Assessment of rabbit spleen size using ultrasonography

Assessment of spleen size using the ultrasonography has become a standard practice in human. However, the assessment is not established method in experimental animals. To establish the index to assess the spleen size using ultrasonography, we measured the cross-section image of rabbit spleen during endotoxin shock. The image of the cross-section was appeared as triangle, and the height of the triangular image was defined as the spleen index. This spleen index showed strong correlation with the spleen weight. In conclusion, this method is suitable for observation of changes in rabbit spleen size and may reduce the number of rabbit in the longitudinal studies.     

A novel culture method of canine peripheral blood lymphocytes with concanavalin a and recombinant human interleukin-2 for adoptive immunotherapy

This study was designed to develop a novel culture method for the efficient proliferation of canine peripheral blood lymphocytes (cPBL) for adoptive immunotherapy. When cPBL were cultured in the presence of concanavalin A (Con A), proliferation of cPBL was induced and expression of interleukin-2 receptor (IL-2R) which enables to respond to exogenously added IL-2 was upregulated. And then, when cPBL were cultured with recombinant human interleukin-2 (rhIL-2) in addition to Con A, proliferation was accelerated and increased to about 10-fold after 1 week. The phenotypic analysis showed that the main population of the cultured cPBL was consisted of CD8+ positive lymphocytes. Among them, CD4+CD8+ double positive (DP) lymphocytes had significantly increased, and the ratio of CD4+ single positive (SP) lymphocytes to CD8+ SP lymphocytes (CD4+SP/CD8+SP) was decreased as compared to before culturing. To evaluate the cytotoxic activity of cPBL cultured with Con A and rhIL-2, furthermore, cytotoxic assay was carried out against xenogeneic melanoma cell line (MeWo), which resulted in MHC-unrestricted cytokilling. These results suggest that the culture method of cPBL by the use of Con A and rhIL-2 may be useful for generating lymphokine activated killer cells, and also this may be beneficial for adoptive immunotherapy of tumor-bearing dogs.     

Phenotype and function of murine discrete Peyer's patch macrophage derived - dendritic cells

The phenotype and function of peritoneal cavity macrophage-derived dendritic cells (PEC-DC) was previously reported. In this study we have gone further in using our established culture system to generated discrete Peyer's patch dendritic cells (DPP-DC) from murine discrete Peyer's patch macrophages (DPP-M?), following stimulation with granulocyte macrophage colony stimulating factor (GM-CSF) plus interleukin 4 (IL-4) for 7 days. DPP-M? from murine small intestines were obtained by mechanical disruption of discrete Peyer's patches (DPP), followed by metrizamide density gradient centrifugation to remove Peyer's patch resident DC and debri, after which an overnight adherent step in tissue culture medium was carried out for macrophage enrichment. Characterization of the generated DPP-DC was carried out using well-established criteria of morphology, expression of membrane antigens and capacity for antigen presentation. Dendritic cells expressed DEC-205, F4/80 and CD34 at high levels, but exhibited very low CD11c levels. They were shown to present soluble protein antigen to CD3(+) spleen T cells. A comparison of the surface antigen expression in the progenitor DPP-M? population and the generated DPP-DC showed a significant decrease in MHC class II levels and a marked down regulation of the co-stimulatory molecule CD86 (B7-2). High expression of the haemopoietic progenitor marker CD34 indicates that the generated DC, possess a haemopoietic rather than myeloid origin. Taken together, these results may provide a better understanding of the complex network regulating mucosal immune responses.     

A role for balance of interferon-gamma and interleukin-4 production in protective immunity against Neospora caninum infection

A suitable balance in the production of Th1/Th2-type cytokines has a crucial role in the control of microbial infections. We investigated cytokine production patterns and effects during Neospora caninum infection, based on two mouse models and an in vitro system. In the acute infection of N. caninum, BALB/c-background IFN-gamma-deficient mice that were sensitive to the N. caninum infection showed high levels of IL-10 production, whereas significant levels of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) production were observed in resistant wild type mice. BALB/c mice vaccinated with recombinant vaccinia virus expressing N. caninum surface protein NcSRS2 resisted parasite spread throughout the body, low levels of IFN-gamma production and high levels of IL-4 production were observed compared to unvaccinated animals. The treatment of N. caninum-infected cells with IFN-gamma or IL-10 decreased the host-cell viability in an in vitro system using mouse macrophage J774A.1 cells. On the other hand, IL-4, but not IL-10 administration, increased the viability of N. caninum-infected and IFN-gamma-treated cells. In the light of the balance of Th1/Th2-type cytokine production, an IFN-gamma/IL-4 balance may have a crucial role for the control of cellular responses against the parasite invasion.     

Seedborne fungi detected on stored solanaceous berry seeds and their biological activities

We isolated 629 fungi from 1296 berry seeds of solanaceous plants, including tomato (Lycopersicon esculentum), eggplant (Solanum melongena), bell pepper (Capsicum annuum), and red pepper (Capsicum annuum var. annuum) preserved for long and short terms. The isolates were classified into 22 genera excluding unidentified fungi, and the fungal floras were divided into two types: the tomato–eggplant and pepper groups. The results of cluster analysis with unweighted pair-group method with arithmetic average also supported these groups. Most tomato seeds infested with Geotrichum candidum germinated and grew the same as uninfested seeds. Cladosporium sphaerospermum and Arthrinium sp. isolated from eggplant seeds strongly suppressed germination, and Penicillium variabile suppressed seminal root elongation on eggplant. Alternaria alternata, Botrytis cinerea, and Myrothecium verrucaria detected from red pepper or bell pepper seeds were pathogenic to the fruits and the seedlings after artificial inoculation.     

Evaluation of three phytoplankton species as food for the pearl oyster <Emphasis Type="Italic">Pinctada fucata</Emphasis>

In the pearl cultivation farms of the Ehime Prefecture, Japan, mass mortalities of the pearl oyster Pinctada fucata have occurred since 1994. The occurrences of mass mortality roughly coincided with a shift of the dominant phytoplankton from Skeletonema and Chaetoceros to Chaetoceros and Nitzschia all of which belong to Bacillariophyceae. Hence, we evaluated Nitzschia, together with Chaetoceros and Isocrysis, as food for the oyster. Wet weights, lengths, widths, glycogen contents, and growth rates in terms of wet weight of the oysters in all the feeding treatments were significantly higher than those in the non-feeding treatment. The highest glycogen content (2.34%) and growth rate (2.21 g month⁻¹) were found in the Chaetoceros treatment. Growth rate in the Isocrysis treatment (1.63 g month⁻¹) was also high, although glycogen content in this treatment (0.41%) was low. In the Nitzschia treatment, growth rate of the oyster (0.94 g month⁻¹) was the lowest and glycogen content (0.83%) was also low relative to that in the Chaetoceros treatment. Chlorophyll a concentration in fecal pellets was lowest in the Nitzschia treatment (<2.7 μg mg⁻¹), suggesting more complete digestion of Nitzschia by the oyster. Thus, Nitzschia was edible and digestible but not assimilated by P. fucata. We propose the following scenario for the relationship between Nitzschia dominance and mass mortality. When Nitzschia dominates in a culture area, the physiological condition of P. fucata deteriorates due to low assimilation of Nitzschia by the oyster, followed by susceptibility of the oyster to infection by agents lethal to the oyster.     

Effects of mithramycin on transplantable canine perianal gland carcinoma (CAC-9) in nude mice: biochemical, histomorphometric, and ultrastructural investigations

A serially transplantable perianal gland carcinoma (CAC-9) was developed in nude mice from a hypercalcemic dog that has been maintained through passage 20. Tumor doubling rate of CAC-9 was 3.1 +/- 0.4 days. Mithramycin (MMC) injected intraperitoneally (8 mg/kg) into nude mice bearing CAC-9 markedly decreased the tumor volume 2 weeks post-injection. MMC returned the elevated serum and urine calcium levels in mice with CAC-9 back to similar values as controls. The few remaining viable tumor cells after MMC were large and had numerous aggregations of intermediate filaments that displaced cytoplasmic organelles. Histomorphometric evaluation of lumbar vertebrae reveled no significant differences in bone resorption of nude mice bearing CAC-9 compared to saline-treated controls. This rapidly growing tumor line in nude mice associated with mild hypercalcemia will be a useful animal model to evaluate combinations of chemotherapy for cancer-associated hypercalcemia.     

Comparison of bacterial endotoxin lipopolysaccharide concentrations in the blood,ovarian follicular fluid and uterine fluid: a clinical case of bovine metritis

We investigated the concentration of the bacterial endotoxin lipopolysaccharide (LPS) in the blood, ovarian follicular fluid and uterine fluid of a clinical case of bovine metritis. A 2-year-old lactating Holstein cow exhibited continuous fever >39.5°C for more than 2 weeks after normal calving. The cow produced a fetid, watery, red-brown uterine discharge from the vagina and was diagnosed with metritis. The LPS concentrations in plasma and uterine fluid were 0.94 and 6.34 endotoxin units (EU)/ml, respectively. One of seven follicles showed an extremely high level of LPS (12.40 EU/ml) compared to the other follicles (0.62–0.97 EU/ml). These results might suggest the presence of high concentration of LPS in follicles in cows with postpartum metritis.     

Growth of the fourspine sculpin <Emphasis Type="Italic">Cottus kazika</Emphasis> in the Gonokawa River,Japan, and effects of water temperature on growth

ABSTRACT:   The fourspine sculpin Cottus kazika is indigenous to Japan and found in Honshu except for the waters facing the Seto Inland Sea, and was also found in southern Shikoku and eastern Kyushu. This species has a catadromous lifestyle and migrates as juveniles from the sea to the middle reaches of rivers to grow. The growth pattern of this fish was investigated by a mark-and-recapture method from July 1994 to December 1996, in the Nigorikawa River, a tributary of the Gonokawa River system, Shimane Prefecture. 0-year-old fish of 50–70 mm total length ( TL ) occurred in the study area from June to July, grew to 90–140 mm  TL by the following April, and attained 160–210 mm  TL by December. This fish grew rapidly in September–November and April–July, almost ceasing to grow in July–September. It seems that this stagnant growth phase in summer is a characteristic of the seasonal growth pattern of C. kazika . A rearing experiment indicated that the growth rate of C. kazika was higher at 16–22°C than at 12–14 and 24–26°C. This result supports the field evidence of a stagnant growth phase in summer in the Nigorikawa River.