Operational lessons from 20 years of the Mectizan Donation Program for the control of onchocerciasis.

The donation of ivermectin (Mectizan, Merck & Co., Inc.) to control onchocerciasis (river blindness) was established in 1987 and has since gradually expanded to provide for >570 million treatments cumulatively over the past 20 years. The Mectizan Donation Program (MDP) operates within a broad partnership in 33 endemic countries in need of mass treatment. Particular operational methods and tools are applied to facilitate ivermectin mass treatment. Drug management has been streamlined, including dosing, tablet size and packaging, and monitoring for adverse events. Much of the experience gained in the development of ivermectin mass treatment can be usefully applied in the recent broader perspective of control of neglected tropical diseases. The most important operational lessons of the MDP include: (i) the need to easily define the target population for treatment using rapid, non-invasive techniques; (ii) the value of a broad partnership; (iii) the great potential of working through community-directed treatment; (iv) the need to streamline all drug management aspects and (v) the importance of operations research to tackle new challenges.

Enhanced acquisition of purine nucleosides and nucleobases by purine-starved Crithidia luciliae.

The effects of purine starvation on the ability of the trypanosomatid Crithidia luciliae to accumulate purines were determined. Kinetic studies showed that the uptake of the nucleoside adenosine by purine-starved organisms was approximately 7-fold faster than by nutrient-replete cells. Further, these studies demonstrated that purine-starved organisms accumulated the nucleobases hypoxanthine and adenine at a rate > 100-fold faster than organisms cultivated under replete conditions. Activities of several intracellular purine-salvage enzymes were measured in organisms from both culture conditions. Of those measured, the activities of adenine deaminase and hypoxanthine phosphoribosyltransferase were elevated approximately 4-fold and approximately 11-fold, respectively, in purine-starved organisms. Competitive substrate specificity studies suggested that these elevated enzyme activities were not responsible for the increased rates of uptake by purine-starved cells. The results are consistent with the induction of novel surface membrane purine transporters expressed in response to purine starvation. These studies using C. luciliae may provide insights into the mechanisms of trypanosomatid adaptation to altered environments encountered during the course of the life cycle.

Crithidia luciliae: effect of purine starvation on S-adenosyl-L-methionine uptake and protein methylation.

The utilization of S-adenosyl-L-[methyl-3H]methionine ([3H-methyl]AdoMet) by Crithidia luciliae was assessed under nutrient-replete and purine-starvation conditions. Uptake experiments with intact cells demonstrated that the radiolabel from this molecule was accumulated by purine-starved organisms at a rate approximately 10-fold greater than that observed in those cultivated in nutrient-replete medium. Purine-starved cells also incorporated the radiolabel into trichloroacetic acid insoluble material at an approximately 10-fold faster rate than nutrient-replete cells. No differences, however, were observed in the intracellular levels of AdoMet and its metabolites between organisms cultivated under the two conditions. Results of comparative labeling studies with [3H-methyl]AdoMet, S-adenosyl-L-[carboxyl-14C]methionine, L-[methyl-3H]methionine and L-[35S]methionine in the presence and absence of cycloheximide demonstrated that the incorporation of label from [3H-methyl]AdoMet was due to transmethylation and was independent of protein synthesis. Further, approximately 15 methylated protein bands were identified by SDS-PAGE analysis. Lysates from both purine-starved and nutrient-replete organisms demonstrated similar levels of activity of three protein methyltransferases (PMI, II, III). The differences observed in [3H-methyl]AdoMet utilization between purine-starved and nutrient-replete C. luciliae may reflect the enhanced purine transport capacity which results from purine starvation.

Identification and characterization of variants of Crithidia luciliae with altered morphological and surface properties.

Variants of a cloned laboratory stock of the trypanosomatid parasite Crithidia luciliae have been distinguished from "parental type" organisms. These variants accumulated spontaneously over time as the protozoan was maintained by continuous passage in a chemically defined medium. Cloned lines of these variants have been isolated by plating on nutrient agar and partially characterized on the basis of their growth characteristics in culture, their colony and cellular morphology as well as their surface protein expression. One cloned line consisted of motile, flagellated forms which, unlike "parental type" organisms, did not adhere to the surface of culture flasks. Another cloned line was composed of non-adherent, nonmotile, amastigote-like forms which were further distinguished from "parental type" cells by virtue of their constitutive expression, in nutrient-replete medium, of high levels of a surface membrane associated 3'-nucleotidase/nuclease (3'-N'ase) activity. Both the motile, flagellated and amastigote-like variants, like the "parental type" organisms, exhibited elevated levels of the 3'-N'ase activity upon exposure to purine starvation conditions. The variants described are of potential importance in elucidating the mechanism of induction of the highly regulated 3'-N'ase activity as well as for understanding the cytoskeletal systems and the surface properties of these protozoa.

Crithidia luciliae: starvation for purines and/or phosphate leads to the enhanced surface expression of a protein responsible for 3'-nucleotidase/nuclease activity.

It has been shown previously that starvation of the trypanosomatid protozoan Crithidia luciliae for purines and/or inorganic phosphate results in increased levels of a surface membrane-associated 3'-nucleotidase/nuclease (3'-N'ase) activity which hydrolyzes both 3'-ribonucleotides and nucleic acids, thereby permitting the organisms to transport these essential nutrients across their cell membranes. A polypeptide with the requisite catalytic properties has been identified by an in situ gel activity assay following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In current studies, differential synthesis of the protein responsible for the 3'-N'ase activity was not demonstrable by comparisons of SDS-PAGE patterns of nutrient-replete or purine-starved parasites metabolically labeled with either [35S]methionine, [3H]leucine, or [3H]tyrosine. However, surface labeling of nutrient-replete and purine-starved cells revealed the enhanced expression of an 125I surface-labeled 43-kDa protein which comigrated with the 3'-N'ase activity in one- and two-dimensional electrophoretic systems. The amount of this surface-labeled peptide correlated with the level of 3'-N'ase activity as measured by test tube assay. Refeeding adenosine to purine-starved cells led to the loss of both the enzyme activity and the surface iodinatable 43-kDa band as a result of renewed cell division. Starvation of these organisms for phosphate also led to the enhanced expression of the 43-kDa radioiodinatable band. The results indicated that the 3'-N'ase protein, itself, is differentially expressed at the cell surface under conditions which lead to increased enzyme activity.