Toluene Exposure Leads to a Change in Expression Patterns of β Defensins in the Mouse Tracheal Epithelium

Defensins are generally implicated in the quick resistance of epithelial surfaces to microbials; however, recent reports have indicated that defensins also have unknown purposes in relation to noninfectious diseases. In this study, the localization patterns of anti-microbial peptides, β defensins (BDs), in the tracheal epithelium of male C3H mice under exposure to toluene were analyzed by immunohistochemistry. Mice were exposed one to ten times to toluene for 30 min by nose-only inhalation. Expression of BDs was revealed by immunohistochemistry in serial sections of trachea after the final exposure. Expression of BD-1 was usually observed at almost the same levels in all exposure groups, and expression of BD-2 was observed in the control group; however, the signals for BD-2 decreased gradually with frequency of exposure. In the group exposed ten times, expression of BD-2 decreased to far lower than that of the control group. No expression of BD-3 was detected in any groups. Interestingly, expression of BD-4 increased to the maximum in the group exposed four times and decreased to a level lower than that of the control in the group exposed ten times. The results of the present study indicated that toluene gas might change the expression pattern of BDs in the tracheal epithelial cells. The oscillation of expression of BD-4 was quite characteristic and might contribute to morphological damage in on the epithelial cells.     

Chemical-induced lung tumor in Tg-rasH2 mice: a novel mouse tumor model to assess immune checkpoint inhibitors combined with a chemotherapy drug

In subcutaneous tumor models, changes in the tumor microenvironment can lead to differences in therapeutic treatment responses between the subcutaneous and parent tumors. Accordingly, we generated a lung carcinogenesis model that combines genetically modified mice (Tg-rasH2 mice) with two-stage chemical carcinogenesis as an alternative to the subcutaneous tumor model. In this model, Tg-rasH2 mice were treated with 1-ethyl-1-nitrosourea, followed by butylhydroxytoluene. Mice developed lung adenomas five weeks after treatment initiation. Subsequently, anti-mouse PD-1 antibody (α-mPD-1) or isotype control was administered intraperitoneally twice a week for 4 weeks. Tumor growth was examined by measuring the relative tumor area in serially sliced lung histopathological specimens. No statistically significant differences were observed in the relative lung tumor areas between treated and control groups. A second experiment then examined the antitumor efficacy of α-mPD-1 combined with gemcitabine in a mouse model. Mice were treated identically as in Experiment 1, except that the treated group received once-weekly intraperitoneal injections of 10 mg/kg gemcitabine. In contrast to Experiment 1, the combined treatment significantly reduced the relative tumor areas in the lungs. This result also resembles that of a phase III clinical trial (ORIENT-12), showing that patients with non-small-cell lung carcinoma benefited from combination treatment with gemcitabine and the anti-human PD-1 antibody sintilimab. Thus, this mouse model could be a feasible means to preclinically evaluate the antitumor efficacy of different immunotherapy and chemotherapy drug combinations.     

Isolation of antifouling substances from the Palauan sponge Protophlitaspongia aga

ABSTRACT: Fouling organisms cause serious problems by settling on ships' hulls, and other marine infrastructures. Organotin compounds have been developed as effective antifoulants. However, environmental problems are associated with these compounds. It is necessary to find antifouling substances that exhibit a lower toxicity than those substances currently used. A new pyrimidine derivative, 3,4,5,6-tetrahydro-6-hydroxymethyl-3,6-dimethyl-4-pyrimidinecarboxylic acid, (compound 1) and zooanemonin (compound 2) were isolated from the marine sponge Protophlitaspongia aga as antifouling substances against the barnacle Balanus amphitrite . We also found that α-nicotinamide ribose (compound 3) inhibited germination and the attachment of Ulva spores.     

Evaluation of developmental changes in the coexpression of myosin heavy chains and metabolic properties of equine skeletal muscle fibers

OBJECTIVE: To determine the growth-related changes in metabolic and anatomic properties in equine muscle fiber type, including hybrid fibers identified with immunohistochemical analysis. ANIMALS: 24 2-, 6-, 12-, and 24-month-old female Thoroughbreds. PROCEDURE: Samples were obtained from the gluteus medius muscle of all horses. Expression of myosin heavy chain (MHC) isoforms MHC-I, -IIa, -IIb, and -IIx in each muscle fiber was detected by use of 4 primary monoclonal antibodies: BA-D5, SC-71, BF-F3, and BF-35, respectively. Five muscle fiber types (types I, I/IIA, IIA, IIA/IIX, and IIX) were immunohistochemically identified. The area and activity of succinic dehydrogenase (SDH) in each fiber type were determined by use of quantitative histochemical staining and image analysis. RESULTS: Although the proportion of type I and IIX fibers did not change with age, the proportion of type IIA and IIA/IIX fibers significantly increased and decreased, respectively, from 2 months to 24 months of age. The increase in proportion of type IIA fibers with growth may have been attributable to muscle fiber-type transition from type IIA/IIX fibers but not from type IIX fibers. Values for SDH activity and fiber area in hybrid fiber types were intermediate to those for their respective pure phenotypes. CONCLUSIONS AND CLINICAL RELEVANCE: Hybrid fibers have an important role for determining the proportion of muscle fiber type in horses < 24 months old, and the metabolic and anatomic properties of the hybrid fibers are well coordinated, as in mature horses.     

Effect of growth and training on muscle adaptation in Thoroughbred horses

OBJECTIVE: To determine the effect of growth and training on metabolic properties in muscle fibers of the gluteus medius muscle in adolescent Thoroughbred horses. ANIMALS: Twenty 2-year-old Thoroughbreds. PROCEDURE: Horses were randomly assigned to 2 groups. Horses in the training group were trained for 16 weeks, and control horses were kept on pasture without training. Samples were obtained by use of a needle-biopsy technique from the middle gluteus muscle of each horse before and after the training period. Composition and oxidative enzyme (succinic dehydrogenase [SDHI) activity of each fiber type were determined by use of quantitative histochemical staining procedures. Whole-muscle activity of SDH and glycolytic enzyme (phosphofructokinase) as well as myosin heavy-chain isoforms were analyzed biochemically and electrophoretically, respectively. RESULTS: The SDH activity of type-I and -IIA fibers increased during growth, whereas whole-muscle activity was unchanged. Percentage of type-IIX/B muscle fibers decreased during training, whereas that of myosin heavy-chain IIa increased. The SDH activity of each fiber type as well as whole-muscle SDH activity increased during training. An especially noticeable increase in SDH activity was found in type-IIX/B fibers. CONCLUSIONS AND CLINICAL RELEVANCE: Changes in muscle fibers of adolescent Thoroughbreds are caused by training and not by growth.The most noticeable change was for the SDH activity of type-IIX/B fibers. These changes in the gluteus medius muscle of adolescent Thoroughbreds were considered to be appropriate adaptations to running middle distances at high speeds.     

Promotion of liver and kidney carcinogenesis by ethyl tertiary-butyl ether (ETBE) in male Wistar rats

Tumor-promoting effects of ethyl tertiary-butyl ether (ETBE) were investigated in a 2-stage carcinogenesis bioassay with regard to hepatic and renal carcinogenesis in rats. Male 6-week-old Wistar rats were given drinking water containing N-ethyl-N-(2-hydroxyethyl)nitrosamine (EHEN), as an initiator, at a dose of 500 ppm for 2 weeks. Starting one week thereafter, the animals were administered ETBE at dose levels of 0 (control), 100, 300, 500 or 1,000 mg/kg/day by gavage for 19 weeks from week 4 to 22. Necropsy of all rats was performed at week 23, and livers and kidneys were examined histopathologically. Incidences of hepatocellular adenomas, and those of combined hepatocellular adenomas and carcinomas were significantly elevated in rats given 1,000 mg/kg/day ETBE, but not 100‒500 mg/kg/day ETBE, and there was a significant increase in the average numbers of lesions. No significant differences in incidences and average numbers of renal tubule neoplasms were found in rats administered 100‒1,000 mg/kg/day ETBE. However, the average numbers of atypical tubule hyperplasias, considered to be preneoplastic lesions, were significantly increased in rats given ETBE at 1,000 mg/kg/day, but not in rats given 500 mg/kg/day or lower doses. Thus, these results imply that ETBE has hepatic and renal tumor-promoting activities that affect EHEN-induced carcinogenesis in male rats, and the no-observed-effect level is 500 mg/kg/day under the present experimental conditions.     

Successful management with CHOP for pulmonary lymphomatoid granulomatosis in a dog

A 3-year-old, spayed female miniature dachshund was presented for vomiting and anorexia. Thoracic radiographs and CT scan revealed abnormal pulmonary opacities at bilateral caudal lobe. Cytological analysis of the pulmonary mass revealed the presence of large lymphohistiocytic cells and small lymphocytes with occasional neutrophils and plasma cells. An open lung biopsy was performed and a diagnosis of pulmonary lymphomatoid granulomatosis (LYG) was made. The dog was administered CHOP based therapy (modified UW-25), and it survived for 1,022 days after admission. Immunohistochemistry revealed pulmonary lesions consisted of many CD79a positive B cells aggregation and proliferation with prominent angiocentric pattern. This was the first case of canine pulmonary LYG managed by CHOP chemotherapy.     

Molecular characterization of Cryptosporidium parvum detected in Japanese black and Holstein calves in Iwate Prefecture and Tanegashima Island,Kagoshima Prefecture,Japan

Cryptosporidium oocysts were found in 43 out of 77 calves from two farms in Iwate Prefecture and nine farms on Tanegashima Island, Kagoshima Prefecture, Japan. The DNA fragments of 18S ribosomal RNA (18S rRNA) gene were amplified by a nested PCR from 43 oocyst-positive as well as one oocyst-negative samples. All of them were precisely identified as C. parvum by analyzing the nucleotide sequences of the 18S rRNA gene. C. parvum oocyst-positive calves ranged in age from 6 to 13 days old and significantly have watery diarrhea (P<0.05). Sequences of the gene encoding the 60-kDa glycoprotein (GP60) in 43 Cryptosporidium oocyst-positive samples were identical to that of the zoonotic IIaA15G2R1 subtype. We therefore suggest that calves could be potential sources of C. parvum infections in humans.     

Centrifuged Liquid and Breadmaking Properties of Frozen‐and‐Thawed Bread Dough

Breadmaking properties (bread height, mm, and specific volume, cm³/g ) showed marked deterioration when bread dough was frozen and stored at ‐20°C for one day. However, these properties of bread dough baked after storage for three to six days were not further deteriorated as compared with that baked after one day of storage. A large amount of liquid was oozed from the frozen‐and‐thawed bread dough. The liquid was separated from the bread dough by centrifugation (38,900 × g for 120 min at 4°C), and collected by tilting the centrifuge tube at an angle of 45° for 30 min. There was a strong correlation between the amount of centrifuged liquid and breadmaking properties (bread height and specific volume). The mechanism responsible for the oozing of liquid in frozen‐and‐ thawed bread dough was studied. The presence of yeast and salt in bread dough was suggested to be closely related to the amount of centrifuged liquid, and fermented products particularly had a large effect on the amount of centrifuged liquid.     

A novel mouse wound model for scar tissue formation in abdominal muscle wall

Hypertrophic scars found on the human body rarely develop in experimental animals, possibly due to their looser skin structure. This makes it difficult to understand the genesis of scar lesions. Therefore, appropriate animal models are urgently needed. In this study, we established a novel experimental model of a scar-forming wound by resecting a small portion of the abdominal muscle wall on the lower center of the abdomen in C57BL/6N mice, which are exposed to contractive forces by the surrounding muscle tissue. As a low-tension control, a back skin excision model was used with a splint fixed onto the excised skin edge, and granulation tissue formed on the muscle fascia supported by the back skeleton. One week after the resection, initial healing reactions, such as fibroblast proliferation, occurred in both models. However, after 21 days, lesions with collagen-rich granulation tissues, which were also accompanied by multiple nodular/spherical-like structures, developed only in the abdominal wall model. These lesions were analogous to scar lesions in humans. Therefore, the animal model developed in this study is unique in that fibrous scar tissues form under physiological conditions without using any artificial factors and is valuable for studying the pathogenesis and preclinical treatment of scar lesions.