Biomedical subjects

R Brun

Publications and source records attributed to R Brun.

At least 19 recordsLinked to original sources

PubMed1992

[Cerebral trypanosomiasis: epidemiology, clinical aspects, therapy and illness course].

More than 20,000 people are infected with one of the two forms of African trypanosomiasis each year. Without treatment the outcome is almost always fatal. There are, however, problems involved in drug treatment. Reactive encephalitis is recorded in up to 18% of all cases treated for cerebral trypanosomiasis. Recently a lack of response to treatment has been reported with increasing frequency from endemic areas. Relapses occur in up to 10% of cases. Reasons for relapses include insufficient treatment due to poor compliance or lack of drugs, reinfection in endemic areas, pharmacokinetic problems, the parasite's evasive mechanisms, and primary and/or secondary drug resistance.

Africa↗

PubMed1992

Difference in the response to PIF/activin between animal caps excised from mid- or late blastula stages of Xenopus laevis.

Animal caps from Xenopus embryos at Stage 7/8 cultured in salt solution, were capable of elongating, and formed various embryonic tissues including axial mesoderm, nervous tissues and pigmented retina. In contrast, animal caps from Stage 9 only developed into permanent blastulae. Following exposure to PIF/activin, however, such animal caps displayed morphogenetic movements. They formed tube-shaped embryoids that frequently had one or more tails but no head. We conclude that animal caps from Stage 9 produce more reliable results than those from Stage 7/8 because the latter are most likely contaminated with mesodermal cells.

Activins↗

PubMed1992

Evolution of the retrotransposons TRS/ingi and of the tubulin genes in trypanosomes.

The African trypanosomes have genomes of high plasticity, as demonstrated for instance by their ability to shuffle their genes around, coding for variant-specific surface glycoproteins (VSGs). Another indication of their genome plasticity is the presence of multiple retro-elements. The retrotransposon-like element TRS/ingi is present in many copies in the genome of trypanosomes. One particular derivative of TRS/ingi, called TUBIS, had previously been found to interrupt a tubulin gene in a particular strain of T. brucei. Here both TRS/ingi and TUBIS were studied by hybridizing genomic DNA of various strains and species of trypanosomes with suitable probes in order to elucidate the evolution of this family of retro-elements. The TSR/ingi elements are highly repeated and have very long open reading frames, while TUBIS clearly is a truncated, inactivated form of this element, found in only one particular chromosomal location. Both elements were shown to be present in several strains and species of the subgenus Trypanozoon, in particular in T. brucei brucei, T. gambiense, T. rhodesiense, T. equiperdum and T. evansi. They could not be detected in species of other subgenera, in particular in T. congolense and T. cruzi. These findings suggest that the retrotransposon TRS/ingi was acquired by trypanosomes only after divergence of present day subgenera. The TUBIS element was found in exactly the same chromosomal location (at the 3' end of the tubulin gene cluster) in many different strains and species of the subgenus Trypanozoon. This shows that the element was transposed to this location before speciation of the subgenus. Although, TRS/ingi is unlikely to be involved directly in VSG switching, it may have contributed to the genome plasticity of trypanosomes.

Animals↗

PubMed1992

Kinetoplast DNA and molecular karyotypes of Trypanosoma evansi and Trypanosoma equiperdum from China.

We compared 12 stocks of Trypanosoma evansi and 1 recently isolated stock of Trypanosoma equiperdum from different regions of China by analysis of kinetoplast DNA (kDNA), nuclear DNA and molecular karyotypes. The T. equiperdum stock was remarkably similar to the T. evansi stocks, except for the possession of kDNA maxi-circles, suggesting a very close evolutionary relationship between T. evansi and T. equiperdum. The maxi-circles of the Chinese T. equiperdum stock were approximately 14.3 kb in size, i.e., about half the size of those of Trypanosoma brucei. This stock is thus similar to an old laboratory stock of T. equiperdum, which also has maxi-circles with a sizeable deletion. Both T. equiperdum and T. evansi kDNA mini-circles hybridised with a T. evansi-specific mini-circle fragment isolated from a Kenyan T. evansi stock. Our results extend the generality that T. evansi and T. equiperdum mini-circles are microheterogeneous rather than homogeneous. Molecular karyotypes obtained by pulsed field gradient gel electrophoresis provided a more sensitive way of distinguishing the T. evansi stocks than isoenzymes or restriction fragment length polymorphisms in kDNA mini-circles, genes for ribosomal RNAs and variant surface glycoproteins. Our results fit the general idea that T. evansi stocks worldwide have a single origin.

Animals↗

PubMed1992

The isoenzyme characteristics of Trypanosoma evansi and Trypanosoma equiperdum isolated from domestic stocks in China.

Twelve stocks of Trypanosoma evansi and one of Trypanosoma equiperdum isolated from domestic animals in China were examined for 16 enzymes using cellulose acetate and thin-layer starch gel electrophoresis. Differences were seen between stocks in only two of the enzymes, MDH and ALAT. Three of the T. evansi stocks, isolated from buffalo, and the T. equiperdum stock had the unusual pattern MDH-3, while all the other Chinese stocks had the common MDH-1. Two other stocks of T. evansi and again the T. equiperdum, all from equines, showed a new pattern ALAT-14. Otherwise the Chinese stocks had the same enzyme profile as T. evansi from elsewhere.

Alanine Transaminase↗

PubMed1992

An in vitro bioassay for quantification of melarsoprol in serum and cerebrospinal fluid.

A biological assay was developed for measuring melarsoprol in serum and cerebrospinal fluid of patients with human African trypanosomiasis. Trypanosomes were cultivated in microtiter plates for 72 hours with melarsoprol (Mel B) in concentrations of 1.25 micrograms/ml to 2.2 ng/ml. The minimum inhibitory concentration of Mel B for a reference Trypanosoma brucei rhodesiense clone was determined by microscopical examination. Samples of serum or cerebrospinal fluid were incubated under the same conditions and the highest dilution determined which caused death of all trypanosomes. The melarsoprol concentration of the sample was then calculated using the sample dilution and the determined minimal inhibitory concentration of the trypanosome population used for the assay. The test was validated using a number of reference samples and it was used for melarsoprol determination in serum- and cerebrospinal fluid samples taken from two treated patients. A sample size of 100 microliters was sufficient to perform the assay. The lower detection limit was 9 ng/ml (22.6 nmol/ml). The assay has potential for measuring other trypanocidal drugs in body fluids.

Animals↗

PubMed1991

Promastigote infectivity in Leishmania infantum.

Different clones and subpopulations of clones of Leishmania infantum were analyzed for their infective potential in vitro. Infectivity for macrophage-like cells (P388D1) and the response to a challenge with normal human serum enabled a clear differentiation between infective and non/low-infective populations. The results were confirmed by determination of parasite burdens in the spleens of infected golden hamsters. Long-term cultivation assays in vitro were used to test the influence of different cultivation conditions on the infectivity of promastigotes. An originally infective clone (LEM 768-A/ST) lost its infectivity during these assays but could regain it after the cultivation conditions had been changed. In addition, an originally non/low-infective clone (LEM 287-D/ST) could be forced to produce highly infective promastigotes. Infective cells were found only among stationary-phase promastigotes, i.e. after the cultures had reached a maximal number of cells per milliliter and the cell volume had clearly decreased.

Animals↗

PubMed1991

In vitro drug sensitivity of Trypanosoma congolense isolates.

The sensitivity of Trypanosoma congolense isolates to diminazene and isometamidium was determined using an incorporation assay based on the uptake of [3H]-hypoxanthine in the presence of serial drug dilutions. The bloodstream forms of the different isolates exhibited variation in their sensitivity to the drugs that correlated well with the in vivo drug response. For diminazene, the sensitivity of the most sensitive population was 40 times that of the least sensitive population. For isometamidium, the IC50 values (the drug concentrations that decreased radiolabel incorporation by 50%) lay in a similar range, except for those found for two isolates from lions, which were 10(3)-10(4) times more sensitive than the isolates from cattle. The sensitivity of procyclic forms differed markedly from that of the bloodstream stages. Therefore, it must be concluded that the procyclic stage does not reflect the sensitivity of the bloodstream forms of T. congolense and that the former should not be used for determinations of in vitro drug sensitivity.

Animals↗

PubMed1991

A new photometric assay for testing trypanocidal activity in vitro.

A new in vitro test was developed to assess the trypanocidal activity of compounds against axenically grown Trypanosoma brucei bloodstream forms. Trypanosomes from continuous culture were exposed to series of drug dilutions in a microtiter plate. After an incubation time of 72 h at 37 degrees C, the pH indicator of the medium had changed its colour in non-inhibited cultures due to the production of pyruvate. Inhibited cultures showed less, if any, colour changes. Plates could be read by eye or using an enzyme-linked immunosorbent assay (ELISA) reader. Within the pH range of 7.0-7.5, the extinction values of phenol red at 560 nm showed a linear correlation with both the pH of the medium and the pyruvate concentration in the medium. By comparison of the decreases in extinction in test cultures with those in control cultures, IC50 values (drug concentrations causing 50% inhibition) could be determined. Minimal inhibitory concentrations (MICs) could be read by eye. The photometric test evaluation was compared with evaluation according to growth inhibition or to incorporation of radiolabelled hypoxanthine. The three methods of evaluation gave similar results.

Animals↗

PubMed1991

Alterations in Krebs cycle enzyme activities and carbohydrate catabolism in two strains of Trypanosoma brucei during in vitro differentiation of their bloodstream to procyclic stages.

A rapid switch from a fermentative to a primarily oxidative type of glucose utilization was observed during in vitro differentiation of Trypanosoma brucei STIB348 and EATRO1244 bloodstream to procyclic trypomastigotes. In accordance with previously published reports bloodstream populations produced pyruvate as the major end product of glucose catabolism, together with very small amounts of CO2, succinate and glycerol. During differentiation pyruvate excretion decreased within 48 h to the low levels produced by 28-day procyclic stages. Concomitant with the decline in pyruvate formation, acetate appeared as a new product and the rates of respiratory CO2 increased considerably. The amount of carbon released with these compounds could account for nearly all of the glucose carbon consumed. Rates of glucose utilization and formation of acetate and CO2 in cells differentiated for 48 h were essentially the same as those found in 28-day procyclics. Succinate and glycerol excretion remained low during the entire transformation process, and no significant difference in the pattern and quantities of end products were found between the two trypanosome strains. During trypanosome differentiation the changes in metabolism were associated with marked alterations in enzyme activity levels. Activities of the tricarboxylic acid (TCA) cycle enzymes citrate synthase, isocitrate dehydrogenase (NAD+), succinate dehydrogenase and fumarase were not detectable in bloodstream trypomastigotes but appeared upon differentiation for 24 h. An exception was citrate synthase whose activity was not demonstrable until 48 h postinoculation into culture. After 48 h the majority of the TCA cycle enzyme activities continued to increase steadily until day 28. Pyruvate kinase activity decreased in differentiating cells after 48 h to about 25% of the level found in bloodstream trypomastigotes.(ABSTRACT TRUNCATED AT 250 WORDS)

Aconitic Acid↗

PubMed1991

In vitro activity of organometallic complexes of Ir, Pt and Rh on Trypanosoma b. gambiense, T. b. rhodesiense and T. b. brucei.

A series of organometallic drugs were tested in a photometric in vitro assay for trypanocidal activity against the three subspecies of the Trypanosoma brucei group. Trypanosoma b. rhodesiense, T. b. gambiense and T. b. brucei displayed a similar sensitivity pattern. The following IC50 values (drug concentration inhibiting decrease of extinction by 50%) could be determined: IrCl6 (IV)-Pentamidine (1.5 ng/ml), IrCl6(IV)-Ro15-0216 (10 micrograms/ml), PtBr6-Metamidium (30 micrograms/ml), PtBr6(IV)-Benznidazole (20 micrograms/ml), PtBr6(IV)-Ro15-0216 (50 micrograms/ml), cis-Pt(II)-Pentamidine (0.1 micrograms/ml), Rh(I)-COD-Pentamidine (5-10 ng/ml), Rh(I)-COD-Benznidazole (10 micrograms/ml), Rh(III)-Ethylxanthate (30 micrograms/ml) and Pt(II)-Pentamidine-cis-Pt(II)(Pentamidine)(Cl)2 (2 ng/ml). The four Pentamidine complexes were the most active compounds and showed activities comparable to that of Pentamidine (IC50: 0.5 to 1 ng/ml). Since the toxicity of the Ir- and Pt-Pentamidine complexes is ten to twenty fold lower (400 to 1000 mg/kg) than that of Pentamidine (50 mg/kg), the complexes might be candidates for investigation as safer trypanocidal drugs.

Animals↗

PubMed1990

Influence of fever and flurbiprofen on trypanosome growth.

The administration of flurbiprofen, a potent non-steroidal anti-inflammatory drug (NSAID), to goats infected with trypanosomes resulted in high elevated parasitaemia and suppression of fever. In contrast to goats, rats infected with trypanosomes do not show febrile reactions. Therefore, the role of body temperature was investigated with yeast-induced fever in Trypanosoma evansi and T. brucei infected rats. These investigations did not support the hypothesis that a high body temperature causes a drop in parasitaemia. In goats infected with trypanosomes, it is also unlikely that fever has an inhibitory influence on the parasitaemia. In these animals, rises in parasitaemia could be provoked by doses of flurbiprofen as low as 1/20 of the normal doses and these doses did not or only partly suppressed fever. No effect on parasite growth could be obtained when flurbiprofen was added in concentrations up to 32 micrograms/ml directly to T. brucei cultures. Moreover, no growth promoting factor(s) could be identified in vitro in serum from flurbiprofen-treated goats.

Animals↗

PubMed1989

Isolation and cultivation in vitro to the infective, metacyclic stage of Trypanosoma (Nannomonas) simiae from Glossina morsitans submorsitans.

Two separate trypanosome isolations were made from a single Nannomonas-infected Glossina morsitans submorsitans from The Gambia. Inoculation of a piglet with the infected hypopharynx produced an infection with Trypanosoma simiae. DNA was isolated from the bloodstream forms to prepare a probe specific for this species. Trypanosomes isolated from the fly midgut were frozen in liquid nitrogen and then cultivated in vitro. Amplification of this population and elimination of a yeast contaminant were achieved by two passages through laboratory G. m. morsitans. Further cultivation in vitro resulted in the production of epimastigotes and, later, metacyclic forms. Two pigs inoculated with cultivated metacyclic forms developed infections with atypical, relapsing parasitaemias and extended survival time. Neither the metacyclic forms, nor bloodstream forms derived from them, infected calves. The identity of various stages of the in vitro cultivated, procyclic-derived stock was confirmed morphologically and with the T. simiae-specific DNA probe.

Animals↗

PubMed1989

In vitro drug sensitivity test for Trypanosoma brucei subgroup bloodstream trypomastigotes.

An in vitro test has been developed to determine drug sensitivities of bloodstream trypomastigotes of Trypanosoma (T.) brucei isolates. The incorporation of [14C]leucine, [3H]thymidine and [3H]hypoxanthine into bloodstream forms in vitro was compared and different sensitivity test procedures with trypanocidal drugs were evaluated. Bloodstream forms were added to a microtiter plate with serial dilutions of trypanocidal drugs containing a cell-free culture medium supplemented with a mammalian serum, which allows continuous cultivation of the bloodstream stages. After a preincubation period of 16 h, [3H]hypoxanthine was added, and after another 8 h, the cells were harvested with a cell harvester. The glass fiber filters on which the cells were collected were counted in a liquid scintillation counter and the percent inhibition determined as percentage of the control counts. This method gave accurate, reproducible results for T. (T.) brucei isolates tested with the trypanocidal drugs suramin, berenil, samorin, lomidine and Mel W.

Animals↗

PubMed1989

In vitro drug sensitivity of Trypanosoma gambiense isolates.

Drug sensitivities of seven Trypanosoma b. gambiense isolates from patients in the Ivory Coast were measured for Mel B, suramin and lomidine using an in vitro incorporation test. Bloodstream forms were isolated from Mastomys natalensis, incubated in serial drug dilutions in a microtiter plate, after 24 h, radiolabeled hypoxanthine was added, and the plate incubated for another 15 h. Trypanosomes were then harvested onto glass fiber filters with a cell harvester and incorporation of label was determined in a liquid scintillation counter. From the incorporation inhibition curves IC50 and IC75 values were calculated. IC50 values for Mel B ranged from 0.5 to 4.6 ng/ml, for suramin from 7.2 to 30.5 micrograms/ml, and for lomidine from 2.1 to 7.0 ng/ml. Isolate TH-1/78E(031) was the least sensitive to all three drugs while TH-1/78E(020), TH-64/78E(020) and TH-31/78E(025) were the most sensitive to the drugs used. THDAL 1030R, an isolate from a patient who relapsed after three consecutive Mel B treatments, showed an IC50 value for Mel B of 1.20 ng/ml. The in vitro drug sensitivity test gave reproducible results for the T b. gambiense isolates tested.

Animals↗

PubMed1988

Ro 15-0216: a nitroimidazole compound active in vitro against human and animal pathogenic African trypanosomes.

In vitro systems for the continuous cultivation of Trypanosoma brucei brucei, T. b. gambiense, T. b. rhodesiense, T. congolense and T. vivax were used to determine the antitrypanosomal activity of the 2-substituted nitroimidazole Ro 15-0216. For all trypanosome species, the concentration which inhibited parasite growth by 50% (IC50 value) was established: 0.0957 microgram ml-1 (T. b. brucei TC221), 0.1327 microgram ml-1 (T. b. gambiense STIB 754-A), 0.0450 microgram ml-1 (T. b. rhodesiense STIB 704-BABA), 0.0896 microgram ml-1 (T. congolense ILNat 3.1) and 0.0109 microgram ml-1 (T. vivax ILRAD 1392). The IC50 value of its major metabolite Ro 19-9638 was 0.0341 microgram ml-1 (T. b. rhodesiense STIB 704-BABA). Furthermore, minimum exposure times required to render T. b. brucei non-infective for mice as well as preventing their growth in vitro have been established to be three, four, six and ten hours at drug concentrations of 30, 10, 3 and 1 microgram ml-1, respectively.

Acetanilides↗

PubMed1988

Production of metacyclic forms by cyclical transmission of west African Trypanosoma (T.) brucei isolates from man and animals.

Fifteen West African Trypanosoma (T.) brucei isolates from man and animals were cyclically transmitted. Five stocks, belonging to the non-gambiense group, could easily be transmitted through Glossina morsitans morsitans or Glossina m. centralis infected on mice, whereas successful transmission of the 10 isolates, identified as Trypanosoma brucei gambiense, was performed using G. palpalis gambiensis as vector. Glossina p. gambiensis was infected with culture-derived procyclic trypanosomes by repeated membrane feeding. In both cases, metacyclic forms could normally be detected in saliva samples of positive flies 3 to 4 weeks after first infection. These forms of major interest were subsequently characterized relative to their resistance/sensitivity against normal human serum in vitro and their antigenic properties, using indirect immunofluorescence: Metacyclic forms of all the T. b. gambiense isolates were determined by a stable human serum resistance and a restricted metacyclic variable antigen type (mVAT) repertoire, whereas representatives of the non-gambiense group (including TH162/78E 021) were sensitive against the trypanolytic factors of normal human serum and expressed a heterogeneous metacyclic antigen profile.

Animals↗

PubMed1987

Human serum resistance of metacyclic forms of Trypanosoma brucei brucei, T. brucei rhodesiense and T. brucei gambiense.

Resistance against the lytic action of human serum has been tested among metacyclic and bloodstream forms of Trypanosoma brucei brucei, T.b. rhodesiense and T.b. gambiense stocks and clones. The resistance was determined by applying an in vitro human serum resistance test. Whereas the majority of T.b. gambiense metacyclic forms exhibited stable human serum resistance, T.b. rhodesiense metacyclics showed inconsistent resistance within a minority of parasites, which tended to diminish completely with prolonged passages in rodents. Infection of tsetse flies with in vivo or in vitro selected human serum resistant forms did not significantly increase the proportion of resistant parasites among extruded metacyclic forms. In a T.b. rhodesiense stock which never showed human serum resistance in the metacyclic forms, human serum resistance reappeared after a 2-day cultivation period in the presence of a mammalian serum. These results reflect important phenotypic dynamics and may lead to a better understanding of the epidemiology of African human sleeping sickness.

Animals↗