Red-Emitting Latex Nanoparticles by Stepwise Entrapment of ß-Diketonate Europium Complexes.

The core-shell structure of poly(St-co-MAA) nanoparticles containing ß-diketonate Eu3+ complexes were synthesized by a step-wise process. The ß-diketonate Eu3+ complexes of Eu (TFTB)2(MAA)P(Oct)3 [europium (III); 4,4,4-Trifluoro-1-(2-thienyl)-1,3-butanedione = TFTB; trioctylphosphine = (P(Oct)3); methacrylic acid = MAA] were incorporated to poly(St-co-MAA). The poly(St-co-MAA) has highly monodispersed with a size of 300 nm, and surface charges of the poly(St-co-MAA) are near to neutral. The narrow particle size distribution was due to the constant ionic strength of the polymerization medium. The activated carboxylic acid of poly(St-co-MAA) further chelated with europium complex and polymerize between acrylic groups of poly(St-co-MAA) and Eu(TFTB)2(MAA)P(Oct)3. The Em spectra of europium complexes consist of multiple bands of Em at 585, 597, 612 and 650 nm, which are assigned to 5D0→7FJ (J = 0-3) transitions of Eu3+, respectively. The maximum Em peak is at 621 nm, which indicates a strong red Em characteristic associated with the electric dipole 5D0→7F2 transition of Eu3+ complexes. The cell-specific fluorescence of Eu(TFTB)2(MAA)P(Oct)3@poly(St-co-MAA) indicated endocytosis of Eu(TFTB)2(MAA)P(Oct)3@poly(St-co-MAA). There are fewer early apoptotic, late apoptotic and necrotic cells in each sample compared with live cells, regardless of the culture period. Eu(TFTB)2(MAA)P(Oct)3@poly(St-co-MAA) synthesized in this work can be excited in the full UV range with a maximum Em at 619 nm. Moreover, these particles can substitute red luminescent organic dyes for intracellular trafficking and cellular imaging agents.

Fractionated Coptis chinensis Extract and Its Bioactive Component Suppress Propionibacterium acnes-Stimulated Inflammation in Human Keratinocytes.

Coptis chinensis (CC) is widely used in Asian countries to treat inflammatory diseases. We investigated the anti-inflammatory activity of the aqueous fraction separated from CC extract and of berberine, its key bioactive component, in human keratinocytes and the possible molecular mechanisms underlying this. Treating HaCaT keratinocytic cells with heat-killed Propionibacterium acnes induced nitric oxide and proinflammatory cytokine (e.g., tumor necrosis factor-α, interleukin (IL)-1ß, and IL-8) production and their mRNA expression; these effects were suppressed by pretreatment with the aqueous fraction or berberine, which also suppressed the phosphorylation of ERK, JNK, and p38 kinases and the nuclear expression of nuclear factor (NF)-κB p65 in P. acnes-stimulated cells. Thus, the aqueous fraction and berberine effectively exerted anti-inflammatory activities by suppressing mitogen-activated protein kinase and NF-κB signaling pathways in human keratinocytes and may be used for treating P. acnes-induced inflammatory skin diseases.

High Endogenous Accumulation of ω-3 Polyunsaturated Fatty Acids Protect against Ischemia-Reperfusion Renal Injury through AMPK-Mediated Autophagy in Fat-1 Mice.

Regulated autophagy is involved in the repair of renal ischemia-reperfusion injury (IRI). Fat-1 transgenic mice produce ω3-Polyunsaturated fatty acids (ω3-PUFAs) from ω6-Polyunsaturated fatty acids (ω6-PUFAs) without a dietary ω3-PUFAs supplement, leading to a high accumulation of omega-3 in various tissues. ω3-PUFAs show protective effects against various renal injuries and it has recently been reported that ω3-PUFAs regulate autophagy. We assessed whether ω3-PUFAs attenuated IR-induced acute kidney injury (AKI) and evaluated its associated mechanisms. C57Bl/6 background fat-1 mice and wild-type mice (wt) were divided into four groups: wt sham (n = 10), fat-1 sham (n = 10), wt IRI (reperfusion 35 min after clamping both the renal artery and vein; n = 15), and fat-1 IRI (n = 15). Kidneys and blood were harvested 24 h after IRI and renal histological and molecular data were collected. The kidneys of fat-1 mice showed better renal cell survival, renal function, and pathological damage than those of wt mice after IRI. In addition, fat-1 mice showed less oxidative stress and autophagy impairment; greater amounts of microtubule-associated protein 1A/1B-light chain 3 (LC3)-II, Beclin-1, and Atg7; lower amounts of p62; and, higher levels of renal cathepsin D and ATP6E than wt kidneys. They also showed more adenosine monophosphate-activated protein kinase (AMPK) activation, which resulted in the inhibition of phosphorylation of the mammalian target of rapamycin (mTOR). Collectively, ω3-PUFAs in fat-1 mice contributed to AMPK mediated autophagy activation, leading to a renoprotective response.

Sequence conservation of Plasmodium vivax glutamate dehydrogenase among Korean isolates and its application in seroepidemiology.

BACKGROUND: Glutamate dehydrogenase of malaria parasites (pGDH) is widely used in rapid diagnostic tests for malaria. Variation in the pGDH gene among Korean isolates of Plasmodium vivax was analysed, and a recombinant pGDH protein was evaluated for use as antigens for the serodiagnosis of vivax malaria. METHODS: Genomic DNA was purified from blood samples of 20 patients and the pGDH gene of P. vivax was sequenced. Recombinant protein was prepared to determine the antigenicity of pGDH by enzyme-linked immunosorbent assay (ELISA). RESULTS: Partial sequence analysis of the P. vivax pGDH gene from the 20 Korean isolates showed that an open reading frame (ORF) of 1410 nucleotides encoded a deduced protein of 470 amino acids. The amino acid and nucleotide sequences were conserved among all the Korean isolates. This ORF showed 100% homology with P. vivax strain Sal-I (GenBank accession No. XP_001616617.1). The full ORF (amino acids 39-503), excluding the region before the intron, was cloned from isolate P. vivax Bucheon 3 (KJ726751) and subcloned into the expression vector pET28b for transformation into Escherichia coli BL21(DE3)pLysS. The expressed recombinant protein had a molecular mass of approximately 55 kDa and showed 84.8% sensitivity (39/46 cases) and 97.2% specificity (35/36 cases) in an ELISA. The efficacy of recombinant pGDH protein in seroepidemiological studies was also evaluated by ELISA using serum samples collected from 876 inhabitants of Gyodong-myeon, Ganghwa County, Incheon Metropolitan City. Of these samples, 91 (10.39%) showed a positive reaction with recombinant pGDH protein. Among the antibody-positive individuals, 13 (14.29%) had experienced malaria infection during the last 10 years. CONCLUSION: The pGDH genes of P. vivax isolates from representative epidemic-prone areas of South Korea are highly conserved. Therefore, pGDH is expected to be a useful antigen in seroepidemiological studies. It was difficult to identify the foci of malaria transmission in Gyodong-myeon based on the patient distribution because of the very low parasitaemia of Korean vivax malaria. However, seroepidemiology with recombinant pGDH protein easily identified regions with the highest incidence of malaria within the study area. Therefore, recombinant pGDH protein may have a useful role in serodiagnosis.

Phlorofucofuroeckol Improves Glutamate-Induced Neurotoxicity through Modulation of Oxidative Stress-Mediated Mitochondrial Dysfunction in PC12 Cells.

Stroke is a complex neurodegenerative disorder with a clinically high prevalence and mortality. Despite many efforts to protect against ischemic stroke, its incidence and related permanent disabilities continue to increase. In this study, we found that pretreatment with phlorofucofuroeckol (PFF), isolated from brown algae species, significantly increased cell viability in glutamate-stimulated PC12 cells. Additionally, glutamate-stimulated cells showed irregular morphology, but PFF pretreatment resulted in improved cell morphology, which resembled that in cells cultured under normal conditions. We further showed that PFF pretreatment effectively inhibited glutamate-induced apoptotic cell death in a caspase-dependent manner. Reactive oxygen species (ROS) induced by oxidative stress are closely associated with ischemia-induced neurological diseases. Exposure of PC12 cells to glutamate induced abundant production of intracellular ROS and mitochondrial dysfunction, which was attenuated by PFF in a dose-dependent manner. In vivo studies revealed that PFF-mediated prevention was achieved predominantly through inhibition of apoptosis and mitochondrial ROS generation. Taken together, these results suggest the possibility of PFF as a neuroprotective agent in ischemic stroke.

Up-regulated S100 calcium binding protein A8 in Plasmodium-infected patients correlates with CD4(+)CD25(+)Foxp3 regulatory T cell generation.

BACKGROUND: The pro-inflammatory S100 calcium binding protein A8 (S100A8) is elevated in the serum of patients with Plasmodium falciparum malaria, but its function in Plasmodium vivax malaria is not yet clear. This function was investigated in P. vivax-infected patients in this study. METHODS: The level of S100A8 in the serum was measured with ELISA. Full amino acids of S100A8 were synthesized to verify the functions for maturation of immature dendritic cell (iDC) and evaluation of CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) generation by mature DC (mDC). RESULTS: A higher amount of S100A8 was detected in vivax-infected patients (141.2 ± 61.849 ng/ml, n = 40) compared with normal control group (48.1 ± 27.384 ng/ml, n = 40). The level of S100A8 did not coincide with that of anti-malarial antibody measured by indirect fluorescent antibody test (IFAT) using parasite-infected red blood cells as antigen. Programmed death-ligand 1 (PD-L1) was up-regulated on the surface of iDCs following treatment with synthetic S100A8, not with synthetic MSP-1, AMA-1 and CSP, as compared to the expression seen for non-treated iDCs. The addition of red blood cells of infected patients to iDCs also elevated their surface expression of CD86. However, the serum levels of S100A8 decreased with increase in parasitaemia. DCs matured by sera containing S100A8 generated Treg cells from naïve T cells. The ratio of Treg cells generated was inversely proportional to the concentration of S100A8 in sera. CONCLUSIONS: Treg cells suppress the activity of cytotoxic T cells, which kill malaria parasites; therefore, the up-regulation of S100A8 in malaria patients may contribute to pathogen immune escape or tolerance.

Relationship between Antibody-Positive Rate against Plasmodium vivax Circumsporozoite Protein and Incidence of Malaria.

The relationship between anti- Plasmodium vivax circumsporozoite protein (CSP) antibody levels and the prevalence of malaria in epidemic areas of South Korea was evaluated. Blood samples were collected from inhabitants of Gimpo-si (city), Paju-si, and Yeoncheon-gun (county) in Gyeonggi-do (province), as well as Cheorwon-gun in Gangwon-do from November to December 2004. Microscopic examinations were used to identify malaria parasites. ELISA was used to quantitate anti-circumsporozoite protein (CSP) antibodies against P. vivax. A total of 1,774 blood samples were collected. The overall CSP-ELISA-positive rate was 7.7% (n=139). The annual parasite incidences (APIs) in these areas gradually decreased from 2004 to 2005 (1.09 and 0.80, respectively). The positive rate in Gimpo (10.4%, 44/425) was the highest identified by CSP-ELISA. The highest API was found in Yeoncheon, followed by Cheorwon, Paju, and Gimpo in both years. The positive rates of CSP-ELISA were closely related to the APIs in the study areas. These results suggest that seroepidemiological studies based on CSP may be helpful in estimating the malaria prevalence in certain areas. In addition, this assay can be used to establish and evaluate malaria control and eradication programs in affected areas.

Current situation of scrub typhus in South Korea from 2001-2013.

BACKGROUND: The bacteria Orientia tsutsugamushi is the causative agent of scrub typhus, mite-borne disease, which causes an acute febrile illness in patients. An epidemiologic study was conducted to understand the characteristics of scrub typhus in South Korea. FINDINGS: Reporting of tsutsugamushi disease is mandatory in South Korea since 1994. To investigate the prevalence of tsutsugamushi disease from 2001 to 2013, medical records from the Korea Center for Disease Control and Prevention were reviewed. In total, 70,914 cases were reported during 2001-2013. Of these, 37.16% (26,349) were male and 62.84% (44,565) were female. The highest number of cases was in the 60-69-year-old age group (19,484; 27.48%), and 72.22% (51,212) were in the 50-79-year-old age group. There were 65,100 cases (91.80%) reported during October (24,964; 35.20%) and November (40,136; 56.60%). An almost four-fold increase in the number of patients was observed in 2013 (10,485 cases) compared to 2001 (2,637 cases). The highest number of patients was reported in the Jeonbuk (9,425; 13.29%) and lowest in the Jeju (362; 0.51%). CONCLUSIONS: A rapid increase in the incidence of patients with tsutsugamushi disease was observed in most areas from 2001 to 2013, with the majority of cases reported in the western and southern coast.

Detection of antibodies against the CB9 to ICB10 region of merozoite surface protein-1 of Plasmodium vivax among the inhabitants in epidemic areas.

BACKGROUND: The purpose of this study was to examine the usefulness of the conserved block 9 (CB9) to interspecies conserved block (ICB10) region of Plasmodium vivax merozoite surface protein-1 (MSP-1 (ICB910)) as a serodiagnostic tool for understanding malaria transmission. METHODS: Antibody titre in the blood samples collected from the inhabitants of Gimpo city, Paju city and Yeoncheon county of Gyeonggi Province, as well as Cheorwon county of Gangwon Province, South Korea were determined by enzyme-linked immunosorbent assay (ELISA). Microscopic examination was performed to identify malarial parasites. RESULTS: MSP-1(ICB910) is encoded by a 1,212-bp sequence, which produced a recombinant protein with a molecular weight of approximately 46 kDa. Antibody titres in 1,774 blood samples were determined with the help of ELISA using purified recombinant MSP-1(ICB910). The overall ELISA-positive rate was 8.08% (n = 146). The annual parasite incidences (APIs) in the regions where the blood sampling was carried out gradually decreased from 2004 to 2005 (1.09 and 0.80, respectively). Yeoncheon county had the highest ELISA-positive rate (10.20%, 46/451). Yeoncheon county also had the highest API both in 2004 and 2005, followed by Cheorwon county, Paju city and Gimpo city. CONCLUSIONS: The MSP-1 (ICB910)-ELISA-positive rates were closely related to API in the geographic areas studied. These results suggest that sero-epidemiological studies employing MSP-1 (ICB910)-ELISA may be helpful in estimating the prevalence of malaria in certain geographic areas. MSP-1(ICB910)-ELISA can be effectively used to establish and evaluate malaria control and eradication programmes in the affected areas.

Seroprevalence of Plasmodium vivax in the Republic of Korea (2003-2005) using indirect fluorescent antibody test.

Plasmodium vivax reemerged in the Republic of Korea (ROK) in 1993, and is likely to continue to affect public health. The purpose of this study was to measure levels of anti-P. vivax antibodies using indirect fluorescent antibody test (IFAT) in border areas of ROK, to determine the seroprevalence of malaria (2003-2005) and to plan effective control strategies. Blood samples of the inhabitants in Gimpo-si, Paju-si, and Yeoncheon-gun (Gyeonggi-do), and Cheorwon-gun (Gangwon-do) were collected and kept in Korea Centers for Disease Control and Prevention (KCDC). Out of a total of 1,774 serum samples tested, the overall seropositivity was 0.94% (n=17). The seropositivity was the highest in Paju-si (1.9%, 7/372), followed by Gimpo-si (1.4%, 6/425), Yeoncheon-gun (0.67%, 3/451), and Cheorwon-gun (0.19%, 1/526). The annual parasite incidence (API) in these areas gradually decreased from 2003 to 2005 (1.69, 1.09, and 0.80 in 2003, 2004, and 2005, respectively). The highest API was found in Yeoncheon-gun, followed by Cheorwon-gun, Paju-si, and Gimpo-si. The API ranking in these areas did not change over the 3 years. The seropositivity of Gimpo-si showed a strong linear relationship with the API of 2005 (r=0.9983, P=0.036). Seropositivity data obtained using IFAT may be useful for understanding malaria prevalence of relevant years, predicting future transmission of malaria, and for establishing and evaluating malaria control programs in affected areas.

Evaluation of circumsporozoite protein of Plasmodium vivax to estimate its prevalence in the Republic of Korea: an observational study of incidence.

BACKGROUND: Plasmodium vivax re-emerged in 1993. Although the number of infections has been steadily decreasing, it is likely to continue to affect public health until it is eradicated. The aim of this study is to measure anti-circumsporozoite protein (CSP) antibody and compare malaria prevalence. As to understand the prevalence, an epidemiology study has to be conducted in the Republic of Korea. METHODS: A total of 1,825 and 1,959 blood samples were collected in 2010 and 2011, respectively, from the inhabitants of Ganghwa and Cheorwon counties. The antibody titers of the inhabitants were measured by enzyme-linked immunosorbent assay (ELISA) using recombinant protein purified from Escherichia coli transformed with a CSP gene-inserted pET-28a(+) expression vector. Microscopic examination was performed to identify malaria parasites. RESULTS: The annual parasite incidence (API) in Ganghwa decreased from 4.28 in 2010 to 2.23 in 2011, and that in Cheorwon decreased from 1.88 in 2010 to 1.15 in 2011. The antibody-positive CSP rate in these areas also decreased from 18.14% (331/1825) in 2010 to 15.36% (301/1959) in 2011. Pearson analysis showed a strong correlation between the API and the antibody-positive CSP rate in these areas (r = 1.000, P < 0.01). The intensity of the immune responses of the inhabitants of Cheorwon, as measured by the mean optical density, decreased from 0.9186 ± 0.0472 in 2010 to 0.7035 ± 0.0457 in 2011 (P = 0.034), but increased in Ganghwa from 0.7649 ± 0.0192 in 2010 to 0.8237 ± 0.1970 in 2011 (P = 0.006). The immune response increased according to age (r = 0.686, P = 0.041). CONCLUSIONS: The positive CSP-ELISA rate was closely related to the API in the study areas. This suggests that seroepidemiological studies based on CSP-ELISA may be helpful in estimating the malaria prevalence. Moreover, such studies can be used to establish and evaluate malaria control and eradication programmes in high-risk areas in Korea.

Identification of human leucocyte antigen (HLA)-A*0201-restricted cytotoxic T lymphocyte epitopes derived from HLA-DOß as a novel target for multiple myeloma.

Despite the recent development of effective therapeutic agents against multiple myeloma (MM), new therapeutic approaches, including immunotherapies, remain to be developed. Here we identified novel human leucocyte antigen (HLA)-A*0201 (HLA-A2)-restricted cytotoxic T lymphocyte (CTL) epitopes from a B cell specific molecule HLA-DOß (DOB) as a potential target for MM. By DNA microarray analysis, the HLA-DOB expression in MM cells was significantly higher than that in normal plasma cells. Twenty-five peptides were predicted to bind to HLA-A2 from the amino acid sequence of HLA-DOB. When screened for the immunogenicity in HLA-A2-transgenic mice immunized with HLA-DOB cDNA, 4 peptides were substantially immunogenic. By mass spectrometry analysis of peptides eluted from HLA-A2-immunoprecipitates of MM cell lines, only two epitopes, HLA-DOB232-240 (FLLGLIFLL) and HLA-DOB185-193 (VMLEMTPEL), were confirmed for their physical presence on cell surface. When healthy donor blood was repeatedly stimulated in vitro with these two peptides and assessed by antigen-specific γ-interferon secretion, HLA-DOB232-240 was more immunogenic than HLA-DOB185-193 . Additionally, the HLA-DOB232-240 -specific CTLs, but not the HLA-DOB185-193 -specific CTLs, displayed an major histocompatibility complex class I-restricted reactivity against MM cell lines expressing both HLA-A2 and HLA-DOB. Taken together, based on the physical presence on tumour cell surface and high immunogenicity, HLA-DOB232-240 might be useful for developing a novel immunotherapy against MM.

Decreasing incidence of Plasmodium vivax in the Republic of Korea during 2010-2012.

BACKGROUND: After the re-emergence of Plasmodium vivax in 1993, a total of 31,254 cases of vivax malaria were reported between 1993-2012 in the Republic of Korea (ROK). The purpose of this study was to review Korea Centers for Disease Control and Prevention records to investigate the transmission of malaria from 2010-2012. METHODS: Reporting of microscopy-diagnosed cases of malaria is mandatory in the ROK. In this study, all available records of malaria cases and malaria vectors collected from 2010 - 2012 in Cheorwon County, Gangwon Province and Ganghwa County, Incheon Metropolitan City, were reviewed. RESULTS: Although the number of cases of malaria peaked a third time in 2010 (1,772 cases) since the re-emergence of P. vivax, the incidence decreased two-fold to 838 in 2011 and three-fold to 555 in 2012. The number of cases decreased 52.7% in 2011 compared with that in 2010 and 33.8% in 2012 compared with that in 2011. However, the number of cases increased in Incheon Metropolitan City (15.3%) and Gyeongnam Province (23.1%) in 2012 compared with 2011. Of the 3,165 cases of vivax malaria in 2010-2012, 798 (25.2%) were in ROK military personnel, 519 (16.4%) in veterans, and 1,848 (58.4%) in civilians. In total, there were 2,666 male patients and 499 female patients, and the ratio of female to male patients increased from 1:7.9 in 2011 to 1:4.1 in 2012. CONCLUSIONS: A rapid decrease in the incidence of malaria was observed in most areas from 2010 to 2012, but the incidence increased again in the western part of the demilitarized zone. Therefore, more intensive surveillance is needed throughout high risk areas to identify factors responsible for increase/decrease in the incidence of malaria in the ROK.

Polymorphism of the parasite lactate dehydrogenase gene from Plasmodium vivax Korean isolates.

BACKGROUND: Assaying for the parasitic lactate dehydrogenase (pLDH) is widely used as a rapid diagnostic test (RDT), but the efficacy of its serological effectiveness in diagnosis, that is antibody detection ability, is not known. The genetic variation of Korean isolates was analysed, and recombinant protein pLDH was evaluated as a serodiagnostic antigen for the detection of Plasmodium vivax malaria. METHODS: Genomic DNA was purified, and the pLDH gene of P. vivax was amplified from blood samples from 20 patients. The samples came from five epidemic areas: Bucheon-si, Gimpo-si, and Paju-si of Gyeonggi Province, Gangwha-gun of Incheon metropolitan city, and Cheorwon-gun of Gangwon Province, South Korea, from 2010 to 2011. The antigenicity of the recombinant protein pLDH was tested by western blot and enzyme-linked immunosorbent assay (ELISA). RESULTS: Sequence analysis of 20 Korean isolates of P. vivax showed that the open reading frame (ORF) of 951 nucleotides encoded a deduced protein of 316 amino acids (aa). This ORF showed 100% identity with the P. vivax Belem strain (DQ060151) and P. vivax Hainan strain (FJ527750), 89.6% homology with Plasmodium falciparum FCC1_HN (DQ825436), 90.2% homology with Plasmodium berghei (AY437808), 96.8% homology with Plasmodium knowlesi (JF958130), and 90.2% homology with Plasmodium reichenowi (AB122147). A single-nucleotide polymorphism (SNP) at nucleotide 456 (T to C) was also observed in the isolate from Bucheon, but it did not change in the amino acid sequence. The expressed recombinant protein had a molecular weight of approximately 32 kDa, as analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Of the 40 P. vivax patients, 34 (85.0%) were positive by ELISA. CONCLUSIONS: The pLDH genes of 19 isolates of P. vivax were identical, except one for SNP at nucleotide 456. This observation indicates that this gene is relatively stable. Based on these results, the relationship between antibody production against pLDH and the pattern of disease onset should be investigated further before using pLDH for serodiagnosis.

Efficient internalization of peptide-conjugated SPIONs in dendritic cells for tumor targeting.

Antigen delivery using nanoparticles becomes useful and novel strategy to develop immunotherapeutic approaches against cancer. In the current study, we examined the feasibility of SPIONs-mediated delivery of antigenic peptides to local tumor for application to cancer immunotherapy. SPIONs carrying murine melanoma antigens, hgp100(25-33) were prepared and used to test its efficacy in mouse model. Efficient uptake of peptide-conjugated SPIONs by murine dendritic cells (DCs) was shown, using NP labeled with the fluorescent dye Furthermore, potential targeting effect of SPIONs carrying tumor antigenic peptide was verified in vitro and in vivo. Our results demonstrate the feasibility of SPIONs-mediated antigen delivery for cancer immunotherapy and highlight the clinical potential of SPIONs for future cancer treatment with high efficacy.

Complete phase transfer of hydrophobic magnetic nanocrystals into aqueous phase.

The effective phase transfer of hydrophobic nanocrystals synthesized in nonpolar solvents into polar solvents remains great challenge for their nanomedicinal applications. To resolve this issue, the exsiting strategies for phase transfer of nanocrystals in organic solvents use amphiphilic compounds or lipids as imperative moieties by ligand exchange and encapsulations. Ligand exchange involves by exchanging the hydrophobic molecules with bifunctional compounds and small size of molecules is coordinated with functional molecules to increase the steric repulsion forces between boundary of water and nanoparticles. However, the yield of phase transferred nanocrystals from hydrophobic nonpolar to hydrophilic polar phase is exceptionally low, and sometime irreversible desorption of replaced ligands leads to agglomeration and aggregation. Also, their intrinsic physiochemical properties are easily influenced by surrounding ion species or pH when the particles are suspended in phosphate saline buffer (PBS). Moreover, conjugation of bioactive molecules often leads to colloidal instability in PBS because of the hydrodynamic nature of the amphiphilic molecules on the surfaces of nanoparticles. Here we report a robust and simple post-synthetic surface modification procedure of hydrophobic nanoparticles to remove the original surface-bound carboxylic acid and increase the water-solubility for nanomedicinal applications.

Impaired tumor antigen processing by immunoproteasome-expressing CD40-activated B cells and dendritic cells.

Professional APCs, such as dendritic cells, are routinely used in vitro for the generation of cytotoxic T lymphocytes specific for tumor antigens. In addition to dendritic cells, CD40-activated B cells and variant K562 leukemic cells can be readily transfected with nucleic acids for in vitro and in vivo antigen presentation. However, the expression of immunoproteasome components in dendritic cells may preclude display of tumor antigens such as Mart1/MelanA. Here, we use three target epitopes, two derived from tumor antigens [Mart1(26-34) (M26) and Cyp1B1(239-247) (Cyp239)] and one derived from the influenza A viral antigen [FluM1(58-66) (FluM58)], to demonstrate that CD40-activated B cells, like dendritic cells, have a limited capability to process certain tumor antigens. In contrast, the K562 HLA-A*0201 transfectant efficiently processes and presents M26 and Cyp239 as well as the influenza FluM58 epitopes to T cells. These results demonstrate that the choice of target APC for gene transfer of tumor antigens may be limited by the relative efficacy of proteasome components to process certain tumor epitopes. Importantly, K562 can be exploited as an artificial APC, efficient in processing both M26 and Cyp239 epitopes and presumably, by extension, other relevant tumor antigens.

Downregulation of erythropoietin receptor by overexpression of phospholipase C-gamma 1 is critical for decrease on focal adhesion in transformed cells.

BACKGROUND: Phospholipase C-γl (PLC-γl) is known to play a critical role in cell adhesion and migration and is highly expressed in metastatic tumors. In the current study, we found that cells transformed by PLC overexpression (PLC-γl cells) exhibited a marked decrease in expression of the Epo receptor (EpoR). Here, we assessed the role of EpoR-dependent signaling pathways in PLC-γl-dependent regulation of cell adhesion and migration. METHODS: Expression and phosphorylation of EpoR and its functional role in PLC-γl cells were evaluated by immunoblot analysis or cell adhesion assay. The mechanism for PLC-γ1-induced EpoR downregulation was analyzed by blockage of proteosomal degradation with MG132. EpoR expression was also confirmed in colorectal cancer tissues in which PLC-γl was highly expressed. RESULTS: EpoR was present on rat fibroblasts, where it functionally active and capable of increasing cell adhesion and migratory activity. However, PLC-γl cells significantly decreased the Epo-dependent effects via ubiquitination-proteosomal degradation of EpoR. A marked decrease of EpoR expression was confirmed in colorectal cancer tissues that showed high-level of PLC-γl expression. CONCLUSION: The Epo/EpoR complex plays a critical role in the adhesion and migration of rat fibroblasts, and its functional inactivation is associated with PLC-γl-dependent reduction of cell-matrix adhesion and this also affects cell migration.

Induction of anti-tumor cytotoxic T cell responses through PLGA-nanoparticle mediated antigen delivery.

Nanotechnology-based antigen delivery has been developing as a vaccine strategy due to its dose-sparing and prolonged antigen presentation features. In the current study, we examined the feasibility of nanoparticle (NP)-mediated delivery of antigenic peptides to efficiently induce cytotoxic T lymphocyte responses against tumor-associated self-antigens in C57BL/6 mouse models. The biodegradable poly(D,L-lactide-co-glycolide) nanoparticle (PLGA-NP) carrying murine melanoma antigenic peptides, hgp100(25-33) and TRP2(180-188), were prepared by double emulsion method. Efficient uptake of PLGA-NP by murine dendritic cells was shown in vitro and in vivo, using NP labeled with the fluorescent dye DiD. Intradermal injection of peptide-loaded PLGA-NP into mice induced antigen-specific T cell responses more strongly than the peptides mixed with Freund's adjuvant. More importantly, vaccination with PLGA-NP carrying both TRP2(180-188) and a toll-like receptor 4 agonist, monophosphoryl lipid A, significantly delayed growth of subcutaneously inoculated B16 melanoma cells in a prophylactic setting. Furthermore, the anti-tumor activity of NP-mediated peptide vaccination was significantly augmented by combined treatment with interferon-γ, which might prevent tumor escape through up-regulation of MHC class I expression on tumor cells. Our findings demonstrate the feasibility of NP-mediated antigen delivery for cancer immunotherapy, in particular when immune escape mechanisms of tumor cells are blocked simultaneously.