Alterations in Plasma Lipid Profile before and after Surgical Removal of Soft Tissue Sarcoma.

Soft tissue sarcoma (STS) is a relatively rare malignancy, accounting for about 1% of all adult cancers. It is known to have more than 70 subtypes. Its rarity, coupled with its various subtypes, makes early diagnosis challenging. The current standard treatment for STS is surgical removal. To identify the prognosis and pathophysiology of STS, we conducted untargeted metabolic profiling on pre-operative and post-operative plasma samples from 24 STS patients who underwent surgical tumor removal. Profiling was conducted using ultra-high-performance liquid chromatography-quadrupole time-of-flight/mass spectrometry. Thirty-nine putative metabolites, including phospholipids and acyl-carnitines were identified, indicating changes in lipid metabolism. Phospholipids exhibited an increase in the post-operative samples, while acyl-carnitines showed a decrease. Notably, the levels of pre-operative lysophosphatidylcholine (LPC) O-18:0 and LPC O-16:2 were significantly lower in patients who experienced recurrence after surgery compared to those who did not. Metabolic profiling may identify aggressive tumors that are susceptible to lipid synthase inhibitors. We believe that these findings could contribute to the elucidation of the pathophysiology of STS and the development of further metabolic studies in this rare malignancy.

Microsatellite DNA analysis revealed a drastic genetic change of Plasmodium vivax population in the Republic of Korea during 2002 and 2003.

BACKGROUND: Vivax malaria was successfully eliminated in the Republic of Korea (South Korea) in the late 1970s, but it was found to have re-emerged from 1993. In order to control malaria and evaluate the effectiveness of malaria controls, it is important to develop a spatiotemporal understanding of the genetic structure of the parasite population. Here, we estimated the population structure and temporal dynamics of the transmission of Plasmodium vivax in South Korea by analyzing microsatellite DNA markers of the parasite. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed 14 microsatellite DNA loci of the P. vivax genome from 163 South Korean isolates collected from 1994 to 2008. Allelic data were used to analyze linkage disequilibrium (LD), genetic differentiation and population structure, in order to make a detailed estimate of temporal change in the parasite population. The LD analysis showed a gradual decrease in LD levels, while the levels of genetic differentiation between successive years and analysis of the population structure based on the Bayesian approach suggested that a drastic genetic change occurred in the South Korean population during 2002 and 2003. CONCLUSIONS/SIGNIFICANCE: Although relapse and asymptomatic parasite carriage might influence the population structure to some extent, our results suggested the continual introduction of P. vivax into South Korea through other parasite population sources. One possible source, particularly during 2002 and 2003, is North Korea. Molecular epidemiology using microsatellite DNA of the P. vivax population is effective for assessing the population structure and temporal dynamics of parasite transmission; information that can assist in the elimination of vivax malaria in endemic areas.

Population structure and transmission dynamics of Plasmodium vivax in the Republic of Korea based on microsatellite DNA analysis.

BACKGROUND: In order to control malaria, it is important to understand the genetic structure of the parasites in each endemic area. Plasmodium vivax is widely distributed in the tropical to temperate regions of Asia and South America, but effective strategies for its elimination have yet to be designed. In South Korea, for example, indigenous vivax malaria was eliminated by the late 1970s, but re-emerged from 1993. We estimated the population structure and temporal dynamics of transmission of P. vivax in South Korea using microsatellite DNA markers. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed 255 South Korean P. vivax isolates collected from 1994 to 2008, based on 10 highly polymorphic microsatellite DNA loci of the P. vivax genome. Allelic data were obtained for the 87 isolates and their microsatellite haplotypes were determined based on a combination of allelic data of the loci. In total, 40 haplotypes were observed. There were two predominant haplotypes: H16 and H25. H16 was observed in 9 isolates (10%) from 1996 to 2005, and H25 in 27 (31%) from 1995 to 2003. These results suggested that the recombination rate of P. vivax in South Korea, a temperate country, was lower than in tropical areas where identical haplotypes were rarely seen in the following year. Next, we estimated the relationships among the 40 haplotypes by eBURST analysis. Two major groups were found: one composed of 36 isolates (41%) including H25; the other of 20 isolates (23%) including H16. Despite the low recombination rate, other new haplotypes that are genetically distinct from the 2 groups have also been observed since 1997 (H27). CONCLUSIONS/SIGNIFICANCE: These results suggested a continual introduction of P. vivax from other population sources, probably North Korea. Molecular epidemiology using microsatellite DNA of the P. vivax population is effective for assessing the population structure and transmission dynamics of the parasites--information that can assist in the elimination of vivax malaria in endemic areas.

Role of aquaporin 3 in development, subtypes and activation of dendritic cells.

Dendritic cells (DCs) uptake soluble antigens and large volumes of fluid through macropinocytosis and migrate for antigen presentation. Aquaporin 3 (AQP3), a water and glycerol transporting protein, is highly expressed in immature DCs. To elucidate the role of AQP3 in DC function, we investigated subtype and activation of DCs in AQP3 knock-out (AQP3(-/-)) mice. Depletion of AQP3 did not affect the development of bone marrow-derived DCs (BM-DCs) by GM-CSF or the Flt3 ligand and the level of expression of CD86 on unstimulated and LPS-stimulated BM-DCs. In addition, the percentage of CD86(+) cells among splenic cDCs after LPS treatment in both in vitro and in vivo conditions was similar in wild type and AQP3(-/-) mice. However, the frequency of CD4(+) cDCs in the spleen of AQP3(-/-) mice was significantly lower than that of wild type mice. There was higher expression of CD103 in the CD8(+) subpopulation of splenic cDCs from AQP3(-/-) mice than wild type mice. In the dermis, more CD103-expressing cells were detected in AQP3(-/-) mice than in wild type mice and the LPS-induced decrease of CD103(+) dermal DCs was impaired in AQP3(-/-) mice. AQP3 depletion did not affect the uptake of either albumin or dextran by CD11c(+) splenic DCs. However, HgCl(2), which is an AQP inhibitor, significantly inhibited the uptake of albumin but not dextran by CD11c(+) splenic DCs. These results suggest that AQP3 may play a role in modulating DC population and migration.

A novel real-time PCR assay for the detection of Plasmodium falciparum and Plasmodium vivax malaria in low parasitized individuals.

The rapid, accurate diagnosis of Plasmodium spp. is essential for the effective control of malaria, especially in asymptomatic infections. In this study, we developed a sensitive, genus-specific, real-time quantitative PCR assay. It was compared with the microscopic examination of Giemsa-stained blood smears and two different molecular diagnostic techniques: nested PCR and multiplex PCR. For the effective quantitative detection of malaria parasites, all reagents were designed with a lyophilized format in one tube. Plasmodium was detected successfully in all 112 clinically suspected malaria patients, including 32 individuals with low parasitemia (1-100 parasites/µl). The sensitivity threshold was 0.2 parasites/µl and no PCR-positive reaction occurred when malaria parasites were not present. This may be a useful method for detecting malaria parasites in endemic areas.

Geographical origin of Plasmodium vivax in the Republic of Korea: haplotype network analysis based on the parasite's mitochondrial genome.

BACKGROUND: The Republic of Korea (South Korea) is one of the countries where vivax malaria had been successfully eradicated by the late 1970s. However, re-emergence of vivax malaria in South Korea was reported in 1993. Several epidemiological studies and some genetic studies using antigenic molecules of Plasmodium vivax in the country have been reported, but the evolutionary history of P. vivax has not been fully understood. In this study, the origin of the South Korean P. vivax population was estimated by molecular phylogeographic analysis. METHODS: A haplotype network analysis based on P. vivax mitochondrial (mt) DNA sequences was conducted on 11 P. vivax isolates from South Korea and another 282 P. vivax isolates collected worldwide. RESULTS: The network analysis of P. vivax mtDNA sequences showed that the coexistence of two different groups (A and B) in South Korea. Groups A and B were identical or close to two different populations in southern China. CONCLUSIONS: Although the direct introduction of the two P. vivax populations in South Korea were thought to have been from North Korea, the results of this analysis suggest the genealogical origin to be the two different populations in southern China.

Porcine aortic endothelial cell genes responsive to selected inflammatory stimulators.

Use of porcine tissues has been suggested as a promising solution for severe shortage of transplantable human organs. The immediate hurdle for xenotransplantation is acute immune/inflammatory vascular rejection of the transplant. Because endothelial cells play a key role in the initiation and the amplification of inflammation, alteration of gene expression in human endothelial cells, by various inflammatory stimulators has been studied extensively. However, transcriptional changes induced by human and other inflammatory stimulators in porcine endothelial cells have thus far not been studied. In this study, we treated porcine endothelial cells with human tumor necrosis factor (TNF)-alpha, porcine interferon (IFN)-gamma, H(2)O(2) and lypopolysaccharide (LPS) and profiled transcriptional change at 1 hr, 6 hr and 24 hr, using pig oligonucleotide 13K microarray. We found that mRNA species such as chemokine (C-X-C motif) ligand 6 (CXCL6) and Cathepsin S were significantly induced in porcine endothelial cells, as was previously reported with human endothelial cell. We also found that mRNA species including secreted frizzled-related protein 2 (SFRP2), radical S-adenosyl methionine domain containing 2 (RSAD2), structure specific recognition protein 1 (SSRP1) also were highly overexpressed in porcine endothelial cells. This result shows clues to understand underlying mechanisms of xenotransplantation rejection and the highly responsive porcine genes may serve as novel targets to be regulated for improving the function of grafted porcine donor organs.

Genetic characteristics of polymorphic antigenic markers among Korean isolates of Plasmodium vivax.

Plasmodium vivax, a protozoan malaria parasite of humans, represents a major public health concern in the Republic of Korea (= South Korea). However, little is known about the genetic properties and population structures of the P. vivax isolates circulating in South Korea. This article reviews known polymorphic genetic markers in South Korean isolates of P. vivax and briefly summarizes the current issues surrounding the gene and population structures of this parasite. The critical genetic characteristics of major antigens of the parasite, such as circumsporozoite protein (CSP), merozoite surface protein 1 (MSP-1) and MSP-3, Duffy binding protein (DBP), apical membrane antigen 1 (AMA-1), and GAM-1, are also discussed.

Molecular genetic characterization of the merozoite surface protein 1 Gene of Plasmodium vivax from reemerging Korean isolates.

Plasmodium vivax merozoite surface protein 1 (PvMSP-1) has been considered a major candidate for the development of an antimalaria vaccine, but the molecule exhibits antigenic diversity among isolates. The extent of genetic polymorphism in the region between interspecies conserved blocks 4 and 5 (ICB4 and ICB5) of the PvMSP-1 gene was analyzed for 30 Korean isolates. Two genotypes, SK-A and SK-B, were identified on the basis of amino acid substitution. Almost all the amino acid sequences of the Korean isolates were nearly identical to those of the Solomon Island isolate Solo-83 (97.8 to 99.9% similarity) and Philippine isolates Ph-79, Ph-52-2, and Ph-49 (97.3 to 99.8% similarity). Also, we report two sequences in the isolates that were characterized on the basis of restriction fragment length polymorphism (RFLP). The RFLP profiles following digestion with the DraI restriction enzyme produced two distinguishable patterns. This study might be the first report of the region between ICB4 and ICB5 of the MSP-1 gene of P. vivax in South Korea.

Plasmodium vivax PCR genotyping of the first malaria case imported from South Korea into Japan.

Although indigenous malaria was successfully eradicated in Japan in 1959, malaria remains one of the most important health concerns in the control of imported infectious diseases. However, in South Korea, the re-emergence of indigenous vivax malaria was reported in 1993 in the Demilitarized Zone (the border region with North Korea), from where a vivax malaria case was imported into Japan in 2002. In this study, we conducted genotyping of the circumsporozoite protein gene, the apical membrane antigen-1 gene, and the merozoite surface protein-1 gene of Plasmodium vivax in one patient, and estimated the geographical origin of the parasites. This estimate was based on the findings of previous studies, which showed the coexistence of at least two distinct genotypes of antigenic molecules of endemic P. vivax in South Korea. One genotype is similar to that of a Chinese strain CH-5, and the other is similar to that of a North Korean isolate. The results of this study showed that the DNA sequences of the patient's P. vivax parasites were similar to those of the North Korean isolate. It may even be possible in the near future for seasonally synchronized North Korean P. vivax parasites to be imported into parts of Japan and to establish breeding populations.

Effects of PEGylated scFv antibodies against Plasmodium vivax duffy binding protein on the biological activity and stability in vitro.

Duffy binding protein (DBP) plays a critical role in Plasmodium vivax invasion of human red blood cells. We previously reported a single-chain antibody fragment (scFv) that was specific to P. vivax DBP (PvDBP). However, the stabilization and the half-life of scFvs have not been studied. Here, we investigated the effect of PEGylated scFvs on their biological activity and stability in vitro. SDS-PAGE analysis showed that three clones (SFDBII-12, -58, and -92) were formed as dimers (about 70 kDa) with PEGylation. Clone SFDBII-58 gave the highest yield of PEGylated scFv. Binding analysis using BIAcore between DBP and scFv showed that both SFDBII-12 and -58 were decreased approximately by two folds at the level of binding affinity to DBP after PEGylation. However, the SFDBII-92 clone still showed a relatively high level of binding affinity (KD=1.02 x 10(-7) M). Binding inhibition assay showed that PEGylated scFv was still able to competitively bind the PvDBP and play a critical role in inhibiting the interactions between PvDBP protein expressed on the surface of Cos-7 cells and Duffy receptor on the surface of erythrocytes. When both scFvs and their PEGylated counterparts were exposed to trypsin, scFv was completely degraded only after 24 h, whereas 35% of PEGylated scFvs remained intact, maintaining their stability against the proteolytic attack of trypsin until 72 h. Taken together, these results suggest that the PEGylated scFvs retain their stability against proteolytic enzymes in vivo, with no significant loss in their binding affinity to target antigen, DBP.

Single-chain antibody fragment specific for Plasmodium vivax Duffy binding protein.

Phage display of single-chain variable fragment (scFv) antibodies is a powerful tool for selecting important, useful, and specific human antibodies. We constructed a library from three patients infected with Plasmodium vivax. Panning on recombinant PvRII enriched a population of scFvs that recognized region II of the P. vivax Duffy binding protein (DBP). Three clones of scFvs that reacted with PvRII were selected, and their biological functions were analyzed. These scFvs inhibited erythrocyte binding to DBP. Clone SFDBII92 had the greatest affinity (dissociation constant = 3.62 x 10(-8) M) and the greatest inhibition activity (50% inhibitory concentration approximately 2.9 microg/ml) to DBP. Thus, we demonstrated that human neutralizing antibody could be made from malaria patients using phage display and that these neutralizing scFvs should prove valuable for developing both passive and active immunization strategies based on DBP.

Epidermal growth factor-like motifs 1 and 2 of Plasmodium vivax merozoite surface protein 1 are critical domains in erythrocyte invasion.

Plasmodium vivax merozoite surface protein 1 (PvMSP1) is believed to be important in erythrocyte invasion. However, the detailed mechanism of PvMSP1-mediated invasion has been unclear. We demonstrate that the C-terminal 19 kDa domain (PvMSP119) of PvMSP1, the 42-kDa fragment of PvMSP1 is further cleaved to a 33 kDa N-terminal polypeptide and a 19 kDa C-terminal fragment in a secondary processing step, is a critical domain in the binding between parasite ligand and erythrocyte receptor. Also, its cytoadherence was successfully blocked by naturally acquired immunity, was partially sensitive to neuraminidase and trypsin. When expressed separately epidermal growth factor (EGF)-like motifs 1 and 2, subunits of the PvMSP119, mediated 64% and 66% of the erythrocyte-binding activity, respectively, relative to their expression together as a single intact ligand domain. These results suggest that the EGF-like motifs 1 and 2 of PvMSP119 function as a core-binding portion in the attachment of PvMSP1 to erythrocytes.

Cloning of a pore-forming subunit of ATP-sensitive potassium channel from Clonorchis sinensis.

A complete cDNA sequence encoding a pore-forming subunit (Kir6.2) of ATP-sensitive potassium channel in the adult worm, Clonorchis sinensis, termed CsKir6.2, was isolated from an adult cDNA library. The cDNA contained a single open-reading frame of 333 amino acids, which has a structural motif (a GFG-motif) of the putative pore-forming loop of the Kir6.2. Peculiarly, the CsKir6.2 shows a lack-sequence structure, which deleted 57 amino acids were deleted from its N-terminus. The predicted amino acid sequence revealed a highly conserved sequence as other known other Kir6.2 subunits. The mRNA was weekly expressed in the adult worm.