Lloyd H. Semprevivo

Thin-layer and liquid column chromatographic analyses of the lipids of adultOnchocerca gibsoni

Lipids were extracted from adultOnchocerca gibsoni with chloroform/methanol and the total lipid content was characterized. Glycolipids were isolated from other lipid classes by Florisil column chromatography and were then fractionated by DEAE-Sephadex ionexchange chromatography. HPTLC revealed the presence of 9 neutral glycolipid bands and of 15 acidic glycolipid bands that stained for sialic acid with resorcinol. Lipids that contained no carbohydrates were analyzed by a combination of TLC and amino column chromatography. Triacylglycerols, cholesterol, cholesterol esters, and free fatty acids were found to be major components of the neutral lipid fraction, and diacylglycerols and monoacylglycerols were minor components. Phosphatidylethanolamine and phosphatidylcholine were the predominant phospholipids. Phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophosphatidylcholine, and lysophosphatidylethanolamine were also present in significant amounts, whereas only traces of cardiolipin and phosphatidic acid were detected. Several minor lipids and phospholipids remained unidentified. These results indicate that adultO. gibsoni have a nonglycosylated lipid composition resembling that of other parasitic nematodes as well as a substantial repertoire of glycolipids, including many with the characteristics of gangliosides.

Leishmania mexicana: Effect of concomitant malaria on cutaneous leishmaniasis. Development of lesions in a Leishmania-susceptible (BALB/c) strain of mouse

Effect of concomitant malaria on cutaneous leishmaniasis. Development of lesions in a Leishmania-susceptible (BALB/c) strain of mouse. Experimental Parasitology 65, 269-276. Symptoms of human leishmaniasis vary greatly, ranging from cryptic infections to cases with fatal sequelae. Factors regulating the severity of the disease are largely undetermined. Malaria coincides geographically with leishmaniasis in many areas and the immunosuppressive effects of malaria are well documented. It is therefore plausible that malaria could enhance the course of concomitant leishmaniasis. Interactions between Leishmania mexicana and Plasmodium yoelii were examined in BALB/c mice. Percentage of blood cells infected with P. yoelii and diameter of footpad lesions caused by L. mexicana were the criteria used to assay for disease severity. L. mexicana and P. yoelii infections were each significantly enhanced in dually infected mice when compared to mice infected with either parasite alone. Mortality rates due to the normally nonlethal P. yoelii were high during concurrent infections.

Characterization of the exometabolite of Leishmaniadonovani as a novel glycopeptidophosphosphingolipid

Abstract Using thin layer chromatography (TLC) and various colorimetric procedures, the exometabolite of Leishmania donovani was shown to be a novel glycopeptidophosphosphingolipid. In aqueous medium the exometabolite aggregated to form micellar structures of high molecular weight. Purity of the various preparations and the novel nature of the micellar structures was demonstrated by TLC. These micelles are unique because they do not break up upon solvation in organic solvents. This indicates that once the supramolecular structure is established, its integrity is maintained by forces other than the apolar ones involved in its formation.

The effect of pentostam and cimetidine on the development of leishmaniasis (Leishmania mexicana amazonensis) and concomitant malaria (Plasmodium yoelii)

BALB/c mice were infected with Leishmania mexicana amazonensis and/or Plasmodium yoelii in order to determine the impact of multiple parasitic infection on the efficacy of chemotherapeutic agents. Uninfected, P. yoelii-infected, L.m. amazonensis-infected, and L.m. amazonensis and P. yoelii-infected mice were inoculated with cimetidine (80 mg kg-1 day-1) or pentostam (200 mg kg-1 day-1) once a day for an initial 20-day period, and once a week thereafter. Leishmania mexicana amazonensis lesion development and P. yoelii parasitaemia were the criteria used to assay disease severity. Mice infected with both P. yoelii and L.m. amazonensis developed more severe disease than did animals infected with either parasite alone. Cimetidine and pentostam each slowed the development of L.m. amazonensis in animals infected with only that parasite and in animals infected with both P. yoelli and L.m. amazonensis. However, mice treated with pentostam developed more severe P. yoelii infections than did control animals, whereas cimetidine significantly reduced P. yoelii parasitaemia in all instances.

Changes in growth rate and infectivity ofLeishmania donovani subjected to various laboratory procedures

Two substrains ofLeishmania donovani strain 3S were used in a study of grwoth rates of promastigote and amastigote stages as well as of infectivity of the latter stages in the course of cultivation, animal passages, and heat adaptation. One of these substrains, 3S-25A, was initiated with amastigotes of strain 3S maintained by serial passages in golden hamsters since 1962. The 3S-25A promastigotes were transferred 24 times (about 142 generations), then passaged once in a hamster. The amastigotes derived from this hamster on postinoculation day 30 were employed for initiation of culture promastigotes designated as substrain 3S-25A′. This culture was transferred 20 times (about 141 generations). The third substrain, 3S-37, was isolated in culture in 1974, then adapted to 37° C. All cultures were grown in modified Tobies medium (Tm). Young C57BL/6J mice were employed in the estimation of generation times (G) of amastigote stages and infectivity of promastigotes.Upon isolation in culture, substrain 3S-25A promastigotes grew poorly and inconsistently during the first five transfers (about 20 generations). Between the fifth and 13th transfers (about 20th and 65th generations), the populations increased from 1.9×107 to 1.2×108 organisms/ml. They remained approximately constant until the 24th serial transfer (about 142nd generation) at which time some of the promastigotes were inoculated into hamsters while others were stabilated in liquid nitrogen. The initial increase in the promastigote yields appeared to depend upon decrease inG (from 12 to 7 h). Promastigotes of substrain 3S-25A′ reached the maximum numbers in the primary cultures.High infectivity was characteristic of the early in vitro transfers of substrain 3S-25A′, 25% for the fifth serial passage (about 30 generations), but the infectivity decreased rapidly on cultivation, reaching a 0.5% level by the tenth transfer (about 100 generations). In contrast, relatively low infectivity levels were observed for 3S-25A promastigotes during the first few passages, e.g., 1.2% for those from cultures obtained after five serial transfers. These levels increased up to the 12th transfer, when they reached 8%. Further cultivation, however, was accompanied by infectivity decrease — after 24 passages in Tm, it was estimated at 4%.TheGs estimated from counts of amastigotes in livers of hamsters inoculated with substrains 3S-25A and 3S-25A′ were closely comparable; for infections with tenth-transfer promastigotes of these strains, the times were, respectively, 34 and 36 h. On the other hand, theG for amastigotes in livers of hamsters inoculated with strain 3S-37 promastigotes from the tenth serial in vitro passage was much longer, 53 h.

Exometabolites of Leishmania donovani promastigotes. II. Spontaneous changes of exometabolite after isolation.

The defined medium of Steiger and Steiger conditioned by growth ofLeishmania donovani strain 3S promastigotes served as the primary source of parasite exometabolites. Four fractions of the conditioned medium were recovered by Sephadex G-25 column chromatography. These fractions shared immunologic determinants, but differed in their molecular weights, affinity for Sephadex G-25, and absorption spectra. Degradation and/or aggregation of one of the fractions (Fraction IV) appeared to yield substances found in the remaining three fractions. Furthermore, by appropriate manipulations, Fraction IV could be transformed into substances resembling those described by previous workers, eg., excretory factor (EF) or antigenically active glycoproteins (AAGP). The present findings suggest that the primary exometabolite released byL. donovani is a small glycopeptide-like molecule; however, in media conditioned by growth of promastigotes and during isolation procedures, aggregation and/or degradation processes yield higher molecular weight substances which vary in size and physico-chemical properties.The defined medium of Steiger and Teiger conditioned by growth oif Leishmania domovani strain 3S promastigotes served as the primary source of parasite exometabolites. Four fractions of the conditioned medium were recovered by Sephadex G-25 column chromatography. These fractions shared immunologic determinants, but differed in their molecular weights, affinity of Sephadex G-25, and absorption spectra. Degradation and/or aggregation of one of the fractions (Fraction IV) appeared to yield substances found in the remaining three fractions. Furthermore, by appropriate manipulations, Fraction IV could be transformed into substances resembling those described by previous workers, eg., excretory factor (EF) or antigenically active glycoproteins (AAGP). The present findings suggest that the primary exometabolite released by L. donovani is a samll glycopeptide-like molecule; however, in media conditioned by growth of promastigotes and during isolation procedures, aggregation and/or degradation processes yield higher molecular weight substances which vary in size and physico-chemical properties.