Beraprost enhances the APC function of B cells by upregulating CD86 expression levels

Lipid mediators are emerging as important regulators of the immune system. Based on our previous result that shows strong expression of prostacyclin synthase in the germinal center, we investigated whether prostacyclin would regulate the APC function of B cells. Owing to the very short half-life of prostacyclin in experimental conditions, we used a more stable analog, beraprost. Beraprost increased the amounts of the costimulatory molecule CD86 but not CD80 on the surface of activated B cells in time- and dose-dependent manners. However, the enhancing effect of beraprost was not observed on memory B cells, centroblasts, and centrocytes. Beraprost required BCR and CD40 signals to upregulate CD86 expression levels. Other prostanoids such as PGE(2), 6-keto-PGF(1α), and PGF(2α) failed to alter CD86 expression levels, whereas other prostacyclin analogs were as potent as beraprost. Results carried out with receptor antagonists revealed that beraprost enhanced CD86 levels by binding to prostacyclin receptor IP and by increasing intracellular cAMP concentrations. Beraprost-treated B cells potently stimulated allogeneic T cells, which was significantly abolished by CD86 neutralization. Our data imply an unrecognized cellular and molecular mechanism about the germinal center reactions.     

Molecular identification of Taenia tapeworms by Cox1 gene in Koh Kong, Cambodia

We collected fecal samples from 21 individuals infected with Taenia tapeworms in Koh Kong Province, Cambodia, and performed nucleotide sequencing of the cox1 gene and multiplex PCR on the eggs for DNA differential diagnosis of human Taenia tapeworms. Genomic DNA was extracted from the eggs of a minimum number of 10 isolated from fecal samples. Using oligonucleotide primers Ta7126F, Ts7313F, Tso7466F, and Rev7915, the multiplex PCR assay proved useful for differentially diagnosing Taenia solium, Taenia saginata, and Taenia asiatica based on 706, 629, and 474 bp bands, respectively. All of the Taenia specimens from Kho Kong, Cambodia, were identified as either T. saginata (n=19) or T. solium (n=2) by cox1 sequencing and multiplex PCR.     

Echinostoma ilocanum infection in Oddar Meanchey Province, Cambodia

Fecal examinations using the Kato Katz technique were performed on a total of 1,287 villagers (945 students and 342 general inhabitants) of Oddar Meanchey Province, Cambodia in May 2007 and November 2009. The overall intestinal helminth egg positive rate was 23.9%, and the most prevalent helminth species was hookworms (21.6%). Other helminth eggs detected included echinostomes (1.0%), Enterobius vermicularis (0.8%), small trematode eggs (0.7%), which may include Opisthorchis viverrini and Haplorchis spp., and Hymenolepis nana (0.4%). In order to recover adult echinostomes, we treated 2 patients with 10-15 mg/kg praziquantel and purged. Total 14 adult echinostomes, 1 and 13 worms from each patient, were collected. The echinostomes characteristically had 49-51 collar spines and 2 round or slightly lobated testes. They were identified as Echinostoma ilocanum (Garrison, 1908) Odhner, 1911. So far as literature are concerned, this is the first record on the discovery of human E. ilocanum infection in Cambodia.     

Three-Step Method for Proliferation and Differentiation of Human Embryonic Stem Cell (hESC)-Derived Male Germ Cells

The low efficiency of differentiation into male germ cell (GC)-like cells and haploid germ cells from human embryonic stem cells (hESCs) reflects the culture method employed in the two-dimensional (2D)-microenvironment. In this study, we applied a three-step media and calcium alginate-based 3D-culture system for enhancing the differentiation of hESCs into male germ stem cell (GSC)-like cells and haploid germ cells. In the first step, embryoid bodies (EBs) were derived from hESCs cultured in EB medium for 3 days and re-cultured for 4 additional days in EB medium with BMP4 and RA to specify GSC-like cells. In the second step, the resultant cells were cultured in GC-proliferation medium for 7 days. The GSC-like cells were then propagated after selection using GFR-α1 and were further cultured in GC-proliferation medium for 3 weeks. In the final step, a 3D-co-culture system using calcium alginate encapsulation and testicular somatic cells was applied to induce differentiation into haploid germ cells, and a culture containing approximately 3% male haploid germ cells was obtained after 2 weeks of culture. These results demonstrated that this culture system could be used to efficiently induce GSC-like cells in an EB population and to promote the differentiation of ESCs into haploid male germ cells.     

Histone Deacetylase-3 Mediates Positive Feedback Relationship between Anaphylaxis and Tumor Metastasis

Allergic inflammation has been known to enhance the metastatic potential of tumor cells. The role of histone deacetylase-3 (HDAC3) in allergic skin inflammation was reported. We investigated HDAC3 involvement in the allergic inflammation-promotion of metastatic potential of tumor cells. Passive systemic anaphylaxis (PSA) induced HDAC3 expression and FcϵRI signaling in BALB/c mice. PSA enhanced the tumorigenic and metastatic potential of mouse melanoma cells in HDAC3- and monocyte chemoattractant protein 1-(MCP1)-dependent manner. The PSA-mediated enhancement of metastatic potential involved the induction of HDAC3, MCP1, and CD11b (a macrophage marker) expression in the lung tumor tissues. We examined an interaction between anaphylaxis and tumor growth and metastasis at the molecular level. Conditioned medium from antigen-stimulated bone marrow-derived mouse mast cell cultures induced the expression of HDAC3, MCP1, and CCR2, a receptor for MCP1, in B16F1 mouse melanoma cells and enhanced migration and invasion potential of B16F1 cells. The conditioned medium from B16F10 cultures induced the activation of FcϵRI signaling in lung mast cells in an HDAC3-dependent manner. FcϵRI signaling was observed in lung tumors derived from B16F10 cells. Target scan analysis predicted HDAC3 to be as a target of miR-384, and miR-384 and HDAC3 were found to form a feedback regulatory loop. miR-384, which is decreased by PSA, negatively regulated HDAC3 expression, allergic inflammation, and the positive feedback regulatory loop between anaphylaxis and tumor metastasis. We show the miR-384/HDAC3 feedback loop to be a novel regulator of the positive feedback relationship between anaphylaxis and tumor metastasis.     

Zoonotic Trematode Metacercariae in Fish from Phnom Penh and Pursat,Cambodia

A survey was performed to investigate the infection status of freshwater fish with zoonotic trematode metacercariae in Phnom Penh and Pursat Province, Cambodia. All collected fish with ice were transferred to our laboratory and examined using the artificial digestion method. In fish from Phnom Penh, 2 kinds of metacercariae (Opisthorchis viverrini and Haplorchis yokogawai) were detected. O. viverrini metacercariae were positive in 37 (50.0%) of 74 fish in 11 species (average no. metacercariae/fish, 18.6). H. yokogawai metacercariae were detected in 23 (57.5%) of 40 fish in 5 species (average no. metacercariae/fish, 21.0). In fish from Pursat Province, 5 kinds of metacercariae (O. viverrini, H. yokogawai, Haplorchis pumilio, Centrocestus formosanus, and Procerovum sp.) were detected; O. viverrini metacercariae (n=3) in 2 fish species (Henicorhynchus lineatus and Puntioplites falcifer), H. yokogawai metacercariae (n=51) in 1 species (P. falcifer), H. pumilio metacercariae (n=476) in 2 species (H. lineatus and Pristolepis fasciata), C. formosanus metacercariae (n=1) in 1 species (H. lineatus), and Procerovum sp. metacercariae (n=63) in 1 species (Anabas testudineus). From the above results, it has been confirmed that various freshwater fish play the role of a second intermediate host for zoonotic trematodes (O. viverrini, H. yokogawai, H. pumilio, C. formosanus, and Procerovum sp.) in Cambodia.     

Prevalence of Intestinal Helminths among Inhabitants of Cambodia (2006-2011)

In order to investigate the status of intestinal helminthic infections in Cambodia, epidemiological surveys were carried out on a national scale, including 19 provinces. A total of 32,201 fecal samples were collected from schoolchildren and adults between 2006 and 2011 and examined once by the Kato-Katz thick smear technique. The overall egg positive rate of intestinal helminths was 26.2%. The prevalence of hookworms was the highest (9.6%), followed by that of Opisthorchis viverrini/minute intestinal flukes (Ov/MIF) (5.7%), Ascaris lumbricoides (4.6%), and Trichuris trichiura (4.1%). Other types of parasites detected were Enterobius vermicularis (1.1%), Taenia spp. (0.4%), and Hymenolepis spp. (0.2%). The northwestern regions such as the Siem Reap, Oddar Meanchey, and Banteay Meanchey Provinces showed higher prevalences (17.4-22.3%) of hookworms than the other localities. The southwestern areas, including Koh Kong and Preah Sihanouk Provinces showed higher prevalences of A. lumbricoides (17.5-19.2%) and T. trichiura (6.1-21.0%). Meanwhile, the central and southern areas, in particular, Takeo and Kampong Cham Provinces, showed high prevalences of Ov/MIF (23.8-24.0%). The results indicate that a considerably high prevalence of intestinal helminths has been revealed in Cambodia, and thus sustained national parasite control projects are necessary to reduce morbidity due to parasitic infections in Cambodia.     

Complete Genome Analysis of Three Live Attenuated Rinderpest Virus Vaccine Strains Derived through Serial Passages in Different Culture Systems

The genomes of three South Korean Rinderpest virus vaccine strains (L72, LA77, and LA96) were analyzed in order to investigate their genetic variability. These three vaccine strains were all derived from the same virus strain origin (Fusan) through repeated passages in different culture systems. The full genome length of the three strains was 15,882 nucleotides, and the sequence similarity between the three South Korean RPV strains at the nucleotide level was 98.1 to 98.9%. The genetic distance between Nakamura III, L72, LA77, LA96, and LATC06 and the Kabete strain was greater than that between the Fusan and Kabete strains for the P, V, and C genes. The difference in pathogenicity among these strains might be due to the V gene, which has a positive (>1) selection ratio based on the analysis of synonymous (dS) and nonsynonymous (dN) substitution rates (dN/dS ratio [ω]).