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E Handman

Publications and source records attributed to E Handman.

At least 91 records · Page 5Linked to original sources
PubMed1987

Lipophosphoglycan of Leishmania major that vaccinates against cutaneous leishmaniasis contains an alkylglycerophosphoinositol lipid anchor.

The major cell surface glycoconjugate of Leishmania major, a putative parasite receptor for macrophages, is a lipophosphoglycan containing 81.6% (wt/wt) carbohydrate, 17.0% (wt/wt) phosphate, and 1.4% (wt/wt) lipid. It has been purified to homogeneity by hydrophobic chromatography and consists of a polydisperse family of molecules with Mr 5000-40,000. It contains galactose, mannose, glucose, arabinose, glucosamine, and inositol in the molar ratio of 51:21:5:6:1:1. The lipophosphoglycan has a complex structure, consisting mainly of tri- and tetrasaccharide units linked by phosphodiester bonds, which are cleaved by HF hydrolysis. The phosphate groups are located on the 6-hydroxyl of both galactose and mannose residues. The lipophosphoglycan is anchored to the parasite surface by a 1-O-alkyl-sn-glycero-3-phosphoinositol moiety. This conclusion is supported by analysis of the products of nitrous acid deamination, HF hydrolysis, and Staphylococcus aureus phosphatidylinositol specific-phospholipase C treatment. The 24:0 and 26:0 alkyl chains accounted for 93% of the ether-linked fatty acids in the lipid anchor. The results are also consistent with a glycosidic linkage between the inositol and a non-N-acetylated glucosamine residue. The lipophosphoglycan membrane anchor shares limited structural homology with the glycosylphosphatidylinositol anchors of several eukaryotic proteins, indicating that this type of membrane anchor is not limited to proteins. Vaccination of mice with the purified L. major lipophosphoglycan in liposomes induced resistance against cutaneous leishmaniasis.

Animals↗
PubMed1987

Leishmania major: a very sensitive dot-blot ELISA for detection of parasites in cutaneous lesions.

There is a need for accurate, rapid and early diagnosis of leishmaniasis, which would distinguish between the benign and severe forms of the disease. We have used a monoclonal antibody directed to a polymorphic, species-specific antigen in diagnostic assays for leishmaniasis. The dot-blot enzyme-linked immunosorbent assay described here can detect as few as 300 culture promastigotes and 20,000 amastigotes of Leishmania major with no cross-reaction with other species and no background from skin macrophages or other cells. This level of sensitivity is sufficient to detect parasite antigen aspirated in a few microliters of liquid from small lesions in mice. This assay could form the basis for a sensitive, rapid and inexpensive dip-stick test for large-scale use for the diagnosis and epidemiology of cutaneous leishmaniasis.

Animals↗
PubMed1986

Passive transfer of Leishmania lipopolysaccharide confers parasite survival in macrophages.

Infection of macrophages by the intracellular protozoan parasite Leishmania involves specific attachment to the host membrane, followed by phagocytosis and intracellular survival and growth. Two parasite molecules have been implicated in the attachment event: Leishmania lipopolysaccharide (L-LPS) and a glycoprotein (gp63). This study was designed to clarify the role of L-LPS in infection and the stage in the process of infection at which it operates. We have recently identified a Leishmania major strain (LRC-L119) which lacks the L-LPS molecule and is not infective for hamsters or mice. This parasite was isolated from a gerbil in Kenya and was identified phenotypically as L. major by isoenzyme and fatty acid analysis. In this study we have confirmed at the genotype level that LRC-L119 is L. major by analyzing and comparing the organization of cloned DNA sequences in the genome of different strains of L. major. Here we show that LRC-L119 promastigotes are phagocytosed rapidly by macrophages in vitro, but in contrast to virulent strains of L. major, they are then killed over a period of 18 hr. In addition, we show that transfer of purified L-LPS from a virulent clone of L. major (V121) into LRC-L119 promastigotes confers on them the ability to survive in macrophages in vitro.

Animals↗
PubMed1986

The glycoconjugate derived from a Leishmania major receptor for macrophages is a suppressogenic, disease-promoting antigen in murine cutaneous leishmaniasis.

Lymphoid cells from genetically-susceptible BALB/c mice immunized against a glycoconjugate of the protozoan parasite, Leishmania major, promote chronic cutaneous disease in BALB/c nude mice. This cell population therefore differs from cells harvested from non-immunized BALB/c mice that are known to be potent mediators of protection against cutaneous leishmaniasis in minimally-reconstituted, syngeneic nude mice. The glycoconjugate when injected into genetically-resistant C57BL/6 mice will increase the size and persistence of cutaneous lesions. Recent studies have established that the water soluble glycoconjugate is derived from a membrane-bound glycolipid that is a receptor used by the parasite in the attachment to macrophages. This glycolipid can protectively immunize mice against cutaneous leishmaniasis. Identification of a vaccinating glycolipid antigen and a suppressogenic component derived from it will greatly facilitate analysis of disease-promoting and resistance-promoting immunity in cutaneous leishmaniasis. However, the fact that a host-protective antigen contains a disease-promoting component may militate against the immediate use of this molecular vaccine in man.

Animals↗
PubMed1985

Nitrocellulose-based assays for the detection of glycolipids and other antigens: mechanism of binding to nitrocellulose.

A variety of simple and rapid assays for the detection of glycolipids by direct binding to nitrocellulose or binding to antibody-coated nitrocellulose, and probing with monoclonal antibodies are described. These include dot-blotting, charge shift electrophoresis and electroblotting. It is shown that the direct binding of the Leishmania major glycolipid to nitrocellulose is dependent on its lipid moiety, indicating that the mechanism of binding is probably via hydrophobic interactions. However, the L. major glycolipid from which the lipid moiety has been removed can still be detected by blotting onto nitrocellulose precoated with a monoclonal antibody directed to a carbohydrate epitope. The general approach of blotting onto antibody-coated nitrocellulose thus extends the usefulness of these techniques to cases in which the antigen to be detected does not bind directly to nitrocellulose.

Antigens, Protozoan↗
PubMed1985

The Leishmania receptor for macrophages is a lipid-containing glycoconjugate.

The glycoconjugate of Leishmania major recognized by the monoclonal antibody WIC-79.3 exists in two forms. The cellular form associated with the promastigote is a population of amphipathic molecules consistent with membrane insertion. In contrast, the extracellular form mainly consists of hydrophilic molecules, and probably arises by cleavage of the cellular form by an endogenous phospholipase. The hydrophilic population of extracellular glycoconjugate molecules binds specifically to macrophages but not to T or B lymphoid cells. Binding of the glycoconjugate and also intact promastigotes to macrophages in vitro is specifically inhibited by Fab fragments of WIC-79.3. These data indicate that the L. major glycoconjugate is the parasite receptor for macrophages, and hence the molecule directly involved in the initiation of infection.

Animals↗
PubMed1985

Phenotypic diversity of cloned lines of Leishmania major promastigotes.

In vitro cultured promastigotes of virulent (V) and avirulent (A) cloned lines of Leishmania major, and the parental isolate LRC-L137, were examined with respect to morphology, cell size, growth rate, and apparent DNA content. Growth rates of all lines were comparable and both virulent (V121, LRC-L137) and avirulent parasites (A12, A52, A59) exhibited a progressive decrease in apparent DNA content with time in culture, as measured by incorporation of Hoechst Dye 33342. The four cloned lines and the parental isolate showed differences in the content of morphological variants and in the mean body length. Morphologically, there were similarities between A12 and A52 and between A59 and V121. Promastigote populations were also examined for the expression of the target antigen of a previously characterized monoclonal antibody, WIC-79.3. This antibody binds to a membrane antigen that is also present in culture supernatants of Leishmania of A1 serotype. Three different assays with culture supernatants all showed that V121, A59, and A12 were high producers with LRC-L137 and A52, low producers. Similar variation in expression of the 79.3 target antigen was detected in intact organisms of the various lines by immunofluorescence with flow cytometry. No simple correlation was found between the expression or release of the WIC-79.3 target antigen and virulence. The virulence or avirulence of all cloned lines for BALB/c mice remained stable. The data are discussed in terms of differentiation stages of L. major promastigotes and the continuing search for morphological and biochemical markers of virulence.

Animals↗
PubMed1985

Immunization with Leishmania receptor for macrophages protects mice against cutaneous leishmaniasis.

The Leishmania major receptor for macrophages is a lipid-containing glycoconjugate that is recognized by the monoclonal antibody WIC-79.3. When L. major promastigotes were incubated with Fab fragments of WIC-79.3 prior to injection into genetically susceptible mice, their infectivity was decreased. Fab fragments from an irrelevant control antibody of the same class had no effect. The L. major glycolipid was purified from detergent-solubilized promastigotes by affinity chromatography on immobilized WIC-79.3 and used to vaccinate mice that are genetically resistant or susceptible to disease. Genetically resistant mice could be protected totally from cutaneous disease with as little as 5 micrograms of glycolipid. A high but not absolute level of resistance was also induced in the susceptible mice, in which the disease is otherwise fatal. No protection was obtained with the carbohydrate fragment of the glycolipid alone or by injection of the glycolipid in the absence of adjuvant. Genetically susceptible mice, immunized and protected from disease as a result of multiple injections of live avirulent cloned promastigotes of L. major, produced antibodies to the glycolipid of L. major. No antibodies were detected in serum from chronically diseased mice. The data suggest that this functionally important antigen of L. major is a candidate vaccine against cutaneous leishmaniasis.

Adjuvants, Immunologic↗
PubMed1984

An amphipathic sulphated glycoconjugate of Leishmania: characterization with monoclonal antibodies.

A major glycoconjugate of Leishmania tropica major identified by two monoclonal antibodies was shown to be an externally oriented, amphipathic membrane antigen shed into the culture medium in which the parasites grow. This molecule could be labelled metabolically with [3H]glucose, [3H]galactose, [32P]phosphate and [35S]sulphate. It migrated as a polydisperse band upon electrophoresis in SDS-polyacrylamide gels, spanning the region of the gel corresponding to an apparent mol. wt. of 20 000-67 000 daltons. An apparently identical family of molecules could be labelled on the surface of living promastigotes using galactose oxidase and [3H]-sodium borohydride. This molecule was shown to be released into the supernatant over a period of several hours. Detection of the 3H- or 35S-labelled molecule required several days exposure of autoradiographs, but a novel blotting technique using nitrocellulose coated with monoclonal antibody allowed rapid detection of the molecule in charge shift electrophoresis, Western blotting and dot blotting. The electrophoretic mobility of the glycoconjugate in agarose relative to its mobility in Triton X-100 was increased in the presence of deoxycholate, and decreased in the presence of cetyl trimethyl-ammonium bromide, indicating amphipathic properties consistent with insertion into the lipid bilayer of the membrane. Using the dot-blotting technique the glycoconjugate was detected in all virulent and avirulent clones of LRC-L137 and in two additional isolates of L. tropica major (LRC-L287 and LRC-L251), but not in L. donovani or L. mexicana, consistent with the previously described specificity of the antibodies. However, the general approaches used in this paper showed that L. donovani (LRC-L52) and L. mexicana (LRC-L94) synthesize a similar, but antigenically distinct glycoconjugate.

Animals↗
PubMed1984

Vaccination against cutaneous leishmaniasis in mice using nonpathogenic cloned promastigotes of Leishmania major and importance of route of injection.

In vaccination studies, mice have been injected by different routes with living promastigotes of nonpathogenic leishmania followed by cutaneous challenge with pathogenic promastigotes. Parasites used for vaccination have been promastigotes of the cloned parasite lines A12 and A52 derived from Leishmania major isolate L137, or long-term cultured promastigotes of the leishmaniasis recidiva isolate, L32 (L.t.tropica). None of these protozoa causes lesions after cutaneous injection to mice. Disease in previously injected mice has been monitored after cutaneous challenge with promastigotes of a virulent cloned line, V121, derived from isolate L137. Mice used were C57BL/6 (genetically resistant), BALB/c and BALB/c.H-2b (genetically susceptible) and BALB/c.H-2k (also genetically susceptible but sometimes less so than BALB/c). C57BL/6 mice were almost completely resistant to subsequent cutaneous disease when challenged after intraperitoneal injection of viable nonpathogenic cloned promastigotes. In contrast, BALB/c, BALB/c.H-2b and BALB/c.H-2k mice challenged after intravenous or intraperitoneal injection were only protected partially against cutaneous leishmaniasis. These vaccinated mice generally showed persistent low grade cutaneous disease for many months after challenge. High doses of viable L32 promastigotes injected intraperitoneally were also able to induce a degree of resistance to subsequent cutaneous leishmaniasis. Using any protocol, subcutaneous injections have been totally without protective effects as have been killed promastigotes injected by any route to mice. Subcutaneous injections appear to be ineffective rather than counterproductive in that mice injected by both the intravenous and subcutaneous routes with nonpathogenic living cloned promastigotes resemble mice injected by the intravenous route in their disease status following challenge.

Animals↗
PubMed1984

Leishmania major: identification of stage-specific antigens and antigens shared by promastigotes and amastigotes.

A battery of antisera to Leishmania major was used to identify stage-specific antigens, or antigens expressed predominantly in amastigotes or promastigotes. At least 30 protein antigens common to amastigotes and promastigotes could be detected in 35S-methionine labelled preparations. They ranged in molecular weight from 25 000 to 165 000. Two amastigote specific antigens and five antigens expressed predominantly in the amastigote were detected in biosynthetically-labelled preparations. Five promastigote specific antigens were also identified. Antibodies from hyperimmunized mice that were resistant to reinfection with L. major recognized mainly antigens shared by the two life-cycle stages of the parasite. Analysis of parasite antigens on 'western blots' provided a different picture from that obtained by immunoprecipitation and gel electrophoresis of 35S-methionine labelled polypeptides. Only 19 antigens were detected and they were all shared by the two parasite forms. However, the abundance and immunogenicity of some of these antigens may be different in the two life-cycle stages of the parasite. Using the various sera, seven shared membrane antigens were identified in radio-iodinated preparations. Antibodies from hyperimmunized resistant mice that recognized shared antigens in 35S-methionine labelled preparations, detected four amastigote membrane antigens not detected by other sera. The function of the stage-specific antigens remains to be established. It is expected that individual antigens produced by recombinant DNA technology will allow these studies to proceed.

Animals↗
PubMed1983

Isolation and characterization of infective and non-infective clones of Leishmania tropica.

Cloning by limit dilution of an isolate of Leishmania tropica (LRC-L137) that is infective for mice resulted in 7 stable clones, only one of which was infective in BALB/c mice. Three of the non-infective clones that were examined for survival in BALB/c macrophages in vitro seemed to be killed more readily, suggesting failure to establish in macrophages as the basis for non-infectivity in vivo. Promastigotes from three non-infective clones and one infective clone were biosynthetically labelled or surface radioiodinated, and the detergent lysates were analyzed by 2-dimensional gel electrophoresis. The pattern of the radiolabelled cytoplasmic and membrane proteins of promastigotes from all L. tropica clones was similar, with minor differences. All clones as well as the uncloned population bound to the same extent to a series of lectins with galactose and N-acetylgalactosamine as specificities. They also bound in a solid-phase radioimmunoassay to 9 monoclonal antibodies raised against the uncloned L. tropica (LRC-L137). The genetic characterization of four L. tropica clones was attempted by analysis of their isolated kinetoplast DNA. The clones from two schizodemes since they possess kinetoplast DNAs which exhibit similar restriction endonuclease fingerprints and show extensive DNA sequence homology, suggesting that the four clones are closely related and that the non-infective variants may be derived from the infective presumptive parental clone L137-7-121. Further characterization of the clones of L. tropica should allow a better understanding of the genetic basis of parasite virulence in cutaneous leishmaniasis.

Animals↗
PubMed1983

Leishmania tropica major in mice: vaccination against cutaneous leishmaniasis in mice of high genetic susceptibility.

BALB/c and BALB/c.H-2b mice are genetically susceptible to development of persistent and severe disease following cutaneous injection of promastigotes of the protozoan parasite, Leishmania tropica major, whereas C57BL/6 are relatively resistant. Resistance in C57BL/6 can be further increased by intraperitoneal injection of living, but not killed, promastigotes prior to cutaneous challenge. Severely diseased BALB/c mice can show resistance to development of a second cutaneous lesion but apparently only in the advanced stages of systemic life-threatening disease. A striking level of resistance to persistent disease has been demonstrated in BALB/c.H-2b mice pre-injected with frozen and thawed L. t. major-infected macrophages of the continuous macrophage cell line IC-21 (H-2b) together with Corynebacterium parvum. No resistance is seen in recipients of either C. parvum or the crude antigen mixture alone. Protection is afforded by intraperitoneal and not subcutaneous injection of crude antigen plus adjuvant. In these vaccination studies all evidence points to the infected macrophage as most appropriate source of 'host-protective' antigens as well as being the most likely target of host-protective immunity. Resistance is expressed in vaccinated mice as minimal signs of cutaneous disease and rapid resolution of any small lesions which do develop. Frozen and thawed promastigotes plus C. parvum will not induce resistance to persistent disease in BALB/c.H-2b mice and preincubation of promastigotes with sera from resistant vaccinated mice does not influence their capacity to cause cutaneous disease. The results provide baseline data for vaccination attempts in genetically susceptible hosts using isolated L. t. major antigens (and, in particular, infected macrophage antigens) and highlight the utility of the intraperitoneal route of injection and the use of the therapeutic biological, C. parvum, as an adjuvant in such studies.

Animals↗