Identification of a Unique Amyloid Sequence in AA Amyloidosis of a Pig Associated With Streptococcus Suis Infection.

Here we report a pig with amyloid A (AA) amyloidosis associated with Streptococcus suis infection and identification of a unique amyloid sequence in the amyloid deposits in the tissue. Tissues from the 180-day-old underdeveloped pig contained foci of necrosis and suppurative inflammation associated with S. suis infection. Congo red stain, immunohistochemistry, and electron microscopy revealed intense AA deposition in the spleen and renal glomeruli. Mass spectrometric analysis of amyloid material extracted from the spleen showed serum AA 2 (SAA2) peptide as well as a unique peptide sequence previously reported in a pig with AA amyloidosis. The common detection of the unique amyloid sequence in the current and past cases of AA amyloidosis in pigs suggests that this amyloid sequence might play a key role in the development of porcine AA amyloidosis. An in vitro fibrillation assay demonstrated that the unique AA peptide formed typically rigid, long amyloid fibrils (10 nm wide) and the N-terminus peptide of SAA2 formed zigzagged, short fibers (7 nm wide). Moreover, the SAA2 peptide formed long, rigid amyloid fibrils in the presence of sonicated amyloid fibrils formed by the unique AA peptide. These findings indicate that the N-terminus of SAA2 as well as the AA peptide mediate the development of AA amyloidosis in pigs via cross-seeding polymerization.

Clinical and pathologic features of neuronal ceroid-lipofuscinosis in a ferret (Mustela putorius furo).

Clinical and pathologic features of neuronal ceroid-lipofuscinosis in a 4-month-old ferret are reported. Clinical signs including neurological symptoms appeared at 3 months of age and progressed rapidly. By magnetic resonance imaging, severe cerebral atrophy was recognized. Histopathologically, there was severe neuronal loss and diffuse astrogliosis with macrophage accumulations; lesions were found predominantly in the cerebral cortex. Intracytoplasmic pigments were observed in surviving neurons and macrophages throughout the brain. The pigments were intensely positive for periodic acid-Schiff, Luxol fast blue, and Sudan black B and exhibited a green autofluorescence. Electron microscopic examination revealed the accumulation of electron-dense granular material within lysosomes of neurons and macrophages. Immunohistochemically, a large number of saposin-positive granules accumulated in the neuronal cells, astrocytes, and macrophages of the lesions, but significant immunoreactivity for subunit c of mitochondrial adenosine triphosphate synthase was not observed. Based on these findings, the animal was diagnosed as affected by neuronal ceroid-lipofuscinosis.

Intracellular Localization of Mycoplasma bovis in the Bronchiolar Epithelium of Experimentally Infected Calves.

Lung tissues from calves infected experimentally with Mycoplasma bovis were examined by immunohistochemistry and electron microscopy. All inoculated calves had dark red areas of consolidation affecting both left and right lungs, which were characterized microscopically by subacute purulent bronchiolitis with hyperplasia of the surrounding lymphoid tissue. Immunohistochemically, M. bovis antigen was detected on the surface and inside the cytoplasm of bronchiolar epithelial cells in the pneumonic foci. The antigen was also found in the cytoplasm of phagocytes at the margin of bronchiolar exudates. Electron microscopically, numerous organisms were demonstrated in the immunohistochemically-positive sites. These findings suggest that M. bovis organisms adhere to the bronchiolar epithelium and at least some of them invade the epithelium.

Cerebral amyloid angiopathy in an aged great spotted woodpecker (Picoides major).

A male great spotted woodpecker (Picoides major), which was at least 16 years old, died due to general weakening. Cerebral vascular walls, including capillaries, were positively stained with Congo red with green-gold birefringence, and some of which showed a severe deposition of the Congophilic materials resulting in a corona-like fibrillar radiating structure. The Congophilic materials were positive for beta amyloid protein, but negative for prion protein. Only a few senile plaque-like structures were observed in the cortex by PAM stain and beta amyloid immunostain. The present case is the first observation of cerebral amyloid angiopathy in avian species and will indicate the presence of such age-related cerebral lesions also in birds.

Prolonged oval cell proliferation with Ito cell activation and extracellular matrix accumulation in galactosamine-induced acute hepatitis in mini rats.

Histopathological and immunohistochemical studies were carried out on D-galactosamine (GalNAc)-induced acute hepatitis in rats of the JCI: Wistar TgN (ARGHGEN) 1 Nts strain (Mini rats), in which expression of the growth hormone gene is suppressed by an antisense transgene. Hepatitis characterized by hepatocellular acidophilic necrosis with inflammatory cell infiltration was most prominent at 2 days after GalNAc (1000 mg/kg)-injection, when proliferation of Ito cells and deposition of fibronectin and laminin were found along the sinusoidal linings. At 72 hours after GalNAc-injection, Ito cell proliferation with deposition of laminin and fibronectin became more prominent, and marked proliferation of small epithelial cells was observed in the periportal area. At 7 days after GalNAc-injection, quite a number of alpha-smooth muscle actin-positive Ito cells, surrounded by abundant fibronectin, laminin and type IV collagen, were still observed in close juxtaposition to rapidly proliferating small epithelial cells. The small epithelial cells were found to be positive for both alpha-fetoprotein and cytokeratin 7 and were therefore considered to be so-called oval cells. The results suggest that there may be some relation between oval cell proliferation, Ito cell activation and extracellular matrix accumulation in GalNAc-induced acute hepatitis in Mini rats.

Apoptotic cell death and cell proliferative activity in the rat fetal central nervous system from dams administered with ethylnitrosourea (ENU).

Ethylnitrosourea (ENU), a well known DNA alkylating agent, induces anomalies in the central nervous system (CNS), craniofacial tissues and male reproductive organs, and the enhancement of apoptosis is found in these tissues immediately after the administration of ENU (Katayama et al., 2000a). In this study, pregnant rats were treated with 60mg/kg of ENU at day 13 of gestation, and kinetics of apoptotic cells, mitotic cells and bromodeoxyuridine (BrdU)-positive cells in the fetal CNS were examined from 3 to 48 hours after the treatment (HAT). From 3 HAT, a significant increase in the number of apoptotic cells and a significant decrease in the number of mitotic cells were detected in the fetal CNS, and BrdU-positive cells significantly decreased in accordance with the increase in the number of apoptotic cells. The present results strongly suggest that both excess cell death by apoptosis and cell growth arrest indicated by decreased number of mitotic cells and BrdU-positive cells may have a close relation to the later occurrence of microencephaly following ENU-administration, and that ENU affects mainly S-phase cells and causes apoptosis.

Ethylnitrosourea (ENU)-induced apoptosis in the rat fetal tissues.

Ethylnitrosourea (ENU), a well known DNA alkylating agent, induces anomalies in the central nervous system (CNS), craniofacial tissues and male reproductive organs. In this study, pregnant rats were treated with 60 mg/kg ENU at day 13 of gestation, and their fetuses were examined from 1 to 48 hours after treatment (HAT) to find a clue for clarifying the mechanisms of the ENU fetotoxicity and teratogenicity. From 3 to 12 HAT, the moderate to marked increase in the number of pyknotic cells was detected in the fetal CNS, craniofacial mesenchymal tissues, gonads and so on. These pyknotic cells had nuclei positively stained by the TUNEL method, which is widely used for the detection of apoptotic nuclei, and they also showed electron microscopic characteristics identical to those of apoptotic cells. The present results strongly suggest that excess cell death by apoptosis in the fetal CNS, craniofacial tissues and gonads may have a close relation to the later occurrence of anomalies reported in these tissues following ENU-administration.

Apoptosis in the developing mouse embryos from T-2 toxin-inoculated dams.

T-2 toxin (3 mg/kg b.w.) was orally inoculated to pregnant mice at 11 days of gestation to examine the effect of T-2 toxin on the developing embryos. At 24 hours after T-2 toxin-inoculation, moderate pyknosis or karyorrhexis was generally observed in some layers of the central nervous system, caudal sclerotomic segment, caudal region of the tongue to pharyngeal- to laryngeal-mesenchyma, trachea and facial mesenchyma. These pyknotic or karyorrhectic nuclei were strongly stained by the modified TUNEL method widely used for the in situ detection of apoptotic nuclei and also showed ultrastructural changes characteristic for apoptosis. This is the first report of mycotoxin-induced apoptosis in embryos.

Hydroxyurea (HU)-induced apoptosis in the mouse fetal tissues.

Hydroxyurea (HU), a ribonucleotide reductase inhibitor, induces morphological anomalies in the central nervous system (CNS), craniofacial tissues and limb buds in animals, and neonatal respiratory distress in humans. In the present study, pregnant mice were treated with 400 mg/kg of HU at day 13 of gestation, and their fetuses were examined from 1 to 48 hours after treatment (HAT) to find a clue to clarify the mechanisms of HU-induced fetotoxicity and teratogenecity. At 6 and 12 HAT, a moderate to marked increase in the number of pyknotic cells was detected in the CNS and lung. A mild increase in the number of pyknotic cells was also found in the craniofacial mesenchymal tissues, limb buds and so on. These pyknotic cells had nuclei positively stained by the TUNEL method, which is widely used for the detection of apoptotic nuclei, and they also showed electron microscopic characteristics identical to those of apoptotic cells. The present results suggest that the HU-induced fetotoxicity is characterized by excess apoptotic cell death in the fetal tissues, and that such excess cell death in the fetal CNS, lung, craniofacial tissue and limb bud may have a certain relation to the later occurrence of morphological or functional anomalies reported in these tissues following HU-administration.

Sunburn reaction in the dorsal skin of hypotrichotic WBN/ILA-Ht rats.

The dorsal skin responses to a single irradiation with a high-dose of UVB (10kJ/m2) were examined histologically and immunohistochemically in UVB-sensitive Wistar-derived hypotrichotic WBN/ILA-Ht rats (HtRs). Sunburn cells (SBCs) which were characterized by pyknotic nuclei and eosinophilic cytoplasm and had ultrastructual characteristics of apoptotic cells were first observed in the epidermis at 3 hours (h) after irradiation. The number peaked at 6 h, and then decreased rapidly. The expressions of p53 protein, which is known to be closely related to the formation of SBCs, and of p21 protein, which is one of the transcriptional target genes of p53, were immunohistochemically detected, and their labeling index (LI) in the epidermis peaked at 12 to 24 h (p53) or at 24h (p21) after irradiation. On the other hand, proliferating cell nuclear antigen (PCNA)-LI in keratinocytes was significantly lower than the control group at 6 h after irradiation and thereafter it increased and became significantly higher than the control group from 24 to 48 h. At 48 h, moderate hyperplasia with moderate numbers of mitotic keratinocytes was first observed in the epidermis. In the dermis, mild edema developed from 12 to 36 h and it accompanied mild lymphocyte infiltration at 36 h. Judging from the present results, it was suggested that some factors other than p53 might be involved in SBC formation, and that p53 might induce p21 protein and play an important role in cell growth arrest in keratinocytes after UVB irradiation.

Hepatitogenicity of three plaque purified variants of hepatotropic mouse hepatitis virus, MHV-2 in athymic nude mice.

Hepatitogenicity of 3 plaque purified variants of hepatotropic mouse hepatitis virus, MHV-2 were examined in athymic BALB/c-nu/nu mice up to 9 weeks post infection (9WPI). All of the MHV-2S- and MHV-2M-infected mice died with severe acute hepatitis in 3WPI. On the other hand, MHV-2L-infected mice did not die until 9WPI and showed signs of slow-developing chronic hepatitis with persistent infection under low serum virus neutralizing antibody titers. This suggests that MHV-2L-infected athymic nude mice may be useful as a new model of chronic viral hepatitis.

Protective effect of recombinant interferon (IFN)-alpha/beta on MHV-2cc-induced chronic hepatitis in athymic nude mice.

The protective effects of recombinant IFN-alpha/beta on MHV-2cc-induced chronic and persistent hepatitis in athymic nude mice were examined. The mice intraperitoneally (ip) inoculated with MHV-2cc at day 0 of experiment were divided into 4 groups. Three of them were administered ip with recombinant IFN-alpha/beta at a daily dose of 1 x 10(3) IU from -1 (-1D-group), 0 (0D-group), and +1 day of experiment (+1D-group), respectively, for 3 consecutive weeks. The remaining one (control group) was given 0.1 ml/mouse of PBS from +1 day of the experiment in the same way. Three mice in each group were killed at 1, 2 and 3 weeks post inoculation (WPI) with MHV, respectively. The liver virus titer in the control group increased gradually and maintained high levels throughout the experimental period. In the IFN-groups, particularly in the -1D- and 0D-groups, the virus titers were significantly lower than that in control group. Histopathologically, focal hepatic lesions were observed at 1WPI and large irregular inflammatory lesions developed at 3WPI in the control group. Similar but somewhat less severe lesions were observed in the +1D-group. In the -1D- and 0D-groups, lesions were not observed at 1WPI and only small organized lesions with mononuclear cell infiltration were seen at 3WPI. In conclusion, it was clarified in the present study that the progression of MHV-2cc-induced chronic hepatitis in athymic nude mice was effectively prevented by extrinsic IFN-alpha/beta when administered from -1 day and 0 day of the virus infection.

Immunohistochemical study on galactosamine-induced subacute hepatitis in rats of JCL: Wistar-TGN (ARGHGEN) 1 Nts strain (Mini rats).

Immunohistochemical study was carried out on D-galactosamine hydrochloride (GaIN)-induced subacute hepatitis in rats of JCL: Wistar-TGN (ARGHGEN) 1Nts strain (Mini rats), in which the expression of growth hormone gene is suppressed by the presence of an antisense transgene. Mini rats were given 1000 mg/kg of GaIN once a week for 4 consecutive weeks and killed at 1, 2, 3 and 4 weeks after the first administration. At 1 week after the first administration, proliferation of small epithelial cells positive for both alpha-fetoprotein and cytokeratin 7, i.e. so-called oval cells, was observed in the whole area of each hepatic lobule, and prominent deposition of fibronectin, laminin and type IV collagen was detected around these oval cells. Together with these extracellular matrix components, many activated Ito cells positive for both desmin and alpha-smooth muscle actin were observed. With time, most of the oval cells formed duct-like structures and lost their positive stainability for alpha-fetoprotein, and many Ito cells became inactive. Deposition of fibronectin decreased rapidly from 2 weeks after the first administration. At 4 weeks after the first administration, deposition of laminin was detected only around the duct-like structures, where that of type IV collagen was also still prominent. These results suggest that a large population of oval cells differentiated into bile duct epithelial cells and that Ito cells and extracellular matrix components might play a role in this process.

Protective effects of a novel quinone derivative, E3330, on mouse hepatitis virus (MHV)-induced chronic hepatitis in athymic nude mice.

In this experiment, we examined the protective effects of a novel quinone derivative, E3330, on MHV-2cc-induced chronic hepatitis in athymic nude mice for up to 3 weeks after virus infection. The daily dose of 25 mg/kg b.w. suppressed the viral replication in the liver and the progression of hepatic lesions. The expansion of small focal lesions at 1 week after viral inoculation (WAI) was suppressed at 2 WAI, and the lesions were still small at 3 WAI in E3330-administered group, whereas small focal lesions at 1 WAI were expanded at 2 WAI to fuse with each other at 3 WAI in the control group. E3330 therefore showed protective effects on MHV-2cc-induced chronic hepatitis in athymic nude mice, but further studies are needed to analyze the mechanism.

Lectin histochemistry of dorsal skin of Wistar-derived hypotrichotic WBN/Ila-Ht rats.

A lectin histochemical study was carried out on the dorsal skin of Wistar-derived hypotrichotic WBN/Ila-Ht rats (HtRs) and Wistar rats (WRs) at 3, 7 and 24 weeks of age to clarify the lectinhistochemical characteristics of the skin during their development. The lectins examined were Concanavalia ensiformis (Con A), Dolichos biflorus agglutinin (DBA), Griffonia simpliciolia (GS-I), Helix pomatia agglutinin (HPA), Arachis hypogaea (PNA), Glycine maximus agglutinin (SBA), Ulex europeus agglutinin (UEA-I) and Triticum vulgaris agglutinin (WGA). None of the nucleated cell layers of the epidermis had DBA-binding sites, but they were all stained intensely with HPA and weakly with Con A irrespective of the strain and age of the rats. As to the other 5 lectins, the intensity of binding activity was generally weaker in HtRs than in WRs and at 3 weeks of age than at 7 or 24 weeks of age, respectively. Among them, UEA-I mainly bound to the spinous cell layer but not to the basal cell layer, suggesting that alpha-L-fucose would be expressed on the cell surface according to the differentiation of keratinocytes. In addition, GS-I, HPA and UEA-I bound to the hair follicle epithelium and many lectins stained sebaceous gland epithelial cells. In conclusion, except for the binding intensity of some lectins, there were no specific differences between HtRs and Wrs in the lectinhistochemical characteristics of the dorsal skin epidermis. The present data on the rat skin would be useful from the viewpoint of comparative lectinhistochemistry.

Process of the development of T-2 toxin-induced apoptosis in the lymphoid organs of mice.

Female ICR:CD-1 mice orally treated with 10 mg/kg b.w. of T-2 toxin were killed at 1, 3, 6, 9, 12, 24 and 48 hr after treatment (HAT) and subjected to examination of the process of the development of T-2 toxin-induced apoptosis in the thymus and spleen. The early ultrastructural changes in lymphocytes characterized by shrinkage of the cell body and condensation of nuclear chromatin were detected at 3HAT in the thymus. The number of apoptotic lymphocytes observed by the in situ detection method for fragmented DNA increased drastically from 9 to 24 HAT in the thymus while it began to increase at 12 HAT in the spleen. The DNA ladder was first detected by agarose gel electrophoresis at 9 HAT and became clearer at 12 and 24 HAT in the thymus but was not clearly detected in the spleen throughout the observation period. Thus T-2 toxin-induced apoptosis developed earlier and was apparently severer in the thymus than in the spleen. Apoptotic was first detected by electron microscopy, then by the in situ detection method for fragmented DNA, and finally by DNA agarose gel electrophoresis.

Glycoconjugate expression in follicle-associated epithelium (FAE) covering the nasal-associated lymphoid tissue (NALT) in specific pathogen-free and conventional rats.

We examined lectin-histochemically the glycoconjugate expression in the follicle-associated epithelium (FAE) covering the nasal-associated lymphoid tissue (NALT) in the rat under specific pathogen-free (SPF) and conventional (CV) conditions and compared the results for SPF and CV rats as well as for membranous (M) cells and adjacent ciliated respiratory epithelial (CRE) cells in FAE. N-acetylgalactosamine-specific lectins, Dolichos biflorus (DBA), Helix pomatia (HPA), Glycine max (SBA) and Vicia villosa (VVA), and alpha-L-fucose-specific lectin, Ulex europaeus (UEA-I), preferentially bound to M cells mainly in the luminal surface compared with CRE cells in SPF rats, whereas DBA and UEA-I showed signs of preferential binding to the apical and basolateral cytoplasm as well as to the luminal surface of M cells in CV rats. In addition, HPA, SBA and VVA more frequently and extensively labeled M cells than CRE cells in CV rats with the same subcellular staining pattern as DBA and UEA-I. On the whole, the changes in lectin binding frequency and strength were more prominent in M cells than in CRE cells in both SPF and CV rats. The present results indicate that DBA and UEA-I are useful as markers of M cells in NALT. Furthermore, the pattern of expression of carbohydrate residues recognized by such lectins in SPF and CV rats suggests that M cells are highly sensitive to environmental changes.

Immunohistochemical study on type II collagen-induced arthritis in DBA/1J mice.

We performed immunohistochemical examinations on type II collagen-induced arthritis (CIA) mice, focusing attention on the changes in distribution of plasma proteins and extracellular matrix materials (ECM) and in expression of adhesion molecules. The limb joints of male DBA/1J mice immunized with bovine type II collagen were obtained at 6 to 20 weeks after the first immunization. In the early stage of CIA, deposition of fibrin, IgG, von Willebrand factor (vWF) and fibronectin was detected on the surface of the synovial lining layer and articular cartilage and in the articular cavity. In the stage of pannus formation, prominent proliferation of ICAM-1-positive capillaries and marked infiltration of LFA-1-positive neutrophils were observed in the pannus. The superficial portion of the pannus and basement membranes of proliferated capillaries were strongly positive for type IV collagen and laminin. In the late stage, the pannus invaded and destroyed articular cartilage and subchondral bone, and strongly positive immunostainabilities for both lysozyme and fibronectin were observed on the surface of the pannus and at the junctional portion between the pannus and the cartilage. The present immunohistochemical findings on the distribution of plasma proteins and ECM materials and the expression of adhesion molecules in CIA mice were similar to those in rheumatoid arthritis (RA) in many aspects. This suggests that CIA is a useful model for the investigation of RA.

Changes in location and number of tartrate-resistant acid phosphatase (TRAP)-positive cells during the development of type II collagen-induced arthritis in DBA/1J mice.

The authors investigated changes in the location and number of osteoclasts and their precursors during the development of articular lesions in type II collagen-induced arthritis in mice using tartrate-resistant acid phosphatase (TRAP) staining. The limb joints were examined at 6 to 15 weeks after the second immunization. The number of TRAP-positive cells increased as the articular lesions progressed. TRAP-positive macrophage-like cells were found in the hyperplastic synovial tissue and bone marrow stroma in the early stage. In the advanced stage, in addition to many TRAP-positive osteoclasts on the bone surface, TRAP-positive macrophage-like cells were observed in the pannus apart from the bone surface in the pannus-joint junctions. The above mentioned TRAP-positive macrophage-like cells are considered to be osteoclast precursors.

Carbon tetrachloride-induced acute liver injury in Mini and Wistar rats.

Acute liver injury induced by CCl4 injection (0.5 ml/kg b.w.) was compared between Mini and Wistar rats. Mini rats (Jcl:Wistar-TgN (ARGHGEN)1Nts strain) are Wistar-derived transgenic animals in which the expression of growth hormone (GH) gene is suppressed by the presence of an antisense transgene. The hepatic lesion appeared earlier and its recovery was delayed in Mini rats compared to in Wistar rats. The degree of the liver injury was more severe in Mini rats than in Wistar rats, and this corresponded well with the changes in serum AST level. Moreover, in accordance with the localization of CYP2E1-positive hepatocytes in the early stage after CCl4 treatment, the initial lesion characterized by ballooning of hepatocytes developed in the centrilobular zone in Wistar rats while it appeared in the middle zone in Mini rats. The changes in the percentage of PCNA-positive cells and the levels of HGF and TGF-beta1 mRNAs were clearly different between the two strains. These results indicate that the response of the liver to CCl4 is different between GH-suppressed Mini rats and Wistar rats.