Kyoung-Hwan Joo

Clinical evaluation of the therapeutic efficacy of praziquantel (Embay 8440) against Clonorchis sinensis infection in man

A total of 63 patients with proven Clonorchis sinensis infection were treated with praziquantel at two dose levels: 35 patients received 3×25·0 mg/kg body weight (bwt) on a single day, and 28 patients were treated with a single dose of 40·0 mg/kg bwt. Follow-up examinations were carried out at about 30 and 60 days after treatment. Where eggs were found at the first or second follow-up examination, patients were treated again with the same dosage of praziquantel and the follow-up continued for another 60 days. Two months after therapy 30 of 35 patients treated with 3×25·0 mg/kg bwt were parasitologically cured. Five patients received the dosage of 3×25·0 mg/kg bwt for a second time and were also cured. After a single dose of 40·0 mg/kg bwt, only seven of 28 patients were cured at 60 days after therapy. Twenty-one patients received 40·0 mg/kg bwt once for a second time and seven of these 21 patients were cured. The remaining 14 patients showed egg reduction rates in excess of 90%. Extended haematological an...

Immunochemical and biological analysis of allergenicity with excretory-secretory products of Anisakis simplex third stage larva

Background:Anisakis simplex third stage larvae (L3) are parasites that frequently give rise to allergic responses. The larvae molt into fourth stage larvae (L4), and at each stage they produce L3-excretory-secretory products (L3-ESP) and L4-ESP, respectively, which are different in their main protein constituents. Although the allergenicity of L4-ESP has been investigated by several research groups, research on the allergenicity of L3-ESP has not been carried out by any researcher. In this investigation, the allergenicity and antigenicity of L3-ESP were investigated in comparison with L4-ESP, using rat sera. Methods: Rat sera were produced by L3 oral infection two times with a 9-week interval. Larvae ESP prepared by culture were concentrated and fractioned using lyophilizer and a centrifugal filter device, respectively. Immunochemical analysis was performed using both indirect ELISA and immunoblot. Biological allergenicity was analyzed by RBL-2H3 exocytosis. Results: With the indirect ELISA, the optical density (OD) value of the nonfractioned (NF)-L3ESP was only one third of that of the NF-L4ESP in both specific IgM and IgG. On measuring specific IgE, the OD of NF-L3ESP was less than one tenth of that of NF-L4ESP. In addition, neither antigen nor allergen was shown in NF-L3ESP, but it was shown in NF-L4ESP with immunoblot. However, the biological allergenicity of NF-L3ESP was comparable to that of NF-L4ESP. To demonstrate the presence of any allergen, L3-ESP was fractioned and found to carry twelve visualized allergen bands from 10 to 186 kDa by immunoblot. Conclusions: These results indicate that L3-ESP may include the important allergens necessary to induce the allergy by L3 oral infection, as compared to L4-ESP.

Immunization with Trichinella spiralis Korean Isolate Larval Excretory–Secretory Antigen Induces Protection and Lymphocyte Subset Changes in Rats

Protection and immune responses were studied in rats immunized with Trichinella spiralis muscle stage larval excretory–secretory antigen (ES Ag) without adjuvant. Protection was assessed by the degree of adult worm burden and the yield of muscle (diaphragmatic) larvae after challenge infection with live larvae. Lymphocyte subsets were identified by flow cytometry in the spleen and peripheral blood. Cytokine production and specific IgG, IgG1 and IgG2a antibody responses were measured. Immunization with ES Ag produced highly significant protection against adult stages (98.4%) and muscle larvae (82.9%). Th2 type cytokines (IL‐10, IL‐4) were predominant. Anti‐muscle stage larval ES Ag antibody was significantly elevated in the order IgG2a > IgG1 > IgG on the 2nd day after final immunization and on the 7th day after challenge infection. Expression of CD4+ and the CD4+/CD8+ ratio from spleen and blood were significantly increased compared to the control. These results demonstrate that immunization with T. spiralis antigen can elicit robust immune response, resulting in complete protection against infective larvae, and this protection can be achieved without the use of any adjuvant.

PURIFICATION AND CHARACTERIZATION OF A 7-KDA PROTEIN FROM CLONORCHIS SINENSIS ADULT WORMS

A 7-kDa protein was purified from extracts of adult Clonorchis sinensis by a combination of ammonium sulfate precipitation, anion exchange chromatography, cation exchange chromatography, gel-filtration chromatography, and reversed-phase FPLC. The 7-kDa protein exists in the excretory–secretory products of adult C. sinensis, but not in extracts of adult Paragonimus westermani. Also, the 7-kDa protein reacted with the sera of patients with clonorchiasis but not with paragonimiasis or normal human sera. To observe the localization of the 7-kDa protein in the tissue of adult C. sinensis, an immunogold labeling method was followed using anti-7-kDa antibody. The gold particles were observed in the basal layer below the tegumental syncytium, in the interstitial matrix of the parenchyma, and in the content of the uterus. The 7-kDa cDNA was obtained through reverse transcription-polymerase chain reaction using a primer designed from N-terminal sequence analysis. Rapid amplification of cDNA ends (5′-RACE) was used to obtain the complete protein coding sequence. The sequence encodes a 90-amino acid polypeptide. The deduced amino acid sequence of the 7-kDa protein revealed no homology with proteins of different organisms reported so far. These results suggest that the 7-kDa protein is a fluid antigen and may be valuable as a tool for the immunodiagnosis of clonorchiasis.

Expression of thioredoxin during progression of hamster and human cholangiocarcinoma.

Thioredoxin (Trx) is a multifunctional redox protein that has growth‐promoting and anti‐apoptotic effects on cells and protects cells from endogenous and exogenous free radicals. Recently, altered expression of Trx has been reported in various cancers. In the present study, we investigated altered expression of Trx at the precancerous and carcinogenic phases during cholangiocarcinogenesis in a hamster cholangiocarcinoma (ChC) model, using semiquantitative immunohistochemical and Western blot analyses. Moreover, to determine if the results correlated well with those in human ChCs, we carried out a comparative immunohistochemical study for Trx in tissue‐arrayed human ChCs with different grades of tumor cell differentiation. Trx was found highly expressed in the cytoplasm of dysplastic bile ducts with highly abnormal growth patterns and ChCs irrespective of tumor type or tumor cell differentiation. Overexpression of Trx at the precancerous and carcinogenic phases was further supported by significant elevation of Trx protein in Western blotting. The results from the hamster ChCs were in good agreement with those from human ChCs. Our results strongly suggested that the redox regulatory function of Trx plays an important role in bile duct cell transformation and tumor progression during cholangiocarcinogenesis. (Cancer Sci 2009)

Enhanced protection against Clonorchis sinensis induced by co-infection with Trichinella spiralis in rats

Although co‐infection with multiple parasites is a frequent occurrence, changes in the humoral immune response against a pre‐existing parasite induced as a result of a subsequent parasitic infection remain undetermined. Here, we utilized enzyme‐linked immunosorbent assay (ELISA) to investigate antibody responses, cytokine production and enhanced resistance in Clonorchis sinensis‐infected rats (Sprague–Dawley) upon Trichinella spiralis infection. Higher levels of C. sinensis‐specific IgG and IgA were elicited upon T. spiralis infection, and these levels remained higher than in rats infected with C. sinensis alone. Upon subsequent infection with T. spiralis, IgG antibodies against C. sinensis appeared to be rapidly boosted at day 3, and IgA antibodies were boosted at day 7. Challenge infection of C. sinensis‐infected rats with T. spiralis induced substantial mucosal IgG and IgA responses in the liver and intestine and increases in antibody‐secreting plasma cells in the spleen and bone marrow. Subsequent infection also appeared to confer effective control of liver C. sinensis loads, resulting in enhanced resistance. Memory B cells generated in response to C. sinensis infection were rapidly amplified into antibody‐secreting cells upon T. spiralis infection. These results indicate that enhanced C. sinensis clearance induced by co‐infection is associated with systemic and mucosal IgG and IgA responses.

Cell Death and Proliferation after Treatment and Reinfection of Clonorchis sinensis in the Sprague-Dawley Rat Bile Duct

The structural change and distribution of mitochondrial enzyme (ATPase, cytochrome-c-oxidase), cell proliferation (proliferating cell nuclear antigen, PCNA), cell death (caspase-3) and cell growth factor (fibroblast growth factor 8, FGF-8) in the Sprague-Dawley rat bile duct during Clonorchis sinensis infection was investigated. Experimental groups were divided into C. sinensis infection, superinfection and reinfection of C. sinensis after `praziquantel` treatment group. As a result, C. sinensis infected rat bile ducts showed the features of chronic clonorchiasis, i.e., connective tissue thickening, ductal fibrosis and epithelial tissue dilatation. PCNA for cell proliferation increased in the infection group, and decreased after praziquantel treatment. Caspase-3 was distributed in reinfection group only. FGF-8 was distributed in the rat bile duct after praziquantel treatment but not distributed in infection and reinfection group. Overall, C. sinensis infection causes physical and chemical irritations and then brings on the abnormalities of intracellular energy metabolism and cellular growth factors, which hinders bile duct tissue from functioning properly, and resultingly, fibrosis occurs and epithelial cells dilated abnormally. More intense infection makes tissue fibrosis chronical and activates apoptosis factors.

Ultrastructure of Capillaria hepatica (Syn. Calodium hepatica) Isolated from the Liver of Mouse Infected with Artificially Embryonated Eggs Collected from House Rats (Rattus norvegicus)

Capillaria hepatica (syn. Calodium hepatica; Bancroft, 1893) is a parasitic nematode living in the liver of rodents and other mammals such as rabbits, dogs, cats, cattle, and humans (Singleton et al., 1991; Roberts et al., 1996; Anderson, 2000; Nabi et al., 2007). About 30 cases of human infections have been reported from Asia, Africa, Europe, North and South America, and Oceania including United States of America, Japan, and Korea (McQuown, 1950; Choe et al., 1993; Kohatsu et al., 1995; Fuehrer et al., 2011). Rat species of the genus Rattus are main primary host of C. hepatica and infection rates of up to 100% have been reported (Farhang-Azad, 1977; Singleton et al., 1991; Ceruti et al., 2001; Claveria et al., 2005). In addition to the principal hosts, rodents, other mammals can be infected through ingestion of water or food contaminated by infectious embryonated eggs. C. hepatica does not require an intermediate host in their life cycle and thus death of the host is only way to release the embedded eggs. When the embryonated eggs are ingested by a new host, they develop into the adult worm in the liver of the host within approximately 20 days. The natural lifespan of the adult female worm is approximately 60 days (Spratt & Singleton, 2001; Kim et al., 2007) and that of male Ultrastructure of Capillaria hepatica (Syn. Calodium hepatica) Isolated from the Liver of Mouse Infected with Artificially Embryonated Eggs Collected from House Rats (Rattus norvegicus)