Ibriosis, which is associated with the ingestion of raw, undercooked, or

Ibriosis, which is associated with the ingestion of raw, undercooked, or contaminated seafood [1,2]. Grimontia CASIN hollisae (previously named V. hollisae) has been frequently reported to cause diseases in humans, including severe gastroenteritis, hypovolemia, and septicemia following the consumption of shellfish or oysters [4?]. However, the mechanism of morbidity due to G. hollisae has not been thoroughly characterized. Although several specific laboratory examinations have detected G. hollisae, its incidence is likely highly underestimated because detection techniques are infrequently used throughout the world [7,8].The toxin thermostable direct hemolysin (TDH) is composed of 165 amino acid residues and exhibits biological activities that include hemolytic activity, cytotoxicity, and enterotoxicity [9]. The toxic effects of TDH have been identified in a variety of Vibrio species, including V. cholera non-O1, V. parahaemolyticus, V. mimicus, V. alginolyticus, and G. hollisae [10?4]. In addition, TDH production has been observed in bacteria with similar tdh gene sequences and pathogenicities [15,16]. The tdh gene is present in all strains of G. hollisae but not in all Vibrio species [10]. The physical characteristics of G. hollisae TDH (Gh-TDH) are not well known. Some studies have reported that Gh-TDH is detoxified by aggregation into fibrils after heating at 60?0uC; the protein can be reversibly refolded into the toxic native form by rapid cooling after unfolding at higher temperatures [17]. The hemolytic activity of Gh-TDH is suppressed by the addition of Congo red [18]. In addition, the lipophilic effect of Gh-TDH has not been clearlyHepatotoxicity of Thermostable Direct Hemolysincharacterized. The toxic effects of TDH have been localized mainly in the intestinal portion of the gastrointestinal tract [19?21]. However, a relationship between the TDH and the liver has not been reported or analyzed. In addition to being absorbed by the intestine, TDH may also cause secondary injury to the liver via effects on the venous return of the portal system. In this study, we analyzed the hepatotoxicity of TDH in vivo and in vitro to provide insights into the acute injury and recovery stages of THD-induced hepatotoxicity in living animals.well) were independently 4EGI-1 treated with 10 mg/ml Gh-rTDHFITC for 20 and 40 min, washed 3 times with PBS, and stained with propidium iodide (PI) for 5 min in the dark. Images were 18325633 acquired by confocal microscopy (wavelength: lex = 488 and lem = 650 nm).In vivo Hepatotoxicity of Gh-rTDH in BALB/c MiceA total of 114 six-week-old female mice were obtained from the National Laboratory Animal Center of Taiwan for the analysis of in vivo hepatotoxicity. All mice were fed normal diets. This study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of National Chiao Tung University (Permit Number: 01001008). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.Materials and Methods Bacterial Strains and MaterialsG. hollisae strain ATCC 33564 was obtained from the Culture Collection and Research Center (Hsin-Chu, Taiwan). Phenyl Sepharose 6 Fast Flow and protein molecular weight standards were purchased from GE Healthcare (Piscataway, NJ). The protein assay kit was obtained from Bio-Rad (.Ibriosis, which is associated with the ingestion of raw, undercooked, or contaminated seafood [1,2]. Grimontia hollisae (previously named V. hollisae) has been frequently reported to cause diseases in humans, including severe gastroenteritis, hypovolemia, and septicemia following the consumption of shellfish or oysters [4?]. However, the mechanism of morbidity due to G. hollisae has not been thoroughly characterized. Although several specific laboratory examinations have detected G. hollisae, its incidence is likely highly underestimated because detection techniques are infrequently used throughout the world [7,8].The toxin thermostable direct hemolysin (TDH) is composed of 165 amino acid residues and exhibits biological activities that include hemolytic activity, cytotoxicity, and enterotoxicity [9]. The toxic effects of TDH have been identified in a variety of Vibrio species, including V. cholera non-O1, V. parahaemolyticus, V. mimicus, V. alginolyticus, and G. hollisae [10?4]. In addition, TDH production has been observed in bacteria with similar tdh gene sequences and pathogenicities [15,16]. The tdh gene is present in all strains of G. hollisae but not in all Vibrio species [10]. The physical characteristics of G. hollisae TDH (Gh-TDH) are not well known. Some studies have reported that Gh-TDH is detoxified by aggregation into fibrils after heating at 60?0uC; the protein can be reversibly refolded into the toxic native form by rapid cooling after unfolding at higher temperatures [17]. The hemolytic activity of Gh-TDH is suppressed by the addition of Congo red [18]. In addition, the lipophilic effect of Gh-TDH has not been clearlyHepatotoxicity of Thermostable Direct Hemolysincharacterized. The toxic effects of TDH have been localized mainly in the intestinal portion of the gastrointestinal tract [19?21]. However, a relationship between the TDH and the liver has not been reported or analyzed. In addition to being absorbed by the intestine, TDH may also cause secondary injury to the liver via effects on the venous return of the portal system. In this study, we analyzed the hepatotoxicity of TDH in vivo and in vitro to provide insights into the acute injury and recovery stages of THD-induced hepatotoxicity in living animals.well) were independently treated with 10 mg/ml Gh-rTDHFITC for 20 and 40 min, washed 3 times with PBS, and stained with propidium iodide (PI) for 5 min in the dark. Images were 18325633 acquired by confocal microscopy (wavelength: lex = 488 and lem = 650 nm).In vivo Hepatotoxicity of Gh-rTDH in BALB/c MiceA total of 114 six-week-old female mice were obtained from the National Laboratory Animal Center of Taiwan for the analysis of in vivo hepatotoxicity. All mice were fed normal diets. This study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of National Chiao Tung University (Permit Number: 01001008). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.Materials and Methods Bacterial Strains and MaterialsG. hollisae strain ATCC 33564 was obtained from the Culture Collection and Research Center (Hsin-Chu, Taiwan). Phenyl Sepharose 6 Fast Flow and protein molecular weight standards were purchased from GE Healthcare (Piscataway, NJ). The protein assay kit was obtained from Bio-Rad (.