Chronic hepatitis C virus (HCV) infection affects approximately 170 million people and is a major global health problem because infected individuals can develop liver cirrhosis and hepatocellular carcinoma. Despite significant improvements in antiviral drugs, only around 50% of treated patients with genotype 1 and 4 demonstrate HCV clearance. Unfortunately, an anti-HCV vaccine is still not available. To progress treatment of HCV, it is necessary to understand the mechanism(s) by which HCV infects hepatocytes, and how the host immune response prevents the spread of the virus. Because HCV infects only humans and chimpanzees, it is difficult to evaluate immune response mechanisms, and the effects of chemicals and new technologies on these response mechanisms. These difficulties underline the importance of establishing a small HCV-infected animal model. This review focuses on the progress made in recent years towards the development of an experimental mouse model for HCV.

View full abstract

To investigate the usefulness of the immunopotentiator from Pantoea agglomerans 1 (IP-PA1) as a supportive drug in melanoma therapy, we analyzed the immunological effects of IP-PA1 on melanoma-inoculated model mice. Oral administration of IP-PA1 increased the serum levels of tumor necrosis factor (TNF)-α at 2 h after the administration and interferon (IFN)-γ and IL-12 at 12 h after the administration in naïve BALB/cCrSlc mice as evaluated by ELISA. IP-PA1 did not affect the proliferation of melanoma cells directly determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Combinatory treatment of IP-PA1 with doxorubicin for 9 days increased the serum levels of IFN-γ and IL-12 by 71.0 and 15.3%, respectively, compared to the treatment of doxorubicin alone in melanoma-bearing C57BL/6NCrSlc mice as evaluated by ELISA. It also increased the proportion of natural killer (NK) cells and the ratio of CD4⁺ to CD8⁺ T cells in the spleen from 6.1 ± 0.3 to 7.4 ± 0.5% and from 1.25 ± 0.03 to 1.38 ± 0.04, respectively, compared to the treatment of doxorubicin alone as analyzed by flow cytometry. The mean survival period of melanoma-bearing, doxorubicin treated mice was prolonged from 31.4 ± 7.1 to 35.3 ± 8.4, 51.1 ± 5.4, and 45.0 ± 8.4 days by combinatory treatment of IP-PA1 at the daily doses of 0.1, 0.5, and 1 mg/kg, respectively. In conclusion, the results of the present study suggest the usefulness of IP-PA1 as a supportive drug in melanoma therapy.

View full abstract

Mood disorders are more frequent in women than men, however, the majority of research has focused on male rodents as animal models. We used a variety of common behavioral tests to look for differences in anxiety-like and social behaviors between and within C57BL/6J and BALB/cJ mice. Our results show that female C57BL/6J mice exhibited lower levels of anxiety-like behavior and higher levels of activity than female BALB/cJ during the open field and elevated plus maze tests. Principal component analysis generated more factors in the behavioral variables of males than females. In the open field, a sex difference was also found and factor 1 emerged as anxiety in males, and motor activity in females. While C57BL/6J mice were found to have higher levels of social exploration and social contacts, differences were found between the sexes (females were more social) in both strains for this measure and also for anxiety-like behaviors. When interacting with animals of the same sex, levels of sniffing body and huddling in both male and female C57BL/6J mice were higher than those in male and female BALB/cJ mice. However, in the between-sex interactions, male C57BL/6J mice sniffed the stimulus mouse less, and female C57BL/6J mice sniffed the stimulus more compared to BALB/cJ mice. This study provides important behavioral phenotypes and confirms the multidimensional behavioral structure of two widely used mice strains.

View full abstract

The condition of hyperglycemia results from multiple genetic and environmental factors. In recent years much progress has been made with regards to the search for candidate genes involved in the expression of various common diseases including type 2 diabetes. However less is known about the specific genetic and environmental connections that are important for the development of the disease. In the present study, we used hyperglycemic congenic rats to address this issue. When given a normal diet, two hyperglycemic QTLs (quantitative trait locus), Nidd2/of and Nidd10/of, showed mild obesity and/or increased blood glucose in the oral glucose tolerance test. In a double congenic strain possessing both loci, these indices were not significantly different from those of either single congenic strain. In contrast, the double congenic strain fed a high-calorie diet showed significantly greater body weight than the single congenic strains or normoglycemic control rats. Although postprandial glucose levels of the double congenic rat were not further aggravated even on the high fat diet, it was notable that the postprandial insulin levels were drastically elevated. From these results, we constructed a novel model animal especially for the study of prediabetic hyperinsulemia, in which two QTLs and an additional dietary condition are involved. This may help to shed light on the genetic basis and gene-to-diet interaction during the early stage of type 2 diabetes.

View full abstract

In the antioxidant defense system, superoxide dismutase (SOD) catalyzes the breakdown of superoxide into hydrogen peroxide and oxygen. In the cecum, the influence of intestinal microflora on SOD activity is unknown. In this study, we used germ-free (GF) mice to examine the effect of intestinal microflora on SOD activity in the cecum, and SOD activity was compared between GF and conventional (CV) mice. The activity of CuZnSOD and MnSOD was determined using the SOD Assay Kit-WST. Expressions of CuZnSOD mRNA and protein were determined by real-time PCR and western blot analyses, respectively. The activities of CuZnSOD and MnSOD were significantly higher in the ceca of GF IQI and FVB/N strain mice than in CV mice (P<0.01–0.05). The gene expressions of CuZnSOD mRNA in the ceca of GF mice were significantly higher than those in CV mice (P<0.05), and CuZnSOD protein expression showed similar tendencies. Consistent with the abovementioned results, the total SOD activity in conventionalized mice decreased to the level of total SOD activity observed in the ceca of CV mice. Furthermore, no differences between GF and CV mice were observed in the SOD activities in the liver and thymus. Our results suggest that the antioxidant defense system in the mouse cecum is influenced by the intestinal microflora that downregulate SOD activity.

View full abstract

Microaspiration due to gastroesophageal reflux (GER) has been suggested as a factor contributing to the development and exacerbation of several respiratory disorders. To explore the relationship between GER and respiratory disorders, we histologically examined the bilateral lungs of a rat gastroduodenal contents reflux model, which was previously used to investigate the histogenesis of Barrett’s esophagus and esophageal carcinoma. GER was surgically induced in male Wistar rats. The bilateral lungs of the reflux rats were examined with hematoxylin and eosin (HE), PAS-Alcian blue, and Azan staining at 10 and 20 weeks after surgery. Immunohistochemical staining of CD68 and α-SMA was also performed. Aspiration pneumonia with severe peribronchiolar neutrophilic and lymphocytic infiltrates, goblet cell hyperplasia, prominence of blood vessels, and increased thickness of the smooth muscle layer were detected. Bronchiolitis obliterans (BO)-like lesions comprising granulation tissue with macrophages, spindle cells, and multinucleated giant cells in the lumen of respiratory bronchioles were observed in the bilateral lungs of the reflux animals. These findings suggest that the severe inflammation and the BO-like lesions may play a role in exacerbation of the forced expiratory volume in 1 second (FEV 1) in human cases. In conclusion, we speculate that repetitive microaspiration due to GER may contribute to the exacerbation of various respiratory diseases, particularly asthma and chronic obstructive pulmonary disease (COPD), and the development of BO syndrome following lung transplantation. The reflux model is a good tool for examining the causal relationships between GER and respiratory disorders.

View full abstract

The Matsumoto Eosinophilia Shinshu (MES) rat strain develops hereditary blood eosinophilia due to the mutant Cyba^mes gene. In contrast, BN.MES-Cyba^mes congenic rats, in which the mutant Cyba^mes gene introduced into the background of the BN strain, have a normal blood eosinophil level despite showing robust proliferation of eosinophils in the bone marrow. However, the congenic rats manifest focal necrosis with eosinophilic infiltration in the liver, a phenotype rarely observed in the original MES rat strain. To elucidate the genetic basis for the strain differences, (MES × BN.MES-Cyba^mes)F₂ rats were bred, and genetic analyses of phenotypes for eosinophilia were performed. Blood and bone marrow eosinophil levels in the F₂ rats showed broad distributions, suggesting that the traits were under the influence of multiple genes. Genetic association studies revealed that BN-derived marker loci on chromosomes 9 and 5 were responsible for the increase in eosinophil level in the bone marrow, decrease in blood eosinophil level, and the induction of focal necrosis with eosinophilic infiltration in the liver. The BN-derived allele of the marker gene on chromosome 1 was responsible for the decrease of both bone marrow and blood eosinophil levels. These data suggest the existence of genes characterizing/distinguishing the eosinophilic phenotypes of MES and BN.MES-Cyba^mes on these chromosomes, and form the basis for positional cloning studies of the genes. These studies will advance the understanding of the mechanisms involved in eosinophil mobilization from the bone marrow and recruitment to the organs.

View full abstract

We found 6 spontaneous mutant mice with long pelage hair in our ICR breeding colony. The abnormal trait was restricted to long hair in these mice, which we named moja. They were fertile and showed the same growth and behavior as wild-type mice. To investigate the manner of the genetic inheritance of the moja allele, offspring were bred by mating the moja mice; all offspring had long pelage hair. Furthermore, we performed a reciprocal cross between moja mice and wild-type ICR mice with normal hair. All offspring exhibited normal hair suggesting an autosomal recessive inheritance of the trait. The moja/moja hair phenotype was maintained in skin grafted onto nude mice, suggesting that circulating or diffusible humoral factors regulating the hair cycle are not involved in the abnormal trait. The phenotype of moja/moja mice is similar to that of Fgf5-deficient mice. Therefore, we examined the expression of Fgf5 by RT-PCR in moja/moja mice. As expected, no Fgf5 expression was found in moja/moja mouse skin. PCR and DNA sequence analyses were performed to investigate the structure of the Fgf5 gene. We found a deletion of a 9.3-kb region in the Fgf5 gene including exon 3 and its 5’ and 3’ flanking sequences. Interestingly, the genomic deletion site showed insertion of a 498-bp early transposon element long terminal repeat. Taken together, these results suggest that the long hair mutation of moja/moja mice is caused by disruption of Fgf5 mediated by insertion of a retrotransposon.

View full abstract

Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL) levels were examined during consecutive reproductive states in Mongolian gerbils. The results indicate that FSH, LH, and PRL levels peak at proestrus, estrus, and diestrus, respectively. During early gestation in primiparous gerbils, gonadotropin levels were the lowest on day 6. This was followed by an increase in FSH and LH levels until days 18 and 15, respectively, with levels remaining constant until day 21. However, in multiparous gerbils, gonadotropin levels were the lowest on day 12 of gestation and were relatively stable between days 15 to 21. In both primiparous and multiparous gerbils, gonadotropin levels increased rapidly from day 21 of gestation to day 3 of lactation, and kept stable between 6–24 days of lactation. PRL peaked during early gestation on days 9 and 6 in the primiparous and multiparous gerbils, respectively, followed by a decline. PRL levels subsequently peaked again on day 21 before parturition. During lactation, PRL levels peaked on days 6 and 9 in primiparous and multiparous gerbils, respectively, followed by a decline until lactation ended. These findings suggest that variations in gonadotropin during the estrous cycle, gestation, and lactation in Mongolian gerbils are similar to those observed in rats, whereas prolactin levels differ. Changes in gonadotropin and prolactin levels during different reproductive states were found to be similar in primiparous and multiparous gerbils, and were correlated with the reproductive stages of Mongolian gerbils.

View full abstract

Japanese macaques bred indoor for laboratory use often show chronic anorexia and intermittent vomiting. In some of our macaques gastric air was observed on physical examination, and we suspected abnormality of gastric motility. We therefore performed contrast radiographic examinations of the gastrointestinal tract without anesthesia of 8 macaques with gastrointestinal symptoms and 9 asymptomatic controls from the same laboratory. Changes of abdominal radiography over time were observed following oral administration of contrast medium. In all control animals, contrast medium had completely passed from the stomach within 150 min after administration. However, all animals with gastrointestinal symptoms retained some contrast medium in the stomach. Gastric emptying time of contrast medium was associated with excessive gastric air in Japanese macaques; therefore, gastric emptying time seems to be associated with decreased gastric motility.

View full abstract

NOD/Shi-scid IL-2Rγ^null (NOG) mice established by introducing the IL-2Rγ^null gene of IL-2Rγ KO mice into NOD/Shi-scid mice by backcross-mating show a high xenograft engraftment level and are therefore well suited as a humanized mouse model. SCID mice bearing the Prkdc^scid gene show a high incidence of thymic lymphoma and a leaky phenomenon in which a few clonal T and B cells develop in aged mice. In the present study, NOG mice were assessed for the presence of a leaky phenomenon such as the one observed in C.B-17-scid and NOD-scid mice. Serum immunoglobulin analysis did not detect IgG or IgM in NOG mice, unlike the findings in C.B-17-scid and NOD-scid mice. Flow cytometry analysis revealed the absence of T and B cells in the peripheral blood and spleens of NOG mice. These results reflect the suppression of the leaky phenomenon in NOG mice through the inactivation of the IL-2Rγ gene, which is commonly expressed in T and B cell growth factor receptors to IL-2, IL-4 and IL-7.

View full abstract

The aim of this study was to investigate the effects of hepatic and renal failure on the pharmacokinetics of flunixin in carbon tetrachloride (CCl₄)- and glycerol-treated rats. After intravenous administration of flunixin (2 mg/kg), the plasma concentration of flunixin was measured by high-performance liquid chromatography. Both acute hepatic and renal failure resulted in significantly increased area under the curve (AUC), prolonged elimination half-life (t_1/2β), and reduced total body clearance (Cl_tot) compared with respective controls (P<0.05). In conclusion, hepatic failure as well as renal failure modified the pharmacokinetics of flunixin.

View full abstract

For accurate protein quantification when using quantitative western blot analysis with chemiluminescence reagents, standard curves are needed because of the narrow quantifiable ranges. However, they are often difficult to obtain because authentic proteins are not always available. Here we present our original and convenient method using a sample mixture as a scale to create standard curves. This method allowed us to determine the quantifiable range of target and loading control proteins, making quantitative comparisons among independent blots more reproducible. Our results indicate that using a sample mixture to create standard curves is a practical method that guarantees the accuracy and reproducibility of quantitative western blot analysis.

View full abstract