Lipo- and apolipoproteins in a population survey subset in MONICA-Israel.

Lipo- and apolipoproteins in a population survey subset in MONICA-Israel. A group of 169 healthy men and women selected at random from the population survey of the MONICA-Israel Project were screened for baseline values of serum lipoproteins and apolipoprotein A-I and B concentrations. The apolipoprotein measurements were obtained using a recently developed immunoturbidimetric assay. The study population was subdivided into age groups of 25-44 years and 45-64 years. It was found that women aged 45-64 years had significantly higher levels of apolipoprotein A-I, apolipoprotein B, total cholesterol (TC), LDL cholesterol (LDL-c) and a lower ratio of HDL-cholesterol (HDL-c) to TC (HDL-c%) than did their younger counterparts. These parameters did not differ significantly between younger and older men. The results suggest that the HDL of women over the age of 45 years contains relatively less cholesterol and more protein than the HDL of younger women. Further studies will show to what degree apolipoprotein concentrations, and their relation to lipoproteins will sharpen the assessment of coronary risk.

Lipid and lipopolysaccharide-like antigens of Leishmania promastigotes.

Extraction of whole promastigotes of Leishmania tropica major and L. donovani with a mixture of hexane and isopropanol (3:2) yielded three fractions containing immunological activity: lipids, where the activity was determined by radioimmunoassay; a lipopolysaccharide-like (LPS-like), water-soluble precipitate, where activity was determined both by radioimmunoassay and double gel diffusion, and the phenol: water extract of the lipid-free promastigotes, where activity was followed by double gel diffusion. The use of a solid state, lipid-based radioimmunoassay for detection of leishmanial antigens provided a sensitive measure of their activity with a considerable degree of species and serotype specificity. We found antibodies to leishmanial lipids in sera from immunized rabbits, convalescent mice, and human patients with confirmed cases of cutaneous leishmaniasis or kala azar. There was very little activity in normal human or animal sera. Analysis by SDS-polyacrylamide gel electrophoresis of fractions from promastigotes surface-labeled with galactose oxidase and sodium borotritiate and preliminary immunochemical characterization of the LPS-like antigen showed that it contained galactose, but otherwise differed immunologically and chemically from excreted factor (EF), the best characterized leishmanial antigen.

Leishmania tropica: protective response in C3H mice vaccinated with excreted factor crosslinked with the synthetic adjuvant, muramyl dipeptide.

Excreted factor, an immunosuppressive, acidic polysaccharide released by promastigotes of Leishmania tropica major in culture, was chemically crosslinked to the synthetic adjuvant muramyl dipeptide via the bifunctional imidoester dimethyladipimidate and poly-L-lysine. This conjugate, an uncrosslinked mixture of the components, or each of the components alone were injected one to three times into different groups of 8- to 12-week-old C3H mice. The mice were challenged 2 weeks after the last injection with 2 X 10(6) promastigotes of L. t. major in the base of the tail. For the next 5 weeks, the animals were monitored for number of parasites and size of the lesion which developed at the site of the challenge. Mice receiving one intraperitoneal injection of the conjugate were partially protected against challenge. Treated animals had higher initial parasite numbers but showed a more rapid clearing of the parasites. Furthermore, the treated animals developed smaller lesions that healed quicker than did those of the control groups. Multiple injections, or injection into a footpad, rather than intraperitoneally, reduced the ability to elicit a protective response. On the other hand, muramyl dipeptide injected into a footpad was partially protective. Antibody production to excreted factor, which was measured by indirect hemagglutination of sensitized erythrocytes, was detected after challenge in mice which had received conjugate or conjugate components. A delayed hypersensitivity reaction (measured by skin testing) was not detected in any of the groups prior to challenge.

Surface reaction of Leishmania. III. Ulex europaeus II lectin affinity for excreted factor (EF) serotype A strains.

Eukaryotic parasites, including species of Leishmania, acquire or synthesize carbohydrate moieties similar to human blood group antigens. Leishmanial strains separate into three serotypes: A, B and AB. All strains containing the A component are agglutinated by Ulex europaeus lectin. Inhibition by haptene sugar suggests that a Ulex II-like receptor is involved. Organic solvents, but not protease treatment, remove its reactivity, suggesting that the receptor is a glycolipid.

Lipid antigens derived from erythrocytes infected with Plasmodium berghei.

Lipids were extracted from red blood cells infected with Plasmodium 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.

Monoclonal antibodies for serotyping Leishmania strains.

Mouse monoclonal antibodies raised against Leishmania tropica major were found to precipitate with the excreted factor produced by this and other leishmanial species. We suggest that a classification system for Leishmania, based on selective precipitation reactions between monoclonal antibodies and the excreted factor, would remove many ambiguities that currently exist.

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

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 non-adherent population (mainly lymphocytes).

Leishmanial excreted factors (EFs): purification by affinity chromatography.

Leishmania species grown in culture excrete a polyanionic, carbohydrate-rich factor (EF) which binds to antibodies produced in rabbits against the parent Leishmania species. EF, previously purified by physical and chemical methods, was purified by affinity chromatography on a Ricinus lectin column. The purified samples were characterised and analysed. The results show a notable proportion of galactose in EF and clarify the reasons for its polyanionic properties. Heterogenicity of EF is demonstrated and discussed.

Effect of leishmanial excreted factor on the activities of adenylate cyclase from hamster liver and Leishmania tropica.

The carbohydrate segment of the excreted factor of culture cells from L. tropica and L. donovani was shown to interact with the adenylate cyclase from hamster liver. At a concentration of 1 mg/ml the excreted factor inhibited the stimulated activity of the mammalian enzyme by about 50%, independent of the activation by glucagon, 5'-guanylylimidodiphosphate and forskolin, respectively. In contrast to the adenylate cyclase system from hamster liver the enzyme from L. tropica-culture cells was not stimulated by addition of glucagon, 5'-guanylylimidodiphosphate and forskolin. In addition, the activity of the adenylate cyclase from L. tropica was not affected by addition of its endogenous excreted factor.

Identification of galactose as the immunodominant sugar of leishmanial excreted factor and subsequent labeling with galactose oxidase and sodium boro[3H]hydride.

Inhibition by low-molecular-weight sugars of precipitin line formation between a polysaccharide (EF) excreted by Leishmania tropica subsp. major, Leishmania enriettii, and rabbit antileishmanial antibodies on double gel diffusion plates revealed that galactose residues, possibly as components of lactosyl groups, were the critical immunodominant sugars mediating antibody recognition of EF. The galactose residues of the EF of L. tropica subsp. major were specifically labeled with tritium via galactose oxidase and sodium boro[3H]hydride. The radioactive EF had an apparent molecular weight of about 85,000 on sodium dodecyl sulfate-polyacrylamide gels and was precipitated by antileishmanial antibodies as well as Ricinus communis lectins I and II (galactose specific). Lectins specific for glucose-mannose residues, fucose, N-acetylglucosamine, and N-acetylgalactosamine did not precipitate the labeled EF. Treatment of [3H]EF with proteolytic (trypsin, papain, protease) or glycosidic (alpha-amylase, beta-galactosidase) enzymes had no effect on either the electrophoretic pattern of the material or on its recognition by antileishmanial antibodies or R. communis lectin. This resistance to enzyme activity suggests that EF may be a useful marker for the presence of the parasite in vivo if it can be detected in minute quantities.

Biochemistry of parasites.

Parasite biochemistry is a field growing in parallel with the new surge of interest in tropical diseases. Whereas previously parasitologists have been required to adopt biochemical methodology in order to stay abreast of developments, today we find a new phenomenon: biochemists abandoning their classical systems (E. coli, red blood cells, etc.) to work on parasites. The 13th annual meeting of the Federation of European Biochemical Societies (FEBS) held in Jerusalem in August 1980 presented the perfect opportunity to summarize work done by parasite biochemists and to introduce this field to workers in classical biochemistry.

Coagglutination and indirect hemagglutination in the detection of an excreted immunologically active substance from Leishmania.

The methods of coagglutination and indirect hemagglutination were used to detect the production of the immunologically active excreted factor (EF) of Leishmania. Staphylococci, rich in protein A and sensitized with specific anti-Leishmania antibodies, coagglutinated with supernatant fractions of cultures, thus enabling continuous monitoring of the excretion of EF by multiplying parasites. Papain-treated human red blood cells, sensitized with crude or purified EF, also agglutinated with the coagglutination reagent. The sensitized papain-treated red blood cells may be employed in indirect hemagglutination to detect specific antibodies to Leishmania in rabbit and human sera. As the EF is specific for each Leishmania serotype group, coagglutination and indirect hemagglutination offer the possibility of rapid, easy, sensitive and specific diagnostic tools in the determinations of both antigen and antibody in specimens from suspected cases of leishmaniasis.

Leishmanial excreted factor: protein-bound and free forms from promastigote cultures of Leishmania tropica and Leishmania donovani.

Leishmania spp. growing in culture produce an immunologically active substance called excreted factor (EF), which precipitates antibodies raised against intact cells and has been implicated as the conditioning agent for parasite infection of host macrophages. An improved method for isolation of the material is described, based on Sephadex column chromatography of growth medium which had been boiled at pH 5.0. This procedure allows the detection of differences among the EF molecules of different species, and it overcomes previous shortcomings through the monitoring of immunological activity throughout. Analysis of the products of this procedure revealed that EFs from Leishmania tropica and Leishmania donovani share a common carrier protein, identified as rabbit serum albumin, and are chemically quite similar. Growth medium from L. tropica boiled at acidic pH contains primarily an EF-albumin complex of 75,000 molecular weight. Treated growth medium from L. donovani, on the other hand, contains both the albumin complex and a smaller molecule (less than 27,000 molecular weight) that is not associated with rabbit protein. This material accounts for nearly 20% of the EF of one L. donovani strain, but constitutes only a minute fraction of L. tropica EF. Treatment of the EF-albumin complex with trichloroacetic acid separates the molecule into two major subunits, one having a molecular weight of about 61,000 (without anti-Leishmania activity) and the other having a molecular weight of about 18,000 (with no anti-rabbit activity). The protein-free EF of L. tropica differs from that released by trichloroacetic acid extraction in that it is capable of precipitating antisera of nonhomologous serotypes, whereas the albumin complex and the trichloroacetic acid-treated EF fragment are not. EFs from both species display pH-dependent affinity for certain lectins.

Cholesterol distribution and movement in the Mycoplasma gallisepticum cell membrane.

The time course and extent of transfer of [14C]-cholesterol from resting Mycoplasma gallisepticum cells or membrane preparations to high-density lipoproteins were studied. More than 90% of the total cholesterol in isolated, unsealed membrane preparations was exchanged in a single kinetic process. In intact cells, however, cholesterol exists in two different environments. Cholesterol in one environment, representing approximately 50% of the total unesterified cholesterol, is readily exchanged with the cholesterol of high-density lipoproteins, with a half-time of about 4 h at 37 degrees C. The rate of exchange of [14C]cholesterol from the other environment was exceedingly slow, with a half-time of about 18 days. The fraction of the total cholesterol in the readily exchangeable cholesterol pool in intact cells increased somewhat upon aging of the culture. Electron spin resonance spectra of nitroxide-labeled stearic acids incorporated into membranes of M. gallisepticum cells indicated increased rigidity at the late exponential phase of growth. These results suggest that cholesterol is present in approximately equal concentrations on both surfaces of the M. gallisepticum membrane and that in resting cells the rate of movement of cholesterol molecules from the inner to outer halves of the lipid bilayer is exceedingly slow or nonexistent.