Mauricio V. Londner

Action of leishmanial excreted factor (EF) on human lymphocyte blast transformation

Summary. The effect of Leishmania tropica major excreted factor (EF) on human immune and normal mononuclear peripheral blood cells has been studied. The response of lymphocytes to stimulation either specifically with leishmanial antigens or non‐specifically with phytohemagglutinin (PHA) in the presence of EF was tested by the uptake of 3[H]thymidine. The results showed that EF inhibits the response of cells from immune donors to leishmanial antigens and from normal donors to PHA or PPD. Adherent and non‐adherent cells were separated and the effect of EF on both populations was analysed. The results showed that EF inhibited blast transformation if both EF and antigen were presented to each of the separate populations. The inhibition of the adherent cells (mainly monocytes) was more marked than the inhibition of the nonadherent population (mainly lymphocytes).

A simple method for separation of uninfected erythrocytes from those infected with Plasmodium berghei and for isolation of artificially released parasites.

Rat erythrocytes infected withPlasmodium berghei were disrupted by gentle passage of Coneanavalin A (ConA) agglutinated cells through a 100 mesh stainless steel grid. The free parasites were separated from cell debris, unbroken infected cells, and from uninfected rat erythrocytes on a Percoll gradient. The free parasites remained morphologically intact, metabolically active, and infective to mice.The parasites were observed by light and electron microscopy. The incorporation of3H-isoleucine and3H-hypoxanthine was compared in intact and infected cells, and the infectivity was measured by the injection of parasites into susceptible mice. It seems that the combination of the two techniques used, produces a high yield of intact free parasites suitable for physiological or immunological studies.

Leishmania major: glycolipid antigens recognized by immune human sera

Extraction of whole promastigotes with a mixture of hexane-isopropanol yielded two carbohydrate-lipid fractions immunologically active against immune sera from patients with cutaneous leishmaniasis (CL): CLF-1 and CLF-2. Thin layer chromatography (TLC) separated both fractions into eight bands labeled A-H. Four of these bands, Rf 0.19, 0.25, 0.39 and 0.48 (A, B, C and E, respectively) were recognized by antibody from patients with CL in a solid phase radioimmunoassay. Antigens were also detected by autoradiography after immunoblotting of TLC. Compound A could be labeled biosynthetically with [3H]oleic acid, [14C]galactose, [14C]mannose, [14C]glucose and [32P]phosphate. B incorporated [14C]galactose, [14C]mannose, [14C]glucose and [14C]myo-inositol. C was labeled with [14C]galactose and [14C]mannose, while E incorporated [14C]glucose, [14C]mannose, [3H]oleic acid and [14C]myoinositol. Two antigens (A and B) could be also labeled on the surface of living promastigotes using galactose oxidase and [3H]sodium borohydride. Experimental data showed that CLF-1 and CLF-2, both carbohydrate-containing fractions, had different chromatographic patterns from excreted factor (EF), a polysaccharide antigen from Leishmania. The present study characterizes glycolipid molecules from L. major promastigotes, able to stimulate the immune system from patients with CL.

Immunoperoxidase method of identification ofLeishmania in routinely prepared histological sections

An indirect immunoperoxidase technique was applied for identification ofleishmania in routinely prepared histological sections. Paraffin embedded, formalin-fixed slide preparations of skin (1 case), bone marrow (2 cases) and lymph node (1 case) were examined. The tissues were obtained from one patient with cutaneous leishmaniasis and from three patients with visceral leishmaniasis. Specific rabbit anti-sera againstL. donovani, L. tropica andL. mexicana were used as primary reagents. Positive controls were performed simultaneously and includedL. tropica cultures in blood-agar (NNN media) - free promastigotes and amastigotes within macrophages. Strongly positive brown staining was localized specifically in Leishman-Donovan (LD) bodies only. This method increases the probability of microscopic diagnosis of leishmaniasis and helps to prevent confusion ofLeishmania with other infective agents in histological sections.

Lipid antigens derived from erythrocytes infected withPlasmodium berghei

Lipids were extracted from red blood cells infected withPlasmodium berghei, from the membranes of infected red cells and from free parasites. A radioimmunoassay was used to detect antibodies to these lipids in sera from convalescent and immune rats. Most of the antigenic activity could be attributed to the parasite although some activity was found in lipids isolated from the membranes of infected red blood cells. Absorption studies showed that the binding was specific for malarial lipid antigens. Immune sera showed no cross-reactivity with lipids from red blood cells of non-infected rats. However, sera from non-infected control rats showed low levels of cross-reactivity with the parasitized red cell-derived lipids. Levels of anti-lipid antibodies were directly correlated with the progress of the infection. The highest antibody level occurred when the parasitaemia reached zero. The malarial lipids had no effect on lymphoblast transformation of immune splenocytes in vitro. However, liposomes prepared from either malarial or non-specific lipids caused an increased response to antigen by the blast cells.

The cellular and humoral immune response in subjects vaccinated against cutaneous leishmaniasis using Leishmania tropica major promastigotes

Summary In vitro lymphocyte transformation was studied in 24 subjects 12 months after vaccination with live Leishmania tropica major vaccine, in 10 normal control subjects and in three controls residing in an endemic area. The vaccines had lesions in various stages of clinical development. Lymphocytes from all the subjects were studied for their response to stimulation with L. tropica major promastigotes. Lymphocytes of vaccinated subjects responded to low concentrations of antigen whereas the lymphocyte response of the controls tended to be depressed by the same concentration of the antigen. Serum from each subject was subjected to a study of the humoral antibody titre against L. tropica major and L. donovani using indirect immunofluorescence. A humoral response to L. donovani was present in a majority of vaccinees who had developed a positive lesion whereas no such response was present in any of the controls. The data suggest that high humoral responses were accompanied by relatively low cell‐mediated responses and vice versa. No significant humoral response to L. tropica major could be demonstrated in any of the subjects. A combination of both the cell‐mediated and humoral mechanisms may participate in the immune response although their usefulness in the assessment of the protectivity of leishmania vaccines has not been established.

Leishmania major: Solid phase radioimmunoassay for antibody detection in human cutaneous leishmaniasis

A radioimmunoassay for the quantitative determination of antileishmanial antibody in sera from patients suffering from cutaneous leishmaniasis was developed. The assay, using as antigen either the soluble fraction from freeze-thawed sonicated Leishmania major (LRC-L137) promastigotes or a carbohydrate-lipid containing fraction obtained by extraction with hexane-isopropanol, was shown to be sensitive and reproducible. The sera of 95 patients were examined. These were from patients with cutaneous leishmaniasis (26 from the Jordan Valley and 13 from Sinai), kala-azar (9), malaria (24), schistosomiasis (10), toxoplasmosis (5), and leprosy (8); controls were 37 normal human sera. No significant antigen dependent differences were observed using sera from cutaneous leishmaniasis patients, although differences in the immunological response were observed between the two populations of these patients. Antileishmanial activity was not detected in sera from patients with malaria, schistosomiasis, or toxoplasmosis. Although sera from leprosy patients crossreacted with the carbohydrate-lipid containing fraction, it was nevertheless more strain specific than freeze thawed sonicated L. major.

Structural analysis of a glycosylphosphatidylinositol glycolipid of Leishmania donovani.

AbstractA glycosylphosphatidylinositol (GPI) glycolipid antigen recognized by sera from patients with visceral leishmaniasis was isolated fromLeishmania donovani promastigotes. The carbohydrate moiety was cleaved from the lipid part by digestion with specific phosphatidylinositol phospholipase C. After separation, structural analysis was carried out on the phosphorylated inositol oligosaccharide and the alkylacyl glycerol. The following major structures were found:

Cell-mediated immunity in rats injected with an antimalaria T-cell line☆

\begin{gathered} Man\alpha 1 - 2Man\alpha 1 \hfill \\ \begin{array}{*{20}c} \backslash \\ 6 \\ { Man\alpha } \\ 3 \\ {Man\alpha 1/} \\ \end{array} 1 - 4GlcN\alpha 1 - inositol - O - \begin{array}{*{20}c} O \\ \parallel \\ P \\ | \\ {O^ - } \\ \end{array} - O - _{\begin{array}{*{20}c} {CH_2 } \\ | \\ {CH} \\ | \\ {CH_2 } \\ \end{array} } \begin{array}{*{20}c} {{\text{ - O - CO - (CH}}_{\text{2}} {\text{)}}_n {\text{ - CH}}_{\text{3}} } \\ {{\text{ - O - (CH}}_{\text{2}} {\text{)}}_m {\text{ - CH}}_{\text{3}} } \\ \end{array} \hfill \\ n = 16,18,20,22,24,26 m = 16,17,18,21 \hfill \\ \end{gathered}