Leishmania tropica and Leishmania mexicana: cross-immunity in mice

The effect of a previous or concurrent Leishmania tropica major infection on a L. mexicana infection was studied. Mice which were recovering from or had recovered from a L. tropica infection were found to be totally resistant to L. mexicana. Infection of mice already carrying a L. mexicana infection with L. tropica resulted in subsequent ulceration and eventual healing of the lesions caused by both Leishmania species. Mice infected with L. mexicana were found normally to be no more susceptible to L. tropica than untreated mice: Only when L. tropica infections were located in the region of a draining lymph node already serving a L. mexicana infection did lesions of the former parasite persist.     

Leishmania (L.) amazonensis: fusion between parasitophorous vacuoles in infected bone-marrow derived mouse macrophages

[Leishmania(L.)] amazonensis amastigotes reside in macrophages within spacious parasitophorous vacuoles (PVs) which may contain numerous parasites. After sporadic fusion events were detected by time-lapse cinemicrography, PV fusion was examined in two different models. In single infections, it was inferred from the reduction in PV numbers per cell. In a reinfection model, macrophages infected with unlabeled amastigotes were reinfected with GFP-transfected- or carboxyfluorescein diacetate succinimidyl ester-labeled parasites, and fusion was detected by the colocalization of labeled and unlabeled amastigotes in the same PVs. The main findings were: (1) as expected, fusion frequency increased with the multiplicity of infection; (2) most fusion events took place in the first 24h of infection or reinfection, prior to the multiplication of incoming parasites; (3) resident and incoming parasites multiplied at similar rates in fused PVs. The model should be useful in studies of parasite and host cell factors and mechanisms involved in PV fusogenicity.     

Leishmania tropica major: effect of paromomycin and pentamidine on polyamine levels in the skin of normal and infected mice

The polyamine content of the skin of BALB/c and C3H mice was determined at intervals, after injecting Leishmania tropica major. In BALB/c mice, putrescine and spermidine levels increased three- to seven-fold; in C3H mice, spontaneous recovery occurred after 3 weeks, accompanied by a reduction in putrescine and spermidine levels. Ornithine decarboxylase activity was negligible in normal, uninfected skin of both BALB/c and C3H mice, but increased steadily during infection. Treatment with drugs that inhibit the growth of leishmanial amastigotes in the skin of mice also reduced polyamine levels and ornithine decarboxylase activity of previously infected skin. There was a close correlation between the therapeutic activity of the drugs and their effect on polyamine content and synthesis. The aminoglycoside paromomycin, which was chemotherapeutically more effective than pentamidine, also had a greater effect on polyamine levels. S-adenosyl-L-Methionine decarboxylase activity in the skin of BALB/c and C3H mice was only slightly affected by the parasites. Polyamine levels and ornithine decarboxylase activity could possibly serve as means for measuring the growth of leishmanial parasites in skin and other tissues and as a measure of the efficacy of anti-leishmanial chemotherapeutics.     

Leishmania tropica: pathogenicity and in vitro macrophage function in strains of inbred mice.

Of seven strains of inbred mice and one hybrid that were infected intracutaneously with 5, 10, or 20 × 10⁶ active promastigotes of Leishmania tropica major, two strains (CBA/Ca and C₃H/He) recovered from the infection and their lesions healed within 3 to 5 months. The other strains, with the possible exception of C57B1/6 animals, remained infected, carrying large cutaneous ulcers throughout their lives. These included DBA/2, A/Jax, Balb/c, athymic nude mice of Balb/c origin (nu/nu) and the heterozygote Balb/c (nu⁺). The responses of C57B1/6 animals were of intermediate type with a tendency toward nonhealing at higher doses of the parasite. The cutaneous infection of athymic nude mice invariably gave rise to fulminating visceral infections and death. This condition was never observed in the other strains tested. Concanavalin A (Con A)-stimulated syngeneic or allogeneic lymphocytes of intact mice activated peritoneal macrophages of both healer and nonhealer mice, resulting in complete destruction of phagocytosed L. enriettii within 24 to 48 hr. The destruction of ingested L. tropica was confined to macrophages of healer mice and required 72 to 96 hr to reach completion. However, removal of Con A-stimulated lymphocytes from macrophage cultures and regular pulsing of the cells with a lymphokine-rich supernatant produced a state of sustained activation, resulting in destruction of L. tropica inside macrophages of both healer and nonhealer mice. The ability of Con A-stimulated lymphocytes of nonhealer animals to induce effective levels of activation in healer macrophages on one hand, and eventual destruction of L. tropica in macrophages of nonhealer mice under condition of sustained activation on the other, had indicated that so far as the in vitro situation is concerned, there is no inherent defect in lymphocytes or macrophages of nonhealer animals, although the threshold of activation necessary for killing of the parasite seems to be higher for cells of nonhealer origin.     

Leishmania tropica and Leishmania donovani: solid phase radioimmunoassay using leishmanial excreted factor

A radioimmunoassay for the quantitative determination of anti-leishmanial excreted factor (EF) antibody in rabbit sera was developed. The assay, using Leishmania tropica and Leishmania donovani promastigotes EF, purified by either extraction with phenol followed by fractionation on a Sephadex G-100 column or by the dissociation of EF antibody complexes, was shown to be sensitive and reproducible. Using monospecific anti-EF antibodies, levels of as low as 0.06-0.12 micrograms/ml of anti-EF IgG could be detected. The specificity of the assay was assessed by inhibition with homologous and heterologous EF. Only minor cross-reactivity with heterologous EF was observed, and as little as 2.5 micrograms/ml of EF could be detected. Sera from kala-azar patients showed only 1.8-3.1 times more anti-EF activity, as compared with uninfected controls. No specificity was observed with sera from kala-azar patients with regard to the type of EF used. Almost the same activity was obtained with both EF from L. tropica and L. donovani. No anti-EF antibodies were detected in sera from patients with cutaneous leishmaniasis.     

Effects of some biologically active compounds on phagosome-lysosome fusion in peritoneal macrophages of mice.

Effects of biologically active compounds bilirubin (BR), farmorubicin (FR), and chelerythrine (CR) on phagosome-lysome (P-L) fusion in mouse peritoneal macrophages were studied using fluorescent dye acridine orange as lysosomal labelling and yeast cells as target. It was found that all three compounds tested enhanced P-L fusion. To investigate mechanisms of these effects, changes in fluidity of rat liver lysosomal membranes under influence of BR, FR and CR were studied by measuring fluorescence intensity, lifetime, and polarization of DPH or TMA-DPH incorporated in isolated rat liver lysosomes. In order to characterize the cytoskeleton changes under the action of these biologically active compounds F-actin content in peritoneal macrophages of mice was determined. Our results demonstrate that BR action induces a decrease in DPH and TMA-DPH polarization, FR increases DPH and TMA-DPH polarization, and CR causes only an increase in TMA-DPH polarization in lysosomal membranes. All three compounds tested increase F-actin content in peritoneal macrophages. Thus, the effect of BR on P-L fusion is connected with increasing fluidity of lysosomal membranes and the cytoskeleton changes. The enhancement of P-L fusion under the action of FR and CR can most likely be explained by changes of the cytoskeleton state.     

Leishmania major: culture media, mouse strains, and promastigote virulence and infectivity

Promastigotes of Leishmania major were isolated from an infected mouse in two media, blood agar and Schneider's medium + 30% fetal calf serum, and maintained continuously for over 1 year. Infectivity studies in two strains of mice, outbred CD1 strain and inbred BALB/c strain, showed that promastigotes grown in Schneider's medium maintained infectivity to BALB/c mice throughout the period of cultivation. Infectivity to CD1 strain mice was progressively lost. Promastigotes grown in blood agar medium, however, lost infectivity to both strains of mice at a faster rate than promastigotes grown in Schneider's medium.     

Leishmania mexicana: Conversion of dihydroorotate to orotate in amastigotes and promastigotes

The specific activity of dihydroorotate dehydrogenase, catalysing the conversion of l-5,6-dihydroorotate (l-DHO) to orotate, in Leishmania mexicana mexicana was found to be 22.1 ± 3.5 nmole/hr/mg protein in the amastigote, and 28.7 ± 4.6 nmole/hr/mg protein in the promastigote. The enzyme was found to be soluble and to require molecular O₂ for activity in both forms of the parasite. Oxygen utilisation was not mediated through the mitochondrial cytochrome-containing respiratory chain, and phenazine methosulphate and ferricyanide could be used as electron acceptors by the enzyme in both aerobic and anaerobic conditions. The enzyme from both amastigote and promastigote had a pH optimum of 7.0, and the K_m values for l-DHO were 11.8 ± 4.9 and 2.3 ± 0.4 μM, respectively. The pyrimidine analogs 5-methylorotate (K_i = 8.8 μM) and 5-aminoorotate (K_i = 57 μM) were shown to be competitive inhibitors of the promastigote enzyme, as was the reaction product orotate (K_i = 10 μM).     

Salmonella typhi does not inhibit phagosome-lysosome fusion in human monocyte-derived macrophages

Abstract We examined phagosome-lysosome fusion in Salmonella typhi -infected human monocyte-derived macrophages and its relevance to the intracellular survival of this bacterium in vitro. S. typhi was found to survive and multiply in human monocyte-derived macrophages, whereas S. typhimurium was killed easily, indicating that the survival of Salmonella serovars is host-specific. Neither S. typhi nor S. typhimurium inhibited phagosome-lysosome fusion in human monocyte-derived macrophages. No difference between the phagosome-lysosome fusibilities of freshly prepared human monocytes and monocyte-derived macrophages was observed. These results suggest that S. typhi may survive by adapting to the conditions within fused phagolysosomes of human monocyte-derived macrophages.     

Plasmodium yoelii: blood oxygen and brain function in the infected mouse

The carriage of oxygen by the blood and the in vivo response of the brain were investigated in mice infected with a lethal strain of Plasmodium yoelii. All mice with parasitaemia exceeding 70% were severely anaemic (Hb 3.5 +/- 1.8 g/dl; mean +/- 1 SD), acidotic (blood pH 7.04 +/- 0.06) and hypoglycaemic (blood glucose 0.6 +/- 0.76 mumol/ml). The oxyhaemoglobin dissociation curve (ODC) of blood from heavily infected mice was shifted right as compared to controls, but the increase in p50 was less than expected from the accompanying acidosis. The reduced shift right was due to a decrease in the 2,3-DPG/Hb ratio in infected animals (0.72 +/- 0.12, n = 17 vs 1.10 +/- 0.09, n = 12 in controls). Despite the severity of terminal infection, the cerebral pH and the relative steady-state concentrations of PCr, ATP and Pi measured in vivo by nuclear magnetic resonance (31P NMR) were normal. Alterations in brain energy status and pH cannot account for cerebral signs or death in this proposed mouse model of cerebral malaria.     

Trypanosoma cruzi, Leishmania donovani, and L. mexicana: extract factor that lyses mammalian cells.

Cell-free extracts of Trypanosoma cruzi, Leishmania donovani, and L. mexicana, cultivated in a medium supplemented with 5% fetal calf serum, contain a factor that induces lysis of mammalian red blood cells and Vero cells. All the lytic activity was found in the insoluble fraction of parasite extracts obtained after centrifugation at 100,000g for 2 hr. The lytic agent is pronase, trypsin, and temperature resistant. The optimum pH of the lytic effect is pH 6.5. Normal red blood cells of several mammalian species had different sensitivities to the lytic agent. The lipid phase of T. cruzi extract contains the total lytic activity. Albumins of different animal species at 1 mg/ml, completely inhibit the lytic activity of parasite extracts.     

Plasmodium falciparum: Drug sensitivity in vitro of isolates before and after adaptation to continuous culture

Fifteen strains of Plasmodium falciparum have been cultivated since 1979 using the Trager and Jensen method of continuous culture on isolates from malaria patients. One hundred and two drug sensitivity studies have been carried out on these strains using a semimicro test. Three isolates, initially resistant to chloroquine, adapted rapidly to in vitro cultivation and maintained their high level of resistance (ED₅₀ above 660 nM). Eleven isolates, initially chloroquine sensitive (ED₅₀ under 90 nM) became resistant to this drug (ED₅₀ = 190 to 1950 nM) after the 2–15 weeks required for their adaptation to continuous culture. The resistance of these strains never decreased during the following 15 months of continuous culture. The sensitivity to quinine varied initially from one strain to another (ED₅₀= 160 to 660 nM) and fluctuated during cultivation in the ratio of 1, 3.5 for a given strain. The sensitivity of mefloquine remained high for all strains (ED₅₀ under 150 nM) but one (ED₅₀ = 560 nM). These results suggest that there might be a relationship between in vitro adaptation to culture of P. falciparum by the Trager-Jensen method and a chloroquine-resistant characteristic of the strain. There is the possibility of the emergence of a drug-resistant subpopulation or of changes in the metabolic pathways.     

Specific inhibition of phagosome-lysosome fusion in murine macrophages mediated by Salmonella typhimurium infection

Phagosome-lysosome fusion in murine macrophages infected with S. typhimurium LT2 or S. typhi 1079 was investigated. Fusion of phagosome containing S. typhimurium LT2 with lysosome was markedly impaired, whereas S. typhi 1079 did not inhibit phagosome-lysosome fusion in murine macrophages. A similar inhibition of fusion was observed with LPS-deficient mutants of S. typhimurium LT2, suggesting that O-antigens do not contribute to the inhibition of fusion. Phagosome-lysosome fusion in macrophages after ingestion of UV-killed S. typhimurium LT2 was much greater than that of live bacteria. Furthermore, treatment of S. typhimurium LT2 with streptomycin, an inhibitor of bacterial protein synthesis, caused an increase in the extent of phagosome-lysosome fusion. Therefore protein synthesis in live bacteria is probably required for the inhibition of phagosome-lysosome fusion. These results suggest that phagosome-lysosome fusion in murine macrophages is impaired by some product(s) of viable S. typhimurium LT2.     

Leishmania donovani: inhibition of phagosomal maturation is rescued by nitric oxide in macrophages

Leishmania donovani promastigotes, the causative agent of visceral leishmaniasis, survive inside macrophages by inhibiting phagosomal maturation. The main surface glycoconjugate on promastigotes, lipophosphoglycan (LPG), is crucial for parasite survival. LPG has several detrimental effects on macrophage function, including inhibition of periphagosomal filamentous actin (F-actin) breakdown during phagosomal maturation. However, in RAW 264.7 macrophages pre-stimulated with lipopolysaccharide (LPS) and interferon gamma (IFNgamma), known to up-regulate inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production, L. donovani promastigotes are unable to inhibit periphagosomal F-actin breakdown and phagosomal maturation proceeds normally. Moreover, the iNOS inhibitor aminoguanidine, blocked the positive effects of LPS/IFNgamma suggesting that NO is a key player in F-actin remodeling. In conclusion, production of NO by stimulated macrophages seems to allow phagosomal maturation following uptake of L. donovani promastigotes, suggesting a novel mechanism whereby NO facilitates killing of an intracellular pathogen.     

Plasmodium berghei: modification of sialic acid on red cells from infected mouse blood

The surface membrane glycoproteins of normal mouse erythrocytes can be labeled by oxidation with either periodate or galactose oxidase in the presence of neuraminidase, followed by reduction with NaB³H₄. Without neuraminidase there is little galactose oxidase-catalyzed labeling of protein. Analysis of labeled proteins by SDS-polyacrylamide gel electrophoresis showed that both methods labeled the same set of glycoproteins. Plasmodium berghei infection dramatically reduced the sialoglycoprotein labeling of red blood cells from infected blood using the periodate/NaB³H₄ method. Provided neuraminidase was present, labeling by the galactose oxidase method gave identical results to normal erythrocytes. We conclude that the glycoprotein sialic acid of uninfected as well as infected red cells is modified during infection such that it is refractory to periodate oxidation. Acylation of the exocyclic hydroxyls of sialic acid is suggested to account for this. Lectin binding and cell agglutination experiments using Limulin, soybean and wheatgerm lectins, and concanavalin A confirmed and extended these observations. The possible implications of these results with regard to anemia induced by malaria are briefly discussed.     

Leishmania donovani: promastigote--macrophage surface interactions in vitro.

The mechanism by which promastigotes of Leismania donovani enter hamster peritoneal macrophages was studied in vitro by light and electron microscopy. Quantitative light microscope studies showed a time-dependent increase of intracellular parasites, which had no preferable orientation during entry. Scanning and transmission electron microscopy revealed striking host-parasite surface interactions marked by the formation of whorled pseudopodia around the promastigotes in the early stage and engulfment of the parasites akin to normal phagocytosis in the later phase. Early host-parasite interactions were categorized quantitatively by scanning electron microscopy into several types, of which “head-first entry” and “tail-first entry” were approximately equal in frequency of occurrence, confirming light microscope observations. Cytochalasin B at 10 μg/ml prevented the intracellular entry of the parasites and the formation of macrophage-originated pseudopodia normally seen to seize the promastigotes. Killed, but morphologically intact, promastigotes were poorly taken up by macrophages and lacked certain types of interactions normally encountered with macrophage pseudopodia. Motility of promastigotes and their affinity to the surface of macrophages are suggested as elements of importance which parasites contribute to aid the process of their entry. The above results indicate that promastigotes of L. donovani depend on phagocytic activity of macrophages to gain intracellular entrance, but parasite-specific activities and/or properties may also play a role. It is suggested that “facilitated phagocytosis” may be used to describe this unique type of endocytosis associated with leishmania-macrophage interactions.     

Effect of γ-interferon on phagosome-lysosome fusion in Salmonella typhimurium-infected murine macrophages

Abstract Effect of recombinant γ-interferon (rIFN-γ) on phagosome-lysosome fusion in Salmonella typhimurium -infected murine macrophages was examined. rIFN-γ enhanced phagosome-lysosome fusion in macrophages infected with S. typhimurium in a dose-dependent manner, and over a range of 10² to 10³ U/ml of rIFN-γ exhibited maximum phagosome-lysosome fusion, although phagocytosis was slightly decreased. The enhancement of phagosome-lysosome fusion occured > 3 h post-treatment with rIFN-γ. Furthermore, the macrophage activation for phagosome-lysosome fusion was found to persist for 4 days even when rIFN-γ had been removed. These results demonstrate that INF-γ may serve as a mediator for the activation of phagosome-lysosome fusion in murine macrophages.     

Depletion of secondary lysosomes in mouse macrophages infected with Leishmania mexicana amazonensis: a cytochemical study

Leishmania amastigotes lodge and multiply within parasitophorous vacuoles, which can fuse with secondary lysosomes of the host macrophages. This study examines the effect of infection with amastigotes of L. mexicana amazonensis on the secondary lysosomes of mouse macrophage cultures. The cultures were stained for the activities of two lysosomal enzyme markers, acid phosphatase and arylsulfatase, and the light microscopic observations were supplemented by electron microscopy. Nearly all noninfected macrophages contained numerous stained secondary lysosomes. The number of such lysosomes was markedly reduced 24 h postinfection, and the reduction persisted for at least 10 days. Stained secondary lysosomes reappeared after the amastigotes were destroyed by exposure of the cultures to phenazine methosulfate or by placing them at 37.5 degrees C. The depletion of lysosomes shown by cytochemical methods may reflect a high rate of fusion of the lysosomes with the parasitophorous vacuoles, exceeding the rate of formation of new secondary lysosomes. Alternatively, the parasites may inhibit the synthesis of lysosomal hydrolases, or the assembly or formation of primary or secondary lysosomes.     

Trypanosoma musculi: Passive hemagglutination technique to measure antibody in mice

A passive hemagglutination assay was developed to measure Trypanosoma musculi-specific antibody in mice. Indicator-erythrocyte donor mice received 550 rad ⁶⁰Co 24 hr before intraperitoneal injection of 3 × 10⁴T. musculi. T. musculi antigen-coated erythrocytes were obtained from these mice on Day 9 postinfection. T. musculi antigen-coated erythrocytes obtained in this manner were used as indicator erythrocytes in a passive hemagglutination procedure. Serum from hyperimmunized mice (three consecutive infections at 21-day intervals) gave titers as high as 1:1024. Titers of 1:256 and 1:512 were obtained from singly infected mice on Days 18 and 28 postinfection, respectively. In marked contrast, nude mice infected with T. musculi did not produce a detectable agglutinating antibody response. Erythrocytes obtained from either irradiated (550 rad ⁶⁰Co) uninfected mice, nonirradiated infected mice, or normal mice did not agglutinate when combined with any of the sera tested. These data suggest the usefulness of this passive hemagglutination assay for the measurement of antibody to T. musculi in the serum of infected mice.     

Leishmania mexicana mexicana: attachment and uptake of promastigotes to and by macrophages in vitro

Promastigotes of Leishmania mexicana mexicana attach to mouse macrophages in vitro in the absence of serum by a wheat germ agglutinin-like ligand on the surface of the promastigote that binds to the N-acetyl glucosamine moiety of a receptor on the surface of the macrophage. The binding is temperature dependent, and the macrophage receptor is trypsin, cytochalasin B, and is assisted or inhibited as for attachment. Treatment of promastigotes with proteolytic enzymes uncovers a receptor for a serum component that binds strongly to a mouse macrophage receptor in vitro. The strain of mice donating the macrophages had little effect upon attachment and uptake except that A strain mouse macrophages attached fewer promastigotes in 10 min than those of outbred mice, but took up as many promastigotes over 90 min as those of outbred mice. Low responder Biozzi mouse macrophages took up more promastigotes than high responder Biozzi mouse macrophages. Normal unheated human, rabbit, and guinea pig sera lysed promastigotes and so inhibited their attachment to macrophages in vitro. Unheated immune serum showed an enhanced inhibition of attachment. Heated normal serum allowed attachment and uptake, while promastigotes treated with heated immune serum showed enhanced attachment to and uptake by macrophages. Treatment of macrophages in vitro with immune serum enhanced their ability to attach promastigotes and to engulf them. Repeated 90-min exposures of a population of promastigotes to uptake by mouse macrophages in vitro did not deplete the population of any sub-population more likely to be taken by macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)