Antitrypanosomal effects of traditional Chinese herbal medicines on bloodstream forms of Trypanosoma brucei rhodesiense in vitro.

The antitrypanosomal activity of traditional Chinese herbal medicines and these crude drug ingredients were determined using axenic cultured bloodstream forms of Trypanosoma b. rhodesiense which is one of the two causative agents of African sleeping sickness in man. The drugs tested were 8 traditional Chinese herbal medicines and these 14 crude drug ingredients. Of these traditional Chinese medicines examined, san'o-shasin-to and oren-gedoku-to showed most potent antitrypanosomal effect. The minimal effective concentration (MEC) which killed all bloodstream form populations within 24 hours of both drug exposure was 125 microg/ml. The 50% effective concentration (EC50) of san'o-shashin-to and oren-gedoku-to was 63 and 74 microg/ml, respectively. In the crude drug ingredients tested, Scutellaria baicalensis G. and Coptis japonica M. which are the main components of san'o-shasin-to and oren-gedoku-to, showed the most powerful antitrypanosomal activity. The MEC and EC50 value of these crude drug ingredients were 30 and 60 microg/ml, and 20 and 36 microg/ml.

Continuous growth of bloodstream forms of Trypanosoma brucei brucei in an axenic culture system containing a low concentration of serum.

An effective axenic culture system for bloodstream forms of Trypanosoma brucei brucei GUT at 3.1 containing a low concentration of serum is described. Bloodstream forms routinely maintained in Iscove's modification of Dulbecco's medium supplemented with 100 microM hypoxanthine, 30 microM thymidine, 40 microM adenosine, 1 mM sodium pyruvate, 50 microM L-glutamine, 100 microM 2-mercaptoethanol and 20% FBS for more than one year were grown in the same medium supplemented with 5% FBS without reducing their growth rate. Then culture adapted trypanosomes in the culture medium containing 5% FBS were transferred into the modified medium supplemented with 0.5% FBS. For the constant growth of bloodstream forms in the medium containing 0.5% FBS, the culture medium was further supplemented with 200 microM L-alanine, 100 microM glycine, 10 microM L-oruithine hydrochloride and 10 microM L-citrullin. The trypanosomes propagated in this culture system for one year retained their infectivity for mice. This culture system was also shown to be useful for cloning of T.b. brucei GUT at 3.1 which is important for separation of mutants.

An axenic culture system for the transformation of bloodstream forms to procyclic forms of Trypanosoma brucei brucei in vitro.

An axenic culture system for continuous cultivation of bloodstream forms of Trypanosoma brucei GUT at 3.1 and subsequent transformation of bloodstream forms to procyclic forms is described. Bloodstream forms were continuously grown at 37 degrees C in Iscove's modification of Dulbecco's medium (M-DMEM, with bovine serum albumin, transferrin and soybean lecithin supplemented with 100 microM hypoxanthine, 30 microns thymidine, 40 microM adenosine, 1mM sodium pyruvate, 50 microM L-glutamine, 100 microM 2-mercaptoethanol and 20% (v/v) heat-inactivated fetal bovine serum. In this system, 2-mercaptoethanol (2-ME) was essential and in the absence of 2-ME, 100 microM L-cysteine and 10 microM bathocuproine sulfonate could not be substituted for 2-ME. This culture system was useful for long-term culture of bloodstream forms of this clone. Axenic cultivation of bloodstream forms at 27 degrees C resulted in transformation to procyclic forms within 5 days in the same medium supplemented with 5 mM L-proline, 8 micrograms/ml hemin and 4 micrograms/ml hematin, respectively and, instead of FBS, 20% (v/v) hemoglobin-poor fraction of fetal bovine serum.

Immunological activities of monoclonal IgG1 antibody against Trypanosoma gambiense.

The present paper deals with the immune reaction between a monoclonal IgG1 antibody and Trypanosoma gambiense. The aggregation of trypanosomes, immune adherence to macrophages and protection against infection are associated with the antibody. IgG1-mediated clumping of trypanosomes is readily dissociated by the addition of complement. Dissociation of the clumped trypanosomes in the equivalence area released approximately fifty percent of previously bound surface antigens. These antigens were capable of binding again to new IgG1 antibody. Complement deposition rendered bivalent IgG1 antibody in the immune complex functionally univalent. Such an event in the presence of complement is of great advantage to the infected host in killing pathogens in vivo, as it allows more antibodies to attach to surface antigens and subsequently to initiate complement activity.

Diversity of complement-mediated immune reaction to Trypanosoma gambiense.

Intact and papain-treated Trypanosoma gambiense clone populations, each expressing special antigens on their cell surfaces, were mixed with rabbit antiserum in the presence of complement. Two distinct types of immune reaction between trypanosomes and antisera were observed: clumping followed by dissociation (CD) and inhibition of aggregation (IA). Special antigens on the cell surface of trypanosomes exposed after papain digestion are implicated in both types of immune reaction. IA was considered to be more effective as an immunological response which would allow the infected host to clear the pathogen without any obstruction of capillaries and impairment of blood flow caused by clumping masses of trypanosomes.

An effective axenic culture system for Trypanosoma brucei rhodesiense blood stream forms in vitro.

An effective axenic culture system for Trypanosoma brucei rhodesiense (ILRAD 1501) bloodstream forms is demonstrated. Bloodstream forms were continuously grown in 25 mM HEPES-buffered D-MEM supplemented with 10 microM bathocuproine sulfonate (BCS), 100 microM cysteine, and 20% heat-inactivated fetal bovine serum at 37 degrees C in vitro. At the initiation of the culture, T. b. rhodesiense bloodstream forms required the presence of 0.2 IU/ml insulin and 1 mM pyruvate, while bloodstream forms were grown in the culture medium without these supplements 4 days after initiation of the culture. Under this culture condition, T. b. rhodesiense bloodstream forms increased in number to 7 to 8 x 10(6) trypanosomes/ml, by day 4 after initiation of the culture. The trypanosomes cultured in this axenic system for 150 days were typically long and slender and retained their virulence for mice. This axenic culture system is extremely useful for in vitro cloning of T. b. rhodesiense bloodstream forms in vitro.

The successful system in long-term cultivation of Trypanosoma gambiense bloodstream forms.

Rat astroglioma cell line (GA-1) was extremely useful for long-term in vitro cultivation of Trypanosoma gambiense blood-stream forms. Parasites could be continuously grown at 37 degrees C for more than 200 days in the culture system, consisted of HEPES-buffered RPMI 1640 (pH 7.2, 300 milliosmole/kg) supplemented with 20% inactivated fetal calf serum in the presence of GA-1 cells. Parasites cultured for more than 200 days still retained not only their virulence for mice but also their original antigenic type. Morphologically, they resembled host infected bloodstream forms by way of having a subterminal kinetoplast and surface coat. The best growth rate of trypanosomes was obtained with 1 X 10(6) GA-1 cells/25 cm2 culture flask. Under this culture condition trypomastigote form populations increased in number up to 7-8 X 10(6) trypanosomes/ml by day 3 after initiation of the culture. The population doubling time in this culture system within the first 24 hours was almost the same as in mice. Most of the cultured trypanosomes were in suspension, but 15-20% of the parasites adhered to the surface of GA-1 cells. The culture system was also shown to be useful for cloning of T. gambiense which is important for separation of mutants.