Experimental "chronic" African trypanosomiasis: endocrine dysfunctions generated by parasitic components released during the tryptanolytic phase in rats.

The disorders of the gonadotropic axis have been studied during the course of a "chronic" african trypanosomiasis induced experimentally in rats inoculated by the variant Trypanosoma brucei brucei AnTat 1.1.E. The levels of serum and pituitary LH as well as serum testosterone and corticosterone have been determined, during the infestation, at a particular period of the circadian cycle, in regard to the parasitemia variations. In addition, the inoculation of trypanosomal component fractions [obtained by concanavalin-A sepharose chromatography (conA-components)], has been performed in an attempt to define more exactly the nature of factor(s) producing the hypotestosteronemia in rats. This work evidenced that the hormonal parameter levels were predominantly decreased at the trypanolytic phase during the evolution of the disease. The action towards the hypothalamo-pituitary gonadal axis was attributed not only to peculiar trypanosomal enzyme(s) [a serine, thiol-dependent, cation sensitive endoprotease with a post-proline cleaving activity (purified from unretained conA fraction)], but also to protein and/or glycoprotein factor(s) released by the trypanosomes (components with affinity to the lectin).

Gonadotropic dysfunction produced by Trypanosoma brucei brucei in the rat.

Hormonal disorders have been frequently observed in humans and animals infected with tsetse-transmitted (African) trypanosomes. We studied the pituitary gonadal axis (plasma concentrations of testosterone, luteinizing hormone (LH), follicle stimulating hormone (FSH) and the pituitary gonadotropin (LH, FSH) concentrations) in rats as an experimental model infected with an acute stock of Trypanosoma brucei brucei (AnTat 1.1A). The same investigations in vivo were carried out with rats inoculated by trypanosomal preparations: surface coat components slowly released at pH 5.5 and the parasitic cellular pellet. The releasing procedure as firstly described by Baltz et al. (1976) was performed in the presence or the absence of protease inhibitors. We noted a testicular hypogonadism produced by the acute infection with the decrease of the testosterone level and an increase of the pituitary LH concentration, although the other circulating FSH and LH hormone levels were stable. The injection of the trypanosomal pellet, obtained in the presence of antiproteases, generated a similar clinical hormonal picture: decrease of testosterone level; increase in pituitary LH, FSH content; absence of significant variation of circulating FSH and LH rates. When the trypanosomal pellet was prepared in absence of antiproteases the circulating gonadostimuline levels were significantly decreased. In the same conditions (absence of antiproteases) the trypanosomal supernatant pH 5.5 induced the decrease of the testosterone and plasma LH levels. These results suggested that component(s) from trypanosomes generated hormonal perturbations.

Variant surface glycoprotein of Trypanosoma brucei brucei AnTat 1.1: influence of the isolation conditions upon the disulfide linked dimer/monomer ratio.

1. Using the variant surface glycoprotein (VSG) isolation procedure described by Baltz et al. ([1976] Ann. Immunol. (Inst. Pasteur) 127 C, 761-774) which involves suspension of the trypanosomes in a pH 5.5 buffer, the Antwerpen trypanozoon antigenic type (AnTat) 1.1 VSG is mainly obtained as a disulfide linked dimeric form with a trace amount of a monomeric form. 2. The use of a parasite suspension buffer at pH 7.0 results in a slight decrease of the VSG dimer/monomer ratio. 3. pH 5.5 and 7.0 supernatants of centrifuged parasite suspensions were submitted to kinetic incubations at different temperatures and pH, and we found conditions involving transformation of the AnTat 1.1 VSG dimer into the AnTat 1.1 VSG monomer (shifting the pH 5.5 supernatant to pH 7.0 and incubation at room temperature). 4. This transformation of the AnTat 1.1 VSG dimer into the AnTat 1.1 VSG monomer is activated by the addition of 1 mM reduced glutathione, and is inhibited by the addition of 1 mM oxidized glutathione or 0.1 mM N-ethylmaleimide or cadmium acetate.

Gonadotropic axis and Trypanosoma brucei gambiense infection.

A gonad endocrine survey on 46 Congolese patients (15 women and 31 men) with parasitologically confirmed trypanosomiasis found amenorrhoea in 60% of the women and impotence in 70% of the men. The basic gonad endocrine examination showed a decrease in oestradiol levels in about 65% of the women. Both amenorrhoea and low oestrogen levels were observed in the second phase (P2) of the disease, but low oestrogen levels were sometimes noted in the first phase of the disease (P1). In the men, about 50% of the cases (P2) showed a decrease in testosterone. However, as in the women, the variation of testosterone was also observed in the first phase (P1). A static and dynamic examination of the hypothalamic-pituitary-gonadal axis was undertaken in order to investigate the origin of these hypogonadisms. A supra - or extra-hypophyseal origin is discussed.

Reversed phase HPLC of different membrane-bound variant surface glycoprotein preparations from Trypanosoma brucei brucei.

The chromatographic behaviour of the membrane-attached variant surface glycoprotein (mfVSG) of bloodstream forms of Trypanosoma brucei brucei AnTat 1.1 A preparations were studied by reversed phase high performance liquid chromatography (RP-HPLC). Among the different preparation procedures used, only the trifluoroacetic acid extraction gave a mfVSG preparation which was eluted from the RP-HPLC column. As well as for the soluble variant surface glycoprotein (sVSG) it was found that different mfVSG forms were eluted at different organic solvent concentrations from the RP-HPLC column. In addition, in preliminary studies, we have attempted to characterize the factors responsible for the trypanosome surface coat assembly. Using chromatographic techniques (RP-HPLC and thin layer chromatography), the results suggest a close relationship between the content of lipid-containing species and the heterogeneity of mfVSG preparation.

Evidence of myristylated disulfide-linked dimer of variant surface glycoprotein of Trypanosoma brucei-brucei.

1. Variant surface glycoprotein (VSGs) of Trypanosoma brucei-brucei may exist as a disulfide-linked dimer in both forms: myristylated (mfVSG) and non-myristylated (sVSG), as judge by fluorography and immunoblotting of SDS-PAGE under non-reducing conditions. 2. The dimeric VSG form is labeled with [3H]-myristic acid in our incorporation conditions. 3. AnTat 1.1 trypanosomes preincubated with tunicamycin and incubated with [3H]-myristic acid synthesized a labeled molecule that has an apparent molecular weight slightly smaller than the native form, and that also corresponds to a disulfide-linked dimer.

Presence of a lipophosphoglycan in two variants of Trypanosoma brucei brucei.

For the family of Trypanosomatidae (Trypanosoma and Leishmania) the organization of the glycoproteins on the cell surface is a well documented structural feature, because their plasma membranes are potential target for chemotherapy. By using metabolic labeling ( [32P] phosphate, [3H]-myristic acid, [3H]-galactose) and by appropriate fractionated extraction, we have found a trypanosomal molecule which has electrophoretic and chromatographic properties consistent with the lipophosphoglycan of Leishmania donovani defined by Turco et al (1987) Biochemistry 26, 6233-6238 (1). In addition, the trypanosomal lipophosphoglycan, appears to have chromatographic behaviour similar to the glycolipid C of Trypanosoma brucei brucei described by Krakow et al (1986) J. Biol. Chem. 261, 12147-12153 (2). Our results suggest that the role of the trypanosomal lipophosphoglycan may take place in the orientation of the glycoproteins in the surface coat and/or corresponds to the glycolipid precursor for the anchor of variant surface glycoprotein.

Molecular heterogeneity of the isolated surface glycoprotein from variant AnTat 1.1 of Trypanosoma brucei brucei.

Variant surface glycoprotein (VSG) of Trypanosoma brucei brucei AnTat 1.1 was released by means of the procedure described by Baltz et al. ([1976], Ann. Immunol. [Inst. Pasteur] 127C, 761-774). The concanavalin-A chromatography yielded 3 VSG fractions according to the addition, in the elution buffer, of alpha-methyl-D-mannopyranoside, beta-mercaptoethanol, and sodium dodecyl sulfate. These VSG fractions showed heterogeneous behaviour on reverse-phase high performance liquid chromatography. The 3 VSG fractions as well as the myristylated VSG of AnTat 1.1 essentially consist of dimer VSG forms linked through a disulfide bridge, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis, under reducing and nonreducing conditions.

[Endocrine function and African trypanosomiasis. Evaluation of 79 cases]. / Fonction endocrine et trypanosomiase africaine. Bilan de 79 cas.

Although the high frequency of hypogonadism was found during the sleeping sickness, the physiological properties of the gonads have been poorly studied. We reported here the exploration of the gonadotropic axis in 79 patients infected by Trypanosoma brucei gambiense. It has been noted a decrease in oestradiol for 50% of women, in testosterone levels for 50% of men whereas no decrease of pituitary gonadotropins (FSH and LH) was found. The results of the gonad adenohypophyseal tests suggested that the dysfunction may be of central origin. The studies of thyreotropic axis were also examined: a decrease in T3 and FT3 (free fraction) even though T4 was normal. The specific relationship between hypogonadism and trypanosomal infection is discussed in this report.

Heterogeneity in high performance liquid chromatography of a variant surface glycoprotein of Trypanosoma brucei.

High performance liquid chromatography (HPLC) procedures have been used to analyze a preparation of the variant surface glycoprotein AnTat 1.1A of Trypanosoma brucei. The native preparation gives several peaks with a high reproducibility both by reverse-phase (RP-) and gel permeation (GP-) HPLC. Under RP-HPLC conditions, nine fractions are fully resolved. The RP-HPLC fractions migrate with the same molecular weight VSG band on polyacrylamide slab gel electrophoresis and no significant differences are observed in amino acid composition among these fractions. The RP-HPLC resolution is found to be related to the ability of the VSG to polymerize as shown using GP-HPLC. These results suggest the existence of a microheterogeneity of the AnTat 1.1A VSG preparation in relation to post-translational modification of the VSG molecule.