sepharose and Trypanosomiasis--African

Differentiation from replicating slender forms to non-dividing stumpy bloodstream forms of T. brucei limits the parasite population size in the mammalian host in addition to and independently of the antibody response. Using a culture system for pleomorphic strains of T. brucei we show that slender forms very efficiently differentiate to stumpy forms in vitro and that the induction of differentiation is correlated to cell density. Differentiation in the host and in culture were compared using a battery of markers including cell morphology and volume, cell cycle position, the kinetics of the differentiation, expression of NADH dehydrogenase (diaphorase), expression of several differentially regulated transcripts and the kinetics of transformation to replicating procyclic forms after induction with cis-aconitate. By all available criteria, differentiation in culture reflects the natural process in the mammalian host. Time course experiments reveal a very tight temporal correlation between cell cycle arrest of bloodstream forms, appearance of a stumpy differentiation marker and the competence of a bloodstream form population to initiate transformation to procyclic forms in response to cis-aconitate. Our results show that induction of bloodstream form differentiation can occur independently of host-derived cues. We suggest a density sensing mechanism which induces differentiation to the non-dividing stumpy stage and thereby enables the parasite population to autoregulate its proliferation.

Topics: Aconitic Acid; Animals; Cell Cycle; Culture Media; Flagella; Gene Expression; Genes, Protozoan; Interphase; Mice; Mice, Inbred BALB C; Rats; Rats, Wistar; Sepharose; Trypanosoma brucei brucei; Trypanosomiasis, African

Primary axenic culture of Trypanosoma brucei bloodstream forms almost invariably requires a period of culture adaptation with cell death and clonal selection. This has been particularly difficult and in many cases unsuccessful for natural pleomorphic strains, which are characterized by their ability to differentiate from replicating long slender bloodstream forms into short stumpy forms. Here we show that a representative set of pleomorphic T. brucei strains can be cultured in vitro on agarose plates without any previous adaptation period and selection. The slender morphology was retained and the growth rate was identical to the growth rate in vivo. Long term in vitro culture for 3 months with this method did not affect the ability of the AnTat 1.1 strain to give rise to pleomorphic infections in mice. Stumpy populations emanating from these infections transformed rapidly and synchronously into dividing procyclic forms when triggered with cis-aconitate and a temperature shift. The growth supporting activity of agarose plates could be traced to high molecular mass polymeric agarose; beta-agarase digestion destroyed the activity. Membrane chamber experiments show that direct physical contact of trypanosomes with the agarose matrix is essential. In the absence of high molecular mass agarose, the cell division process is grossly impaired. We suggest that agarose mimics an interaction of trypanosomes with the host's extracellular matrix. Applications of the culture method are discussed.

Topics: Animals; Cell Division; Culture Media; DNA, Protozoan; Extracellular Matrix; Mice; Mice, Inbred BALB C; Molecular Weight; Parasitemia; Rats; Rats, Wistar; Sepharose; Time Factors; Trypanosoma brucei brucei; Trypanosomiasis, African

Activities of a range of macromolecular conjugates of daunorubicin against Trypanosoma brucei rhodesiense in vitro and in vivo are described and compared to those of free daunorubicin. Conjugates tested were daunorubicin attached to bovine serum albumin by (i) a labile 'glutaraldehyde' linkage (D-BSAG), and (ii) a stable succinyl linkage (D-BSAS), daunorubicin covalently linked to agarose beads (D-AG), and daunorubicin adsorbed onto polyisobutylcyanoacrylate nanoparticles (D-PICA). Trypanocidal activity in vitro was retained in all except D-BSAS, whereas in vivo only D-BSAG had any activity. The results indicate that daunorubicin must be released from the conjugate before it can exert its activity.

Topics: Animals; Cyanoacrylates; Daunorubicin; Drug Carriers; Enbucrilate; Mice; Polymers; Sepharose; Serum Albumin, Bovine; Trypanosoma brucei brucei; Trypanosomiasis, African

Topics: Acrylamides; Animals; Cerebrospinal Fluid; Humans; Immunodiffusion; Immunoglobulin A; Immunoglobulin G; Immunoglobulin M; Macroglobulins; Molecular Weight; Rats; Sepharose; Trypanosoma brucei gambiense; Trypanosomiasis, African