Yoshimasa Kaneda

Identification of the 150-kDa surface antigen of Entamoeba histolytica as a galactose- and N-acetyl-D-galactosamine-inhibitable lectin

Abstract A monoclonal antibody that reacts with a 150-kDa protein of Entamoeba histolytica on Western immunoblotting under nonreducing conditions inhibits the adherence and cytotoxicity of the ameba to mammalian cells in vitro. Affinity purification of solubilized trophozoites using the monoclonal antibody and electophoresis yielded three glycoproteins with molecular masses of 150, 170, and 260 kDa, suggesting the existence of either a common epitope or the close association of these proteins. The 260-kDa fraction was identified as the well-known galactose (Gal)- and N-acetyl-d-galactosamine (GalNAc)-inhibitable lectin. The 150- and 170-kDa fractions seemed to exist as part of a 380-kDa native protein with an isoelectric point of pH␣6.9. The N-terminal amino acid sequence of the 150-kDa protein was unique, indicating that the protein was not a degraded product of the 260-kDa lectin. By gel filtration, the 260-kDa lectin and the 150/170-kDa protein could be separated. When Chinese hamster ovary cells were pretreated with the fraction consisting of the 150/170-kDa protein the adherence of trophozoites to Chinese hamster ovary cells was competitively inhibited to a level equivalent to that observed for the 260-kDa lectin. The inhibitory effect was lost in the presence of Gal and GalNAc but was not influenced by the presence of glucose. These results demonstrate that the 150/170-kDa protein is a Gal/GalNAc-inhibitable lectin. The existence of a sugar-binding domain in the protein was confirmed by Gal-affinity chromatography.

Asymptomatic cyst passers of Entamoeba histolytica but not Entamoeba dispar in institutions for the mentally retarded in Japan.

Entamoeba histolytica/Entamoeba dispar was isolated from 50 asymptomatic amebic cyst passers in three institutions for the mentally retarded in Kanagawa Prefecture, Japan. To distinguish between E. histolytica and E. dispar, the isolates were analyzed by PCR, reactivity to monoclonal antibodies, and zymodemes. All isolates were identified as E. histolytica. The results lead us to conceive that, in Japan, E. histolytica is predominant even in asymptomatic cyst passers.

Alteration of the cell surface acid phosphatase concomitant with the morphological transformation in Trypanosoma cruzi.

Acid phosphatase activity in Trypanosoma cruzi was found to be located on the external surface of the plasma membranes. Both specific activity and activity per cell of this bound enzyme were significantly higher in the cells of amastigote (an intracellular form) than that of trypomastigote (a bloodstream form) and epimastigote (culture form). During the transformation of epimastigotes to amastigotes in vitro the activity of surface acid phosphatase was elevated concomitant with the increase in population of amastigotes. These results were interpreted as that the elevated enzyme activity is required for the intracellular parasitization of this organism or is a consequence of the morphological transformation.

Reproductive failure in mice chronically infected withToxoplasma gondii

Nya: NYLAR female mice infected withToxoplasma gondii for 1 and 2 months were cohabited with normal males for 1 week, then sequestered individually to monitor their reproductive performance. Mice bred 1 month postinfection (p.i.) exhibited reproductive failure, with 1 of 16 females delivering 2 sickly pups; in others, interruption of pregnancy and fetal wastage occurred. Mice infected for 2 months were uniformly infertile. Vaginal lavage showed cessation of estrus cycling and constant diestrus cytology at as early as 1 month p.i. Histologic examination of the ovaries revealed impaired folliculogenesis and few corpora lutea, if any. Uterine atrophy was marked. Coronal sections of the cerebrum disclosed widespread vasculitis, focal disruptions of the ependymal cell layer lining the lateral and third ventricles, and periventricular edema. We suggest that the reproductive failure of the infected mice is due to an acquired hypogonadotropic hypogonadism secondary to hypothalamic dysfunction.

Lipid composition of three morphological stages of Trypanosoma cruzi

The neutral and phospholipid content at each of the three morphological stages of the parasite, Trypanosoma cruzi, was analyzed by thin-layer chromatography. Total lipid fatty acid composition at each stage was analyzed by gas-liquid chromatography and the results were compared. Change in lipid composition at each stage was observed.

Protection of Toxoplasma gondii-infected mice by stearylamine-bearing liposomes.

The cytotoxic activity of stearylamine-bearing liposomes against Toxoplasma gondii (RH strain) was examined. When tachyzoites were treated in vitro with liposomes consisting of 20 mol% stearylamine and 80 mol% phosphatidylcholine (130 micrograms/ml total lipids), more than 95% of the parasites were killed within 90 min. Intraperitoneal injection of 10 mg of 30 mol% stearylamine/70 mol% phosphatidylcholine-liposomes in mice shortly before or after T. gondii challenge afforded protection from death for more than 30 days to 70-80% of the treated mice, whereas all untreated mice succumbed within 9 days. The liposome-injected mice that survived remained symptom-free and behaved normally.

Protection of hamsters from amebic liver abscess formation by a monoclonal antibody to a 150-kDa surface lectin of Entamoeba histolytica

Abstract We examined the effects of passive immunization with a monoclonal antibody (EH3015) that recognizes a 150-kDa surface lectin of Entamoeba histolytica on amebic liver-abscess formation in hamsters. The hamsters were inoculated i.p with 0.1, 1.0, or 10 mg of EH3015 at 24 h prior to an intrahepatic challenge with 105 trophozoites of E. histolytica. In hamsters treated with 1.0 and 10 mg of EH3015 the incidence of liver abscesses was significantly reduced. These results demonstrate that monoclonal antibody EH3015 can prevent the development of amebic liver abscesses and that the 150-kDa lectin may be a protective antigen on the surface of E. histolytica.

VH3 Gene Usage in Neutralizing Human Antibodies Specific for the Entamoeba histolytica Gal/GalNAc Lectin Heavy Subunit

ABSTRACT A combinatorial human immunoglobulin gene library was constructed from peripheral lymphocytes of an asymptomatic Entamoeba histolytica cyst passer and screened for the production of Fab antibody to the parasite. One of the Fab clones, CP33, recognized the 260-kDa galactose- and N-acetyl-d-galactosamine (Gal/GalNAc)-specific lectin of E. histolytica. By shuffling the heavy and light chains of CP33 with the heavy and light chains of two libraries derived from the cyst passer and a liver abscess patient, 18 additional clones were obtained. Sequence analysis of the heavy-chain genes, including CP33-H, revealed that all the nearest V-segment germ lines belonged to the VH3 family (VH3-21, VH3-30, VH3-48, and VH3-53), but the levels of homology were only 85 to 95%. The closest D-segment germ line was D2-2 or D6-6, and for the J-segment the closest germ line was JH4b or JH6b. On the other hand, all the light-chain genes, including CP33-L, belonged to the Vκ1 family, in which the closest Vκ germ line gene was 02/012 or L5, with the Jκ1, Jκ2, Jκ4, or Jκ5 segment. CP33 and three other Fabs obtained by light-chain shuffling were purified and analyzed further. All of these Fabs recognized the cysteine-rich domain of the 170-kDa heavy subunit of the Gal/GalNAc lectin. Preincubation of E. histolytica trophozoites with these Fabs significantly inhibited amebic adherence to Chinese hamster ovary cells and also inhibited erythrophagocytosis. The ability of the neutralizing antibodies to block erythrophagocytosis for the first time implicates the lectin in phagocytosis and VH3 antibodies in defense against parasitic infections. These results demonstrate the utility of a combinatorial human immunoglobulin gene library for identifying and characterizing neutralizing antibodies from humans with amebiasis.

Etiology of ovarian dysfunction in chronic murine toxoplasmosis

Ovarian dysfunction develops in Nya:NYLAR mice chronically infected withToxoplasma gondii. To differentiate between primary ovarian failure and pituitary gonadotropin insufficiency, we (a) monitored ovarian responsiveness to pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) and (b) assessed endogenous pituitary gonadotropin capacity by the degree of ovarian compensatory hypertrophy (OCH) developing after unilateral ovariectomy (ULO). PMSG stimulated vigorous folliculogenesis and estrogen synthesis, but not ovulation. HCG given 3 days after PMSG induced “superovulation” within 16 h. These observations indicate the absence of the critical preovulatory surge of endogenous luteinizing hormone (LH) from the pituitary. In addition, ULO did not result in compensatory hypertrophy of the contralateral ovary, an indication of follicle-stimulating hormone (FSH) insufficiency. We hypothesize that cytokines released peripherally in response to the parasite reached the hypothalamus and initiated a sequence of events that inhibited the pulsatile release of gonadotropin-releasing hormone (GnRH), leading to the subsequent impairment of the pituitary-ovarian axis.

Leishmania braziliensis: Localization of glycoproteins in promastigotes

Two species of glycoproteins from Leishmania braziliensis promastigotes of apparent molecular weights of 53,000 (glycoprotein 53) and 47,000 (glycoprotein 47) were localized. Four lectins with different sugar specificities bound to the blotting sheet to which the electrophoretically separated materials were transferred. Concanavalin A and Ricinus communis agglutinin bound to the band of glycoprotein 53 and the lectin from Dolichos biflorus bound to the band of glycoprotein 47. Wheat germ agglutinin bound to the bands of both glycoproteins. Histochemical examinations using fluorescence labeled lectins demonstrated that the glycoproteins 53 and 47 were located on the cell surface and in the cytoplasm of promastigotes, respectively. The results are consistent with the result of agglutination test.