Alteration of free serum amino acids in voles infected with Trypanosoma brucei gambiense.

Free serum amino acid pools of field voles, Microtus montanus, were determined over a 24 hr period, and compared to values obtained from voles infected with Trypanosoma brucei gambiense. The majority of amino acids in the control animals demonstrated a diurnal variation, peaking predominantly during the dark portion of the photoperiod. This trend was not evident in the infected animals. In addition, infected voles possessed an apparent state of hypoaminoacidemia, with levels of threonine, serine, valine, isoleucine, leucine, tryosine, and tryptophan typically below uninfected values. Alanine and proline, in contrast, were markedly increased at certain time points. Tyrosine (reduced by approximately 50%) and tryptophan (reduced to levels below detection) underwent the most pronounced drop in trypanosome-infected animals, indicating the possibility of a related alteration in pools of derivative biogenic amines in other tissues. This suggests a role for the latter 2 amino acids in the neuropsychiatric syndromes of African trypanosomiasis.     

Inhibition of protein kinase activity from Trypanosoma cruzi and Trypanosoma gambiense by 3'-deoxyadenosine.

3'-Deoxydadenosine was found to be a potent inhibitor of nucleoside-stimulated protein kinase activity from culture forms of Trypanosoma cruzi and bloodstream forms of Trypanosoma gambiense. The type of inhibition by 3'-deoxyadenosine was competitive with respect to ATP. The inhibition constants for 3'-deoxyadenosine were determined to be 0.11mM and 0.085mM for the enzyme from T. cruzi and T. gambiense, respectively. The apparent Km value for ATP was 0.2mM for both enzymes. 2'-Deoxyadenosine was less effective as inhibitor of the protein kinase activity. The inhibition constants were calculated to be 0.8mM and 0.67mM, respectively.     

Elevated serum and hepatic tyrosine aminotransferase in voles chronically infected with Trypanosoma brucei gambiense.

Chronic infections of voles (Microtus montanus) with Trypanosoma brucei gambiense result in marked elevations in both serum and hepatic tyrosine aminotransferase. Serum enzyme levels correlate poorly with hepatic levels but well with parasitemia. Because the trypanosome itself contains high levels of this same enzyme, it is hypothesized that lysis of extravascular parasites, possibly due to agglutination by variant-specific antibody, releases parasite enzymes into host tissues, resulting in the observed increase in serum enzyme levels. The possible role of increased tyrosine transamination in the behavioral syndrome of trypanosome-infected voles is considered.     

Effect of hydroxystilbamidine on the kinetoplast of Trypanosoma gambiense in mice.

Hydroxystilbamidine (OHSA), an inhibitor of DNA synthesis, was shown to induce the dyskinetoplastic forms (akinetoplastic forms, AK forms) of Trypanosoma gambiense. The mode of appearance of the AK forms after injection of various doses of OHSA into infected mice was examined. The results suggested that production of the AK form is due to the selective inhibition of kinetoplast duplication of the drug without any effect on nuclear and cytoplasmic multiplication. When the parasites were treated with moderate doses of OHSA, segmenting forms without stainable kinetoplasts, were occasionally seen but attempts to establish a clone of akinetoplastic parasite were unsuccessful. Electron microscopy of parasites obtained after OHSA treatment showed not only irregular division of the kinetoplast but also the disorganization of kinetonucleus with disappearance of its envelope. Therefore, it was concluded that the AK forms were also produced by OHSA through disappearance of the kinetoplast.     

Effect of neocarzinostatin on the nucleus, kinetoplast and microtubules of Trypanosoma gambiense and Trypanosoma evansi.

The antibiotic neocarzinostatin (NCS) induces the anucleate form, not the dyskinetoplastic form, of Trypanosoma gambiense and Trypanosoma evansi. Light and electron microscopic studies indicated that production of the anucleate form is due to delay or inhibition of nuclear division. Excess pellicular microtubules are formed after treatment of trypanosomes with NCS, suggesting that in trypanosomes the microtubules replicate by induction, not by division. NCS also causes deformation of the axonemal and spindle microtubules. The K clone of T. evansi is more sensitive than the AK clone to the effects of NCS in inhibiting nuclear division and inducing the anucleate form.     

Preparation and study of an exoantigen of Histoplasma capsulatum for serological reactions.

The aim of this study was to determine the usefulness of a yeast-phase exo-antigen of Histoplasma capsulatum in standard serologic reactions. Three native strains of H.capsulatum which belong to Mycology Center collection were employed. They were maintained in their yeast-phase by weekly subcultures in 2% dextrose broth agar at 37 degrees C. After one week incubation yeast cells were suspended in distilled water containing thimerosal and phenylmethyl sulfonyl fluoride at a concentration of 1:5000. This suspension was left at room temperature for 72 h, then the supernatant was separated by centrifugation and it was lyophilized. Proteins and polysaccharides concentrations were determined. Immunodiffusion (ID) tests were carried out with an antigenic dilution containing 1.4 mg/ml of proteins. This exo-antigen was submitted to SDS-PAGE. Seven protein fractions were detected but only two of them showed antigenic activity against a pool of positive human sera; the molecular weights of these two proteins were 97 kDa and 66 kDa respectively. A metabolic antigen from the mycelial phase of H. capsulatum was used as control. A rabbit gammaglobulin anti-H. capsulatum was prepared and employed as positive control in serologic reactions. The antigenic capacity of ten batches of this exo-antigen was studied by ID and counterimmunoelectrophoresis (CIE) tests using serum samples of 20 hamsters experimentally infected by intracardiac inoculation of the yeast-phase of H. capsulatum. All tests presented positive results after three weeks of the infection. Fifty sera from patients suffering progressive histopasmosis were analyzed: ID, CIE and complement fixation (CF) tests were performed in all cases. HIV negative patients presented 7/7 (100%) positive reactions with the yeast-phase exoantigen and 5/7 (71.4%) with histoplasmin. In HIV positive patients CIE and CF were the most sensitive serologic tests, they gave positive results in 15/43 cases (34.8%) with the yeast-phase exo-antigen and in 7/43 cases (13.9%) with histoplasmin. Sera from 10 patients with paracoccidioidomycosis, aspergillosis and candidiasis respectively were studied by ID with the aim of detecting serologic cross reactions. No cross reaction was detected in these serum samples. This yeast-phase exo-antigen of H. capsulatum is more sensitive than and equally as specific as control histoplasmin.     

Immunological purification and partial characterization of variant-specific surface antigen messenger RNA of Trypanosoma brucei brucei.

Polyadenylated RNA isolated from total polyribosomes of two variable antigen types (VATs) of T. brucei brucei were shown to program the synthesis, in mRNA-dependant reticulocyte lysates, of a wide variety of polypeptides. After immunoprecipitation of these cell-free products with an homologous antiserum raised against purified variant-specific surface antigen (VSSA), a major electrophoretic band was apparent on fluorography. It was confirmed that this band corresponds to the variable antigen since only an excess of purified homologous antigen will provoke competition. The apparent molecular weight of the in vitro synthesized antigen is about 63,000 daltons. The VSSA mRNA has been found in membrane-bound polyribosomes and a 15 fold immunological purification of this mRNA has been obtained, using partially purified anti-VSSA IgG in conjunction with inactivated Staphylococcus aureus.     

Isolation and purification of a preparation possessing leukocytosis-stimulating properties from pertussis bacteria]

A homogeneous protein LSF-2 preparation was extracted from the cultural fluid of Bordetella pertussis strains of the 1.0.3 serological type by means of precipitation with ammonium sulphate and electrofocussing; this preparation proved to produce a marked leukocytosis-stimulating and a weak toxic action of delayed type in experiments on animals. Intraperitoneal administration of 5 mug of the LSF-2 preparation caused a rise of leukocytosis in mice to 100,000 cells per 1 mm3, a delay in the gain in weight beginning from the 3rd day of the administration and a late death of the animals in 5% of cases. The LSF-2 preparation protected the mice in infection with a virulent pertussis strain No. 18323 in the amount of from 12 to 91%, depending on the immunizing dose; its ImD50 was equal to 2.0 -2.4 mug of protein. The results of investigations carried out permitted to assess the role of this substance in the formation of specific immunity in pertussis infection.     

A preparation and purification of [1-14C]acetylcarnitine.

[1-14C]Acetylcarnitine was prepared from [1-14C]acetate and L-carnitine using acetyl-CoA synthetase and carnitine acetyltransferase. The product was purified by ion-exchange and thin-layer chromatography. Conversion of [1-14C]acetate to [1-14C]acetylcarnitine was better than 90% and overall recovery of the pure product was greater that 80%.     

An experimental study of a lactoglobulin preparation against opportunistic bacteria and salmonellae]

A preclinical study of seven batches of lactoglobulin, a new biological preparation against opportunistic bacteria and salmonellae, has been carried out. High antibacterial activity of the preparation with respect to the virulent forms of Salmonella typhimurium, Salmonella enteritidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus vulgaris, Proteus mirabilis has been established. The preparation has been shown to be safe and nontoxic. The 4-year term of its storage at a temperature of 6 degrees +/- 4 degrees C has been substantiated.     

Trypanosoma brucei gambiense: HMI-9 medium containing methylcellulose and human serum supports the continuous axenic in vitro propagation of the bloodstream form

Trypanosoma brucei (T.b.) gambiense causes the chronic form of human African trypanosomiasis or sleeping sickness. One of the major problems with studying T.b. gambiense is the difficulty to isolate it from its original host and the difficult adaptation to in vivo and in vitro mass propagation. The objective of this study was to evaluate if an established method for axenic culture of pleomorphic bloodstream form T.b. brucei strains, based on methylcellulose containing HMI-9 medium, also facilitated the continuous in vitro propagation of other bloodstream form Trypanozoon strains, in particular of T.b. gambiense. Bloodstream form trypanosomes from one T.b. brucei, two T.b. rhodesiense, one T. evansi and seven T.b. gambiense strains were isolated from mouse blood and each was concurrently cultivated in liquid and methylcellulose-containing HMI-9 based medium, either with or without additional human serum supplementation, for over 10 consecutive sub passages. Although HMI-9 based medium supplemented with 1.1% (w/v) methylcellulose supported the continuous cultivation of all non-gambiense strains better than liquid media could, the in vitro cultivation of all gambiense strains was only achieved in HMI-9 based medium containing 1.1% (w/v) methylcellulose, 15% (v/v) fetal calf serum and 5% (v/v) heat-inactivated human serum.