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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Biologic activity of irradiated, autologous, GM-CSF-secreting leukemia cell vaccines early after allogeneic stem cell transplantation.

Through an immune-mediated graft-versus-leukemia effect, allogeneic hematopoietic stem cell transplantation (HSCT) affords durable clinical benefits for many patients with hematologic malignancies. Nonetheless, subjects with high-risk acute myeloid leukemia or advanced myelodysplasia often relapse, underscoring the need to intensify tumor immunity within this cohort. In preclinical models, allogeneic HSCT followed by vaccination with irradiated tumor cells engineered to secrete GM-CSF generates a potent antitumor effect without exacerbating the toxicities of graft-versus-host disease (GVHD). To test whether this strategy might be similarly active in humans, we conducted a Phase I clinical trial in which high-risk acute myeloid leukemia or myelodysplasia patients were immunized with irradiated, autologous, GM-CSF-secreting tumor cells early after allogeneic, nonmyeloablative HSCT. Despite the administration of a calcineurin inhibitor as prophylaxis against GVHD, vaccination elicited local and systemic reactions that were qualitatively similar to those previously observed in nontransplanted, immunized solid-tumor patients. While the frequencies of acute and chronic GVHD were not increased, 9 of 10 subjects who completed vaccination achieved durable complete remissions, with a median follow-up of 26 months (range 12-43 months). Six long-term responders showed marked decreases in the levels of soluble NKG2D ligands, and 3 demonstrated normalization of cytotoxic lymphocyte NKG2D expression as a function of treatment. Together, these results establish the safety and immunogenicity of irradiated, autologous, GM-CSF-secreting leukemia cell vaccines early after allogeneic HSCT, and raise the possibility that this combinatorial immunotherapy might potentiate graft-versus-leukemia in patients.

An active CD8alpha/pMHCI interaction is required for CD8 single positive thymocyte differentiation.

Recognition of viral antigenic peptides bound to major histocompatibility complex class I molecules (MHCI) by TCR is critical for initiating the responses of CD8(+) T cells that ultimately lead to elimination of virus-infected cells. This antigen recognition is enhanced by the CD8 coreceptor through its interaction with the peptide-MHCI complexes (pMHCI). Mouse CD8alphabeta can form two different complexes with pMHCI via either the CD8alpha- or CD8beta-dominated interaction. To understand the functional significance of these complexes in vivo, we generated Tg mice carrying a variant CD8alphabeta (CD8alpha(m3)beta) capable of forming only the CD8beta-dominated CD8alphabeta/pMHCI complex. These mice show sub-optimal thymic differentiation with reduced populations of CD8(+) single-positive thymocytes. Tg CD8(+) T cells exhibit a compromised developmental capacity when competing with CD8(+) T cells from B6 mice in mixed bone marrow chimera experiments. However, once these CD8(+) T cells have emigrated to the peripheral lymphoid organs, they exhibit normal effector function against viral infection. Our observations indicate that, in addition to the CD8 activity conferred by CD8beta-dominated CD8alphabeta/pMHCI complexes, full thymocyte differentiation requires additional coreceptor activities conferred by CD8alphaalpha and/or CD8alphabeta with CD8alpha-dominated CD8/pMHCI complexes.

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.

The role of Pvs28 in sporozoite development in Anopheles sinensis and its longevity in BALB/c mice.

To develop a vivax malaria vaccine for blocking malarial transmission, the ookinete surface protein Pvs28 was cloned from Korean malaria patients using polymerase chain reaction. The Pvs28 gene consists of 726bp and encodes 241 amino acids. It was subcloned into the expression vector pQE30 and expressed in Escherichia coli. The expressed recombinant protein, rPvs28, has a molecular weight of about 28 kDa in SDS-PAGE analysis. A monoclonal antibody against rPvs28 was produced using BALB/c mice. It inhibited sporozoite development in Anopheles sinensis mosquitoes (n = 81) which is one of the malaria vectors in Korea, with relatively high antibody titer against rPv28 persisting for more than 6 months. These results indicate that rPvs28 induces an immune response in mice that effectively blocks sporozoite development in mosquitoes. Therefore it could be a vaccine candidate for preventing vivax malaria in Korea.

Prevalence of Plasmodium vivax VK210 and VK247 subtype in Myanmar.

BACKGROUND: Plasmodium vivax is divided into two subtypes, a dominant form, VK210 and a variant form, VK247. This division is dependent on the amino acid composition of the circumsporozoite (CS) protein. In this study, the prevalence of the VK247 variant form of P. vivax was investigated in Myanmar. METHODS: The existence of malaria parasites in blood samples was determined by microscopic examination, polymerase chain reaction (PCR) and DNA hybridization assays. To test for antibodies against P. vivax and Plasmodium falciparum in blood samples, an indirect immunofluorescence antibody test (IFAT) was performed using asexual blood antigens. An enzyme-linked immunosorbent assay with synthetic VK210 and VK247 antigens was carried out to discriminate between the P. vivax subtypes. RESULTS: By thick smear examination, 73 (n=100) patients were single infected with P. vivax, one with P. falciparum and 13 with both species. By thin smear, 53 patients were single infected with P. vivax, eight with only P. falciparum and 16 with both. Most of the collected blood samples were shown to be P. vivax positive (n=95) by PCR. All cases that were positive for P. falciparum by PCR (n=43) were also positive for P. vivax. However, 52 cases were single infected with P. vivax. IFAT showed antibody titres from 1:32 to 1:4,096. Additionally, using specific antibodies for VK210 and VK247, ELISA showed that 12 patients had antibodies for only the VK210 subtype, 4 patients had only VK247 subtype antibodies and 21 patients had antibodies for both subtypes. Using a DNA hybridization test, 47 patients were infected with the VK210 type, one patient was infected with VK247 and 23 patients were infected with both subtypes. CONCLUSIONS: The proportion of the VK247 subtype in Myanmar was 43.1% (n=25) among 58 positive cases by serodiagnosis and 25.6% (n=24) among 94 positive cases by genetic diagnosis. In both diagnostic methods, the infection status of malaria patients is highly diverse with respect to malaria species, and multiple clonal infections are prevalent in Myanmar. Therefore, the complexity of the infection should be considered carefully when diagnosing malaria in Myanmar.

Comparative analysis of the expression patterns of various TNFSF/TNFRSF in atherosclerotic plaques.

Members of the TNFSF/TNFRSF are involved in the immunoregulation of various immune reactions and diseases. Recently, LIGHT/TR2, GITRL/GITR, and TL1A/DR3 have been reported as playing roles in the inflammatory reactions in atherosclerosis, but a comparative analysis of these molecules has not been conducted. In order to compare their expression patterns, immunohistochemical analyses were performed using six human carotid endoarterectomy samples. The expression of these molecules was detected in the various cell types that constitute atherosclerotic plaques. The expression of all analyzed molecules was detected, albeit at various levels, mainly in foamy macrophages in all tested samples. The strong expression of these molecules in endothelial and smooth muscle cells was also detected in 2 and 1 plaque samples, respectively, while others express only some of the tested molecules. Flow cytometry analyses of human monocyte/macrophage cell lines, U937 and THP-1, detected the expression of the tested molecules while a relatively undifferentiated monocytic cell line, TF-1A, failed to express them. These data indicate that activated and differentiated macrophages are the main cell type expressing tested molecules in atherosclerotic plaques while endothelial and smooth muscle cells can express them in limited cases. Pro-inflammatory activities of the tested molecules may contribute to the atherogenesis by stimulating the cells expressing them in atherosclerotic plaques and the successful treatment of atherosclerosis may require cooperative regulation of these activities.