Identification of the transcriptome profile of Miamiensis avidus after mebendazole treatment / 고신대학교의과대학학술지

Background@#The scuticociliate Miamiensis avidus is a major pathogenic agent that causes significant economic losses in the flounder aquaculture industry. Many different types of drugs are being tested to control this disease, including mebendazole, which is a broad-spectrum antiprotozoal agent. The purpose of this study was to determine whether mebendazole worked in vitro against M. avidus and to explore its mechanism of action. @*Methods@#Transcriptome and gene ontology analyses were conducted to investigate the specifically expressed gene profile. We confirmed the cytotoxic effect of mebendazole against M. avidus when it was applied intermittently for a total of three times. We also identified differentially expressed genes using transcriptome analysis. @*Results@#Most of the upregulated genes were membrane transport-related genes, including Na+/K+-ATPase. Most of the downregulated genes were categorized into three groups: tubulin-related, metabolism-related, and transport-related genes. The expression levels of glucose uptake-related genes decreased due to the inhibition of tubulin polymerization, but this was not statistically significant. @*Conclusions@#Our results demonstrate that intermittent treatment with mebendazole has a significant cytotoxic effect on M. avidus. Furthermore, mebendazole induces downregulation of the tubulin-alpha chain and metabolism-related genes. It is presumed that this leads to a glucose shortage and the death of M. avidus. Transcriptome analysis will provide useful clues for further studies on mebendazole applications for scutica control.

Correlation of long interspersed element-1 open reading frame 1 and c-Met proto-oncogene protein expression in primary and recurrent colorectal cancers / 고신대학교의과대학학술지

Background@#Colorectal cancer is one of the most common cancers worldwide. Colorectal cancer that has recurred and metastasized to other organs also has a very poor prognosis. According to recent studies, the long interspersed element-1 (LINE-1) retrotransposon open reading frame (ORF) is located in the intron of the c-Met proto-oncogene, which is involved in cancer progression and metastasis, and regulates its expression. However, no study has compared the expression patterns of LINE-1 ORF1 and c-Met, which are closely related to cancer progression and metastasis, and their correlation in primary and recurrent cancers. @*Methods@#In the present study, we compared the expression patterns of LINE-1 ORF1 and c-Met in both primary and recurrent colorectal cancer tissues from 10 patients. Expression patterns and correlations between LINE-1 ORF1 and c-Met proto-oncogene proteins were analyzed by immunofluorescence staining using both LINE-1 ORF1 and c-Met antibodies. @*Results@#The expression patterns of LINE-1 ORF1 and c-Met showed significant individual differences, and the expression of both proteins was correlated in all colorectal cancer patients. However, the expression levels of LINE-1 ORF1 and c-Met were not significantly different between primary and recurrent colorectal cancers. @*Conclusions@#The protein expression levels of LINE-1 ORF1 and c-Met were correlated, but did not change significantly in cases of recurrent colorectal cancer in the same patient.

Induction of Angiogenesis by Malarial Infection through Hypoxia Dependent Manner

Malarial infection induces tissue hypoxia in the host through destruction of red blood cells. Tissue hypoxia in malarial infection may increase the activity of HIF1α through an intracellular oxygen-sensing pathway. Activation of HIF1α may also induce vascular endothelial growth factor (VEGF) to trigger angiogenesis. To investigate whether malarial infection actually generates hypoxia-induced angiogenesis, we analyzed severity of hypoxia, the expression of hypoxia-related angiogenic factors, and numbers of blood vessels in various tissues infected with Plasmodium berghei. Infection in mice was performed by intraperitoneal injection of 2×10⁶ parasitized red blood cells. After infection, we studied parasitemia and survival. We analyzed hypoxia, numbers of blood vessels, and expression of hypoxia-related angiogenic factors including VEGF and HIF1α. We used Western blot, immunofluorescence, and immunohistochemistry to analyze various tissues from Plasmodium berghei-infected mice. In malaria-infected mice, parasitemia was increased over the duration of infection and directly associated with mortality rate. Expression of VEGF and HIF1α increased with the parasitemia in various tissues. Additionally, numbers of blood vessels significantly increased in each tissue type of the malaria-infected group compared to the uninfected control group. These results suggest that malarial infection in mice activates hypoxia-induced angiogenesis by stimulation of HIF1α and VEGF in various tissues.

Preventive and Therapeutic Effects of Anisakis simplex Larval Protein in a Mouse Model of Crohn'S Disease / 고신대학교의과대학학술지

OBJECTIVES: Some helminths have been known to have a treatment effect in inflammatory bowel diseases, including Crohn's disease (CD); however, live parasite therapy can cause unwanted side effects. To develop a safe therapeutic, we investigated the preventive or therapeutic potential of proteins from the third stage larva of A. simplex in a mouse model. We also analyzed the cytokine profile from splenic and mesenteric lymph node lymphocytes to elucidate the underlying immunological mechanism. METHODS: CD was induced in mice with DSS, and the effect of an A. simplex larval protein on CD was assessed. A change in body weight and DAI (disease activity index) were observed in mice. The expression levels of cytokines from mesenteric lymph nodes (MLN) compared to splenic lymphocytes were measured with ELISA. RESULTS: Peritoneal administration of preventive and therapeutic A. simplex larval proteins attenuated DSS-induced CD by a reduction of the DAI and weight loss. A shortening of colon length was more definitely observed in the therapeutic group than in the preventive group. The cytokine expression levels were more obvious in lymphocytes from mesenteric lymph nodes than from splenic lymphocytes. CONCLUSIONS: Taken together, these results suggest that A. simplex proteins can change cytokine profiles and may have a preventive effect in DSS-induced CD mice.

Anisakidosis in Korea; Changes Over the Last Decade / 고신대학교의과대학학술지

Anisakidosis is caused by Anisakis simplex and other anisakids larvae parasitizing marine fish and cephalopods. A lot of case reports about anisakidosis have been published in Korea because of raw fish eating habits. Recently seafood consumption has continued to increase due to health concerns and thus, it increases the risk for infectious diseases including anisakidosis. The aim of this review is to analyze the clinical and epidemiological characteristics of anisakidosis during the last 10 years in Korea, based on the case reports published from 2000 to 2010. The incidence age was changed from 30s and 40s to 50s. The young generation was considered to consume seafood in various ways, including raw fish as well. The most noticeable change was the appearance of Anisakis allergy patients over the last decade. The patients showed abdominal pain, urticaria after eating sea food. It reaffirmed that anisakid infection induces not only gastric and intestinal anisakidosis but also cause allergic reaction. Anisakid should be considered as a possible causative food allergen provoking allergic responses after eating raw fish.

A 24 kDa Excretory-Secretory Protein of Anisakis simplex Larvae Could Elicit Allergic Airway Inflammation in Mice

We have reported that a 24 kDa protein (22U homologous; As22U) of Anisakis simplex larvae could elicit several Th2-related chemokine gene expressions in the intestinal epithelial cell line which means that As22U may play a role as an allergen. In order to determine the contribution of As22U to allergic reactions, we treated mice with 6 times intra-nasal application of recombinant As22U (rAs22U). In the group challenged with rAs22U and ovalbumin (OVA), the number of eosinophils in the bronchial alveolar lavage fluid (BALF) was significantly increased, as compared to the group receiving only OVA. In addition, mice treated with rAs22U and OVA showed significantly increased airway hyperresponsiveness. Thus, severe inflammation around the airway and immune cell recruitment was observed in mice treated with rAs22U plus OVA. The levels of IL-4, IL-5, and IL-13 cytokines in the BALF increased significantly after treatment with rAs22U and OVA. Similarly, the levels of anti-OVA specific IgE and IgG1 increased in mice treated with rAs22U and OVA, compared to those treated only with OVA. The Gro-alpha (CXCL1) gene expression in mouse lung epithelial cells increased instantly after treatment with rAs22U, and allergy-specific chemokines eotaxin (CCL11) and thymus-and-activation-regulated-chemokine (CCL17) gene expressions significantly increased at 6 hr after treatment. In conclusion, rAs22U may induce airway allergic inflammation, as the result of enhanced Th2 and Th17 responses.

Anisakis simplex Larvae: Infection Status in Marine Fish and Cephalopods Purchased from the Cooperative Fish Market in Busan, Korea

The infection status of marine fish and cephalopods with Anisakis simplex third stage larva (L3) was studied over a period of 1 year. A total of 2,537 specimens, which consisted of 40 species of fish and 3 species of cephalopods, were purchased from the Cooperative Fish Market in Busan, Korea, from August 2006 to July 2007. They were examined for A. simplex L3 from the whole body cavity, viscera, and muscles. A. simplex L3 were confirmed by light microscopy. The overall infection rate reached 34.3%, and average 17.1 larvae were parasitized per infected fish. Fish that recorded the highest infection rate was Lophiomus setigerus (100%), followed by Liparis tessellates (90%), Pleurogrammus azonus (90%), and Scomber japonicus (88.7%). The intensity of infection was the highest in Gadus macrocephalus (117.7 larvae per fish), followed by S. japonicus (103.9 larvae) and L. setigerus (54.2 larvae). Although abundance of A. simplex L3 was not seasonal in most of the fish species, 10 of the 16 selected species showed the highest abundance in February and April. A positive correlation between the intensity of L3 infection and the fish length was obvious in S. japonicus and G. macrocephalus. It was likely that A. simplex L3 are more frequently infected during the spring season in some species of fish. Our study revealed that eating raw or undercooked fish or cephalopods could still be a source of human infection with A. simplex L3 in Korea.

Cloning of a Family of Serine Protease Genes Expressed in German Cockroach(Blattella germanica) and Possibility as Allergens / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: The serine protease allergens have been known as one of the most important inhalent allergens of insects as well as house dust mites. There is no known serine protease allergen of German cockroach, which is a well-known etiological agent of allergic diseases. We identified the serine protease genes of cockroach, Blattella germanica, and examined its possibility as an allergen. MATERIALS AND METHOD: We have isolated three partial genes, Bg3, Bg5 and Bg6, of serine protease from the Blattella germanica cDNA library using the degenerate oligonucleotide PCR primers specific for the conserved regions. RESULTS: The genes, Bg3, Bg5 and Bg6, were composed of 396 bp 131 amino acids, 513 bp 170 amino acids, and 410 bp 136 amino acids, respectively. Northern hybridization analysis indicated that the size of Bg5 and Bg6 transcripts was approximately 0.7 kb. The gene, Bg5, was also identified as a multiple gene by the Southern blot analysis. The gene, Bg3, showed significant homology to trypsin-like serine protease of various insects. The deduced amino acids sequence of the gene Bg5 was matched to the mite group III allergen as well as various species of insect serine protease sequences. The gene Bg6 also showed high homology to the amino acids sequences of insect serine proteases and mite group III allergens. Three Blattella germanica serine protease gene fragments revealed close genetic relationships with Dermatophagoides farinae group III allergen in the phylogenetic analysis. CONCLUSION: The high homology and close phylogenetic relationship raise the possibility of three serine protease genes as being an allergen of German cockroach.

A serological survey of Dirofilaria immitis infection in pet dogs of Busan, Korea, and effects of chemoprophylaxis

The status of Dirofilaria immitis infection was assessed in pet dogs of Busan, Korea, and chemoprophylactic effects of microfilaricidal medication were evaluated. A total of 294 pet dogs older than 6 mo were examined, 217 of which had been maintained indoors, and 77 had been kept outdoors. The Snap(R) kit and direct microscopic examinations of the peripheral blood were used. The mean overall parasite positive rates were 10.2% and 6.5%, respectively. Outdoor dogs evidenced adult worm infection rate of 31.2% and microfilaria infection rate of 18.2%. The indoor dogs, however, evidenced adult worm infection rate of 2.8% and microfilaria infection rate of 2.3%. The prevalence in males was more than 2 times that of females. The changing pattern of infection rates by age evidenced a gradual increase, from 2- to 6-year-old dogs, after which, a decrease in infection rates was noted. With regard to chemoprophylaxis, the infection rates of complete and incomplete chemoprophylaxis groups were found to be 2-3 times lower than that of the non-chemoprophylaxis group. The results of the present study indicate that the risk of exposure to D. immitis in pet dogs is quite high, particularly in male outdoor dogs, and chemoprophylactic measures were quite effective.

Molecular characterization of Acanthamoeba isolated from amebic keratitis related to orthokeratology lens overnight wear

In an effort to characterize, on the molecular scale, the Acanthamoeba initially isolated from the cornea of an amoebic keratitis patient associated with overnight-wear orthokeratology lens in Korea, we conducted mitochondrial DNA restriction fragment length polymorphism, 18S rDNA sequencing, and drug sensitivity analyses on the isolate (KA/PE1). The patient was treated with polyhexamethylene biguanide, chlorhexidine and oral itraconazole, which resulted in resolution of the patient's ocular inflammation. The majority of the molecular characteristics of the KA/PE1 were determined to be identical, or quite similar, to those of A. castellanii Ma strain, which had been isolated also from amoebic keratitis. The risk of Acanthamoeba keratitis as a potential complication of overnight orthokeratology is briefly discussed.

Expressed sequence tags analysis of Blattella germanica

Four hundred and sixty five randomly selected clones from a cDNA library of Blattella germanica were partially sequenced and searched using BLAST as a means of analyzing the transcribed sequences of its genome. A total of 363 expressed sequence tags (ESTs) were generated from 465 clones after editing and trimming the vector and ambiguous sequences. About 42% (154/363) of these clones showed significant homology with other data base registered genes. These new B. germanica genes constituted a broad range of transcripts distributed among ribosomal proteins, energy metabolism, allergens, proteases, protease inhibitors, enzymes, translation, cell signaling pathways, and proteins of unknown function. Eighty clones were not well-matched by database searches, and these represent new B. germanica-specific ESTs. Some genes which drew our attention are discussed. The information obtained increases our understanding of the B. germanica genome.

Cloning and expression of trypsin-encoding cDNA from Blattella germanica and its possibility as an allergen

In this study, the trypsin gene (bgtryp-1) from the German cockroach, Blattella germanica, was cloned via the immunoscreening of patients with allergies to cockroaches. Nucleotide sequence analysis predicted an 863 bp open reading frame which encodes for 257 amino acids. The deduced amino acid sequence exhibited 42-57% homology with the serine protease from dust mites, and consisted of a conserved catalytic domain (GDSGGPLV). bgtryp-1 was determined by both Northern and Southern analysis to be a 0.9 kb, single-copy gene. SDS-PAGE and Western blotting analyses of the recombinant protein (Bgtryp-1) over-expressed in Escherichia coli revealed that the molecular mass of the expressed protein was 35 kDa, and the expressed protein was capable of reacting with the sera of cockroach allergy patients. We also discussed the possibility that trypsin excreted by the digestive system of the German cockroach not only functions as an allergen, but also may perform a vital role in the activation of PAR-2.

A Case of Echinococcal Hydatid Cyst as Misdiagnosed to the Bladder Cancer / 대한비뇨기과학회지

Hydatid disease is a parasitic infection that is caused by the larval stage of Echinococcus granulosus. This disease is widely distributed in a temperate and subtropical countries, and it is especially prevalent in sheep and cattle raising countries. The most common infection site is the liver, and the second most common site is the lung. We report here on our experience with Echinococcal hydatid cysts that were found in the bladder of a 38 year old worker from Uzbekistan. The cysts were surgically removed and the patient was managed postoperatively with albendazole.

A Case of Echinococcal Hydatid Cyst as Misdiagnosed to the Bladder Cancer / 대한비뇨기과학회지

Hydatid disease is a parasitic infection that is caused by the larval stage of Echinococcus granulosus. This disease is widely distributed in a temperate and subtropical countries, and it is especially prevalent in sheep and cattle raising countries. The most common infection site is the liver, and the second most common site is the lung. We report here on our experience with Echinococcal hydatid cysts that were found in the bladder of a 38 year old worker from Uzbekistan. The cysts were surgically removed and the patient was managed postoperatively with albendazole.

A case of acute gastric anisakiasis provoking severe clinical problems by multiple infection

Acute gastric anisakiasis with multiple anisakid larvae infection is reported. A 68-year-old woman residing in Busan, Korea, had epigastric pain with severe vomiting about 5 hours after eating raw anchovies. Four nematode larvae penetrating the gastric mucosae in the great curvature of the middle body and fundus were found and removed during gasteroendoscopic examination. Another one thread-like moving larva was found in the great curvature of upper body on the following day. On the basis of their morphology, the worms were identified as the 3rd stage larvae of Anisakis simplex. This case is acute gastric anisakiasis provoking severe clinical problems by the multiple infection and the greatest number of anisakid larvae found in a patient in Korea.

The intestinal parasite infection status of inhabitants in the Roxas city, the Philippines

We carried out a small-scale survey to investigate the status of intestinal protozoa and helminthes infection of inhabitants in Roxas city, Mindoro, the Philippines. Total 301 stool samples were subjected to the formalin-ether concentration method for the detection of helminth ova and protozoan cysts. The overall positive rate was 64.5%, and that of male and female were 56.6% and 72.5%, respectively. The highest infected helminth was Ascaris lumbricoudes (51.2%), followed by Trichuris trichiura (27.6%), hookworm (8.0%) and Enterobius vermicularis (0.3%). The protozoa infection status revealed that Entamoeba coli was the most frequent (15.0%). Iodoamoeba buetschlii and E. histolytica were found but few. The multiple infection more than two parasites was 29.6%, and double infection with A. lumbricoides and T. trichiura was most common. The intestinal helminth infections were highly prevalent in this area, according to this result, and we concluded that anthelminthic drugs should be given to inhabitants, especially to children of 1 to 15 years-old.

Egg positive rate of Enterobius vermicularis of primary school children in Geoje island

The status of pinworm (Enterobius vermicularis) egg positive rate of primary school children in Geoje island was investigated by using adhesive cellotape anal swap method, in September, 2002. Total egg positive rates of E. vermicularis were 9.8% (74/754) and those of male and female were 10.8% and 8.7%, respectively. Among three schools examined, Myeongsa primary school showed the highest egg positive rate (12.6%) followed by Yeoncho [9.8% (26/266)] and Geoje [9.1% (35/385)]. As for the age groups, the 2nd grade had the highest egg positive rate (15.3%), whereas the 5th grade showed the lowest egg positive rate (2.6%). The above result led us to confirm that the egg positive rates of E. vermicularis in primary school children in Geoje island were not significantly different from the those in the whole country including urban and rural areas, showing more than 10%.

Infection status of Clonorchis sinensis in residents of Hamyang-gun, Gyeongsangnam-do, Korea

Oriental liver fluke (Clonorchis sinensis) infection was surveyed among residents of Hamyang-gun, Gyeongsangnam-do, Korea during the period of January 2001 to March 2002. Total 1,041 stool samples were collected from residents who visited Public Health Center and its branches in Hamyang-gun and examined using formalin-ether sedimentation method. The overall egg positive rate was 16%, male showing higher positive rate (21%) than female (10%). The age group of 30 to 50 years had the highest egg positive rate of C. sinensis from 20% to 22%. The positive examinees were treated with praziquantel and educated individually to prevent reinfection. Egg positive rate in this area was decreased when compared with results recorded in the past, however, still remained more than 10%. This study suggests that periodic examination, treatment as well as education of residents should be continued and systematized.

Close relatedness of Acanthamoeba pustulosa with Acanthamoeba palestinensis based on isoenzyme profiles and rDNA PCR-RFLP patterns

The taxonomic validity of morphological group III Acanthamoeba spp. is uncertain. In the present study, six type strains of group III Acanthamoeba spp., Acanthamoeba culbertsoni, A. healyi, A. pustulosa, A. palestinensis, A. royreba and A. lenticulata were subjected for the evaluation of their taxonomic validity by comparison of the isoenzyme patterns by isoelectic focusing on polyacrylamide gels, mitochondrial DNA (Mt DNA) restriction fragment length polymorphism (RFLP), and small subunit ribosomal DNA (ssu rDNA) PCR-RFLP patterns. The Mt DNA RFLP patterns were heterogeneous between species. The type strains of A. palestinensis and A. pustulosa showed almost identical patterns of isoenzymes and rDNA PCR-RFLP with an estimated sequence divergence of 2.6%. The other species showed heterogeneous patterns of isoenzymes and rDNA PCR-RFLP. It is likely that A. pustulosa is closely related with A. palestinensis and that the former may be regarded as a junior synonym of the latter.

Change in incidence of parasite eggs and larvae from vegetable in the markets of Taegu

A survey on vegetables, which consisted of lettuce (Lactuca sativa), young radish (Raphanus sapivus), and chinese cabbage (Brassica pekinensis) collected from 6 markets in the Taegu city, was conducted for the discovery of helminth eggs and larvae, from July 1982 to June 1983. The results were compared with the data obtained from vegetables collected at the same markets by Lee (1969) and Choi and Lee (1972). Both sides of vegetable leaves were washed carefully with a hard brush, and the species of parasites and the mean number of parasites per 200 g of vegetable were determined. When vegetable were examined, 4 species of parasite eggs (ascarid, Trichostrongylus, liver fluke, and hookworms) and 2 larvae (filariform and rhabditoid larvae) were found. Of the parasite discovered, ascarid egg was found to be highest (4.2 percent), followed by hookworm egg (3.6 percent). The mean number of ascarid egg per 200 grams of vegetable was 0.6 in young radishes, and 0.3 in both lettuces and chinese cabbages. Similarly, the number of filariform larva of hookworm was 0.4 in young radishes, 0.3 in lettuces, and 0.l in chinese cabbages, and the number of rhabditoid larva was 0.2 in young radishes, 0.1 in lettuces, and 0.05 in chinese cabbages. In the results obtained by Lee (1969) and Choi and Lee (1972), the mean number of ascarid egg per 200 grams of vegetable was 7.5 in young radishes, 3.l in lettuces, and 0.5 in chinese cabbages. By contrast, in the present study it was 0.6 in young radishes and 0.3 in both lettuces and chinese cabbages. These results suggest that there has been significant reduction in the incidence of parasite egg and larva on vegetable leaves during the past 10 years.