Primary epididymis squamous cell carcinoma in a CB6F1-Tg rasH2 mouse.

In a 26-week carcinogenicity study in rasH2-Tg mice, squamous cell carcinoma on the epididymis was observed in a male mouse in the positive control group treated with N-methyl-N-nitrosourea. A 29-week-old male rasH2-Tg mouse that was euthanized 21 weeks after the administration of N-methyl-N-nitrosourea had a white-grayish mass on the left caput epididymis. The mass was nodular and consisted of pleomorphic tumor cells forming alveolar, sheeted, and trabecular structures suggesting epithelial tumor growth. These cells presented a cobblestone-like arrangement and formed intercellular bridges. Keratinization was infrequently observed. Periodic acid-methenamine-silver staining revealed argyrophilic fibrous structures around the alveolar structure of the tumor cells. Immunohistochemically, the tumor cells were positive for cytokeratin AE1/AE3 and cytokeratin 14 and negative for cytokeratin 5, p63, uroplakin III, vimentin, desmin, and αSMA. These immunohistochemical results suggested the tumor cells originated from the epididymal ducts. Metastatic lesions were observed in the mesenteric, inguinal, and pancreaticoduodenal lymph nodes. Based on these results, this tumor was diagnosed to be a primary squamous cell carcinoma of the epididymis. This is the first report of primary squamous cell carcinoma of the epididymis in a rasH2-Tg mouse.

Data on long-term survival of the NOD/Shi-scid IL-2Rγnull (NOG) mouse in two facilities.

The objective of this study was to investigate an appropriate observation period for an evaluation of tumorigenicity in NOD/Shi-scid IL-2 Rγnull (NOG) mice. At SNBL, 19 male and 19 female NOG mice were observed the general condition from 7 weeks old up to 68 weeks old and at FBRI, 7 male and 16 female NOG mice were observed the general condition throughout the lifespan from 7 weeks old. The survival rate started to decline rapidly around 54 to 56 weeks of age in both facilities without a facility difference. Based on these survival data, it seems reasonable to terminate a tumorigenicity study at 52 weeks of age.

A spontaneous basal cell carcinoma with pulmonary metastasis in a 6-week-old SD rat.

To our knowledge, this is the first report on basal cell carcinoma with lung metastasis in a rat. A 6-week-old male Sprague-Dawley rat presented ulceration of the oral mucosa with surrounding tumor growth and white nodules in the lung. Microscopically, the mass showed solid, sheet-like growth with a partially lobular pattern and invaded the gingival mucosa, maxilla, and nasal submucosa. The nuclei of tumor cells were round to oval in shape with basophilic cytoplasm and a large number of mitotic figures. The pulmonary nodules were almost identical to the maxillary tumor in histopathological characteristics. Immunohistochemically, tumor cells were positive for cytokeratin, vimentin, PCNA, and p63 and negative for desmin, S-100, and αSMA. Based on these results, we diagnosed the tumor as a maxillary basal cell carcinoma with pulmonary metastasis.

Establishment of disseminated intravascular coagulation (DIC) model by a single iv administration of Escherichia coli-derived lipopolysaccharide (LPS) to cynomolgus monkeys and evaluation of its pathophysiological status.

We prepared a DIC model by administrating LPS to cynomolgus monkeys, and investigated its potential for evaluations of new medicines for DIC therapy. Peripheral blood mononuclear cells (PBMC) collected from cynomolgus monkeys were incubated with LPS (8 types), and TNF-α levels in the media were measured. LPS from Escherichia coli (K-235) was most appropriate in terms of larger increases and smaller variation in TNF-α levels. PBMC from rats, cynomolgus monkeys or humans were incubated with LPS (K-235), and the TNF-α response to LPS was investigated. The response was comparable between cynomolgus monkeys and humans but small in rats. In an in vivo experiment, LPS (K-235) was administered once intravenously to cynomolgus monkeys with or without recombinant human thrombomodulin (rhTM) to investigate any changes in coagulation and fibrinolysis biomarkers and the suppressive effect of rhTM. The liver, kidney, and lung were examined histopathologically. Almost all of the changes resembled the pathophysiological status of human DIC and were suppressed by co-administration of rhTM. The DIC model resembling human DIC was established by LPS (K-235) treatment in cynomolgus monkeys, and therapeutic effect of rhTM was noted, suggesting that this model is useful in evaluations of the efficacy of new medicines for DIC therapy.

Neuroblastoma at the trigeminal nerve in a cynomolgus monkey (Macaca fascicularis).

A male cynomolgus monkey (Macaca fascicularis) of 5 years and 11 months of age from the vehicle control group of a 4-week repeated oral dose toxicity study had a spontaneously occurring mass lesion directly attached to the proximal part of the left trigeminal nerve. Histologically, the mass was characterized by a multifocal nodular appearance. Nodular zones showed low to moderate cellularity and were composed of small round cells exhibiting nuclear uniformity. On the other hand, inter-nodular zones were composed of nerve fiber containing septa and closely aggregated highly pleomorphic cells. Immunohistochemically, the small round cells were strongly immunopositive for synaptophysin, neuN, and class III beta-tubulin, while the highly pleomorphic cells were weakly immunopositive for neuN and occasionally immunopositive for class III beta-tubulin and doublecortin, suggesting that the tumor had originated from a neuronal lineage cell. Based on these findings, the mass was diagnosed as a neuroblastoma at the trigeminal nerve.

Dysferlin and animal models for dysferlinopathy.

Dysferlin (DYSF) is involved in the membrane-repair process, in the intracellular vesicle system and in T-tubule development in skeletal muscle. It interacts with mitsugumin 53, annexins, caveolin-3, AHNAK, affixin, S100A10, calpain-3, tubulin and dihydropyridine receptor. Limb-girdle muscular dystrophy 2B (LGMD2B) and Miyoshi myopathy (MM) are muscular dystrophies associated with recessively inherited mutations in the DYSF gene. The diseases are characterized by weakness and muscle atrophy that progress slowly and symmetrically in the proximal muscles of the limb girdles. LGMD2B and MM, which are collectively termed "dysferlinopathy", both lead to abnormalities in vesicle traffic and membrane repair at the plasma membrane in muscle fibers. SJL/J (SJL) and A/J mice are naturally occurring animal models for dysferlinopathy. Since there has been no an approach to therapy for dysferlinopathy, the immediate development of a therapeutic method for this genetic disorder is desirable. The murine models are useful in verification experiments for new therapies and they are valuable tools for identifying factors that accelerate dystrophic changes in skeletal muscle. It could be possible that the genetic or immunological background in SJL or A/J mice could modify muscle damage in experiments involving these models, because SJL and A/J mice show differences in the progress and prevalent sites of skeletal muscle lesions as well as in the gene-expression profiles of their skeletal muscle. In this review, we provide up-to-date information on the function of dysferlin, the development of possible therapies for muscle dystrophies (including dysferlinopathy) and the detection of new therapeutic targets for dysferlinopathy by means of experiments using animal models for dysferlinopathy.

Comparative Gene Expression Analysis in the Skeletal Muscles of Dysferlin-deficient SJL/J and A/J Mice.

Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was conducted to determine whether or not there are interstrain or site-dependent differences in the gene expression profiles of skeletal muscles in SJL/J and A/J mice as dysferlinopathy models. Upon analysis by qRT-PCR, SJL/J mice showed a trend of increased gene expression level of uncoupling protein 2 in the rectus femoris and longissimus lumborum at 30 weeks of age when dystrophic lesions became histopathologically pronounced. Heme oxygenase 1 and S100 calcium binding protein A4 were upregulated in the rectus femoris, longissimus lumborum and abdominal muscles, in which dystrophic lesions occur more commonly in SJL mice. The gene expression levels of heat shock protein 70 in most muscles of A/J mice were lower than those of BALB/c mice as control. SJL/J mice exhibited a marked lowering of decay-accelerating factor 1/CD55 gene expression level in all studied muscles except for the heart at all ages compared with that of BALB/c mice. This study showed that there were some interstrain differences in the gene expres sion profiles of skeletal muscles between SJL/J and A/J mice. Further investigation is required to reveal whether these alterations of the expression levels are the cause of dystrophic changes or occur subsequent to muscle damage.

The distribution and characterization of skeletal muscle lesions in dysferlin-deficient SJL and A/J mice.

The pathogenesis of limb-girdle muscular dystrophy type 2B (LGMD2B) dysferlinopathy remains to be investigated. The distribution and characterization of skeletal muscle lesions were examined in two different LGMD2B mouse models, SJL and A/J mice (at 10 and 35 weeks old), in association with the endoplasmic reticulum (ER) stress. SJL mice showed an earlier age of onset and a faster progression of skeletal muscle lesions as compared with those of A/J mice; the sensitivity difference to muscular dystrophic lesions between SJL and A/J mice was observed in the lumbar muscles (particularly, lumbar longissimus and sublumbar muscles); the lesions seen mainly in SJL mice at 35 weeks old consisted of degeneration, necrosis, fatty infiltration, variation in muscle fiber size and atrophy in muscle fibers. Enzyme-histochemically, the fast-twitch muscle fiber was predominant for the degenerative changes seen in the rectus femoris and lateral longissimus muscles of SJL mice. Immunohistochemically, the main reactive cell type observed in and around degenerative and/or necrotic muscle fibers was macrophages, demonstrable with an anti-F4/80 antibody. Because the analyses of spliced XBP1 mRNA, a marker of ER stress, did not show the increased expression, it was considered that ER stress did not affect the progression of skeletal muscle lesions in SJL mice with the advanced stage of dysferlinopathy.

Isolation and characterization of embryonic stem-like cells from canine blastocysts.

Embryonic stem (ES) cells are pluripotent cells with the capacity to generate any type of cell. Here we describe the isolation of ES-like cells from canine blastocysts. Canine embryos were collected from beagle bitches at day 11-16 of first estrus. A total of 80 normal embryos were obtained from 15 dogs. Of the embryos, 13 were at the morulae stage, 39 at the blastocyst stage, and 28 at the hatched blastocyst stage. The inside of morulae or inner cell masses (ICMs) of blastocysts were isolated mechanically, and cultured onto mouse embryonic fibroblasts (MEF) as feeder layers. Primary cell colonies were formed in 0% (0/13) of morulae, 25.6% (10/39) of blastocysts, and 67.9% (19/28) of hatched blastocysts. These colonies were separated either by enzymatic dissociation or by mechanical disaggregation. Dissociation with collagenase resulted in immediate differentiation, but with mechanical disaggregation these cells remained undifferentiated, and two ES-like cell lines (cES1, cES2) continued to grow in culture after eight passages. These cells had typical stem cell-like morphology and expressed specific markers such as alkaline phosphatase activity, stage specific embryonic antigen-1 and Oct-4. These cells formed embryoid bodies (EBs) in a suspension culture; extended culture of EBs resulted in the formation of cystic EBs. When the simple EBs were cultured on tissue culture plates, they differentiated into several types of cells including neuron-like, epithelium-like, fibroblast-like, melanocyte-like, and myocardium-like cells. These observations indicate that we successfully isolated and characterized canine ES-like cells.

Na+/Ca2+ exchanger-deficient mice have disorganized myofibrils and swollen mitochondria in cardiomyocytes.

The Na(+)/Ca(2+) exchanger (NCX1) plays a key role in maintaining Ca(2+) homeostasis in cardiomyocytes. Disruption of Ncx1 gene in mice results in embryonic lethality between embryonic day 9 and 10, with the mice lacking spontaneous heartbeats. We examined the mechanism of lack of heartbeats in Ncx1-deficient mice. Ultrastructual analysis demonstrated that Ncx1-deficient mice showed severe disorganization of myofibrils, a lack of Z-lines and swelling of mitochondria in cardiomyocytes. However, the expressions of cardiac-specific genes including transcription factor genes and contractile protein genes were not changed in Ncx1-deficient mice. Abnormal Ca(2+) handling itself or the lack of heartbeats due to the inactivation of Ncx1 gene may cause the disorganization of myofibrillogenesis. Although NCX1 protein levels were decreased in heterozygous mice, there were no changes in NCX2 and NCX3 protein levels between wild type and heterozygous mice.

Twenty-six week carcinogenicity study of ampicillin in CB6F1-TgrasH2 mice.

As a part of the ILSI-HESI Alternative to Carcinogenicity Testing (ACT) program, we performed a 26-week carcinogenetic study of nonmutagenic drug, ampicillin (ABPC) in Tg-rasH2 mice. ABPC was given to Tg-rasH2 mice (0, 350, 1000, 3000 mg/kg, p.o.) and Non-Tg mice (0, 3000 mg/kg, p.o.) daily for 26 weeks. As a positive control, a single dose of MNU was administered once to Tg-rasH2 mice (75 mg/kg, i.p.). In this study, Tg-rasH2 mice did not demonstrate any increases in tumor development in response to ABPC. Thus, ABPC had no carcinogenicity in the 26-week carcinogenesis study in Tg-rasH2 mice or in a 2-year carcinogenesis study in B6C3F1 mice.

Targeted disruption of LIG-1 gene results in psoriasiform epidermal hyperplasia.

The gene encoding a transmembrane glycoprotein LIG-1, of which the extracellular region was organized with the leucine-rich repeats and immunoglobulin-like domains, was disrupted in mice by gene targeting. LIG-1-deficient mice developed a skin change on the tail and facial area after birth. The affected skin was histologically reminiscent of the epidermis in human common skin disease 'psoriasis'. LIG-1 was expressed in basal cells of the epidermis and outer root sheath cells of hair follicles in mice. Interestingly, the LIG-1 expression was apparently down-regulated in the psoriatic lesions, suggesting that LIG-1 inversely correlates with proliferative ability of epidermal keratinocytes.

Monkey embryonic stem cell lines expressing green fluorescent protein.

The major limitation of nonhuman primate (NHP) embryonic stem (ES) cell research is inefficient genetic modification and limited knowledge of differentiation mechanisms. A genetically modified NHP-ES cell with biomarkers, such as green fluorescent protein (GFP), that allow noninvasive monitoring of transgenic cells, is a useful tool to study cell differentiation control during preimplantation and fetal development, which also plays a crucial role in the development of cell transplantation medicine. Here we report the establishment of transgenic NHP-ES cell lines that express GFP without jeopardizing their pluripotency, which was confirmed by in vitro and in vivo differentiation. These GFP-expressing ES cells reproducibly differentiated into embryoid bodies, neural cells, and cardiac myocytes. They formed teratoma composed of tissues derived from the three embryonic germ layers when transplanted into severe combined immunodeficient disease (SCID) mice. GFP expression was maintained in these differentiated cells, suggesting that these cells were useful for cell transplantation experiments. Furthermore, we showed that these ES cells have the ability to form chimeric blastocysts by introducing into the early preimplantation stage NHP embryo.