Young-Ha Lee

Inhibition of Lewis Lung Carcinoma Growth by Toxoplasma gondii through Induction of Th1 Immune Responses and Inhibition of Angiogenesis

Toxoplasma gondii is an obligate intracellular protozoan parasite that induces antitumor activity against certain types of cancers. However, little information is available regarding the immunologic mechanisms that regulate these effects. For this purpose, C57BL/6 mice were administered either the T. gondii Me49 strain orally or Lewis lung carcinoma (LLC) cells intramuscularly. Survival rates, tumor size, histopathology, and immune responses were determined for each group, and angiogenesis was evaluated by in vivo Matrigel plug assay. Toxoplasma-infected (TG-injected) mice survived the entire experimental period, whereas cancer cell-bearing (LLC-injected) mice died within six weeks. Mice injected with both T. gondii and cancer cells (TG/LLC-injected group) showed significantly increased survival rates, CD8+ T-cell percentages, IFN-γ mRNA expression levels, serum IgG2a titers, and CTL responses as compared to the LLC-injected mice. In addition, angiogenesis in the TG/LLC-injected mice was notably inhibited. These effects in TG/LCC-injected mice were similar or were increased by the addition of an adjuvant, Quil-A. However, TG/LLC-injected mice showed decreased percentages of CD4+ and CD8+ T-cells, IFN-γ mRNA expression levels, and serum IgG1 and IgG2a titers as compared to TG-injected mice. Taken together, our results demonstrate that T. gondii infection inhibits tumor growth in the Lewis lung carcinoma mouse model through the induction of Th1 immune responses and antiangiogenic activity.

Seroprevalence of Toxoplasma gondii Infection and Risk Factors Associated with Seropositivity of Pregnant Women in Korea

A total of 351 serum samples was examined for anti–T. gondii antibody titers; the overall seroprevalence was 3.7%. The Sabin– Feldman dye test (DT), latex agglutination test (LAT), and IgG enzyme-linked immunosorbent assay (IgG ELISA) indicated seroprevalences of 3.7%, 3.4%, and 4.0%, respectively. Compared with the DT, the sensitivities of the LAT and IgG ELISA were 84.6% and 100.0%, respectively, and the specificities of the LAT and IgG ELISA were both 99.7%. An increase in T. gondii seroprevalence with increasing age was detected, but the difference was not significant. The overall seroprevalence of T. gondii–antibody titers in pregnant Korean women was relatively low compared to those of Europeans and Americans. A multivariate analysis of risk factors showed that T. gondii infection was positively correlated with eating raw meat, but was not associated with the consumption of unwashed vegetables, drinking untreated water, a history of raising a cat, or blood transfusion. The consumption of raw or undercooked meat may, therefore, be the main route of T. gondii infection in Korea.

Induction of Protective Immune Responses by a Multiantigenic DNA Vaccine Encoding GRA7 and ROP1 of Toxoplasma gondii

ABSTRACT Toxoplasma gondii is distributed worldwide and infects most species of warm-blooded animals, including humans. The heavy incidence and severe or lethal damage caused by T. gondii infection clearly indicates the need for the development of a vaccine. To evaluate the protective efficacy of a multiantigenic DNA vaccine expressing GRA7 and ROP1 of T. gondii with or without a plasmid encoding murine interleukin-12 (pIL12), we constructed DNA vaccines using the eukaryotic plasmids pGRA7, pROP1, and pGRA7-ROP1. Mice immunized with pGRA7, pROP1, or pGRA7-ROP1 showed significantly increased serum IgG2a titers; production of gamma interferon (IFN-γ), IL-10, and tumor necrosis factor alpha (TNF-α); in vitro T cell proliferation; and survival, as well as decreased cyst burdens in the brain, compared to mice immunized with either the empty plasmid, pIL12, or vector with pIL12 (vector+pIL12). Moreover, mice immunized with the multiantigenic DNA vaccine pGRA7-ROP1 had higher IgG2a titers, production of IFN-γ and TNF-α, survival time, and cyst reduction rate compared to those of mice vaccinated with either pGRA7 or pROP1 alone. Furthermore, mice immunized with either a pGRA7-ROP1+pIL12 or a single-gene vaccine combined with pIL12 showed greater Th1 immune response and protective efficacy than the single-gene-vaccinated groups. Our data suggest that the multiantigenic DNA antigen pGRA7-ROP1 was more effective in stimulating host protective immune responses than separately injected single antigens, and that IL-12 serves as a good DNA adjuvant.

Genotyping of a Korean isolate of Toxoplasma gondii by multilocus PCR-RFLP and microsatellite analysis

Although the Korean isolate KI-1 of Toxoplasma gondii has been considered to be a virulent type I lineage because of its virulent clinical manifestations, its genotype is unclear. In the present study, genotyping of the KI-1 was performed by multilocus PCR-RFLP and microsatellite sequencing. For 9 genetic markers (c22-8, c29-2, L358, PK1, SAG2, SAG3, GRA6, BTUB, and Apico), the KI-1 and RH strains exhibited typical PCR-RFLP patterns identical to the type I strains. DNA sequencing of tandem repeats in 5 microsatellite markers (B17, B18, TUB2, W35, and TgM-A) of the KI-1 also revealed patterns characteristic of the type I. These results provide strong genetic evidence that KI-1 is a type I lineage of T. gondii.

Involvement of PI 3 kinase/Akt-dependent Bad phosphorylation in Toxoplasma gondii-mediated inhibition of host cell apoptosis.

Toxoplasma gondii-infected cells are resistant to various apoptotic stimuli, however, the role of the pro-apoptotic BH3-only Bad protein in T. gondii-imposed inhibition of host cell apoptosis in connection with the phosphoinositide 3-kinase (PI3K)-PKB/Akt pathway was not well delineated. Here, we investigated the signaling patterns of Bad, Bax and PKB/Akt in T. gondii-infected and uninfected THP-1 cells treated with staurosporine (STS) or PI3K inhibitors. STS treatment, without T. gondii infection, reduced the viability of THP-1 cells in proportion to STS concentration and triggered many cellular death events such as caspase-3 and -9 activation, Bax translocation, cytochrome c release from host cell mitochondria into cytosol, and PARP cleavage in the host cell. However, T. gondii infection eliminated the STS-triggered mitochondrial apoptotic events described above. Additionally, T. gondii infection in vitro and in vivo induced the phosphorylation of PKB/Akt and Bad in a parasite-load-dependent manner which subsequently inhibited Bax translocation. The PI3K inhibitors, LY294002 and Wortmannin, both blocked parasite-induced phosphorylation of PKB/Akt and Bad. Furthermore, THP-1 cells pretreated with these PI3K inhibitors showed reduced phosphorylation of Bad in a dose-dependent manner and subsequently failed to inhibit the Bax translocation, also these cells also failed to overcome the T. gondii-imposed inhibition of host cell apoptosis. These data demonstrate that the PI3K-PKB/Akt pathway may be one of the major route for T. gondii in the prevention of host cell apoptosis and T. gondii phosphorylates the pro-apoptotic Bad protein to prevent apoptosis.

Intestinal Parasite Infections in Pigs and Beef Cattle in Rural Areas of Chungcheongnam-do, Korea

The present study was performed to investigate the infection status of intestinal parasites in pigs and beef cattle in rural areas of Chungcheongnam-do, Korea. From November 2009 to April 2010, a total of 241 fecal samples of pigs and beef cattle (136 and 105, respectively) were examined by direct smear and centrifugal sedimentation methods. The overall positive rates of intestinal parasites among pigs and beef cattle were 73.5% and 4.8%, respectively, and the double-infection rate was 10.3% in pigs. Of 136 specimens from pigs, Balantidium coli, Ascaris suum, and Entamoeba spp. infections were found in 88 (64.7%), 24 (17.6%), and 5 cases (3.7%), respectively. Of 105 beef cattle, Entamoeba spp. infections were detected in 5 cases (4.8%). From these results, it is shown that pigs raised on rural farms in Chungcheongnam-do had a high B. coli infection rate and a moderate A. suum infection rate. These results demonstrate that environmentally resistant cysts or eggs could be widespread on the farms examined, and thus an effective hygienic management system is needed to prevent them from serving as the source of infection for human beings.

Toxoplasma gondii Proliferation Require Down-Regulation of Host Nox4 Expression via Activation of PI3 Kinase/Akt Signaling Pathway

Toxoplasma gondii results in ocular toxoplasmosis characterized by chorioretinitis with inflammation and necrosis of the neuroretina, pigment epithelium, and choroid. After invasion, T. gondii replicates in host cells before cell lysis, which releases the parasites to invade neighboring cells to repeat the life cycle and establish a chronic retinal infection. The mechanism by which T. gondii avoids innate immune defense, however, is unknown. Therefore, we determined whether PI3K/Akt signaling pathway activation by T. gondii is essential for subversion of host immunity and parasite proliferation. T. gondii infection or excretory/secretory protein (ESP) treatment of the human retinal pigment epithelium cell line ARPE-19 induced Akt phosphorylation, and PI3K inhibitors effectively reduced T. gondii proliferation in host cells. Furthermore, T. gondii reduced intracellular reactive oxygen species (ROS) while activating the PI3K/Akt signaling pathway. While searching for the main source of these ROS, we found that NADPH oxidase 4 (Nox4) was prominently expressed in ARPE-19 cells, and this expression was significantly reduced by T. gondii infection or ESP treatment along with decreased ROS levels. In addition, artificial reduction of host Nox4 levels with specific siRNA increased replication of intracellular T. gondii compared to controls. Interestingly, these T. gondii-induced effects were reversed by PI3K inhibitors, suggesting that activation of the PI3K/Akt signaling pathway is important for suppression of both Nox4 expression and ROS levels by T. gondii infection. These findings demonstrate that manipulation of the host PI3K/Akt signaling pathway and Nox4 gene expression is a novel mechanism involved in T. gondii survival and proliferation.

Prevalence, risk factors, and clinical manifestations of schistosomiasis among school children in the White Nile River basin, Sudan

BackgroundWe investigated the prevalence, risk factors, and clinical manifestations of schistosomiasis in White Nile State, Sudan, to determine the local characteristics of schistosomiasis in the White Nile River basin.MethodsUrine and stool samples were collected from 338 students (176 boys, 162 girls) at three primary schools and were examined using the urine filtration method and the Kato-Katz technique, respectively. Of the students, 200 were interviewed using a semi-structured questionnaire to assess water-contact patterns and health conditions related with urinary schistosomiasis.ResultsOf the 338 students, egg-positive rates for S. haematobium and S. mansoni were 45.0% and 5.9%, respectively, and 4.4% were mixed. The intensities of S. haematobium and S. mansoni infection were 1.091 ± 0.744 log EP10 (eggs per 10 mL of urine, mean ± SD = 57 ± 172 EP10) and 1.787 ± 0.844 log EPG (eggs per gram of stool, mean ± SD = 156 ± 176 EPG), respectively. The prevalence and intensity of S. haematobium infection differed significantly among the three schools, but not by gender or age. Urinary schistosomiasis was significantly associated with the frequencies of contaminated water contact, taking baths, swimming, and wading the stream; however, frequencies of these events were not significantly correlated with infection intensity. Self-reported hematuria and dysuria also correlated significantly with urinary schistosomiasis.ConclusionsThe overall prevalence of schistosomiasis, especially urinary schistosomiasis, is high in the White Nile River basin, Sudan, and is closely associated with frequencies of water contact, taking baths, swimming, and wading the stream. We strongly recommend implementation of an integrated schistosomiasis control program in this area.

Toxoplasma gondii infection inhibits the mitochondrial apoptosis through induction of Bcl-2 and HSP70

Heat-shock protein 70 (HSP70) is highly expressed in Toxoplasma gondii-infected cells. However, the role of this protein is not well understood, especially during apoptosis. This study addresses the mechanism behind the antiapoptotic chaperone activity of HSP70 in Toxoplasma-infected host cells using a human macrophage cell line, THP-1 by Western blot, DNA fragmentation assay, immunoprecipitation, and a caspase-3/7 activity assay based on cleavage of the colorimetric substrate DEVD-pNA. Apoptosis induced by arsenic trioxide (As2O3) was inhibited in T. gondii-infected THP-1 cells, but not in uninfected cells. Without As2O3 induction of apoptosis, T. gondii infection caused increased expression of Bcl-2 and HSP70, but not caspase-3. However, active form caspase-3 levels were lower in As2O3-treated infected cells as compared with As2O3-treated uninfected cells. Bcl-2 expression in As2O3-treated infected cells was similar to that in cells infected with T. gondii. Translocation of apoptosis-inducing factor (AIF) and release of cytochrome c from mitochondria were inhibited in As2O3-treated infected cells as compared with As2O3-treated uninfected cells. Increased parasite loads in Toxoplasma-infected macrophages caused higher HSP70 and Bcl-2 expression in whole-cell extracts and fractionated components, respectively. However, expression of AIF and cytochrome c was unaffected. Toxoplasma dose-dependently inhibited caspase-3 activation, thus revealing an anti-apoptotic parasite activity on cytochrome c-mediated caspase activation in subcellular components. In addition, immunoprecipitation analysis suggested that HSP70 is capable of binding to the pro-apoptotic factors AIF and Apaf-1, but not to cytochrome c or procaspase-9. Taken together, these data demonstrate that T. gondii infection inhibits mitochondrial apoptosis through overproduction of anti-apoptotic Bcl-2 as well as HSP70, which are increased parasite loads dependently.

Intracellular Networks of the PI3K/AKT and MAPK Pathways for Regulating Toxoplasma gondii-Induced IL-23 and IL-12 Production in Human THP-1 Cells

Interleukin (IL)-23 and IL-12 are closely related in structure, and these cytokines regulate both innate and adaptive immunity. However, the precise signaling networks that regulate the production of each in Toxoplasma gondii-infected THP-1 monocytic cells, particularly the PI3K/AKT and MAPK signaling pathways, remain unknown. In the present study, T. gondii infection upregulated the expression of IL-23 and IL-12 in THP-1 cells, and both cytokines increased with parasite dose. IL-23 secretion was strongly inhibited by TLR2 monoclonal antibody (mAb) treatment in a dose-dependent manner and by TLR2 siRNA transfection, whereas IL-12 secretion was strongly inhibited by TLR4 mAb treatment dose-dependently and by TLR4 siRNA transfection. IL-23 production was dose-dependently inhibited by the PI3K inhibitors LY294002 and wortmannin, whereas IL-12 production increased dose-dependently. THP-1 cells exposed to live T. gondii tachyzoites underwent rapid p38 MAPK, ERK1/2 and JNK activation. IL-23 production was significantly upregulated by the p38 MAPK inhibitor SB203580 dose-dependently, whereas pretreatment with 10 μM SB203580 significantly downregulated IL-12 production. ERK1/2 inhibition by PD98059 was significantly downregulated IL-23 production but upregulated IL-12 production. JNK inhibition by SP600125 upregulated IL-23 production, but IL-12 production was significantly downregulated dose-dependently. T. gondii infection resulted in AKT activation, and AKT phosphorylation was inhibited dose-dependently after pretreatment with PI3K inhibitors. In T. gondii-infected THP-1 cells, ERK1/2 activation was regulated by PI3K; however, the phosphorylation of p38 MAPK and JNK was negatively modulated by the PI3K signaling pathway. Collectively, these results indicate that IL-23 production in T. gondii-infected THP-1 cells was regulated mainly by TLR2 and then by PI3K and ERK1/2; however, IL-12 production was mainly regulated by TLR4 and then by p38 MAPK and JNK. Our findings provide new insight concerning the intracellular networks of the PI3K/AKT and MAPK signaling cascades for regulating T. gondii-induced IL-23 and IL-12 secretion in human monocytic cells.