Therapeutic Effect and Immune Changes after Treatment of Hymenolepis nana-Infected BALB/c Mice with Compounds Isolated from Leucaena leucocephala.

BACKGROUND/PURPOSE: Hymenolepis nana is globally distributed. Leucaena leucocephala has been studied as a treatment, including the nematodes and protozoa, but no research results are related to cestodes. Therefore, the aim of this study was to target H. nana. METHODS: The natural components of L. leucocephala were isolated and added to H. nana, which was cultured in vitro, to observe changes in the mortality, motility, and morphology. BALB/c male mice infected with H. nana were treated with effective components of L. leucocephala for 10 days, and the changes were recorded. After the mice were sacrificed, the spleen weight was measured, and a primary culture was performed for the subsequent cytokine and chemokine testing. RESULTS: The experiment found that 132-hydroxy-(132-S)-pheophytin a and aristophyll-C have clear cestocidal effects in vitro. 132-hydroxy-(132-S)-pheophytin a has been shown to be effective at reducing parasite populations and eliciting host immune responses in vivo. IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, MCP-1, IFN-γ, TNF-α, MIP-1α, and GM-CSF in 132-hydroxy-(132-S)-pheophytin a were significantly increased after stimulation, while IL-1α, IL-1ß, IL-3, IL-12p70, and RANTES were unchanged. CONCLUSIONS: The investigation shows that components of L. leucocephala have actual cestocidal activity against H. nana.

Cestocidal activities of bioactive garlic compounds against Hymenolepis nana.

Hymenolepis nana, a parasitic tapeworm distributed worldwide, is very prevalent in countries with poor sanitary conditions. Garlic is widely used as a seasoning and medicinal plant all over the world, and its derivatives have proven anti-microbial and anti-inflammatory effects. Our study explored the cestocidal and therapeutic effects of allicin derivatives against H. nana in vitro and in vivo. Worms taken from a host were cultured in vitro, and the effects of allyl sulfide (DAS), allyl disulfide (DADS) and dimethyl sulfoxide (DMSO) treatments were observed. Male BALB/c mice were then fed eggs to produce infection, given drugs for ten days and dissected. The results of this study showed that DADS in garlic exhibited good cestocidal effects in vitro and in vivo. DADS and DATS reduced motility, induced mortality and damaged body segments of worms in vitro. In vivo, the number of worms in the low-dose and high-dose DADS groups was significantly less than the infected control group. DADS effected cytokine changes in BALB/c mice after infection. IFN-γ increased, IL-2, 4, 6 and 13 decreased, and IL-5, 10 and IL-12 p70 did not change significantly. As a medicinal plant, garlic has many active ingredients that can developed as anti-microbial or parasite-related drugs.

A school-based soil-transmitted helminths survey in the Guadalcanal Province, the Solomon Islands.

Infections by soil-transmitted helminths are a major public health problem worldwide, especially among schoolchildren in low-income countries. Little information is described about their prevalence in the Solomon Islands. From 2017 to 2018, a school-based soil-transmitted helminths survey in the Guadalcanal Province was conducted. A total of 454 schoolchildren were selected; the Merthiolate-iodine-formaldehyde concentration and stain was used. The prevalence was 17% of one or more parasites, including hookworm (8.8%), Strongyloides stercoralis (5.7%), Ascaris lumbricoides (4.2%) and Trichuris trichiura (3.5%). STH infection was significantly correlated with parents' occupations, hand washing, shoe wearing as well as gastrointestinal symptoms. To prevent STH transmission for schoolchildren in the Solomon Islands completely, combined preventive strategies seem necessary.

Galectin-9-like from Angiostrongylus cantonensis young adult worms modulates eosinophil chemotaxis in vitro.

BACKGROUND/PURPOSE: Eosinophils are recruited to the brain of mice after infection with Angiostrongylus canonensis. Several factors produced by infected mice are well known playing the role to chemoattract eosinophils from the blood into the brain. The purpose of this study is to investigate whether Angiostronylus cantonensis young-adult worms (AcYA) have components which have eosinophilic chemotactic activity. METHODS: Eosinophil chemotactic activity of AcYA was tested by Boyden blind-well chamber technique. The components of AcYA were analysed by SDS-PAGE and Mass spectrometry. Furthermore, galectin-9 in the cerebrospinal fluid (CSF) of infected mice and galectin-9-like in AcYA were measured by ELISA technic and also were recognized by western blot analysis respectively. RESULTS: Excretory-secretory products of AcYA did not show eosinophil chemotactic activity. However, the extracts of AcYA showed protein concentration-dependent eosinophil chemotactic activity and reached the peak at the 24 µg/ml. The eosinophil chemotactic activity was significantly reduced by lactose. The components of AcYA at molecular weights of approximatively 15 kDa and 35 kDa showed several galectins component in Mass spectrometric analysis. Furthermore, galectin-9-like in AcYA was recognized by ELISA and western blot analysis. In parallel with increase of galectin-9 in the CSF, eosinophils were also significantly increased in mouse after infected with A. cantonensis. CONCLUSION: Galectin-9-like in AcYA and galectin-9 in mouse CSF were confirmed demonstrating eosinophil chemotactic activity both in vitro study and in the infection of mouse in this study.

Dexamethasone Downregulates Expressions of 14-3-3ß and γ-Isoforms in Mice with Eosinophilic Meningitis Caused by Angiostrongylus cantonensis Infection.

Steroids are commonly used in patients with eosinophilic meningitis caused by A. cantonensis infections. The mechanism steroids act on eosinophilic meningitis remains unclear. In this mouse experiments, expressions of 14-3-3 isoform ß and γ proteins significantly increased in the CSF 2-3 weeks after the infection, but not increasedin the dexamethasone-treated group. Expression of 14-3-3 ß, γ, ε, and θ isoforms increased in brain meninges over the 3-week period after infection and decreased due to dexamethasone treatment. In conclusion, administration of dexamethasone in mice with eosinophilic meningitis decreased expressions of 14-3-3 isoform proteins in the CSF and in brain meninges.

Increased 14-3-3ß and γ protein isoform expressions in parasitic eosinophilic meningitis caused by Angiostrongylus cantonensis infection in mice.

The 14-3-3 proteins are cerebrospinal fluid (CSF) markers of neuronal damage during infectious meningitis and Creutzfeldt-Jakob disease. Little is known about dynamic changes in the individual isoforms in response to parasitic eosinophilic meningitis. The purposes of this study were to determine the 14-3-3 protein isoform patterns, examine the kinetics and correlate the severity of blood brain barrier (BBB) damage with the expressions of these markers in mice with eosinophilic meningitis. Mice were orally infected with 50 A. cantonensis L3 via an oro-gastric tube and sacrificed every week for 3 consecutive weeks after infection. The Evans blue method and BBB junctional protein expressions were used to measure changes in the BBB. Hematoxylin and eosin staining was used to analyze pathological changes in the mice brains following 1-3 weeks of infection with A. cantonensis. The levels of 14-3-3 protein isoforms in serum/CSF and brain homogenates were analyzed by Western blot, and immunohistochemistry (IHC) was used to explore the different isoform distributions of 14-3-3 proteins and changes in BBB junctional proteins in the mice brain meninges. Dexamethasone was injected intraperitoneally from the seventh day post infection (dpi) until the end of the study (21 dpi) to study the changes in BBB junctional proteins. The amounts of Evans blue, tight junction and 14-3-3 protein isoforms in the different groups of mice were compared using the nonparametric Kruskal-Wallis test. There were significant increases in 14-3-3 protein isoforms ß and γ in the CSF in the second and third weeks after infection compared to the controls and first week of infection, which were correlated with the severity of BBB damage in brain histology, and Evans blue extravasation. Using IHC to assess the distribution of 14-3-3 protein isoforms and changes in BBB junctional proteins in the mice brain meninges, the expressions of isoforms ß, γ, ε, and θ and junctional proteins occludin and claudin-5 in the brain meninges increased over a 3-week period after infection compared to the controls and 1 week after infection. The administration of dexamethasone decreased the expressions of BBB junctional proteins occludin and claudin-5 in the mice brain meninges. Our findings support that 14-3-3 proteins ß and γ can potentially be used as a CSF marker of neuronal damage in parasitic eosinophilic meningitis caused by A. cantonensis.

Enterobius vermicularis infection and its risk factors among pre-school children in Taipei, Taiwan.

BACKGROUND: The prevalence of pinworm infection is extremely low in Taipei, Taiwan. This population study was designed to determine the current status and the associated risk factors of this infection among pre-school children. METHODS: Perianal swab specimens were obtained from the parents or guardians using a two-consecutive-day adhesive cellophane perianal swab kit. Information of family background, personal hygiene, and household sanitary conditions were collected by asking the parents or guardians to complete a questionnaire. RESULTS: Of 44,163 children, 0.21% was found to infect with pinworm. The positive rate was highest in Datong (0.59%) and Nangang (0.58%) Districts and lowest in Neihu District (0.02%). There was no significant difference in the rates by gender (boys 0.24% and girls 0.19%) or school (kindergartens 0.25% and nurseries 0.17%). Significantly higher positive rates were found in children having parent with lower educational level and elder brother(s)/sister(s). Children taking bath by themselves and those sleeping in bed with matting had significantly higher positive rates. Five significant independent predictors of pinworm infection were determined by multivariate analysis: having elder brother(s), having elder sister(s), infrequent washing hands after using toilet facilities, bathing without the help of family members, and sleeping on bed with matting. CONCLUSION: The prevalence of pinworm infection in the pre-school children of Taipei is extremely low and decreasing. Good hand washing habit should be an important preventive measure. Transmission of this infection in pre-school children may occur in the family through their school-age siblings.

Excretory/secretory proteases and mechanical movement of Anisakis pegreffii infective larvae in the penetration of BALB/c mice gastrointestine.

Anisakiasis is a human parasitic disease caused by infection with the infective larvae of Anisakis. Accidental infection in humans causes the gastrointestinal pathophysiological effects of mechanical tissue damage by migrating larvae. The mechanism of the infective larval invasion and migration is suspected to involve larval excretory/secretory proteases and motility. This study demonstrates the penetration rate of the infective larvae of Anisakis pegreffii in mouse gastrointestine depends on the time after infection, and that only 15% of larvae remain in the gastrointestinal tract 3 h after infection. Strong activities of matrix metalloproteinases (MMPs) and serine proteases, especially plasmin, were found in the excretory/secretory products of A. pegreffii; these can be inhibited by ONO-4817 and phenylmethylsulfonyl fluoride, respectively. The protease activity was also significantly decreased in another 1 h of cultivation of larvae in fresh 0.9% normal saline (NS) after previous cultivation for 48 h in NS. The motility scores of larvae were significantly lower after 48 h of cultivation in NS. The penetration rate of A. pegreffii larvae in the gastrointestine of infected mice sequentially were 90% in the freshly prepared, 68% in serine protease inhibited, 55% in MMPs inhibited larvae, and 16% in larvae cultivated in NS for 48 h. Therefore, this study demonstrates that MMPs and serine proteases excreted and secreted by A. pegreffii and the mechanical movement of infective larvae participate in the penetration of the gastrointestine of mice after infection.

Dexamethasone inhibits brain apoptosis in mice with eosinophilic meningitis caused by Angiostrongylus cantonensis infection.

BACKGROUND: Angiostrongylus cantonensis, the rat lungworm, is the major cause of eosinophilic meningitis worldwide. Rats serve as the definitive host of the nematode, but humans can be infected incidentally, leading to eosinophilic meningitis. A previous BALB/c animal study has demonstrated increased apoptotic proteins and decreased anti-apoptotic proteins in mice infected with A. cantonensis. Steroids may be an effective treatment option for eosinophilic meningitis caused by A. cantonensis, but the involved mechanism is unclear. This study hypothesized that the beneficial effects of steroids on eosinophilic meningitis are mediated by decreased apoptosis. METHODS: In a BALB/c animal model, mice were orally infected with 50 A. cantonensis L3 via an oro-gastric tube and were sacrificed every week for 3 consecutive weeks after infection or until the end of the study. Dexamethasone was injected intra-peritoneally from the 7(th) day post-infection until the end of the 21-day study. Evans blue method was used to measure changes in the blood brain barrier, while western blotting, immuno-histochemistry, and TUNEL assay were used to analyze brain homogenates expression of apoptotic and anti-apoptotic proteins. RESULTS: There were increased amounts of Evans blue, apoptotic proteins (caspase-3, -8, and -9 and cytochrome C), and decreased anti-apoptotic proteins (bcl-2) after 2-3 weeks of infection. Dexamethasone administration significantly decreased Evans blue extravasations and apoptotic protein expressions. CONCLUSIONS: Apoptosis of mice brain homogenates can be repressed by dexamethasone treatment.

Comparative studies on the proteomic expression patterns in the third- and fifth-stage larvae of Angiostrongylus cantonensis.

Angiostrongylus cantonensis is an important zoonotic parasite causing eosinophilic meningitis and eosinophilic meningoencephalitis in humans. In this study, the protein expression profiles of the infective third- and pathogenic fifth-stage larvae (L3 and L5) of this parasite were compared by proteomic techniques. Isolated protein samples were separated by two-dimensional gel electrophoresis (2-DE), stained with silver nitrate, and analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Proteins from L5 were mainly at pH 5-7 and with molecular weight (MW) 40-100 kDa, whereas those from L3 were at pH 5-6 and with 5-35 kDa. Of 100 protein spots identified, 33 were from L3 whereas 67 from L5 and 63 had known identities, whereas 37 were hypothetical proteins. There were 15 spots of stress proteins, and HSP60 was the most frequently found heat stress proteins in L5. More binding and protein transport-related proteins were found in L5 including peptidylprolyl isomerase (cyclophilin)-like 2, serum albumin, preproalbumin precursor, and dilute class unconventional myosin. L3 had a higher expression of cytoskeleton and membrane proteins than L5. In addition, four protein spots were identified in the sera of the rat host by Western blot analysis. The present proteomic study revealed different protein expression profiles in L3 and L5 of A. cantonensis. These changes may reflect the development of L3 from the poikilothermic snails to L5 in the homoeothemic rats. This information may be useful for the finding of stage-specific proteins and biomarker for diagnosis of angiostrongyliasis.

Anthelmintic constituents from ginger (Zingiber officinale) against Hymenolepis nana.

This study investigated the anthelmintic activity of gingerenone A, [6]-dehydrogingerdione, [4]-shogaol, 5-hydroxy-[6]-gingerol, [6]-shogaol, [6]-gingerol, [10]-shogaol, [10]-gingerol, hexahydrocurcumin, 3R,5S-[6]-gingerdiol and 3S,5S-[6]-gingerdiol, a constituent isolate from the roots of ginger, for the parasite Hymenolepis nana. The cestocidal activity or ability to halt spontaneous parasite movement (oscillation/peristalsis) in H. nana of above constituents was reached from 24 to 72h in a time- and dose-dependent manner, respectively. The [10]-shogaol and [10]-gingero1 have maximum lethal efficacy and loss of spontaneous movement than the others at 24-72h. In addition, worms treated with 1 and 10µM [10]-gingero1, more than 30% had spontaneous movement of oscillation at 72h but [10]-shogaol at 72h only about 15-20% of oscillation. This showing that [10]-gingero1 had less loss of spontaneous movement efficacy than [10]-shogaol. After exposure to 200µM [10]-shogaol, 100% of H. nana had died at 12h rather than died at 24h for [10]-gingerol, showing that [10]-gingero1 had less lethal efficacy than [10]-shogaol. In addition, these constituents of ginger showed effects against peroxyl radical under cestocidal activity. In order to evaluate the cestocidal activity and cytokine production caused by ginger's extract R0 in the H. nana infected mice, we carried out in vivo examination about H. nana infected mice BALB/c mice were inoculated orally with 500 eggs. After post-inoculation, R0 (1g/kg/day) was administered orally for 10 days. The R0 exhibited cestocidal activity in vivo of significantly reduced worms number and cytokines production by in vitro Con A-stimulated spleen cells showed that INF-γ and IL-2 were significantly increases by R0. IL-4, IL-5, IL-6, IL-10 and IL-13 were significantly decreases and Murine KC and IL-12 were not significantly changes by R0. Together, these findings first suggest that these constituents of ginger might be used as cestocidal agents against H. nana.

Anthelmintic activities of aporphine from Nelumbo nucifera Gaertn. cv. Rosa-plena against Hymenolepis nana.

Nelumbo nucifera Gaertn. cv. Rosa-plena (Nelumbonaceae), commonly known as lotus, is a perennial aquatic plant grown and consumed throughout Asia. All parts of N. nucifera have been used for various medicinal purposes in oriental medicine. From the leaves of Nelumbo nucifera Gaertn. cv. Rosa-plena (an aquatic plant), liriodenine (1), lysicamine (2), (-)-anonaine (3), (-)-asimilobine (4), (-)-caaverine (5), (-)-N-methylasimilobine (6), (-)-nuciferine (7), (-)-nornuciferine (8), (-)-roemerine (9), 7-hydroxydehydronuciferine (10) and cepharadione B (11) were isolated and identification and anthelmintic activities of aporphine was evaluated against Anisakis simplex and Hymenolepis nana. This study found that the above constituents killed H. nana or reduced their spontaneous movements (oscillation/peristalsis). However, the above constituents at various concentrations demonstrated no larvicidal effect or ability to halt spontaneous parasite movement for 72 h against A. simplex, respectively. In addition, according to an assay of cestocidal activity against H. nana and nematocidal activity against A. simplex, we found that the above compounds showed greater lethal efficacy on H. nana than against A. simplex. Further investigation showed that these above constituents have effects against peroxyl radicals under cestocidal effect. Together, these findings suggest that these constituents of Nelumbo nucifera Gaertn. cv. Rosa-plena might be used as anthelmintic agents against H. nana.

14-3-3ß protein expression in eosinophilic meningitis caused by Angiostrongylus cantonensis infection.

BACKGROUND: Angiostrongylus cantonensis is a parasite endemic in the Southeast Asian and Pacific regions. Humans are incidentally infected either by eating uncooked intermediate hosts or by consuming vegetables containing the living third-stage larvae. The 14-3-3ß protein is a cerebrospinal fluid (CSF) marker of neuronal damage during the development of Creutzfeldt-Jakob disease. In addition, increased 14-3-3ß protein is also found in CSF from patients with a variety of neurological disorders. The goal of this study is to determine the roles of serum/CSF14-3-3ß protein in patients with eosinophilic meningitis. METHODS: In a cohort study among nine Thai laborers with eosinophilic meningitis due to eating raw snails (Pomacea canaliculata), we examined the CSF weekly while patients were still hospitalized and followed up the serum for 6 months. The levels of 14-3-3ß protein in CSF were analyzed by western blot and an in-house 14-3-3ß enzyme-linked immunosorbent assay (ELISA) measurement was established and tested in an animal model of eosinophilic meningitis. RESULTS: The elevated 14-3-3ß level was detected in the CSF from eight out of nine (81%) patients After 2 weeks of treatment, all patients showed a declined level or cleared of 14-3-3ß protein in the CSF. By developing an in-house ELISA for measurement of 14-3-3ß protein, it was found that the serum 14-3-3ß level was significantly increased in patients during initial visit. . This finding was consistent to the animal experiment result in which there was severe blood brain barrier damage three weeks after infection and increased 14-3-3ß protein expression in the CSF and serum by western blot and in house ELISA. After treatment, the serum 14-3-3ß level in meningitis patients was rapidly returned to normal threshold. There was a correlation between initial CSF 14-3-3ß level with severity of headache (r = 0.692, p = 0.039), CSF pleocytosis (r = 0.807, p = 0.009) and eosinophilia (r = 0.798, p = 0.01) in the CSF of patients with eosinophilic meningitis (Spearman's correlation test). CONCLUSIONS: The serum 14-3-3ß concentrations may constitute a useful marker for blood brain barrier damage severity and follow up in patients with eosinophilic meningitis caused by A. cantonensis.

Sequence analysis in partial genes of five isolates of Angiostrongylus cantonensis from Taiwan and biological comparison in infectivity and pathogenicity between two strains.

Angiostrongylus cantonensis is the most common infectious agent causing eosinophilic meningitis and is present in Taiwan, Thailand and the Pacific islands. Clinical symptoms vary within different endemic regions, and their severity is probably dependent on the number of ingested parasites and the diversity among strains. The experimentally definitive host is the rat, and non-permissive hosts are certain mammals such as humans and mice. In this study, the partial gene sequences of two A. cantonensis strains isolated from five different regions in Taiwan were selected and molecularly analyzed. The internal transcribed spacer gene and cytochrome-c oxidase subunit I gene sequences of the Hualien (H) strain of A. cantonensis differed from those of the Pingtung (P) strain and the other three strains by 19% and 11%, respectively. We analyzed the infectivity, fecundity, and development of the H and P strain in rats and host pathogenicity in mice inoculated with both strains. The number of the emerged first-stage larvae, adult recovery, and average length of adults in Sprague-Dawley rats significantly differed between rats inoculated with the H and P strain. Young adult recovery, average length of young adults, eosinophil counts in the cerebrospinal fluid (CSF), glutathione peroxidase concentration, levels of reactive oxygen species as well as malondialdehyde concentration in the CSF, and the survival of mice significantly differed between BALB/c mice inoculated with the H and P strain. The H strain of A. cantonensis had lower infectivity, delayed fecundity, and poor development in rats, and caused milder pathology and lower mortality in mice than the P strain. These data clearly indicate that the H strain of A. cantonensis is a pathogenically distinct strain with lower infectivity to its definitive host, and causing mild pathogenic symptoms to its non-permissive host.

Dexamethasone downregulated the expression of CSF 14-3-3ß protein in mice with eosinophilic meningitis caused by Angiostrongylus cantonensis infection.

Angiostrongylus cantonensis is the main causative agent of human eosinophilic meningitis in Southeast Asia and the Pacific Islands. A previous study demonstrated that the 14-3-3ß protein is a neuropathological marker in monitoring neuronal damage in meningitis. Steroids are commonly used in patients with eosinophilic meningitis caused by A. cantonensis infection. However, the mechanism by which steroids act in eosinophilic meningitis is unknown. We hypothesized that the beneficial effect of steroids on eosinophilic meningitis is partially mediated by the down-regulation of 14-3-3ß protein expression in the cerebrospinal fluid (CSF). In this animal study, we determined the dynamic changes of 14-3-3ß protein in mice with eosinophilic meningitis. The 14-3-3ß protein in serum and CSF was increased in week 2 and 3 after infections. Dexamethasone administration significantly decreased the amounts of CSF 14-3-3ß protein. By developing an in-house ELISA to measure 14-3-3ß protein, it was found that the amounts of 14-3-3ß protein in the CSF and serum increased over a three-week period after infection. There was a remarkable reduction of 14-3-3ß protein in the CSF after 2 weeks of dexamethasone treatment. In conclusion, the administration of corticosteroids in mice with eosinophilic meningitis decreased the expression of 14-3-3ß protein in the CSF.

A transcriptomic analysis on gene expressions in the infective third and pathogenic fifth larval stages of Angiostrongylus cantonensis.

Although Angiostrongylus cantonensis is a parasite of rats, it is an important etiologic agent of eosinophilic meningitis and eosinophilic meningoencephalitis in humans. This study was designed to compare the gene expression in the third- and fifth-stage (L3 and L5) by analysis of expressed sequence tags (ESTs). After removing low quality sequences, vector trimming, clustering and contig assembly, there remained 1437 clusters (285 contigs and 1152 singletons). Among these clusters, 779 (54.2%) showed significant similarity to the known proteins in the non-redundant protein database of GenBank (E-value<1×10(-10)) and species of the best hit sequences mainly belonged to nematodes. These clusters included 869 (60.5%) that were entirely comprised of ESTs from L3 (L3-biased clusters), 518 (36.0%) entirely from L5 (L5-biased clusters) and 50 (3.5%) from both stages (stage-shared clusters). Functional annotations by the Gene Ontology (GO) comparing with the eukaryotic clusters of orthologous groups of proteins (KOG) indicate that the L3-biased clusters significantly related to metabolism and the L5-biased clusters to growth, development, sexual differentiation and reproduction. Moreover, L3 were found to have expressions of metalloproteases, aspartic proteases, and cysteine proteases whereas expressions of cysteine, aspartic and serine proteases were revealed in L5. The results indicate that earlier developmental stages of nematodes may have a gene expression profile towards metabolism and later stages towards growth and development.

Antioxidant, anti-adipocyte differentiation, antitumor activity and anthelmintic activities against Anisakis simplex and Hymenolepis nana of yakuchinone A from Alpinia oxyphylla.

BACKGROUND: Alpinia oxyphylla is a common remedy in traditional Chinese medicine. Yakuchinone A is a major constituent of A. oxyphylla and exhibits anti-inflammatory, antitumor, antibacterial, and gastric protective activities. METHODS: Antioxidant and antitumor characteristics of yakuchinone A in skin cancer cells as well as novel mechanisms for the inhibition of adipocyte differentiation, cestocidal activities against Hymenolepis nana adults, and nematocidal activities against Anisakis simplex larvae are investigated. RESULTS: Yakuchinone A presents the ability of the removal of DPPH·and ABTS+ free radicals and inhibition of lipid peroxidation. Yakuchinone A suppresses intracellular lipid accumulation during adipocyte differentiation in 3 T3-L1 cells and the expressions of leptin and peroxisome proliferator-activated receptor γ (PPARγ). Yakuchinone A induces apoptosis and inhibits cell proliferation in skin cancer cells. The inhibition of cell growth by yakuchinone A is more significant for non-melanoma skin cancer (NMSC) cells than for melanoma (A375 and B16) and noncancerous (HaCaT and BNLCL2) cells. Treatment BCC cells with yakuchinone A shows down-regulation of Bcl-2, up-regulation of Bax, and an increase in cleavage poly (ADP-ribose) polymerase (PARP). This suggests that yakuchinone A induces BCC cells apoptosis through the Bcl-2-mediated signaling pathway. The anthelmintic activities of yakuchinone A for A. simplex are better than for H. nana. CONCLUSIONS: In this work, yakuchinone A exhibits antioxidative properties, anti-adipocyte differentiation, antitumor activity, and anthelmintic activities against A. simplex and H. nana.

Human parasitic meningitis caused by Angiostrongylus cantonensis infection in Taiwan.

The major cause of eosinophilic meningitis in Taiwan is Angiostrongylus cantonensis. Humans are infected by ingesting terrestrial and freshwater snails and slugs. In 1998 and 1999, two outbreaks of eosinophilic meningitis caused by A. cantonensis infection were reported among 17 adult male immigrant Thai laborers who had eaten raw golden apple snails (Pomacea canaliculata). Another outbreak associated with consuming a health drink consisting of raw vegetable juice was reported in 2001. These adult cases differed from reports in the 1970s and 1980s, in which most of the cases were in children. With improvements in public health and education of foreign laborers, there have since been only sporadic cases in Taiwan. Review of clinical research indicates inconsistent association of Magnetic Resonance Imaging (MRI) results with clinical features of eosinophilic meningitis. MRI features were nonspecific but there was an association between the presence of high brain MRI signal intensities and severity of peripheral and cerebrospinal fluid (CSF) eosinophilia. Inflammatory markers have been identified in the CSF of patients with eosinophilic meningitis caused by A. cantonensis infection, and vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and the matrix metalloproteinase system may be associated with blood-brain barrier disruption. Eosinophilic meningitis caused by A. cantonensis infection is not a reportable disease in Taiwan. It is important that a public advisory and education program be developed to reduce future accidental infection.

Angiostrongylus cantonensis (Nematode: Metastrongiloidea): in vitro cultivation of infective third-stage larvae to fourth-stage larvae.

The present study to attempt to cultivate Angiostrongylus cantonensis from third-stage larvae (AcL3) to fourth-stage larvae (AcL4) in vitro in defined complete culture medium that contained with Minimum Essential Medium Eagle (MEM), supplemented amino acid (AA), amine (AM), fatty acid (FA), carbohydrate (CA) and 20% fetal calf serum (FCS) was successful. When AcL3 were cultured in the defined complete culture medium at 37°C in a 5% CO2 atmosphere, the larvae began to develop to AcL4 after 30 days of cultivation, and were enclosed within the sheaths of the third molts of the life cycle. Under these conditions, the larvae developed uniformly and reached to the fourth-stage 36 days. The morphology of AcL3 develop to AcL4 were recording and analyzing. Then comparison of A. cantonensis larval morphology and development between in vitro cultivation in defined complete culture medium and in vivo cultivation in infective BALB/c mice. The larvae that had been cultivated in vitro were smaller than AcL4 of infective BALB/c mice. However the AcL3 that were cultured using defined incomplete culture medium (MEM plus 20% FCS with AA+AM, FA, CA, AA+AM+FA, FA+CA, CA+AA+AM or not) did not adequately survive and develop. Accordingly, the inference is made that only the defined complete medium enable AcL3 develop to AcL4 in vitro. Some nematodes have been successfully cultured into mature worms but only a few researches have been made to cultivate A. cantonensis in vitro. The present study is the first to have succeeded in developing AcL3 to AcL4 by in vitro cultivation. Finally, the results of in vitro cultivation studies herein contribute to improving media for the effective development and growth of A. cantonensis. The gap in the A. cantonensis life cycle when the larvae are cultivated in vitro from third-stage larvae to fourth-stage larvae can thus be solved.