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1.
Parasitol Int ; 63(2): 479-84, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24157443

RESUMO

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects humans and animals. Ingested parasites cross the intestinal epithelium, invade leukocytes and are then disseminated to peripheral organs. However, the mechanism of extravasation of the infected leukocytes remains poorly understood. In this study, we demonstrate that T. gondii-invaded human and mouse leukocytes express higher level of CD44, a ligand of hyaluronan (HA), and its expression on myeloid and non-myeloid leukocytes causes T. gondii-invaded human and mouse leukocyte to adhere to HA more effectively than non-invaded leukocytes. The specific adherence of parasite-invaded leukocytes was inhibited by anti CD44 antibody. Leukocytes of CD44 knockout mice did not show parasite-invaded leukocyte specific adhesion. Our results indicate that parasite-invaded leukocytes, regardless of whether myeloid or not, gain higher ability to adhere to HA than non-invaded leukocytes, via upregulation of CD44 expression and/or selective invasion to CD44 highly expressing cells. The difference in ability to adhere to HA between parasite-invaded cells and non-invaded neighboring cells might facilitate effective delivery of parasite-invaded leukocytes to the HA-producing endothelial cell surface and/or HA-rich extra cellular matrix.


Assuntos
Adesão Celular/fisiologia , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , Leucócitos Mononucleares/parasitologia , Toxoplasma/fisiologia , Animais , Regulação da Expressão Gênica , Humanos , Leucócitos Mononucleares/fisiologia , Camundongos , Baço/citologia
2.
Exp Parasitol ; 134(2): 160-4, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23538031

RESUMO

The intracellular parasite Toxoplasma gondii is thought to disseminate throughout the host by circulation of tachyzoite-infected leukocytes in the blood, and adherence and migration of such leukocytes into solid tissues. However, it is unclear whether T. gondii-infected leukocytes can migrate to solid organs via the general circulation. In this study, we developed a real-time quantitative PCR (qRT-PCR) method to determine the rate of infection of peripheral blood mononuclear cells (PBMCs) flowing into and remaining within solid organs in mice. A transgenic T. gondii parasite line derived from the PLK strain that expresses DsRed Express, and transgenic green fluorescent protein-positive PBMCs, were used for these experiments. Tachyzoite-infected PBMCs were injected into mouse tail veins and qRT-PCR was used to measure the infection rates of the PBMCs remaining in the lungs, liver, spleen and brain. We found that the PBMCs in the lungs and liver had statistically higher infection rates than that of the original inoculum; this difference was statistically significant. However, the PBMC infection rate in the spleen showed no such enhancement. These results show that tachyzoite-infected PBMCs in the general circulation remain in the lungs and liver more effectively than non-infected PBMCs.


Assuntos
Leucócitos Mononucleares/parasitologia , Fígado/patologia , Pulmão/patologia , Toxoplasma/fisiologia , Toxoplasmose Animal/patologia , Animais , Chlorocebus aethiops , DNA de Protozoário/isolamento & purificação , Proteínas de Fluorescência Verde , Ventrículos do Coração/parasitologia , Ventrículos do Coração/patologia , Leucócitos Mononucleares/citologia , Fígado/parasitologia , Substâncias Luminescentes , Pulmão/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Baço/parasitologia , Baço/patologia , Toxoplasma/genética , Toxoplasma/isolamento & purificação , Toxoplasmose Animal/sangue , Toxoplasmose Animal/parasitologia , Células Vero
3.
J Vet Med Sci ; 72(11): 1399-403, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20567093

RESUMO

In mammals with a hemochorial placenta (e.g., primates and rodents), the maternal and fetal bloodstreams are separated by the blood-placenta barrier. However, a few maternal cells in the general circulation pass through the barrier during normal pregnancy. So far, the transfer mechanism has not been investigated. In this study, we established a chemokine (C-C motif) ligand 3 (CCL3)-deficient mouse model to examine the effect of fetus-derived chemokine(s) on the migration of maternal cells through the blood-placenta barrier. Using this model, we obtained CCL3-positive and -negative littermates from a mother expressing both CCL3 and green fluorescent protein (GFP). The numbers of GFP positive maternal cells in the lung, liver, spleen and heart of CCL3-positive and -negative fetuses were compared. A few GFP-positive cells were detected in the lung and liver of both types of fetus. These results indicate that maternal cells can migrate through the blood-placenta barrier even in the absence of fetal CCL3.


Assuntos
Quimiocina CCL3/metabolismo , Placenta/metabolismo , Animais , Quimiocina CCL3/sangue , Quimiocina CCL3/deficiência , Quimerismo/embriologia , DNA/genética , DNA/isolamento & purificação , Primers do DNA , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Proteínas de Fluorescência Verde/sangue , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fígado/embriologia , Fígado/metabolismo , Pulmão/embriologia , Pulmão/metabolismo , Masculino , Troca Materno-Fetal/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miocárdio/metabolismo , Reação em Cadeia da Polimerase , Gravidez
4.
Biochem Biophys Res Commun ; 391(1): 477-80, 2010 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19914206

RESUMO

The apicomplexan, obligate intracellular parasite Toxoplasma gondii orally infects humans and animals. The parasites cross the intestinal epithelium, invade leukocytes in the general circulation and then disseminate into the peripheral organs. The mechanism of extravasation of the infected leukocytes, however, remains poorly understood. It is known that adhesion of leukocytes to extracellular matrix (ECM) is an important factor in extravasation, and CD44 and ICAM-1 on the leukocyte surface are known receptors for hyaluronan (HA), an ECM component. In this study, we demonstrated up-regulation of CD44 and ICAM-1 expression on the surface of T. gondii-infected human monocytic THP-1 cells and fresh isolated human monocyte. T. gondii-infected THP-1 cells adhered more efficiently to immobilized HA than did non-infected cells. T. gondii-infected monocytes in the general circulation might preferentially adhere to the ECM and migrate out from blood vessels, so transporting parasites into the peripheral organs.


Assuntos
Receptores de Hialuronatos/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Monócitos/imunologia , Toxoplasma , Toxoplasmose/imunologia , Adesão Celular , Humanos , Monócitos/parasitologia , Regulação para Cima
5.
Parasitol Int ; 57(4): 515-8, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18652914

RESUMO

Toxoplasma gondii is an intracellular parasite. It has been thought that T. gondii can disseminate throughout the body by circulation of tachyzoite-infected leukocytes (intracellular parasite) in the blood flow. However, a small number of parasites exist as free extracellular tachyzoites in the blood flow (extracellular parasite). It is still controversial whether the extracellular parasites in the blood flow disseminate into the peripheral tissues. In this study, we evaluated the dissemination efficiency of the extracellular and intracellular parasites in the blood flow using GFP-expressing transgenic parasite (PLK/GFP) and DsRed Express-expressing transgenic parasite (PLK/RED). When PLK/GFP and PLK/RED tachyzoites were injected, as intracellular and extracellular forms respectively, at the same time into the tail vein of a mouse, many disseminated green fluorescent PLK/GFP tachyzoites were observed in the lung, the spleen, the liver and the brain. However, only a few red fluorescent PLK/RED tachyzoites were detected in these organs. When PLK/GFP and PLK/RED tachyzoites were injected in the opposite manner, that is, as extracellular and intracellular forms respectively, the majority of tachyzoites in these tissues were PLK/RED tachyzoites. Collectively, these results indicate that intracellular tachyzoites mainly disseminate throughout the body and that extracellular tachyzoites hardly contribute to parasite dissemination.


Assuntos
Circulação Sanguínea , Espaço Extracelular/parasitologia , Leucócitos/parasitologia , Toxoplasma/fisiologia , Toxoplasma/patogenicidade , Toxoplasmose Animal/fisiopatologia , Animais , Chlorocebus aethiops , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Interações Hospedeiro-Parasita , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Toxoplasma/genética , Toxoplasma/metabolismo , Toxoplasmose Animal/parasitologia , Células Vero
6.
Int J Parasitol ; 38(5): 601-7, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18022177

RESUMO

Detection of the initial site of Toxoplasma gondii reactivation in brain tissue is difficult because the number of latent cysts is small and reactivation is a transient event. To detect the early stage of reactivation in mouse brain tissue, we constructed a cyst-forming strain of T. gondii in the tachyzoite stage, specifically expressing red fluorescence. The PLK strain of T. gondii was stably transfected with a red fluorescent protein gene, DsRed Express, under the control of a tachyzoite-specific SAG-1 promoter and the resulting parasite was designated as PLK/RED. Tachyzoites of PLK/RED growing in Vero cells showed red fluorescence. When C57BL/6J mice were i.p. infected with tachyzoites of PLK/RED, red fluorescent tachyzoites were detected in their brains at the fourth day p.i. However, red fluorescent tachyzoites were not detected in BALB/c mice latently infected with PLK/RED, although non-fluorescent cysts were detected in their brains. After treatment of latently infected mice with dexamethasone for 1 month, the mice showed neurological symptoms. In mice with symptoms, red fluorescent tachyzoites were again detected in their brains and in other organs. To detect the initial site of reactivation, BALB/c mice latently infected with the strain were treated with dexamethasone for 3 weeks, and brains were excised before any symptoms appeared. Excised brains were examined for red fluorescence-positive sites. By a histological study of red fluorescent-positive sites, we detected a cyst containing red fluorescent zoites, which still had a PAS stain-positive cyst wall. A few red fluorescent zoites breaking away from the cyst were also observed. The stage-specific expression of fluorescent protein facilitates detection of a rare transient event and makes it possible to detect the initial site of reactivation.


Assuntos
Encéfalo/parasitologia , Toxoplasma/fisiologia , Toxoplasmose Animal/parasitologia , Toxoplasmose Cerebral/parasitologia , Animais , Dexametasona/farmacologia , Feminino , Substâncias Luminescentes , Proteínas Luminescentes , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Toxoplasma/efeitos dos fármacos , Toxoplasma/isolamento & purificação , Toxoplasma/patogenicidade , Virulência , Proteína Vermelha Fluorescente
7.
Anal Chem ; 75(11): 2775-83, 2003 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-12948149

RESUMO

A new on-column derivatization method based on the redox activity of porous graphitic carbon (PGC) packing materials was presented for enhancement of separation selectivity of HPLC. Two PGC packing materials were used as the solid redox agents as well as the stationary phase, and their redox activities were investigated using trans-1,2-diaminocyclohexanetetraacetate (DCTA) complexes of some metal ions as probe compounds. It was found that the redox property of PGC was modified by treating them with a solution containing a reducing agent, sodium sulfite or hydroxylammonium chloride. The original PGC packings oxidized Co(II)-DCTA to Co(III)-DCTA during elution, while the PGC treated with a reducing agent showed reduction activity converting Co(III)-DCTA to Co(II)-DCTA. These two cobalt complexes do not form their individual chromatographic zones but migrate as a single zone of their mixture on the PGC column contrary to the chromatographic behavior on a C18 bonded silica, on which Co(II)-DCTA and Co(III)-DCTA can be separated. Treatment of the PGC column with a reducing agent solution transforms the oxidative activity of the original PGC packing to a reductive one from the upper part of the column, so that the retention time of the cobalt complex can be controlled by changing the volume of the reducing agent solution to be used for treatment of the PGC column. The selective separation and determination of cobalt in a reference manganese nodule sample by the developed method was demonstrated.

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