Search PubMed⌕ Search

Biomedical subjects

R Brun

Publications and source records attributed to R Brun.

At least 91 records · Page 5Linked to original sources
PubMed1995

Evaluation of the human cell line RPMI 2650 as an in vitro nasal model.

We have investigated a human nasal cell line, RPMI 2650 (ATCC), as an in vitro model to assess the absorption and tolerability of nasally administrated peptides. This cell line was cultured on different filters (i.e. Nunc, Costar, Falcon, Millipore, and Becton) which were uncoated or coated with collagen types I or IV, laminin, fibronectin and extracellular matrix. Cell line morphology, capability of forming a cell layer and phenotype were analyzed by light-, fluorescence- and electron microscopy. The morphological analysis showed that RPMI 2650 cells were forming cell clusters on one group of filters (i.e., Nunc, Costar, Falcon and Becton filters coated with collagen types I or IV, laminin and extracellular matrix). On the second group of filters (i.e., Becton and Millipore filters, coated with fibronectin) the cells had the tendency to spread in a cell layer. In both groups of filters, the cells never showed cell polarization, nor microvilli and tight junctions. Phenotyping of this cell line was performed by indirect immunofluorescence using monoclonal antibodies against human cytokeratins 10, 17 and 18 (markers of epithelial cells), desmin (markers of mesothelial cells) and vimentin (marker of mesothelial cells). Vimentin was strongly expressed, cytokeratins 10, 17 and 18 were weakly expressed, and desmin was not expressed. The human nasal cell line RPMI 2650 was not able to form a tight cell layer under these cell culture conditions. The limits of this cell line as an in vitro nasal model for drug absorption is discussed.

Absorption↗
PubMed1994

Studies on compounds promoting the in vitro transformation of Trypanosoma brucei from bloodstream to procyclic forms.

In vitro differentiation of pleomorphic blood-stream forms of Trypanosoma brucei to procyclic culture forms occurred rapidly and at high rates at 27 degrees C in a culture medium containing 1 mM cis-aconitate as the transformation-inducing agent. Citrate was required at a much higher concentration (10 mM) to produce a similar transformation rate. The highest percentage of transformed cells was obtained when bloodstream-form trypanosomes were treated with pronase in the absence of a feeder-cell layer. However, under these conditions, the amount of procyclic forms obtained after 72 h was lower than that obtained in the presence of cis-aconitate. Trypsin was also capable of inducing transformation in the absence of a feeder-cell layer, but this treatment again resulted in low numbers of transformed cells. Blood-stream-form trypanosomes were incapable of taking up citrate to any significant extent and the citrate content of these stages was negligible. After 72 h of exposure to citrate (3 mM), intracellular levels of this compound remained very low (< 1 nmol (10(9) cells)-1), increasing in established procyclic stages to approximately 1.7 nmol (10(9) cells)-1. These observations suggest that the tricarboxylic acid (TCA)-cycle metabolite-dependent transformation may be initiated externally to the trypanosome cell membrane. The ability of both citrate and cis-aconitate to bind calcium and, thus, to reduce the concentration of this cation in the culture medium was found not to be responsible for the triggering effect on trypanosome transformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Aconitic Acid↗
PubMed1994

Induction of resistance to melarsenoxide cysteamine (Mel Cy) in Trypanosoma brucei brucei.

A population of Trypanosoma brucei brucei with reduced sensitivity to melarsenoxide cysteamine (Mel Cy) was produced in immunosuppresed mice using subcurative drug treatment. Melarsenoxide cysteamine resistance was stable after cyclical transmission through Glossina morsitans centralis. In vitro, the blood-stream forms showed 15-fold higher values for the minimal inhibitory concentration as compared with the parental clone. Cross-resistance could be determined with another arsenical drug, melarsoprol (14-fold) and to two different diamidines (diminazene aceturate: 47-fold; pentamidine methanesulphonate: 34-fold), but not to suramin. When cells were transformed to procyclic forms and tested in vitro, the sensitivity of the resistant population to melarsenoxide cysteamine was only 6-fold lower than that of the parent, but comparatively high cross-resistance could be shown to other drugs (melarsoprol; 85-fold; pentamidine methanesulphonate: 17-fold; quinapyramine sulphate: 40-fold). Selection of the resistant trypanosomes from non-resistant ones was possible under pentamidine methanesulphonate pressure in cell culture.

Animals↗
PubMed1994

Pharmacokinetics of melarsoprol in uninfected vervet monkeys.

The level of the trypanocidal drug melarsoprol was determined in serum and cerebrospinal fluid (CSF) of six healthy vervet monkeys after intravenous application of the drug following a standard treatment schedule and a recently suggested alternative protocol. The maximum serum levels measured were about 3 micrograms/ml. A three-compartment model was used to analyze the serum data. The mean residence time calculated for melarsoprol in serum was 18 h, the volume of distribution was 3.6 l/kg and the clearance was 3.5 ml/min*kg. In the CSF the drug levels were generally very low, not exceeding 55 ng/ml, and the adaptation of the drug levels was found to be very low. The comparison of the drug concentrations required to eliminate trypanosomes in vitro and the drug concentrations reached in the CSF during treatment revealed that the latter might be insufficient in some cases to eliminate all trypanosomes from this site. The peak serum levels during alternative application of the drug were lower compared to those during empirical treatment. No evidence for drug cumulation in the body was found. The results of this study are compared with recent pharmacokinetic data from human patients, and discussed in the context of the problem of relapses and reactive encephalopathy occurring after treatment of sleeping sickness.

Animals↗
PubMed1994

Drug sensitivity of Chinese Trypanosoma evansi and Trypanosoma equiperdum isolates.

The drug sensitivities of eleven Trypanosoma evansi isolates from China were examined using two different in vitro assays, a 3H-hypoxanthine incorporation assay and a long incubation low inoculation test (LILIT). Better discrimination of the drug susceptibility of the strains was observed with the LILIT. The drug responses of all the isolates to the arsenical melarsoprol were very similar. In contrast, for suramin, minimal inhibitory concentrations (MIC) varied within a 27-fold range and for diminazene within a 55-fold range. Comparison of MIC values with expected drug levels in the host as well as in vivo experiments with selected isolates and drugs indicated that all the isolates examined would be sensitive to melarsoprol, diminazene and suramin under in vivo conditions. For isometamidium, the difference in MIC values between the most and the least sensitive isolate was 724-fold. Neither of two isolates tested in mice--the most resistant and the second most sensitive--was cured with the highest acceptable dose of 10 mg kg-1 isometamidium chloride. Comparison of our results with blood levels of drug to be expected in cattle support the assumption that the Chinese T. evansi isolates have more or less innate resistance to isometamidium under in vivo conditions. One Trypanosoma equiperdum isolate was tested in the 3H-hypoxanthine incorporation assay. The results indicated that this isolate was highly sensitive to melarsoprol, isometamidium and suramin; with regard to diminazene, T. equiperdum was not as sensitive as the most sensitive T. evansi strains.

Animals↗
PubMed1993

In vitro assays to determine drug sensitivities of African trypanosomes: a review.

In vitro test methods have been developed both for determining the sensitivity of isolates of pathogenic African trypanosomes and for evaluating new compounds for antitrypanosomal activity. The principles of the assays and their main advantages or drawbacks are presented and discussed. In vitro assays which do not require trypanosomes pre-adapted to culture conditions are the Drug Incubation Infectivity Test (DIIT) and the [3H]Hypoxanthine Incorporation Assay. Chemosensitivity tests which do require continuous growth of trypanosomes in vitro include photometric, fluorescence, growth inhibition, long-term viability and metacyclic incubation assays. Evidence is presented to use metacyclic or bloodstream forms in such assays but to avoid procyclic trypanosomes. The drug sensitivity of a homogeneous trypanosome population can be quantified by using photometric, fluorescence or growth inhibition assays lasting 1-3 days. Small numbers of resistant organisms hidden in a sensitive population can be detected employing long-term viability assays (7-10 days). Final selection of the assay to be employed will depend on the parameter to be investigated, and the equipment available.

Animals↗
PubMed1993

Trypanosomiasis of domestic animals in China.

Trypanosomiasis caused by Trypanosoma evansi (surra) is a problem of great economic importance in livestock in China because it affects important working animals: buffaloes in the south and east, and horses and cattle in the north and west. In addition, buffaloes are an important source of meat and leather for the Chinese population. In the north and west, T. evansi is found mainly in camels, whereas in the east, the south and the southwest, it is primarily buffaloes, cattle and horses that are affected by this parasitic flagellate. Although trypanosomiasis is one of the most important parasitic diseases of domestic animals in China, little information on this disease has been published. In this article Zhao-Rong Lun, Yuang Fang, Chang-Ji Wang and Reto Brun evaluate trypanosomiasis of domestic animals in China and review epidemiological studies of the past 40 years.

Journal Article↗
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↗