Constrained randomization in a therapeutic efficacy trial.

The use of valid constrained randomization is presented for a therapeutic efficacy trial. The construction and evaluation of randomization schemes are studied for two treatment replicates per block and for hypothesized linear gradients. Two different isomorphism classes of designs are compared using the variance of the mean squared error criterion. The results indicate that valid constrained randomization schemes can be constructed that are superior to ordinary randomization. A mastitis efficacy trial provided the specific problem to be solved.

Epidemiologic investigation of an outbreak of cutaneous leishmaniasis in a defined geographic focus of transmission.

An outbreak of cutaneous leishmaniasis occurred in a unit of 608 Puerto Rican national guardsmen conducting jungle warfare training in the Panama Canal Area in July 1984. An epidemiologic investigation of reported nonhealing, ulcerating skin lesions was conducted among 540 (89%) unit members in November and December 1984. Fifteen (88%) of 17 individuals with chronic, ulcerating skin lesions were confirmed as cases of cutaneous leishmaniasis by culture or histopathology. Twelve cases yielded positive Leishmania cultures, identified as L. braziliensis panamensis by cellulose acetate electrophoresis. Evaluation of different diagnostic techniques revealed that direct examination of tissues by Giemsa-stained histological examination was the most sensitive test (87% sensitivity), with an indirect immunofluorescent antibody test being rather insensitive (67%). All but one of the confirmed cases operated in small units that trained and slept overnight at a mortar firing site for a period of three days, yielding a site-specific attack rate of 22% (14 of 64). This contrasted with a much lower attack rate of 0.2% (1 of 476), experienced by unit members who trained at other locations during the same time frame (P less than 0.001). The median incubation period calculated from day of arrival at the mortar firing site was 17 days (range 2-78) for the 15 confirmed cases. Available personal protection methods, such as the use of insect repellents, were not appropriately implemented by unit personnel and thus, were not found to effectively protect against Leishmania infection. This is the largest reported outbreak of cutaneous leishmaniasis in military personnel associated with a single geographic focus of infection and contrasts with the usual sporadic disease experience in Panama.

Development of Leishmania (Viannia) panamensis lesions and relationship of numbers of amastigotes to lesion area on antimony-treated and untreated hamsters.

Young adult (60-70-g) male golden hamsters (Mesocricetus auratus) each were injected intradermally at the dorsal base of the tail with 15 x 10(6) promastigotes of Leishmania (Viannia) panamensis (MHOM/PA/83/WR539), and progression and regression of subsequent lesions were evaluated for up to 17 wk postinfection (PI) as to area, weight, and number of amastigotes within lesions in untreated hamsters and in hamsters treated with meglumine antimoniate (Glucantime). In untreated hamsters total area of lesion, weight, and numbers of amastigotes generally increased rapidly and concomitantly up to 3-4 wk PI. Amastigote numbers tended to decrease from 4 to 11 wk PI and subsequently the numbers of amastigotes within the lesions decreased rapidly, whereas relatively little change occurred in the area and weight of the lesions. Meglumine antimoniate treatment of cutaneous hamster lesions resulted in marked concomitant decrease in size of the lesions and numbers of amastigotes within the lesions examined 1 wk after treatment. Measurement of the area of cutaneous leishmanial lesions thus would appear to be a valid method of evaluating the efficacy of promising compounds against L. panamensis in hamsters when measurements are taken 3-5 wk after experimental infection and reflects the number of amastigotes present in the lesion.

Berberine derivatives as antileishmanial drugs.

Berberine, a quaternary alkaloid, and several of its derivatives were tested for efficacy against Leishmania donovani and Leishmania braziliensis panamensis in golden hamsters. Tetrahydroberberine was less toxic and more potent than berberine against L. donovani but was not as potent as meglumine antimonate (Glucantime), a standard drug for the treatment of leishmaniasis. Only berberine and 8-cyanodihydroberberine showed significant activity (greater than 50% suppression of lesion size) against L. braziliensis panamensis.

Comparison of the protein kinase and acid phosphatase activities of five species of Leishmania.

Promastigotes from log phase and stationary phase cultures of Leishmania donovani, L. braziliensis panamensis, L. tropica, L. major, and L. mexicana amazonensis were analyzed for their content of protein kinase and acid phosphatase activities. Cell surface, histone-specific protein kinase activity was 1.3- to 2.8-fold higher in stationary phase cells of all species except for L. tropica in which the activities of stationary and log phase cells were equal; L. mexicana amazonensis had the highest histone-specific protein kinase activity and L. donovani the lowest. When viable, motile promastigotes of all five species were incubated for 10 min with [gamma-32P]ATP and Mg2+ (10 mM) in the absence of exogenous histone acceptor; about one dozen proteins were phosphorylated in each case. Both log phase and stationary phase promastigotes of all five species extensively phosphorylated a 50-kDa protein that had the mobility of tubulin. Incubation of pure calf brain tubulin with [gamma-32P]ATP and purified L. donovani protein kinase resulted in extensive phosphorylation of the former. Highly infective metacyclic forms (PNA-) of L. major, isolated from a stationary culture using the peanut agglutinin (PNA), contained eight times more histone-specific protein kinase activity than noninfective log phase cells (PNA+). The PNA- and PNA+ forms of L. major both phosphorylated a 50-kDa protein when incubated with [gamma-32P]ATP and magnesium or manganese ions (10 mM); the 50-kDa protein was precipitated by anti-tubulin rabbit antibodies. Extracts of all five species contained large amounts of acid phosphatase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for phosphorylation of the extracellular acid phosphatase of Leishmania donovani.

The presence of two phosphorylated molecular species in the culture supernatants of axenically cultivated Leishmania donovani promastigotes was demonstrated by biosynthetically labeling cultures with [32P]phosphate. One of these species was resolved into two bands with Mr's of 149,000 and 97,000 by dissociating polyacrylamide gel electrophoresis and copurified with the extracellular acid phosphatase activity produced by the promastigotes. The site of phosphorylation of the extracellular acid phosphatase is not yet known.

Effect of tunicamycin on the extracellular acid phosphatase of Leishmania donovani promastigotes.

Inhibition of replication of Leishmania donovani promastigotes in axenic culture medium by tunicamycin, an inhibitor of asparagine linked protein glycosylation, depends upon the cell density of the culture at the time of addition of tunicamycin as well as upon the concentration of tunicamycin itself. Parasite multiplication in cultures with initial densities of less than or equal to 1 X 10(6) cells ml-1 and a tunicamycin concentration of 1 microgram ml-1 was limited to 2-3 replications, but this limitation was not observed in cultures with initial densities greater than or equal to 2 X 10(6) cells ml-1. Under conditions in which tunicamycin inhibited parasite growth and protein synthesis by only 15% and 6%, respectively, there was a greater than 90% reduction in the level of secreted acid phosphatase activity in comparison to control cultures. The extracellular acid phosphatase activity remaining in tunicamycin treated cultures was electrophoretically distinct from that found in control cultures. No significant decrease in the amount of [35S]methionine incorporated into acid insoluble products in the supernatant of tunicamycin treated cultures was observed, and a radiolabeled protein with an electrophoretic Mr of 97,000 was immunoprecipitated from this supernatant by an anti extracellular acid phosphatase antiserum. It was concluded that the L. donovani extracellular acid phosphatase, previously shown to be a mannose containing glycoprotein, contains N-linked oligosaccharides which are necessary for maintenance of its catalytic activity, but not its secretion.

Activity of purine analogs against Leishmania donovani in vivo.

The dose of orally administered 9-deazainosine calculated to suppress 50% of Leishmania donovani amastigotes in hamster livers was 19 mg/kg (body weight) per day; 96 to 99% of Leishmania organisms were eliminated from the liver and spleen of squirrel monkeys by 50 mg/kg per day. Because these activities were greater than that of the experimental clinical agent allopurinol and comparable to that of the classical agent parenteral pentavalent antimony, 9-deazainosine should be considered for clinical development for visceral leishmaniasis.

Comparison of extracellular acid phosphatases from various isolates of Leishmania.

Forty isolates of Leishmania, representing all major species infecting humans and one parasite of lizards, were examined for their ability to secrete an extracellular acid phosphatase activity. This enzyme, which was originally described and characterized from a Sudanese strain of L. donovani, was detected in the culture supernatants of all species of promastigotes examined except for L. major and L. tarentolae. There were quantitative differences among species in their levels of enzyme activity and in the sensitivity of the exoenzyme to inhibition by L(+) tartrate. Upon electrophoresis in nondenaturing polyacrylamide gels, extracellular acid phosphatase from L. braziliensis panamensis, L. tropica, and L. mexicana showed distinctive patterns which were similar for all isolates of a given species, while enzymes from L. donovani isolates differed from one another in relative electrophoretic mobility. Enzymes from all species appeared heterogeneous, showing either discrete multiple bands or single diffuse bands on gels stained for enzyme activity. Although the biological function of the extracellular acid phosphatase is presently unknown, the exoenzyme may be of value as a diagnostic or taxonomic characteristic.

Purification and characterization of the extracellular acid phosphatase of Leishmania donovani.

A tartrate sensitive acid phosphatase activity was purified from culture supernatants of Leishmania donovani promastigotes grown in a macromolecule-free defined medium. Purification was accomplished by ultrafiltration, lentil lectin affinity chromatography, and sucrose density gradient ultracentrifugation. This enzyme was determined to be an acid glycoprotein containing 0.37 mg hexose per mg protein. A molecular weight of 134 000 by sucrose density gradient centrifugation was observed, although on molecular sieve chromatography the enzyme eluted with an apparent molecular weight of greater than 700 000. The specific activity of the purified enzyme was greater than 200 mumol min-1(mg protein)-1 when assayed with 4-methylumbelliferylphosphate as the substrate. In addition to various hexose phosphates, the enzyme hydrolyzed phosphorylated amino acids, in particular phosphotyrosine. The purified enzyme was heterodisperse with respect to both protein and activity staining patterns upon polyacrylamide gel electrophoresis.

Toxoplasmosis among the Ticuna Indians in the state of Amazonas, Brazil.

Results of a serologic survey for Toxoplasma gondii among 408 Ticuna Indians from five villages in western Brazil are presented and compared with the results of 61 non-Indian inhabitants of the town of Codajas, Amazonas. Indirect hemagglutination antibody titers greater than or equal to 64 were found in 39% of the Ticuna population as compared to 77% of the Codajas population. Prevalence rates of titers greater than or equal to 256 were 20.3% for Ticunas and 39.3% for Codajas. Prevalence rates of titers greater than or equal to 256 in Ticuna villages where dietary habits were most variable were higher and more similar to those of non-Indian populations than were the prevalence rates of this titer range in villages where the animal food source was predominantly fish.