acid synthase(regulate fatty acid synthesis), and glycerol-3-phosphate acyltransferase(regulates triacylglycerol synthesis)-were more markedly reduced in B6 than BALB/c mice. In conclusion, fasting may modify hepatic lipid accumulation in HFD-fed B6 and BALB/c mice differently. The difference may be partly owing to a marked downregulation of the expression of some lipid-metabolism-related genes in B6 mice. These results suggest that fasting per se has a significant effect on hepatic lipid accumulation in mouse strains. SREBP1 might play a role in this fasting effect.

Incorporation of beta-amyloid protein through the bovine ileal epithelium before and after weaning: model for orally transmitted amyloidoses.

To determine the mechanism of bovine intestinal incorporation of the pathogen, and the pathogenesis of prion protein in the early stage, cows suckling and weaning were orally given a fusion protein of Abeta-EGFP. Abeta-EGFP was incorporated through the villous columnar epithelial cells and accumulated in crypt patches in the ileum of suckling cows. The sites of the uptake and accumulation of Abeta-EGFP are very close to the peripheral nervous system; however, such uptake of Abeta-EGFP was not observed in 6-month-old post-weaning cows. The present study, therefore, suggests that the weaning period is very important for the risk of transmission.

Neural progenitor cells are protected against MPTP by MAO-B inhibitors.

Neurotoxic effects of MPTP on the nigrostriatal dopaminergic system are thought to be initiated by 1-methyl-4-phenylpyridinium (MPP+), a metabolite formed by the monoamine oxidase (MAO)-B-mediated oxidation of MPTP. We previously reported that the administration of MPTP induced apoptosis in migrating neuroblasts (neural progenitor cells, NPCs) in adult mice. To determine whether MAO-B is also involved in the neurotoxicity of MPTP to NPCs, this study looked at the effects of MAO B inhibitors, R(-)-deprenyl (deprenyl) and N-(2-aminoethyl)-4-chlorobenzamide (Ro 16-6491), both of which protect the dopaminergic system against MPTP. Few apoptotic cells were found in saline- or MAO-B inhibitor-treated animals but MPTP markedly induced apoptosis in the subventricular zone (SVZ) and rostral migratory stream (RMS) after 1 day. When mice were pretreated with deprenyl or Ro 16-6491, not only nigrostriatal dopamine levels but also NPCs were significantly protected against MPTP. In addition, MPTP-induced apoptosis was found in both juvenile (postnatal 21 days) and older (12 months old) mice, suggesting NPCs to be different from the dopamine system, which has been thought to exhibit age-dependent susceptibility to MPTP.

Intracranial meningioma with polygonal granular cell appearance in a Chihuahua.

A menigioma with polygonal granular cell proliferation in an 11-year and 8-month-old male Chihuahua is described. The tumor was observed under the dura matter of the right cerebrum. Microscopically, the tumor consisted of solid growth foci of small- or large- sized polygonal cells, with pale-stained nuclei, prominent nucleoli, and fine granular to foamy eosinophilic cytoplasm. Some of the proliferating cells contained variable amounts of cytoplasmic PAS-positive granules. Immunohistochemical analysis revealed that neoplastic cells were positive for vimentin and S-100 protein. Ultrastructurally, the neoplastic cells contained vesicular structures with a few small round-shaped bodies in the cytoplasm. We diagnosed the case as canine meningioma with granular cell appearance.

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.

Neurotoxicity of MPTP to migrating neuroblasts: studies in acute and subacute mouse models of Parkinson's disease.

The acute or subacute administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been widely used in C57BL/6 mice to develop models of Parkinson's disease (PD). The loss of dopaminergic neurons is suggested to be mediated by a mechanism of nonapoptotic cell death or by apoptosis. In recent years, the notion that the neurotoxicity of MPTP is restricted to dopaminergic neurons in the substantia nigra (SN) has been challenged. Here, we provide evidence of rapid cell death in the subventricular zone (SVZ) and rostral migratory stream (RMS) in the adult C57BL/6 mouse brain in response to acute or subacute treatment with MPTP. Significant terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) of fragmented DNA was observed at 24 h (or 1 day) after the last injection in the acute model or after the first injection in the subacute model. Ultrastructural analysis confirmed that dying cells displayed an apoptotic morphology. Using a double labeling method, we demonstrated that the phenotype of the cells undergoing apoptosis is that of migrating neuroblasts. This is further supported by evidence of a subsequent loss of migrating neuroblasts. The results raise the possibility that migrating neuroblasts in the SVZ and RMS may be more vulnerable to MPTP than nigrostriatal dopaminergic neurons in the SN, and the death of migrating neuroblasts may be a primary event in the mouse model of PD. Furthermore, our data suggests that the death and subsequent loss of migrating neuroblasts in the acute or subacute model probably lead to a decreased potential for neurogenesis to some extent.