Alagille-like syndrome with surprising karyotype: a case report.

BACKGROUND: Chromosome 5p partial monosomy (5p-syndrome) and chromosome 6p partial trisomy are chromosomal abnormalities that result in a variety of symptoms, but liver dysfunction is not normally one of them. Alagille syndrome (OMIM #118450) is a multisystem disorder that is defined clinically by hepatic bile duct paucity and cholestasis, in association with cardiac, skeletal, and ophthalmologic manifestations, and characteristic facial features. Alagille syndrome is caused by mutations in JAG1 on chromosome 20 or NOTCH2 on chromosome 1. Here, we report a preterm infant with karyotype 46,XX,der(5)t(5,6)(p15.2;p22.3) and hepatic dysfunction, who was diagnosed as having incomplete Alagille syndrome. CASE PRESENTATION: The Japanese infant was diagnosed based on the cardiac abnormalities, ocular abnormalities, characteristic facial features, and liver pathological findings. Analysis of the JAG1 and NOTCH sequences failed to detect any mutations in these genes. CONCLUSIONS: These results suggest that, besides the genes that are known to be responsible for Alagille syndrome, other genetic mutations also may cause Alagille syndrome.

A Case of Feline T-cell Lymphoma with Tropism for Striated Muscle and Peripheral Nerve.

An 11-year-old female American shorthair cat was presented with a 3-month history of hindlimb ataxia and knuckling of the left forelimb. Clinical abnormalities included weight loss, hyperaesthesia of the neck and back, cardiac murmur and systemic muscle atrophy. The cat died 10 days after the initial presentation and a necropsy examination was performed. Grossly, extensive pale lesions were seen in the wall of the left ventricle and the septum of the heart. There were no detectable masses in the heart, skeletal muscles or peripheral nerves. Histopathological examination revealed diffuse, extensive infiltration of atypical lymphoid cells in the heart; the cardiac muscles were markedly degenerate and atrophic and were replaced by the neoplastic cells. Neoplastic cells with similar morphology were seen in all specimens of the skeletal muscles and peripheral nerves. Clonality analysis of the paraffin wax-embedded heart tissue revealed a monoclonal rearrangement of the gene encoding the T-cell receptor γ chain. Based on these findings, the case was diagnosed as T-cell lymphoma with tropism for striated muscle and peripheral nerve.

Participation of Tumor-Associated Myeloid Cells in Progression of Amelanotic Melanoma (RMM Tumor Line) in F344 Rats, with Particular Reference to MHC Class II- and CD163-Expressing Cells.

Tumor progression is often influenced by infiltration of myeloid cells; depending on the M1- or M2-like activation status, these cells may have either inhibitory or promoting effects on tumor growth. We investigated the properties of tumor-associated myeloid cells in a previously established homotransplantable amelanotic melanoma (RMM tumor line) in F344 rats. RMM tumor nodules were allowed to reach the sizes of 0.5, 1, 2 and 3 cm, respectively. Immunohistochemistry and flow cytometry was performed for macrophage markers CD68 and CD163, and for the antigen-presenting cell marker, MHC class II. Although no significant change was observed in the number of CD68+ and CD163+ macrophages during RMM progression, the number of MHC class II+ antigen-presenting cells was reduced in 3 cm nodules. Real-time RT-PCR of laser microdissection samples obtained from RMM regions rich in MHC class II+ cells demonstrated high expressions of M1-like factors: IFN-γ, GM-CSF and IL-12a. Furthermore, fluorescence-activated cell sorting, followed by real-time RT-PCR for CD11b+ MHC class II+ (myeloid antigen-presenting cells), CD11b+ CD163+ (M2 type myeloid cells), CD11b+ CD80+ (M1 type myeloid cells) and CD11b+ CD11c+ (dendritic cells) cells was performed. Based on the levels of inflammation- and tumor progression-related factors, MHC class II+ antigen-presenting cells showed polarization towards M1, while CD163+ macrophages, towards M2. CD80+ and CD11c+ myeloid cells did not show clear functional polarization. Our results provide novel information on tumor-associated myeloid cells in amelanotic melanoma, and may become useful in further research on melanoma immunity.

Cutaneous Histiocytic Sarcoma with Regional Lymph Node Metastasis in a Netherland Dwarf Rabbit (Oryctolagus cuniculus).

A 10-year-old male Netherland dwarf rabbit (Oryctolagus cuniculus) was presented with a red nodular mass (1 cm in diameter) with ulceration and hair loss in the skin of the left upper lip. Cytological examination revealed atypical round cells. The mass was excised surgically. Histologically, the mass was composed of large round to polyhedral neoplastic cells with marked cytological atypia. The neoplastic cells were often binucleated or multinucleated. Immunohistochemically, the neoplastic cells were intensely positive for Iba1 and vimentin, but fewer neoplastic cells expressed E-cadherin. Nuclear immunoreactivity for Ki67 was detected in approximately 41% of the neoplastic cells. Metastasis to the left cervical lymph nodes was detected 6 months after the surgical excision. Based on clinical, histopathological and immunohistochemical findings, the present case was diagnosed as cutaneous histiocytic sarcoma. To the authors' knowledge cutaneous histiocytic disease has not been reported previously in lagomorphs.

Metastatic Liposarcoma in a South African Fur Seal (Arctocephalus pusillus).

A 14-year-old female South African fur seal (Arctocephalus pusillus) was presented with a large skin mass on the right shoulder. At necropsy examination, multiple white nodules were found in the lungs, liver, spleen and right axillary lymph nodes. Histologically, the skin mass was composed of round to polygonal neoplastic cells with round to oval nuclei and variably sized cytoplasmic vacuoles. Cellular and nuclear atypia were prominent. Immunohistochemically, the neoplastic cells expressed vimentin, but not cytokeratins, S100 protein, adipophilin or desmin. The cytoplasmic lipid droplets stained positively with oil red O. Metastasis was seen in the lungs, liver, spleen and right axillary lymph nodes, with similar morphological features to the skin mass. Based on these findings, a diagnosis of a pleomorphic liposarcoma with systemic metastasis was made. No previous reports of metastatic liposarcomas have been published in marine mammals.

Multiple Histiocytic Foam Cell Nodules in the Tongue of Miniature Dachshund Dogs.

Miniature dachshund dogs are a common breed in Japan and are known to be predisposed to granulomatous diseases. Here we report the pathologic features of multiple lingual nodules in 7 miniature dachshunds. Seven dogs had multiple nodules of variable sizes mainly on the ventral and lateral surface of the tongue. In addition, 1 dog also had masses on the left oral mucosa. Three cases had recurrence after surgical resection. Histologically, the lingual nodules were composed of aggregates of foam cells with clear vacuolated cytoplasm that were negative for oil red O, PAS, and alcian blue. They stained positively for CD204 (macrophage scavenger receptor) and MHC class II and negatively for Iba-1, E-cadherin, adipophilin, cytokeratins, S-100, and nestin. These findings indicate that the multiple lingual nodules in miniature dachshunds are an unusual, unique lesion consisting of macrophage-derived foam cells, which does not correspond to canine lingual diseases reported to date.

Expression of nestin in remodelling of α-naphthylisothiocyanate-induced acute bile duct injury in rats.

The function of the intermediate filament protein nestin is poorly understood. The significance of nestin expression was assessed in α-naphthylisothiocyanate (ANIT)-induced cholangiocyte injury lesions in F344 rats. Liver samples obtained from rats injected intraperitoneally with ANIT (75 mg/kg) on post-injection days 0 (control) and 1-12 were labelled immunohistochemically for expression of nestin and markers specific for mesenchymal cells (vimentin), hepatic stellate cells (HSCs) (desmin and glial fibrillary acidic protein [GFAP]), endothelial cells (rat endothelial cell antigen [RECA]-1), cholangiocytes (cytokeratin [CK] 19) and cellular proliferation (Ki67). Cholangiocyte injury led to infiltration of neutrophils and macrophages followed by aggregation of mesenchymal cells and regeneration of bile ducts. Nestin expression was detected in mesenchymal cells (vimentin positive) on days 1-7 with a peak on days 3-5 and in newly-formed RECA-1-positive endothelial cells on day 3. Nestin expression was also observed in regenerating CK19-positive cholangiocytes on days 2-5, with a peak on day 3. Labelling for Ki67 showed proliferation of cholangiocytes, mesenchymal cells and HSCs. Real-time reverse transcriptase polymerase chain reaction with microdissected samples showed significantly elevated nestin mRNA on day 3. The findings suggest an association between nestin expression and cellular proliferation. Based on these findings, it was considered that nestin-expressing mesenchymal cells, HSCs and endothelial cells may be possible progenitors of repopulating cholangiocytes. Nestin expression may serve as an indicator for cellular remodelling and behaviour of injured and repopulating bile ducts.

Myoepithelioma of the gland of the third eyelid in a dog.

A 12-year-old female miniature dachshund was presented with a tan-white, firm mass (4 × 3 × 2 cm) occupying the left medial canthus. The mass compressed and displaced the left eye dorsally, and it was surgically removed. Microscopically, the mass was composed of interlacing bundles of spindle cells with clear cytoplasm and a small number of atypical glandular epithelial cells. Immunohistochemically, the spindle cells expressed p63, α-smooth muscle actin and calponin, and were negative for cytokeratin AE1/AE3. The glandular epithelial cells expressed cytokeratin AE1/AE3. Based on these findings, this case was diagnosed as a myoepithelioma of the gland of the third eyelid.

A collision tumour consisting of malignant trichoblastoma and melanosarcoma in a rabbit.

A 7-year-old mixed breed neutered female rabbit (Orytolagus cuniculus) developed a solitary black nodular mass (1 cm in diameter) in the skin of the right flank. Microscopically, the mass consisted of an admixture of neoplastic trichoblasts and melanocytes. The former were arranged as solid, trabecular, island-like and gland-like structures and the cells had oval nuclei with prominent nucleoli and lightly eosinophilic scant cytoplasm. The latter population exhibited prominent nuclear atypia and high mitotic index in the clusters of a few cells or single cells. Immunohistochemically, the neoplastic trichoblasts expressed cytokeratins and E-cadherin, while the neoplastic melanocytes expressed vimentin, S100 protein, melan-A and melanoma antigen. A diagnosis of collision tumour involving malignant trichoblastoma and melanosarcoma was made.

Distribution of cells labelled by a novel somatic stem cell-recognizing antibody (A3) in pulmonary genesis and bleomycin induced pulmonary fibrosis in rats.

Stem cells play important roles in organogenesis and remodelling after tissue injury. A monoclonal antibody (A3) has been produced against rat somatic stem cells. The present study investigated the distribution of cells labelled by A3 in the lung of fetal, neonatal and adult rats, as well as in the lung of rats with bleomycin (BLM) induced pulmonary fibrosis. In developing fetal lungs, A3(+) interstitial cells were present around the bronchi/bronchioles and arterioles, while in neonatal and adult lungs, the A3 reactivity of the interstitial cells gradually disappeared and instead, vascular endothelial cells in alveolar capillaries and arterioles expressed A3. By double immunofluorescence labelling, the A3(+) interstitial cells also expressed vimentin (a mesenchymal marker) and CD34 (a marker of immature mesenchymal cells), indicating that the interstitial cells were immature mesenchymal cells concentrated in organs as precursors to cells of connective tissues. A3(+)endothelial cells were co-expressed RECA-1 (a marker of rat endothelial cells) and A3 was localized to the cell membrane and cytoplasm of these cells by immunoelectron microscopy. In BLM induced fibrotic lesions, there were many A3(+) cells, which also expressed vimentin or RECA-1 by dual immunofluorescence labelling. There were few CD34(+)/A3(+) double positive cells. No cells co-expressed A3 and α-smooth muscle actin (a marker of well-differentiated myofibroblastic cells). Although the detailed properties of cells labelled by A3 remain to be discovered, A3 would appear to be a useful marker of immature mesenchymal cells and vascular endothelial cells in developing lungs and in pulmonary fibrosis.

Immunophenotypical characterization of macrophages in rat bleomycin-induced scleroderma.

Scleroderma is a skin disorder characterized by persistent fibrosis. Macrophage properties influencing cutaneous fibrogenesis remain to be fully elucidated. In this rat (F344 rats) model of scleroderma, at 1, 2, 3, and 4 weeks after initiation of daily subcutaneous injections of bleomycin (BLM; 100 µl of 1 mg/ml daily), skin samples were collected for histological and immunohistochemical evaluations. Immunohistochemically, the numbers of cells reacting to ED1 (anti-CD68; phagocytic activity) and ED2 (anti-CD163; inflammatory factor production) began to increase at week 1, peaked at week 2, and decreased thereafter. In contrast, the increased number of cells reacting to OX6 (anti-MHC class II molecules) was seen from week 2 and remained elevated until week 4. α-Smooth muscle actin-positive myofibroblasts were increased for 4 weeks. Double labeling revealed that galectin-3, a regulator of fibrogenic factor TGF-ß1, was expressed in CD68+, CD163+, and MHC class II+ macrophages and myofibroblasts. mRNA expression of TGF-ß1, as well as MCP-1 and CSF-1 (both macrophage function modulators), were significantly elevated at weeks 1 to 4. This study shows that the increased number of macrophages with heterogeneous immunophenotypes, which might be induced by MCP-1 and CSF-1, could participate in the sclerotic lesion formation, presumably through increased fibrogenic factors such as galectin-3 and TGF-ß1; the data may provide useful information to understand the pathogenesis of the human scleroderma condition.

Mutational analysis of the feline CLN3 gene and an ultrastructural evaluation of lysosomal storage materials in a cat with neuronal ceroid lipofuscinosis: an investigation into the molecular basis of the disease.

Neuronal ceroid lipofuscinosis (NCL) is a neurodegenerative disease caused by a number of different genes. A mutational analysis of the feline CLN3 gene was performed in a cat with NCL that had vacuolated lymphocytes, which is a feature of human NCL caused by defects of the CLN3 gene. To determine the candidate gene(s) responsible for this case, NCL-specific ultrastructures of storage materials were analysed. A sequence analysis indicated that the CLN3 gene was not likely to be responsible for this case of feline NCL because no deleterious mutation was detected. An ultrastructural analysis did not reveal any candidate gene because of inconsistency with any pattern found in human NCL. These findings suggest that the diagnostic criteria for human NCL are not directly applicable to feline NCL.

Immunophenotypical analyses of myofibroblasts in rat excisional wound healing: possible transdifferentiation of blood vessel pericytes and perifollicular dermal sheath cells into myofibroblasts.

Cutaneous fibrosis after wound is evoked by myofibroblasts capable of producing collagen; the derivation and features remain to be investigated. Immunophenotypical characteristics of myofibroblasts were analysed in excisional rat wound healing, of which samples were obtained on post-wounding (PW) days 1 to 26. Myofibroblasts were characterized for expressions of intermediate cytoskeletons such as vimentin, desmin, and α-smooth muscle actin (α-SMA). To pursue the progenitor, immunolabeling analyses were performed using stromal-/bone marrow-stem cell markers (Thy-1 and A3). Myofibroblasts reacting to vimentin and α-SMA were first seen on PW day 5, then peaked on PW day 9 in granulation tissues, and gradually decreased in remodeling tissues; these immunopositive cells reacted simultaneously to Thy-1. Desmin-reacting cells were limited to newly-formed blood vessels in wound bed. The single/double immunolabelings revealed that pericytes (identified by positive reaction to PDGFR-ß and negative reaction to endothelial markers) in newly-developing blood vessels reacted to vimentin, α-SMA, Thy-1 and A3, and occasionally to desmin, and that perifollicular dermal sheath cells in the wound periphery showed increased expressions for vimentin, Thy-1 and A3. There is considerable immunophenotypical similarity between myofibroblasts (expressing vimentin, α-SMA and Thy-1), pericytes (reacting to vimentin, α-SMA, Thy-1 and A3) in newly-developing blood vessels, and perifollicular dermal sheath cells (reacting to vimentin, Thy-1 and A3). Collectively, myofibroblasts in rat cutaneous fibrosis are characterized by vimentin, α-SMA and Thy-1 expressions, and the cells might be generated from the pericytes or perifollicular dermal sheath cells in the lineage of stroma-/bone marrow-stem cells.

Heterogeneity of macrophage populations and expression of galectin-3 in cutaneous wound healing in rats.

The aim of this study was to investigate the properties of macrophages that infiltrated the sites of cutaneous wound healing in rats between 1 and 26 days post wounding (dpw). During the inflammation phase (1-3 dpw), ED1(+) (CD68(+)) macrophages with enhanced lysosomal activity dominated. From 5 to 7 dpw there was formation of granulation tissue as indicated by the presence of myofibroblasts expressing α-smooth muscle actin. At this stage, ED2(+) (CD163(+)) macrophages, capable of producing inflammatory factors, were dominant. The majority of ED1(+) macrophages expressed galectin-3, a regulator of fibrosis. Corresponding to the increased numbers of ED1(+) and ED2(+) macrophages at 3-9 dpw, there was increased expression of genes encoding transforming growth factor-ß1 (a major fibrogenic factor), monocyte chemoattractant protein-1 and colony stimulating factor-1. These macrophage-related factors might contribute to inflammation and formation of granulation tissue. OX6(+) macrophages expressing class II molecules of the major histocompatibility complex became predominant in the healing stages (15-26 dpw), indicating important roles for antigen-presenting cells in tissue remodelling. The OX6(+) macrophages were most likely derived from ED1(+) macrophages. The results of this study show that infiltration of phenotypically- and functionally-distinct macrophage populations characterizes different stages of the wound healing process.

Cardiac hamartoma in a young squirrel monkey who died suddenly.

A case of cardiac hamartoma in a 2-month-old squirrel monkey is reported. The monkey showed a loss of appetite and died suddenly. Microscopically, an encapsulated nodular lesion was found at the right atrial wall. The lesion consisted of irregularly shaped, slender myocytes intermingled with a few fibroblasts and collagen fibers. Neither nuclear atypia nor inflammatory cell infiltrate was seen. The constituting cells had stratified striations in the cytoplasm and reacted immunohistochemically for desmin, indicating the nature of myocytes. Based on the above findings, a diagnosis of cardiac hamartoma was made. This is the first case of cardiac hamartoma in this species.

Late onset cerebellar cortical degeneration in a koala.

A 10-year-old male koala started to fall from the tree while sleeping. Subsequently, the koala often fell down while walking and showed a gait abnormality, abnormal nystagmus and hypersalivation. At 12 years of age, the koala became ataxic and seemed blind. At 13 years of age, the koala exhibited signs of dysstasia and was euthanased. Necropsy revealed marked symmetrical atrophy of the cerebellum. Histopathologically, a severe loss of Purkinje and granule cells was evident in the cerebellum, while the molecular layer was more cellular than normal with cells resembling small neurons, which were positively stained with parvalbumin immunohistochemistry. Reactive Bergmann glial cells (astrocytes) were present adjacent to the depleted Purkinje cell zone. The very late onset and slow progression of the cerebellar cortical degeneration in this case is particularly interesting and appears to be the first report in the koala.

Malignant seminoma with multiple visceral metastases in a guinea fowl (Numida meleagris) kept in a zoo.

A case of seminoma found in an adult guinea fowl (Numida meleagris) that has been exhibited in a zoo is reported. The right testis was extremely enlarged and replaced by round and polyhedral pleomorphic neoplastic cells showing nest, sheet, and diffuse growth patterns. The neoplastic cells had acidophilic cytoplasm and hyperchromatic and eccentrically placed nuclei. Metastatic lesions composed of diffuse growth of neoplastic cells similar to those of the primary tumor were seen in the liver, lungs, kidneys, and heart, and neoplastic emboli were often detected within blood vessels of these organs, indicating hematogenous metastasis. This is the first report of malignant seminoma with multiple metastases in the visceral organs in the guinea fowl.

Vasoformative disorder, resembling littoral cell angioma, of the spleen in a geriatric Japanese macaque (Macaca fuscata).

A 30-year-old female Japanese macaque showed marked splenomegaly. The enlarged spleen consisted of neoplastic proliferation of anastomosing vascular channels resembling morphologic structures of red pulp sinuses; occasionally, papillary fronds were seen in dilated channels. Immunohistochemically, the lining cells reacted to both endothelial cell (von Willebrand factor) and macrophage (macrophage scavenger receptor class A) markers, indicating features of littoral cells of the spleen. Based on the pathologic characteristics, particularly the presence of neoplastic cells with macrophage/histiocyte-like attributes, this tumor was regarded as littoral cell angioma; this is a rare benign splenic vascular tumor.